Compositions and methods for transformation of embryonic explant populations

Abstract

The present disclosure provides novel methods for en bloc transformation or genetic modification of a population of embryonic explants derived from different genetic resources or having different genotypes, which may include one or more steps of explant preparation, explant rehydration, inoculation and co-cultivation with Rhizobiales bacteria or particle bombardment, germination induction, expansion germination induction, and / or regeneration or development of a genetically modified plant or plant part. The methods provided herein may include transforming at least one plant cell of the embryonic explant with a heterologous polynucleotide. The methods provided herein also include methods for regenerating or growing a plurality of genetically modified plants or plant parts from a population of transformed or edited plant cells or explants, and comparing, selecting, or screening for one or more genotypes having improved phenotypes or other culture characteristics.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 328,567, filed April 7, 2022, U.S. Provisional Patent Application No. 63 / 441,369, filed January 26, 2023, and U.S. Provisional Patent Application No. 63 / 492,279, filed March 27, 2023, the entire disclosures of each of which are incorporated herein by reference.

[0002] The present disclosure relates to compositions and methods for genetically modifying populations of plant embryonic explants having different genotypes. [Background technology]

[0003] Crop plants such as corn, wheat, rice, barley, sorghum, soybean, cotton, and canola are important crops and a major source of food in many parts of the world. Genetic modification of embryonic explants has been used to produce such crop plants with improved traits or characteristics. However, there is a continuing need for improved methods for genetically modifying multiple different plant genotypes at once that reduce or eliminate the use of costly and time-consuming plant breeding techniques.

[0004] The present disclosure provides novel compositions and methods for the genetic modification of populations of embryonic explants having different plant genotypes and the regeneration of genetically modified plants or plant parts, thereby reducing or eliminating the use of plant breeding or gene transfer techniques and overcoming many of the challenges and limitations in the art. Summary of the Invention

[0005] In one aspect, the present disclosure provides a method for genetically modifying a population of plant embryo explants, comprising introducing a heterologous polynucleotide molecule en bloc into at least two embryo explants of the population, wherein the at least two plant embryo explants each comprise a meristem, and the population comprises embryo explants of at least two different plant genotypes. In some embodiments, the population is defined as a population of monocotyledonous plant embryo explants. The population of monocotyledonous plant embryo explants is, in further embodiments, defined as a population of corn, wheat, rice, barley, sorghum, or turfgrass embryo explants. In certain embodiments, the population is defined as a population of dicotyledonous plant embryo explants. The population of dicotyledonous plant embryo explants is, in further embodiments, defined as a population of soybean, cotton, or canola embryo explants. In certain embodiments, introducing the heterologous polynucleotide molecule en bloc comprises introducing the heterologous polynucleotide molecule into at least two explants of the population via bacterial-mediated transformation. In other embodiments, introducing the heterologous polynucleotide molecules en bloc comprises introducing the heterologous polynucleotide molecules into at least two explants of the population via Rhizobiales bacteria-mediated transformation. The Rhizobiales bacteria, in some embodiments, are selected from the group consisting of a) Rhizobiaceae, Phyllobacteriaceae, Brucellaceae, Bradyrhizobiaceae, and Xanthobacteraceae bacteria, or b) Agrobacterium, Rhizobium, Sinorhizobium, Mesorhizobium, Phyllobacterium, Ochrobactrum, Bradyrhizobium, and Azorhizobium bacteria. In further embodiments, introducing the heterologous polynucleotide molecules en bloc comprises introducing the heterologous polynucleotide molecules via Agrobacterium-mediated transformation. In certain embodiments, introducing the heterologous polynucleotide molecules en bloc comprises inoculating at least two embryonic explants with an inoculation medium comprising RHIZOBIALES bacteria capable of transforming the at least two embryonic explants with the heterologous polynucleotide molecules.In certain embodiments, a force treatment is applied to the population in contact with the inoculation medium. In other embodiments, the force treatment is applied prior to introducing the heterologous polynucleotide molecules en bloc. In certain embodiments, the force treatment comprises a gravity treatment within a range of about 3,000 x g to about 6,000 x g, about 3,500 x g to about 5,000 x g, or about 3,500 x g to about 4,500 x g. In some embodiments, the method may further comprise contacting at least two embryo explants with a co-culture medium to co-cultivate them with the Rhizobiales bacteria. In certain embodiments, the population of embryo explants is a population of monocotyledonous seed embryo explants, and the method comprises contacting at least two embryo explants of the population with the co-culture medium to co-cultivate them over a co-culture surface area of ​​1 square centimeter (cm). 2 ) or less. In some embodiments, the population of embryo explants is a population of monocotyledonous seed embryo explants, and the method further comprises contacting at least two embryo explants of the population with a co-culture medium and co-culturing them for a period ranging from about 6 days to about 8 days. In additional embodiments, introducing the heterologous polynucleotide molecule en bloc comprises introducing the heterologous polynucleotide molecule into at least two explants of the population by particle bombardment. In certain embodiments, the methods provided by the present disclosure may further comprise introducing a site-specific nuclease en bloc to at least two embryo explants of the population. In certain embodiments, the heterologous polynucleotide molecule comprises a guide RNA molecule, and introducing en bloc comprises introducing the site-specific nuclease into at least two embryo explants. The site-specific nuclease is, in some embodiments, a ribonucleoprotein, and the ribonucleoprotein comprises the site-specific nuclease and the guide RNA molecule.

[0006] In further embodiments, the heterologous polynucleotide molecule may comprise a first expression cassette and a second expression cassette, wherein the first expression cassette may, in certain embodiments, comprise a first selectable marker gene, a first screenable marker gene, a first gene of interest, a nucleotide sequence encoding a first site-specific nuclease, or a nucleotide sequence encoding a first guide RNA, and the second expression cassette may, in certain embodiments, comprise a second selectable gene, a second screenable marker gene, a second gene of interest, a nucleotide sequence encoding a second site-specific nuclease, or a nucleotide sequence encoding a second guide RNA.

[0007] In certain embodiments, introducing heterologous polynucleotide molecules en bloc comprises introducing at least two heterologous polynucleotides into at least two explants of the population, wherein the at least two heterologous polynucleotides are different. In further embodiments, the at least two heterologous polynucleotides comprise a first heterologous polynucleotide comprising a first expression cassette and a second heterologous polynucleotide comprising a second expression cassette. The first expression cassette, in some embodiments, may comprise a nucleotide sequence encoding a first selectable marker gene, a first screenable marker gene, a first gene of interest, a first site-specific nuclease, or a first guide RNA, and the second expression cassette, in some embodiments, may comprise a nucleotide sequence encoding a second selectable marker gene, a second screenable marker gene, a second gene of interest, a second site-specific nuclease, or a second guide RNA.

[0008] In certain embodiments, the methods of genetically modifying a population of plant embryo explants provided by the present disclosure may further include culturing at least two embryo explants in contact with a first emergence-inducing medium comprising a first auxin and a first cytokinin, which in certain embodiments comprises a high cytokinin-to-auxin ratio. In some embodiments, the first auxin in the first emergence-inducing medium is selected from the group consisting of 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid or p-chlorophenoxyacetic acid (4-CPA or pCPA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,3,5-triiodobenzoic acid (TIBA), phenylacetic acid (PAA), and 3,6-dichloro-2-methoxybenzoic acid (dicamba). In further embodiments, the concentration of the first auxin in the first emergence-inducing medium is from about 0.02 mg / L to about 25 mg / L, or from about 1 mg / L to about 2 mg / L. In certain embodiments, the first cytokinin in the first germination induction medium is selected from the group consisting of 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-topolin). The concentration of the first cytokinin in the first germination induction medium, in certain embodiments, ranges from about 0.1 mg / L to about 50 mg / L. In some embodiments, the population of embryo explants is a population of monocotyledonous seed embryo explants, and the method includes placing at least two embryo explants in a first germination induction medium and a first germination induction medium on a surface area of ​​1 square centimeter (cm). 2 ) or less than about 3.9 embryonic explants per 1000 cells / well.

[0009] In certain embodiments, the method of genetically modifying a population of plant embryo explants provided by the present disclosure may further include culturing at least two embryo explants in contact with a second germination-inducing medium comprising a first auxin or a second auxin and a first cytokinin or a second cytokinin. The embryo explants are contacted with the second germination-inducing medium and cultured, in some embodiments, at a temperature ranging from about 20°C to about 32°C, from about 25°C to about 29°C, or from about 27°C to about 28°C. In certain embodiments, the second germination-inducing medium comprises a high cytokinin-to-auxin ratio. In specific embodiments, the second germination-inducing medium comprises: a) the first auxin and the first cytokinin; b) the first auxin and the second cytokinin; c) the second auxin and the first cytokinin; or d) the second auxin and the second cytokinin. In certain embodiments, the population of embryo explants is a population of monocotyledonous seed embryo explants, and the method includes cultivating at least two embryo explants in a second germination induction medium and a second germination induction surface area of ​​1 square centimeter (cm 2In some embodiments, the first auxin or the second auxin in the second germination-inducing medium is selected from the group consisting of 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid or p-chlorophenoxyacetic acid (4-CPA or pCPA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,3,5-triiodobenzoic acid (TIBA), phenylacetic acid (PAA), and 3,6-dichloro-2-methoxybenzoic acid (dicamba). In some embodiments, the first or second cytokinin in the second germination-inducing medium is selected from the group consisting of 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-topolin). In specific embodiments, the concentration of the first or second cytokinin in the second germination-inducing medium is in the range of about 0.1 mg / L to about 1 mg / L, about 0.1 mg / L to about 5 mg / L, about 0.1 mg / L to about 50 mg / L, about 0.1 mg / L to about 25 mg / L, about 0.5 mg / L to about 25 mg / L, or about 2 mg / L to about 10 mg / L. In a specific embodiment, the concentration of the first auxin or the second auxin in the second germination-inducing medium is about 0.01 mg / L to about 25 mg / L, about 0.02 mg / L to about 10 mg / L, or about 1 mg / L to about 2 mg / L. In a specific embodiment, the heterologous polynucleotide molecule comprises a selectable marker gene, the second germination-inducing medium comprises a selective agent, and the selectable marker gene confers resistance to the selective agent to the plant.

[0010] In specific embodiments, the methods of genetically modifying a population of plant embryo explants provided by the present disclosure can further include contacting at least two embryo explants, or any progeny generation of cells thereof, with a regeneration medium to regenerate or grow a plurality of genetically modified plants or plant parts. In some embodiments, the population is a population of monocotyledonous seed embryo explants, and the method includes contacting at least two embryo explants with a regeneration medium and a regeneration surface area of ​​1000 square centimeters (cm 2(a) contacting the cultured monocotyledonous seed embryo explants with a first regeneration medium at a density of about 2.6 or less embryo explants per cultured monocotyledonous seed embryo explants to regenerate or grow a plurality of genetically modified plants or plant parts. In still other embodiments, the disclosed method may further include contacting a population of cultured monocotyledonous seed embryo explants with a first regeneration medium to regenerate, transferring the population of cultured monocotyledonous seed embryo explants or a subset of the population of cultured monocotyledonous seed embryo explants to a second regeneration medium, and contacting the plurality of genetically modified monocotyledonous plants or plant parts with the second regeneration medium to regenerate. The regeneration medium, the first regeneration medium, or the second regeneration medium, in some embodiments, has a low salt concentration. In some embodiments, the heterologous polynucleotide molecule comprises a selectable marker gene, the regeneration medium comprises a selective agent, and the selectable marker gene confers resistance to the plant to the selective agent. In certain embodiments, the genetically modified plant or plant part comprises at least one genetic modification. In certain embodiments, the genetic modification can include the integration or insertion of a heterologous polynucleotide molecule or a fragment thereof into the genome of a plurality of genetically modified plants or plant parts, where the integration or insertion includes at least one expression cassette or at least one transgene. In some embodiments, the genetic modification can include edits introduced into the genome of a plurality of genetically modified plants or plant parts by genome editing techniques using site-specific nucleases and / or guide RNA molecules. In certain embodiments, the heterologous polynucleotide molecule includes at least one expression cassette, where the at least one expression cassette encodes a site-specific nuclease or a guide RNA molecule, or the heterologous polynucleotide molecule includes at least two expression cassettes, where the first expression cassette encodes a site-specific nuclease and the second expression cassette encodes a guide RNA molecule. In certain embodiments, the genetically modified plant part includes a shoot or root. In certain embodiments, the genetically modified plant part includes a seed. In further embodiments, the genetically modified plant or plant part is non-chimeric. The genetically modified plants or plant parts are, in certain embodiments, cultured and / or regenerated without producing a callus tissue culture.

[0011] In further embodiments, the methods of genetically modifying a population of plant embryo explants provided by the present disclosure may further include contacting at least two embryo explants, or any progeny generations of cells thereof, with a regeneration medium to regenerate or grow a plurality of genetically modified plants or plant parts, wherein each of the plurality of genetically modified plants or plant parts comprises at least one genetic modification. In certain embodiments, the methods provided by the present disclosure may additionally include selecting a genetically modified plant comprising at least one genetic modification and crossing the plant with itself or a second plant to obtain progeny plants or seeds. The selecting, in some embodiments, includes identifying the genotype of the genetically modified plant and selecting plants comprising the genotype. In further embodiments, the methods provided by the present disclosure may further include selecting a first genetically modified plant comprising at least a first genetic modification and a second genetically modified plant comprising at least a second genetic modification; crossing the first genetically modified plant with itself or a first different plant to obtain a first progeny plant or seed; and crossing the second genetically modified plant with either itself, the first genetically modified plant, or a second different plant to obtain a second progeny plant or seed. The first different plant and / or the second different plant may, in certain embodiments, have a different genotype from the first genetically modified plant and / or the second genetically modified plant. In certain embodiments, the selecting step includes identifying a first genotype of the first genetically modified plant and selecting a first genetically modified plant comprising the first genotype; and identifying a second genotype of the second genetically modified plant and selecting a second genetically modified plant comprising the second genotype.

[0012] In certain embodiments, the population of embryo explants provided by the present disclosure comprises embryo explants having an internal moisture content ranging from about 3% to about 25% prior to introduction of a heterologous polynucleotide molecule. In specific embodiments, the population comprises embryo explants comprising an apical portion of the hypocotyl lacking roots, with the remainder of the seed from which the embryo explant was prepared being substantially removed from the embryo explant. In certain embodiments, the population is defined as a population of dried, dry-excised, wet-excised, or wet seed embryo explants. In other embodiments, the population is defined as a population of mature or immature seed embryo explants. In further embodiments, the population comprising embryo explants is prepared from a population of monocotyledonous seeds under conditions in which the embryo explants do not germinate, remain viable, and remain capable of genetic modification. The population may in certain embodiments be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 40 0, at least 450, or at least 500 different plant genotypes, or from 2 to about 750, from 2 to about 600, from about 10 to about 500, from about 15 to about 400, from about 20 to about 300, from about 25 to about 200, from about 10 to about 150, from about 10 to about 100, from about 10 to about 90, from about 10 to about 80, from about 10 to about 70, from about 10 to about 60, from about 10 to about 50, from about 10 to about 40, from about 10 to about 30, from about 10 to about 20, from 2 to about 50, from 2 to about 40, from 2 to about 30, from 2 to about 20, from 2 to about 15, or from 2 to about 10 different plant genotypes.At least two embryonic explants of the population may in certain embodiments be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least At most, the number of different plant genotypes is 350, at least 400, at least 450, or at least 500, or 2 to about 600, 2 to about 600, about 10 to about 500, about 15 to about 400, about 20 to about 300, about 25 to about 200, about 10 to about 150, about 10 to about 100, about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, 2 to about 50, 2 to about 40, 2 to about 30, 2 to about 20, 2 to about 15, or 2 to about 10 different plant genotypes.The population may in some embodiments be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, or at least 50,000 embryonic explants, or from about 2 to about 50,000. 00, about 1,000 to about 50,000, about 1,000 to about 40,000, about 1,000 to about 30,000, about 1,000 to about 20,000, about 1,000 to about 10,000, about 1,000 to about 9,000, about 1,000 to about 8,000, about 1,000 to about 7,000, about 1,000 to about 6,000, about 1,000 to about 5,000, about 1,000 to about 4,000, about 1,000 to about 3,000, about 5,000 to about 50,000, about 5,000 to about 40,000, about 5,000 to about 30,000, about 5,000 to about 20,0 00, about 5,000 to about 10,000, about 2 to about 1000, about 5 to about 900, about 5 to about 800, about 5 to about 700, about 5 to about 600, about 5 to about 500, about 10 to about 500, about 15 to about 400, about 20 to about 300, about 25 to about 200, about 10 to about 150, about 10 to about 100, about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, 2 to about 50, 2 to about 40, 2 to about 30, 2 to about 20, 2 to about 15, or 2 to about 10 embryonic explants.In some embodiments, the population includes at least one embryonic explant with at least one unidentified genotype prior to the collective introduction of the heterologous polynucleotide molecule. In other embodiments, at least two different genotypes of the embryonic explants are known. In further embodiments, each embryonic explant in the population is of known genotype.

[0013] In certain embodiments, the heterologous polynucleotide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, The vectors are introduced en bloc into embryonic explants having at least 400, at least 450, or at least 500 different plant genotypes, or from 2 to about 600, from 2 to about 600, from about 10 to about 500, from about 15 to about 400, from about 20 to about 300, from about 25 to about 200, from about 10 to about 150, from about 10 to about 100, from about 10 to about 90, from about 10 to about 80, from about 10 to about 70, from about 10 to about 60, from about 10 to about 50, from about 10 to about 40, from about 10 to about 30, from about 10 to about 20, from 2 to about 50, from 2 to about 40, from 2 to about 30, from 2 to about 20, from 2 to about 15, or from 2 to about 10 different plant genotypes.In a further embodiment, a heterologous polynucleotide molecule is introduced into an embryonic explant and a plurality of genetically modified plants or plant parts are regenerated therefrom, wherein the plurality of genetically modified plants or plant parts is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 15 0, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 different plant genotypes, or 2 to about 600, 2 to about 600, about 10 to about 500, about 15 to about 400, about 20 to about 300, about 25 to about 200, about 10 to about 150, about 10 to about 100, about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, 2 to about 50, 2 to about 40, 2 to about 30, 2 to about 20, 2 to about 15, or 2 to about 10 different plant genotypes.In additional embodiments, the plurality of genetically modified plants or plant parts comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, At least 350, at least 400, at least 450, or at least 500 different plant genotypes, or 2 to about 600, 2 to about 600, about 10 to about 500, about 15 to about 400, about 20 to about 300, about 25 to about 200, about 10 to about 150, about 10 to about 100, about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, 2 to about 50, 2 to about 40, 2 to about 30, 2 to about 20, 2 to about 15, or 2 to about 10 different plant genotypes.

[0014] In certain embodiments, the method of genetically modifying a population of plant embryo explants provided by the present disclosure may further include identifying the genotype of at least one of the embryo explants of the population or at least one of the genetically modified plants or plant parts. In some embodiments, identifying the genotype includes detecting at least one genetic marker in at least one embryo explant or at least one genetically modified plant or plant part, wherein the at least one genetic marker comprises a polynucleotide sequence characteristic of the genotype. In specific embodiments, the polynucleotide sequence is exclusively characteristic of the genotype. Identifying the genotype, in further embodiments, comprises detecting at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or about 2 to about 26, about 5 to about 26, about 10 to about 26, about 15 to about 26, or about 20 to about 26 genetic markers in at least one embryonic explant or at least one genetically modified plant or plant part, wherein each of the genetic markers comprises a polynucleotide sequence characteristic of that genotype. In certain embodiments, each of the genetic markers comprises a polynucleotide sequence exclusively characteristic of that genotype. In some embodiments, identifying the genotype comprises identifying the genotype before or after introducing the heterologous polynucleotide molecule into at least two embryo explants of the population; identifying the genotype before or after co-culturing at least two embryo explants of the population; identifying the genotype before or after contacting and culturing at least two embryo explants of the population with a first germination-inducing medium; identifying the genotype before or after contacting and culturing at least two embryo explants of the population with a second germination-inducing medium; or identifying the genotype before or after regenerating or growing a plurality of genetically modified plants or plant parts from at least two embryo explants of the population or any progeny generation of cells thereof.Identifying the genotype, in certain embodiments, comprises performing genetic sequencing on a sample comprising polynucleotide molecules derived from or obtained from at least one embryonic explant or at least one genetically modified plant or plant part, and detecting at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 genetic markers in the sample, wherein the polynucleotide molecules are, or are derived from, genomic DNA molecules or fragments thereof or mRNA molecules or fragments thereof, and each of the genetic markers comprises a polynucleotide sequence characteristic of the genotype. In other embodiments, genotyping comprises contacting a sample comprising polynucleotide molecules derived from or obtained from at least one embryonic explant or at least one genetically modified plant or plant part with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 polynucleotide probe(s), each of which is specific for one genetic marker, and subjecting the sample and polynucleotide probe(s) to stringent hybridization. and detecting hybridization of the polynucleotide probe(s) to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 genetic markers in the sample, wherein the polynucleotide molecules are, or are derived from, genomic DNA molecules or fragments thereof or mRNA molecules or fragments thereof, and each of the genetic markers comprises a polynucleotide sequence characteristic of the genotype.In various embodiments, identifying the genotype comprises amplifying or restricting the polynucleotide molecule. In specific embodiments, identifying the genotype comprises identifying the genotype of a plurality of embryonic explants or a plurality of genetically modified plants or plant parts. In certain embodiments, identifying the genotype comprises identifying the genotype of at least one of the embryonic explants or at least one of the genetically modified plants or plant parts of the population. In some embodiments, identifying the genotype comprises identifying the genotype of a plurality of embryonic explants or a plurality of genetically modified plants or plant parts.

[0015] In certain embodiments, identifying the genotype comprises detecting at least two genetic markers in at least two of the embryonic explants or at least two of the genetically modified plants or plant parts of the population. In further embodiments, the at least two genetic markers comprise a first genetic marker and a second genetic marker, wherein the first genetic marker comprises a first polynucleotide sequence characteristic of the first genotype and the second genetic marker comprises a second polynucleotide sequence characteristic of the second genotype. In certain embodiments, the first polynucleotide sequence is exclusively characteristic of the first genotype, or the second polynucleotide sequence is exclusively characteristic of the second genotype. In some embodiments, the identifying comprises detecting at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 genetic markers in at least two embryonic explants or at least two genetically modified plants or plant parts, wherein at least two of the genetic markers comprise a polynucleotide sequence characteristic of a first genotype and at least one of the genetic markers comprises a polynucleotide sequence characteristic of a second genotype.Identifying, in a further embodiment, comprises performing genetic sequencing on at least a first sample and a second sample, wherein the first sample comprises a first polynucleotide molecule derived from or obtained from a first embryonic explant or a first genetically modified plant or plant part, and the second sample comprises a second polynucleotide molecule derived from or obtained from a second embryonic explant or a second genetically modified plant or plant part, and detecting at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 genetic markers in the first sample and the second sample, wherein the first polynucleotide molecule is , the first polynucleotide molecule is a genomic DNA molecule or a fragment thereof or an mRNA molecule or a fragment thereof, or is derived from a genomic DNA molecule or a fragment thereof or an mRNA molecule or a fragment thereof, or the second polynucleotide molecule is a genomic DNA molecule or a fragment thereof or an mRNA molecule or a fragment thereof, or is derived from a genomic DNA molecule or a fragment thereof or an mRNA molecule or a fragment thereof, and each of the genetic markers comprises a polynucleotide sequence characteristic of the genotype, or the genetic markers comprise a first genetic marker and a second genetic marker, and the first genetic marker comprises a first polynucleotide sequence specific to the first genotype, and the second genetic marker comprises a second polynucleotide sequence specific to the second genotype.

[0016] In certain embodiments, the method of genetically modifying a population of plant embryo explants provided by the present disclosure may further include identifying a genetic modification present in at least one embryo explant or at least one genetically modified plant or plant part of the population, and selecting an embryo explant or genetically modified plant or plant part of the population that contains the genetic modification, wherein the selected embryo explant or selected genetically modified plant or plant part further contains at least one genetic marker characteristic of its genotype, or the selected embryo explant or selected genetically modified plant or plant part further does not contain at least one genetic marker characteristic of its genotype. In some embodiments, the method of genetically modifying a population of plant embryo explants provided by the present disclosure may further include regenerating or growing a regenerated genetically modified plant or plant part from any progeny generation of the selected embryo explant or its cells, or crossing the selected genetically modified plant with itself or a different plant to obtain progeny plants or seeds. In these embodiments, the method may further include identifying the genetic modification present in at least two embryo explants or at least two genetically modified plants or plant parts of the population, and selecting a first embryo explant or first genetically modified plant or plant part of the population that comprises the genetic modification and a second embryo explant or second genetically modified plant or plant part of the population that comprises the genetic modification, wherein the first selected embryo explant or first selected genetically modified plant or plant part further comprises the first genetic marker and / or the second genetic marker, or wherein the first selected embryo explant or first selected genetically modified plant or plant part does not further comprise the first genetic marker and / or the second genetic marker, and wherein the second selected embryo explant or second selected genetically modified plant or plant part further comprises the first genetic marker and / or the second genetic marker, or wherein the second selected embryo explant or second selected genetically modified plant or plant part does not further comprise the first genetic marker and / or the second genetic marker.In certain embodiments, methods provided by the present disclosure may include identifying at least two genetic modifications present in at least two embryo explants or at least two genetically modified plants or plant parts of the population, wherein the at least two genetic modifications comprise a first genetic modification and a second genetic modification, and selecting a first embryo explant or first genetically modified plant or plant part of the population that comprises the first genetic modification and a second embryo explant or second genetically modified plant or plant part of the population that comprises the second genetic modification, wherein the first selected embryo explant or first selected genetically modified plant or plant part is selected from the population. The selected plant or plant part further comprises the first genetic marker and / or the second genetic marker, or the first selected embryo explant or the first selected genetically modified plant or plant part does not further comprise the first genetic marker and / or the second genetic marker, and the second selected embryo explant or the second selected genetically modified plant or plant part further comprises the first genetic marker and / or the second genetic marker, or the second selected embryo explant or the second selected genetically modified plant or plant part does not further comprise the first genetic marker and / or the second genetic marker.

[0017] In some embodiments, the methods of genetically modifying a population of plant embryo explants provided by the present disclosure can further include regenerating or growing a first regenerated genetically modified plant or plant part from any progeny generation of the first selected embryo explant or cells thereof, or regenerating or growing a second regenerated genetically modified plant or plant part from any progeny generation of the second selected embryo explant or cells thereof. In some embodiments, the present disclosure provides methods of genetically modifying a population of plant embryo explants, further comprising observing the cultural characteristics of the first selected embryo explant and the second selected embryo explant, observing the phenotype of the first regenerated genetically modified plant or plant part and the second regenerated genetically modified plant or plant part, or observing the phenotype of the first selected genetically modified plant or plant part and the second selected genetically modified plant or plant part. Methods of genetically modifying a population of plant embryo explants provided by the present disclosure may, in some embodiments, further include comparing the culture characteristics of the first selected embryo explant and the second selected embryo explant and determining whether the culture characteristics of the first selected embryo explant or the second selected embryo explant are superior; comparing the phenotype of the first regenerated genetically modified plant or plant part and the second regenerated genetically modified plant or plant part and determining whether the phenotype of the first regenerated genetically modified plant or plant part or the second regenerated genetically modified plant or plant part is superior; or comparing the phenotype of the first selected genetically modified plant or plant part and the second selected genetically modified plant or plant part and determining whether the phenotype of the first selected genetically modified plant or plant part or the second selected genetically modified plant or plant part is superior.In certain embodiments, these methods may further include regenerating or growing a first regenerated genetically modified plant or plant part from any progeny generation of the first selected embryonic explant or cells thereof, or regenerating or growing a second regenerated genetically modified plant or plant part from any progeny generation of the second selected embryonic explant or cells thereof, based on the culture characteristics of the first selected embryonic explant and the second selected embryonic explant. In certain embodiments, the methods provided by the present disclosure may further include crossing the first selected genetically modified plant with itself or a different plant to obtain a first progeny plant or seed, crossing the second selected genetically modified plant with itself or a different plant to obtain a second progeny plant or seed, crossing the first regenerated genetically modified plant with itself or a different plant to obtain a third progeny plant or seed, or crossing the second regenerated genetically modified plant with itself or a different plant to obtain a fourth progeny plant or seed.

[0018] In certain embodiments, the present disclosure provides a method for genetically modifying a population of plant embryo explants, further comprising introducing a second heterologous polynucleotide molecule into at least one explant of a second population of embryo explants, wherein at least one embryo explant of the second population has the same genotype or a different genotype compared to at least one embryo explant of the population; at least one genetically modified plant or plant part; selected embryo explant; or selected genetically modified plant or plant part. In certain embodiments, the method provided by the present disclosure may comprise detecting a genetic modification of at least one embryo explant of the population or at least one genetically modified plant or plant part.

[0019] In further embodiments, the method of genetically modifying a population of plant embryo explants provided by the present disclosure may further include identifying the genotype of at least one of the modified plants or plant parts and correlating the genotype with at least one cultural trait or phenotype. Identifying, in certain embodiments, may further include identifying a genetic marker or quantitative trait locus (QTL) associated with the at least one cultural trait or phenotype. In certain embodiments, the at least one cultural trait or phenotype is selected from the group consisting of explant excision efficiency, regeneration efficiency, genetic modification efficiency, transformation efficiency, shoot development, and ability to regenerate into a genetically modified plant or plant part. In certain embodiments, the phenotype is an observable plant trait or results from the expression of a selectable or screenable marker. The plant trait can be observed using any method known in the art. Non-limiting examples of observable plant traits include plant height, panicle height, stilt root color, internode orientation, internode length, leaf color, leaf length, leaf width, leaf sheath pubescence, leaf margin waviness, tassel length, anther color, glume color, silk color, silk position, husk opening, husk color, rachis diameter, kernel row number, kernel per row number, endosperm type, endosperm color, relative maturity, flower color, hilum color, seed coat color, seed shape, leaf shape, and growth habit. Selectable marker genes that can be used include, but are in no way limited to, aroA, EPSPS, aadA, pat, bar, hph (hygromycin B phosphotransferase), DMO (dicamba monooxygenase), CAT, and NPT II. Plant selectable marker genes are generally used to confer resistance to selective agents, but additional screenable or scorable marker gene(s) can also be used in addition to the selectable marker, optionally in conjunction with a gene of agronomic interest.Such screenable marker genes can include, for example, uidA for β-glucuronidase (GUS; e.g., as described in U.S. Pat. No. 5,599,670; incorporated herein by reference), or gfp for green fluorescent protein and its variants (GFP; described in U.S. Pat. Nos. 5,491,084 and 6,146,826; all of which are incorporated herein by reference), or crtB for phytoene synthase (e.g., as described in U.S. Pat. Nos. 8,237,016 and 10,240,165; all of which are incorporated herein by reference). Additional examples of screenable markers can include secreted markers, the expression of which results in the secretion of a molecule(s) that can be detected as a means of identifying transformed cells.

[0020] In additional embodiments, the phenotype is a genetic modification phenotype, where the genetic modification results from integration of a heterologous polynucleotide molecule, or a fragment thereof, into the genome of at least one genetically modified plant or plant part, and the heterologous polynucleotide molecule comprises an expression cassette encoding a gene of interest, a site-specific nuclease, or a guide RNA molecule. In some embodiments, the methods provided by the present disclosure may further include introgressing a chromosomal segment that confers at least one cultural trait or at least one phenotype into a plant having a plant genotype that lacks the cultural trait or phenotype in the absence of the chromosomal segment, or crossing a genetically modified plant containing a chromosomal segment that confers at least one cultural trait or at least one phenotype with a self or second plant to produce a progeny plant or seed containing the chromosomal segment.

[0021] In some embodiments, the present disclosure provides a method for genetically modifying a population of plant embryo explants, comprising introducing a heterologous polynucleotide molecule into at least two embryo explants of the population en bloc, wherein the at least two different genotypes of embryo explants include embryo explants of a first genotype and embryo explants of a second genotype, and the embryo explants of the first genotype and the embryo explants of the second genotype are present in the population at a predetermined ratio. In specific embodiments, the predetermined ratio is determined based on at least one culture characteristic associated with the first genotype, the second genotype, or the first genotype and the second genotype. In certain embodiments, the at least one culture characteristic is selected from the group consisting of explant excision efficiency, regeneration efficiency, shoot development efficiency, genetic modification efficiency, transformation efficiency, and ability to regenerate into a genetically modified plant or plant part. In some embodiments, the predetermined ratio of embryo explants of the first genotype and the second genotype comprises approximately equal numbers of embryo explants of the first genotype and the second genotype. In further embodiments, a predetermined ratio of embryo explants of a first genotype and a second genotype results in an approximately equal number of regenerated genetically modified plants or plant parts of the first genotype and the second genotype. In some embodiments, the first genotype is associated with favorable cultural characteristics relative to the second genotype, and the method comprises modifying the predetermined ratio to include an increased number of embryo explants of the second genotype relative to the first genotype in the population, or the second genotype is associated with favorable cultural characteristics relative to the first genotype, and the method comprises modifying the predetermined ratio to include an increased number of embryo explants of the first genotype relative to the second genotype in the population. In certain embodiments, a predetermined ratio of embryo explants of a first genotype and a second genotype results in an approximately equal number of regenerated genetically modified plants or plant parts of the first genotype and the second genotype. Favorable cultural properties may, in certain embodiments, result in an increase in explant excision efficiency, regeneration efficiency, shoot development efficiency, genetic modification efficiency, transformation efficiency, or ability to regenerate into a genetically modified plant or part.

[0022] In specific embodiments, the method of genetically modifying a population of plant embryo explants provided by the present disclosure may further include contacting at least two embryo explants, or any progeny generations of cells thereof, with a regeneration medium to regenerate or grow a plurality of genetically modified plants or plant parts, and observing at least one culture characteristic or phenotype of at least one of the embryo explants or at least one of the genetically modified plants or plant parts of the population. The at least one culture characteristic or phenotype, in some embodiments, is related to the genetic modification of the at least one embryo explant or at least one genetically modified plant or plant part. In further embodiments, the method provided by the present disclosure may further include observing a first culture characteristic or phenotype of at least one embryo explant of a first genotype or at least one genetically modified plant or plant part of a first genotype, and observing a second culture characteristic or phenotype of at least one embryo explant of a second genotype or at least one genetically modified plant or plant part of a second genotype. In certain embodiments, the first culture characteristic or phenotype and the second culture characteristic or phenotype are the same. In additional embodiments, the first culture characteristic or phenotype and the second culture characteristic or phenotype are different. In some embodiments, the methods provided by the present disclosure may further include evaluating at least one embryo explant or at least one genetically modified plant or plant part of a first genotype and at least one embryo explant or at least one genetically modified plant or plant part of a second genotype by comparing the first culture characteristic or phenotype with the second culture characteristic or phenotype. In specific embodiments, each of the multiple genetically modified plants or plant parts comprises at least one genetic modification. In certain embodiments, the methods provided by the present disclosure may further include selecting a genetically modified plant comprising at least one genetic modification and crossing the genetically modified plant with itself or a second plant to obtain a progeny plant or seed, wherein the second plant has the same or a different genotype as the genetically modified plant.In certain embodiments, the selecting comprises identifying the genotype of the genetically modified plant and selecting a genetically modified plant comprising that genotype.

[0023] In certain embodiments, the present disclosure provides a method for genetically modifying a population of plant embryo explants, the method comprising simultaneously introducing a heterologous polynucleotide molecule into at least two embryo explants of the population. In certain embodiments, the simultaneously introducing comprises site-directed integration of the heterologous polynucleotide or a fragment thereof. In some embodiments, the heterologous polynucleotide molecule comprises at least one expression cassette, wherein the at least one expression cassette comprises a selectable marker gene, a screenable marker gene, a gene of interest, a nucleotide sequence encoding a guide RNA molecule, or a nucleotide sequence encoding a site-specific nuclease. In certain embodiments, the heterologous polynucleotide molecule comprises or encodes a guide RNA. In further embodiments, the simultaneously introducing comprises simultaneously introducing the heterologous polynucleotide molecule into the population. In certain embodiments, the simultaneously introducing comprises introducing the heterologous polynucleotide molecule into the population of embryo explants while the population resides together in a single container.

[0024] In some embodiments, the present disclosure provides a method for genetically modifying a population of plant embryo explants, comprising simultaneously introducing a heterologous polynucleotide molecule into at least two embryo explants of the population, wherein the population comprises embryo explants of at least two different plant genotypes, and the population is defined as a population of dried dicotyledonous plant embryo explants. In certain embodiments, the population is defined as a population of soybean, cotton, or canola embryo explants.

[0025] In some embodiments, the present disclosure provides a method for genetically modifying a population of plant embryo explants, comprising introducing a ribonucleoprotein or a site-specific nuclease into at least two plant embryo explants of the population, each of the at least two plant embryo explants comprising a meristem, and the population comprising plant embryo explants of at least two different plant genotypes. In certain embodiments, the ribonucleoprotein comprises a site-specific nuclease and a guide RNA molecule.

[0026] In certain embodiments, the methods provided by the present disclosure may further include excising a population of embryo explants from the population of plant seeds, wherein the excision is performed before introducing the heterologous polynucleotide molecule, ribonucleoprotein, or site-specific nuclease en bloc, and the population of plant seeds comprises plant seeds of at least two different plant genotypes. In certain embodiments, the methods provided by the present disclosure may further include sorting the population of plant seeds into at least two batches of plant seeds according to plant seed size, plant seed shape, or a combination thereof, prior to excising the population of embryo explants. In a further embodiment, the at least two batches of plant seeds comprise a first batch of plant seeds and a second batch of plant seeds, wherein the population of plant embryo explants comprises a first batch of embryo explants and a second batch of embryo explants, and the excising comprises excising the first batch of embryo explants from the first batch of plant seeds and the second batch of embryo explants from the second batch of plant seeds using the same excision method; or excising the first batch of embryo explants from the first batch of plant seeds and the second batch of embryo explants from the second batch of plant seeds using different excision methods.

[0027] In some embodiments, the present disclosure provides a method for sorting a first population of plant seeds having a first genotype into at least two batches of plant seeds, including a first batch of plant seeds and a second batch of plant seeds, wherein the first batch of plant seeds comprises a first plant seed size, a first plant seed shape, or a combination thereof, and the second batch of plant seeds comprises a second plant seed size, a second plant seed shape, or a combination thereof; and sorting a second population of plant seeds having a second genotype into at least two batches of plant seeds, including the first batch of plant seeds and the second batch of plant seeds. and sorting the first batch of plant seeds into batches, wherein the first batch of plant seeds comprises a first plant seed size, a first plant seed shape, or a combination thereof, and the second batch of plant seeds comprises a second plant seed size, a second plant seed shape, or a combination thereof, wherein the population of plant seeds comprises a first predetermined ratio of plant seeds derived from the first batch of plant seeds of the first genotype and the second batch of plant seeds of the first genotype, and a second predetermined ratio of plant seeds derived from the first batch of plant seeds of the second genotype and the second batch of plant seeds of the second genotype. In some embodiments, the first predetermined ratio of plant seeds of the first genotype and the second predetermined ratio of plant seeds of the second genotype are approximately equal. In certain embodiments, the number of plant seeds from the first batch of plant seeds of the first genotype is approximately equal to the number of plant seeds from the first batch of plant seeds of the second genotype, and / or the number of plant seeds from the second batch of plant seeds of the first genotype is approximately equal to the number of plant seeds from the second batch of plant seeds of the second genotype. In some embodiments, the at least two different genotypes include the first genotype and the second genotype, and the excising results in the excision of approximately equal numbers of embryo explants of the first genotype and the second genotype.

[0028] In certain embodiments, the plant seeds of at least two different genotypes may include plant seeds of a first genotype and plant seeds of a second genotype, wherein the plant seeds of the first genotype and the plant seeds of the second genotype are present in the population at a predetermined ratio. In certain embodiments, the predetermined ratio is determined based on at least one cultural characteristic associated with the first genotype or the second genotype. Non-limiting examples of such cultural characteristics include explant excision efficiency, regeneration efficiency, shoot development efficiency, genetic modification efficiency, transformation efficiency, and regeneration ability into genetically modified plants or plant parts. In some embodiments, the predetermined ratio of plant seeds of the first genotype and the second genotype results in approximately equal numbers of regenerated genetically modified plants or plant parts of the first genotype and the second genotype. In certain embodiments, the first genotype is associated with a favorable cultural trait and the method comprises modifying the population to include a predetermined ratio of plant seeds of the second genotype relative to the first genotype, or the second genotype is associated with a favorable cultural trait and the method comprises modifying the population to include a predetermined ratio of plant seeds of the first genotype relative to the second genotype, wherein the predetermined ratio of plant seeds of the first genotype and the second genotype results in approximately equal numbers of regenerated genetically modified plants or plant parts of the first genotype and the second genotype. Non-limiting examples of such favorable cultural traits include increased explant excision efficiency, regeneration efficiency, shoot development efficiency, genetic modification efficiency, transformation efficiency, or ability to regenerate into genetically modified plants or parts.

[0029] In some embodiments, the population is defined as a population of dicotyledonous plant embryonic explants, and the co-culture medium comprises at least one cytokinin or lipoic acid. Non-limiting examples of cytokinins that may be present in the co-culture medium include 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-topolin). In a specific embodiment, the concentration of the cytokinin in the co-culture medium is about 0.1 mg / L to about 50 mg / L. In a specific embodiment, the at least one cytokinin is thidiazuron (TDZ), and the concentration of thidiazuron (TDZ) in the co-culture medium is about 0.1 mg / L to about 10 mg / L. In a specific embodiment, the at least one cytokinin is 6-benzylaminopurine (BAP), and the concentration of 6-benzylaminopurine (BAP) in the co-culture medium is about 0.1 mg / L to about 15 mg / L. In some embodiments, the concentration of lipoic acid in the co-culture medium is about 0.1 mg / L to about 500 mg / L.

[0030] In certain embodiments, the population is defined as a population of dicotyledonous plant embryonic explants. In some embodiments, the heterologous polynucleotide molecule comprises a selectable marker gene, the regeneration medium comprises a selective agent, and the selectable marker gene confers resistance to the selective agent to the plant. In certain embodiments, the plurality of genetically modified plants or plant parts comprises at least one genetic modification. In certain embodiments, the at least one genetic modification comprises the integration or insertion of a heterologous polynucleotide molecule or a fragment thereof into the genome of the plurality of genetically modified plants or plant parts, wherein the integration or insertion comprises at least one expression cassette or at least one transgene. In some embodiments, the at least one genetic modification comprises an edit introduced into the genome of the plurality of genetically modified plants or plant parts by genome editing techniques using site-specific nucleases or guide RNA molecules. In certain embodiments, the heterologous polynucleotide molecule comprises at least one expression cassette, wherein the at least one expression cassette encodes a site-specific nuclease or a guide RNA molecule, or the heterologous polynucleotide molecule comprises at least two expression cassettes, including a first expression cassette encoding a site-specific nuclease and a second expression cassette encoding a guide RNA molecule. In certain embodiments, the regeneration medium comprises at least one cytokinin. Non-limiting examples of cytokinins that may be present in the regeneration medium include 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-topolin). In some embodiments, the concentration of the cytokinin in the regeneration medium is from about 0.1 mg / L to about 50 mg / L. In some embodiments, the at least one cytokinin is zeatin or 6-benzylaminopurine (BAP), and the concentration of zeatin or 6-benzylaminopurine (BAP) in the regeneration medium is from about 0.1 mg / L to about 15 mg / L.In certain embodiments, the methods of the present disclosure may comprise contacting a plurality of genetically modified plants or plant parts with a regeneration medium and regenerating or growing them at about 15°C to about 40°C. In certain embodiments, the population of embryo explants is a population of cotton embryo explants, and the method comprises contacting a plurality of genetically modified plants or plant parts with a regeneration medium and regenerating or growing them at about 30°C to about 40°C for a first regeneration period. The first regeneration period, in some embodiments, is from about 1 hour to about 14 days. In certain embodiments, the method may further comprise contacting a plurality of genetically modified cotton plants or cotton plant parts with a regeneration medium and regenerating or growing them at about 20°C to about 33°C for a second regeneration period. The second regeneration period, in certain embodiments, is from about 7 days to about 56 days. In certain embodiments, the method comprises contacting a plurality of genetically modified plants or plant parts with a regeneration medium and regenerating or growing them for about 5 days to about 70 days or from about 14 days to about 50 days. In one embodiment, the population of embryo explants is a population of soybean embryo explants, and the method comprises contacting a plurality of genetically modified plants or plant parts with a regeneration medium and regenerating or growing for about 5 days to about 70 days. In another embodiment, the population of embryo explants is a population of cotton embryo explants, and the method comprises contacting a plurality of genetically modified plants or plant parts with a regeneration medium and regenerating or growing for about 14 days to about 70 days. In yet another embodiment, the population of embryo explants is a population of canola embryo explants, and the method comprises contacting a plurality of genetically modified plants or plant parts with a regeneration medium and regenerating or growing for about 14 days to about 70 days.

[0031] In certain aspects, the population of embryo explants is a population of dicotyledonous plant embryo explants, and the methods of the present disclosure further include contacting the plurality of genetically modified plants or plant parts with a second regeneration medium and regenerating or growing them for an extended regeneration period. In some embodiments, the second regeneration medium comprises at least one auxin, at least one cytokinin, or at least one auxin and at least one cytokinin. Non-limiting examples of auxins that may be present in the second regeneration medium include 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid or p-chlorophenoxyacetic acid (4-CPA or pCPA), 2,4,5-trichloro-phenoxyacetic acid (2,4,5-T), 2,3,5-triiodobenzoic acid (TIBA), phenylacetic acid (PAA), and 3,6-dichloro-2-methoxybenzoic acid (dicamba). Non-limiting examples of cytokinins that may be present in the second regeneration medium include 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-topolin). The concentration of the auxin in the second regeneration medium, in certain embodiments, is from about 0.1 mg / L to about 15 mg / L. The concentration of at least one cytokinin in the second regeneration medium, in certain embodiments, is from about 0.1 mg / L to about 50 mg / L. In some embodiments, the method comprises contacting a plurality of genetically modified plants or plant parts with the second regeneration medium and regenerating or growing them at about 15°C to about 40°C. In certain embodiments, the expanded regeneration period is from about 7 days to about 56 days. In certain embodiments, the population of embryo explants is a population of cotton embryo explants, and the method further comprises transferring selected portions of the plurality of genetically modified cotton plants or cotton plant portions to a second regeneration medium prior to contacting the plurality of genetically modified cotton plants or cotton plant portions with the second regeneration medium and regenerating or growing the plurality of genetically modified cotton plants or cotton plant portions.

[0032] In certain aspects, the population of embryo explants is a population of dicotyledonous plant embryo explants, and the methods of the present disclosure may further include contacting the plurality of genetically modified plants or plant parts with a first elongation medium and regenerating or growing them for a first elongation period. In some embodiments, the first elongation medium comprises at least one auxin, at least one cytokinin, or at least one auxin and at least one cytokinin. In certain embodiments, the population of embryo explants is a population of canola embryo explants. Non-limiting examples of auxins that may be present in the first elongation medium include 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid or p-chlorophenoxyacetic acid (4-CPA or pCPA), 2,4,5-trichloro-phenoxyacetic acid (2,4,5-T), 2,3,5-triiodobenzoic acid (TIBA), phenylacetic acid (PAA), and 3,6-dichloro-2-methoxybenzoic acid (dicamba). Non-limiting examples of cytokinins that may be present in the first extension medium include 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-topolin). The concentration of the at least one auxin in the first extension medium, in some embodiments, is from about 0.1 mg / L to about 15 mg / L. The concentration of the at least one cytokinin in the first extension medium, in certain embodiments, is from about 0.1 mg / L to about 50 mg / L. In some embodiments, methods of the present disclosure may include contacting a plurality of genetically modified plants or plant parts with the first extension medium and regenerating or growing them at about 15°C to about 40°C. The first extension period, in certain embodiments, is from about 7 days to about 56 days.

[0033] In some aspects, the population of embryo explants is a population of dicotyledonous embryo explants, and the methods of the present disclosure can further include contacting the plurality of genetically modified plants or plant parts with a second elongation medium and regenerating or growing them for a second elongation period. The second elongation medium, in certain embodiments, comprises at least one auxin, at least one cytokinin, or at least one auxin and at least one cytokinin. Non-limiting examples of auxins that may be present in the second elongation medium include 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid or p-chlorophenoxyacetic acid (4-CPA or pCPA), 2,4,5-trichloro-phenoxyacetic acid (2,4,5-T), 2,3,5-triiodobenzoic acid (TIBA), phenylacetic acid (PAA), and 3,6-dichloro-2-methoxybenzoic acid (dicamba). Non-limiting examples of cytokinins that may be present in the second elongation medium include 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-topolin). The concentration of the at least one auxin in the second elongation medium, in certain embodiments, is from about 0.1 mg / L to about 15 mg / L. The concentration of the at least one cytokinin in the second elongation medium, in certain embodiments, is from about 0.1 mg / L to about 50 mg / L. In some embodiments, the methods of the present disclosure comprise contacting a plurality of genetically modified plants or plant parts with a second elongation medium and regenerating or growing them at about 15°C to about 40°C. In certain embodiments, the second elongation period is from about 7 days to about 56 days.

[0034] In certain embodiments, the present disclosure provides a combination of a robust genomic platform, a genome editing toolbox, and a high-throughput, genotype-independent transformation system, enabling genome editing at any target site in any genotype and species.

[0035] The present disclosure also provides methods for simultaneously transforming and editing multiple genotypes in a single experiment under identical conditions.

[0036] Additionally, the present disclosure provides specific embodiments for bulk transformation of mixed germplasm, demonstrating genotype-flexible regeneration and genome editing of up to 100 elite genotypes via seed embryo explant-based meristem transformation in soybean and maize. In specific embodiments, over 800 different edits were recovered in soybean at conserved target sites, such as near loci of interest, and the individuals represented nearly all transformed germplasm.

[0037] In additional embodiments in maize, more variation between inbred lines was observed, but transformants were recovered from 23 of 40 female inbred lines. Thus, the present disclosure describes the accelerated implementation and development of genome editing strategies in breeding and product development for precision breeding.

[0038] Any embodiment or aspect of the present disclosure may be used in combination with any other embodiment or aspect described herein. [Brief explanation of the drawings]

[0039] [Figure 1] 1 shows a graph of similarity scores for soybean lines sampled after bulk transformation in genotyping. [Figure 2]Figure 1 shows a comparison of transformation and editing frequencies in soybean bulk by maturity group. (A) shows the frequency of transformation events in each maturity group compared with the proportion of bulk consisting of that maturity group. (B) shows the total number of editing and non-editing events by maturity group. [Figure 3] The percentage of heritable editing reads by T-DNA copy number after soybean bulk transformation is shown. DETAILED DESCRIPTION OF THE INVENTION

[0040] The following is a detailed description provided to aid those skilled in the art in practicing embodiments of the present disclosure. Modifications and variations on the embodiments described herein can be made without departing from the spirit or scope of the present disclosure. The compositions and methods provide for the en bloc transformation or genetic modification of a population of embryonic explants, and may include one or more steps of explant preparation, explant rehydration, inoculation and co-cultivation with RHIZOBIALES bacteria, particle bombardment, germination induction, expansion germination induction, and / or regeneration or development of the genetically modified plant or plant part, as described herein. The present disclosure further provides methods for identifying and selecting individual members of a population containing a desired genotype, genetic modification, cultural trait, and / or phenotype.

[0041] The present disclosure provides compositions and methods for introducing heterologous polynucleotides en masse into a population of embryonic explants having different genotypes to introduce one or more genetic modifications. As described herein, the genetic modifications may include a transgene, or site-directed integration of a DNA segment or transgene, and / or the genetic modifications may include mutations or editing. The present disclosure represents a substantial advance in the art by providing compositions and methods for simultaneously or en masse producing multiple genetically modified plants having different genotypes without the use of time-consuming, costly, and inefficient plant breeding or introgression techniques. The present disclosure further provides methods and compositions for investigating or screening for variable phenotypes that arise when the same genetic modification is introduced into different plant genetic resources or genetic backgrounds, which may be described as "germline x transgene / edit (or trait) interactions." Prior to the present disclosure, analysis of such genetic resource x transgene / edit (or trait) interactions typically involved creating a transgenic event, edit, and / or trait in a single genetic resource, followed by breeding or introgression of that event, edit, and / or trait into other genetic resources, a time-consuming, expensive, and inefficient process. Prior to the present disclosure, any genetic background- or genetic resource-dependent differences in the phenotype for a given transgenic event, edit, and / or trait could not be tested, observed, screened, or selected for until the event, edit, and / or trait was bred or introgressed into multiple genetic resources over several generations. The present disclosure further provides methods and compositions for investigating or screening different cultural traits of different plant genetic resources or genetic backgrounds when introducing heterologous polynucleotides and / or genetic modification(s) into the different plant genetic resources or genetic backgrounds en masse.

[0042] A. Preparation of Explant Populations According to an embodiment of the present disclosure, a group of seed embryo explants can be produced from plant seeds to produce genetically modified plants or plant parts.The group of seed embryo explants can be produced from seeds by applying mechanical force, for example, by cutting, crushing, scraping, crushing or damaging the seeds.The seeds used according to the present disclosure can be collected from plants grown in fields or greenhouses, and can be mature or immature seeds, but preferably mature seeds.Examples of seeds used according to the present disclosure include, but are not limited to, monocotyledonous seeds, dicotyledonous seeds, corn seeds, wheat seeds, barley seeds, rice seeds, oat seeds, sorghum seeds, rye seeds, millet seeds, soybean seeds, cotton seeds, canola seeds and Brassica seeds. Examples of embryo explants for use in accordance with the present disclosure include, but are not limited to, mature embryo explants, immature embryo explants, monocotyledonous embryo explants, dicotyledonous embryo explants, corn embryo explants, wheat embryo explants, barley embryo explants, rice embryo explants, oat embryo explants, sorghum explants, rye embryo explants, millet embryo explants, soybean embryo explants, cotton embryo explants, canola embryo explants, and Brassica embryo explants. The use of mature seeds may provide benefits or advantages due to improved seed storage, explant preparation, and / or culture.Examples of monocotyledonous plants, seeds, or explants that may be used in accordance with this embodiment include those derived from any plant species of monocotyledonous plants or cereal plants and grasses of the families Poaceae or Gramineae, including any corn or maize species of the genus Zea, such as Zea mays, any rice species of the genus Oryza, such as Oryza sativa, any wheat species of the genus Triticum, such as Triticum aestivum or Triticum turgidum var durum, any barley species of the genus Hordeum, such as Hordeum vulgare, any oat species of the genus Avena, such as Avena sativa, any sorghum species of the genus Sorghum, such as Sorghum bicolor or Sorghum vulgare, any rye species of the genus Secale, such as Secale cereale, any Saccharum sugarcane species, or any Setaria, Pennisetum, Eleusine, Echinochloa, or Panicum millet species, such as Setaria virdis, Setaria italica, Pennisetum glaucum, Eleusine coracana, Echinochloa frumentacea, Panicum sumatrense, or Panicum miliaceum.Examples of dicotyledonous plants, seeds, and explants that may be used in accordance with the present embodiments include those derived from any plant species in the families Fabaceae, Malvaceae, or Brassicaceae, and may include any Glycine species, such as Glycine max, any Gossypium species, such as Gossypium arboretum, Gossypium herbaceum, Gossypium raimondii, Gossypium thurberi, Gossypium barbadense, Gossypium hirsutum, Gossypium darwinii, Gossypium mustelinum, Gossypium tomentosum, Gossypioides brevilanatum, or Gossypioides kirkii, or any Brassica species, such as Brassica napus, Brassica rapa, or Brassica juncea.

[0043] According to some embodiments, methods and compositions for preparing, culturing, selecting, and using explant populations, as well as the resulting explant populations or cultured explants, are provided. As used herein, the term "explant" or "seed embryo explant" refers to a plant part or plant tissue that has been genetically modified and can subsequently be generated / regenerated into a genetically modified plant or plant part. An "explant" or "seed embryo explant" may refer to a plant seed or any part of a plant seed, and in the case of a seed embryo explant, includes at least a portion of the plant seed embryo. An "explant" or "seed embryo explant" may include an embryo explant excised from a plant seed, which may include at least a portion of the embryonic meristem. Alternatively, an "explant" or "seed embryo explant" may refer to a whole or intact plant seed, or a fractured, deformed, or partially opened plant seed, which may be produced by any suitable mechanical process. Various methods for preparing plant seed explants and plant seed embryo explants are known in the art. When used with respect to an explant or seed embryo explant, "partially opened" refers to an altered state of a plant seed having one or more openings or fissures in the plant seed. Such openings or fissures may be introduced by mechanical forces such as squeezing, crushing, rolling, pressing, or extrusion. Explants or seed embryo explants, which are whole or intact plant seeds or broken, deformed, or partially opened plant seeds, often have their seed coats removed. In one aspect or embodiment, an explant may be defined as comprising a meristematic tissue or embryonic meristem containing plant cells that can differentiate or develop into multiple plant structures, including, but not limited to, stems, roots, leaves, germline tissue, and seeds. In certain embodiments, an embryonic explant may be defined as comprising all or a portion of a seed embryo removed from other non-embryonic seed tissues, and further comprising all or a portion of a meristematic tissue or embryonic meristem. In some embodiments, the present disclosure provides embryo explants comprised of the apical portion of the hypocotyl lacking roots, wherein the remainder of the seed has been substantially removed from the embryo explant, hi further embodiments, the present disclosure provides embryo explants that do not germinate, are viable, and maintain the capacity for genetic modification.As used herein, the term "cultured embryo explant" refers to an embryo explant that is in culture but has not yet regenerated into a plant or plant part. In specific embodiments, the cultured embryo explant may be a genetically modified embryo explant. The cultured embryo explant may, in some embodiments, be cultured in contact with a co-culture medium, a germination induction medium, an expansion germination induction medium, or a regeneration medium. Cultured embryo explants derived from monocotyledonous seeds may, in some embodiments, be cultured in contact with a co-culture medium, a germination induction medium, an expansion germination induction medium, and a regeneration medium after an optional inoculation step, while cultured embryo explants derived from dicotyledonous seeds may, in some embodiments, be cultured in contact with a co-culture medium and a regeneration medium after an optional inoculation step. As used herein, a "population of embryo explants" refers to a group of explants derived from the same plant species. A population of explants may, in some embodiments, include explants having the same or different genotypes, germplasm, and / or genetic backgrounds. In certain embodiments, the genotypes of the explants in the population may be known or unknown. In specific embodiments, a population of embryo explants may refer to a group of embryo explants that includes embryo explants of at least two different plant genotypes.In specific embodiments, the present disclosure provides at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least Populations of embryo explants are provided having at least 450, or at least 500 different plant genotypes, or from 2 to about 750, from 2 to about 600, from about 10 to about 500, from about 15 to about 400, from about 20 to about 300, from about 25 to about 200, from about 10 to about 150, from about 10 to about 100, from about 10 to about 90, from about 10 to about 80, from about 10 to about 70, from about 10 to about 60, from about 10 to about 50, from about 10 to about 40, from about 10 to about 30, from about 10 to about 20, from 2 to about 50, from 2 to about 40, from 2 to about 30, from 2 to about 20, from 2 to about 15, or from 2 to about 10 different plant genotypes.In certain embodiments, the present disclosure provides a method for producing a polymerizable compound having at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 10,000, at least 15,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, at least 10,000, at least 15 ... 00, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, at least 100,000, at least 150,000, or at least 200,000 embryonic explants, or about 2 to about 1000, about 5 to about 900, about 5 to about 800, about 5 to about 700, about 5 to about 60 Populations of embryo explants are provided, including 0, about 5 to about 500, about 10 to about 500, about 15 to about 400, about 20 to about 300, about 25 to about 200, about 10 to about 150, about 10 to about 100, about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, 2 to about 50, 2 to about 40, 2 to about 30, 2 to about 20, 2 to about 15, or 2 to about 10 embryo explants. In certain embodiments, explants according to the present disclosure can be produced manually or using automated processes. For example, seed tissue can be removed from the seeds by cutting, grinding, scraping, crushing, wounding, or any other similar process. Manual or automated methods of removing unwanted seed parts can also be performed. By way of non-limiting example, fluids including compressed air, other gases, and liquids can be used to separate the explants from debris during explant purification.

[0044] In certain embodiments provided by the present disclosure, embryo explants can be excised from a population of plant seeds, wherein the population of plant seeds comprises plant seeds of at least two different plant genotypes. In specific embodiments, the population of plant seeds from which the population of embryo explants is excised comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10000, at least 1500, at least 2000, at least 15 ... The plant genotype may have at least 350, at least 400, at least 450, or at least 500 different plant genotypes, or from 2 to about 750, from 2 to about 600, from about 10 to about 500, from about 15 to about 400, from about 20 to about 300, from about 25 to about 200, from about 10 to about 150, from about 10 to about 100, from about 10 to about 90, from about 10 to about 80, from about 10 to about 70, from about 10 to about 60, from about 10 to about 50, from about 10 to about 40, from about 10 to about 30, from about 10 to about 20, from 2 to about 50, from 2 to about 40, from 2 to about 30, from 2 to about 20, from 2 to about 15, or from 2 to about 10 different plant genotypes.In certain embodiments, the present disclosure provides a method for producing a polymerizable compound having at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 10,000, at least 15,000 , at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, at least 100,000, at least 150,000, or at least 200,000 embryonic explants, or about 2 to about 1000, about 5 to about 900, about 5 to about 800, about 5 to about 700, about 5 to about 600, about 5 to about The present invention provides a population of plant seeds from which embryonic explants can be excised, the population comprising about 500, about 10 to about 500, about 15 to about 400, about 20 to about 300, about 25 to about 200, about 10 to about 150, about 10 to about 100, about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, 2 to about 50, 2 to about 40, 2 to about 30, 2 to about 20, 2 to about 15, or 2 to about 10 plant seeds.

[0045] In some embodiments, the present disclosure provides a method comprising sorting a population of plant seeds into at least two batches of plant seeds according to plant seed size and / or plant seed shape prior to excision of the population of embryo explants. Sorting the population of seeds according to seed size and / or seed shape prior to excision can, in some embodiments, result in approximately equal numbers of explants being excised from seeds of each genotype present in the population. Different batches of seeds sorted according to seed size and / or seed shape can, in certain embodiments, be excised using different excision methods and / or settings to maximize explant excision efficiency. In certain embodiments, different batches of seeds sorted according to seed size and / or seed shape can be excised using the same excision method and / or settings. In some embodiments, two or more different batches of seeds sorted according to seed size and / or seed shape can be mixed prior to excision of the explants. In some embodiments, two or more different batches of seeds sorted according to seed size and / or shape can be mixed in equal numbers or in different proportions or percentages prior to explant excision, taking into account their relative transformation, culture, and / or regeneration efficiencies. For example, a batch of seeds with a higher frequency of explant transformation, culture, and / or regeneration can be added to the population of seeds for explant excision at a lower percentage than another batch of seeds with a lower frequency of explant transformation, culture, and / or regeneration. Alternatively, for example, explants can be excised separately from different batches of seeds and then mixed in equal numbers or in different proportions or percentages prior to the subsequent rehydration, culture, and / or transformation steps, taking into account their relative transformation, culture, and / or regeneration efficiencies.

[0046] Embryo explants can be excised from dry seeds, dried seeds, or wet seeds. Mature plant seeds can become dry as part of the normal maturation process, but the seeds can be further dried before explant excision and / or the explants can be dried after excision from the seeds. The dried or desiccated excised plant embryo explants can be used immediately for genetic modification or stored for a period of time for later use. Explant preparation can further include drying the seeds and / or explants to a desired moisture content. Drying the seeds and / or explants to such a desired moisture content can improve the excision, storage, and / or use of the seeds and / or explants, depending on the initial moisture content of the seeds or explants. After excision, the explants can be purified or separated from other seed materials and debris by rinsing, flotation, or other methods known in the art. In certain embodiments, the present disclosure provides seeds or explants having an internal moisture content of about 3% to about 25%, about 3% to about 20%, about 3% to about 15%, about 3% to about 10%, about 3% to about 11%, about 4% to about 16%, about 4% to about 12%, or about 5% to about 10%, for example, about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or about 25% (including all derivable values ​​and ranges therebetween). Explants can be produced from mature seeds having the moisture contents described herein. In certain embodiments, the moisture content of the seed or explant can be measured before or after explant excision, before or after explant storage, during explant storage, before rehydration of the explant, and / or before genetic modification or transformation. A seed embryo explant, in one aspect or embodiment, can be defined as comprising a meristematic tissue or embryonic meristem that contains plant cells that can differentiate or develop into multiple plant structures, including, but not limited to, stems, roots, leaves, germline tissue, and seeds. Indeed, an embryonic explant can be defined as comprising all or a portion of a seed embryo that has been removed from other non-embryonic seed tissues, and further comprising all or a portion of a meristematic tissue or embryonic meristem.

[0047] In one aspect, any embryo explant can be prepared or used in accordance with embodiments of the present disclosure. In certain embodiments, the embryo explant may be a mature or immature embryo and / or may include meristem, callus tissue, or any other tissue that is transformable and regenerable. In some embodiments, the mature embryo explant is a dried explant. The dried explant is harvested from a seed and can be used substantially intact as a target for transformation or genetic modification. In some embodiments, the dried explant or desiccated explant is harvested from a mature, dried seed and can often be used as a target for transformation or genetic modification with only minimal wetting, hydration, or pre-culture steps. In further embodiments, wet embryo explants, dried wet embryo explants, or wet excised embryo explants can be used as a target for transformation or genetic modification. In further embodiments, immature or mature embryo explants or excised embryo explants can be dried prior to transformation or genetic transformation. As used herein, a "wet" embryo explant refers to a dried explant that has been subjected to wetting, hydration, imbibition, or other minimal culture steps prior to transformation or genetic modification. As used herein, a "dried" embryo explant refers to an excised explant that has been subjected to a drying step prior to transformation or genetic modification. As used herein, a "dried wet" embryo explant refers to an embryo explant that has been prepared for germination by wetting and then dried to stop germination. As used herein, a "wet excised" explant refers to an explant excised from an imbibed or hydrated seed. A wet embryo explant is one that has been hydrated or imbibed after being excised from a seed, whereas a wet excised embryo explant is one that has already been hydrated or imbibed. As used herein, a "callus" refers to a growth mass of unorganized, undifferentiated, and / or dedifferentiated plant cells or tissues.

[0048] In accordance with this embodiment, a population explant may, in some embodiments, be defined as comprising a meristematic or embryonic meristem tissue containing plant cells that can differentiate or develop into a plurality of plant structures, including, but not limited to, a stem, root, leaf, germline tissue, and seed. In certain embodiments, a population embryonic explant may be defined as comprising all or a portion of a seed embryo that has been removed from other non-embryonic seed tissue, and further comprising all or a portion of a meristematic or embryonic meristem tissue.

[0049] Explants for use in accordance with the present disclosure can be genetically modified at various time points after isolation, excision, and / or removal from the seed. In one embodiment, the explants can be removed from the seed in less than one day, e.g., about 1 to about 24 hours, e.g., about 1, 2, 3, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, before use. In further embodiments, the explants can be stored for extended periods of time, e.g., days, weeks, months, or years, before use. Methods and parameters for desiccation, storage, transformation, regeneration, and / or germination of seeds or seed embryo explants are known in the art (see, e.g., US 7,402,734, US 8,044,260, US 8,030,544, US 8,362,317, US 2022 / 0340916, and US 2022 / 0340925; which are expressly incorporated by reference herein in their entireties; Senaratna et al., 1983, Pl. Physiol. 72:620-624, 1983; Vertucci and Roos, 1990, Pl. Physiol. 90:1019-1023, 1990; Chai et al., 1998, Seed Science Research 8(Supplement 1):23-28, 1998). Any conditions can be used as desired, including, for example, incubation or storage at temperatures from about −80° C. to about 60° C. If the explants or seeds are stored in a freezer, they can be thawed before use in a subsequent step, where such thawing step can be for a period of about 20 minutes to about 4 hours or more, or about 30 minutes to about 2 hours, depending on the storage temperature and the number of seeds or explants to be thawed and returned to room temperature for use in explant excision or transformation.

[0050] In certain aspects, the present disclosure may include sterilization of seeds or explants. Sterilization may involve contacting the seed or explant material with various liquids or gases, which serve to reduce or eliminate the presence of viable bacterial or fungal contaminants that may interfere with seed or embryo viability. Sterilization by applying a liquid may also hydrate or partially hydrate the plant seed, explant, embryo, or tissue, serving the purpose of priming the seed, explant, embryo, or tissue. Sterilization methods include, but are not limited to, the use of chlorine gas, ozone, bleach or alcohol solutions, ultraviolet light, exposure to temperatures below -20°C, and temperatures above 40°C. In some embodiments, the sterilization medium may include polyethylene glycol and / or an antifungal or antibacterial agent.

[0051] In one aspect of the present disclosure, explants can be rehydrated prior to transformation or genetic modification. Rehydration media or solutions are known in the art and can include, for example, water, basal salts, macronutrients, micronutrients, and / or vitamins. In certain embodiments, the rehydration solution can include polyethylene glycol, an antibacterial agent, and / or an antifungal agent. In some embodiments, the rehydration medium can be water. In some embodiments, the rehydration medium can be an inoculation medium. The rehydration medium typically does not contain any plant hormones, such as auxins or cytokinins. It can be important to provide an optimal time for rehydration. If the explants remain in the rehydration medium(s) for too long, they may become "mushy" and become non-regenerative or non-viable; if the explants remain in the rehydration medium(s) for too short a time, they may not be fully rehydrated and therefore may not be transformed or may undergo low transformation efficiency. Embryo explants of some plant species are more or less sensitive to rehydration, which may depend on their relative size or composition. Embryo explants that are more sensitive to rehydration can be placed in the rehydration medium for shorter periods of time, but can tolerate rehydration for up to about 1 hour to about 2 hours. In one embodiment, seed embryo explants are rehydrated for a period of time ranging from about 15 minutes to about 24 hours, or from about 15 minutes to about 12 hours, or from about 15 minutes to about 6 hours, or from about 15 minutes to about 4 hours, or from about 30 minutes to about 2 hours, or any length of time within such ranges, such as about 15 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3.5 hours, or about 4 hours, or less than about 4 hours, or may be carried out for about 3 hours or less, or about 2.5 hours or less, or about 2 hours or less, or in the range of about 20 minutes to about 4 hours, or in the range of about 20 minutes to about 3 hours, or in the range of about 20 minutes to about 2 hours, or in the range of about 20 minutes to about 1.5 hours, or in the range of about 1 hour to about 3 hours, or in the range of about 1 hour to about 2.5 hours, or in the range of about 1 hour to about 2.0 hours, or in the range of about 1.5 hours to about 2.5 hours (including all derivable values ​​and ranges therebetween).Rehydration of embryo explants prior to transformation or genetic modification may improve the frequency of transformation or editing or the recovery of transformed or edited plants compared to explants that are not rehydrated. In some embodiments, the rehydration medium may be shaken or rocked, such as on a shaker or rocker, or otherwise physically or mechanically agitated, moved, or inverted during this step to improve rehydration and / or reduce the time required for explant rehydration. In certain embodiments, the embryo explant may be a monocotyledonous plant embryo explant, and rehydration of the seed embryo explant may be performed for at least about 2 hours prior to transformation or genetic modification, which may improve the frequency of transformation or editing or the recovery of transformed or edited plants compared to explants that are rehydrated for about 1 hour or less. In certain embodiments, the embryo explant may be a dicotyledonous plant embryo explant, and rehydration of the embryo explant may be performed for about 1 hour or for about 15 minutes to about 4 hours. In certain embodiments, the embryonic explants can be rinsed to remove the rehydration medium prior to subsequent steps.

[0052] In certain embodiments, the embryo explants can be rinsed to remove the rehydration medium prior to subsequent steps. In certain embodiments, the embryo explants can be rinsed for about 20 seconds to about 10 minutes, about 20 seconds to about 9 minutes, about 20 seconds to about 8 minutes, about 20 seconds to about 7 minutes, about 20 seconds to about 6 minutes, about 20 seconds to about 5 minutes, about 20 seconds to about 4 minutes, about 20 seconds to about 3 minutes, about 20 seconds to about 2 minutes, about 20 seconds to about 1 minute, about 1 minute to about 10 minutes, about 1 minute to about 6 minutes, about 2 minutes to about 6 minutes, or about 3 minutes to about 5 minutes (including all derivable values ​​and ranges therebetween). In certain embodiments, embryo explants may be subjected to about 1 to about 10 rounds of rinsing, about 1 to about 9 rounds, about 1 to about 8 rounds, about 1 to about 7 rounds, about 1 to about 6 rounds, about 1 to about 5 rounds, about 1 to about 4 rounds, about 1 to about 3 rounds, about 1 to about 2 rounds, about 2 to about 6 rounds, or about 3 to about 5 rounds (including all derivable values ​​and ranges therebetween). In certain embodiments, embryo explants may be rinsed in a container or by placing them in a strainer, fishing net, or the like and then allowing a rinsing solution to flow over the explants. In certain embodiments, embryo explants may be rinsed in a container by removing, decanting, and / or aspirating the rehydration solution or liquid and then adding a rinsing solution or liquid. The first volume of rinse solution or liquid can be removed, decanted, and / or aspirated and then replaced with new rinse solution or liquid, which may be repeated one or more times (i.e., a total of two or more rounds of rinsing). In some embodiments, the rehydration solution or liquid and the rinse solution or liquid are the same solution or liquid. In certain embodiments, the rehydration solution or liquid and the rinse solution or liquid are different solutions or liquids. In some embodiments, the rinse solution is water.After rehydration and / or rinsing of the explants, the rehydration and / or rinsing medium can be removed by decanting, pipetting, vacuuming, and / or aspirating the medium, etc., and the explants can be dried by blotting, wicking, or otherwise contacting filter paper or other absorbent material, etc., with the rehydration or rinsing solution or liquid to remove at least excess amounts of the rehydration or rinsing solution or liquid. Blotting, etc., can be particularly useful or necessary for explants that are sensitive to excessive rehydration.

[0053] As used herein, a "genetically modified" plant, plant part, plant tissue, explant, or plant cell includes a genetic modification, such as a mutation, edit, or transgene, introduced into the genome of the plant, plant part, plant tissue, explant, or plant cell by genetic engineering, which may be via genetic transformation, mutagenesis, or genome editing techniques. As used herein, "genetic modification" refers to one or more transgenic event(s), mutation(s), and / or edit(s) introduced into the genome of the plant, plant part, or plant cell using transformation, mutagenesis, or genome editing techniques. Aside from genome editing techniques, mutagenesis techniques can include any chemical, physical, radiological, or biological (e.g., transposon-mediated) mutagenesis technique or mutagen. As used herein, a "transgenic" plant, plant part, plant tissue, explant, or plant cell has an exogenous nucleic acid sequence, polynucleotide, expression cassette, or transgene integrated into the genome of the plant, plant part, plant tissue, explant, or plant cell. In certain embodiments, genetically modified plants, plant parts, plant tissues, explants, or plant cells may contain heritable or non-heritable edits. In some embodiments, heritable or non-heritable edits may be identified by gene sequencing. When gene sequencing is performed on a sample containing polynucleotide molecules derived from or obtained from a genetically modified plant, plant part, plant tissue, explant, or plant cell, and more than 10% of the sequencing reads contain the expected edit, the edit is likely to be heritable and may therefore be described as a "heritable edit." When gene sequencing is performed on a sample containing polynucleotide molecules derived from or obtained from a genetically modified plant, plant part, plant tissue, explant, or plant cell, and more than 1-10%, or possibly 0.1%-10%, 0.1%-5%, 0.1%-1%, or 0% of the sequencing reads contain the expected edit, the edit is likely not heritable and may therefore be described as a "non-heritable edit."

[0054] Transformation or editing of embryo explants or plants, in some embodiments of the present disclosure, can be measured by genotyping, the number of shoots that developed after Agrobacterium-mediated inoculation (or number of shoot initiation), the percentage of shoots that developed after Agrobacterium-mediated inoculation (or percentage of shoot initiation), relative shoot abundance, number of transformants, percent of transformants, relative transformation rate, number of edits, percent editing, relative editing rate, number of edits (heritable edits), percent editing (heritable edits), relative editing rate (heritable), number of edits (non-heritable edits), percent editing (non-heritable edits), or relative editing rate (non-heritable), as described herein.

[0055] B. Introduction of a heterologous polynucleotide molecule, ribonucleoprotein, or nuclease Provided herein are methods and compositions for the collective genetic transformation or modification of a population of embryonic explants. In specific embodiments, a heterologous polynucleotide molecule, ribonucleoprotein, and / or site-specific nuclease is introduced into at least two embryonic explants of the population at the same time. As used herein, the term "introducing en bloc" refers to introducing a heterologous polynucleotide molecule, ribonucleoprotein, and / or site-specific nuclease into one or more explants of the population at approximately the same time and / or when the explants of the population are in the same or approximately the same region or in one or more containers. In some embodiments, the heterologous polynucleotide molecule, ribonucleoprotein, and / or site-specific nuclease can be introduced into the explants of the population contemporaneously, simultaneously, or approximately simultaneously. In specific embodiments, a population of embryonic explants can include one or more groups or batches of embryonic explants. Groups or batches of populations may, in certain embodiments, be present in separate containers which may have approximately the same cross-sectional area or volume, and the heterologous polynucleotide molecule, ribonucleoprotein, and / or site-specific nuclease may be introduced collectively to the groups or batches of populations at approximately the same time. In further embodiments, the heterologous polynucleotide molecule, ribonucleoprotein, and / or site-specific nuclease may be introduced collectively to the population of embryonic explants while the individual explants of the population reside together in a single container.

[0056] Embodiments of the present disclosure may include genetically transforming or modifying at least one cell of each of at least two embryonic explants of the population by introducing heterologous polynucleotide molecules, ribonucleoproteins, and / or site-specific nucleases en bloc via any suitable method or technique known in the art, such as electroporation, microprojectile or particle bombardment, microinjection, PEG-mediated transformation, Rhizobiales- or Agrobacterium-mediated transformation, and other modes of direct DNA uptake. All or part of the heterologous polynucleotide can then be transformed or integrated into the genome of the plant cell to express one or more editing molecules or tools (such as guide RNAs and / or site-specific nucleases) and / or provide a template for editing or site-directed integration. According to many embodiments, the heterologous polynucleotide is introduced into the population via Rhizobiales- or Agrobacterium-mediated transformation.

[0057] In certain embodiments, Rhizobiales bacteria or Agrobacterium can be cultured in a medium optionally containing one or more cytokinins and / or lipoic acid prior to Rhizobiales- or Agrobacterium-mediated transformation. Alternatively, the medium for culturing Rhizobiales bacteria or Agrobacterium may not contain or may be free of cytokinins and / or lipoic acid. Such a culture medium may include, for example, water, basal salts, macronutrients, micronutrients, and / or vitamins. In certain embodiments, the culture medium may include polyethylene glycol and / or an antibacterial agent and / or an antifungal agent. According to some embodiments, the culture medium comprises an inoculation medium. Non-limiting examples of cytokinins that can be used in bacterial culture media include 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-topolin).In some embodiments, the concentration of cytokinin in the bacterial culture medium is from about 0.1 mg / L to about 50 mg / L, from about 0.1 mg / L to about 45 mg / L, from about 0.1 mg / L to about 40 mg / L, from about 0.1 mg / L to about 35 mg / L, from about 0.1 mg / L to about 30 mg / L, from about 0.1 mg / L to about 25 mg / L, from about 0.1 mg / L to about 20 mg / L, from about 0.1 mg / L to about 15 mg / L, Approximately 0.1 mg / L to approximately 10 mg / L, approximately 0.1 mg / L to approximately 5 mg / L, approximately 0.1 mg / L to approximately 4 mg / L, approximately 0.1 mg / L to approximately 3 mg / L, approximately 0.1 mg / L to approximately 2 mg / L, approximately 0. 1mg / L~Approx. 1mg / L, Approx. 0.5mg / L~Approx. 5mg / L, Approx. 0.5mg / L~Approx. 4mg / L, Approx. 0.5mg / L~Approx. 3mg / L, Approx. 1mg / L~Approx. 5mg / L, Approx. 1mg / L~Approx. 4m g / L, about 1 mg / L to about 3 mg / L, about 5 mg / L to about 40 mg / L, about 5 mg / L to about 30 mg / L, about 10 mg / L to about 30 mg / L, or about 20 mg / L to about 30 mg / L, or about 0.1 mg / L, about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, about 1 mg / L, about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, about 10 mg / L, about 15 mg / L, about 20 mg / L, about 25 mg / L, about 30 mg / L, about 35 mg / L, about 40 mg / L, about 45 mg / L, or about 50 mg / L (including all derivable values ​​and ranges therebetween).In certain embodiments, lipoic acid is present in an amount of from about 0.1 mg / L to about 500 mg / L, from about 0.1 mg / L to about 400 mg / L, from about 0.1 mg / L to about 300 mg / L, from about 0.1 mg / L to about 200 mg / L, from about 10 mg / L to about 200 mg / L, from about 10 mg / L to about 180 mg / L, from about 10 mg / L to about 160 mg / L, from about 10 mg / L to about 140 mg / L, from about 10 mg / L to about 120 mg / L, from about 10 mg / L to about 100 mg / L, from about 20 mg / L to about 80 mg / L, from about 40 mg / L to about 60 mg / L, or from about 50 mg / L. g / L to about 60 mg / L, or about 50 mg / L to about 55 mg / L, or about 5 mg / L, about 10 mg / L, about 15 mg / L, about 20 mg / L, about 25 mg / L, about 30 mg / L, about 35 mg / L, about 40 mg / L, about 45 mg / L, about 50 m g / L, approximately 55 mg / L, approximately 60 mg / L, approximately 65 mg / L, approximately 70 mg / L, approximately 75 mg / L, approximately 80 mg / L, approximately 85 mg / L, approximately 90 mg / L, approximately 95 mg / L, approximately 100 mg / L, approximately 105 mg / L, approximately 110 mg / L, approximately 115 mg / L, approximately 1 20mg / L, about 125mg / L, about 130mg / L, about 135mg / L, about 140mg / L, about 145mg / L, about 150mg / L, about 155mg / L, about 160mg / L, about 165mg / L, about 170mg / L, about 175mg / L, about 180mg / L L, about 185 mg / L, about 190 mg / L, about 195 mg / L, about 200 mg / L, about 210 mg / L, about 220 mg / L, about 230 mg / L, about 240 mg / L, about 250 mg / L, about 260 mg / L, about 270 mg / L, about 280 mg / L, about 290

[0033] The present invention may be present in a bacterial culture medium at a concentration of about 300 mg / L, 310 mg / L, 320 mg / L, 330 mg / L, 340 mg / L, 350 mg / L, 360 mg / L, 370 mg / L, 380 mg / L, 390 mg / L, 400 mg / L, about 410 mg / L, about 420 mg / L, about 430 mg / L, about 440 mg / L, about 450 mg / L, about 460 mg / L, about 470 mg / L, about 480 mg / L, about 490 mg / L, or about 500 mg / L (including all derivable ranges and values ​​therebetween).In certain embodiments, including one or more cytokinins and / or lipoic acid in bacterial culture media can improve survival, regrowth, and / or transformation / editing frequency after Rhizobiales- or Agrobacterium-mediated transformation. In certain embodiments, including lipoic acid in bacterial culture media reduces stress during Rhizobiales- or Agrobacterium-mediated transformation. In some embodiments, including one or more cytokinins and / or lipoic acid in bacterial culture media improves survival, regrowth, and / or transformation / editing frequency of dicotyledonous embryo explants. In certain embodiments, including one or more cytokinins and / or lipoic acid in bacterial culture media improves survival, regrowth, and / or transformation / editing frequency of soybean embryo explants.

[0058] In some embodiments, thidiazuron (TDZ) is about 0.1 mg / L to about 10 mg / L, about 0.1 mg / L to about 9 mg / L, about 0.1 mg / L to about 8 mg / L, about 0.1 mg / L to about 7 mg / L, about 0.1 mg / L to about 6 mg / L, about 0.1 mg / L to about 5 mg / L, about 0.1 mg / L to about 4 mg / L, about 0.1mg / L to about 3mg / L, about 0.1mg / L to about 2mg / L, about 0.1mg / L to about 1mg / L, about 0.25mg / L to about 1.75m g / L, about 0.5 mg / L to about 1.5 mg / L, about 0.1 mg / L to about 5 mg / L, about 0.5 mg / L to about 4 mg / L, about 0.5 mg / L to about 3 mg / 1.5 mg / L, about 1 mg / L to about 5 mg / L, about 1 mg / L to about 4 mg / L, about 1 mg / L to about 3 mg / L, or about 0.1 mg / L, about 0.25 mg / L, about 0.5 mg / L, about 0.75 mg / L, about 1.0 mg / L, about 1.25 mg / L, about 1.5 mg / L, about 1.75 mg / L, about 2.0 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, or about 10 mg / L (including all derivable ranges and values ​​therebetween). In some embodiments, cytokinins other than thidiazuron (TDZ), such as 6-benzylaminopurine (BAP), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-topolin), may be included in bacterial culture media at concentrations adjusted and set depending on their relative activities.For example, 6-benzylaminopurine (BAP), kinetin, zeatin, and / or 6-(3-hydroxybenzylamino)purine (meta-topolin) may be present in concentrations of about 0.1 mg / L to about 10 mg / L, about 0.1 mg / L to about 9 mg / L, about 0.1 mg / L to about 8 mg / L, about 0.1 mg / L to about 7 mg / L, about 0.1 mg / L to about 6 mg / L, about 0.1 mg / L to about 5 mg / L, about 0.1 mg / L to about 4 mg / L, about 0.1 mg / L to about 3 mg / L, about 0.1 mg / L to about 2 mg / L, about 1 mg / L to about 10 mg / L, about 2 mg / L to about 10 mg / L, about 2 mg / L to about 8 mg / L, about 2.5 mg / L to about 7.5 mg / L, or about 3 mg / L. The compound may be present in the bacterial culture medium at a concentration of about 10 mg / L, about 4 mg / L to about 6 mg / L, about 1.0 mg / L, about 1.25 mg / L, about 1.5 mg / L, about 1.75 mg / L, about 2.0 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, or about 10 mg / L (including all derivable ranges and values ​​therebetween), which may improve the survival rate, regrowth potential, and / or transformation / editing frequency of embryo explants, e.g., dicotyledonous embryo explants or soybean, cotton, or canola embryo explants, following Rhizobiales- or Agrobacterium-mediated transformation. In some embodiments, 6-(gamma,gamma-dimethylallylamino)purine (2iP) is in the range of about 5 mg / L to about 50 mg / L, about 10 mg / L to about 50 mg / L, about 10 mg / L to about 40 mg / L, about 15 mg / L to about 40 mg / L, about 15 mg / L to about 35 mg / L, about 15 mg / L to about 30 mg / L, about 20 mg / L to about 30 mg / L, about 22.5 mg / L to about 30 mg / L, about 22.5 mg / L to about 27.5 mg / L, or about 5 mg / L. The compound may be present in the bacterial culture medium at a concentration of about 7.5 mg / L, about 10 mg / L, about 15 mg / L, about 20 mg / L, about 25 mg / L, about 30 mg / L, about 35 mg / L, about 40 mg / L, about 45 mg / L, or about 50 mg / L (including all derivable ranges and values ​​therebetween), which may improve the survival rate, regrowth potential, and / or transformation / editing frequency of soybean embryo explants following Rhizobiales- or Agrobacterium-mediated transformation.

[0059] Embodiments of the present disclosure may include genetically transforming or modifying embryonic explants, such as at least one cell of each of at least two embryonic explants of a population, by introducing a heterologous polynucleotide molecule en bloc via Rhizobiales- or Agrobacterium-mediated transformation, which generally involves contacting the explants with a Rhizobiales bacterium or Agrobacterium containing the heterologous polynucleotide. Such an introduction step may involve placing, adding, and / or contacting the explant or a population of explants with an inoculation medium containing the Rhizobiales bacterium or Agrobacterium, and performing the inoculation step for a period of time before removing the Rhizobiales bacterium or Agrobacterium from the explants. The inoculation medium may include, for example, water, basal salts, macronutrients, micronutrients, and / or vitamins. In certain embodiments, the inoculation may include polyethylene glycol and / or an antibacterial and / or antifungal agent. The concentration of Rhizobiales bacteria or Agrobacterium in the inoculation medium can be measured and / or defined in terms of optical density (OD). Increasing the concentration of Agrobacterium in the inoculation and / or co-cultivation medium can improve or increase shoot and plugging frequencies, and therefore transformation frequencies. The OD concentration of Rhizobiales bacteria or Agrobacterium in the inoculation medium can range from about 0.1 to about 2.0, about 0.1 to about 1.0, about 0.1 to about 0.75, about 0.1 to about 0.5, about 0.2 to about 2.0, about 0.2 to about 1.0, about 0.2 to about 0.5, about 0.25 to about 2.0, about 0.25 to about 1.0, about 0.25 to about 0.5, and about 0.5 to about 2. It may be in the range of 0, about 0.5 to about 1.5, about 0.75 to about 1.5, or about 0.75 to about 1.25, or about 0.1, about 0.2, about 0.25, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.75, about 0.8, about 0.9, about 1.0, about 1.25, about 1.5, or about 2.0 (including all derivable ranges and values ​​therebetween).According to some embodiments, transformation may be improved in corn germplasm, including explants of some monocotyledonous or male corn lines, by lowering the OD concentration of Rhizobiales bacteria or Agrobacterium in the inoculation medium, for example, to a range of about 0.25 to about 1.0 or about 0.25 to about 0.5, or to about 0.1, about 0.2, about 0.25, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.75, about 0.8, about 0.9, or about 1.0 (including all derivable ranges and values ​​therebetween).

[0060] In certain embodiments, the inoculation medium may optionally contain thiabedazole (TBZ) and / or nystatin, which may be used as antibacterial or antifungal agents. The TBZ concentration may range from about 0 g / L to about 100 g / L, or more preferably from about 5 g / L to about 15 g / L, or about 10 g / L, and / or the nystatin concentration may range from about 0 g / L to about 300 g / L, or more preferably from about 25 g / L to about 75 g / L, or about 50 g / L. In certain embodiments, the inoculation medium may contain the Rhizobiales or Agrobacterium culture medium described above, which may be transferred in a volumetric manner to contact the explants. In certain embodiments, the inoculation medium may optionally contain or include cytokinin and / or lipoic acid. According to some embodiments, the explant (or group or plurality of explants) can be transferred or placed into a tube or container, to which a quantity of Rhizobiales or Agrobacterium culture medium can be added. The quantity of inoculum (or inoculation medium containing Rhizobiales or Agrobacterium) can be added to the tube or container in an amount or volume sufficient to cover and / or submerge the explants.

[0061] According to some embodiments, the tube or container containing the explants in the inoculation medium may optionally be sonicated, vortexed, or otherwise physically or mechanically agitated. Such agitation may aid in improving transformation and / or delivery or introduction of heterologous polynucleotides into the cell(s) of the explant, such as by wounding and / or increasing penetration or penetration of Rhizobiales bacteria into the meristem or explant tissue. According to some embodiments, the explants may be sonicated or agitated for a period ranging from about 1 second to about 10 minutes, or from about 5 seconds to about 5 minutes, or from about 10 seconds to about 4 minutes, or from about 10 seconds to about 3 minutes, or for about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3 minutes, or about 4 minutes (including all derivable ranges and values ​​therebetween). With or without a physical or mechanical agitation step, the explant may be gently agitated, moved, or inverted (rather than the preceding, more vigorous agitation or injury), such as by shaking or rocking, as part of this inoculation step to improve transformation and / or delivery or introduction of the heterologous polynucleotide into the cell(s) of the explant. This gentle agitation may last for about 5 seconds to about 2 hours, or about 5 seconds to about 20 minutes, or about 10 seconds to about 15 minutes, or about 1 minute to about 15 minutes, or about 5 minutes to about 15 minutes, or about 7.5 minutes to about 12.5 minutes, or about 1 minute to about 2 hours, or about 5 minutes to about 2 hours, or about 10 minutes to about 2 hours, or about 20 minutes to about 1.5 hours, or about 30 minutes to about 1.5 hours, or about 45 minutes to about 1.25 hours, or about 1 minute to about 1 hour, or about 5 minutes to about 45 minutes. minutes, or a period ranging from about 15 minutes to about 45 minutes, or from about 20 minutes to about 40 minutes, or about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 1.5 hours, or about 2 hours (including all derivable ranges and values ​​therebetween).

[0062] In some embodiments, the introducing or inoculating step may be performed under ambient light conditions. In some embodiments, the introducing or inoculating step, such as for monocotyledonous plant embryo explants, may include subjecting the population of embryo explants to a force treatment, such as centrifugation and / or pressure treatment(s). According to some embodiments, the heterologous polynucleotide molecule, ribonucleoprotein, and / or site-specific nuclease is introduced into the population via particle bombardment.

[0063] According to embodiments of the present disclosure, a force treatment is applied to a population of seed embryo explants either before, during, or both before and during inoculation of the population with Rhizobiales or Agrobacterium bacteria containing the heterologous polynucleotide molecule. In certain embodiments, the force treatment is applied during and / or after rehydration of the seed embryo explants. In certain embodiments, the force treatment may be applied during the inoculation step while the population is in contact with the inoculation medium. In one embodiment, explants "in contact" with the medium may be placed completely or partially in or on the medium. Non-limiting examples of media with which the explants may be in contact include liquid media, solid media, and media-containing substrates. The population may be immersed in a volume of inoculation medium when the force treatment is applied. Alternatively, the force treatment may be applied to the embryo explants of the population after excess inoculation medium has been removed. The inoculation medium may be, for example, decanted, poured, aspirated, or blotted from the explants prior to application of the force treatment. When force treatment is applied during the inoculation step, even if an amount or volume of inoculation medium is removed from the explants before force treatment, the inoculation medium is not completely removed from contact with the explants of the population.

[0064] As used herein, the term "heterologous polynucleotide molecule" refers to a polynucleotide molecule that does not naturally occur in the transformed or modified cell or that does not naturally occur in the same form or structure without human intervention. For example, the heterologous polynucleotide molecule may not naturally occur in the transformed or modified plant species, or may be expressed in a manner or genomic background that differs from the natural expression pattern or genomic background found in the transformed or modified species; for example, in some embodiments, the heterologous polynucleotide molecule may be overexpressed. In certain embodiments, the heterologous polynucleotide molecule may be a combination of two or more polynucleotide molecules, such a combination not normally found in nature. In certain embodiments, the two polynucleotide molecules may be derived from different species or from different genes, such as different genes from the same species or the same gene from different species. In some embodiments, the heterologous polynucleotide molecule may comprise two polynucleotide sequences that are not juxtaposed or operably linked in any naturally occurring polynucleotide molecule. A heterologous polynucleotide molecule, in further embodiments, may comprise a promoter or other regulatory sequence operably linked to a transcribable polynucleotide sequence, wherein the promoter or other regulatory sequence and the transcribable polynucleotide sequence are not operably linked in any naturally occurring polynucleotide molecule. As used herein, the term "polynucleotide molecule" refers to a linear or circular, single- or double-stranded DNA or RNA polynucleotide molecule or sequence, which may be derived from any source. For example, a polynucleotide molecule may comprise a polynucleotide sequence in which one or more nucleic acid sequences are linked to each other in a functionally operative manner. As used herein, the term "nucleic acid sequence" refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence.

[0065] As used herein, the term "ribonucleoprotein" refers to a protein that can interact with nucleic acids or polynucleotide molecules. The ribonucleoprotein may be, for example, a site-specific nuclease known in the art to associate with nucleic acids or polynucleotide molecules. Non-limiting examples of site-specific nucleases that may be ribonucleoproteins include RNA-guided endonucleases, such as those in the CRISPR / Cas system (see, for example, U.S. Patent Nos. 8,697,359, 8,771,945, and 9,790,490 and U.S. Patent Application Publication No. 2014 / 0068797), and CRISPR-associated transposases or CASTs (see, for example, U.S. Patent Application Publication No. 2020 / 0190487), the contents and disclosures of which are incorporated herein by reference in their entirety. In some embodiments, the polynucleotide molecule, heterologous polynucleotide molecule, or ribonucleoprotein may be a recombinant polynucleotide molecule or recombinant protein. In some embodiments, a polynucleotide molecule or heterologous polynucleotide molecule can be a recombinant polynucleotide molecule. As used herein, the term "recombinant," when used with respect to a polynucleotide (DNA or RNA) molecule, protein, construct, vector, etc., refers to a polynucleotide or protein molecule or sequence that does not occur in nature or that does not occur in nature in the same form and / or structure without human intervention. In certain embodiments, a recombinant polynucleotide (DNA or RNA) molecule, protein, construct, vector, etc. can include a combination of two or more polynucleotide or protein sequences that do not occur together in nature in the same manner, such as, for example, a polynucleotide molecule, protein, construct that includes at least two polynucleotide or protein sequences that are operably linked but heterologous with respect to each other.In additional embodiments, recombinant polynucleotide (DNA or RNA) molecules, proteins, constructs, vectors, etc., can include, for example, any combination of two or more polynucleotide or protein sequences in the same molecule (e.g., a plasmid, construct, vector, chromosome, protein, etc.), where such combinations are artificial and not normally found in nature. As used herein, the phrase "not normally found in nature" means not found in nature without human intervention. Recombinant polynucleotide or protein molecules, constructs, etc., can include polynucleotide or protein sequence(s) that are (i) separated from other polynucleotide or protein sequence(s) that are naturally present in close proximity to each other and / or (ii) adjacent to (or contiguous with) other polynucleotide or protein sequence(s) that are not naturally present in close proximity to each other. Such recombinant polynucleotide molecules, proteins, constructs, etc., can also refer to polynucleotide or protein molecules or sequences that have been genetically engineered and / or constructed extracellularly. For example, a recombinant polynucleotide molecule can include any engineered or artificial plasmid, vector, etc., and can include linear or circular polynucleotide molecules. Such plasmids, vectors, etc. can contain various maintenance elements, including, for example, a prokaryotic origin of replication and a selectable marker, and often one or more transgenes or expression cassettes in addition to a plant selectable marker gene.

[0066] To improve the transformation or editing of a population of explants, a variety of different force treatments can be used or applied to the population before and / or during the inoculation step, such as centrifugation, gravity, vacuum, sonication, vortexing, shearing, mechanical force, pressure, or any combination thereof. In some embodiments, the force treatment can include pressure and / or gravity (or centrifugal) force treatment. In specific embodiments, the force treatment can include pressure treatment. In further embodiments, the force treatment can include gravity (or centrifugal) force treatment. In certain embodiments, the methods described herein can further include applying mechanical force, vortexing, shaking or shearing, sonication, and / or vacuum treatment in addition to pressure and / or gravity (or centrifugal) force treatment. Without being bound by theory, applying force treatments before or during inoculation may improve transformation by increasing contact and attachment of Rhizobiales bacteria to the explants of the population, wounding the explants, and / or increasing penetration or penetration of Rhizobiales bacteria into the meristem or explant tissue.

[0067] In some embodiments, the force treatment is from about 100 pounds per square inch (psi) to about 20,000 psi, from about 100 psi to about 18,000 psi, from about 100 psi to about 16,000 psi, from about 100 to about 14,000 psi, from about 100 to about 12,000 psi, from about 100 to about 10,000 psi, from about 100 to about 8,000 psi, from about 100 to about 6,000 psi, or from about 100 to about 4,000 psi. , about 100 to about 2,000 psi, about 100 to about 1,000 psi, or about 100 psi to about 500 psi, e.g., about 100 psi, about 150 psi, about 200 psi, about 250 psi, about 300 psi, about 350 psi, about 400 psi, or about 500 psi (including all derivable values ​​and ranges therebetween). Other pressure units are known in the art. Pressure in psi may be expressed in other units, such as standard atmospheres (atm) and Newtons (N) per square meter (N / m 2Methods for converting pressure in atm to pressure in psi are known in the art. Pressure in atm can be accurately calculated using the formula: atm = pressure (psi) / 14.6959488, where 1 psi is approximately 6894.76 N / m 2 Thus, 100 psi corresponds to approximately 6.80 atm, and 20,000 psi corresponds to approximately 1360.9 atm. Pressure treatments can also be converted to amounts of force if the surface area is known or fixed. For example, the piston / cell cavity surface area of ​​the French Press 40K pressure cell (Thermo® IEC, FA-032) used in the examples herein is approximately 0.88 in. 2 Therefore, 3,334 psi applied using the French Press 40K pressure cell is approximately 13,000 N (3,334 psi x 0.88 in) 2 )] / [0.225 lb / N]. Pressure treatment, in some embodiments, may be applied for about 10 seconds to about 10 minutes, about 15 seconds to about 8 minutes, about 30 seconds to about 6 minutes, about 2 minutes to about 4 minutes, or about 3 minutes (including all derivable values ​​and ranges therebetween).

[0068] The methods described herein may be used with a range of about 100×g to about 10,000×g, about 100×g to about 5,000×g, about 250×g to about 5,000×g, about 500×g to about 5,000×g, about 500×g to about 3,000×g, about 600×g to about 2,700×g, for example, about 500×g, about 550×g, about 600×g, about 650×g, about 700×g, or about 800×g. The gravity or centrifugal force may be applied to the population of embryonic explants, including applying a gravity or centrifugal force of about 750×g, about 800×g, about 850×g, about 900×g, about 950×g, about 1000×g, about 1500×g, about 2000×g, about 2500×g, about 3000×g, about 3500×g, or about 4000×g (including all derivable values ​​and ranges therebetween). Non-limiting examples of gravity treatments that may be applied to the population include centrifugal force or relative centrifugal force, which may be applied using a suitable centrifuge. Methods for converting gravity or centrifugal force, such as the relative centrifugal force (RCF) generated by a centrifuge, to other units, such as revolutions per minute (rpm) and Newtons (N), are known in the art. Relative centrifugal force can be calculated using the following formula: rpm = √[RCF / (r×1.118)×1×10 5 ] (where r = radius of rotation in centimeters) can be used to calculate based on rpm and the known dimensions of the device. For the Sorvall™ RC3BP centrifuge (Thermo Fisher Scientific, Waltham, MA, USA) used in the examples described herein, the radius of rotation is approximately 24.67 cm. Therefore, 2620 × g is approximately 3,082 rpm [√[2620 / (24.67 × 1.118)] × 1 × 10 5 Similarly, centrifugal force in Newtons is calculated using the formula: Force (N) = RCF x Mass of contents of centrifuge tube (kg) x 9.82 m / s 2 In a particular embodiment, if the mass of the contents of a centrifuge tube can be approximately 0.05 kg, then 2620×g is approximately 1286 N [(2620×g)×0.05 kg×9.82 m / s 2Gravity or centrifugal force treatment, in some embodiments, may be applied for a period in the range of about 1 minute to about 2 hours, about 2 minutes to about 110 minutes, about 5 minutes to about 90 minutes, about 10 minutes to about 90 minutes, about 10 minutes to about 80 minutes, about 10 minutes to about 70 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 50 minutes, about 15 minutes to about 45 minutes, or about 20 minutes to about 40 minutes, e.g., about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes (1 hour), including all derivable values ​​and ranges therebetween.

[0069] According to some embodiments, the methods described herein can achieve a yield in the range of about 2,500×g to about 10,000×g, about 2,500×g to about 5,000×g, about 2,500×g to about 4,500×g, about 2,800×g to about 5,000×g, about 2,800×g to about 4,500×g, about 3,000×g to about 8,000×g, about 3,000×g to about 7,000×g, about 3,000×g to about 6,000×g, about 3,000×g to about 5,000×g, about 3,500×g to about 5,000×g, or about 3,500×g to about 4,500×g, e.g., about 2,500×g, about 3,000×g, about 3,5 ... The method includes applying a higher gravity or centrifugal force or relative centrifugal force (RCF) to the monocotyledonous seed embryo explants, such as about 3,000×g, about 3,600×g, about 3,700×g, about 3,800×g, about 3,900×g, about 4,000×g, about 4,100×g, about 4,200×g, about 4,300×g, about 4,400×g, about 4,500×g, about 4,600×g, about 4,700×g, about 4,800×g, about 4,900×g, about 5,000×g, about 6,000×g, about 7,000×g, about 8,000×g, about 9,000×g, or about 10,000×g (including all derivable ranges and values ​​therebetween). According to some embodiments, application of higher gravity or centrifugal force or relative centrifugal force (RCF) may improve the transformation and / or regeneration of genetically modified plants of certain monocotyledonous plants or maize lines that are more amenable to efficient transformation and / or regeneration of monocotyledonous plants or maize embryo explants or genetically modified plants, e.g., certain male germplasm maize lines.

[0070] Force treatments, such as gravity (or centrifugal force) and / or pressure treatment(s), can be applied at temperatures of about 0.5°C to about 28°C, about 2°C to about 28°C, about 4°C to about 28°C, about 10°C to about 28°C, about 10°C to about 25°C, or about 15°C to about 23°C (including all derivable values ​​and ranges therebetween).

[0071] In one aspect of the methods provided herein, the force treatment may include applying both pressure and gravity treatment to a population of embryonic explants. Pressure and / or gravity treatment may be applied before, during, or both before and during inoculation of the population with a bacterium from the order Rhizobiales, wherein the Rhizobiales bacterium comprises a heterologous polynucleotide for transforming, editing, or genetically modifying at least one plant cell of the explants of the population. In some embodiments, pressure treatment is applied before gravity treatment. In some embodiments, gravity treatment is applied before pressure treatment. The order of application of pressure and gravity treatment may be selected based on improving the efficiency or frequency of transformation or editing, or based on ease of handling. In some embodiments, when a combination of pressure and gravity treatment is applied to a population, pressure treatment may be applied before gravity treatment, which may be, at least in part, because force treatment can be applied more uniformly before pelleting the explants by gravity or centrifugation. Alternatively, centrifuged or pelleted explants may be resuspended before the subsequent pressure treatment, or pressure treatment may be applied to centrifuged or pelleted explants without resuspension. In one aspect of the present disclosure, applying pressure and gravity treatments either before, during, or before and during inoculation may improve plant transformation or editing compared to applying pressure or gravity treatments alone.

[0072] In another aspect, the methods described herein may further include applying a vacuum treatment to the population of embryo explants. Vacuum treatment may include, for example, immersing the population in a liquid inoculation medium containing Rhizobiales bacteria and subjecting the population to reduced pressure followed by rapid or gradual re-pressurization. Alternatively, vacuum treatment may be applied to a population of embryo explants that is not immersed in a liquid inoculation medium. In some embodiments, vacuum treatment may be applied before a force treatment is applied, after a force treatment is applied, before a gravity treatment is applied, after a gravity treatment is applied, before a pressure treatment is applied, and / or after a pressure treatment is applied. In certain embodiments, when force treatment includes applying a pressure treatment and a gravity treatment, vacuum treatment may be applied between the application of the pressure treatment and the application of the gravity treatment, regardless of whether the gravity treatment or the pressure treatment is applied first. In one embodiment, the mass may be subjected to a vacuum treatment at a pressure of about 0.05 atm to about 0.50 atm, about 0.05 atm to about 0.40 atm, about 0.05 atm to about 0.30 atm, about 0.05 atm to about 0.20 atm, about 0.05 atm to about 0.10 atm, about 0.10 atm to about 0.50 atm, about 0.10 atm to about 0.40 atm, about 0.10 to about 0.30 atm, or about 0.10 atm to about 0.20 atm (including all derivable values ​​and ranges therebetween).

[0073] Once the Rhizobiales or Agrobacterium have been inoculated into a population of embryonic explants to introduce the heterologous polynucleotide into at least one cell of the explants, the inoculation medium containing the Rhizobiales or Agrobacterium, or most or at least an excess amount of the inoculation medium, can be removed prior to any subsequent transformation or culturing step. The inoculation medium can be removed by any combination of decanting (often using a strainer or net), filtration, aspiration, pipetting, vacuuming, and / or blotting or wicking. Blotting or wicking of the inoculation medium can be performed by contacting the medium with filter paper or other absorbent material. Removal of the inoculation medium can be performed prior to any subsequent co-cultivation step.

[0074] C. Embryonic Explant Co-cultivation After inoculating a population of embryonic explants with Rhizobiales or Agrobacterium containing the heterologous polynucleotide to introduce the heterologous polynucleotide into at least one cell of the explants, and optionally after removing the inoculation medium, the explants can be contacted with a co-culture medium and co-cultured. According to this embodiment, the seed embryo explant(s) can be transferred to or contacted with a co-culture medium, or the seed embryo explant(s) can be transferred to one or more (co-culture) plates containing a co-culture medium, or the co-culture medium can be added after the explants are transferred to the plates. The co-culture medium can include, for example, water, basal salts, macronutrients, micronutrients, and / or vitamins. According to some embodiments, the co-culture medium may be free of or may be free of any plant hormones, such as auxins and / or cytokinins, and / or any surfactants or wetting agents. Alternatively, the co-culture medium may contain or may include plant hormones, such as auxins and / or cytokinins, and / or surfactants or wetting agents. In certain embodiments, the co-culture medium includes one or more cytokinins. Non-limiting examples of cytokinins that can be used in the co-culture medium include 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-topolin). According to some embodiments, the co-cultivation medium may optionally contain an antibacterial or antifungal agent, such as nystatin and / or thiabendazole (TBZ), which may be present at the concentration(s) described above for the bacterial culture medium and / or inoculation medium. Inclusion of one or more cytokinins and / or lipoic acid in the co-cultivation medium may, in certain embodiments, improve survival, regrowth potential, and / or transformation / editing frequency following Rhizobiales- or Agrobacterium-mediated transformation. In certain embodiments, inclusion of lipoic acid in the co-cultivation medium reduces stress during Rhizobiales- or Agrobacterium-mediated transformation (e.g., during the inoculation and co-cultivation steps).

[0075] The concentration of cytokinin in the co-cultivation medium can be or be within the concentration(s) described above for the bacterial culture medium and / or inoculation medium, and in some embodiments, the concentration of cytokinin in the co-cultivation medium can be from about 0.1 mg / L to about 50 mg / L, from about 0.1 mg / L to about 45 mg / L, from about 0.1 mg / L to about 40 mg / L, from about 0.1 mg / L to about 35 mg / L, from about 0.1 mg / L to about 30 mg / L, from about 0.1 mg / L to about 25 mg / L, from about 0.1 mg / L to about 20 mg / L, from about 0.1 mg / L to about 15 mg / L, Approximately 0.1 mg / L to approximately 10 mg / L, approximately 0.1 mg / L to approximately 5 mg / L, approximately 0.1 mg / L to approximately 4 mg / L, approximately 0.1 mg / L to approximately 3 mg / L, approximately 0.1 mg / L to approximately 2 mg / L, approximately 0. 1mg / L to about 1mg / L, about 0.5mg / L to about 5mg / L, about 0.5mg / L to about 4mg / L, about 0.5mg / L to about 3mg / L, about 1mg / L to about 5mg / L, about 1mg / L to about 4 mg / L, about 1 mg / L to about 3 mg / L, about 5 mg / L to about 40 mg / L, about 5 mg / L to about 30 mg / L, about 10 mg / L to about 30 mg / L, or about 20 mg / L to about 30 mg / L L, or about 0.1mg / L, about 0.2mg / L, about 0.3mg / L, about 0.4mg / L, about 0.5mg / L, about 0.6mg / L, about 0.7mg / L, about 0.8mg / L, about 0.9mg / L, about 1 mg / L, about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, about 10 mg / L, about 15 mg / L, about 20 mg / L, about 25 mg / L, about 30 mg / L, about 35 mg / L, about 40 mg / L, about 45 mg / L, or about 50 mg / L (including all derivable ranges and values ​​therebetween).

[0076] In certain embodiments, lipoic acid is present in an amount of from about 0.1 mg / L to about 500 mg / L, from about 0.1 mg / L to about 400 mg / L, from about 0.1 mg / L to about 300 mg / L, from about 0.1 mg / L to about 200 mg / L, from about 10 mg / L to about 200 mg / L, from about 10 mg / L to about 180 mg / L, from about 10 mg / L to about 160 mg / L, from about 10 mg / L to about 140 mg / L, from about 10 mg / L to about 120 mg / L, from about 10 mg / L to about 100 mg / L, from about 20 mg / L to about 80 mg / L, from about 40 mg / L to about 60 mg / L, from about 50 mg / L to about 60 mg / L, or from about 70 mg / L to about 80 mg / L. mg / L to about 60 mg / L, or about 50 mg / L to about 55 mg / L, or about 5 mg / L, about 10 mg / L, about 15 mg / L, about 20 mg / L, about 25 mg / L, about 30 mg / L, about 35 mg / L, about 40 mg / L, about 45 mg / L, about 50 mg / L L, about 55 mg / L, about 60 mg / L, about 65 mg / L, about 70 mg / L, about 75 mg / L, about 80 mg / L, about 85 mg / L, about 90 mg / L, about 95 mg / L, about 100 mg / L, about 105 mg / L, about 110 mg / L, about 115 mg / L, about 12 0mg / L, about 125mg / L, about 130mg / L, about 135mg / L, about 140mg / L, about 145mg / L, about 150mg / L, about 155mg / L, about 160mg / L, about 165mg / L, about 170mg / L, about 175mg / L, about 180mg / L , about 185 mg / L, about 190 mg / L, about 195 mg / L, about 200 mg / L, about 210 mg / L, about 220 mg / L, about 230 mg / L, about 240 mg / L, about 250 mg / L, about 260 mg / L, about 270 mg / L, about 280 mg / L, about 290

[0039] The co-cultivation medium may be present at a concentration of about 300 mg / L, 310 mg / L, 320 mg / L, 330 mg / L, 340 mg / L, 350 mg / L, 360 mg / L, 370 mg / L, 380 mg / L, 390 mg / L, 400 mg / L, about 410 mg / L, about 420 mg / L, about 430 mg / L, about 440 mg / L, about 450 mg / L, about 460 mg / L, about 470 mg / L, about 480 mg / L, about 490 mg / L, or about 500 mg / L (including all derivable ranges and values ​​therebetween).

[0077] In some embodiments, thidiazuron (TDZ) is about 0.1 mg / L to about 10 mg / L, about 0.1 mg / L to about 9 mg / L, about 0.1 mg / L to about 8 mg / L, about 0.1 mg / L to about 7 mg / L, about 0.1 mg / L to about 6 mg / L, about 0.1 mg / L to about 5 mg / L, about 0.1 mg / L to about 4 mg / L, about 0.1mg / L to about 3mg / L, about 0.1mg / L to about 2mg / L, about 0.1mg / L to about 1mg / L, about 0.25mg / L to about 1.75m g / L, about 0.5 mg / L to about 1.5 mg / L, about 0.1 mg / L to about 5 mg / L, about 0.5 mg / L to about 4 mg / L, about 0.5 mg / L to about 3 mg / L, about 0.5 mg / L to about 2 mg / L, about 0.5 mg / L to about 1.5 mg / L, about 1 mg / L to about 5 mg / L, about 1 mg / L to about 4 mg / L, about 1 mg / L to about 3 mg / L, or about 0.1 mg / L, about 0.25 mg / L, about 0.5 mg / L, about 0.75 mg / L, about 1.0 mg / L, about 1.25 mg / L, about 1.5 mg / L, about 1.75 mg / L, about 2.0 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, or about 10 mg / L (including all derivable ranges and values ​​therebetween). Inclusion of thidiazuron (TDZ) in the co-culture medium can improve the survival rate, regenerability, and / or transformation / editing frequency after Rhizobiales- or Agrobacterium-mediated transformation. In some embodiments, inclusion of one or more cytokinins and / or lipoic acid in the co-culture medium improves the survival rate, regenerability, and / or transformation / editing frequency of dicotyledonous embryo explants. In particular embodiments, inclusion of one or more cytokinins and / or lipoic acid in the co-culture medium improves the survival rate, regenerability, and / or transformation / editing frequency of soybean, cotton, or canola embryo explants.

[0078] In some embodiments, cytokinins other than thidiazuron (TDZ), such as 6-benzylaminopurine (BAP), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-topolin), may be included in the co-cultivation medium at concentrations adjusted and set depending on their relative activities. For example, 6-benzylaminopurine (BAP), kinetin, zeatin, and / or 6-(3-hydroxybenzylamino)purine (meta-topolin) may be present in concentrations of about 0.1 mg / L to about 10 mg / L, about 0.1 mg / L to about 9 mg / L, about 0.1 mg / L to about 8 mg / L, about 0.1 mg / L to about 7 mg / L, about 0.1 mg / L to about 6 mg / L, about 0.1 mg / L to about 5 mg / L, about 0.1 mg / L to about 4 mg / L, about 0.1 mg / L to about 3 mg / L, about 0.1 mg / L to about 2 mg / L, about 1 mg / L to about 10 mg / L, about 2 mg / L to about 10 mg / L, about 2 mg / L to about 8 mg / L, about 2.5 mg / L to about 7.5 mg / L, or about 3 mg / L. The compound may be present in the bacterial culture medium at a concentration of about 10 mg / L, about 4 mg / L to about 6 mg / L, about 1.0 mg / L, about 1.25 mg / L, about 1.5 mg / L, about 1.75 mg / L, about 2.0 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, or about 10 mg / L (including all derivable ranges and values ​​therebetween), which may improve the survival rate, regrowth potential, and / or transformation / editing frequency of embryo explants, e.g., dicotyledonous embryo explants or soybean, cotton, or canola embryo explants, following Rhizobiales- or Agrobacterium-mediated transformation.

[0079] In some embodiments, 6-(gamma,gamma-dimethylallylamino)purine (2iP) is in the range of about 5 mg / L to about 50 mg / L, about 10 mg / L to about 50 mg / L, about 10 mg / L to about 40 mg / L, about 15 mg / L to about 40 mg / L, about 15 mg / L to about 35 mg / L, about 15 mg / L to about 30 mg / L, about 20 mg / L to about 30 mg / L, about 22.5 mg / L to about 30 mg / L, about 22.5 mg / L to about 27.5 mg / L, or about 5 mg / L, about 7.5 mg / L, about 10 mg / L , about 15 mg / L, about 20 mg / L, about 25 mg / L, about 30 mg / L, about 35 mg / L, about 40 mg / L, about 45 mg / L, or about 50 mg / L (including all derivable ranges and values ​​therebetween), which may improve the survival, regrowth potential, and / or transformation / editing frequency of embryo explants, e.g., dicotyledonous embryo explants or soybean, cotton, or canola embryo explants, following Rhizobiales- or Agrobacterium-mediated transformation.

[0080] In certain embodiments, the co-cultivation medium may contain a surfactant, which may be particularly applicable to the transformation or editing of monocotyledonous embryonic explants, and may include any suitable surfactant or combination of surfactants known in the art, such as a detergent, wetting agent, emulsifier, foaming agent, or dispersing agent. In some embodiments, the surfactant may be Silwet® or a similar surfactant.

[0081] According to some embodiments, the methods described herein include transferring monocotyledonous seed embryo explant(s) to co-cultivation plate(s) or container(s) at a reduced density of visible or total explants per container or plate. According to some embodiments, reducing the density of visible or total explants per co-cultivation plate may improve the transformation and / or regeneration of genetically modified plants of certain monocotyledonous plants or maize lines, e.g., certain male germplasm maize lines, that are more resistant to efficient transformation and / or regeneration of monocotyledonous or maize embryo explants or genetically modified plants. As described herein, transformation of male maize lines, and possibly other monocotyledonous germplasm, can often be difficult or less efficient compared to female maize lines or other monocotyledonous germplasm, which may be due to variable or low germination rates, low Agrobacterium infection rates, crowding and tissue death (especially at high densities) in culture and selection media, and / or minimal shoot regeneration. These differences may be due to the different genetics and characteristics of male corn lines or other germplasm and embryo explants derived from seeds of male corn lines or other germplasm compared to female corn lines or other germplasm. Given that seed embryo explants of certain male germplasm or lines or other monocotyledonous germplasm may have a lower regeneration rate, which may be associated with reduced, decreased, or diminished viability and / or regrowth potential, a greater number of visible (or total) explants may be required to have an equivalent number of regrowable explants and produce an equivalent number of regenerated explants, plantlets, or plants compared to female corn lines or other monocotyledonous germplasm with a relatively higher or increased regeneration rate (or higher or increased viability and / or regrowth potential).However, placing a greater number of visible (or total) explants in the same number of co-culture plate(s) or container(s) may cause or even result in the co-culture plate(s) or container(s) becoming overcrowded, resulting in a reduced, decreased, or diminished regeneration rate (or a reduced, decreased, or diminished viability and / or regeneration potential). In some embodiments, the overcrowding may reduce drying of the embryonic explants, which may result in reduced delivery of the heterologous polynucleotide molecule to at least one cell of the embryonic explant. Reduced delivery of the heterologous polynucleotide molecule may, in some embodiments, result in a reduced, decreased, or diminished regeneration rate (or a reduced, decreased, or diminished viability and / or regeneration potential) when the co-culture medium, sprouting induction medium, expansion sprouting induction medium, and / or regeneration medium comprises a selection agent. Thus, increasing the number of co-culture plate(s) of a given size and / or decreasing, lowering, or reducing the number of visible (or total) explants per plate or co-culture plate area may result in a relatively higher or increased regeneration rate (or higher or increased viability and / or regeneration potential) per number of visible (or total) explants in the co-culture plate(s) or container(s) and / or per number of inoculated explants in contact with the co-culture medium, or a relatively higher or increased number of regenerable explants. In some embodiments, the number of visible explants may be defined as the total number of explants, including regenerable and non-regenerable explants. In certain embodiments, the percent regenerable explants may be calculated as the number of regenerable explants per gram or the number of viable explants before transformation divided by the total number of visible explants per gram, multiplied by 100.According to some embodiments, the saturation index is from about 500 to about 50,000, from about 500 to about 25,000, from about 500 to about 20,000, from about 500 to about 15,000, from about 500 to about 14,000, from about 500 to about 13,000, from about 500 to about 12,000, from about 500 to about 11,000, from about 500 to about 10,000, from about 500 to about 9,000, from about 500 to about 8,000, from about 500 to about 7,000, from about 500 to about 6,000, from about 500 to about 5,000, from about 500 to about 4,000, from about 500 to about 3,000, from about 500 to about 2,500, from about 500 to about 2,000, or from about 500 to about 6,000. A predetermined number of visible (or total) monocotyledonous seed embryo explants, such as in the range of about 1,500, about 1,000 to about 3,000, about 1,000 to about 2,500, about 1,000 to about 2,500, about 1,000 to about 2,000, or about 1,000 to about 1,500 (including all derivable ranges and values ​​therebetween), can be transferred from the inoculation medium to a relatively larger or greater number of co-culture plate(s) or container(s), e.g., 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more co-culture plate(s) or container(s).

[0082] According to some embodiments, a certain number of visible monocotyledonous seed embryo explants of a given germplasm or lineage, corresponding to a target number of regenerable seed embryo explants based on known or theorized regeneration rates for such germplasm or lineage, are added or transferred to a greater number of co-culture plate(s) or container(s), or to a greater total surface area of ​​the co-culture plate(s) or container(s), such that the density of visible (or total) explants per plate or container, or per total surface area of ​​the co-culture plate(s) or container(s) is lower.According to some embodiments, the number of visible (or total) monocotyledonous seed embryo explants transferred or added to the co-cultivation plate(s) or container(s) can be at a density of about 800 or less seed embryo explants per plate, about 700 or less seed embryo explants per plate, or about 600 or less seed embryo explants per plate, or at a density of about 50 to about 800 seed embryo explants per plate, about 50 to about 700 seed embryo explants per plate, or about 600 or less seed embryo explants per plate. About 50 to about 600 seed embryo explants, about 50 to about 500 seed embryo explants per plate, about 50 to about 400 seed embryo explants per plate, about 100 to about 700 seed embryo explants per plate, about 100 to about 600 seed embryo explants per plate, about 100 to about 500 seed embryo explants per plate, about 100 to about 400 seed embryo explants per plate, about 150 to about 700 seed embryo explants per plate, about 150 to about 600 Seed embryo explants, a density range of about 150 to about 500 seed embryo explants per plate, about 150 to about 400 seed embryo explants per plate, about 200 to about 500 seed embryo explants per plate, about 200 to about 400 seed embryo explants per plate, about 300 to about 700 seed embryo explants per plate, about 400 to about 700 seed embryo explants per plate, or about 500 to about 700 seed embryo explants per plate, or about 100 , about 150, about 175, about 200, about 225, about 250, about 255, about 260, about 265, about 270, about 275, about 280, about 285, about 290, about 295, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 570, about 575, about 600, about 650, about 700, about 750, or about 800 seed embryo explants (including all derivable ranges and values ​​therebetween). For these density values ​​and ranges, the surface area of ​​each co-culture plate is approximately 11.9 square inches (in. 2 ) or 76.8 square centimeters (cm 2Therefore, all of the above density values ​​and ranges for seed embryo explants per plate can be easily converted to density values ​​and ranges for seed embryo explants per co-culture surface area (e.g., a density of 100 seed embryo explants per plate divided by the surface area per plate yields approximately 8.4 seed embryo explants per square inch (in 2 ) or approximately 1.3 seed embryo explants per square centimeter (cm 2 ) yields a density of seed embryo explants per co-culture surface area; similar conversions can be easily made for other density values ​​and ranges.) The value and range of density of seed embryo explants per co-culture surface area is a more universal definition for seed embryo explant densities in a variety of different co-culture plate(s) or container(s), each of which may have a different surface area.

[0083] In some embodiments, the number of visible (or total) monocot seed embryo explants transferred or added to the co-cultivation plate(s) or container(s) is determined by the square centimeter (cm) of co-cultivation surface area. 2) per approximately 11.0, approximately 10.9, approximately 10.8, approximately 10.7, approximately 10.6, approximately 10.5, approximately 10.4, approximately 10.3, approximately 10.2, approximately 10.1, approximately 10.0, approximately 9.9, approximately 9.8, approximately 9.7, approximately 9.6, approximately 9.5, approximately 9.4, approximately 9.3, approximately 9.2, approximately 9.1, approximately 9.0, approximately 8.9, approximately 8.8, approximately 8.7, approximately 8.6, approximately 8.5, approximately 8.4, approximately 8.3, about 8.2, about 8.1, about 8.0, about 7.9, about 7.8, about 7.7, about 7.6, about 7.5, about 7.4, about 7.3, about 7.2, about 7.1, about 7.0, about 6.9, about 6.8, about 6.7, about 6.6, about 6.5, about 6.4, about 6.3, about 6.2, about 6.1, about 6.0, about 5.9, about 5.8, about 5.7, about 5.6, about 5.5, about 5.4, about 5 .3, approximately 5.2, approximately 5.1, approximately 5.0, approximately 4.9, approximately 4.8, approximately 4.7, approximately 4.6, approximately 4.5, approximately 4.4, approximately 4.3, approximately 4.2, approximately 4.1, approximately 4.0, approximately 3.9, approximately 3.8, approximately 3.7, approximately 3.6, approximately 3.5, approximately 3.4, approximately 3.3, approximately 3.2, approximately 3.1, approximately 3.0, approximately 2.9, approximately 2.8, approximately 2.7, approximately 2.6, approximately 2.5, approximately 2.4, approximately 2. In certain embodiments, the number of visible (or total) monocotyledonous seed embryo explants transferred or added to the co-cultivation plate(s) or container(s) is / are determined based on the density of the embryo explants per square centimeter (cm) of co-cultivation surface area. 2) about 0.1 to about 11.0, about 0.1 to about 10.5, about 0.1 to about 10.0, about 0.1 to about 9.5, about 0.1 to about 9.0, about 0.1 to about 8.5, about 0.1 to about 8.0, about 0.1 to about 7.5, about 0.1 to about 7.0, about 0.1 to about 6.5, about 0.1 to about 6.0, about 0.1 to about 5 0.5, about 0.1 to about 5.0, about 0.1 to about 4.5, about 0.1 to about 4.0, about 0.1 to about 3.5, about 0.1 to about 3.0, about 0.1 to about 2.5, about 0.1 to about 2.0, about 0.1 to about 1.5, about 0.1 to about 1.0, about 0.1 to about 0.5, about 0.2 to about 11.0, about 0.2 to about 10.5, About 0.2 to about 10.0, about 0.2 to about 9.5, about 0.2 to about 9.0, about 0.2 to about 8.5, about 0.2 to about 8.0, about 0.2 to about 7.5, about 0.2 to about 7.0, about 0.2 to about 6.5, about 0.2 to about 6.0, about 0.2 to about 5.5, about 0.2 to about 5.0, about 0.2 to about 4.5, about 0. 2 to about 4.0, about 0.2 to about 3.5, about 0.2 to about 3.0, about 0.2 to about 2.5, about 0.2 to about 2.0, about 0.2 to about 1.5, about 0.2 to about 1.0, about 0.2 to about 0.5, about 0.3 to about 11.0, about 0.3 to about 10.5, about 0.3 to about 10.0, about 0.3 to about 9.5, about 0.3 to About 9.0, about 0.3 to about 8.5, about 0.3 to about 8.0, about 0.3 to about 7.5, about 0.3 to about 7.0, about 0.3 to about 6.5, about 0.3 to about 6.0, about 0.3 to about 5.5, about 0.3 to about 5.0, about 0.3 to about 4.5, about 0.3 to about 4.0, about 0.3 to about 3.5, about 0.3 to about 3.0 , about 0.3 to about 2.5, about 0.3 to about 2.0, about 0.3 to about 1.5, about 0.3 to about 1.0, about 0.3 to about 0.5, about 0.4 to about 11.0, about 0.4 to about 10.5, about 0.4 to about 10.0, about 0.4 to about 9.5, about 0.4 to about 9.0, about 0.4 to about 8.5, about 0.4 to about 8.0, about 0.4 to about 7.5, about 0.4 to about 7.0, about 0.4 to about 6.5, about 0.4 to about 6.0, about 0.4 to about 5.5, about 0.4 to about 5.0, about 0.4 to about 4.5, about 0.4 to about 4.0, about 0.4 to about 3.5, about 0.4 to about 3.0, about 0.4 to about 2.5, about 0.4 to about 2.0, about 0.4 to about 1.5, about 0.4 to about 1.0, about 0.4 to about 0.5, about 0.5 to about 11.0, about 0.5 to about 10.5, about 0.5 to about 10.0, about 0.5 to about 9.5, about 0.5 to about 9.0, about 0.5 to about 8.5, about 0.5 to about 8.0, about 0.5 to about 7.5, about 0.5 to about 7.0, about 0.5 to about 6.5, about 0.5 to about 6.0, about 0.5 to about 5.5, about 0.5 to about 5.0, about 0.5 to about 4.5, about 0.5 to about 4.0, about 0.5 to about 3.5, about 0.5 to about 3.0, about 0.5 to about 2.5, about 0.5 to about 2.0, about 0.5 to about 1.5, about 0.5 to about 1.0, about 0.6 to about 11.0, about 0 0.6 to about 10.5, about 0.6 to about 10.0, about 0.6 to about 9.5, about 0.6 to about 9.0, about 0.6 to about 8.5, about 0.6 to about 8.0, about 0.6 to about 7.5, about 0.6 to about 7.0, about 0.6 to about 6.5, about 0.6 to about 6.0, about 0.6 to about 5.5, about 0.6 to about 5.0, about 0.6 to About 4.5, about 0.6 to about 4.0, about 0.6 to about 3.5, about 0.6 to about 3.0, about 0.6 to about 2.5, about 0.6 to about 2.0, about 0.6 to about 1.5, about 0.6 to about 1.0, about 0.7 to about 11.0, about 0.7 to about 10.5, about 0.7 to about 10.0, about 0.7 to about 9.5, about 0.7 to about 9 .0, about 0.7 to about 8.5, about 0.7 to about 8.0, about 0.7 to about 7.5, about 0.7 to about 7.0, about 0.7 to about 6.5, about 0.7 to about 6.0, about 0.7 to about 5.5, about 0.7 to about 5.0, about 0.7 to about 4.5, about 0.7 to about 4.0, about 0.7 to about 3.5, about 0.7 to about 3.0, about 0 0.7 to about 2.5, about 0.7 to about 2.0, about 0.7 to about 1.5, about 0.7 to about 1.0, about 0.8 to about 11.0, about 0.8 to about 10.5, about 0.8 to about 10.0, about 0.8 to about 9.5, about 0.8 to about 9.0, about 0.8 to about 8.5, about 0.8 to about 8.0, about 0.8 to about 7.5, about 0.8 to about 7.0, about 0.8 to about 6.5, about 0.8 to about 6.0, about 0.8 to about 5.5, about 0.8 to about 5.0, about 0.8 to about 4.5, about 0.8 to about 4.0, about 0.8 to about 3.5, about 0.8 to about 3.0, about 0.8 to about 2.5, about 0.8 to about 2.0, about 0.8 to about 1.5, about 0.8 to about 1.0 , about 0.9 to about 11.0, about 0.9 to about 10.5, about 0.9 to about 10.0, about 0.9 to about 9.5, about 0.9 to about 9.0, about 0.9 to about 8.5, about 0.9 to about 8.0, about 0.9 to about 7.5, about 0.9 to about 7.0, about 0.9 to about 6.5, about 0.9 to about 6.0, about 0.9 to about 5.5, about 0.9 to about 5.0, about 0.9 to about 4.5, about 0.9 to about 4.0, about 0.9 to about 3.5, about 0.9 to about 3.0, about 0.9 to about 2.5, about 0.9 to about 2.0, about 0.9 to about 1.5, about 0.9 to about 1.0, about 1.0 to about 11.0, about 1.0 to about 10.5, about 1.0 to about 10.0, about 1.0 to about 9.5, about 1.0 to about 9.0, about 1.0 to about 8.5, about 1.0 to about 8.0, about 1.0 to about 7.5, about 1.0 to about 7.0, about 1.0 to about 6.5, about 1.0 to about 6.0, about 1.0 to about 5.5, about 1.0 to about 5.0, about 1.0 to about 4.5, about 1.0 to about 4.0, about 1.0 to about 3. 5, about 1.0 to about 3.0, about 1.0 to about 2.5, about 1.0 to about 2.0, about 1.0 to about 1.5, about 1.5 to about 11.0, about 1.5 to about 10.5, about 1.5 to about 10.0, about 1.5 to about 9.5, about 1.5 to about 9.0, about 1.5 to about 8.5, about 1.5 to about 8.0, about 1.5 to about 7.5, About 1.5 to about 7.0, about 1.5 to about 6.5, about 1.5 to about 6.0, about 1.5 to about 5.5, about 1.5 to about 5.0, about 1.5 to about 4.5, about 1.5 to about 4.0, about 1.5 to about 3.5, about 1.5 to about 3.0, about 1.5 to about 2.5, about 1.5 to about 2.0, about 2.0 to about 11.0, about 2.0 to about 10.5, about 2.0 to about 10.0, about 2.0 to about 9.5, about 2.0 to about 9.0, about 2.0 to about 8.5, about 2.0 to about 8.0, about 2.0 to about 7.5, about 2.0 to about 7.0, about 2.0 to about 6.5, about 2.0 to about 6.0, about 2.0 to about 5.5, about 2.0 to about 5.0, about 2.0 to about 4 0.5, about 2.0 to about 4.0, about 2.0 to about 3.5, about 2.0 to about 3.0, about 2.0 to about 2.5, about 2.5 to about 11.0, about 2.5 to about 10.5, about 2.5 to about 10.0, about 2.5 to about 9.5, about 2.5 to about 9.0, about 2.5 to about 8.5, about 2.5 to about 8.0, about 2.5 to about 7.5 , about 2.5 to about 7.0, about 2.5 to about 6.5, about 2.5 to about 6.0, about 2.5 to about 5.5, about 2.5 to about 5.0, about 2.5 to about 4.5, about 2.5 to about 4.0, about 2.5 to about 3.5, about 2.5 to about 3.0, about 3.0 to about 11.0, about 3.0 to about 10.5, about 3.0 to about 10.0, about 3.0 to about 9.5, about 3.0 to about 9.0, about 3.0 to about 8.5, about 3.0 to about 8.0, about 3.0 to about 7.5, about 3.0 to about 7.0, about 3.0 to about 6.5, about 3.0 to about 6.0, about 3.0 to about 5.5, about 3.0 to about 5.0, about 3.0 to about 4.5, about 3.0 to about 4.0, about 3.0 to about 3.5, about 3.5 to about 11.0, about 3.5 to about 10.5, about 3.5 to about 10.0, about 3.5 to about 9.5, about 3.5 to about 9.0, about 3.5 to about 8.5, about 3.5 to about 8.0, about 3.5 to about 7.5, about 3.5 to about 7.0, about 3.5 to about 6.5, about 3.5 to about 6.0, about 3.5 to about 5.5, about 3.5 to about 5.0, about 3.5 to about 4.5, about 3.5 to about 4.0, about 4.0 to about 11.0, about 4.0 to about 10.5, about 4.0 to about 10.0, about 4.0 to about 9.5, about 4.0 to about 9.0, about 4.0 to about 8.5, about 4.0 to about 8.0, about 4.0 to about 7.5, about 4.0 to about 7.0, About 4.0 to about 6.5, about 4.0 to about 6.0, about 4.0 to about 5.5, about 4.0 to about 5.0, about 4.0 to about 4.5, about 4.5 to about 11.0, about 4.5 to about 10.5, about 4.5 to about 10.0, about 4.5 to about 9.5, about 4.5 to about 9.0, about 4.5 to about 8.5, about 4.5 to about 8.0, about 4 0.5 to approximately 7.5, approximately 4.5 to approximately 7.0, approximately 4.5 to approximately 6.5, approximately 4.5 to approximately 6.0, approximately 4.5 to approximately 5.5, approximately 4.5 to approximately 5.0 embryo, approximately 5.0 to approximately 11.0, approximately 5.0 to approximately 10.5, approximately 5.0 to approximately 10.0, approximately 5.0 to approximately 9.5, approximately 5.0 to approximately 9.0, approximately 5.0 to approximately 8.5, approximately 5. 0 to about 8.0, about 5.0 to about 7.5, about 5.0 to about 7.0, about 5.0 to about 6.5, about 5.0 to about 6.0, about 5.0 to about 5.5, about 5.5 to about 11.0, about 5.5 to about 10.5, about 5.5 to about 10.0, about 5.5 to about 9.5, about 5.5 to about 9.0, about 5.5 to about 8.5, about 5.5 to About 8.0, about 5.5 to about 7.5, about 5.5 to about 7.0, about 5.5 to about 6.5, about 5.5 to about 6.0, about 6.0 to about 11.0, about 6.0 to about 10.5, about 6.0 to about 10.0, about 6.0 to about 9.5, about 6.0 to about 9.0, about 6.0 to about 8.5, about 6.0 to about 8.0, about 6.0 to about 7 .5, approximately 6.0 to approximately 7.0, approximately 6.0 to approximately 6.5, approximately 6.5 to approximately 11.0, approximately 6.5 to approximately 10.5, approximately 6.5 to approximately 10.0, approximately 6.5 to approximately 9.5, approximately 6.5 to approximately 9.0, approximately 6.5 to approximately 8.5, approximately 6.5 to approximately 8.0, approximately 6.5 to approximately 7.5, approximately 6.5 to approximately 7.0, approximately 7.0 to approximately 11. 0, about 7.0 to about 10.5, about 7.0 to about 10.0, about 7.0 to about 9.5, about 7.0 to about 9.0, about 7.0 to about 8.5, about 7.0 to about 8.0, about 7.0 to about 7.5, about 7.5 to about 11.0, about 7.5 to about 10.5, about 7.5 to about 10.0, about 7.5 to about 9.5, about 7.5 to about 9. 0, about 7.5 to about 8.5, about 7.5 to about 8.0, about 8.0 to about 11.0, about 8.0 to about 10.5, about 8.0 to about 10.0, about 8.0 to about 9.5, about 8.0 to about 9.0, about 8.0 to about 8.5, about 8.5 to about 11.0, about 8.5 to about 10.5, about 8.5 to about 10.0, about 8.5 to about 9.The density may be in the range of about 8.5 to about 9.0 explants, including all derivable ranges and values ​​therebetween.

[0084] In certain embodiments, the embryonic explants may be contacted with the co-culture medium at a temperature ranging from about 15°C to about 25°C, or from about 17°C to about 23°C, or from about 18°C ​​to about 20°C, or at a temperature of about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, or about 25°C. Explants may, in some embodiments, be cultured for about 1 day to about 14 days, about 1 day to about 13 days, about 1 day to about 12 days, about 1 day to about 11 days, about 1 day to about 10 days, about 1 day to about 9 days, about 1 day to about 8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 10 days, about 2 days to about 8 days, about 2 days to about 4 days, about 2 days to about 5 days. The explants may be contacted with the co-culture medium for a period ranging from about 3 days to about 8 days, about 4 days to about 8 days, about 4 days to about 5 days, about 5 days to about 7 days, or about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days (including all derivable ranges and values ​​therebetween). In further embodiments, the explants may be contacted with the co-culture medium for at least 5 days or at least 6 days. In this embodiment, the co-culture medium contacted with the explants may be a solid, liquid, or semi-solid medium.

[0085] According to some embodiments, the monocotyledonous seed embryo explant(s) may be contacted with the co-cultivation medium for a period ranging from about 1 day to about 10 days, or from about 2 days to about 10 days, or from about 2 days to about 8 days, or from about 3 days to about 8 days, or from about 4 days to about 8 days, or from about 5 days to about 7 days, e.g., about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days (including all derivable ranges and values ​​therebetween). According to some embodiments, the monocotyledonous seed embryo explant(s) may be contacted with the co-cultivation medium for a longer period to improve the frequency of transformation, shoots, and / or regeneration, which may be particularly useful for seed embryo explants of certain male germplasm or lines or other monocotyledonous germplasm that exhibit lower frequencies of transformation, shoots, and / or regeneration. According to some embodiments, the monocotyledonous seed embryo explant(s) may be contacted with the co-cultivation medium for a period ranging from about 5 days to about 10 days, or from about 5 days to about 9 days, or from about 5 days to about 8 days, or from about 5 days to about 7 days, or from about 5 days to about 6 days, or from about 6 days to about 7 days, e.g., about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days (including all derivable ranges and values ​​therebetween).

[0086] According to some embodiments, the co-cultivation medium may contain Rhizobiales bacteria or Agrobacterium capable of transforming at least one cell of the explant with a heterologous polynucleotide molecule. Increasing the concentration of Agrobacterium in the inoculation and / or co-cultivation medium may improve or increase the shoot and plugging frequency, and thus the transformation frequency. The OD of the Rhizobiales bacteria or Agrobacterium in the inoculation and / or co-cultivation medium may be increased by 20% or more. 660The concentration can be in the range of about 0.1 to about 2.0, about 0.1 to about 1.0, about 0.1 to about 0.75, about 0.1 to about 0.5, about 0.2 to about 2.0, about 0.2 to about 1.0, about 0.2 to about 0.5, about 0.25 to about 2.0, about 0.5 to about 2.0, about 0.5 to about 1.5, about 0.75 to about 1.5, or about 0.75 to about 1.25, or about 0.1, 0.2, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, or 2.0 (including all derivable ranges and values ​​therebetween). However, in some cases, the OD may be in the range of about 0.1 to about 1.0, or about 0.25 to about 0.5, or about 0.1, about 0.2, about 0.25, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.75, about 0.8, about 0.9, or about 1.0 (including all derivable ranges and values ​​therebetween), which may depend on the plant genetic resource or genetic line of the monocot being transformed. 660 Lower concentrations may improve the survival and / or regrowth potential of the explants.

[0087] In certain embodiments, explants may be in contact with a matrix, paper, or mesh material or substrate, e.g., Whatman or other filter paper, moistened, saturated, or saturated with liquid co-culture medium. In certain embodiments, explants may be in contact with, but not submerged in, the co-culture medium. In certain embodiments, explants may be co-cultured at a relative humidity of about 20% to about 90%, about 25% to about 65%, about 30% to about 60%, about 35% to about 55%, about 40% to about 50%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, or about 65%, including all derivable ranges and values ​​therebetween.

[0088] The co-cultivation step can also be carried out under various lighting conditions. While some degree of illumination is generally used, all or part of the co-cultivation step can alternatively be carried out in the dark. Light treatments can be quantified in terms of light-dark cycles and light intensity, and can be expressed in photosynthetic photon flux density (PPFD) (units: μE / m 2 In some embodiments, the co-cultivation step is performed at a mean or set light intensity of about 0 μg / m2 ·s~approx. 200μE / m 2 ·s, 20μE / m 2 ·s~approx. 200μE / m 2 ·s, 20μE / m 2 ·s~approx. 180μE / m 2 ·s, 30μE / m 2 ·s~approx. 180μE / m 2 ·s, 30μE / m 2 ·s~approx. 150μE / m 2 ·s, 30μE / m 2 ·s~approx. 120μE / m 2 ·s, 60μE / m 2 ·s~approx. 120μE / m 2 ·s, 70μE / m 2 ·s~approx. 110μE / m 2 ·s, or 80 μE / m 2 ·s~approx. 100μE / m 2 In certain embodiments, the co-cultivation step may be carried out at a mean or set light intensity of about 0 μE / m 2 ·s, approximately 10μE / m 2 ·s, approx. 20μE / m 2 ·s, approx. 30μE / m 2 ·s, approx. 40μE / m 2 ·s, approx. 50μE / m 2 ·s, approximately 60μE / m 2 ·s, approx. 70μE / m 2 ·s, approx. 80μE / m 2 ·s, approximately 90μE / m 2 ·s, approximately 100μE / m 2 ·s, approximately 110μE / m 2 ·s, approximately 120μE / m 2 ·s, approximately 130μE / m 2 ·s, approximately 140μE / m 2 ·s, approximately 150μE / m 2 ·s, approximately 160μE / m 2 ·s, approximately 170μE / m 2 ·s, approx. 180μE / m 2 ·s, approximately 190μE / m 2 ·s, or approximately 200 μE / m 2Light-dark cycles of different amounts may be used during the co-cultivation step in some embodiments, which may include illumination present at lengths of about 0 to about 24 hours light, about 2 to about 22 hours light, about 4 to about 20 hours light, about 8 to about 20 hours light, about 12 to about 20 hours light, and about 16 to about 20 hours light, each of which has a corresponding amount of relative darkness for a corresponding length of time based on the length of a 24-hour day. According to some embodiments, the amount of light and dark cycle during the co-cultivation step is about 0 hours light and about 24 hours dark, about 1 hour light and about 23 hours dark, about 2 hours light and about 22 hours dark, about 3 hours light and about 21 hours dark, about 4 hours light and about 20 hours dark, about 5 hours light and about 19 hours dark, about 6 hours light and about 18 hours dark, about 7 hours light and about 17 hours dark, about 8 hours light and about 16 hours dark, about 9 hours light and about 15 hours dark, about 10 hours light and about 14 hours dark, about 11 hours light and about 13 hours dark. The light and dark periods may be between about 12 hours of light and about 12 hours of darkness, about 13 hours of light and about 11 hours of darkness, about 14 hours of light and about 10 hours of darkness, about 15 hours of light and about 9 hours of darkness, about 16 hours of light and about 8 hours of darkness, about 17 hours of light and about 7 hours of darkness, about 18 hours of light and about 6 hours of darkness, about 19 hours of light and about 5 hours of darkness, about 20 hours of light and about 4 hours of darkness, about 21 hours of light and about 3 hours of darkness, about 22 hours of light and about 2 hours of darkness, about 23 hours of light and about 1 hour of darkness, or about 24 hours of light and about 0 hours of darkness.

[0089] After the introduction and / or inoculation step and optional co-cultivation step, embryo explants derived from dicotyledonous plant species can, in certain embodiments, be transferred to a regeneration medium for regenerating a plant or part thereof. However, embryo explants derived from monocotyledonous plant species can, in some embodiments, instead be transferred to an emergence induction medium after the introduction and / or inoculation step and optional co-cultivation step, and an emergence induction step and optionally an expansion induction step can be performed. After the emergence induction and optionally an expansion induction step, the monocotyledonous plant embryo explant can then be transferred to a regeneration medium for regenerating a plant or part thereof, as further described below.

[0090] D. Budding induction and expansion budding induction According to this embodiment, a population of monocotyledonous plant embryo explants transformed or edited by introducing a heterologous polynucleotide molecule into at least one cell of the embryo explants can be cultured in contact with at least a first germination induction medium containing an auxin and a cytokinin. The monocotyledonous plant embryo explants can be inoculated with an inoculation medium containing a Rhizobiales or Agrobacterium containing the heterologous polynucleotide molecule, and the monocotyledonous plant seed embryo explants can also be co-cultured in contact with a co-culture medium prior to the germination induction step.

[0091] As provided herein, monocotyledonous embryo explants can be further cultured in contact with a second or extended germination-induction medium comprising an auxin and a cytokinin, and then cultured in contact with a regeneration medium to produce genetically modified plants or plant parts. In some embodiments, the methods described herein comprise culturing the monocotyledonous embryo explant in contact with a second germination-induction medium after culturing the monocotyledonous embryo explant in contact with the germination-induction medium (or the first germination-induction medium) and before contacting the cultured monocotyledonous embryo explant with a regeneration medium to regenerate or grow a genetically modified monocotyledonous plant or plant part. In further embodiments, the (first) germination-induction medium and / or the second (or extended) germination-induction medium can each comprise a high cytokinin-to-auxin ratio.

[0092] In certain embodiments, the germination induction medium (or first germination induction medium) and the second germination induction medium (or expanded germination induction medium) may each contain various standard culture medium or solution components or ingredients, such as basal salts, macronutrients, micronutrients, sugars, antibiotics, and / or vitamins. The germination induction medium (or first germination induction medium) and the second germination induction medium (or expanded germination induction medium) may each contain an auxin and a cytokinin. The germination induction medium (or first germination induction medium) and the second germination induction medium (or expanded germination induction medium) may each contain one or more selective agents, although according to many embodiments, a selective agent is not present in the first germination induction medium. The absence of a selective agent in the first germination induction medium may allow the first germination induction medium to function as a delay medium. The nature of the selective agent typically depends on the selectable marker gene present in the heterologous polynucleotide molecule introduced into the population of monocotyledonous embryo explants. The sprout-induction medium (or first sprout-induction medium) and / or the second sprout-induction medium (or expansion sprout-induction medium) can each be a solid, semi-solid, or liquid medium, although each of these media will typically be a solid medium. A solid medium may include a gelling or polymerizing agent or component, such as agarose, that can solidify to form a solid medium.

[0093] As used herein, a "high cytokinin to auxin ratio" generally refers to a condition in which the level of cytokinin activity is relatively high compared to the level of auxin activity present in the medium, and typically can be a cytokinin:auxin ratio of at least about 1:1 or greater in terms of weight / volume provided. However, as is known in the art, different auxins and cytokinins have different activities and / or modes of action, so the exact cytokinin:auxin ratio will depend on the exact chemical nature of the auxin and cytokinin. The levels of cytokinin and auxin in a medium having a high cytokinin to auxin ratio can be present in the medium (measured by weight / volume) in a ratio of, for example, about 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1, 12:1, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, or 15:1 (including all derivable values ​​and ranges therebetween).

[0094] The levels of cytokinin and auxin in a culture medium having a high cytokinin to auxin ratio can be, for example, about 1:1 or greater or at least about 1:1 or greater, about 1.5:1 or greater or at least about 1.5:1 or greater, about 2:1 or greater or at least about 2:1 or greater, about 2.5:1 or greater or at least about 2.5:1 or greater, about 3:1 or greater or at least about 3:1 or greater, about 3.5:1 or greater or at least about 3.5:1 or greater, about 4:1 or greater or at least about 4:1 or greater, about 4.5:1 or greater or at least about 5:1 or greater, about 5.5:1 or greater or at least about 5.5:1 or greater, about 6:1 or greater or at least about 6.5:1 or greater. or at least about 6.5:1 or greater, about 7:1 or greater, about 7.5:1 or greater, about 8:1 or greater, about 8.5:1 or greater, about 9:1 or greater, about 9.5:1 or greater, about 10:1 or greater, about 10.5:1 or greater, about 11:1 or greater, about 11.5:1 or greater, or about 12:1 or greater (including all derivable values ​​and ranges therebetween).

[0095] The levels of cytokinin and auxin in a culture medium having a high cytokinin to auxin ratio can be, for example, about 1:1 to about 12:1, about 2:1 to about 12:1, about 4:1 to about 12:1, about 6:1 to about 12:1, about 8:1 to about 12:1, about 1:1 to about 10:1, about 2:1 to about 10:1, about 4:1 to about 10:1, about 6:1 to about 10:1, about 8:1 to about 10:1, about 1:1 to about 8:1, about 2 The ratio may be in the range of about 1:1 to about 8:1, about 4:1 to about 8:1, about 6:1 to about 8:1, about 1:1 to about 6:1, about 2:1 to about 6:1, about 4:1 to about 6:1, about 1:1 to about 5:1, about 2:1 to about 5:1, about 3:1 to about 5:1, about 1:1 to about 4:1, about 2:1 to about 4:1, about 3:1 to about 4:1, about 1:1 to about 3:1, or about 1:1 to about 2:1 (including all derivable values ​​and ranges therebetween).

[0096] Non-limiting examples of cytokinins that may be used in accordance with the present disclosure include, but are not limited to, 6-benzylaminopurine (BAP), thidiazuron (TDZ), N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-topolin). Auxins that may be used in accordance with the present disclosure may include, but are not limited to, 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid or p-chlorophenoxyacetic acid (4-CPA or pCPA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,3,5-triiodobenzoic acid (TIBA), phenylacetic acid (PAA), and 3,6-dichloro-2-methoxybenzoic acid (dicamba).

[0097] In this embodiment, the germination induction medium (or the first germination induction medium) may contain the same or a different auxin and / or the same or a different cytokinin as the second germination induction medium (or the expansion germination induction medium). The germination induction medium (or the first germination induction medium) may contain a first auxin and a first cytokinin, and the second germination induction medium (or the expansion germination induction medium) may contain a first auxin or a second auxin and a first cytokinin or a second cytokinin. In some embodiments, the second germination induction medium (or the expansion germination induction medium) may contain the same or a different auxin as the germination induction medium (or the first germination induction medium). In further embodiments, the second germination induction (or the expansion germination induction medium) may contain the same or a different cytokinin as the germination induction medium (or the first germination induction medium).

[0098] In certain embodiments, the concentration of the cytokinin (or two or more cytokinins) or the total cytokinin concentration in the first germination induction medium and / or the second (or expansion) germination induction medium is from about 0.1 mg / L to about 100.0 mg / L, 1 mg / L to about 90.0 mg / L, 1 mg / L to about 80.0 mg / L, 1 mg / L to about 75.0 mg / L, 2 mg / L to about 90.0 mg / L, 2 mg / L to about 80.0 mg / L, 2 mg / L to about 75.0 mg / L, 5 mg / L to about 90.0 mg / L, 5 mg / L to about 80.0 mg / L, 5 mg / L to about 75.0 mg / L / L, 5mg / L~approx. 70.0mg / L, 10mg / L~approx. 90.0mg / L, 10mg / L~approx. 80.0mg / L, 10mg / L~approx. 75.0mg / L, 10mg / L~approx. 70.0mg / L, 15mg / L~approx. 90.0mg / L, 15mg / L~approx. 80.0mg / L, 15m g / L~about 75.0mg / L, 15mg / L~about 70.0mg / L, 20mg / L~about 90.0mg / L, 20mg / L~about 80.0mg / L, 20mg / L~about 75.0mg / L, 20mg / L~about 70.0mg / L, 20mg / L~about 60.0mg / L, 30mg / L~about 9 0.0mg / L, 30mg / L~about 80.0mg / L, 30mg / L~about 75.0mg / L, 30mg / L~about 70.0mg / L, 30mg / L~about 60.0mg / L, 40mg / L~about 90.0mg / L, 40mg / L~about 80.0mg / L, 40mg / L~about 75.0mg / L, 40mg / L~Approx. 70.0mg / L, 40mg / L~Approx. 60.0mg / L, Approx. 0.1mg / L~Approx. 25.0mg / L, Approx. 0.1mg / L~Approx. 20.0mg / L, Approx. 0.1mg / L~Approx. 15.0mg / L, Approx. 0.2mg / L~Approx. 25.0mg / L, Approx. 0.2mg / L~Approx. 2 0.0mg / L, about 0.2mg / L to about 15.0mg / L, about 0.5mg / L to about 25.0mg / L, about 0.5mg / L to about 20.0mg / L, about 0.5mg / L to about 15.0mg / L, about 0.5mg / L to about 12.5mg / L, about 1.0mg / L to about 25.0mg / L, about 1 .0mg / L~about 20.0mg / L, about 1.0mg / L~about 15.0mg / L, about 1.0mg / L~about 12.5mg / L, about 2.0mg / L~about 25.0mg / L, about 2.0mg / L~about 20.0mg / L, about 2.0mg / L~about 15.0mg / L, about 2.0mg / L~about 12.5mg / L, about 5.0mg / L to about 25.0mg / L, about 5.0mg / L to about 20.0mg / L, about 5.0mg / L to about 15.0mg / L, about 5.0mg / L to about 12.5mg / L, about 7.5mg / L ~25.0mg / L, approximately 7.5mg / L~20.0mg / L, approximately 7.5mg / L~15.0mg / L, approximately 7.5mg / L~12.5mg / L, approximately 0.1mg / L~15.0mg / L, approximately 0. 1mg / L~Approx. 12.5mg / L, Approx. 0.1mg / L~Approx. 10.0mg / L, Approx. 0.1mg / L~Approx. 7.5mg / L, Approx. 0.1mg / L~Approx. 5.0mg / L, Approx. 0.1mg / L~Approx. 4.0mg / L, Approx. 0.1mg / L~about 3.0mg / L, about 0.2mg / L~about 15.0mg / L, about 0.2mg / L~about 12.5mg / L, about 0.2mg / L~about 10.0mg / L, about 0.2mg / L~about 7.5mg / L , about 0.2 mg / L to about 5.0 mg / L, about 0.2 mg / L to about 4.0 mg / L, about 0.2 mg / L to about 3.0 mg / L, about 0.5 mg / L to about 15.0 mg / L, about 0.5 mg / L to about 12.5 mg / L, about 0.5mg / L to about 10.0mg / L, about 0.5mg / L to about 7.5mg / L, about 0.5mg / L to about 5.0mg / L, about 0.5mg / L to about 4.0mg / L, about 0.5mg / L to about 3.0mg / L, about 1.0 mg / L to about 15.0 mg / L, about 1.0 mg / L to about 12.5 mg / L, about 1.0 mg / L to about 10.0 mg / L, about 1.0 mg / L to about 7.5 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 4.0 mg / L, or about 1.0 mg / L to about 3.0 mg / L (including all derivable values ​​and ranges therebetween). In some embodiments, the concentration of cytokinin in the first germination-inducing medium or the second germination-inducing medium is, for example, about 0.1 mg / L, about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, about 10.0 mg / L, about 11.0 mg / L, or about 12.The cytokinin concentration in the first and second germination-inducing media may be about 0 mg / L, about 13.0 mg / L, about 14.0 mg / L, about 15.0 mg / L, about 16.0 mg / L, about 17.0 mg / L, about 18.0 mg / L, about 19.0 mg / L, about 20.0 mg / L, about 21.0 mg / L, about 22.0 mg / L, about 23.0 mg / L, about 24.0 mg / L, about 25.0 mg / L, about 30 mg / L, about 40 mg / L, about 50 mg / L, about 60 mg / L, about 70 mg / L, about 75 mg / L, about 80 mg / L, about 90 mg / L, or about 100 mg / L (including all derivable values ​​and ranges therebetween). The cytokinins in the first and second germination-inducing media may be the same or different, and each of these germination-inducing media may contain one or more cytokinins.

[0099] In some embodiments, the concentration of the auxin (or two or more auxins) or the total auxin concentration in the first germination-inducing medium and / or the second (or expanded) germination-inducing medium is from about 0.01 mg / L to about 25.0 mg / L, from about 0.05 mg / L to about 25 mg / L, from about 0.1 mg / L to about 25.0 mg / L, from about 0.1 mg / L to about 20.0 mg / L, from about 0.1 mg / L to about 15.0 mg / L, from about 0.2 mg / L to about 25.0 mg / L, from about 0.2 mg / L to about 20.0 mg / L, from about 0.2 mg / L to about 15.0 mg / L, or from about 0.5 mg / L to about 25.0 mg / L. L, about 0.5mg / L~about 20.0mg / L, about 0.5mg / L~about 15.0mg / L, about 0.5mg / L~about 12.5mg / L, about 1.0mg / L~about 25.0mg / L, about 1.0mg / L~about 20.0mg / L, about 1.0mg / L~about 15.0mg / L, about 1.0mg / L~ Approx. 12.5 mg / L, Approx. 2.0 mg / L ~ Approx. 25.0 mg / L, Approx. 2.0 mg / L ~ Approx. 20.0 mg / L, Approx. 2.0 mg / L ~ Approx. 15.0 mg / L, Approx. 2.0 mg / L ~ Approx. 12.5 mg / L, Approx. 5.0mg / L~about 15.0mg / L, about 5.0mg / L~about 12.5mg / L, about 7.5mg / L~about 25.0mg / L, about 7.5mg / L~about 20.0mg / L, about 7.5mg / L~about 15.0mg / L, about 7.5mg / L~about 12.5mg / L, about 8.0mg / L~about 12 .0mg / L, about 9.0mg / L to about 11.0mg / L, about 0.1mg / L to about 10.0mg / L, about 0.1mg / L to about 7.5mg / L, about 0.1mg / L to about 7.0mg / L, about 0.1mg / L to about 6.0mg / L, about 0.2mg / L to about 10.0mg / L, about 0.2mg / L L ~ about 7.5mg / L, about 0.2mg / L - about 7.0mg / L, about 0.2mg / L - about 6.0mg / L, about 0.5mg / L - about 10.0mg / L, about 0.5mg / L - about 7.5mg / L, about 0.5mg / L - about 7.0mg / L, about 0.5mg / L - about 6.0mg / L, about 1.0m g / L ~ approx. 10.0 mg / L, approx. 1.0 mg / L ~ approx. 7.5 mg / L, approx. 1.0 mg / L ~ approx. 7.0 mg / L, approx. 1.0 mg / L ~ approx. 6.0 mg / L, approx. 2.0 mg / L ~ approx. 10.0 mg / L, approx. 2.0 mg / L ~ approx. 7.5 mg / L, approx. 2.0 mg / L ~ approx.0 mg / L to about 6.0 mg / L, about 3.0 mg / L to about 10.0 mg / L, about 3.0 mg / L to about 7.5 mg / L, about 3.0 mg / L to about 7.0 mg / L, about 3.0 mg / L to about 6.0 mg / L, about 4.0 mg / L to about 10.0 mg / L, about 4.0 mg / L to about 7.5 mg / L, about 4.0 mg / L to about 7.0 mg / L, about 4.0 mg / L to about 6.0 mg / L, about 0.1 mg / L to about 15.0 mg / L, about 0.1 mg / L to about 12.5 mg / L, about 0.1 mg / L to about 10.0 mg / L, about 0.1 mg / L to about 7.5 mg / L, about 0.1 mg / L to about 5.0 mg / L, about 0.1 mg / L to about 4.0 mg / L, about 0.1 mg / L to about 3.0 mg / L, about 0.2 mg / L to about 15.0 mg / L, about 0.2 mg / L to about 12.5 mg / L, about 0.2 mg / L to about 10.0 mg / L, about 0.2 mg / L to about 7.5 mg / L, about 0.2 mg / L to about 5.0 mg / L, about 0.2 mg / L to about 4.0 mg / L, about 0.2 mg / L to about 3.0 mg / L, about 0.5 mg / L to about 15.0 mg / L, about 0.5 mg / L to about 12.5 mg / L, about 0.5 mg / L to about 10.0 mg / L, about 0.5 mg / L to about 7.5 mg / L, about 0.5 mg / L g / L to about 5.0 mg / L, about 0.5 mg / L to about 4.0 mg / L, about 0.5 mg / L to about 3.0 mg / L, about 1.0 mg / L to about 15.0 mg / L, about 1.0 mg / L to about 12.5 mg / L, about 1.0 mg / L to about 10.0 mg / L, about 1.0 mg / L to about 7.5 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 4.0 mg / L, about 1.0 mg / L to about 3.0 mg / L, about 1.5 mg / L to about 2.5 mg / L, about 0.1 mg / L to about 2.0 mg / L, about 0.1 mg / L to about 1.5 mg / L, About 0.1 mg / L to about 1.25 mg / L, about 0.1 mg / L to about 1.2 mg / L, about 0.1 mg / L to about 1.1 mg / L, about 0.2 mg / L to about 2.0 mg / L, about 0.2 mg / L to about 1.5 mg / L, about 0.2 mg / L to about 1.25 mg / L, about 0.2 mg / L to about 1.2 mg / L, about 0.2 mg / L to about 1.1 mg / L, about 0.5 mg / L to about 2.0 mg / L, about 0.5 mg / L to about 1.5 mg / L, about 0.5 mg / L to about 1.25 mg / L, about 0.5 mg / L to about 1.2 mg / L, about 0.5 mg / L to about 1.1 mg / L, about 0.75 mg / L to about 2.0mg / L, about 0.75mg / L to about 1.5mg / L, about 0.75mg / L to about 1.25mg / L, about 0.75mg / L to about 1.2mg / L, about 0.75mg / L to about 1.1mg / L, about 0.8mg / L to about 2.0mg / L, about 0.8mg / L to about 1.5mg / L, about 0.8mg / L to about 1.25mg / L, about 0.8mg / L to about 1.2mg / L, about 0.8 mg / L~about 1.1mg / L, about 0.9mg / L~about 2.0mg / L, about 0.9mg / L~about 1.5mg / L, about 0.9mg / L~about 1.25mg / L, about 0.9mg / L~about 1 .2mg / L, about 0.9mg / L to about 1.1mg / L, about 0.1mg / L to about 1.0mg / L, about 0.1mg / L to about 0.75mg / L, about 0.1mg / L to about 0.7mg / L, about 0.1mg / L~Approx. 0.6mg / L, Approx. 0.2mg / L~Approx. 1.0mg / L, Approx. 0.2mg / L~Approx. 0.75mg / L, Approx. 0.2mg / L~Approx. 0.7mg / L, Approx. 0.2mg / L ~0.6mg / L, approximately 0.3mg / L~1.0mg / L, approximately 0.3mg / L~0.75mg / L, approximately 0.3mg / L~0.7mg / L, approximately 0.3mg / L~0.6mg / L, about 0.4 mg / L to about 1.0 mg / L, about 0.4 mg / L to about 0.75 mg / L, about 0.4 mg / L to about 0.7 mg / L, about 0.4 mg / L to about 0.6 mg / L, about 0.05 mg / L to about 7.5 mg / L, about 0.02 mg / L to about 5 mg / L, or about 0.75 mg / L to about 2.5 mg / L (including all derivable values ​​and ranges therebetween). In some embodiments, the concentration of auxin in the first germination induction medium or the second germination induction medium is, for example, about 0.1 mg / L, about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, or about 3.5 mg / L. L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, about 10.0 mg / L, about 11.0 mg / L, about 12. 0mg / L, about 13.0mg / L, about 14.0mg / L, about 15.0mg / L, about 16.0mg / L, about 17.0mg / L, about 18.0mg / L, about 19.0mg / L, about 20.0mg / L, about 21.The auxin concentration in the first and second germination-inducing media may be about 0 mg / L, about 22.0 mg / L, about 23.0 mg / L, about 24.0 mg / L, or about 25.0 mg / L (including all derivable values ​​and ranges therebetween). The auxin in the first and second germination-inducing media may be the same or different, and each of these media may contain one or more auxins.

[0100] As used herein, the "total cytokinin concentration" of a medium refers to the total concentration of all cytokinins present in the medium. For example, if the TDZ concentration in the medium is 1 mg / L, the BAP concentration in the medium is 2 mg / L, and no other cytokinins are present in the medium, the total cytokinin concentration in the medium is 3 mg / L. As used herein, the "total auxin concentration" of a medium refers to the total concentration of all auxins present in the medium. For example, if the 2,4-D concentration in the medium is 0.5 mg / L, the IAA concentration in the medium is 1 mg / L, and no other auxins are present in the medium, the total auxin concentration in the medium is 1.5 mg / L. For clarity, if only one cytokinin is present in the medium, the total cytokinin concentration of the medium is equal to the concentration of that single cytokinin in the medium, and if only one auxin is present in the medium, the total auxin concentration of the medium is equal to the concentration of that single auxin in the medium.

[0101] As used herein, "co-culture surface area," "sprouting induction surface area," "first sprouting induction surface area," "expanded sprouting induction surface area," "second sprouting induction surface area," and "regeneration surface area" refer to the surface area of ​​a solid or semi-solid medium, which may be a co-culture medium, a sprouting induction medium, a first sprouting induction medium, an expanded sprouting induction medium, a second sprouting induction medium, or a regeneration medium, respectively.

[0102] In certain embodiments, the first germination induction medium and / or the second (or extended) germination induction medium may contain a cytokinin, where the cytokinin is 6-benzylaminopurine (BAP). In some embodiments, the concentration of BAP in the first germination induction medium and / or the second (or extended) germination induction medium is about 1.0 mg / L to about 25.0 mg / L, about 1.0 mg / L to about 20.0 mg / L, about 1.0 mg / L to about 15.0 mg / L, about 1.0 mg / L to about 12.5 mg / L, about 2.0 mg / L to about 25.0 mg / L, about 2.0 mg / L to about 20.0 mg / L, about 2.0 mg / L to about 15.0 mg / L, about 2.0 mg / L to about 12.5 mg / L, or about 5.0 mg / L to about 15.0 mg / L. It may be in the range of about 25.0 mg / L, about 5.0 mg / L to about 20.0 mg / L, about 5.0 mg / L to about 15.0 mg / L, about 5.0 mg / L to about 12.5 mg / L, about 7.5 mg / L to about 25.0 mg / L, about 7.5 mg / L to about 20.0 mg / L, about 7.5 mg / L to about 15.0 mg / L, about 7.5 mg / L to about 12.5 mg / L, about 8.0 mg / L to about 12.0 mg / L, or about 9.0 mg / L to about 11.0 mg / L (including all derivable values ​​and ranges therebetween). In some embodiments, the concentration of BAP in the first sprouting induction medium and / or the second (or expansion) sprouting induction medium is about 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, about 10.0 mg / L, It can be about 11.0 mg / L, about 12.0 mg / L, about 13.0 mg / L, about 14.0 mg / L, about 15.0 mg / L, about 16.0 mg / L, about 17.0 mg / L, about 18.0 mg / L, about 19.0 mg / L, about 20.0 mg / L, about 21.0 mg / L, about 22.0 mg / L, about 23.0 mg / L, about 24.0 mg / L, or about 25.0 mg / L (including all derivable values ​​and ranges therebetween).

[0103] In certain embodiments, the first germination induction medium and / or the second (or extended) germination induction medium may contain a cytokinin, where the cytokinin is thidiazuron (TDZ). In some embodiments, the concentration of TDZ in the first germination induction medium and / or the second (or extended) germination induction medium is from about 0.1 mg / L to about 10.0 mg / L, from about 0.1 mg / L to about 7.5 mg / L, from about 0.1 mg / L to about 7.0 mg / L, from about 0.1 mg / L to about 6.0 mg / L, from about 0.1 mg / L to about 5.0 mg / L, or from about 0.1 mg / L to about 4.0 mg / L. mg / L, about 0.1 mg / L to about 3.0 mg / L, about 0.2 mg / L to about 10.0 mg / L, about 0.2 mg / L to about 7.5 mg / L, about 0.2 mg / L to about 7.0 mg / L, about 0.2mg / L~Approx. 6.0mg / L, Approx. 0.2mg / L~Approx. 5.0mg / L, Approx. 0.2mg / L~Approx. 4.0mg / L, Approx. 0.2mg / L~Approx. 3.0mg / L, Approx. 0.5mg / L ~10.0mg / L, 0.5mg / L~7.5mg / L, 0.5mg / L~7.0mg / L, 0.5mg / L~6.0mg / L, 0.5mg / L~5.0 mg / L, about 0.5 mg / L to about 4.0 mg / L, about 0.5 mg / L to about 3.0 mg / L, about 1.0 mg / L to about 10.0 mg / L, about 1.0 mg / L to about 7.5 mg / L, about It may be in the range of 1.0 mg / L to about 7.0 mg / L, about 1.0 mg / L to about 6.0 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 4.0 mg / L, about 1.0 mg / L to about 3.0 mg / L, or about 1.5 mg / L to about 2.5 mg / L (including all derivable values ​​and ranges therebetween).In some embodiments, the concentration of TDZ in the first sprouting induction medium and / or the second (or expansion) sprouting induction medium can be about 0.1 mg / L, about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, or about 10.0 mg / L (including all derivable values ​​and ranges therebetween).

[0104] In some embodiments, the first germination induction medium and / or the second (or expansion) germination induction medium can contain a cytokinin, where the cytokinin is N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU). In specific embodiments, the concentration of 4-CPPU in the first germination induction medium and / or the second (or expansion) germination induction medium is from about 0.1 mg / L to about 10.0 mg / L, from about 0.1 mg / L to about 7.5 mg / L, from about 0.1 mg / L to about 7.0 mg / L, from about 0.1 mg / L to about 6.0 mg / L, from about 0.1 mg / L to about 5.0 mg / L, or from about 0.1 mg / L to about 4. 0mg / L, about 0.1mg / L to about 3.0mg / L, about 0.2mg / L to about 10.0mg / L, about 0.2mg / L to about 7.5mg / L, about 0.2mg / L to about 7.0mg / L, Approx. 0.2mg / L~Approx. 6.0mg / L, Approx. 0.2mg / L~Approx. 5.0mg / L, Approx. 0.2mg / L~Approx. 4.0mg / L, Approx. 0.2mg / L~Approx. 3.0mg / L, Approx. 0.5mg / L L ~ approx. 10.0 mg / L, approx. 0.5 mg / L ~ approx. 7.5 mg / L, approx. 0.5 mg / L ~ approx. 7.0 mg / L, approx. 0.5 mg / L ~ approx. 6.0 mg / L, approx. 0.5 mg / L ~ approx. 5.0 mg / L, about 0.5 mg / L to about 4.0 mg / L, about 0.5 mg / L to about 3.0 mg / L, about 1.0 mg / L to about 10.0 mg / L, about 1.0 mg / L to about 7.5 mg / L, about It may be in the range of 1.0 mg / L to about 7.0 mg / L, about 1.0 mg / L to about 6.0 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 4.0 mg / L, about 1.0 mg / L to about 3.0 mg / L, or about 1.5 mg / L to about 2.5 mg / L (including all derivable values ​​and ranges therebetween).In further embodiments, the concentration of 4-CPPU in the first sprouting induction medium and / or the second (or expansion) sprouting induction medium can be about 0.1 mg / L, about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, or about 10.0 mg / L (including all derivable values ​​and ranges therebetween).

[0105] In specific embodiments, the first germination induction medium and / or the second (or expanded) germination induction medium may contain a cytokinin, where the cytokinin is kinetin. In some embodiments, the concentration of kinetin in the first germination induction medium and / or the second (or expanded) germination induction medium is about 1.0 mg / L to about 25.0 mg / L, about 1.0 mg / L to about 20.0 mg / L, about 1.0 mg / L to about 15.0 mg / L, about 1.0 mg / L to about 12.5 mg / L, about 2.0 mg / L to about 25.0 mg / L, about 2.0 mg / L to about 20.0 mg / L, about 2.0 mg / L to about 15.0 mg / L, about 2.0 mg / L to about 12.5 mg / L, or about 5.0 mg / L. to about 25.0 mg / L, about 5.0 mg / L to about 20.0 mg / L, about 5.0 mg / L to about 15.0 mg / L, about 5.0 mg / L to about 12.5 mg / L, about 7.5 mg / L to about 25.0 mg / L, about 7.5 mg / L to about 20.0 mg / L, about 7.5 mg / L to about 15.0 mg / L, about 7.5 mg / L to about 12.5 mg / L, about 8.0 mg / L to about 12.0 mg / L, or about 9.0 mg / L to about 11.0 mg / L (including all derivable values ​​and ranges therebetween). In further embodiments, the concentration of kinetin in the first sprouting induction medium and / or the second (or expansion) sprouting induction medium is about 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, about 10.0 mg / L, It can be about 11.0 mg / L, about 12.0 mg / L, about 13.0 mg / L, about 14.0 mg / L, about 15.0 mg / L, about 16.0 mg / L, about 17.0 mg / L, about 18.0 mg / L, about 19.0 mg / L, about 20.0 mg / L, about 21.0 mg / L, about 22.0 mg / L, about 23.0 mg / L, about 24.0 mg / L, or about 25.0 mg / L (including all derivable values ​​and ranges therebetween).

[0106] In certain embodiments, the first germination induction medium and / or the second (or extended) germination induction medium may contain a cytokinin, wherein the cytokinin is zeatin. In some embodiments, the concentration of zeatin in the first germination induction medium and / or the second (or extended) germination induction medium may be about 1.0 mg / L to about 25.0 mg / L, about 1.0 mg / L to about 20.0 mg / L, about 1.0 mg / L to about 15.0 mg / L, about 1.0 mg / L to about 12.5 mg / L, about 2.0 mg / L to about 25.0 mg / L, about 2.0 mg / L to about 20.0 mg / L, about 2.0 mg / L to about 15.0 mg / L, about 2.0 mg / L to about 12.5 mg / L, or about 5.0 mg / L. to about 25.0 mg / L, about 5.0 mg / L to about 20.0 mg / L, about 5.0 mg / L to about 15.0 mg / L, about 5.0 mg / L to about 12.5 mg / L, about 7.5 mg / L to about 25.0 mg / L, about 7.5 mg / L to about 20.0 mg / L, about 7.5 mg / L to about 15.0 mg / L, about 7.5 mg / L to about 12.5 mg / L, about 8.0 mg / L to about 12.0 mg / L, or about 9.0 mg / L to about 11.0 mg / L (including all derivable values ​​and ranges therebetween). In further embodiments, the concentration of zeatin in the first sprouting induction medium and / or the second (or expansion) sprouting induction medium is about 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, about 10.0 mg / L, It can be about 11.0 mg / L, about 12.0 mg / L, about 13.0 mg / L, about 14.0 mg / L, about 15.0 mg / L, about 16.0 mg / L, about 17.0 mg / L, about 18.0 mg / L, about 19.0 mg / L, about 20.0 mg / L, about 21.0 mg / L, about 22.0 mg / L, about 23.0 mg / L, about 24.0 mg / L, or about 25.0 mg / L (including all derivable values ​​and ranges therebetween).

[0107] In certain embodiments, the first germination induction medium and / or the second (or expanded) germination induction medium can contain a cytokinin, where the cytokinin is 6-(gamma,gamma-dimethylallylamino)purine (2iP). In some embodiments, the concentration of 2iP in the first germination induction medium and / or the second (or expanded) germination induction medium is 5 mg / L to about 100.0 mg / L, 5 mg / L to about 90.0 mg / L, 5 mg / L to about 80.0 mg / L, 5 mg / L to about 75.0 mg / L, 5 mg / L to about 70.0 mg / L, 10 mg / L to about 100.0 mg / L, 10 ... 0.0mg / L, 10mg / L~Approx. 80.0mg / L, 10mg / L~Approx. 75.0mg / L, 10mg / L~Approx. 70.0mg / L, 15mg / L~Approx. 100.0mg / L, 15m g / L~about 90.0mg / L, 15mg / L~about 80.0mg / L, 15mg / L~about 75.0mg / L, 15mg / L~about 70.0mg / L, 20mg / L~about 100.0mg / L L, 20mg / L~Approx. 90.0mg / L, 20mg / L~Approx. 80.0mg / L, 20mg / L~Approx. 75.0mg / L, 20mg / L~Approx. 70.0mg / L, 20mg / L~Approx. 60 .0mg / L, 30mg / L~Approx. 100.0mg / L, 30mg / L~Approx. 90.0mg / L, 30mg / L~Approx. 80.0mg / L, 30mg / L~Approx. 75.0mg / L, 30mg / L The range may be from 30 mg / L to about 70.0 mg / L, 30 mg / L to about 60.0 mg / L, 40 mg / L to about 100.0 mg / L, 40 mg / L to about 90.0 mg / L, 40 mg / L to about 80.0 mg / L, 40 mg / L to about 75.0 mg / L, 40 mg / L to about 70.0 mg / L, or 40 mg / L to about 60.0 mg / L (including all derivable values ​​and ranges therebetween).In further embodiments, the concentration of 2iP in the first germination induction medium and / or the second (or expansion) germination induction medium is about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, about 10.0 mg / L, about 11.0 mg / L, about 12.0 mg / L, about 13.0 mg / L, about 14.0 mg / L, about 15.0 mg / L, about 16.0 mg / L, about 17.0 mg / L, about 18.0 mg / L, about 19.0 mg / L, about 20.0 mg / L, about 21.0 mg / L, about 22.0 mg / L, about 23.0 mg / L, about 24.0 mg / L, about 25.0 mg / L, about 26.0 mg / L, about 27.0 mg / L, about 28.0 mg / L, about 29.0 mg / L, about 30.0 mg / L, about 31.0 mg / L, about 32.0 mg / L, about 33.0 mg / L, about 34.0 mg / L, about 35.0 mg / L, about 36.0 mg / L, about 37.0 mg / L, about 38.0 mg / L, about 39.0 mg / L, about 40.0 mg / L, about 41.0 mg / L, about 42.0 mg / L, about 43.0 mg / L, about 44.0 mg / L, about 45.0 mg / L, about 46.0 mg / L, about 47.0 mg / L, about 48.0 mg / L, about 49.0 mg / L, about 50.0 mg / L, about 51.0 mg / L, about 52.0 mg / L, about mg / L, about 19.0 mg / L, about 20.0 mg / L, about 21.0 mg / L, about 22.0 mg / L, about 23.0 mg / L, about 24.0 mg / L, or about 25.0 mg / L, about 30 mg / L, about 40 mg / L, about 50 mg / L, about 60 mg / L, about 70 mg / L, about 75 mg / L, about 80 mg / L, about 90 mg / L, or about 100 mg / L (including all derivable values ​​and ranges therebetween).

[0108] In certain embodiments, the first germination-inducing medium and / or the second (or expanded) germination-inducing medium can contain a cytokinin, where the cytokinin is 6-(3-hydroxybenzylamino)purine (meta-topolin). In some embodiments, the concentration of meta-topolin in the first germination-inducing medium and / or the second (or expanded) germination-inducing medium is about 1.0 mg / L to about 25.0 mg / L, about 1.0 mg / L to about 20.0 mg / L, about 1.0 mg / L to about 15.0 mg / L, about 1.0 mg / L to about 12.5 mg / L, about 2.0 mg / L to about 25.0 mg / L, about 2.0 mg / L to about 20.0 mg / L, about 2.0 mg / L to about 15.0 mg / L, about 2.0 mg / L to about 12.5 mg / L, or about 5.0 mg / L. The concentration may be in the range of about 7.5 mg / L to about 25.0 mg / L, about 5.0 mg / L to about 20.0 mg / L, about 5.0 mg / L to about 15.0 mg / L, about 5.0 mg / L to about 12.5 mg / L, about 7.5 mg / L to about 25.0 mg / L, about 7.5 mg / L to about 20.0 mg / L, about 7.5 mg / L to about 15.0 mg / L, about 7.5 mg / L to about 12.5 mg / L, about 8.0 mg / L to about 12.0 mg / L, or about 9.0 mg / L to about 11.0 mg / L (including all derivable values ​​and ranges therebetween). In further embodiments, the concentration of meta-topolin in the first sprouting induction medium and / or the second (or expansion) sprouting induction medium is about 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, about 10.0 mg / L, or about 12.0 mg / L. , about 11.0 mg / L, about 12.0 mg / L, about 13.0 mg / L, about 14.0 mg / L, about 15.0 mg / L, about 16.0 mg / L, about 17.0 mg / L, about 18.0 mg / L, about 19.0 mg / L, about 20.0 mg / L, about 21.0 mg / L, about 22.0 mg / L, about 23.0 mg / L, about 24.0 mg / L, or about 25.0 mg / L (including all derivable values ​​and ranges therebetween).

[0109] According to some embodiments, the second (or expanded) germination induction medium may have a lower concentration of cytokinin(s) to improve transformation, shoot, and / or regeneration frequency, which may be particularly useful for monocot seed embryo explants of certain male germplasm or lines or other monocot germplasm that have lower transformation, shoot, and / or regeneration frequencies. According to some embodiments, the cytokinin (or two or more cytokinins) may be present at a lower concentration in the second (or expanded) germination induction medium, or the total cytokinin concentration in the second (or expanded) germination induction medium may be lower, such as from about 0.1 mg / L to about 20 mg / L, from about 0.1 mg / L to about 15 mg / L, from about 0.1 mg / L to about 10 mg / L, from about 0.1 mg / L to about 9 mg / L, or from about 0.1 mg / L to about 10 mg / L. g / L~about 8mg / L, about 0.1mg / L~about 7mg / L, about 0.1mg / L~about 6mg / L, about 0.1mg / L~about 5mg / L, about 0.1mg / L~about 4mg / L, about 0.1mg / L~about 3mg / L, Approx. 0.1mg / L~Approx. 2mg / L, Approx. 0.1mg / L~Approx. 1mg / L, Approx. 0.2mg / L~Approx. 20mg / L, Approx. 0.2mg / L~Approx. 15mg / L, Approx. 0.2mg / L~Approx. 10mg / L, Approx. 0.2mg / L ~9mg / L, 0.2mg / L~8mg / L, 0.2mg / L~7mg / L, 0.2mg / L~6mg / L, 0.2mg / L~5mg / L, 0.2mg / L~4mg / L, 0. 2mg / L~about 3mg / L, about 0.2mg / L~about 2mg / L, about 0.2mg / L~about 1mg / L, about 0.5mg / L~about 20mg / L, about 0.5mg / L~about 15mg / L, about 0.5mg / L~about 10 mg / L, about 0.5 mg / L to about 9 mg / L, about 0.5 mg / L to about 8 mg / L, about 0.5 mg / L to about 7 mg / L, about 0.5 mg / L to about 6 mg / L, about 0.5 mg / L to about 5 mg / L, about 0.5 mg / L to about 4 mg / L, about 0.5 mg / L to about 3 mg / L, about 0.5 mg / L to about 2 mg / L, or about 0.5 mg / L to about 1 mg / L (including all derivable ranges and values ​​therebetween).In some embodiments, the lower concentration of cytokinin in the second (or expanded) seedling induction medium, or the lower total cytokinin concentration in the second (or expanded) seedling induction medium, is, for example, about 0.1 mg / L, about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, It can be about 3.5 mg / L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, about 10.0 mg / L, about 11.0 mg / L, about 12.0 mg / L, about 13.0 mg / L, about 14.0 mg / L, about 15.0 mg / L, about 16.0 mg / L, about 17.0 mg / L, about 18.0 mg / L, about 19.0 mg / L, or about 20.0 mg / L (including all derivable ranges and values ​​therebetween).

[0110] In some embodiments, the second (or expanded) germination induction medium may contain a lower concentration of cytokinin, where the cytokinin is 6-benzylaminopurine (BAP), kinetin, zeatin, or 6-(3-hydroxybenzylamino)purine (meta-topolin).In some embodiments, the lower concentration of BAP, kinetin, zeatin, or 6-(3-hydroxybenzylamino)purine (meta-topolin) in the second (or expansion) sprouting induction medium is from about 0.1 mg / L to about 20 mg / L, from about 0.1 mg / L to about 15 mg / L, from about 0.1 mg / L to about 10 mg / L, from about 0.1 mg / L to about 9 mg / L, from about 0.1 mg / L to about 8 mg / L, from about 0.1 mg / L to about 7 mg / L, from about 0.1 mg / L to about 6 mg / L, from about 0.1 mg / L to about 5 mg / L, from about 0.1 mg / L to about 4 mg / L, Approximately 0.1 mg / L to approximately 3 mg / L, approximately 0.1 mg / L to approximately 2 mg / L, approximately 0.1 mg / L to approximately 1 mg / L, approximately 0.5 mg / L to approximately 20 mg / L, approximately 0.5 mg / L to approximately 15 mg / L, approximately 0.5 mg / L to approximately 10 mg / L, approximately 0.5 mg / L to approximately 9 mg / L, approximately 0 .5mg / L~about 8mg / L, about 0.5mg / L~about 7mg / L, about 0.5mg / L~about 6mg / L, about 0.5mg / L~about 5mg / L, about 0.5mg / L~about 4mg / L, about 0.5mg / L~about 3mg / L, about 0.5mg / L~about 2mg / L, about 0.5mg / L ~1mg / L~20mg / L, 1mg / L~15mg / L, 1mg / L~10mg / L, 1mg / L~9mg / L, 1mg / L~8mg / L, 1mg / L~7mg / L, 1mg / L~6mg / L, 1mg / L~10mg / L 5mg / L, about 1mg / L to about 4mg / L, about 1mg / L to about 3mg / L, about 1mg / L to about 2mg / L, about 2mg / L to about 20mg / L, about 2mg / L to about 15mg / L, about 2mg / L to about 10mg / L, about 2mg / L to about 9mg / L, about 2mg / L to about 8m g / L, about 2 mg / L to about 7 mg / L, about 2 mg / L to about 6 mg / L, about 2 mg / L to about 5 mg / L, about 2 mg / L to about 4 mg / L, or about 2 mg / L to about 3 mg / L, or about 0.5 mg / L, about 1 mg / L, about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, about 10 mg / L, about 11 mg / L, about 12 mg / L, about 13 mg / L, about 14 mg / L, or about 15 mg / L (including all derivable ranges and values ​​therebetween).

[0111] In some embodiments, the second (or extended) germination induction medium may contain a lower concentration of cytokinin, where the cytokinin is thidiazuron (TDZ). In some embodiments, the lower concentration of TDZ in the second (or extended) germination induction medium may be from about 0.1 mg / L to about 10 mg / L, from about 0.1 mg / L to about 9 mg / L, from about 0.1 mg / L to about 8 mg / L, from about 0.1 mg / L to about 7 mg / L, from about 0.1 mg / L to about 6 mg / L, from about 0.1 mg / L to about 5 mg / L, from about 0.1 mg / L to about 4 mg / L, from about 0.1 mg / L to about 3 mg / L, or from about 0.1 mg / L to about 2mg / L, about 0.1mg / L to about 1mg / L, about 0.5mg / L to about 10mg / L, about 0.5mg / L to about 9mg / L, about 0.5mg / L to about 8mg / L, about 0.5mg / L to about 7mg / L, about 0. 5mg / L~about 6mg / L, about 0.5mg / L~about 5mg / L, about 0.5mg / L~about 4mg / L, about 0.5mg / L~about 3mg / L, about 0.5mg / L~about 2mg / L, about 0.5mg / L~about 1mg / L L, about 1 mg / L to about 10 mg / L, about 1 mg / L to about 9 mg / L, about 1 mg / L to about 8 mg / L, about 1 mg / L to about 7 mg / L, about 1 mg / L to about 6 mg / L, about 1 mg / L to about 5 mg / L, about 1 m g / L~about 4mg / L, about 1mg / L~about 3mg / L, about 1mg / L~about 2mg / L, about 2mg / L~about 10mg / L, about 2mg / L~about 9mg / L, about 2mg / L~about 8mg / L, about 2mg / L~about 7 mg / L, about 2 mg / L to about 6 mg / L, about 2 mg / L to about 5 mg / L, about 2 mg / L to about 4 mg / L, or about 2 mg / L to about 3 mg / L, or about 0.5 mg / L, about 1 mg / L, about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, or about 10 mg / L (including all derivable ranges and values ​​therebetween).

[0112] In some embodiments, the second (or expanded) germination-inducing medium can contain a lower concentration of cytokinin, where the cytokinin is 6-(gamma,gamma-dimethylallylamino)purine (2iP). In some embodiments, the lower concentration of 2iP in the second (or expanded) germination-inducing medium is about 0.5 mg / L to about 40 mg / L, 0.5 mg / L to about 30 mg / L, 0.5 mg / L to about 25 mg / L, 0.5 mg / L to about 20 mg / L, about 0.5 mg / L to about 15 mg / L, about 0.5 mg / L to about 10 mg / L, about 0.5 mg / L to about 5 mg / L, or about 1 mg / L to about 40 mg / L. L, 1mg / L to approx. 30mg / L, 1mg / L to approx. 25mg / L, 1mg / L to approx. 20mg / L, approx. 1mg / L to approx. 15mg / L, approx. 1mg / L to approx. 10mg / L, approx. 1mg / L to approx. 5mg / L, about 2mg / L to about 40mg / L, 2mg / L to about 30mg / L, 2mg / L to about 25mg / L, 2mg / L to about 20mg / L, about 2mg / L to about 15mg / L, about 2mg / L to about 10m g / L, about 2 mg / L to about 5 mg / L, about 5 mg / L to about 40 mg / L, 5 mg / L to about 30 mg / L, 5 mg / L to about 25 mg / L, 5 mg / L to about 20 mg / L, about 5 mg / L to about 15 mg / L, about 5 mg / L to about 10 mg / L, about 10 mg / L to about 40 mg / L, 10 mg / L to about 30 mg / L, 10 mg / L to about 25 mg / L, 10 mg / L to about 20 mg / L, or about It can be in the range of 10 mg / L to about 15 mg / L, or about 1 mg / L, about 2 mg / L, about 3 mg / L, about 4 mg / L, about 5 mg / L, about 6 mg / L, about 7 mg / L, about 8 mg / L, about 9 mg / L, about 10 mg / L, about 15 mg / L, about 20 mg / L, about 25 mg / L, about 30 mg / L, about 35 mg / L, or about 40 mg / L (including all derivable ranges and values ​​therebetween).

[0113] In specific embodiments, the first germination induction medium and / or the second (or expanded) germination induction medium may contain an auxin, where the auxin is 2,4-dichlorophenoxyacetic acid (2,4-D). In some embodiments, the concentration of 2,4-D in the first germination induction medium and / or the second (or expanded) germination induction medium is about 0.1 mg / L to about 10.0 mg / L, about 0.1 mg / L to about 7.5 mg / L, about 0.1 mg / L to about 7.0 mg / L, about 0.1 mg / L to about 6.0 mg / L, about 0.1 mg / L to about 5.0 mg / L, about 0.1 mg / L to about 4.0 mg / L, about 0.1 mg / L to about 3.0 mg / L, about 0.1 mg / L to about 2.0 mg / L, about 0.1 mg / L to about 1.5 mg / L, about 0.1 mg / L to about 1.5 mg / L, or about 0.1 mg / L to about 2.0 mg / L. Approximately 1.25 mg / L, approximately 0.1 mg / L ~ approximately 1.2 mg / L, approximately 0.1 mg / L ~ approximately 1.1 mg / L, approximately 0.2 mg / L ~ approximately 10.0 mg / L, approximately 0.2 mg / L ~ approximately 7.5 mg / L, approximately 0.2 mg / L ~ approximately 7.0 mg / L, approximately 0.2 mg / L ~ approximately 6. 0mg / L, about 0.2mg / L to about 5.0mg / L, about 0.2mg / L to about 4.0mg / L, about 0.2mg / L to about 3.0mg / L, about 0.2mg / L to about 2.0mg / L, about 0.2mg / L to about 1.5mg / L, about 0.2mg / L to about 1.25mg / L , about 0.2 mg / L to about 1.2 mg / L, about 0.2 mg / L to about 1.1 mg / L, about 0.5 mg / L to about 10.0 mg / L, about 0.5 mg / L to about 7.5 mg / L, about 0.5 mg / L to about 7.0 mg / L, about 0.5 mg / L to about 6.0 mg / L, about 0. 5mg / L~about 5.0mg / L, about 0.5mg / L~about 4.0mg / L, about 0.5mg / L~about 3.0mg / L, about 0.5mg / L~about 2.0mg / L, about 0.5mg / L~about 1.5mg / L, about 0.5mg / L~about 1.25mg / L, about 0.5mg / L ~1.2mg / L, 0.5mg / L~1.1mg / L, 0.75mg / L~2.0mg / L, 0.75mg / L~1.5mg / L, 0.75mg / L~1.25mg / L, 0.75mg / L~1.2mg / L, 0.75mg / L L ~ about 1.1mg / L, about 0.8mg / L - about 2.0mg / L, about 0.8mg / L - about 1.5mg / L, about 0.8mg / L - about 1.25mg / L, about 0.8mg / L - about 1.2mg / L, about 0.8mg / L - about 1.1mg / L, about 0.9mg / L - about 2.The concentration may be in the range of 0 mg / L, about 0.9 mg / L to about 1.5 mg / L, about 0.9 mg / L to about 1.25 mg / L, about 0.9 mg / L to about 1.2 mg / L, or about 0.9 mg / L to about 1.1 mg / L (including all derivable values ​​and ranges therebetween). In further embodiments, the concentration of 2,4-D in the first germination induction medium and / or the second (or expansion) germination induction medium can be about 0.1 mg / L, about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, or about 10.0 mg / L (including all derivable values ​​and ranges therebetween).

[0114] In some embodiments, the first germination induction medium and / or the second (or expanded) germination induction medium may contain an auxin, wherein the auxin is 2,4,5-trichloro-phenoxyacetic acid (2,4,5-T). In certain embodiments, the concentration of 2,4,5-T in the first germination induction medium and / or the second (or expanded) germination induction medium is from about 0.1 mg / L to about 10.0 mg / L, from about 0.1 mg / L to about 7.5 mg / L, from about 0.1 mg / L to about 7.0 mg / L, from about 0.1 mg / L to about 6.0 mg / L, from about 0.1 mg / L to about 5.0 mg / L, from about 0.1 mg / L to about 4.0 mg / L, from about 0.1 mg / L to about 3.0 mg / L, from about 0.1 mg / L to about 2.0 mg / L, from about 0.1 mg / L to about 1.5 mg / L, or from about 0.1 mg / L to about 1.5 mg / L. g / L ~ approx. 1.25 mg / L, approx. 0.1 mg / L ~ approx. 1.2 mg / L, approx. 0.1 mg / L ~ approx. 1.1 mg / L, approx. 0.2 mg / L ~ approx. 10.0 mg / L, approx. 0.2 mg / L ~ approx. 7.5 mg / L, approx. 0.2 mg / L ~ approx. 7.0 mg / L, approx. 0.2 mg / L L~6.0mg / L, 0.2mg / L~5.0mg / L, 0.2mg / L~4.0mg / L, 0.2mg / L~3.0mg / L, 0.2mg / L~2.0mg / L, 0.2mg / L~1.5mg / L, 0.2mg / L~1. 25mg / L, about 0.2mg / L to about 1.2mg / L, about 0.2mg / L to about 1.1mg / L, about 0.5mg / L to about 10.0mg / L, about 0.5mg / L to about 7.5mg / L, about 0.5mg / L to about 7.0mg / L, about 0.5mg / L to about 6.0m g / L, about 0.5 mg / L to about 5.0 mg / L, about 0.5 mg / L to about 4.0 mg / L, about 0.5 mg / L to about 3.0 mg / L, about 0.5 mg / L to about 2.0 mg / L, about 0.5 mg / L to about 1.5 mg / L, about 0.5 mg / L to about 1.25 mg / L, Approximately 0.5mg / L to approximately 1.2mg / L, approximately 0.5mg / L to approximately 1.1mg / L, approximately 0.75mg / L to approximately 2.0mg / L, approximately 0.75mg / L to approximately 1.5mg / L, approximately 0.75mg / L to approximately 1.25mg / L, approximately 0.75mg / L to approximately 1.2mg / L, Approx. 0.75mg / L~Approx. 1.1mg / L, Approx. 0.8mg / L~Approx. 2.0mg / L, Approx. 0.8mg / L~Approx. 1.5mg / L, Approx. 0.8mg / L~Approx. 1.25mg / L, Approx. 0.8mg / L~Approx. 1.2mg / L, Approx. 0.8mg / L~Approx. 1.1mg / L, Approx.The range may be from about 9 mg / L to about 2.0 mg / L, from about 0.9 mg / L to about 1.5 mg / L, from about 0.9 mg / L to about 1.25 mg / L, from about 0.9 mg / L to about 1.2 mg / L, or from about 0.9 mg / L to about 1.1 mg / L (including all derivable values ​​and ranges therebetween). In further embodiments, the concentration of 2,4,5-T in the first germination induction medium and / or the second (or expansion) germination induction medium can be about 0.1 mg / L, about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, or about 10.0 mg / L (including all derivable values ​​and ranges therebetween).

[0115] In some embodiments, the first germination induction medium and / or the second (or expanded) germination induction medium can contain an auxin, where the auxin is 4-amino-3,5,6-trichloropicolinic acid (picloram). In further embodiments, the concentration of picloram in the first germination induction medium and / or the second (or expanded) germination induction medium is from about 0.1 mg / L to about 10.0 mg / L, from about 0.1 mg / L to about 7.5 mg / L, from about 0.1 mg / L to about 7.0 mg / L, from about 0.1 mg / L to about 6.0 mg / L, from about 0.1 mg / L to about 5.0 mg / L, or from about 0.1 mg / L to about 4.0 mg / L. mg / L, about 0.1 mg / L to about 3.0 mg / L, about 0.2 mg / L to about 10.0 mg / L, about 0.2 mg / L to about 7.5 mg / L, about 0.2 mg / L to about 7.0 mg / L, about 0.2mg / L~Approx. 6.0mg / L, Approx. 0.2mg / L~Approx. 5.0mg / L, Approx. 0.2mg / L~Approx. 4.0mg / L, Approx. 0.2mg / L~Approx. 3.0mg / L, Approx. 0.5mg / L ~10.0mg / L, 0.5mg / L~7.5mg / L, 0.5mg / L~7.0mg / L, 0.5mg / L~6.0mg / L, 0.5mg / L~5.0 mg / L, about 0.5 mg / L to about 4.0 mg / L, about 0.5 mg / L to about 3.0 mg / L, about 1.0 mg / L to about 10.0 mg / L, about 1.0 mg / L to about 7.5 mg / L, about It may be in the range of 1.0 mg / L to about 7.0 mg / L, about 1.0 mg / L to about 6.0 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 4.0 mg / L, about 1.0 mg / L to about 3.0 mg / L, or about 1.5 mg / L to about 2.5 mg / L (including all derivable values ​​and ranges therebetween).In specific embodiments, the concentration of picloram in the first germination induction medium and / or the second (or expansion) germination induction medium can be about 0.1 mg / L, about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, or about 10.0 mg / L (including all derivable values ​​and ranges therebetween).

[0116] In certain embodiments, the first germination induction medium and / or the second (or expanded) germination induction medium may contain auxin, where the auxin is indole-3-acetic acid (IAA). In some embodiments, the concentration of IAA in the first germination induction medium and / or the second (or expanded) germination induction medium is about 0.1 mg / L to about 25.0 mg / L, about 0.1 mg / L to about 20.0 mg / L, about 0.1 mg / L to about 15.0 mg / L, about 0.2 mg / L to about 25.0 mg / L, about 0.2 mg / L to about 20.0 mg / L, about 0.2 mg / L to about 15.0 mg / L, about 0.2 mg / L to about 25.0 mg / L, about 0.2 mg / L to about 1 ... g / L ~ approx. 15.0 mg / L, approx. 0.5 mg / L ~ approx. 25.0 mg / L, approx. 0.5 mg / L ~ approx. 20.0 mg / L, approx. 0.5 mg / L ~ approx. 15.0 mg / L, approx. 0.5 mg / L ~ approx. 12.5 mg / L, 1.0 mg / L ~ approx. 25.0 mg / L, approx. 1.0 mg / L ~ approx. 20.0 mg / L, approx. 1.0 mg / L ~ approx. 15.0 mg / L, approx. 1.0 mg / L ~12.5mg / L, 2.0mg / L~25.0mg / L, 2.0mg / L~20.0mg / L, 2.0mg / L~15.0mg / L, 2.0mg / L ~12.5mg / L, approx. 5.0mg / L~25.0mg / L, approx. 5.0mg / L~20.0mg / L, approx. 5.0mg / L~15.0mg / L, approx. 5.0mg / L~ It may be in the range of about 12.5 mg / L, about 7.5 mg / L to about 25.0 mg / L, about 7.5 mg / L to about 20.0 mg / L, about 7.5 mg / L to about 15.0 mg / L, about 7.5 mg / L to about 12.5 mg / L, about 8.0 mg / L to about 12.0 mg / L, or about 9.0 mg / L to about 11.0 mg / L (including all derivable ranges therebetween).In further embodiments, the concentration of IAA in the first sprouting induction medium and / or the second (or expansion) sprouting induction medium is about 0.1 mg / L, about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, about 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, about 10.0 mg / L, about 11.0 mg / L, about 12.0 mg / L, about 13.0 mg / L, about 14.0 mg / L, about 15.0 mg / L, about 16.0 mg / L, about 17.0 mg / L, about 18.0 mg / L, about 19.0 mg / L, about 20.0 mg / L, about 21.0 mg / L, about 22.0 mg / L, about 23.0 mg / L, about 24.0 mg / L, about 25.0 mg / L, about 26.0 mg / L, about 27.0 mg / L, about 28.0 mg / L, about 29.0 mg / L, about 30.0 mg / L, about 31.0 mg / L, about 32.0 mg / L, about 33.0 mg / L, about 34.0 mg / L, about 35.0 mg / L, about 36.0 mg / L, about 37.0 mg / L, about / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, about 10.0 mg / L, about 11.0 mg / L, about 12.0 mg / L, about 13.0 mg / L, about 14.0 mg / L, about 15.0 mg / L, about 16.0 mg / L, about 17.0 mg / L, about 18.0 mg / L, about 19.0 mg / L, about 20.0 mg / L, about 21.0 mg / L, about 22.0 mg / L, about 23.0 mg / L, about 24.0 mg / L, or about 25.0 mg / L (including all derivable values ​​and ranges therebetween).

[0117] In specific embodiments, the first germination induction medium and / or the second (or expanded) germination induction medium may contain an auxin, where the auxin is indole-3-butyric acid (IBA). In some embodiments, the concentration of IBA in the first germination induction medium and / or the second (or expanded) germination induction medium is from about 0.1 mg / L to about 10.0 mg / L, from about 0.1 mg / L to about 7.5 mg / L, from about 0.1 mg / L to about 7.0 mg / L, from about 0.1 mg / L to about 6.0 mg / L, from about 0.1 mg / L to about 5.0 mg / L, or from about 0.1 mg / L to about 4.0 mg / L. mg / L, about 0.1 mg / L to about 3.0 mg / L, about 0.2 mg / L to about 10.0 mg / L, about 0.2 mg / L to about 7.5 mg / L, about 0.2 mg / L to about 7.0 mg / L, about 0.2mg / L~Approx. 6.0mg / L, Approx. 0.2mg / L~Approx. 5.0mg / L, Approx. 0.2mg / L~Approx. 4.0mg / L, Approx. 0.2mg / L~Approx. 3.0mg / L, Approx. 0.5mg / L ~10.0mg / L, 0.5mg / L~7.5mg / L, 0.5mg / L~7.0mg / L, 0.5mg / L~6.0mg / L, 0.5mg / L~5.0 mg / L, about 0.5 mg / L to about 4.0 mg / L, about 0.5 mg / L to about 3.0 mg / L, about 1.0 mg / L to about 10.0 mg / L, about 1.0 mg / L to about 7.5 mg / L, about It may be in the range of 1.0 mg / L to about 7.0 mg / L, about 1.0 mg / L to about 6.0 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 4.0 mg / L, about 1.0 mg / L to about 3.0 mg / L, or about 1.5 mg / L to about 2.5 mg / L (including all derivable values ​​and ranges therebetween).In further embodiments, the concentration of IBA in the first germination induction medium and / or the second (or expanded) germination induction medium can be about 0.1 mg / L, about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, or about 10.0 mg / L (including all derivable values ​​and ranges therebetween).

[0118] In certain embodiments, the first germination induction medium and / or the second (or expanded) germination induction medium may contain auxin, where the auxin is naphthalene acetic acid (NAA). In further embodiments, the concentration of NAA in the first germination induction medium and / or the second (or expanded) germination induction medium may be from about 0.1 mg / L to about 25.0 mg / L, from about 0.1 mg / L to about 20.0 mg / L, from about 0.1 mg / L to about 15.0 mg / L, from about 0.1 mg / L to about 10.0 mg / L, from about 0.1 mg / L to about 7.5 mg / L, from about 0.1 mg / L to about 7.0 mg / L, from about 0.1 mg / L to about 6.0 mg / L, from about 0.2 mg / L to about 25.0 mg / L, from about 0.2 mg / L to about 20.0 mg / L, or from about 0.2 mg / L to about 1. 5.0mg / L, about 0.2mg / L to about 10.0mg / L, about 0.2mg / L to about 7.5mg / L, about 0.2mg / L to about 7.0mg / L, about 0.2mg / L to about 6.0mg / L, about 0.5mg / L to about 25.0mg / L, about 0.5mg / L to about 20.0m g / L, about 0.5 mg / L to about 15.0 mg / L, about 0.5 mg / L to about 12.5 mg / L, about 0.5 mg / L to about 10.0 mg / L, about 0.5 mg / L to about 7.5 mg / L, about 0.5 mg / L to about 7.0 mg / L, about 0.5 mg / L to about 6.0 mg / L, Approximately 1.0mg / L to approximately 25.0mg / L, approximately 1.0mg / L to approximately 20.0mg / L, approximately 1.0mg / L to approximately 15.0mg / L, approximately 1.0mg / L to approximately 12.5mg / L, approximately 1.0mg / L to approximately 10.0mg / L, approximately 1.0mg / L to approximately 7.5mg / L, approximately 1 .0mg / L~Approx. 7.0mg / L, Approx. 1.0mg / L~Approx. 6.0mg / L, Approx. 2.0mg / L~Approx. 25.0mg / L, Approx. 2.0mg / L~Approx. 20.0mg / L, Approx. 2.0mg / L~Approx. 15.0mg / L, Approx. 2.0mg / L~Approx. 12.5mg / L, Approx. 2.0m g / L ~ approx. 10.0 mg / L, approx. 2.0 mg / L ~ approx. 7.5 mg / L, approx. 2.0 mg / L ~ approx. 7.0 mg / L, approx. 2.0 mg / L ~ approx. 6.0 mg / L, approx. 3.0 mg / L ~ approx. 25.0 mg / L, approx. 3.0 mg / L ~ approx. 20.0 mg / L, approx. 3.0 mg / L ~ Approximately 15.0 mg / L, approximately 3.0 mg / L to approximately 12.5 mg / L, approximately 3.0 mg / L to approximately 10.0 mg / L, approximately 3.0 mg / L to approximately 7.5 mg / L, approximately 3.0 mg / L to approximately 7.0 mg / L, approximately 3.0 mg / L to approximately 6.0 mg / L, approximately 4.0 mg / L to approximately 25.The range may be 0 mg / L, about 4.0 mg / L to about 20.0 mg / L, about 4.0 mg / L to about 15.0 mg / L, about 4.0 mg / L to about 12.5 mg / L, about 4.0 mg / L to about 10.0 mg / L, about 4.0 mg / L to about 7.5 mg / L, about 4.0 mg / L to about 7.0 mg / L, or about 4.0 mg / L to about 6.0 mg / L (including all derivable values ​​and ranges therebetween). In specific embodiments, the concentration of NAA in the first sprouting induction medium and / or the second (or expansion) sprouting induction medium is 0.1 mg / L, about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, about 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, or , about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, about 10.0 mg / L, about 11.0 mg / L, about 12.0 mg / L, about 13.0 mg / L, about 14.0 mg / L, about 15.0 mg / L, about 16.0 mg / L, about 17.0 mg / L, about 18.0 mg / L, about 19.0 mg / L, or about 20.0 mg / L (including all derivable values ​​and ranges therebetween).

[0119] In some embodiments, the first germination induction medium and / or the second (or expanded) germination induction medium can contain auxin, where the auxin is 2,3,5-triiodobenzoic acid (TIBA). In further embodiments, the concentration of TIBA in the first germination induction medium and / or the second (or expanded) germination induction medium is from about 0.1 mg / L to about 25.0 mg / L, from about 0.1 mg / L to about 20.0 mg / L, from about 0.1 mg / L to about 15.0 mg / L, from about 0.1 mg / L to about 10.0 mg / L, from about 0.1 mg / L to about 7.5 mg / L, from about 0.1 mg / L to about 7.0 mg / L, from about 0.1 mg / L to about 6.0 mg / L, from about 0.2 mg / L to about 25.0 mg / L, from about 0.2 mg / L to about 20.0 mg / L, or from about 0.2 mg / L to about 25.0 mg / L. g / L~about 15.0mg / L, about 0.2mg / L~about 10.0mg / L, about 0.2mg / L~about 7.5mg / L, about 0.2mg / L~about 7.0mg / L, about 0.2mg / L~about 6.0mg / L, about 0.5mg / L~about 25.0mg / L, about 0.5mg / L L~about 20.0mg / L, about 0.5mg / L~about 15.0mg / L, about 0.5mg / L~about 12.5mg / L, about 0.5mg / L~about 10.0mg / L, about 0.5mg / L~about 7.5mg / L, about 0.5mg / L~about 7.0mg / L, about 0.5mg / L ~6.0mg / L, 1.0mg / L~25.0mg / L, 1.0mg / L~20.0mg / L, 1.0mg / L~15.0mg / L, 1.0mg / L~12.5mg / L, 1.0mg / L~10.0mg / L, 1.0mg / L L ~ approx. 7.5 mg / L, approx. 1.0 mg / L ~ approx. 7.0 mg / L, approx. 1.0 mg / L ~ approx. 6.0 mg / L, approx. 2.0 mg / L ~ approx. 25.0 mg / L, approx. 2.0 mg / L ~ approx. 20.0 mg / L, approx. 2.0 mg / L ~ approx. 15.0 mg / L, approx. 2.0 mg / L Approximately 12.5 mg / L, approximately 2.0 mg / L to approximately 10.0 mg / L, approximately 2.0 mg / L to approximately 7.5 mg / L, approximately 2.0 mg / L to approximately 7.0 mg / L, approximately 2.0 mg / L to approximately 6.0 mg / L, approximately 3.0 mg / L to approximately 25.0 mg / L, approximately 3.0 mg / L to approximately 2 0.0mg / L, about 3.0mg / L to about 15.0mg / L, about 3.0mg / L to about 12.5mg / L, about 3.0mg / L to about 10.0mg / L, about 3.0mg / L to about 7.5mg / L, about 3.0mg / L to about 7.0mg / L, about 3.0mg / L to about 6.The range may be about 0 mg / L, about 4.0 mg / L to about 25.0 mg / L, about 4.0 mg / L to about 20.0 mg / L, about 4.0 mg / L to about 15.0 mg / L, about 4.0 mg / L to about 12.5 mg / L, about 4.0 mg / L to about 10.0 mg / L, about 4.0 mg / L to about 7.5 mg / L, about 4.0 mg / L to about 7.0 mg / L, or about 4.0 mg / L to about 6.0 mg / L (including all derivable values ​​and ranges therebetween). In additional embodiments, the concentration of TIBA in the first germination induction medium and / or the second (or expansion) germination induction medium is 0.1 mg / L, about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, about 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, or about 5.0 mg / L. , about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, about 10.0 mg / L, about 11.0 mg / L, about 12.0 mg / L, about 13.0 mg / L, about 14.0 mg / L, about 15.0 mg / L, about 16.0 mg / L, about 17.0 mg / L, about 18.0 mg / L, about 19.0 mg / L, or about 20.0 mg / L (including all derivable values ​​and ranges therebetween).

[0120] In certain embodiments, the first germination induction medium and / or the second (or expanded) germination induction medium may contain an auxin, where the auxin is phenylacetic acid (PAA). In further embodiments, the concentration of PAA in the first germination induction medium and / or the second (or expanded) germination induction medium is from about 0.1 mg / L to about 25.0 mg / L, from about 0.1 mg / L to about 20.0 mg / L, from about 0.1 mg / L to about 15.0 mg / L, from about 0.2 mg / L to about 25.0 mg / L, from about 0.2 mg / L to about 20.0 mg / L, or from about 0.2 mg / L to about 15.0 mg / L. L ~ approx. 15.0 mg / L, approx. 0.5 mg / L ~ approx. 25.0 mg / L, approx. 0.5 mg / L ~ approx. 20.0 mg / L, approx. 0.5 mg / L ~ approx. 15.0 mg / L, approx. 0.5 mg / L L ~ approx. 12.5 mg / L, 1.0 mg / L ~ approx. 25.0 mg / L, approx. 1.0 mg / L ~ approx. 20.0 mg / L, approx. 1.0 mg / L ~ approx. 15.0 mg / L, approx. 1.0 mg / L ~ Approx. 12.5mg / L, Approx. 2.0mg / L~Approx. 25.0mg / L, Approx. 2.0mg / L~Approx. 20.0mg / L, Approx. 2.0mg / L~Approx. 15.0mg / L, Approx. 2.0mg / L~ Approx. 12.5 mg / L, approx. 5.0 mg / L ~ approx. 25.0 mg / L, approx. 5.0 mg / L ~ approx. 20.0 mg / L, approx. 5.0 mg / L ~ approx. 15.0 mg / L, approx. 5.0 mg / L ~ approx. The range may be 12.5 mg / L, about 7.5 mg / L to about 25.0 mg / L, about 7.5 mg / L to about 20.0 mg / L, about 7.5 mg / L to about 15.0 mg / L, about 7.5 mg / L to about 12.5 mg / L, about 8.0 mg / L to about 12.0 mg / L, or about 9.0 mg / L to about 11.0 mg / L (including all derivable ranges therebetween).In some embodiments, the concentration of PAA in the first sprouting induction medium and / or the second (or expansion) sprouting induction medium is about 0.1 mg / L, about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, about 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, about 10.0 mg / L, about 11.0 mg / L, about 12.0 mg / L, about 13.0 mg / L, about 14.0 mg / L, about 15.0 mg / L, about 16.0 mg / L, about 17.0 mg / L, about 18.0 mg / L, about 19.0 mg / L, about 20.0 mg / L, about 21.0 mg / L, about 22.0 mg / L, about 23.0 mg / L, about 24.0 mg / L, about 25.0 mg / L, about 26.0 mg / L, about 27.0 mg / L, about 28.0 mg / L, about 29.0 mg / L, about 30.0 mg / L, about 31.0 mg / L, about 32.0 mg / L, about 33.0 mg / L, about 34.0 mg / L, about 35.0 mg / L, about 36.0 mg / L, about 37.0 mg / L, about g / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, about 10.0 mg / L, about 11.0 mg / L, about 12.0 mg / L, about 13.0 mg / L, about 14.0 mg / L, about 15.0 mg / L, about 16.0 mg / L, about 17.0 mg / L, about 18.0 mg / L, about 19.0 mg / L, about 20.0 mg / L, about 21.0 mg / L, about 22.0 mg / L, about 23.0 mg / L, about 24.0 mg / L, or about 25.0 mg / L (including all derivable values ​​and ranges therebetween).

[0121] In certain embodiments, the first emergence induction medium and / or the second (or extended) emergence induction medium can contain an auxin, where the auxin is 3,6-dichloro-2-methoxybenzoic acid (dicamba). In further embodiments, the concentration of dicamba in the first emergence induction medium and / or the second (or extended) emergence induction medium can be from about 0.1 mg / L to about 10.0 mg / L, from about 0.1 mg / L to about 7.5 mg / L, from about 0.1 mg / L to about 7.0 mg / L, from about 0.1 mg / L to about 6.0 mg / L, from about 0.1 mg / L to about 5.0 mg / L, or from about 0.1 mg / L to about 4.0 mg / L. g / L, about 0.1 mg / L to about 3.0 mg / L, about 0.2 mg / L to about 10.0 mg / L, about 0.2 mg / L to about 7.5 mg / L, about 0.2 mg / L to about 7.0 mg / L, about 0.2mg / L~Approx. 6.0mg / L, Approx. 0.2mg / L~Approx. 5.0mg / L, Approx. 0.2mg / L~Approx. 4.0mg / L, Approx. 0.2mg / L~Approx. 3.0mg / L, Approx. 0.5mg / L ~10.0mg / L, 0.5mg / L~7.5mg / L, 0.5mg / L~7.0mg / L, 0.5mg / L~6.0mg / L, 0.5mg / L~5.0 mg / L, about 0.5 mg / L to about 4.0 mg / L, about 0.5 mg / L to about 3.0 mg / L, about 1.0 mg / L to about 10.0 mg / L, about 1.0 mg / L to about 7.5 mg / L, about It may be in the range of 1.0 mg / L to about 7.0 mg / L, about 1.0 mg / L to about 6.0 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 4.0 mg / L, about 1.0 mg / L to about 3.0 mg / L, or about 1.5 mg / L to about 2.5 mg / L (including all derivable values ​​and ranges therebetween).In additional embodiments, the concentration of dicamba in the first emergence induction medium and / or the second (or expansion) emergence induction medium can be about 0.1 mg / L, about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, 1.0 mg / L, about 1.5 mg / L, about 2.0 mg / L, about 2.5 mg / L, about 3.0 mg / L, about 3.5 mg / L, about 4.0 mg / L, about 4.5 mg / L, about 5.0 mg / L, about 6.0 mg / L, about 7.0 mg / L, about 8.0 mg / L, about 9.0 mg / L, or about 10.0 mg / L (including all derivable values ​​and ranges therebetween).

[0122] According to some embodiments, the first germination induction medium comprises a first auxin and a first cytokinin, wherein the first auxin is 2,4-dichlorophenoxyacetic acid (2,4-D) and the first cytokinin is 6-benzylaminopurine (BAP).According to these embodiments, the concentration of 2,4-D in the first germination induction medium is about 0.1 mg / L to about 10.0 mg / L, about 0.1 mg / L to about 7.5 mg / L, about 0.1 mg / L to about 7.0 mg / L, about 0.1 mg / L to about 6.0 mg / L, about 0.1 mg / L to about 5.0 mg / L, about 0.1 mg / L to about 4.0 mg / L, about 0.1 mg / L to about 3.0 mg / L, about 0.1 mg / L to about 2.0 mg / L, about 0.1 mg / L to about 1.5 mg / L, about 0.1 mg / L to about 1.25 mg / L, about 0.1 mg / L to about 1.2 mg / L, about 0.1 mg / L to about 1.1 mg / L, / L, about 0.2 mg / L to about 10.0 mg / L, about 0.2 mg / L to about 7.5 mg / L, about 0.2 mg / L to about 7.0 mg / L, about 0.2 mg / L to about 6.0 mg / L, about 0.2 mg / L to about 5.0 mg / L, about 0.2 mg / L to about 4.0 mg / L, about 0.2 mg / L to about 3. 0mg / L, about 0.2mg / L~about 2.0mg / L, about 0.2mg / L~about 1.5mg / L, about 0.2mg / L~about 1.25mg / L, about 0.2mg / L~about 1.2mg / L, about 0.2mg / L~about 1.1mg / L, about 0.5mg / L~about 10.0mg / L, about 0.5mg / L~ Approximately 7.5 mg / L, approximately 0.5 mg / L ~ approximately 7.0 mg / L, approximately 0.5 mg / L ~ approximately 6.0 mg / L, approximately 0.5 mg / L ~ approximately 5.0 mg / L, approximately 0.5 mg / L ~ approximately 4.0 mg / L, approximately 0.5 mg / L ~ approximately 3.0 mg / L, approximately 0.5 mg / L ~ approximately 2.0 mg / L, approximately 0.5 mg / L L ~ approx. 1.5 mg / L, approx. 0.5 mg / L ~ approx. 1.25 mg / L, approx. 0.5 mg / L ~ approx. 1.2 mg / L, approx. 0.5 mg / L ~ approx. 1.1 mg / L, approx. The range may be from 0.75 mg / L to about 1.2 mg / L, from about 0.75 mg / L to about 1.1 mg / L, from about 0.8 mg / L to about 2.0 mg / L, from about 0.8 mg / L to about 1.5 mg / L, from about 0.8 mg / L to about 1.25 mg / L, from about 0.8 mg / L to about 1.2 mg / L, from about 0.8 mg / L to about 1.1 mg / L, from about 0.9 mg / L to about 2.0 mg / L, from about 0.9 mg / L to about 1.5 mg / L, from about 0.9 mg / L to about 1.25 mg / L, from about 0.9 mg / L to about 1.2 mg / L, or from about 0.9 mg / L to about 1.1 mg / L (including all derivable values ​​and ranges therebetween).According to these embodiments, the concentration of 6-benzylaminopurine (BAP) in the first germination induction medium is about 1.0 mg / L to about 25.0 mg / L, about 1.0 mg / L to about 20.0 mg / L, about 1.0 mg / L to about 15.0 mg / L, about 1.0 mg / L to about 12.5 mg / L, about 2.0 mg / L to about 25.0 mg / L, about 2.0 mg / L to about 20.0 mg / L, about 2.0 mg / L to about 15.0 mg / L, about 2.0 mg / L to about 12.5 mg / L, or about 5.0 mg / L to about 2 It may be in the range of 5.0 mg / L, about 5.0 mg / L to about 20.0 mg / L, about 5.0 mg / L to about 15.0 mg / L, about 5.0 mg / L to about 12.5 mg / L, about 7.5 mg / L to about 25.0 mg / L, about 7.5 mg / L to about 20.0 mg / L, about 7.5 mg / L to about 15.0 mg / L, about 7.5 mg / L to about 12.5 mg / L, about 8.0 mg / L to about 12.0 mg / L, or about 9.0 mg / L to about 11.0 mg / L (including all derivable ranges therebetween).

[0123] According to certain embodiments, the second (or expanded) germination-inducing medium comprises a second auxin and a second cytokinin, wherein the second auxin is 4-amino-3,5,6-trichloropicolinic acid (picloram) and the second cytokinin is thidiazuron (TDZ). According to these embodiments, the concentration of picloram in the second (or expanded) germination-inducing medium is from about 0.1 mg / L to about 10.0 mg / L, from about 0.1 mg / L to about 7.5 mg / L, from about 0.1 mg / L to about 7.0 mg / L, from about 0.1 mg / L to about 6.0 mg / L, from about 0.1 mg / L to about 5.0 mg / L, from about 0.1 mg / L to about 4.0 mg / L, or from about 0.1 mg / L to about 10.0 mg / L. mg / L ~ approx. 3.0 mg / L, approx. 0.2 mg / L ~ approx. 10.0 mg / L, approx. 0.2 mg / L ~ approx. 7.5 mg / L, approx. 0.2 mg / L ~ approx. 7.0 mg / L, approx. 0.2 mg / L ~6.0mg / L, 0.2mg / L~5.0mg / L, 0.2mg / L~4.0mg / L, 0.2mg / L~3.0mg / L, 0.5mg / L~10.0 mg / L, about 0.5 mg / L to about 7.5 mg / L, about 0.5 mg / L to about 7.0 mg / L, about 0.5 mg / L to about 6.0 mg / L, about 0.5 mg / L to about 5.0 mg / L, Approx. 0.5 mg / L ~ approx. 4.0 mg / L, approx. 0.5 mg / L ~ approx. 3.0 mg / L, approx. 1.0 mg / L ~ approx. 10.0 mg / L, approx. 1.0 mg / L ~ approx. 7.5 mg / L, approx. 1.0 The range may be from about 1.0 mg / L to about 7.0 mg / L, from about 1.0 mg / L to about 6.0 mg / L, from about 1.0 mg / L to about 5.0 mg / L, from about 1.0 mg / L to about 5.0 mg / L, from about 1.0 mg / L to about 4.0 mg / L, from about 1.0 mg / L to about 3.0 mg / L, or from about 1.5 mg / L to about 2.5 mg / L (including all derivable values ​​and ranges therebetween).According to these embodiments, the concentration of TDZ in the second (or expanded) germination-inducing medium is about 0.1 mg / L to about 10.0 mg / L, about 0.1 mg / L to about 7.5 mg / L, about 0.1 mg / L to about 7.0 mg / L, about 0.1 mg / L to about 6.0 mg / L, about 0.1 mg / L to about 5.0 mg / L, about 0.1 mg / L to about 4.0 mg / L, about 0.1 mg / L to about 5 ... / L ~ approx. 3.0 mg / L, approx. 0.2 mg / L ~ approx. 10.0 mg / L, approx. 0.2 mg / L ~ approx. 7.5 mg / L, approx. 0.2 mg / L ~ approx. 7.0 mg / L, approx. 0.2 mg / L ~ approx. 6.0mg / L, about 0.2mg / L to about 5.0mg / L, about 0.2mg / L to about 4.0mg / L, about 0.2mg / L to about 3.0mg / L, about 0.5mg / L to about 10.0m g / L, about 0.5 mg / L to about 7.5 mg / L, about 0.5 mg / L to about 7.0 mg / L, about 0.5 mg / L to about 6.0 mg / L, about 0.5 mg / L to about 5.0 mg / L, about 0.5mg / L~Approx. 4.0mg / L, Approx. 0.5mg / L~Approx. 3.0mg / L, Approx. 1.0mg / L~Approx. 10.0mg / L, Approx. 1.0mg / L~Approx. 7.5mg / L, Approx. 1.0m g / L to about 7.0 mg / L, about 1.0 mg / L to about 6.0 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 5.0 mg / L, about 1.0 mg / L to about 4.0 mg / L, about 1.0 mg / L to about 3.0 mg / L, or about 1.5 mg / L to about 2.5 mg / L (including all derivable values ​​and ranges therebetween).

[0124] According to some embodiments, the methods described herein include achieving a lower density of visible or total monocotyledonous seed embryo explant(s) in the germination (or first germination) induction medium per container or plate, which may be achieved by transferring cultured monocotyledonous seed embryo explant(s) from a predetermined number of co-cultivation plates(s) or container(s) to a relatively larger number of germination (or first germination) induction plates(s) or container(s). As described herein, transformation of male corn lines, and possibly other monocotyledonous germplasm, can often be difficult or less efficient than transformation of female corn lines or other monocotyledonous germplasm. Compared to germination induction plates for female corn embryo explants, germination induction plates for male corn embryo explants can frequently exhibit contamination and tissue necrosis. According to some embodiments, reducing the density of visible or total explants per germination (or first germination) induction plate or container may improve the frequency of transformation, shoot, and / or regeneration of monocotyledonous or maize embryo explants, or genetically modified plants of particular monocotyledonous plants or maize lines that are more resistant to transformation and / or regeneration of genetically modified plants, e.g., particular male germplasm maize lines. According to some embodiments, monocotyledonous seed embryo explants can be transferred from one or more co-cultivation plate(s) or container(s) to a relatively smaller or fewer number of germination induction plate(s) or container(s), for example, at a 2:1 or 1:1 ratio in terms of number of co-cultivation plate(s) or container(s):number of germination induction plate(s) or container(s), although the number of explants per germination induction plate may be overcrowded.

[0125] According to some embodiments, monocotyledonous seed embryo explants can be transferred from one or more co-culture induction plate(s) or container(s) to a relatively larger or greater number of germination induction plate(s) or container(s), for example, at a ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, or 1:15 in terms of number of co-culture plate(s) or container(s):number of germination induction plate(s) or container(s).According to some embodiments, the range is from about 10 to about 2,000, from about 20 to about 2,000, from about 30 to about 2,000, from about 40 to about 2,000, from about 50 to about 2,000, from about 10 to about 1,500, from about 10 to about 1,000, from about 10 to about 1,500, from about 10 to about 1,000, from about 10 to about 750, from about 10 to about 500, from about 10 to about 600, from about 10 to about 500, from about 10 to about 400, from about 10 to about 300, from about 50 to about 200, from about 20 to about 2,000, from about 20 to about 1,500, from about 20 to about 1,000. , about 20 to about 1,500, about 20 to about 1,000, about 20 to about 750, about 20 to about 500, about 20 to about 600, about 20 to about 500, about 20 to about 400, about 20 to about 300, about 20 to about 200, about 30 to about 2,000, about 30 to about 1,500, about 30 to about 1,000, about 30 to about 1,500, about 30 to about 1,000, about 30 to about 750, about 30 to about 500, about 30 to about 600, about 30 to about 500, about 30 to about 400, about 30 to about 300, about 30 to about 200, about 40 to About 2,000, about 40 to about 1,500, about 40 to about 1,000, about 40 to about 1,500, about 40 to about 1,000, about 40 to about 750, about 40 to about 500, about 40 to about 600, about 40 to about 500, about 40 to about 400, about 40 to about 300, about 40 to about 200, about 50 to about 2,000, about 50 to about 1,500, about 50 to about 1,000, about 50 to about 1,500, about 50 to about 1,000, about 50 to about 750, about 50 to about 500, about 50 to about 600, about 50 to about 500, about 50 A predetermined number of visible (or total) monocotyledonous seed embryo explants, such as in the range of about 400, about 50 to about 300, about 50 to about 200 (including all derivable ranges and values ​​therebetween), can be transferred from the co-cultivation plate or medium to a relatively larger or greater number of emergence induction plate(s) or container(s), for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more emergence induction plate(s) or container(s).

[0126] According to some embodiments, the number of visible (or total) monocotyledonous seed embryo explants transferred or added to the germination induction plate(s) or container(s) is about 500 or less seed embryo explants per plate, about 400 or less seed embryo explants per plate, about 300 or less seed embryo explants per plate, about 200 or less seed embryo explants per plate, about 150 or less seed embryo explants per plate, about 100 or less seed embryo explants per plate, about 75 or less seed embryo explants per plate, about 50 or less seed embryo explants per plate. The following seed embryo explants may be grown at a density of about 30 or less seed embryo explants per plate, or about 10 to about 300 seed embryo explants per plate, about 10 to about 250 seed embryo explants per plate, about 10 to about 200 seed embryo explants per plate, about 10 to about 150 seed embryo explants per plate, about 10 to about 100 seed embryo explants per plate, about 10 to about 75 seed embryo explants per plate, about 10 to about 50 seed embryo explants per plate, about 10 to about 25 embryo explants per plate, about 25 to about About 300 seed embryo explants, about 25 to about 250 seed embryo explants per plate, about 25 to about 200 seed embryo explants per plate, about 25 to about 150 seed embryo explants per plate, about 25 to about 100 seed embryo explants per plate, about 25 to about 75 seed embryo explants per plate, about 25 to about 50 seed embryo explants per plate, about 50 to about 300 seed embryo explants per plate, about 50 to about 250 seed embryo explants per plate, about 50 to about 200 seed embryo explants per plate, about 50 to about 1 The density range can be 50 seed embryo explants, about 50 to about 100 seed embryo explants per plate, about 50 to about 75 seed embryo explants per plate, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, or about 300 embryo explants per plate (including all derivable ranges and values ​​therebetween). For these density values ​​and ranges, the surface area of ​​each germination induction plate is approximately 11.9 square inches (in 2 ) or 76.8 square centimeters (cm2 Therefore, all of the above density values ​​and ranges for seed embryo explants per plate can be easily converted to density values ​​and ranges for seed embryo explants per germination-inducing surface area (e.g., a density of 100 seed embryo explants per plate divided by the surface area per plate yields approximately 8.4 seed embryo explants per square inch (in 2 ) or approximately 1.3 seed embryo explants per square centimeter (cm 2 ) yields a density of seed embryo explants per germination induction surface area, and similar conversions can be easily made for other density values ​​and ranges.) The value and range of density of seed embryo explants per germination induction surface area is a more universal definition for the density of seed embryo explants in a variety of different germination induction plate(s) or container(s), each of which may have a different surface area.

[0127] In some embodiments, the number of visible (or total) monocotyledonous seed embryo explants transferred or added to the germination induction plate(s) or container(s) is determined by the square centimeter (cm) of germination induction surface area. 2 , about 3.9, about 3.8, about 3.7, about 3.6, about 3.5, about 3.4, about 3.3, about 3.2, about 3.1, about 3.0, about 2.9, about 2.8, about 2.7, about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about 2.1, about 2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5, about 1.4, about 1.3, about 1.2, about 1.1, about 1.0, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 2, or about 0.1 or less embryo explants per 1000 cells / well (including all derivable ranges and values ​​therebetween). In certain embodiments, the number of visible (or total) monocotyledonous seed embryo explants transferred or added to the germination induction plate(s) or container(s) is determined by the square centimeter (cm) of germination induction surface area. 2) about 0.1 to about 6.0, about 0.1 to about 5.5, about 0.1 to about 5.0, about 0.1 to about 4.5, about 0.1 to about 4.0, about 0.1 to about 3.5, about 0.1 to about 3.0, about 0.1 to about 2.5, about 0.1 to about 2.0, about 0.1 to about 1.5, about 0.1 to about 1.0, about 0.1 to about 0.5, About 0.2 to about 6.0, about 0.2 to about 5.5, about 0.2 to about 5.0, about 0.2 to about 4.5, about 0.2 to about 4.0, about 0.2 to about 3.5, about 0.2 to about 3.0, about 0.2 to about 2.5, about 0.2 to about 2.0, about 0.2 to about 1.5, about 0.2 to about 1.0, about 0.2 to about 0.5, about 0.3 to About 6.0, about 0.3 to about 5.5, about 0.3 to about 5.0, about 0.3 to about 4.5, about 0.3 to about 4.0, about 0.3 to about 3.5, about 0.3 to about 3.0, about 0.3 to about 2.5, about 0.3 to about 2.0, about 0.3 to about 1.5, about 0.3 to about 1.0, about 0.3 to about 0.5, about 0.4 to about 6.0 , about 0.4 to about 5.5, about 0.4 to about 5.0, about 0.4 to about 4.5, about 0.4 to about 4.0, about 0.4 to about 3.5, about 0.4 to about 3.0, about 0.4 to about 2.5, about 0.4 to about 2.0, about 0.4 to about 1.5, about 0.4 to about 1.0, about 0.4 to about 0.5, about 0.5 to about 6.0, about 0.5 to about 5.5, about 0.5 to about 5.0, about 0.5 to about 4.5, about 0.5 to about 4.0, about 0.5 to about 3.5, about 0.5 to about 3.0, about 0.5 to about 2.5, about 0.5 to about 2.0, about 0.5 to about 1.5, about 0.5 to about 1.0, about 0.6 to about 6.0, about 0.6 to about 5.5, about 0.6 to about 5. 0, about 0.6 to about 4.5, about 0.6 to about 4.0, about 0.6 to about 3.5, about 0.6 to about 3.0, about 0.6 to about 2.5, about 0.6 to about 2.0, about 0.6 to about 1.5, about 0.6 to about 1.0, about 0.7 to about 6.0, about 0.7 to about 5.5, about 0.7 to about 5.0, about 0.7 to about 4.5, about 0. 7 to about 4.0, about 0.7 to about 3.5, about 0.7 to about 3.0, about 0.7 to about 2.5, about 0.7 to about 2.0, about 0.7 to about 1.5, about 0.7 to about 1.0, about 0.8 to about 6.0, about 0.8 to about 5.5, about 0.8 to about 5.0, about 0.8 to about 4.5, about 0.8 to about 4.0, about 0.8 to about 3. 5, about 0.8 to about 3.0, about 0.8 to about 2.5, about 0.8 to about 2.0, about 0.8 to about 1.5, about 0.8 to about 1.0, about 0.9 to about 6.0, about 0.9 to about 5.5, about 0.9 to about 5.0, about 0.9 to about 4.5, about 0.9 to about 4.0, about 0.9 to about 3.5, about 0.9 to about 3.0, about 0.9 to about 2.5, about 0.9 to about 2.0, about 0.9 to about 1.5, about 0.9 to about 1.0, about 1.0 to about 6.0, about 1.0 to about 5.5, about 1.0 to about 5.0, about 1.0 to about 4.5, about 1.0 to about 4.0, about 1.0 to about 3.5, about 1.0 to about 3.0, about 1.0 to about 2.5, about 1.0 to about 2.0, about 1.0 to about 1 The density may be in the range of embryo explants from about 0.5, about 1.5 to about 6.0, about 1.5 to about 5.5, about 1.5 to about 5.0, about 1.5 to about 4.5, about 1.5 to about 4.0, about 1.5 to about 3.5, about 1.5 to about 3.0, about 1.5 to about 2.5, or about 1.5 to about 2.0 (including all derivable ranges and values ​​therebetween).

[0128] According to embodiments of the present disclosure, a population of monocotyledonous plant embryo explants may be contacted with a first germination-inducing medium and cultured for about 2 to about 14 days, about 4 to about 12 days, about 5 to about 10 days, or about 6 to about 8 days (including all derivable ranges therebetween). In some embodiments, the monocotyledonous plant embryo explants are contacted with a first germination-inducing medium and cultured for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days (or about 1 week), about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days (or about 2 weeks) (including all derivable ranges therebetween). In further embodiments, monocotyledonous embryo explants may be contacted with the first germination-inducing medium and cultured at a temperature ranging from about 20° C. to about 30° C., about 22° C. to about 28° C., about 25° C. to about 30° C., about 25° C. to about 29° C., or about 25° C. to about 28° C. (including all derivable ranges therebetween). According to specific embodiments, monocotyledonous embryo explants may be contacted with the first germination-inducing medium and cultured at a temperature ranging from about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., or about 30° C. (including all derivable ranges therebetween). According to aspects of the present disclosure, monocotyledonous plant embryo explants can be contacted with a first germination induction medium and cultured at an elevated temperature that can be in the range of about 30°C to about 40°C, about 30°C to about 38°C, about 30°C to about 36°C, about 30°C to about 35°C, about 31°C to about 40°C, about 31°C to about 38°C, about 31°C to about 36°C, about 31°C to about 35°C, about 32°C to about 40°C, about 32°C to about 38°C, about 32°C to about 36°C, about 32°C to about 35°C, about 33°C to about 40°C, about 33°C to about 38°C, about 33°C to about 36°C, or about 33°C to about 35°C (including all derivable ranges therebetween). According to further embodiments, monocotyledonous embryo explants may be contacted with a first germination induction medium and cultured at an elevated temperature of about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., or about 40° C. (including all derivable ranges therebetween). A selective agent may generally be absent from the first germination induction medium, although the first germination induction medium may alternatively include a selective agent.

[0129] In another aspect, contacting a monocotyledonous seed embryo explant with a first germination induction medium and culturing it at an elevated temperature, for example, at a temperature in the range of about 30°C to about 40°C, for about one week may improve transformation during the first germination induction step compared to contacting the explant with a first germination induction medium and culturing it at a lower temperature, for example, at a temperature in the range of about 20°C to about 30°C.

[0130] The first germination induction step can also be carried out under various lighting conditions. While some illumination is generally used, all or part of the first germination induction step can alternatively be carried out in the dark. According to some embodiments, the first germination induction step is carried out under conditions where the average or set light intensity of photosynthetically active radiation (PAR) is about 0 μE / m 2 ·s~approx. 200μE / m 2 ·s, 20μE / m 2 ·s~approx. 200μE / m 2 ·s, 20μE / m 2 ·s~approx. 180μE / m 2 ·s, 30μE / m 2 ·s~approx. 180μE / m 2 ·s, 50μE / m 2 ·s~approx. 180μE / m 2 ·s, 50μE / m 2 ·s~approx. 150μE / m 2 ·s, 60μE / m 2 ·s~approx. 150μE / m 2 ·s, 70μE / m 2 ·s~approx. 140μE / m 2 ·s, 80μE / m 2 ·s~approx. 130μE / m 2 ·s, or 90 μE / m 2 ·s~approx. 120μE / m 2 According to a further embodiment, the first germination induction step may be carried out at an average or set light intensity of photosynthetically active radiation (PAR) in the range of about 0 μE / m 2 ·s, approximately 10μE / m 2 ·s, approx. 20μE / m 2 ·s, approx. 30μE / m 2 ·s, approx. 40μE / m 2 ·s, approx. 50μE / m 2 ·s, approximately 60μE / m2 ·s, approx. 70μE / m 2 ·s, approx. 80μE / m 2 ·s, approximately 90μE / m 2 ·s, approximately 100μE / m 2 ·s, approximately 110μE / m 2 ·s, approximately 120μE / m 2 ·s, approximately 130μE / m 2 ·s, approximately 140μE / m 2 ·s, approximately 150μE / m 2 ·s, approximately 160μE / m 2 ·s, approximately 170μE / m 2 ·s, approx. 180μE / m 2 ·s, approximately 190μE / m 2 ·s, or approximately 200 μE / m 2 In a specific embodiment, different amounts of light / dark cycles may be used during the first germination induction step, which may include illumination with lengths of time from about 0 hours to about 24 hours of light, from about 2 hours to about 22 hours of light, from about 4 hours to about 20 hours of light, from about 8 hours to about 20 hours of light, from about 12 hours to about 20 hours of light, and from about 16 hours to about 20 hours of light, each of which has a corresponding amount of relative darkness for a corresponding length of time based on the length of a 24-hour day.

[0131] According to some embodiments, the amount of light and dark cycles during the first germination induction step is about 0 hours light and about 24 hours dark, about 1 hour light and about 23 hours dark, about 2 hours light and about 22 hours dark, about 3 hours light and about 21 hours dark, about 4 hours light and about 20 hours dark, about 5 hours light and about 19 hours dark, about 6 hours light and about 18 hours dark, about 7 hours light and about 17 hours dark, about 8 hours light and about 16 hours dark, about 9 hours light and about 15 hours dark, about 10 hours light and about 14 hours dark, about 11 hours light and about 1 The period may be 3 hours of darkness, about 12 hours of light and about 12 hours of darkness, about 13 hours of light and about 11 hours of darkness, about 14 hours of light and about 10 hours of darkness, about 15 hours of light and about 9 hours of darkness, about 16 hours of light and about 8 hours of darkness, about 17 hours of light and about 7 hours of darkness, about 18 hours of light and about 6 hours of darkness, about 19 hours of light and about 5 hours of darkness, about 20 hours of light and about 4 hours of darkness, about 21 hours of light and about 3 hours of darkness, about 22 hours of light and about 2 hours of darkness, about 23 hours of light and about 1 hour of darkness, or about 24 hours of light and about 0 hours of darkness.

[0132] According to embodiments of the present disclosure, the monocotyledonous plant embryo explants of the population may be contacted with a second (or expanded) germination induction medium and cultured for about 4 days to about 28 days, about 4 days to about 25 days, about 4 days to about 21 days, about 5 days to about 25 days, about 5 days to about 23 days, about 7 days to about 21 days, about 5 days to about 15 days, about 7 days to about 14 days, about 12 days to about 23 days, or about 14 days to about 21 days (including all derivable ranges therebetween). In some embodiments, monocotyledonous embryo explants may be contacted with the second (or expanded) germination-inducing medium and cultured for about 4 days, about 5 days, about 6 days, about 7 days (or about 1 week), about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days (or about 2 weeks), about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days (or about 3 weeks), about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, or about 28 days (or about 4 weeks), including all derivable ranges therebetween. In further embodiments, monocotyledonous seed embryo explants may be contacted with the second (or expanded) germination-inducing medium and cultured at a temperature ranging from about 20°C to about 32°C, about 20°C to about 30°C, about 22°C to about 28°C, about 25°C to about 30°C, about 25°C to about 29°C, about 26°C to about 29°C, about 25°C to about 28°C, or about 27°C to about 28°C (including all derivable ranges therebetween). According to certain embodiments, monocotyledonous seed embryo explants may be contacted with the second (or expanded) germination-inducing medium and cultured at a temperature ranging from about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, or about 30°C (including all derivable ranges therebetween). In certain embodiments, monocotyledonous seed embryo explants may be contacted with a first germination induction medium and cultured at a temperature ranging from about 20° C. to about 30° C. or at an elevated temperature ranging from about 30° C. to about 40° C. for a period ranging from about 2 days to about 14 days, and then subsequently contacted with a second (or extended) germination induction medium and cultured at a temperature ranging from about 20° C. to about 32° C. for a period ranging from about 4 days to about 28 days. The second (or extended) germination induction medium may also contain a selection agent.In a specific embodiment, culturing the explant in contact with the second germination-inducing medium may improve transformation compared to culturing the explant in contact with only the first germination-inducing medium.

[0133] The second (or extended) germination induction step can also be carried out under various lighting conditions. Some illumination is generally used during the second (or extended) germination induction step. According to some embodiments, the second (or extended) germination induction step is carried out under conditions where the average or set light intensity of photosynthetically active radiation (PAR) is about 30 μE / m 2 ·s~approx. 200μE / m 2 ·s, 30μE / m 2 ·s~approx. 180μE / m 2 ·s, 50μE / m 2 ·s~approx. 180μE / m 2 ·s, 50μE / m 2 ·s~approx. 150μE / m 2 ·s, 60μE / m 2 ·s~approx. 150μE / m 2 ·s, 70μE / m 2 ·s~approx. 140μE / m 2 ·s, 80μE / m 2 ·s~approx. 130μE / m 2 ·s, or 90 μE / m 2 ·s~approx. 120μE / m 2 According to a further embodiment, the second (or extended) germination induction step may be carried out at an average or set light intensity of photosynthetically active radiation (PAR) in the range of about 10 μE / m 2 ·s, approx. 20μE / m 2 ·s, approx. 30μE / m 2 ·s, approx. 40μE / m 2 ·s, approx. 50μE / m 2 ·s, approximately 60μE / m 2 ·s, approx. 70μE / m 2 ·s, approx. 80μE / m 2 ·s, approximately 90μE / m 2 ·s, approximately 100μE / m 2 ·s, approximately 110μE / m 2 ·s, approximately 120μE / m 2 ·s, approximately 130μE / m 2·s, approximately 140μE / m 2 ·s, approximately 150μE / m 2 ·s, approximately 160μE / m 2 ·s, approximately 170μE / m 2 ·s, approx. 180μE / m 2 ·s, approximately 190μE / m 2 ·s, or approximately 200 μE / m 2 In certain embodiments, different amounts of light / dark cycles may be used during the second (or expanded) budding induction step, which may include illumination present at lengths of about 2 hours to about 24 hours of light, about 2 hours to about 22 hours of light, about 4 hours to about 20 hours of light, about 8 hours to about 20 hours of light, about 12 hours to about 20 hours of light, and about 16 hours to about 20 hours of light, each of which has a corresponding amount of relative darkness for a corresponding length of time based on the length of a 24-hour day.

[0134] According to some embodiments, the amount of light / dark cycle during the second (or expanded) budding induction step is about 2 hours light and about 22 hours dark, about 3 hours light and about 21 hours dark, about 4 hours light and about 20 hours dark, about 5 hours light and about 19 hours dark, about 6 hours light and about 18 hours dark, about 7 hours light and about 17 hours dark, about 8 hours light and about 16 hours dark, about 9 hours light and about 15 hours dark, about 10 hours light and about 14 hours dark, about 11 hours light and about 13 hours dark, about 12 hours light, and about 12 hours of darkness, about 13 hours of light and about 11 hours of darkness, about 14 hours of light and about 10 hours of darkness, about 15 hours of light and about 9 hours of darkness, about 16 hours of light and about 8 hours of darkness, about 17 hours of light and about 7 hours of darkness, about 18 hours of light and about 6 hours of darkness, about 19 hours of light and about 5 hours of darkness, about 20 hours of light and about 4 hours of darkness, about 21 hours of light and about 3 hours of darkness, about 22 hours of light and about 2 hours of darkness, about 23 hours of light and about 1 hour of darkness, or about 24 hours of light and about 0 hours of darkness.

[0135] According to some embodiments, the methods described herein include achieving a lower density of visible or total monocotyledonous seed embryo explant(s) in the second (or expanded) germination induction medium per container or plate, which may be achieved by transferring cultured monocotyledonous seed embryo explant(s) from a predetermined number of germination induction plate(s) or container(s) to a relatively larger number of expanded germination induction plate(s) or container(s). As described herein, transformation of male corn lines, and possibly other monocotyledonous germplasm, can often be difficult or less efficient than transformation of female corn lines or other monocotyledonous germplasm. Compared to expanded germination induction plates for female corn embryo explants, expanded germination induction plates for male corn embryo explants can frequently exhibit contamination and tissue necrosis. According to some embodiments, reducing the density of visible or total explants per expansion induction plate or container may improve the frequency of transformation, shoot, and / or regeneration of monocotyledonous or maize embryo explants, or genetically modified plants of particular monocotyledonous plants or maize lines that are more resistant to transformation and / or regeneration, for example, particular male germplasm maize lines. According to some embodiments, monocotyledonous seed embryo explants can be transferred from one or more expansion induction plate(s) or container(s) to a relatively smaller or fewer number of expansion induction plate(s) or container(s), for example, at a 2:1 or 1:1 ratio in terms of number of expansion induction plate(s) or container(s):number of expansion induction plate(s) or container(s), although the number of explants per expansion induction plate may be overcrowded.

[0136] According to some embodiments, monocotyledonous seed embryo explants can be transferred from one or more germination induction plate(s) or container(s) to a relatively larger or greater number of expanded germination induction plate(s) or container(s), for example, in a ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 in terms of number of germination induction plate(s) or container(s):number of expanded germination induction plate(s) or container(s).According to some embodiments, the saturation rate is from 10 to about 2,000, from about 20 to about 2,000, from about 30 to about 2,000, from about 40 to about 2,000, from about 50 to about 2,000, from about 10 to about 1,500, from about 10 to about 1,000, from about 10 to about 1,500, from about 10 to about 1,000, from about 10 to about 750, from about 10 to about 500, from about 10 to about 600, from about 10 to about 500, from about 10 to about 400, from about 10 to about 300, from about 50 to about 200, from about 20 to about 2,000, from about 20 to about 1,500, from about 20 to about 1 ,000, about 20 to about 1,500, about 20 to about 1,000, about 20 to about 750, about 20 to about 500, about 20 to about 600, about 20 to about 500, about 20 to about 400, about 20 to about 300, about 20 to about 200, about 30 to about 2,000, about 30 to about 1,500, about 30 to about 1,000, about 30 to about 1,500, about 30 to about 1,000, about 30 to about 750, about 30 to about 500, about 30 to about 600, about 30 to about 500, about 30 to about 400, about 30 to about 300, about 30 to about 200, about 40 to about 2,000, about 40 to about 1,500, about 40 to about 1,000, about 40 to about 1,500, about 40 to about 1,000, about 40 to about 750, about 40 to about 500, about 40 to about 600, about 40 to about 500, about 40 to about 400, about 40 to about 300, about 40 to about 200, about 50 to about 2,000, about 50 to about 1,500, about 50 to about 1,000, about 50 to about 1,500, about 50 to about 1,000, about 50 to about 750, about 50 to about 500, about 50 to about 600, A predetermined number of visible (or total) monocotyledonous seed embryo explants, such as in the range of about 50 to about 500, about 50 to about 400, about 50 to about 300, or about 50 to about 200, can be transferred from the germination induction plate or medium to a relatively larger or greater number of expanded germination induction plate(s) or container(s), for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more expanded germination induction plate(s) or container(s).

[0137] According to some embodiments, the number of visible (or total) monocotyledonous seed embryo explants transferred or added to the expansion germination induction plate(s) or container(s) is about 300 or less seed embryo explants per plate, about 200 or less seed embryo explants per plate, about 150 or less seed embryo explants per plate, about 100 or less seed embryo explants per plate, about 75 or less seed embryo explants per plate, about 50 or less seed embryo explants per plate, about 30 or less seed embryo explants per plate, about 40 or less seed embryo explants per plate, about 50 or less seed embryo explants per plate, about 60 or less seed embryo explants per plate, about 75 or less seed embryo explants per plate, about 50 or less seed embryo explants per plate, about 60 or less seed embryo explants per plate, about 7 ... The density of seed embryo explants below or about 10 to about 300 seed embryo explants per plate, about 10 to about 250 seed embryo explants per plate, about 10 to about 200 seed embryo explants per plate, about 10 to about 150 seed embryo explants per plate, about 10 to about 100 seed embryo explants per plate, about 10 to about 75 seed embryo explants per plate, about 10 to about 50 seed embryo explants per plate, about 25 to about 300 seed embryo explants per plate, about 25 to about 25 0 seed embryo explants, about 25 to about 200 seed embryo explants per plate, about 25 to about 150 seed embryo explants per plate, about 25 to about 100 seed embryo explants per plate, about 25 to about 75 seed embryo explants per plate, about 25 to about 50 seed embryo explants per plate, about 50 to about 300 seed embryo explants per plate, about 50 to about 250 seed embryo explants per plate, about 50 to about 200 seed embryo explants per plate, about 50 to about 150 seed embryo explants per plate The density range can be about 50 to about 100 seed embryo explants per plate, about 50 to about 75 seed embryo explants per plate, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, or about 300 (including all derivable ranges and values ​​therebetween). For these density values ​​and ranges, the surface area of ​​each expanded germination induction plate is approximately 11.9 square inches (in 2 ) or 76.8 square centimeters (cm 2Therefore, all of the above density values ​​and ranges for seed embryo explants per plate can be easily converted to density values ​​and ranges for seed embryo explants per expanded germination-inducing surface area (e.g., a density of 100 seed embryo explants per plate divided by the surface area per plate yields approximately 8.4 seed embryo explants per square inch (in 2 ) or approximately 1.3 seed embryo explants per square centimeter (cm 2 ) yields a density of seed embryo explants per expanded germination induction surface area, and similar conversions can be easily made for other density values ​​and ranges.) The value and range of density of seed embryo explants per expanded germination induction surface area is a more universal definition for the density of seed embryo explants in a variety of different expanded germination induction plates or containers, each of which may have a different surface area.

[0138] In some embodiments, the number of visible (or total) monocotyledonous seed embryo explants transferred or added to the expansion germination induction plate(s) or container(s) is determined by the square centimeter (cm) of the expansion germination induction surface area. 2 , about 3.9, about 3.8, about 3.7, about 3.6, about 3.5, about 3.4, about 3.3, about 3.2, about 3.1, about 3.0, about 2.9, about 2.8, about 2.7, about 2.6, about 2.5, about 2.4, about 2.3, about 2.2, about 2.1, about 2.0, about 1.9, about 1.8, about 1.7, about 1.6, about 1.5, about 1.4, about 1.3, about 1.2, about 1.1, about 1.0, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, or about 0.1 or less embryo explants per 1000 cells / well (including all derivable ranges and values ​​therebetween). In certain embodiments, the number of visible (or total) monocotyledonous seed embryo explants transferred or added to the expansion germination induction plate(s) or container(s) is determined by the square centimeter (cm) of the expansion germination induction surface area. 2) about 0.1 to about 6.0, about 0.1 to about 5.5, about 0.1 to about 5.0, about 0.1 to about 4.5, about 0.1 to about 4.0, about 0.1 to about 3.5, about 0.1 to about 3.0, about 0.1 to about 2.5, about 0.1 to about 2.0, about 0.1 to about 1.5, about 0.1 to about 1.0, about 0.1 to about 0.5, About 0.2 to about 6.0, about 0.2 to about 5.5, about 0.2 to about 5.0, about 0.2 to about 4.5, about 0.2 to about 4.0, about 0.2 to about 3.5, about 0.2 to about 3.0, about 0.2 to about 2.5, about 0.2 to about 2.0, about 0.2 to about 1.5, about 0.2 to about 1.0, about 0.2 to about 0.5, about 0.3 to About 6.0, about 0.3 to about 5.5, about 0.3 to about 5.0, about 0.3 to about 4.5, about 0.3 to about 4.0, about 0.3 to about 3.5, about 0.3 to about 3.0, about 0.3 to about 2.5, about 0.3 to about 2.0, about 0.3 to about 1.5, about 0.3 to about 1.0, about 0.3 to about 0.5, about 0.4 to about 6.0 , about 0.4 to about 5.5, about 0.4 to about 5.0, about 0.4 to about 4.5, about 0.4 to about 4.0, about 0.4 to about 3.5, about 0.4 to about 3.0, about 0.4 to about 2.5, about 0.4 to about 2.0, about 0.4 to about 1.5, about 0.4 to about 1.0, about 0.4 to about 0.5, about 0.5 to about 6.0, about 0.5 to about 5.5, about 0.5 to about 5.0, about 0.5 to about 4.5, about 0.5 to about 4.0, about 0.5 to about 3.5, about 0.5 to about 3.0, about 0.5 to about 2.5, about 0.5 to about 2.0, about 0.5 to about 1.5, about 0.5 to about 1.0, about 0.6 to about 6.0, about 0.6 to about 5.5, about 0.6 to about 5. 0, about 0.6 to about 4.5, about 0.6 to about 4.0, about 0.6 to about 3.5, about 0.6 to about 3.0, about 0.6 to about 2.5, about 0.6 to about 2.0, about 0.6 to about 1.5, about 0.6 to about 1.0, about 0.7 to about 6.0, about 0.7 to about 5.5, about 0.7 to about 5.0, about 0.7 to about 4.5, about 0. 7 to about 4.0, about 0.7 to about 3.5, about 0.7 to about 3.0, about 0.7 to about 2.5, about 0.7 to about 2.0, about 0.7 to about 1.5, about 0.7 to about 1.0, about 0.8 to about 6.0, about 0.8 to about 5.5, about 0.8 to about 5.0, about 0.8 to about 4.5, about 0.8 to about 4.0, about 0.8 to about 3. 5, about 0.8 to about 3.0, about 0.8 to about 2.5, about 0.8 to about 2.0, about 0.8 to about 1.5, about 0.8 to about 1.0, about 0.9 to about 6.0, about 0.9 to about 5.5, about 0.9 to about 5.0, about 0.9 to about 4.5, about 0.9 to about 4.0, about 0.9 to about 3.5, about 0.9 to about 3.0, about 0.9 to about 2.5, about 0.9 to about 2.0, about 0.9 to about 1.5, about 0.9 to about 1.0, about 1.0 to about 6.0, about 1.0 to about 5.5, about 1.0 to about 5.0, about 1.0 to about 4.5, about 1.0 to about 4.0, about 1.0 to about 3.5, about 1.0 to about 3.0, about 1.0 to about 2.5, about 1.0 to about 2.0, about 1.0 to about 1 The density may be in the range of embryo explants from about 0.5, about 1.5 to about 6.0, about 1.5 to about 5.5, about 1.5 to about 5.0, about 1.5 to about 4.5, about 1.5 to about 4.0, about 1.5 to about 3.5, about 1.5 to about 3.0, about 1.5 to about 2.5, or about 1.5 to about 2.0 (including all derivable ranges and values ​​therebetween).

[0139] Without being bound by theory, the budding induction step(s) may result in differentiation and / or proliferation of the cells of the explant, causing the explant to form multiple buds, which may then be regenerated into plants. According to some preferred embodiments, the first auxin and cytokinin are different from the second auxin and cytokinin and affect the formation and development of multiple buds through somewhat different activities and / or mechanisms of action. Without being bound by theory, the first budding induction step may cause the cells of the explant to differentiate into multiple buds, while the second (or expansion) budding induction step may promote the proliferation or expansion of the multiple buds, resulting in more compact or robust multiple budding explant(s) for further culture and regeneration into plant(s). The inclusion of the second (or expansion) budding induction step may have the additional benefit of reducing chimerism in the resulting genetically modified plant or plant part. According to certain embodiments, culturing monocotyledonous seed embryo explants on a first germination induction medium followed by culturing on a second (or extended) germination induction medium may result in reduced chimerism in the regenerated plants compared to monocotyledonous seed embryo explants cultured on the first germination induction medium and regenerated without culturing on the second (or extended) germination induction medium.

[0140] E. Regeneration of Transformed or Edited Plants In another aspect of the present disclosure, a plurality of genetically modified plants or plant parts are regenerated from a population of cultured embryo explants by contacting them with a regeneration medium. According to this embodiment, the regeneration medium may contain various standard culture medium or solution components or ingredients, such as basal salts, macronutrients, micronutrients, sugars, antibiotics, and / or vitamins. The regeneration medium may be free of auxin or cytokinin, which may be particularly applicable to the regeneration of or from monocotyledonous embryo explants. Alternatively, auxin and / or cytokinin may be present in the regeneration medium, which may in some embodiments be particularly applicable to the regeneration of or from dicotyledonous embryo explants, depending on the dicotyledonous plant species. The regeneration medium may typically contain at least one selection agent that may correspond to a selection marker present in the heterologous polynucleotide molecule. The regeneration medium may be a solid, semi-solid, or liquid medium, but typically is a solid medium. A solid medium may include a gelling or polymerizing agent or component, such as agarose, that can solidify to form a solid medium. As used herein, the term "regeneration" refers to the process of growing a plant or part thereof from one or more plant cells or tissues of an explant or any progeny generation of those cells, and the term "regeneration medium" refers to a plant tissue culture medium formulated for regenerating a plant from an explant. In some embodiments, regeneration or a regeneration step may refer to one or more regeneration step(s) that may include culturing an explant or cultured explant in contact with two or more regeneration media. These two or more regeneration media may be the same or different regeneration medium(s). Explants of a population may, in some embodiments, be subcultured and transferred from a first regeneration medium to a second regeneration medium, and optionally to a third regeneration medium.

[0141] According to some embodiments, the methods described herein include achieving a lower density of visible (or total) monocotyledonous seed embryo explant(s) in the regeneration medium per container or plate, which may be achieved by transferring cultured monocotyledonous seed embryo explant(s) from a predetermined number of expansion induction plate(s) or container(s) to a larger number of regeneration plate(s) or container(s). As described herein, transformation of male corn lines, and possibly other monocotyledonous germplasm, can often be difficult or less efficient than transformation of female corn lines or other monocotyledonous germplasm. Compared to regeneration plates for female corn embryo explants, regeneration plates for male corn embryo explants can frequently exhibit contamination and tissue necrosis. According to some embodiments, lowering the density of visible or total explants per regeneration plate or container may improve the frequency of transformation, shoots, and / or regeneration of monocotyledonous or maize embryo explants, or genetically modified plants of particular monocotyledonous plants or maize lines that are more resistant to transformation and / or regeneration of genetically modified plants, e.g., particular male germplasm maize lines.

[0142] According to some embodiments, monocotyledonous seed embryo explants can be transferred from one or more expansion induction plates(s) or container(s) to a relatively small number of regeneration plates(s) or container(s), for example, at a ratio of 3:1 or 2:1 in terms of the number of expansion induction plates(s) or container(s):number of regeneration plates(s) or container(s), although the number of explants per regeneration plate may be overcrowded. After expansion induction, transferring the explants to solid regeneration medium at a ratio of one regeneration plate for every three expansion induction plates may increase contamination and tissue necrosis during regeneration, leading to loss of transformed tissue and regenerated plants.

[0143] According to some embodiments, monocotyledonous seed embryo explants can be transferred from one or more expansion germination induction plate(s) or container(s) to a larger or greater number of regeneration plate(s) or container(s), for example, in a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 in terms of number of expansion germination induction plate(s) or container(s):number of regeneration plate(s) or container(s). According to some embodiments, a predetermined number of visible (or total) monocotyledonous seed embryo explants, such as in the range of about 10 to about 400, about 10 to about 300, about 10 to about 250, about 10 to about 200, about 10 to about 175, about 10 to about 150, about 10 to about 125, about 10 to about 100, about 10 to about 75, about 10 to about 50, or about 10 to about 25 (including all derivable ranges and values ​​therebetween), can be transferred from the expansion germination induction plate or medium to regeneration plate(s) or container(s), e.g., 1...

Claims

1. A method for genetically modifying a population of plant embryo explants that includes a mixture of different genotypes, This involves introducing heterologous polynucleotide molecules collectively into at least two embryonic explants of the aforementioned population, wherein each of the at least two plant embryonic explants contains meristematic tissue. The method wherein the at least two embryonic explants comprise embryonic explants of at least two different plant genotypes.

2. The method according to claim 1, a) Whether the aforementioned group is defined as a group of monocotyledonous plant embryo explants, b) The population is defined as a population of exoplants of maize, wheat, rice, barley, sorghum, or turfgrass, or c) The method wherein the at least two different plant genotypes include at least two distinguishable genetic markers.

3. The method according to claim 1, further comprising identifying at least two distinguishable genotypes and at least two plant tissues containing the heterologous polynucleotide molecules.

4. The method according to claim 1, a) The heterogeneous polynucleotide molecule comprises an expression cassette, the expression cassette comprising a selection marker gene, a screenable marker gene, a target gene, a nucleotide sequence encoding a site-specific nuclease, or a nucleotide sequence encoding a guide RNA, b) The collectively introduced means introducing the heterologous polynucleotide molecules into at least two explants of the population via bioristic particle delivery-mediated transformation, bacterial-mediated transformation, or Rhizobiales bacterial-mediated transformation, c) The aforementioned collectively introduced includes introducing the heterologous polynucleotide molecules via Agrobacterium-mediated transformation, d) The collectively introducing the heterologous polynucleotide molecules includes inoculating the at least two embryonic explants with an inoculation medium containing Rhizobiales bacteria capable of transforming the at least two embryonic explants with the heterologous polynucleotide molecules, e) The collectively introduced method includes introducing the heterologous polynucleotide molecules into the at least two explants of the population via particle bombardment, f) The aforementioned collectively introduced includes introducing site-specific nucleases into at least two embryonic explants of the population, g) The heterogeneous polynucleotide molecule includes a guide RNA molecule, and the collective introduction includes introducing the site-specific nuclease into the at least two embryonic explants of the population, h) The collectively introduced includes introducing at least two heterologous polynucleotides into at least two explants of the population, wherein the at least two heterologous polynucleotides are different, i) Whether the aforementioned introduction in a single package includes site-directed incorporation of the heterogeneous polynucleotide or its fragments, j) The heterogeneous polynucleotide molecule contains or encodes guide RNA, k) Whether the aforementioned introduction in a single batch includes simultaneously introducing the heterogeneous polynucleotide molecules into the population, l) Whether the group of embryonic explants is a group of dicotyledonous plant embryonic explants, m) Whether the group of embryonic explants is a group of soybean, cotton, or canola embryonic explants, n) The collectively introduced method includes introducing the heterologous polynucleotide molecules into the group of embryonic explants while the group is present together in a single container, o) The group is a group of dried, dried, dried excised, moist excised, or moist seed embryo explants, or p) The method wherein the population is a population of mature or immature seed embryo explants.

5. The aforementioned Rhizobiales bacteria, a) Bacteria of the order Rhizobiaceae, Phyllobacteriaceae, Brucellaceae, Bradyrhizobiaceae, and Xanthobacteriaceae, or b) Bacteria of Agrobacterium, Rhizobium, Sinorhizobium, Mesorhizobium, Phyllobacterium, Ochrobactrum, Bradyrhizobium, and Azorhizobium The method according to claim 4, selected from the group consisting of the following.

6. The method according to claim 4, a) The population in contact with the inoculation medium is subjected to force treatment, or the heterologous polynucleotide molecules are subjected to force treatment before being introduced collectively. b) The embryo explant in contact with the inoculation medium is subjected to force treatment, the force treatment including gravity treatment in the range of approximately 3,000 × g to approximately 6,000 × g, approximately 3,500 × g to approximately 5,000 × g, or approximately 3,500 × g to approximately 4,500 × g, c) The method further comprises bringing the at least two embryonic explants into contact with a co-culture medium and co-culturing them with the Rhizobiales bacteria, d) The group of embryonic explants is a group of monocotyledonous plant seed embryonic explants, and the method further includes a) co-culturing at least two of the embryonic explants of the group in contact with a co-culture medium at a density of about 9.1 or fewer embryonic explants per square centimeter (cm²) of co-culture surface area, or b) co-culturing at least two of the embryonic explants of the group in contact with a co-culture medium for a period ranging from about 6 to about 8 days. e) The site-specific nuclease is a ribonucleoprotein, and the ribonucleoprotein includes the site-specific nuclease and the guide RNA molecule, f) The group of embryonic explants is a group of dicotyledonous plant embryonic explants, and the co-culture medium contains at least one cytokinin or lipoic acid, g) The group of embryonic explants is a group of dicotyledonous plant embryonic explants, and the co-culture medium contains at least one cytokinin or lipoic acid, and the at least one cytokinin is selected from the group consisting of 6-benzylaminopurine (BAP), thidiazurone (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-toporin), h) The group of embryonic explants is a group of dicotyledonous plant embryonic explants, the co-culture medium contains at least one cytokinin or lipoic acid, and the concentration of the cytokinin in the co-culture medium is about 0.1 mg / L to about 50 mg / L. i) The group of embryonic explants is a group of dicotyledonous plant embryonic explants, and the co-culture medium contains at least one cytokinin or lipoic acid, i) the at least one cytokinin is thidiazurone (TDZ) and the concentration of thidiazurone (TDZ) in the co-culture medium is about 0.1 mg / L to about 10 mg / L, or ii) the at least one cytokinin is 6-benzylaminopurine (BAP) and the concentration of 6-benzylaminopurine (BAP) in the co-culture medium is about 0.1 mg / L to about 15 mg / L, or j) The method wherein the group of embryonic explants is a group of dicotyledonous plant embryonic explants, the co-culture medium contains at least one cytokinin or lipoic acid, and the concentration of lipoic acid in the co-culture medium is about 0.1 mg / L to about 500 mg / L.

7. The at least two heterologous polynucleotides include a first heterologous polynucleotide comprising a first expression cassette and a second heterologous polynucleotide comprising a second expression cassette, wherein the first expression cassette comprises a first selection marker gene, a first screenable marker gene, a first target gene, a nucleotide sequence encoding a first site-specific nuclease, or a nucleotide sequence encoding a first guide RNA. The second expression cassette includes a second selection marker gene, a second screenable marker gene, a second target gene, a nucleotide sequence encoding a second site-specific nuclease, or a nucleotide sequence encoding a second guide RNA. The method according to claim 4.

8. The heterogeneous polynucleotide molecule comprises a first expression cassette and a second expression cassette, The first expression cassette comprises a first selection marker gene, a first screenable marker gene, a first target gene, a nucleotide sequence encoding a first site-specific nuclease, or a nucleotide sequence encoding a first guide RNA. The second expression cassette includes a second select gene, a second screenable marker gene, a second target gene, a nucleotide sequence encoding a second site-specific nuclease, or a nucleotide sequence encoding a second guide RNA. The method according to claim 1.

9. The method according to claim 1, further comprising culturing the at least two embryonic explants in contact with a first budding-inducing medium containing a first auxin and a first cytokinin.

10. The method according to claim 9, a) The first budding-inducing medium contains a high cytokinin-to-auxin ratio, b) The first auxin in the first budding medium is 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid or p-chlorophenoxyacetic acid (4-CPA or pCPA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,3,5-triiodobenzoic acid (TIB) A) selected from the group consisting of phenylacetic acid (PAA) and 3,6-dichloro-2-methoxybenzoic acid (dicamba), or the first cytokinin in the first budding-inducing medium is selected from the group consisting of 6-benzylaminopurine (BAP), thidiazurone (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-toporin), c) The concentration of the first auxin in the first budding-inducing medium is approximately 0.02 mg / L to approximately 25 mg / L or approximately 1 mg / L to approximately 2 mg / L, d) The concentration of the first cytokinin in the first budding-inducing medium is in the range of about 0.1 mg / L to about 50 mg / L, or e) The method wherein the group of embryonic explants is a group of monocotyledonous plant seed embryonic explants, and the method further comprises culturing the at least two embryonic explants in contact with the first budding-inducing medium at a density of about 3.9 embryonic explants or less per square centimeter (cm²) of the first budding-inducing surface area.

11. The method according to claim 9, further comprising culturing the at least two embryonic explants in contact with a second budding-inducing medium containing the first or second auxin and the first or second cytokinin.

12. The method according to claim 11, a) The embryo explant is cultured in contact with the second budding-inducing medium at a temperature in the range of approximately 20°C to approximately 32°C, approximately 25°C to approximately 29°C, or approximately 27°C to approximately 28°C, b) The second budding-inducing medium is i) A high cytokinin-to-auxin ratio; ii) The first auxin and the first cytokinin; iii) The first auxin and the second cytokinin; iv) the second auxin and the first cytokinin; or v) The second auxin and the second cytokinin Does it include, c) The group of embryonic explants is a group of monocotyledonous plant seed embryonic explants, and the method further includes culturing the at least two embryonic explants in contact with the second budding-inducing medium at a density of about 2.6 or fewer embryonic explants per square centimeter (cm²) of the second budding-inducing surface area, d) The first auxin or the second auxin in the second budding-inducing medium is selected from the group consisting of 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid or p-chlorophenoxyacetic acid (4-CPA or pCPA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,3,5-triiodobenzoic acid (TIBA), phenylacetic acid (PAA), and 3,6-dichloro-2-methoxybenzoic acid (dicamba), e) The first cytokinin or the second cytokinin in the second budding-inducing medium is selected from the group consisting of 6-benzylaminopurine (BAP), thidiazurone (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-toporin), f) The concentration of the first cytokinin or the second cytokinin in the second budding-inducing medium is in the range of approximately 0.1 mg / L to approximately 1 mg / L, approximately 0.1 mg / L to approximately 5 mg / L, approximately 0.1 mg / L to approximately 50 mg / L, approximately 0.1 mg / L to approximately 25 mg / L, approximately 0.5 mg / L to approximately 25 mg / L, or approximately 2 mg / L to approximately 10 mg / L. g) The concentration of the first auxin or the second auxin in the second budding medium is approximately 0.01 mg / L to approximately 25 mg / L, approximately 0.02 mg / L to approximately 10 mg / L, or approximately 1 mg / L to approximately 2 mg / L, or h) The method wherein the heterogeneous polynucleotide molecule comprises a selection marker gene, the second budding medium comprises a selectant, and the selection marker gene confers resistance to the selectant to the plant.

13. The method according to claim 1, further comprising regenerating or growing a plurality of genetically modified plants or plant parts by contacting at least two embryonic explants or any offspring generations of their cells with a regeneration medium.

14. The method according to claim 13, a) The population is a population of monocotyledonous plant seed embryo explants, and the method further includes regenerating or growing the plurality of genetically modified plants or plant parts by bringing at least two of the embryo explants into contact with the regeneration medium at a density of about 2.6 or fewer embryo explants per square centimeter (cm²) of regeneration surface area, b) The population is a population of monocotyledonous plant seed embryo explants, and the method further includes: regenerating the population of cultured monocotyledonous plant seed embryo explants by contacting them with a first regeneration medium; transferring the population of cultured monocotyledonous plant seed embryo explants or a subset of the population of cultured monocotyledonous plant seed embryo explants to a second regeneration medium; and regenerating the plants or plant parts of the plurality of genetically modified monocotyledonous plants by contacting them with the second regeneration medium. c) The regenerated culture medium has a low salt concentration, d) The heterogeneous polynucleotide molecule contains a selection marker gene, the regeneration medium contains a selectant, and the selection marker gene confers resistance to the selectant to the plant. e) The plurality of genetically modified plants or plant parts include at least one genetic modification, f) The plurality of genetically modified plants or plant parts are at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, and at least or include 350, at least 400, at least 450, or at least 500 different plant genotypes, or 2 to about 600, 2 to about 600, about 10 to about 500, about 15 to about 400, about 20 to about 300, about 25 to about 200, about 10 to about 150, about 10 to about 100, about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, 2 to about 50, 2 to about 40, 2 to about 30, 2 to about 20, 2 to about 15, or 2 to about 10 different plant genotypes, g) Whether the group of embryonic explants is a group of dicotyledonous plant embryonic explants, h) The regeneration medium contains at least one cytokinin, i) The regeneration medium comprises at least one cytokinin, i) the at least one cytokinin is selected from the group consisting of 6-benzylaminopurine (BAP), thidiazurone (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-toporin), ii) the concentration of the cytokinin in the regeneration medium is about 0.1 mg / L to about 50 mg / L, or iii) the at least one cytokinin is zeatin or 6-benzylaminopurine (BAP), and the concentration of the zeatin or 6-benzylaminopurine (BAP) in the regeneration medium is about 0.1 mg / L to about 15 mg / L. j) The method includes bringing the plurality of genetically modified plants or plant parts into contact with the regeneration medium and regenerating or growing them at a temperature of approximately 15°C to approximately 40°C, k) The group of embryo explants is a group of cotton embryo explants, and the method includes bringing the plurality of genetically modified plants or plant parts into contact with the regeneration medium and regenerating or growing them at a temperature of approximately 30°C to approximately 40°C for a first regeneration period, l) The group of embryo explants is a group of cotton embryo explants, and the method includes bringing the plurality of genetically modified plants or plant parts into contact with the regeneration medium and regenerating or growing them at a temperature of approximately 30°C to approximately 40°C for a first regeneration period, wherein the first regeneration period is approximately 1 hour to approximately 14 days. m) The group of embryo explants is a group of cotton embryo explants, and the method includes bringing the plurality of genetically modified plants or plant parts into contact with the regeneration medium and regenerating or growing them at a temperature of approximately 30°C to approximately 40°C for a first regeneration period, and bringing the plurality of genetically modified cotton plants or cotton plant parts into contact with the regeneration medium and regenerating or growing them at a temperature of approximately 20°C to approximately 33°C for a second regeneration period, n) The group of embryo explants is a group of cotton embryo explants, and the method includes bringing the plurality of genetically modified plants or plant parts into contact with the regeneration medium and regenerating or growing them at a temperature of approximately 30°C to approximately 40°C for a first regeneration period, and bringing the plurality of genetically modified cotton plants or cotton plant parts into contact with the regeneration medium and regenerating or growing them at a temperature of approximately 20°C to approximately 33°C for a second regeneration period, wherein the second regeneration period is approximately 7 days to approximately 56 days. o) The method includes contacting the plurality of genetically modified plants or plant parts with the regeneration medium and regenerating or growing them for about 5 to about 70 days or about 14 to about 50 days, p) The group of embryo explants is a group of soybean embryo explants, and the method includes contacting the plurality of genetically modified plants or plant parts with the regeneration medium for about 5 to 70 days to regenerate or grow them, q) The group of embryo explants is a group of cotton embryo explants, and the method includes contacting the plurality of genetically modified plants or plant parts with the regeneration medium for about 14 to about 70 days to regenerate or grow them, r) The group of embryo explants is a group of canola embryo explants, and the method includes contacting the plurality of genetically modified plants or plant parts with the regeneration medium for about 14 to about 70 days to regenerate or grow them, s) The method further includes bringing the plurality of genetically modified plants or plant parts into contact with a second regeneration medium and regenerating or growing them over an extended regeneration period, t) The method further comprises bringing the plurality of genetically modified plants or plant parts into contact with a second regeneration medium and regenerating or growing them over an extended regeneration period, wherein the second regeneration medium comprises at least one auxin, at least one cytokinin, or at least one auxin and at least one cytokinin. u) The method further comprises contacting the plurality of genetically modified plants or plant parts with a second regeneration medium and regenerating or growing them over an extended regeneration period, wherein the second regeneration medium comprises at least one auxin, at least one cytokinin, or at least one auxin and at least one cytokinin, i) the at least one auxin is 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid or ii) At least one cytokinin is selected from the group consisting of p-chlorophenoxyacetic acid (4-CPA or pCPA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,3,5-triiodobenzoic acid (TIBA), phenylacetic acid (PAA), and 3,6-dichloro-2-methoxybenzoic acid (dicamba), or ii) at least one cytokinin is selected from the group consisting of 6-benzylaminopurine (BAP), tidiazurone (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-toporin), v) The method further comprises bringing the plurality of genetically modified plants or plant parts into contact with a second regeneration medium and regenerating or growing them over an extended regeneration period, wherein the second regeneration medium comprises at least one auxin, at least one cytokinin, or at least one auxin and at least one cytokinin, and the concentration of the at least one auxin in the second regeneration medium is about 0.1 mg / L to about 15 mg / L, or the concentration of the at least one cytokinin in the second regeneration medium is about 0.1 mg / L to about 50 mg / L. w) The method further includes bringing the plurality of genetically modified plants or plant parts into contact with a second regeneration medium and regenerating or growing them at a temperature of approximately 15°C to approximately 40°C over an extended regeneration period, x) The method further comprises bringing the plurality of genetically modified plants or plant parts into contact with a second regeneration medium and regenerating or growing them over an extended regeneration period, wherein the extended regeneration period is approximately 7 to 56 days. y) The group of embryo explants is a group of cotton embryo explants, and the method further includes transferring selected portions of the group of genetically modified cotton plants or cotton plant portions to the second regeneration medium before the group of genetically modified cotton plants or cotton plant portions are brought into contact with the second regeneration medium for regeneration or growth, z) The method further includes bringing the plurality of genetically modified plants or plant parts into contact with a first elongation medium and allowing them to regenerate or grow over a first elongation period, aa) The method further comprises bringing the plurality of genetically modified plants or plant parts into contact with a first elongation medium and regenerating or growing them over a first elongation period, wherein the population of embryonic explants is a population of canola embryonic explants, bb) The method further comprises bringing the plurality of genetically modified plants or plant parts into contact with a first elongation medium and regenerating or growing them over a first elongation period, wherein the first elongation medium comprises at least one auxin, at least one cytokinin, or at least one auxin and at least one cytokinin. cc) The method further comprises contacting the plurality of genetically modified plants or plant parts with a first elongation medium to regenerate or grow over a first elongation period, wherein the first elongation medium comprises at least one auxin, at least one cytokinin, or at least one auxin and at least one cytokinin, i) the at least one auxin is 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid or is selected from the group consisting of p-chlorophenoxyacetic acid (4-CPA or pCPA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,3,5-triiodobenzoic acid (TIBA), phenylacetic acid (PAA), and 3,6-dichloro-2-methoxybenzoic acid (dicamba), or ii) at least one cytokinin is selected from the group consisting of 6-benzylaminopurine (BAP), tidiazurone (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-toporin), dd) The method further comprises contacting the plurality of genetically modified plants or plant parts with a first elongation medium to regenerate or grow over a first elongation period, wherein the first elongation medium comprises at least one auxin, at least one cytokinin, or at least one auxin and at least one cytokinin, and the concentration of the at least one auxin in the first elongation medium is about 0.1 mg / L to about 15 mg / L, or the concentration of the at least one cytokinin in the first elongation medium is about 0.1 mg / L to about 50 mg / L. ee) The method further includes bringing the plurality of genetically modified plants or plant parts into contact with a first elongation medium and regenerating or growing them at a temperature of about 15°C to about 40°C over a first elongation period, ff) The method further comprises bringing the plurality of genetically modified plants or plant parts into contact with a first elongation medium and regenerating or growing them over a first elongation period, wherein the first elongation period is approximately 7 to 56 days. gg) The method further includes bringing the plurality of genetically modified plants or plant parts into contact with a first elongation medium to regenerate or grow over a first elongation period, and bringing the plurality of genetically modified plants or plant parts into contact with a second elongation medium to regenerate or grow over a second elongation period, hh) The method further comprises contacting the plurality of genetically modified plants or plant parts with a first elongation medium to regenerate or grow them over a first elongation period, and contacting the plurality of genetically modified plants or plant parts with a second elongation medium to regenerate or grow them over a second elongation period, wherein the second elongation medium comprises at least one auxin, at least one cytokinin, or at least one auxin and at least one cytokinin. ii) The method further comprises contacting the plurality of genetically modified plants or plant parts with a first elongation medium to regenerate or grow them over a first elongation period, and contacting the plurality of genetically modified plants or plant parts with a second elongation medium to regenerate or grow them over a second elongation period, wherein the second elongation medium comprises at least one auxin, at least one cytokinin, or at least one auxin and at least one cytokinin, wherein the at least one auxin is 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) ), selected from the group consisting of naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid or p-chlorophenoxyacetic acid (4-CPA or pCPA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,3,5-triiodobenzoic acid (TIBA), phenylacetic acid (PAA), and 3,6-dichloro-2-methoxybenzoic acid (dicamba), or at least one cytokinin selected from the group consisting of 6-benzylaminopurine (BAP), tidiazurone (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(gamma,gamma-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (meta-toporin), jj) The method further comprises contacting the plurality of genetically modified plants or plant parts with a first elongation medium to regenerate or grow them over a first elongation period, and contacting the plurality of genetically modified plants or plant parts with a second elongation medium to regenerate or grow them over a second elongation period, wherein the second elongation medium comprises at least one auxin, at least one cytokinin, or at least one auxin and at least one cytokinin, and the concentration of the at least one auxin in the second elongation medium is about 0.1 mg / L to about 15 mg / L, or the concentration of the at least one cytokinin in the second elongation medium is about 0.1 mg / L to about 50 mg / L. k) The method further includes bringing the plurality of genetically modified plants or plant parts into contact with a first elongation medium and regenerating or growing them over a first elongation period, and bringing the plurality of genetically modified plants or plant parts into contact with a second elongation medium and regenerating or growing them at approximately 15°C to approximately 40°C over a second elongation period, ll) The method further comprises bringing the plurality of genetically modified plants or plant parts into contact with a first elongation medium to regenerate or grow over a first elongation period, and bringing the plurality of genetically modified plants or plant parts into contact with a second elongation medium to regenerate or grow over a second elongation period, wherein the second elongation period is approximately 7 to 56 days. mm) The plant part includes a shoot, root, or seed, nn) The genetically modified plant or plant part is non-chimera, or oo) The method by which the genetically modified plant or plant part is cultured or regenerated without producing callus tissue culture.

15. The method according to claim 14, a) The first regeneration medium or the second regeneration medium has a low salt concentration, b) The heterogeneous polynucleotide molecule contains a selection marker gene, the first regeneration medium or the second regeneration medium contains a selectant, and the selection marker gene confers resistance to the selectant to the plant. c) The at least one gene modification comprises the incorporation or insertion of the heterologous polynucleotide molecule or fragment thereof into the genome of the plurality of gene modified plants or plant parts, wherein the incorporation or insertion comprises at least one expression cassette or at least one transgene, d) The at least one gene modification includes editing introduced into the genome of the plurality of gene-modified plants or plant parts by genome editing technology using site-specific nucleases or guide RNA molecules, e) The heterogeneous polynucleotide molecule comprises at least one expression cassette, the at least one expression cassette encoding a site-specific nuclease or guide RNA molecule, or f) The method comprising at least two expression cassettes, wherein the heterogeneous polynucleotide molecule comprises a first expression cassette encoding a site-specific nuclease and a second expression cassette encoding a guide RNA molecule.

16. The method according to claim 1, a) The population includes embryonic explants having an internal water content in the range of approximately 3% to approximately 25% before the introduction of the heterologous polynucleotide molecules, b) The population includes an exoplant containing the apical portion of the hypocotyl lacking roots, or the remaining portion of the seed from which the exoplant was prepared is substantially removed from the exoplant. c) The population is a population of monocotyledonous plant embryo explants, and the population of monocotyledonous plant embryo explants is prepared from a population of monocotyledonous plant seeds under conditions in which the embryo explants do not germinate, are viable, and maintain their ability to undergo genetic modification. d) The group comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, and at least The embryo contains an exoplant having at least 450 or 500 different plant genotypes, or 2 to about 750, 2 to about 600, about 10 to about 500, about 15 to about 400, about 20 to about 300, about 25 to about 200, about 10 to about 150, about 10 to about 100, about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, 2 to about 50, 2 to about 40, 2 to about 30, 2 to about 20, 2 to about 15, or 2 to about 10 different plant genotypes. e) The at least two embryo explants of the population contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 different plant genotypes, or including 2 to about 600, 2 to about 600, about 10 to about 500, about 15 to about 400, about 20 to about 300, about 25 to about 200, about 10 to about 150, about 10 to about 100, about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, 2 to about 50, 2 to about 40, 2 to about 30, 2 to about 20, 2 to about 15, or 2 to about 10 different plant genotypes, f) The group comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, and at least 45 0, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, or at least 50,000 embryo explants, or about 2 to about 50,000, about 1 ,000 to approximately 50,000, approximately 1,000 to approximately 40,000, approximately 1,000 to approximately 30,000, approximately 1,000 to approximately 20,000, approximately 1,000 to approximately 10,000, approximately 1,000 to approximately 9,000, approximately 1,000 to approximately 8,000, approximately 1,000 to approximately 7,000, approximately 1,000 to approximately 6,000, approximately 1,000 to approximately 5,000, approximately 1,000 to approximately 4,000, approximately 1,000 to approximately 3,000, approximately 5,000 to approximately 50,000, approximately 5,000 to approximately 40,000, approximately 5,000 to approximately 30,000, approximately 5,000 to approximately 20,000, Contains approximately 5,000 to 10,000, approximately 2 to 1,000, approximately 5 to 900, approximately 5 to 800, approximately 5 to 700, approximately 5 to 600, approximately 5 to 500, approximately 10 to 500, approximately 15 to 400, approximately 20 to 300, approximately 25 to 200, approximately 10 to 150, approximately 10 to 100, approximately 10 to 90, approximately 10 to 80, approximately 10 to 70, approximately 10 to 60, approximately 10 to 50, approximately 10 to 40, approximately 10 to 30, approximately 10 to 20, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 15, or 2 to 10 embryonic explants. g) The population includes at least one exoplant having at least one unidentified genotype before the heterologous polynucleotide molecules are introduced collectively, h) At least two different genotypes of the embryonic explant are known, or i) The method wherein each embryonic explant in the population is an embryonic explant of a known genotype.

17. The method according to claim 1, further comprising identifying at least one genotype of the embryonic explants of the population.

18. The method according to claim 17, a) Identifying the genotype includes detecting at least one genetic marker of the at least one explant, wherein the at least one genetic marker includes a polynucleotide sequence characteristic of the genotype, b) Identifying the genotype includes detecting at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least 20, or at least 25 gene markers of the at least one embryonic explant, each of which contains a polynucleotide sequence characteristic of the genotype, c) Identifying the genotype includes identifying the genotype before or after introducing the heterologous polynucleotide molecule into the at least two embryonic explants of the population, d) Identifying the genotype comprises i) performing gene sequencing on a sample containing a polynucleotide molecule derived from or obtained therefrom the at least one embryonic explant or the at least one genetically modified plant or plant part, and ii) detecting at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, or at least twenty genetic markers in the sample, wherein the polynucleotide molecule is a genomic DNA molecule or a fragment thereof or an mRNA molecule or a fragment thereof, or the polynucleotide molecule is derived from a genomic DNA molecule or a fragment thereof or an mRNA molecule or a fragment thereof, and each of the genetic markers contains a polynucleotide sequence characteristic of the genotype. e) Identifying the genotype comprises i) contacting a sample containing a polynucleotide molecule derived from or obtained therefrom the at least one exoplant or the at least one genetically modified plant or plant part with at least one polynucleotide probe, wherein the polynucleotide probe is specific to one gene marker; ii) subjecting the sample and the polynucleotide probe to stringent hybridization conditions; and iii) detecting the hybridization of the polynucleotide probe to at least one gene marker in the sample, wherein the polynucleotide molecule is a genomic DNA molecule or a fragment thereof or an mRNA molecule or a fragment thereof, or the polynucleotide molecule is derived from a genomic DNA molecule or a fragment thereof or an mRNA molecule or a fragment thereof, and the gene marker contains a polynucleotide sequence characteristic of the genotype. f) Identifying the genotype includes identifying the genotypes of multiple embryonic explants, g) Identifying the genotypes includes identifying at least one or more genotypes of the embryonic explants of the population, h) Identifying the genotype includes identifying multiple genotypes of multiple embryonic explants, i) Identifying the genotype includes detecting at least two genetic markers in at least two of the embryonic explants of the population, j) Identifying the genotype includes detecting at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 genetic markers of the at least two embryonic explants, wherein at least two of the genetic markers include polynucleotide sequences characteristic of the first genotype, and at least one of the genetic markers includes polynucleotide sequences characteristic of the second genotype. k) Identifying the genotype comprises i) performing gene sequencing on at least a first sample and a second sample, wherein the first sample comprises a first polynucleotide molecule derived from or obtained therefrom a first embryonic explant, and the second sample comprises a second polynucleotide molecule derived from or obtained therefrom a second embryonic explant, and ii) detecting at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 gene markers in the first sample and the second sample, wherein the first polynucleotide molecule comprises a genomic DNA molecule or a fragment thereof or mR The first gene marker is an NA molecule or a fragment thereof, or derived from a genomic DNA molecule or a fragment thereof, or from an mRNA molecule or a fragment thereof, or the second polynucleotide molecule is a genomic DNA molecule or a fragment thereof, or derived from a genomic DNA molecule or a fragment thereof, or each of the gene markers contains a polynucleotide sequence characteristic of the genotype, or the gene marker comprises a first gene marker and a second gene marker, the first gene marker contains a first polynucleotide sequence characteristic of the first genotype, and the second gene marker contains a second polynucleotide sequence characteristic of the second genotype, l) The method further includes associating the genotype with at least one culture characteristic, m) The method further includes identifying a gene marker or quantitative trait locus (QTL) associated with at least one culture characteristic, n) The method further comprises associating the genotype with at least one culture characteristic, wherein the at least one culture characteristic is selected from the group consisting of explant cutting efficiency, regeneration efficiency, genetic modification efficiency, transformation efficiency, and regeneration capacity to a genetically modified plant or plant part, or o) The method further comprises identifying a gene marker or quantitative trait locus (QTL) associated with at least one culture characteristic, wherein the at least one culture characteristic is selected from the group consisting of explant cutting efficiency, regeneration efficiency, gene modification efficiency, transformation efficiency, and regeneration capacity to a genetically modified plant or plant part.

19. The method according to claim 13, further comprising identifying at least one genotype of the genetically modified plant or plant part.

20. The method according to claim 19, a) Identifying the genotype includes detecting at least one genetic marker of the at least one genetically modified plant or plant part, wherein the at least one genetic marker includes a polynucleotide sequence characteristic of the genotype, b) Identifying the genotype involves detecting at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least 20, or at least 25 gene markers of the at least one genetically modified plant or plant part, each of which contains a polynucleotide sequence characteristic of the genotype, c) The identification includes identifying the genotype before or after regenerating or growing the plurality of genetically modified plants or plant parts from any offspring generation of at least two embryonic explants or cells of the population, d) Identifying the genotype comprises i) performing gene sequencing on a sample containing a polynucleotide molecule derived from or obtained therefrom the at least one embryonic explant or the at least one genetically modified plant or plant part, and ii) detecting at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, or at least twenty genetic markers in the sample, wherein the polynucleotide molecule is a genomic DNA molecule or a fragment thereof or an mRNA molecule or a fragment thereof, or the polynucleotide molecule is derived from a genomic DNA molecule or a fragment thereof or an mRNA molecule or a fragment thereof, and each of the genetic markers contains a polynucleotide sequence characteristic of the genotype. e) Identifying the genotype comprises i) contacting a sample containing a polynucleotide molecule derived from or obtained therefrom the at least one exoplant or the at least one genetically modified plant or plant part with at least one polynucleotide probe, wherein the polynucleotide probe is specific to one gene marker; ii) subjecting the sample and the polynucleotide probe to stringent hybridization conditions; and iii) detecting the hybridization of the polynucleotide probe to at least one gene marker in the sample, wherein the polynucleotide molecule is a genomic DNA molecule or a fragment thereof or an mRNA molecule or a fragment thereof, or the polynucleotide molecule is derived from a genomic DNA molecule or a fragment thereof or an mRNA molecule or a fragment thereof, and the gene marker contains a polynucleotide sequence characteristic of the genotype. f) Identifying the genotype includes identifying the genotypes of multiple genetically modified plants or plant parts, g) Identifying the genotypes includes identifying at least one or more genotypes of the genetically modified plant or plant part, h) Identifying the genotypes includes identifying multiple genotypes of multiple genetically modified plants or plant parts, i) Identifying the genotype includes detecting at least two genetic markers in the genetically modified plant or plant part, j) Identifying the genotype includes detecting at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 genetic markers of the at least two genetically modified plants or plant parts, wherein at least two of the genetic markers include polynucleotide sequences characteristic of the first genotype, and at least one of the genetic markers includes polynucleotide sequences characteristic of the second genotype, or k) Identifying the genotype comprises i) performing gene sequencing on at least a first sample and a second sample, wherein the first sample comprises a first polynucleotide molecule derived from or obtained therefrom a first genetically modified plant or plant part, and the second sample comprises a second polynucleotide molecule derived from or obtained therefrom a second genetically modified plant or plant part, and ii) detecting at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 gene markers in the first sample and the second sample, wherein the first polynucleotide molecule comprises a genomic DNA molecule or a fragment thereof, or an mRNA molecule or a fragment thereof, or derived from a genomic DNA molecule or a fragment thereof, or the second polynucleotide molecule is a genomic DNA molecule or a fragment thereof, or derived from a genomic DNA molecule or a fragment thereof, or each of the gene markers contains a polynucleotide sequence characteristic of the genotype, or the gene marker comprises a first gene marker and a second gene marker, the first gene marker contains a first polynucleotide sequence characteristic of the first genotype, and the second gene marker contains a second polynucleotide sequence characteristic of the second genotype. l) The method further includes associating the genotype with at least one phenotype, m) The method further includes identifying a gene marker or quantitative trait locus (QTL) associated with at least one phenotype, n) The method further comprises associating the genotype with at least one phenotype, wherein the at least one phenotype is an observable plant trait, results from the expression of a selection marker or a screenable marker, or results from a genetic modification, wherein the genetic modification results from the incorporation of the heterologous polynucleotide molecule or a fragment thereof into the genome of the at least one genetically modified plant or plant part, wherein the heterologous polynucleotide molecule includes an expression cassette encoding a target gene, a site-specific nuclease, or a guide RNA molecule, or o) The method further comprises identifying a genetic marker or quantitative trait locus (QTL) associated with at least one phenotype, wherein the at least one phenotype is an observable plant trait, results from the expression of a selection marker or a screenable marker, or results from a genetic modification, wherein the genetic modification results from the incorporation of the heterologous polynucleotide molecule or a fragment thereof into the genome of the at least one genetically modified plant or plant part, wherein the heterologous polynucleotide molecule comprises an expression cassette encoding a target gene, a site-specific nuclease, or a guide RNA molecule.

21. The method according to claim 20, a) Whether the polynucleotide sequence is exclusively characteristic of the genotype, b) Each of the gene markers contains a polynucleotide sequence that is exclusively characteristic of the genotype, c) The at least two gene markers include a first gene marker and a second gene marker, wherein the first gene marker includes a first polynucleotide sequence characteristic of a first genotype, and the second gene marker includes a second polynucleotide sequence characteristic of a second genotype, or d) The method wherein the at least two gene markers include a first gene marker and a second gene marker, the first gene marker includes a first polynucleotide sequence exclusively characteristic of a first genotype, and the second gene marker includes a second polynucleotide sequence exclusively characteristic of a second genotype.

22. To identify the genetic modifications present in at least one embryonic explant of the aforementioned population, The further includes selecting an embryonic explant from the population containing the aforementioned gene modification, The method according to claim 17, wherein the selected embryonic explant further comprises at least one gene marker characteristic of the genotype, or the selected embryonic explant further comprises at least one gene marker characteristic of the genotype.

23. A method according to claim 22, a) Further comprising regenerating or growing a regenerated genetically modified plant or plant part from the selected embryonic explant or any offspring generation of its cells, b) Identifying the genetic modifications present in at least two embryonic explants of the population, The method further includes selecting a first embryonic explant from the population containing the gene modification and a second embryonic explant from the population containing the gene modification. The first selected embryo explant further comprises the first gene marker or the second gene marker, or the first selected embryo explant further comprises the first gene marker or the second gene marker. The second selected embryonic explant further contains the first gene marker or the second gene marker, or the second selected embryonic explant further does not contain the first gene marker or the second gene marker, or c) Identifying at least two gene modifications present in at least two embryonic explants of the population, wherein the at least two gene modifications include a first gene modification and a second gene modification. The further includes selecting a first embryonic explant from the population containing the first gene modification and a second embryonic explant from the population containing the second gene modification, The first selected embryo explant further comprises the first gene marker or the second gene marker, or the first selected embryo explant further comprises the first gene marker or the second gene marker. The method wherein the second selected embryo explant further comprises the first gene marker or the second gene marker, or the second selected embryo explant further comprises neither the first gene marker nor the second gene marker.

24. Identifying the genetic modification present in at least one genetically modified plant or plant part, The further includes selecting a genetically modified plant or plant part that includes the aforementioned genetic modification, The method according to claim 20, wherein the selected genetically modified plant or plant part further comprises the at least one gene marker characteristic of the genotype, or the selected genetically modified plant or plant part does not further comprise the at least one gene marker characteristic of the genotype.

25. The method according to claim 24, a) Further includes crossbreeding the selected genetically modified plant with itself or a different plant to obtain offspring plants or seeds, b) Identifying the gene modifications present in at least two of the genetically modified plants or plant parts, The method further includes selecting a first genetically modified plant or plant part containing the aforementioned genetic modification and a second genetically modified plant or plant part containing the aforementioned genetic modification. The first selected genetically modified plant or plant part further contains the first genetic marker or the second genetic marker, or the first selected genetically modified plant or plant part does not further contain the first genetic marker or the second genetic marker. The second selected genetically modified plant or plant part further contains the first genetic marker or the second genetic marker, or the second selected genetically modified plant or plant part further does not contain the first genetic marker or the second genetic marker, or c) Identifying at least two genetic modifications present in the at least two genetically modified plants or plant parts, wherein the at least two genetic modifications include a first genetic modification and a second genetic modification. The further includes selecting a first genetically modified plant or plant part that includes the first genetic modification and a second genetically modified plant or plant part that includes the second genetic modification, The first selected genetically modified plant or plant part further contains the first genetic marker or the second genetic marker, or the first selected genetically modified plant or plant part does not further contain the first genetic marker or the second genetic marker. The method wherein the second selected genetically modified plant or plant part further comprises the first genetic marker or the second genetic marker, or the second selected genetically modified plant or plant part further comprises the first genetic marker or the second genetic marker.

26. The method according to claim 23, a) further comprising regenerating or growing a first regenerated genetically modified plant or plant part from the first selected embryonic explant or any offspring generation of its cells, or regenerating or growing a second regenerated genetically modified plant or plant part from the second selected embryonic explant or any offspring generation of its cells, b) further comprising regenerating or growing a first regenerated genetically modified plant or plant part from the first selected embryonic explant or any offspring generation of its cells, or regenerating or growing a second regenerated genetically modified plant or plant part from the second selected embryonic explant or any offspring generation of its cells, and observing the phenotypes of the first regenerated genetically modified plant or plant part and the second regenerated genetically modified plant or plant part, or c) The method further comprising observing the culture characteristics of the first selected embryo explant and the second selected embryo explant.

27. The method according to claim 26, a) further comprising comparing the culture characteristics of the first selected embryo explant and the second selected embryo explant, and determining which of the first selected embryo explant or the second selected embryo explant has superior culture characteristics, b) further comprising comparing the culture characteristics of the first selected embryo explant and the second selected embryo explant, determining which of the culture characteristics of the first selected embryo explant or the second selected embryo explant is superior, and regenerating or growing a first regenerated genetically modified plant or plant part from any offspring generation of the first selected embryo explant or its cells, or regenerating or growing a second regenerated genetically modified plant or plant part from any offspring generation of the second selected embryo explant or its cells, based on the culture characteristics of the first selected embryo explant and the second selected embryo explant, c) further comprising comparing the phenotype of the first regenerated genetically modified plant or plant part with that of the second regenerated genetically modified plant or plant part, and determining which of the phenotypes is superior. d) Further comprising crossbreeding the first regenerated genetically modified plant with the same plant or a different plant to obtain a third offspring plant or seed, e) further comprising crossbreeding the second regenerated genetically modified plant with the same plant or a different plant to obtain a fourth offspring plant or seed, or f) The method further comprising introducing a second heterologous polynucleotide molecule into at least one explant of a second population of embryonic explants, wherein the at least one embryonic explant of the second population has the same genotype as or a different genotype from the at least one embryonic explant of the population or the selected embryonic explant.

28. The method according to claim 25, further comprising observing the phenotypes of the first selected genetically modified plant or plant part and the second selected genetically modified plant or plant part.

29. The method according to claim 28, a) further comprising comparing the phenotype of the first selected genetically modified plant or plant part with the second selected genetically modified plant or plant part, and determining which of the phenotypes is superior, b) further comprising crossing the first selected genetically modified plant with the same plant or a different plant to obtain a first offspring plant or seeds, c) Further comprising crossing the second selected genetically modified plant with the same plant or a different plant to obtain a second offspring plant or seeds, or d) The method further comprising introducing a second heterologous polynucleotide molecule into at least one explant of a second population of embryonic explants, wherein the at least one embryonic explant of the second population has the same genotype as or a different genotype from the at least one genetically modified plant or plant part or the selected genetically modified plant or plant part.

30. The method according to claim 1, further comprising detecting a genetic modification in at least one of the embryonic explants of the population.

31. The method according to claim 13, a) The method further includes detecting at least one genetic modification in the genetically modified plant or plant part, or b) The method wherein each of the plurality of genetically modified plants or plant parts comprises at least one genetic modification.

32. The method according to claim 31, a) The method further comprises i) selecting a genetically modified plant containing the at least one genetic modification, and ii) crossing the genetically modified plant with the same plant or a second plant to obtain offspring plants or seeds, wherein the second plant has the same genotype as the genetically modified plant, or has a different genotype, or b) The method further comprising: i) selecting a first genetically modified plant comprising at least a first genetic modification and a second genetically modified plant comprising at least a second genetic modification; ii) crossing the first genetically modified plant with the first genetically modified plant or with a first different plant to obtain a first offspring plant or seed; and iii) crossing the second genetically modified plant with the first genetically modified plant or with a second different plant to obtain a second offspring plant or seed.

33. The method according to claim 32, a) Whether the selection includes identifying the genotype of the genetically modified plant and selecting the genetically modified plant containing the genotype, b) The selection includes identifying a first genotype of the first genetically modified plant and selecting the first genetically modified plant containing the first genotype, or c) The method wherein the selection includes identifying a second genotype of the second genetically modified plant and selecting the second genetically modified plant comprising the second genotype.

34. The method according to claim 33, a) further comprising associating the genotype with at least one culture characteristic or at least one phenotype, b) further comprising associating the genotype with at least one culture characteristic or at least one phenotype, wherein the at least one culture characteristic is selected from the group consisting of explant excision efficiency, regeneration efficiency, gene modification efficiency, transformation efficiency, and regenerative capacity to a gene modified plant or plant part, or the at least one phenotype is an observable plant trait, results from the expression of a selection marker or a screenable marker, or results from gene modification, wherein the gene modification results from the incorporation of the heterologous polynucleotide molecule or a fragment thereof into the genome of the at least one gene modified plant or plant part, wherein the heterologous polynucleotide molecule includes an expression cassette encoding a target gene, a site-specific nuclease, or a guide RNA molecule. c) further comprising identifying a genetic marker or quantitative trait locus (QTL) associated with at least one phenotype, or d) The method further comprising identifying a genetic marker or quantitative trait locus (QTL) associated with at least one culture characteristic or at least one phenotype, wherein the at least one culture characteristic is selected from the group consisting of explant excision efficiency, regeneration efficiency, gene modification efficiency, transformation efficiency, and regeneration ability to a genetically modified plant or plant part, or the at least one phenotype is an observable plant trait, results from the expression of a selection marker or a screenable marker, or results from a gene modification, wherein the gene modification results from the incorporation of the heterologous polynucleotide molecule or a fragment thereof into the genome of the at least one genetically modified plant or plant part, wherein the heterologous polynucleotide molecule comprises an expression cassette encoding a target gene, a site-specific nuclease, or a guide RNA molecule.

35. Transferring a chromosome segment that confers the aforementioned at least one culture characteristic or the aforementioned at least one phenotype into a plant having a plant genotype that lacks the aforementioned culture characteristic or the aforementioned phenotype when the aforementioned chromosome segment is absent, or To produce offspring plants or seeds containing the chromosome segment by crossing a genetically modified plant containing a chromosome segment that confers at least one culture characteristic or at least one phenotype with the plant itself or a different plant. The method according to claim 34, further comprising:

36. The method according to claim 1, wherein the embryonic explants of at least two different genotypes include embryonic explants of a first genotype and embryonic explants of a second genotype, and the embryonic explants of the first genotype and the embryonic explants of the second genotype are present in the population in a predetermined ratio.

37. The method according to claim 36, a) The predetermined ratio is determined based on the first genotype, the second genotype, or at least one culture characteristic related to the first and second genotypes, b) The predetermined ratio is determined based on the first genotype, the second genotype, or at least one culture characteristic related to the first and second genotypes, wherein the at least one culture characteristic is selected from the group consisting of explant cutting efficiency, regeneration efficiency, shoot development efficiency, genetic modification efficiency, transformation efficiency, and regeneration capacity to genetically modified plants or plant parts. c) The embryonic explants in the predetermined ratio include approximately equal numbers of embryonic explants of the first genotype and the second genotype, d) The embryo explants in the predetermined ratio yield approximately equal numbers of regenerated genetically modified plants or plant parts of the first and second genotypes, e) The first genotype relates to preferred culture characteristics compared to the second genotype, and the method includes modifying the predetermined ratio to include an increased number of embryonic explants of the second genotype compared to the first genotype in the population, f) The first genotype relates to a preferred culture characteristic compared to the second genotype, and the method includes modifying the predetermined ratio to include an increased number of embryonic explants of the second genotype compared to the first genotype in the population, wherein the preferred culture characteristic results in an increase in explant cleavage efficiency, regeneration efficiency, shoot development efficiency, genetic modification efficiency, transformation efficiency, or regeneration capacity to a genetically modified plant or part. g) The second genotype relates to preferred culture characteristics compared to the first genotype, and the method includes modifying the predetermined ratio to include an increased number of embryonic explants of the first genotype compared to the second genotype in the population, or h) The method wherein the second genotype relates to a preferred culture characteristic compared to the first genotype, and the method comprises modifying the predetermined ratio to include an increased number of embryonic explants of the first genotype compared to the second genotype in the population, wherein the preferred culture characteristic results in an increase in explant cutting efficiency, regeneration efficiency, shoot development efficiency, genetic modification efficiency, transformation efficiency, or regeneration capacity to a genetically modified plant or part.

38. The method according to claim 1, a) further comprising observing at least one culture characteristic or at least one phenotype of at least one of the embryo explants of the population, b) further comprising observing at least one culture characteristic or at least one phenotype of at least one of the embryonic explants of the population, wherein the at least one culture characteristic or at least one phenotype is related to the genetic modification of the at least one embryonic explant, c) further comprising observing a first culture characteristic or first phenotype of at least one embryonic explant of the first genotype, and observing a second culture characteristic or second phenotype of at least one embryonic explant of the second genotype, d) further comprising observing a first culture characteristic or first phenotype of at least one embryonic explant of a first genotype, and observing a second culture characteristic or second phenotype of at least one embryonic explant of a second genotype, wherein the first culture characteristic or first phenotype is the same as the second culture characteristic or second phenotype, or the first culture characteristic or first phenotype is different from the second culture characteristic or phenotype, or e) The method further comprising evaluating at least one embryonic explant of a first genotype and at least one embryonic explant of a second genotype by comparing the first culture characteristics or first phenotype with the second culture characteristics or second phenotype.

39. The method according to claim 13, a) further comprising observing at least one culture characteristic or at least one phenotype of at least one of the genetically modified plants or plant parts, b) further comprising observing at least one culture characteristic or at least one phenotype of at least one of the genetically modified plant or plant part, wherein the at least one culture characteristic or at least one phenotype is related to the genetic modification of the at least one genetically modified plant or plant part. c) further comprising observing a first culture characteristic or first phenotype of at least one genetically modified plant or plant part of the first genotype, and observing a second culture characteristic or second phenotype of at least one genetically modified plant or plant part of the second genotype, d) further comprising observing a first culture characteristic or first phenotype of at least one genetically modified plant or plant part of a first genotype, and observing a second culture characteristic or second phenotype of at least one genetically modified plant or plant part of a second genotype, wherein the first culture characteristic or first phenotype and the second culture characteristic or second phenotype are the same, or the first culture characteristic or first phenotype and the second culture characteristic or phenotype are different, or e) The method further comprising evaluating a genetically modified plant or plant part having at least one genetically modified plant or plant part having at least one genetically modified plant or plant part having a second genotype by comparing a first culture characteristic or first phenotype with a second culture characteristic or second phenotype.

40. A method for genetically modifying a population of plant embryo explants that includes a mixture of different genotypes, The method comprising introducing a ribonucleoprotein or site-specific nuclease collectively into at least two plant embryo explants of the population, wherein each of the at least two plant embryo explants comprises a meristematic tissue, and the at least two embryo explants comprise plant embryo explants of at least two different plant genotypes.

41. The method according to claim 40, wherein the ribonucleoprotein comprises a site-specific nuclease and a guide RNA molecule.

42. The method according to claim 1, a) further comprising excising the group of exophyte fragments from a group of plant seeds, wherein the excision is performed before introducing the heterologous polynucleotide molecule, the ribonucleoprotein, or the site-specific nuclease collectively, and the group of plant seeds comprises plant seeds of at least two different plant genotypes, or b) The method further comprising sorting the group of plant seeds into batches of at least two plant seeds according to the size of the plant seeds, the shape of the plant seeds, or a combination thereof, before excising the group of exophytes from the group of plant seeds, wherein the excision is performed before introducing the heterologous polynucleotide molecule, the ribonucleoprotein, or the site-specific nuclease in a batch, and the group of plant seeds comprises plant seeds of at least two different plant genotypes.

43. The method according to claim 42, a) The batch of at least two plant seeds comprises a first batch of plant seeds and a second batch of plant seeds, and the group of plant embryo explants comprises a first batch of embryo explants and a second batch of embryo explants, and the excision is i) Dissecting a batch of first embryonic explants from a batch of first plant seeds, and a batch of second embryonic explants from a batch of second plant seeds, using the same dissection method, or ii) Dissecting a first batch of embryonic explants from the first batch of plant seeds, and a second batch of embryonic explants from the second batch of plant seeds, using different dissection methods. Does it include, b) The method described above, i) sorting a population of first plant seeds having a first genotype into at least two batches of plant seeds, comprising a first batch of plant seeds and a second batch of plant seeds, wherein the first batch of plant seeds comprises the size of the first plant seeds, the shape of the first plant seeds, or a combination thereof, and the second batch of plant seeds comprises the size of the second plant seeds, the shape of the second plant seeds, or a combination thereof. ii) sorting a population of second plant seeds having a second genotype into at least two batches of plant seeds, comprising a batch of first plant seeds and a batch of second plant seeds, wherein the batch of first plant seeds comprises the size of the first plant seeds, the shape of the first plant seeds, or a combination thereof, and the batch of second plant seeds comprises the size of the second plant seeds, the shape of the second plant seeds, or a combination thereof, further comprising the sorting, The aforementioned group of plant seeds A first predetermined ratio of plant seeds derived from a batch of first plant seeds of the first genotype and a batch of second plant seeds of the first genotype, The present invention comprises a second predetermined ratio of plant seeds derived from a batch of the first plant seeds of the second genotype and a batch of the second plant seeds of the second genotype, c) The at least two distinct genotypes include a first genotype and a second genotype, and the excision results in the excision of approximately equal numbers of exoembryonic explants of the first genotype and the second genotype, or d) The method wherein the plant seeds of at least two different genotypes include plant seeds of a first genotype and plant seeds of a second genotype, and the plant seeds of the first genotype and the plant seeds of the second genotype are present in the population in a predetermined ratio.

44. The method according to claim 43, a) Plant seeds of the first genotype in the first predetermined ratio and plant seeds of the second genotype in the second predetermined ratio are approximately equal, b) The number of plant seeds derived from the first batch of plant seeds of the first genotype is approximately equal to the number of plant seeds derived from the first batch of plant seeds of the second genotype, and / or the number of plant seeds derived from the second batch of plant seeds of the first genotype is approximately equal to the number of plant seeds derived from the second batch of plant seeds of the second genotype, c) The predetermined ratio is determined based on at least one culture characteristic related to the first genotype or the second genotype, d) The predetermined ratio is determined based on at least one culture characteristic related to the first or second genotype, wherein the at least one culture characteristic is selected from the group consisting of explant cutting efficiency, regeneration efficiency, shoot development efficiency, genetic modification efficiency, transformation efficiency, and regeneration capacity to genetically modified plants or plant parts. e) The plant seeds in the predetermined ratio of the first genotype and the second genotype yield approximately equal numbers of regenerated genetically modified plants or plant parts of the first genotype and the second genotype, f) The first genotype relates to preferred culture characteristics compared to the second genotype, and the method includes modifying the predetermined ratio to include an increased number of plant seeds of the second genotype compared to the first genotype in the population, such that the predetermined ratio of plant seeds of the first and second genotypes results in approximately equal numbers of regenerated genetically modified plants or plant parts of the first and second genotypes. g) The second genotype relates to preferred culture characteristics compared to the first genotype, and the method includes modifying the predetermined ratio to include an increased number of plant seeds of the first genotype compared to the second genotype in the population, such that the predetermined ratio of plant seeds of the first and second genotypes results in approximately equal numbers of regenerated genetically modified plants or plant parts of the first and second genotypes. h) The first genotype relates to a preferred culture characteristic compared to the second genotype, and the method comprises modifying the predetermined ratio to include an increased number of plant seeds of the second genotype compared to the first genotype in the population, wherein the predetermined ratio of plant seeds of the first and second genotypes results in approximately equal numbers of regenerated genetically modified plants or plant parts of the first and second genotypes, and the preferred culture characteristic results in an increase in explant cutting efficiency, regeneration efficiency, shoot development efficiency, genetic modification efficiency, transformation efficiency, or regeneration capacity to genetically modified plants or parts, or i) The method wherein the second genotype relates to a preferred culture characteristic compared to the first genotype, and the method comprises modifying the predetermined ratio to include an increased number of plant seeds of the first genotype compared to the second genotype in the population, wherein the predetermined ratio of plant seeds of the first and second genotypes results in substantially equal numbers of regenerated genetically modified plants or plant parts of the first and second genotypes, and the preferred culture characteristic results in an increase in explant cutting efficiency, regeneration efficiency, shoot development efficiency, genetic modification efficiency, transformation efficiency, or regeneration capacity to genetically modified plants or parts.

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