Compositions and methods for transforming embryo explant populations

Abstract

The present disclosure provides novel methods for co-transformation or genetic modification of populations of embryo explants of different germplasm or of different genotypes, it may include one or more steps of explant preparation, explant rehydration, rhizobium bacterial inoculation and co-culture or particle bombardment, bud induction, extended bud induction, and / or regeneration or development of a genetically modified plant or plant part. The methods provided herein can include transforming at least one plant cell of an embryo explant with a heterologous polynucleotide. The methods provided herein also include methods of 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 one or more genotypes with improved phenotypes or other culture characteristics.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 328,567, filed on April 7, 2022, U.S. Provisional Application Serial No. 63 / 441,369, filed on January 26, 2023, and U.S. Provisional Application Serial No. 63 / 492,279, filed on March 27, 2023, the entire disclosure of each of which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to compositions and methods for genetically modifying a population of plant embryonic explants having different genotypes. Background Art

[0004] Crop plants such as corn, wheat, rice, barley, Chinese sorghum, soybean, cotton and rape are important crops, and are the main food source in many areas of the world.The genetic modification of embryo explants has been used to produce this type of crop plants with improved proterties or characteristics.Yet this area still needs improved method to carry out genetic modification to multiple different plant genotypes simultaneously, which can reduce or get rid of adopting expensive and time-consuming plant breeding technology.

[0005] The present disclosure provides novel compositions and methods for genetically modifying a population of embryonic explants having different plant genotypes and regenerating genetically modified plants or plant parts therefrom, which reduces or eliminates the use of plant breeding or introgression techniques to overcome many challenges and limitations in the art. Summary of the Invention

[0006] On the one hand, the disclosure provides the method that the colony of plant embryo explant is carried out to genetic modification, comprising: heterologous polynucleotide molecule is introduced jointly into at least two embryo explants of described colony, described at least two plant embryo explants comprise meristematic tissue separately, wherein said colony comprises the embryo explant of at least two different plant genotypes.In some embodiments, colony is limited to the colony of monocotyledonous plant embryo explant.In further embodiment, the colony of monocotyledonous plant embryo explant can be limited to the colony of corn, wheat, rice, barley, Chinese sorghum or lawn grass embryo explant.In specific embodiment, colony is limited to the colony of dicotyledonous plant embryo explant.In further embodiment, the colony of dicotyledonous plant embryo explant can be limited to the colony of soybean, cotton or rape embryo explant.In certain embodiments, introducing heterologous polynucleotide molecule jointly comprises that the heterologous polynucleotide molecule is introduced into at least two explants of described colony via bacteria-mediated transformation. In other embodiments, co-introduction of the heterologous polynucleotide molecule comprises introducing the heterologous polynucleotide molecule into at least two explants of the population via transformation mediated by a Rhizobiales bacterium. In some embodiments, the Rhizobiales bacterium is 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 molecule together comprises introducing the heterologous polynucleotide molecule via Agrobacterium-mediated conversion. In specific embodiments, introducing the heterologous polynucleotide molecule together comprises inoculating at least two embryo explants with inoculation medium, and said inoculation medium comprises the Rhizobiales bacterium that has the ability to transform at least two embryo explants with the heterologous polynucleotide molecule. In certain embodiments, the colony contacted with the inoculation medium is subjected to force processing. In other embodiments, before introducing the heterologous polynucleotide molecule together, force processing is applied. In specific embodiments, force processing comprises approximately 3,000xg to approximately 6,000xg, approximately 3,500xg to approximately 5,000xg or approximately 3,500xg to approximately 4,500xg scope gravity processing.In some embodiments, the method may further comprise co-culturing the at least two embryo explants with a Rhizobiales bacterium contacted with a co-culture medium. In certain embodiments, the colony of embryo explants is a colony of monocotyledonous plant seed embryo explants, and the method further comprises co-culturing the at least two embryo explants with a Rhizobiales bacterium contacted with a co-culture medium. 2 ) co-cultivation surface area is less than or equal to the density of about 9.1 embryo explants at least two embryo explants of the colony are contacted with the co-culture medium for co-cultivation. In some embodiments, the colony of embryo explants is the colony of monocotyledonous plant seed embryo explants, and the method further comprises contacting the co-culture medium with at least two embryo explants of the colony for co-cultivation for a period of time within the range of about 6 days to about 8 days. In other embodiments, jointly introducing heterologous polynucleotide molecules comprises introducing heterologous polynucleotide molecules into at least two explants of the colony via particle bombardment. In certain embodiments, the method provided by the present disclosure may further comprise jointly introducing site-specific nucleases into at least two embryo explants of the colony. In specific embodiments, the heterologous polynucleotide molecules comprise guide RNA molecules, and jointly introducing comprises introducing site-specific nucleases into at least two embryo explants. In some embodiments, the site-specific nuclease is a ribonucleoprotein, and the ribonucleoprotein comprises site-specific nucleases and guide RNA molecules.

[0007] In further embodiments, the heterologous polynucleotide molecule may comprise a first expression cassette and a second expression cassette. In certain embodiments, the first expression cassette may 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 in specific embodiments, the second expression cassette may 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.

[0008] In certain embodiments, co-introduction of heterologous polynucleotide molecules 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. In some embodiments, the first expression cassette may 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 in certain embodiments, the second expression cassette may comprise a second selectable marker 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.

[0009] In specific embodiments, the method that the colony of plant embryo explant provided by the present disclosure is carried out genetic modification may also include contacting and cultivating at least two embryo explants with the first bud inducing culture medium comprising the first auxin and the first cytokinin.In certain embodiments, the first bud inducing culture medium comprises high cytokinin and auxin ratio.In some embodiments, the first auxin in the first bud inducing culture medium is selected from the group consisting of: 2,4-dichlorophenoxy-acetic acid (2,4-D), 4-amino-3,5,6-trichloro-picolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid or para-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-methoxy-benzoic acid (dicamba). In further embodiments, the concentration of the first auxin in the first bud induction 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 bud induction medium is selected from the group consisting of: 6-benzylaminopurine (BAP), thiadiazole (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino)purine (2iP) and 6-(3-hydroxybenzylamino)purine (topolin). In specific embodiments, the concentration of the first cytokinin in the first bud induction medium is in the range of from about 0.1 mg / L to about 50 mg / L. In some embodiments, the population of embryo explants is a population of monocot seed embryo explants, and the method further comprises administering the first cytokinin to the first bud induction medium at a rate of 100 μg / L per square centimeter (cm 2 ) The first bud induction surface area is less than or equal to a density of about 3.9 embryo explants. At least two embryo explants are cultured in contact with the first bud induction medium.

[0010] In certain embodiments, the method for genetically modifying a population of plant embryo explants provided by the present disclosure may further comprise contacting and culturing at least two embryo explants with a second bud induction medium comprising a first auxin or a second auxin and a first cytokinin or a second cytokinin. In some embodiments, the embryo explants are contacted and cultured with a second bud induction medium at a temperature in the range of about 20°C to about 32°C, about 25°C to about 29°C, or about 27°C to about 28°C. In specific embodiments, the second bud induction medium comprises a high cytokinin to auxin ratio. In specific embodiments, the second bud induction medium comprises: a) a first auxin and a first cytokinin; b) a first auxin and a second cytokinin; c) a second auxin and a first cytokinin; or d) a second auxin and a second cytokinin. In certain embodiments, the population of embryo explants is a population of monocot seed embryo explants, and the method further comprises contacting and culturing the embryo explants with a second bud induction medium at a temperature in the range of about 20°C to about 32°C, about 25°C to about 29°C, or about 27°C to about 28°C. 2) The second bud induction surface area is less than or equal to a density of about 2.6 embryo explants, and at least two embryo explants are contacted with the second bud induction medium. In some embodiments, the first auxin or the second auxin in the second bud induction medium is selected from the group consisting of: 2,4-dichlorophenoxy-acetic acid (2,4-D), 4-amino-3,5,6-trichloro-picolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthylacetic 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-methoxy-benzoic acid (dicamba). In some embodiments, the first cytokinin or the second cytokinin in the second bud induction medium is selected from the group consisting of 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino) purine (2iP) and 6-(3-hydroxybenzylamino) purine (topolin). In specific embodiments, the concentration of the first cytokinin or the second cytokinin in the second bud induction 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 certain embodiments, the concentration of the first auxin or the second auxin in the second shoot induction medium is from about 0.01 mg / L to about 25 mg / L, from about 0.02 mg / L to about 10 mg / L, or from about 1 mg / L to about 2 mg / L. In specific embodiments, the heterologous polynucleotide molecule comprises a selectable marker gene, the second shoot induction medium comprises a selective agent, and the selectable marker gene confers resistance to the selective agent in the plant.

[0011] In a specific embodiment, the method for genetically modifying a population of plant embryo explants provided herein may further comprise regenerating or growing a plurality of genetically modified plants or plant parts by contacting at least two embryo explants or any progeny of their cells with a regeneration medium. In some embodiments, the population is a population of monocotyledonous plant seed embryo explants, and the method comprises: 2) regeneration surface area is less than or equal to the density of about 2.6 embryo explants by at least two embryo explants in contact with the regeneration medium to regenerate or grow a plurality of genetically modified plants or plant parts. Still in other embodiments, the method of the present disclosure may also include regenerating a culture population of monocotyledonous plant seed embryo explants in contact with a first regeneration medium, transferring the culture population of monocotyledonous plant seed embryo explants or a subpopulation of the culture population of monocotyledonous plant seed embryo explants to a second regeneration medium, and contacting the second regeneration medium to regenerate a plurality of genetically modified monocotyledonous plants or plant parts. In some embodiments, the regeneration, first regeneration medium or second regeneration medium has a low salt concentration. In some embodiments, the heterologous polynucleotide molecule comprises a selectable marker gene, the regeneration medium comprises a selection agent, and the selectable marker gene provides resistance to the selection agent in the plant. In certain embodiments, the genetically modified plant or plant part comprises at least one genetic modification. In specific embodiments, the genetic modification may include integrating or inserting a heterologous polynucleotide molecule or a fragment thereof into the genome of a 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 genetic modification may comprise editing of the genomes of multiple genetically modified plants or plant parts using site-specific nucleases and / or guide RNA molecules introduced by genome editing techniques. In certain embodiments, the heterologous polynucleotide molecule comprises at least one expression cassette, and 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, the at least two expression cassettes comprising a first expression cassette encoding a site-specific nuclease and a second expression cassette encoding a guide RNA molecule. In specific embodiments, the genetically modified plant part comprises a shoot or a root. In certain embodiments, the genetically modified plant part comprises a seed. In further embodiments, the genetically modified plant or plant part is non-chimeric. In specific embodiments, the genetically modified plant or plant part is cultured and / or regenerated without producing a callus culture.

[0012] In a further embodiment, the method for genetically modifying a population of plant embryo explants provided by the present disclosure may also include contacting at least two embryo explants or any progeny of their cells 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 method provided by the present disclosure may additionally include: selecting a genetically modified plant comprising at least one genetic modification; and hybridizing the plant with itself or a second plant to obtain a progeny plant or seed. In some embodiments, the selection includes confirming the genotype of the genetically modified plant and selecting a plant comprising the genotype. In a further embodiment, the method provided by the present disclosure may also 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; hybridizing the first genetically modified plant with itself or a first different plant to obtain a first progeny plant or seed; and hybridizing the second genetically modified plant with itself, the first genetically modified plant or a second different plant to obtain a second progeny plant or seed. In certain embodiments, the first different plant and / or the second different plant may have a genotype different from the first genetically modified plant and / or the second genetically modified plant. In certain embodiments, selecting comprises identifying a first genotype of a first genetically modified plant and selecting a first genetically modified plant comprising said first genotype; and identifying a second genotype of a second genetically modified plant and selecting a second genetically modified plant comprising said second genotype.

[0013] In specific embodiments, the colony of embryo explants provided by the disclosure is included in the embryo explants with an internal moisture content of about 3% to about 25% in the scope before introducing the heterologous polynucleotide molecule. In specific embodiments, the colony comprises embryo explants, and the embryo explants comprise the top portion of the embryonic axis that lacks the radicle, and wherein the remainder of the seed of the embryo explant has been removed substantially from the embryo explants. In certain embodiments, the colony is limited to the colony of dry, dry excised, wet excised or wet seed embryo explants. In other embodiments, the colony is limited to the colony of mature or immature seed embryo explants. In further embodiments, the colony that comprises the embryo explants is prepared by a colony of monocotyledonous plant seeds under the condition that the embryo explants do not germinate and keep viable and genetic modification ability is arranged. In some embodiments, the population comprises plants 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, or at least 500 different plant genotypes or 250 different plant genotypes. or about 10 different plant genotypes.In certain embodiments, at least two of the embryonic explants of the population comprise 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 or 2 to about 600, about 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 some embodiments, the population 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, at least 450, at least 500, at least 600, at least 700, at least 800, at least 90 0, 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 about 2 to about 50,000, 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 20,000, about 5,000 to about 10,000, about 20,000 to about 90,000. In some embodiments, the colony comprises at least one embryo explant with at least one unconfirmed genotype before introducing the heterologous polynucleotide molecule together.In some embodiments, the colony comprises at least one embryo explant with at least one unconfirmed genotype before introducing the heterologous polynucleotide molecule together.In other embodiments, at least two different genotypes of embryo explant are known. In a further embodiment, each embryonic explant of the population is an embryonic explant of known genotype.

[0014] In some embodiments, heterologous polynucleotides are co-introduced into 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 plants. genotypes, or in embryo explants of 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 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 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.In other 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.

[0015] In specific embodiment, the colony of plant embryo explant that the disclosure provides is carried out the method for genetic modification and can also comprise the genotype of at least one in the embryo explant of confirming described colony or described genetic modification plant or plant part.In some embodiments, confirm that genotype comprises at least one genetic marker that detects at least one embryo explant or at least one genetic modification plant or plant part, wherein said at least one genetic marker comprises the polynucleotide sequence of described genotype characteristic.In specific embodiment, polynucleotide sequence is the proprietary characteristic of genotype.In further embodiment, confirm that genotype comprises at least 2 kinds, at least 3 kinds, at least 4 kinds, at least 5 kinds, at least 6 kinds, at least 7 kinds, at least 8 kinds, at least 9 kinds, at least 10 kinds, at least 15 kinds, at least 20 kinds, at least 25 kinds or approximately 2 to approximately 26 kinds, approximately 5 to approximately 26 kinds, approximately 10 to approximately 26 kinds, approximately 15 to approximately 26 kinds or approximately 20 to approximately 26 kinds of genetic markers that detect at least one embryo explant or at least one genetic modification plant or plant part, wherein every kind of genetic marker comprises the polynucleotide sequence of described genotype characteristic. In certain embodiments, each genetic marker comprises a polynucleotide sequence that is a genotype-specific characteristic. In some embodiments, confirming genotype is included in confirming the genotype before or after a heterologous polynucleotide molecule is introduced into at least two embryo explants of the population; before or after co-culturing at least two embryo explants of the population, confirming the genotype; before or after contacting at least two embryo explants of the population with a first bud induction culture medium, confirming the genotype; before or after contacting at least two embryo explants of the population with a second bud induction culture medium, confirming the genotype; or before or after regenerating or growing a plurality of genetically modified plants or plant parts by any progeny of at least two embryo explants of the population or its cells, confirming the genotype. In a specific embodiment, confirming the genotype comprises performing genetic sequencing on a sample comprising polynucleotide molecules from or derived 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 genomic DNA molecules or fragments thereof or mRNA molecules or fragments thereof, or wherein the polynucleotide molecules are derived from genomic DNA molecules or fragments thereof or mRNA molecules or fragments thereof, and wherein each genetic marker comprises a polynucleotide sequence characteristic of the genotype.In other embodiments, confirming genotype comprises contacting a sample comprising a polynucleotide molecule from or derived 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 probes, wherein each polynucleotide probe is specific to a genetic marker; subjecting the sample and the polynucleotide probe to stringent hybridization conditions; and detecting hybridization of the polynucleotide probe and 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 molecule is a genomic DNA molecule or a fragment thereof or an mRNA molecule or a fragment thereof, or wherein the polynucleotide molecule is derived from a genomic DNA molecule or a fragment thereof or an mRNA molecule or a fragment thereof, and wherein each genetic marker comprises a polynucleotide sequence characteristic of the genotype. In various embodiments, confirming genotype comprises amplifying the polynucleotide molecule or restriction enzyme mapping. In a specific embodiment, confirming the genotype comprises confirming the genotype of a plurality of embryonic explants or a plurality of genetically modified plants or plant parts. In certain embodiments, confirming the genotype comprises confirming multiple genotypes of at least one of the embryonic explants of the colony or at least one of the genetically modified plants or plant parts. In some embodiments, confirming the genotype comprises confirming multiple genotypes of a plurality of embryonic explants or a plurality of genetically modified plants or plant parts.

[0016] In certain embodiments, confirm that genotype comprises at least two or at least two genetic markers in at least two of the embryo explants detecting said colony or said genetically modified plant or plant part.In further embodiment, said at least two genetic markers comprise the first genetic marker and the second genetic marker, wherein the first genetic marker comprises the first polynucleotide sequence characteristic of the first genotype, and the second genetic marker comprises the second polynucleotide sequence characteristic of the second genotype.In specific embodiment, the first polynucleotide sequence is that the first genotype is proprietary and characteristic, or the second polynucleotide sequence is that the second genotype is proprietary and characteristic.In some embodiments, confirm that comprise at least 3 kinds, at least 4 kinds, at least 5 kinds, at least 6 kinds, at least 7 kinds, at least 8 kinds, at least 9 kinds, at least 10 kinds, at least 15 kinds or at least 20 kinds of genetic markers detecting at least two embryo explants or at least two genetically modified plants or plant part, wherein at least two genetic markers comprise the polynucleotide sequence characteristic of the first genotype, and at least one genetic marker comprises the polynucleotide sequence characteristic of the second genotype. In a further embodiment, the confirming comprises performing genetic sequencing on at least a first sample and a second sample, wherein the first sample comprises a first polynucleotide molecule from or derived from a first embryonic explant or a first genetically modified plant or plant part, and the second sample comprises a second polynucleotide molecule from or derived 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 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 wherein 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 wherein 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, the first genetic marker comprising a first polynucleotide sequence characteristic of a first genotype, and the second genetic marker comprising a second polynucleotide sequence characteristic of a second genotype.

[0017] In certain embodiments, the method for genetically modifying a population of plant embryonic explants provided herein may further comprise confirming the genetic modification present in at least one embryonic explant or at least one genetically modified plant or plant part of the population; and selecting the embryonic explant or genetically modified plant or plant part of the population that comprises the genetic modification, wherein the selected embryonic explant or the selected genetically modified plant or plant part further comprises at least one genetic marker characteristic of the genotype, or wherein the selected embryonic explant or the selected genetically modified plant or plant part does not further comprise at least one genetic marker characteristic of the genotype. In some embodiments, the method for genetically modifying a population of plant embryonic explants provided herein may further comprise regenerating or growing a regenerated genetically modified plant or plant part from any progeny of the selected embryonic explant or its cells; or hybridizing the selected genetically modified plant with itself or a different plant to obtain a progeny plant or seed. In these embodiments, the method may further comprise confirming the genetic modification present in at least two embryonic explants or at least two genetically modified plants or plant parts of the population; and selecting a first embryonic explant or a first genetically modified plant or plant part of the population comprising the genetic modification and a second embryonic explant or a second genetically modified plant or plant part of the population comprising the genetic modification, wherein the first selected embryonic explant or the 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 embryonic 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 wherein the second selected embryonic explant or the 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 embryonic 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.In specific embodiments, the methods provided by the present disclosure may further include: confirming the presence of at least two genetic modifications in at least two embryonic explants or at least two genetically modified plants or plant parts of the population, the at least two genetic modifications comprising a first genetic modification and a second genetic modification; and selecting a first embryonic explant or a first genetically modified plant or plant part of the population comprising the first genetic modification and a second embryonic explant or a second genetically modified plant or plant part of the population comprising the second genetic modification, wherein the first selected embryonic explant or the 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 embryonic 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 wherein the second selected embryonic explant or the 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 embryonic 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.

[0018] In some embodiments, the present disclosure provides a method for genetically modifying a population of plant embryonic explants, further comprising regenerating or growing a first regenerated genetically modified plant or plant part from any progeny of a first selected embryonic explant or a cell thereof, or regenerating or growing a second regenerated genetically modified plant or plant part from any progeny of a second selected embryonic explant or a cell thereof. In some embodiments, the present disclosure provides a method for genetically modifying a population of plant embryonic explants, further comprising observing the culture characteristics of the first selected embryonic explant and the second selected embryonic 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. In some embodiments, the methods provided herein for genetically modifying a population of plant embryonic explants may further comprise comparing the culture characteristics of a first selected embryonic explant and a second selected embryonic explant, and determining whether the culture characteristics of the first selected embryonic explant or the second selected embryonic explant are superior; comparing the phenotype of a first regenerated genetically modified plant or plant part and a 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 a first selected genetically modified plant or plant part and a 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 comprise regenerating or growing a first regenerated genetically modified plant or plant part from any progeny of the first selected embryonic explant or its cells, or regenerating or growing a second regenerated genetically modified plant or plant part from any progeny of the second selected embryonic explant or its cells, based on the culture characteristics of the first selected embryonic explant and the second selected embryonic explant. In certain embodiments, the methods provided herein may further comprise crossing a first selected genetically modified plant with itself or a different plant to obtain a first progeny plant or seed; crossing a 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.

[0019] In certain embodiments, the present disclosure provides methods for genetically modifying a population of plant embryonic explants, 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 or a different genotype compared to: at least one embryonic explant of the population; the at least one genetically modified plant or plant part; a selected embryonic explant; or a selected genetically modified plant or plant part. In specific embodiments, the present disclosure provides methods that may include detecting a genetic modification in at least one of the embryonic explants of the population or at least one of the genetically modified plants or plant parts.

[0020] In further embodiments, the method that the colony of plant embryo explant that the disclosure provides is carried out genetic modification can also comprise confirming the genotype of at least one in the embryo explant of described colony or described genetically modified plant or plant part; And described genotype is associated with at least one culture characteristic or phenotype.In specific embodiment, confirmation can comprise confirming the genetic marker or quantitative trait locus (QTL) relevant to described at least one culture characteristic or phenotype.In certain embodiments, at least one culture characteristic or phenotype are selected from the group consisting of: explant excision efficiency, regeneration efficiency, genetic modification efficiency, conversion efficiency, bud generation and the ability that regenerates into genetically modified plant or plant part.In specific embodiment, phenotype is observable plant trait or is produced by the expression of selectable or screenable marker.Can adopt any method as known in the art to observe plant trait. The limiting examples of observable plant traits comprise plant height, fringe height, supporting root color, internode direction, internode length, leaf color, leaf length, leaf width, leaf sheath pubescence, leaf margin corrugation, tassel length, anther color, glume color, fringe silk color, fringe silk position, bract dehiscence, bract color, cob diameter, kernel row number, every row kernel number, endosperm type, endosperm color, relative maturity, flower color, hilum color, seed coat color, seed shape, leaf shape and growth habit.Operable selectable marker gene includes but not limited to aroA, EPSPS, aadA, pat, bar, hph (hygromycin B phosphotransferase), DMO (dicamba monooxygenase) CAT and NPT II.Although plant selectable marker gene is generally used for giving the tolerance to selection agent, except selectable marker, can also use other screenable or scoreable marker gene, perhaps can also use together with the gene with agronomic value. Such selectable marker genes may include, for example, uidA for beta-glucuronidase (GUS; e.g., as described in U.S. Pat. No. 5,599,670, which is hereby incorporated by reference), gfp for green fluorescent protein and its variants (GFP as described in U.S. Pat. Nos. 5,491,084 and 6,146,826, each of which is hereby incorporated by reference), or crtB for phytoene synthase (e.g., as described in U.S. Pat. Nos. 8,237,016 and 10,240,165, each of which is hereby incorporated by reference). Additional examples of selectable markers may include secretable markers, the expression of which results in the secretion of a molecule that can be detected as a means of confirming transformed cells.

[0021] In other embodiments, the phenotype is a genetically modified phenotype, wherein the genetic modification is produced by integration of a heterologous polynucleotide molecule or fragment thereof into the genome of at least one genetically modified plant or plant part, wherein 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 herein may further comprise introgressing a chromosome segment conferring at least one culture characteristic or at least one phenotype into a plant that, in the absence of the chromosome segment, would have a plant genotype lacking the culture characteristic or phenotype; or crossing a genetically modified plant comprising a chromosome segment conferring at least one culture characteristic or at least one phenotype with itself or a second plant to produce a progeny plant or seed comprising the chromosome segment.

[0022] In some embodiments, present disclosure provides the method that the colony of plant embryo explant is carried out genetic modification, comprising: heterologous polynucleotide molecule is introduced jointly into at least two embryo explants of described colony, wherein the embryo explants of at least two different genotypes comprise the embryo explant of the first genotype and the embryo explant of the second genotype, and wherein the embryo explant of the first genotype and the embryo explant of the second genotype are present in described colony with predetermined ratio.In specific embodiment, predetermined ratio is based on and the first genotype, with the second genotype or with at least one culture characteristic relevant to the first genotype and the second genotype and determine.In certain embodiments, at least one culture characteristic is selected from the group consisting of: explant excision efficiency, regeneration efficiency, bud generation efficiency, genetic modification efficiency, conversion efficiency and the ability to be regenerated into genetically modified plant or plant part.In some embodiments, the predetermined ratio of the embryo explant of the first genotype and the second genotype comprises the embryo explant of the first genotype and the second genotype of roughly equal quantity. In further embodiments, the predetermined ratio of the embryo explants of the first genotype and the second genotype causes the genetically modified plants or plant parts of the regeneration of the first genotype and the second genotype to be approximately equal. In some embodiments, the first genotype is associated with a preferred culture characteristic relative to the second genotype, and the method includes changing the predetermined ratio to include the embryo explants of the second genotype whose quantity is increased compared to the first genotype in the colony; or the second genotype is associated with a preferred culture characteristic relative to the first genotype, and the method includes changing the predetermined ratio to include the embryo explants of the first genotype whose quantity is increased compared to the second genotype in the colony. In certain embodiments, the predetermined ratio of the embryo explants of the first genotype and the second genotype causes the genetically modified plants or plant parts of the regeneration of the first genotype and the second genotype to be approximately equal. In certain embodiments, the preferred culture characteristic can cause explant excision efficiency, regeneration efficiency, bud generation efficiency, genetic modification efficiency, conversion efficiency or the ability to regenerate into genetically modified plants or parts to improve.

[0023] In a specific embodiment, the method that the colony of plant embryo explants is genetically modified provided by the present disclosure may also include contacting regeneration medium with a regeneration medium to regenerate or grow multiple genetically modified plants or plant parts by at least two embryo explants or any progeny of their cells; and observing at least one culture characteristic or phenotype of at least one of the embryo explants of the colony or at least one of the genetically modified plants or plant parts. In some embodiments, at least one culture characteristic or phenotype is associated with the genetic modification of at least one embryo explant or at least one genetically modified plant or plant part. In a further embodiment, the method provided by the present disclosure may also 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 a specific embodiment, the first culture characteristic or phenotype and the second culture characteristic or phenotype are identical. In another embodiment, the first culture characteristic or phenotype and the second culture characteristic or phenotype are different. In some embodiments, the method that the disclosure provides can also comprise by comparing the first culture characteristic or phenotype and the second culture characteristic or phenotype at least one embryo explant or at least one genetically modified plant or plant part of the first genotype and at least one embryo explant or at least one genetically modified plant or plant part of the second genotype.In specific embodiments, each in a plurality of genetically modified plants or plant parts comprises at least one genetic modification.In specific embodiments, the method that the disclosure provides can also comprise selecting to comprise the genetically modified plant of at least one genetic modification, and described genetically modified plant and itself or second plant are hybridized to obtain progeny plant or seed, and wherein the second plant has the genotype identical with described genetically modified plant or different genotypes.In certain embodiments, selection comprises the genotype of confirming genetically modified plant, and selects the genetically modified plant that comprises described genotype.

[0024] In certain embodiments, present disclosure provides the method that the colony of plant embryo explant is carried out genetic modification, comprising: heterologous polynucleotide molecule is introduced jointly in at least two embryo explants of described colony.In specific embodiment, introduce jointly and comprise heterologous polynucleotide or its fragmentary fixed-site integration.In some embodiments, heterologous polynucleotide molecule comprises at least one expression cassette, and wherein said at least one expression cassette comprises selectable marker gene, screenable marker gene, the nucleotide sequence of gene of paid close attention, coding guide RNA molecule or the nucleotide sequence of coding site-specific nuclease.In certain embodiments, heterologous polynucleotide molecule comprises or codes guide RNA.In further embodiment, introduce jointly and comprise heterologous polynucleotide molecule is introduced in described colony simultaneously.In specific embodiment, introduce jointly and comprise heterologous polynucleotide molecule is introduced in the colony of embryo explant when described colony is present in single container jointly.

[0025] In some embodiments, the present disclosure provides a method for genetically modifying a population of plant embryo explants, comprising: co-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 wherein the population is defined as a population of dicotyledonous plant stem embryo explants. In a specific embodiment, the population is defined as a population of soybean, cotton, or rapeseed embryo explants.

[0026] In some embodiments, the present disclosure provides a method for genetically modifying a population of plant embryo explants, the method comprising: co-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 meristems, wherein the population comprises 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.

[0027] In certain embodiments, the method provided by the present disclosure may also include excising a colony of embryo explants from a colony of plant seeds, wherein the excision is carried out before jointly introducing heterologous polynucleotide molecules, ribonucleoproteins or site-specific nucleases, and wherein the colony of plant seeds comprises plant seeds of at least two different plant genotypes. In certain embodiments, the method provided by the present disclosure may also include sorting the colony of plant seeds into at least two batches of plant seeds according to plant seed size, plant seed shape or its combination before excising the colony 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 colony of the plant embryo explant comprises a first batch of embryo explants and a second batch of embryo explants, and the excision comprises: adopting identical excision methods to excise the first batch of embryo explants from the first batch of plant seeds, and excising the second batch of embryo explants from the second batch of plant seeds; or adopting different excision methods to excise the first batch of embryo explants from the first batch of plant seeds, and excising the second batch of embryo explants from the second batch of plant seeds.

[0028] In some embodiments, the present disclosure provides a method further comprising: sorting a first population of plant seeds having a first genotype into at least two batches of plant seeds, the 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 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, the 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 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 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 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 wherein 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 a first genotype and a second genotype, and the excision results in the excision of approximately equal numbers of embryonic explants of the first genotype and the second genotype.

[0029] In certain embodiments, the plant seeds of at least two different genotypes can comprise the plant seeds of the first genotype and the plant seeds of the second genotype, wherein the plant seeds of the first genotype and the plant seeds of the second genotype are present in the colony with a predetermined ratio. In certain embodiments, the predetermined ratio is based on determining at least one culture characteristic relevant to the first genotype or the second genotype. The limiting examples of this type of culture characteristic comprises explant excision efficiency, regeneration efficiency, bud generation efficiency, genetic modification efficiency, conversion efficiency and the ability to be regenerated into genetically modified plants or plant parts. In some embodiments, the predetermined ratio of the plant seeds of the first genotype and the second genotype causes the genetically modified plants of the regeneration of the first genotype and the second genotype or the quantity of plant parts to be roughly equal. In certain embodiments, the first genotype is associated with a preferred culture characteristic of, and the method comprises altering the predetermined ratio to include an increased number of plant seeds of the second genotype compared to the first genotype in the population; or the second genotype is associated with a preferred culture characteristic of, and the method comprises altering 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 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 preferred culture characteristics include increased explant excision efficiency, regeneration efficiency, shoot formation efficiency, genetic modification efficiency, transformation efficiency, or the ability to regenerate into a genetically modified plant or plant part.

[0030] In some embodiments, the colony is defined as a colony of dicotyledonous plant embryo explants, and the co-culture medium includes 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-(γ,γ-dimethylallylamino) purine (2iP) and 6-(3-hydroxybenzylamino) purine (topolin). In certain embodiments, the concentration of the cytokinin in the co-culture medium is about 0.1 mg / L to about 50 mg / L. In specific embodiments, 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 specific embodiments, 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 from about 0.1 mg / L to about 500 mg / L.

[0031] In a specific embodiment, the colony is defined as a colony of dicotyledonous plant embryo explants. In some embodiments, the heterologous polynucleotide molecule comprises a selectable marker gene, the regeneration medium comprises a selection agent, and the selectable marker gene provides resistance to the selection agent in the plant. In certain embodiments, a plurality of genetically modified plants or plant parts comprise at least one genetic modification. In a specific embodiment, at least one genetic modification comprises integrating or inserting a heterologous polynucleotide molecule or a fragment thereof into the genome of a 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, at least one genetic modification comprises editing introduced into the genome of a plurality of genetically modified plants or plant parts using a site-specific nuclease or a guide RNA molecule through genome editing technology. In certain embodiments, the heterologous polynucleotide molecule comprises at least one expression cassette, and 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, the at least two expression cassettes comprising 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), thiadiazole (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino) purine (2iP), and 6-(3-hydroxybenzylamino) purine (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, 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 include regenerating or growing a plurality of genetically modified plants or plant parts in contact with a regeneration medium at about 15°C to about 40°C. In certain embodiments, the colony of embryo explant is a colony of cotton embryo explant, and described method comprises contacting regeneration medium at about 30 ℃ to about 40 ℃ and regenerating or growing multiple genetic modification plants or plant parts and continuing the first regeneration period.In some embodiments, the first regeneration period is about 1 hour to about 14 days.In specific embodiments, method may also comprise contacting regeneration medium at about 20 ℃ to about 33 ℃ and regenerating or growing multiple genetic modification cotton plants or cotton plant parts and continuing the second regeneration period.In certain embodiments, the second regeneration period is about 7 days to about 56 days.In specific embodiments, described method comprises contacting regeneration medium at about 5 days to about 70 days or about 14 days to about 50 days and regenerating or growing multiple genetic modification plants or plant parts.In one embodiment, the colony of embryo explants is a colony of soybean embryo explants, and the method comprises contacting a regeneration medium to regenerate or grow a plurality of genetically modified plants or plant parts for about 5 days to about 70 days. In another embodiment, the colony of embryo explants is a colony of cotton embryo explants, and the method comprises contacting a regeneration medium to regenerate or grow a plurality of genetically modified plants or plant parts for about 14 days to about 70 days. In yet another embodiment, the colony of embryo explants is a colony of rapeseed embryo explants, and the method comprises contacting a regeneration medium to regenerate or grow a plurality of genetically modified plants or plant parts for about 14 days to about 70 days.

[0032] In some aspects, the colony of embryo explant is the colony of dicotyledonous plant embryo explant, and the method of the present disclosure also includes contacting regeneration with the second regeneration medium or growing multiple genetically modified plants or plant parts for the regeneration period of continuous extension. In some embodiments, the second regeneration medium includes at least one auxin, at least one cytokinin or at least one auxin and at least one cytokinin. The limiting examples of the auxin that may exist in the second regeneration medium include 2,4-dichlorophenoxy-acetic acid (2,4-D), 4-amino-3,5,6-trichloro-picolinic 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-methoxy-benzoic acid (medamba). Non-limiting examples of cytokinins that may be present in the second regeneration medium include 6-benzylaminopurine (BAP), thiadiazole (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino) purine (2iP) and 6-(3-hydroxybenzylamino) purine (topolin). In specific embodiments, the concentration of the plant growth hormone in the second regeneration medium is about 0.1 mg / L to about 15 mg / L. In certain embodiments, the concentration of at least one cytokinin in the second regeneration medium is about 0.1 mg / L to about 50 mg / L. In some embodiments, the method includes contacting the second regeneration medium at about 15 ° C to about 40 ° C to regenerate or grow multiple genetically modified plants or plant parts. In certain embodiments, the extended regeneration period is about 7 days to about 56 days. In a specific embodiment, the population of embryonic explants is a population of cotton embryonic explants, and the method further comprises transferring selected portions of the plurality of genetically modified cotton plants or cotton plant parts to a second regeneration medium prior to contacting with the second regeneration medium to regenerate or grow the plurality of genetically modified cotton plants or cotton plant parts.

[0033] In certain aspects, the population of embryonic explants is a population of dicotyledonous embryonic explants, and the methods of the present disclosure may further comprise regenerating or growing the plurality of genetically modified plants or plant parts in contact with a first elongation medium 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 embryonic explants is a population of rapeseed embryonic explants. Non-limiting examples of auxins that may be present in the first elongation medium include 2,4-dichlorophenoxy-acetic acid (2,4-D), 4-amino-3,5,6-trichloro-picolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthylacetic 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-methoxy-benzoic acid (dicamba). Non-limiting examples of cytokinins that may be present in the first elongation medium include 6-benzylaminopurine (BAP), thiadiazole (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (topolin). In some embodiments, the concentration of at least one auxin in the first elongation medium is from about 0.1 mg / L to about 15 mg / L. In certain embodiments, the concentration of at least one cytokinin in the first elongation medium is from about 0.1 mg / L to about 50 mg / L. In some embodiments, the method of the present disclosure may include regenerating or growing a plurality of genetically modified plants or plant parts in contact with the first elongation medium at about 15°C to about 40°C. In specific embodiments, the first elongation period is from about 7 days to about 56 days.

[0034] In some aspects, the colony of embryo explant is the colony of dicotyledonous plant embryo explant, and method of the present disclosure may also include contacting regeneration or growing multiple genetically modified plants or plant parts with the second elongation culture medium for the second elongation period. In certain embodiments, the second elongation culture medium comprises at least one auxin, at least one cytokinin or at least one auxin and at least one cytokinin. The limiting examples of the auxin that may exist in the second elongation culture medium include 2,4-dichlorophenoxy-acetic acid (2,4-D), 4-amino-3,5,6-trichloro-picolinic 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-methoxy-benzoic acid (dicamba). Non-limiting examples of cytokinins that may be present in the second elongation medium include 6-benzylaminopurine (BAP), thiadiazole (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (topolin). In certain embodiments, the concentration of at least one auxin in the second elongation medium is from about 0.1 mg / L to about 15 mg / L. In specific embodiments, the concentration of at least one cytokinin in the second elongation medium is from about 0.1 mg / L to about 50 mg / L. In some embodiments, the method of the present disclosure includes regenerating or growing a plurality of genetically modified plants or plant parts in contact with the second elongation medium at about 15°C to about 40°C. In specific embodiments, the second elongation period is from about 7 days to about 56 days.

[0035] 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, and is capable of genome editing at any target site in any genotype and species.

[0036] The present disclosure also provides methods for simultaneously transforming and editing many genotypes in a single experiment under the same conditions.

[0037] In addition, the present disclosure provides certain embodiments of batch transformation of mixed germplasm, which demonstrates flexible genotype regeneration and genome editing of up to 100 elite genotypes in soybean and corn via meristem-based transformation of seed embryo explants. In a specific embodiment, in soybean, more than 800 different edits were recovered at conserved target sites (such as near the locus of interest), with individuals representing almost every transformed germplasm.

[0038] In another embodiment, although more inbred-to-inbred variation was observed in corn, transformants were recovered from 23 of 40 female inbred lines. The present disclosure thus describes accelerating the implementation and deployment of genome editing strategies in breeding and product development for precision breeding.

[0039] Any embodiment or aspect of the present disclosure may be used in combination with any other embodiment or aspect described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Shown are graphical representations of similarity scores for soybean lines sampled after bulk transformation during genotyping.

[0041] Figure 2 Shown is a comparison of conversion and editing frequencies among groups by maturity in soybean batches. Figure 2 A shows the frequency of transformation events in each maturity group compared to the proportion of the batch consisting of that maturity group. Figure 2 B shows the total number of editing and non-editing events for groups by maturity.

[0042] Figure 3 Shown are the proportion of heritable editable reads by T-DNA copy number after bulk soybean transformation. DETAILED DESCRIPTION

[0043] Provide detailed description below to help those skilled in the art to practice the embodiment of the present disclosure.Can modify and change the embodiment described herein without departing from the essence or scope of the present disclosure.The invention provides for the colony of embryonic explants to be carried out the composition and method of co-transformation or genetic modification, it can comprise one or more following steps as described herein: explant preparation, explant rehydration, Rhizobiales bacteria inoculation and co-cultivation, particle bombardment, bud induction, prolonged bud induction and / or genetic modification plant or plant part regeneration or growth.The disclosure further provides the method for confirming and selecting the individual member comprising desired genotype, genetic modification, culture characteristic and / or phenotype in the described colony.

[0044] The present disclosure provides for the composition and method for introducing heterologous polynucleotides into a colony of embryo explants with different genotypes to introduce one or more genetic modifications. As described herein, genetic modification can include the site-directed integration of transgenic or DNA segments or transgenics, and / or genetic modification can include mutation or editing. The present disclosure represents a major advancement in the art, because it provides for once or jointly producing a composition and method for multiple genetically modified plants with different genotypes, without the need to adopt time-consuming, expensive and inefficient plant breeding or gene infiltration technology. The present disclosure further provides for studying or screening the method and composition of the variable phenotype produced when the same genetic modification is introduced into different plant germplasms or genetic backgrounds, which can be described as "germplasm x transgenic / editing (or prosthesis) interaction". Prior to the present disclosure, the analysis of this type of germplasm x transgenic / editing (or prosthesis) interaction generally involves producing transgenic events, editing and / or prostheses in a single germplasm, followed by hybridization or infiltration of the event, editing and / or prostheses into other germplasms, and this process is time-consuming, expensive and inefficient. Prior to the present disclosure, any genetic background or germplasm-dependent phenotypic differences could not be tested, observed, screened, or selected for a given transgenic event, edit, and / or trait until the event, edit, and / or trait had been crossed or introgressed into multiple germplasms over several generations. The present disclosure further provides methods and compositions for studying or screening for variable culture characteristics of different plant germplasms or genetic backgrounds when heterologous polynucleotides and / or genetic modifications are co-introduced into different plant germplasms or genetic backgrounds.

[0045] A. Explant Population Preparation

[0046] According to embodiments of the present disclosure, the colony of seed embryo explant can be produced by plant seeds, for producing genetically modified plants or plant parts. The colony of embryo explant can be produced by seed by applying mechanical force, for example, by cutting, grinding, scraping, crushing or destroying seed. The seed used according to the present disclosure can be gathered in the crops by the plant grown in fields or greenhouse, and can be mature or immature seed, but preferably can be mature seed. The example of the seed used according to the present disclosure includes but is not limited to monocotyledonous plant seeds, dicotyledonous plant seeds, corn seeds, wheat seeds, barley seeds, rice seeds, oat seeds, sorghum seeds, rye seeds, millet seeds, soybean seeds, cotton seeds, rapeseed seeds and Brassica (Brassica) seeds. The example of the embryo explant used according to the disclosure includes but is not limited to ripe embryo explant, immature embryo explant, monocotyledon embryo explant, dicotyledon embryo explant, corn embryo explant, wheat embryo explant, barley embryo explant, rice embryo explant, oat embryo explant, Chinese sorghum explant, rye embryo explant, millet embryo explant, soybean embryo explant, cotton embryo explant, rape embryo explant and Brassica embryo explant. Using ripe seed can provide the benefit or advantage of improving seed storage, explant preparation and / or cultivation.Examples of monocotyledonous plants, seeds, or explants that can be used according to embodiments of the present invention include those derived from any plant species within the Poaceae / Gramineae family of monocotyledonous plants or cereals and grasses, which can include any Zea corn or maize species, such as maize, any Oryza rice species, such as Oryza sativa, any Triticum wheat species, such as Triticum aestivum or Triticum turgidum var durum, any Hordeum barley species, such as Hordeum vulgare, any Avena oat species, such as Avenasativa, any Sorghum sorghum species, such as Sorghum bicolor or Sorghum vulgare, any Secale rye species, such as Secale cereale. cereale, any sugarcane species of the genus Saccharum, 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 can be used according to embodiments of the present invention include those derived from any plant species within the Fabaceae, Malvaceae, or Cruciferae families, which can 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, Brassica rapa, or Brassica rapa. rapa) or mustard (Brassica juncea).

[0047] According to some embodiments, methods and compositions for preparing, cultivating, selecting and using a population of explants, as well as explants or populations of cultured explants produced therefrom, are provided. As used herein, the term "explant" or "seed embryo explant" refers to a plant part or plant tissue that can be genetically modified and subsequently generated / regenerated into a genetically modified plant or plant part. An "explant" or "seed embryo explant" can refer to a plant seed or any part of a plant seed. In the case of a seed embryo explant, it includes at least a portion of a plant seed embryo. An "explant" or "seed embryo explant" can include an embryo explant excised from a plant seed, which can include at least a portion of an embryonic meristem. Alternatively, an "explant" or "seed embryo explant" can refer to a whole or complete plant seed, or a crushed, deformed, or partially opened plant seed that can be produced by any suitable mechanical method. Various methods for preparing plant seed explants and plant seed embryo explants are known in the art. When used to refer to an explant or seed embryo explant, "partially opened" refers to a change in the state of a plant seed, with one or more openings or cracks in the plant seed. In one embodiment, the explant of the plant seed of the present invention can be defined as the plant explant that comprises meristematic tissue or embryo meristematic tissue, and it contains the plant cell that can break up or grow to produce multiple plant structure (including but not limited to stem, root, leaf, germline tissue and seed).In certain embodiments, embryo explant can be defined as the whole or part of seed embryo that takes out from other non-embryo seed tissue, and also comprises whole or part of meristematic tissue or embryo meristematic tissue.In some embodiments, the present disclosure provides the embryo explant that is made up of the top part of the hypocotyl that lacks radicle, wherein has removed the remainder of seed basically from embryo explant.In further embodiment, the present disclosure provides the embryo explant that does not germinate and keeps survival and can carry out genetic modification. As used herein, term " embryo explant of cultivation " refers to be in cultivation but not yet be regenerated into the embryo explant of plant or plant part.In specific embodiment, the embryo explant of cultivation can be the embryo explant of genetic modification.In some embodiments, the embryo explant of cultivation can contact with co-culture medium, bud inducing culture medium, prolongation bud inducing culture medium or regeneration medium and cultivate.In some embodiments, can contact the embryo explant of cultivation from the cultivation of monocotyledonous plant seed with co-culture medium, bud inducing culture medium, prolongation bud inducing culture medium and regeneration medium after any inoculation step, and in some embodiments, can contact the embryo explant of cultivation from the cultivation of dicotyledonous plant seed with co-culture medium and regeneration medium after any inoculation step.As used herein, " colony of embryo explant " refers to one group of explant from same plant species.In some embodiments, the colony of explant can include the explant with identical or different genotype, germplasm and / or genetic background.In certain embodiments, the genotype of the explant in the colony can be known, or can be unknown.In specific embodiments, the colony of embryo explant can refer to a group of embryo explant comprising the embryo explant of at least two different plant genotypes.In specific embodiments, the colony of embryo explant provided by the disclosure has at least 3 kinds, at least 4 kinds, at least 5 kinds, at least 6 kinds, at least 7 kinds, at least 8 kinds, at least 9 kinds, at least 10 kinds, at least 15 kinds, at least 20 kinds, at least 25 kinds, at least 30 kinds, at least 35 kinds, at least 40 kinds, at least 45 kinds, at least 50 kinds, at least 60 kinds, at least 70 kinds, at least 80 kinds, at least 90 kinds, at least 100 kinds, at least 150 kinds, at least 200 kinds, at least 250 kinds, at least 300 kinds, at least 350 kinds, at least 400 kinds, at least 450 kinds or at least 500 kinds of 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.In certain embodiments, the present disclosure provides a population of embryonic explants comprising 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, or more. 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 4 0,000, 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 500, about 10 to about 500, about 15 to about 200, or about 200,000 embryonic explants. In some embodiments, the present invention provides the explant of at least one embryo of the present invention.Approximately 400, approximately 20 to approximately 300, approximately 25 to approximately 200, approximately 10 to approximately 150, approximately 10 to approximately 100, approximately 10 to approximately 90, approximately 10 to approximately 80, approximately 10 to approximately 70, approximately 10 to approximately 60, approximately 10 to approximately 50, approximately 10 to approximately 40, approximately 10 to approximately 30, approximately 10 to approximately 20, 2 to approximately 50, 2 to approximately 40, 2 to approximately 30, 2 to approximately 20, 2 to approximately 15 or 2 to approximately 10 embryo explants.In certain embodiments, can manually or adopt automated process to produce according to explant of the present disclosure.For example, can from seed, remove seed tissue by cutting, grinding, scraping, crushing, destruction or any other similar method.Also can implement manually or automated method and remove unnecessary seed part.During the explant purification period, can use fluid that explant is separated with fragment, the limiting examples of said fluid comprises compressed air, other gas and liquid.

[0048] In certain embodiments provided herein, embryonic 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 embryonic explants are excised can have 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 5 or about 10 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.In certain embodiments, the present disclosure provides a population of plant seeds from which embryonic explants can be excised, comprising 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, or more. , 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 500, 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, 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.

[0049] In some embodiments, the method provided by the present disclosure includes sorting the plant seed population into at least two batches of plant seeds according to plant seed size and / or plant seed shape before excision of the embryo explant population. In some embodiments, sorting the seed population according to seed size and / or seed shape before excision can result in the number of explants excised from the seeds of each genotype present in the population being roughly equal. In certain embodiments, different excision methods and / or settings can be adopted to excise different batches of seeds sorted out according to seed size and / or seed shape to maximize explant excision efficiency. In specific embodiments, the same excision method and / or setting can be adopted to excise different batches of seeds sorted out according to seed size and / or seed shape. In some embodiments, two or more different batches of seeds sorted out according to seed size and / or shape can be merged before excision of the explant. In some embodiments, considering the relative transformation, cultivation and / or regeneration efficiency of two or more different batches of seeds sorted out according to seed size and / or shape, they can be merged in equal quantities or in different ratios or percentages before excision of the explant. For example, a batch of seeds having a higher frequency of conversion, cultivation and / or regeneration of explants can be added to a population of explant-excised seeds at a lower percentage than another batch of seeds having a lower frequency of conversion, cultivation and / or regeneration of explants. Alternatively, for example, explants can be excised separately from different batches of seeds and then combined in equal quantities or in different ratios or percentages before subsequent rehydration, cultivation and / or conversion steps, taking into account their relative efficiency of conversion, cultivation and / or regeneration.

[0050] In some embodiments, the embryo explant of the present invention can be excised from the seed that is dry, dry or wet. The mature plant seed can become drier as a part of its normal maturation process, although seed can be further dried before the explant excision, and / or the explant can be dried after excision from the seed. The plant embryo explant that is dry or dry excision can be used for genetic modification immediately, or can store a period of time for future use. The explant preparation can also comprise seed and / or explant being dried to required moisture content. According to the initial moisture content of seed or explant, seed and / or explant being dried to this required moisture content can improve the excision of seed and / or explant, storage and / or use. After the excision, can be through flushing, flotation or additive method as known in the art with the explant purification or with other seed material and fragment separation. In certain embodiments, the internal moisture of seed or explant provided by the disclosure is 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%, about 5% to about 10%, comprise 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% internal moisture, comprise all values ​​therebetween and derivable scope.Explant can be produced by the mature seed with moisture content as described herein.In specific embodiments, the moisture content of seed or explant can be measured before or after explant excision, before or after explant storage, during explant storage, before explant rehydration and / or before genetic modification or conversion. In one aspect or embodiment, a seed embryo explant can be defined as comprising meristem or embryo meristems, which contain plant cells that can differentiate or develop to produce a variety of plant structures, including but not limited to stems, roots, leaves, germline tissues, and seeds. In fact, an embryo explant can be defined as comprising all or part of a seed embryo removed from other non-embryonic seed tissues and also comprising all or part of the meristem or embryo meristems.

[0051] On the one hand, can prepare or use any embryo explant according to embodiment of the present disclosure.In specific embodiment, embryo explant can be mature embryo or immature embryo, and / or can comprise meristematic tissue, callus or any other tissue that can be transformed and regenerated.In some embodiments, mature embryo explant is the explant of dry excision.Can take out the explant of dry excision from seed, and almost directly be used as the target of conversion or genetic modification.In some embodiments, can take out the explant of dry excision or the explant of desiccation from ripe dry seed, and be used as the target of conversion or genetic modification, may only need the moistening, aquation or pre-cultivation step of minimum degree.In further embodiment, can use wet, dry wet or wet excision embryo explant as the target of conversion or genetic modification.In further embodiment, can dry immature or mature embryo explant or the embryo explant of excision before conversion or genetic transformation.As used herein, " wet " embryo explant refers to the explant of dry excision through moistening, aquation, imbibition or other minimum degree culture step before conversion or genetic modification. As used herein, " dry " embryo explant refers to the explant of the excision of the drying step before transformation or genetic modification.As used herein, " dry wet " embryo explant refers to the embryo explant that is prepared and then dried to stop germination by wetting for germination.As used herein, " wet excision " explant refers to the explant excised from the seed of imbibition or hydration.Wet embryo explant is hydrated or imbibed after excision from seed, and the embryo explant of wet excision is excised from the seed of hydration or imbibition.As used herein, " callus " refers to the propagation mass of unorganized, undifferentiated and / or dedifferentiated plant cells or tissue.

[0052] According to embodiments of the present invention, in some embodiments, the explants of the colony can be defined as comprising meristem or embryo meristem, which contains plant cells that can differentiate or develop to produce a variety of plant structures (including but not limited to stem, root, leaf, germline tissue and seed). In specific embodiments, the embryo explants of the colony can be defined as comprising all or part of a seed embryo taken out from other non-embryonic seed tissues, and also comprising all or part of the meristem or embryo meristem.

[0053] The explant used according to the disclosure can be genetically modified at different times after being separated, excised and / or taken out from the seed. In one embodiment, before use, the explant can be taken out from the seed less than one day, for example, from about 1 to about 24 hours, such as 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. In a further embodiment, before use, the explant can be stored for a long time, including several days, weeks, months or years. Methods and parameters for drying, storing, transforming, regenerating and / or germinating seeds or seed embryo explants are known in the art (see, for example, 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 specifically incorporated herein by reference in their entirety; 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 (Suppl 1):23-28, 1998). Any conditions may be employed as desired, including incubation or storage at, for example, a temperature of about -80°C to about 60°C. If the explants or seeds are stored in a refrigerator, they can be thawed prior to use in a subsequent step, wherein such thawing step can last for a period of time between about 20 minutes and about 4 hours or longer, or between about 30 minutes and about 2 hours, depending on the storage temperature and the number of seeds or explants to be thawed and brought to room temperature for explant excision or for transformation.

[0054] The present disclosure may relate in certain aspects to the sterilization of seeds or explants. Sterilization may include contacting the seed or explant material with various liquids or gases that are used to reduce or eliminate the presence of viable bacterial or fungal contaminants that might otherwise interfere with the developmental capacity of the seed or embryo. Sterilization by applying a liquid may also hydrate or partially hydrate the plant seed, explant, embryo, or tissue and serve the purpose of priming the seed, explant, embryo, or tissue. Methods of sterilization include, but are not limited to, the use of chlorine, ozone, bleach or alcohol solutions, ultraviolet light, temperatures of -20°C or lower, and exposure to temperatures above 40°C. In some embodiments, the sterilizing medium may contain polyethylene glycol and / or an antifungal or antimicrobial agent.

[0055] In one aspect of the present disclosure, the explant can be rehydrated before transformation or genetic modification. Rehydration medium or solution is 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 antimicrobial 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 will generally not contain any plant hormones, such as auxins or cytokinins. Having an optimal rehydration time period may be important. If the explant is in the rehydration medium for too long, the explant may become "mushy" and unable to regenerate or survive, but if the explant is in the rehydration medium for too short a period of time, the explant may not be fully rehydrated and therefore may not be transformed, or the transformation efficiency may be low. The embryonic 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 a shorter period of time, but can tolerate rehydration for a maximum of about 1 hour to about 2 hours. In one embodiment, prior to transformation or genetic modification, the rehydration of seed embryo explants can be carried out 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 for 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 or equal to about 4 hours, or less than about 4 hours. In some embodiments, the rehydration medium may be shaken or agitated during this step, such as on a shaker or shaker, or otherwise physically or mechanically agitated, moved, or turned over to improve rehydration and / or reduce the time it takes for the explant to be rehydrated.In a specific embodiment, the embryo explant can be a monocotyledonous plant embryo explant, and before conversion or genetic modification, the seed embryo explant can be rehydrated at least about 2 hours, compared with the explant of about 1 hour or shorter time of rehydration, this can improve the rate of recovery of the plant of conversion or editing frequency or conversion or editing. In a specific embodiment, the embryo explant can be a dicotyledonous plant embryo explant, and the rehydration of the embryo explant can be carried out about 1 hour or about 15 minutes to about 4 hours. In certain embodiments, the embryo explant can be rinsed to remove the rehydration culture medium before subsequent steps.

[0056] In certain embodiments, can be before subsequent step, rinse embryo explant to remove rehydration culture medium.In certain embodiments, embryo explant can be rinsed approximately 20 seconds to approximately 10 minutes, approximately 20 seconds to approximately 9 minutes, approximately 20 seconds to approximately 8 minutes, approximately 20 seconds to approximately 7 minutes, approximately 20 seconds to approximately 6 minutes, approximately 20 seconds to approximately 5 minutes, approximately 20 seconds to approximately 4 minutes, approximately 20 seconds to approximately 3 minutes, approximately 20 seconds to approximately 2 minutes, approximately 20 seconds to approximately 1 minute, approximately 1 minute to approximately 10 minutes, approximately 1 minute to approximately 6 minutes, approximately 2 minutes to approximately 6 minutes or approximately 3 minutes to approximately 5 minutes, comprise all values ​​and scope that can be derived therebetween. In certain embodiments, embryo explant can be made to stand approximately 1 round to approximately 10 rounds, approximately 1 round to approximately 9 rounds, approximately 1 round to approximately 8 rounds, approximately 1 round to approximately 7 rounds, approximately 1 round to approximately 6 rounds, approximately 1 round to approximately 5 rounds, approximately 1 round to approximately 4 rounds, approximately 1 round to approximately 3 rounds, approximately 1 round to approximately 2 rounds, approximately 2 rounds to approximately 6 rounds or approximately 3 rounds to approximately 5 rounds of flushing, comprise all values ​​and scopes that can be derived therebetween.In specific embodiments, the flushing of embryo explant can be carried out in container, or by being placed in filter, mesh cloth etc., makes rinsing solution flow through explant and flush.In specific embodiments, can by shifting out, decant and / or suction rehydration solution or liquid, then add rinsing solution or liquid, rinse embryo explant thus in container. Can shift out, decant and / or suction rinsing solution or liquid of first volume, be replaced with new rinsing solution or liquid then, these operations can be repeated one or many (that is, carry out two rounds or more of the flushing altogether).In some embodiments, rehydration solution or liquid and rinsing solution or liquid are identical solution or liquid. In certain embodiments, rehydration solution or liquid and flushing solution or liquid are different solutions or liquids.In some embodiments, flushing solution is water.After the rehydration and / or the flushing of explant, can remove rehydration and / or flushing culture medium, as by decantation, pipetting, vacuum treatment and / or suction culture medium, and can by blotting, wicking or other method dry explant, as contact with rehydration or flushing solution or liquid with filter paper or other absorbing material, to remove at least excessive rehydration or flushing solution or liquid.For the more responsive explant to excessive rehydration, blotting etc. may be particularly useful or necessary.

[0057] As used herein, "genetically modified" plants, plant parts, plant tissues, explants or plant cells comprise genetic modifications, such as mutations, edits or transgenes introduced into the genome of plants, plant parts, plant tissues, explants or plant cells by genetic engineering, which can be carried out via genetic transformation, mutagenesis or genome editing techniques. As used herein, "genetic modification" refers to one or more transgenic events, mutations and / or edits introduced into the genome of plants, plant parts or plant cells using transformation, mutagenesis or genome editing techniques. In addition to genome editing techniques, mutagenesis techniques can include any chemical, physical, radiological or biological (e.g., transposon-mediated) mutagenesis techniques or mutagens. As used herein, "transgenic" plants, plant parts, plant tissues, explants or plant cells have exogenous nucleic acid sequences, polynucleotides, expression cassettes or transgenes that are integrated into the genome of plants, plant parts, plant tissues, explants or plant cells. In certain embodiments, genetically modified plants, plant parts, plant tissues, explants or plant cells can comprise heritable edits or non-heritable edits. In some embodiments, heritable or non-heritable edits can be confirmed by genetic sequencing. When a sample comprising polynucleotide molecules from or derived from a genetically modified plant, plant part, plant tissue, explant, or plant cell is subjected to genetic sequencing, and 10% of the sequencing reads contain the expected edit, then the edit is likely heritable and, therefore, can be described as a "heritable edit." When a sample comprising polynucleotide molecules from or derived from a genetically modified plant, plant part, plant tissue, explant, or plant cell is subjected to genetic sequencing, and 1-10% or, perhaps, 0.1%-10%, 0.1%-5%, or 0.1%-1%, or more than 0% of the sequencing reads contain the expected edit, then the edit is likely not heritable and, therefore, can be described as a "non-heritable edit."

[0058] In some embodiments of the present disclosure, transformation or editing of embryonic explants or plants can be measured by genotyping, number of shoots produced after Agrobacterium-mediated inoculation (or number of regenerated shoots), % of shoots produced after Agrobacterium-mediated inoculation (or % of regenerated shoots), relative shoot presence, number of transformants, % of transformants, relative transformation rate, number edited, % edited, relative editing rate, number edited (heritable editing), % edited (heritable editing), relative editing rate (heritable), number edited (non-heritable editing), % edited (non-heritable editing), or relative editing rate (non-heritable), as described herein.

[0059] B. Introduction of Heterologous Polynucleotide Molecules, Ribonucleoproteins, or Nucleases

[0060] This paper provides the method and composition for the common genetic transformation of the colony of embryo explant or modification.In specific embodiments, heterologous polynucleotide molecule, ribonucleoprotein and / or site-specific nuclease are introduced into at least two embryo explants of described colony together.As used herein, term " introducing together " refers to heterologous polynucleotide molecule, ribonucleoprotein and / or site-specific nuclease being introduced into one or more explants of colony at roughly the same time and / or when the explant of colony is in identical or roughly the same region or in one or more containers.In some embodiments, heterologous polynucleotide molecule, ribonucleoprotein and / or site-specific nuclease can be synchronous, simultaneously or roughly simultaneously introduced into the explant of described colony together.In specific embodiments, the colony of embryo explant can comprise one or more groups or many batches of embryo explants.In certain embodiments, each group or each batch of described colony can be present in independent container, and these independent containers can have identical approximate cross-sectional area or volume, and heterologous polynucleotide molecule, ribonucleoprotein and / or site-specific nuclease can be introduced into each group or each batch of described colony together roughly simultaneously. In further embodiments, heterologous polynucleotide molecules, ribonucleoproteins, and / or site-specific nucleases can be co-introduced into a population of embryonic explants while the individual explants of the population are present together in a single container.

[0061] The embodiment of the present disclosure may include introducing heterologous polynucleotide molecules, ribonucleoproteins and / or site-specific nucleases together by any suitable method or technology as known in the art such as electroporation, micro-bullet or particle bombardment, microinjection, PEG-mediated transformation, rhizobium or Agrobacterium-mediated transformation and other direct DNA absorption modes, thereby genetically transforming or modifying at least one cell of each of at least two embryo explants of the colony.Then all or part of the heterologous polynucleotides can be transformed or incorporated into the genome of the plant cell, expressed in one or more editing molecules or tools (such as guide RNA and / or site-specific nuclease), and / or provide a template for editing or site-specific integration.According to many embodiments, heterologous polynucleotides are introduced into the colony via rhizobium or Agrobacterium-mediated transformation.

[0062] In a specific embodiment, before rhizobium or agrobacterium-mediated transformation, Rhizobiumales bacteria or Agrobacterium can be cultured in a culture medium comprising optional one or more cytokinins and / or lipoic acid. Alternatively, the culture medium for cultivating Rhizobiumales bacteria or Agrobacterium may not contain or comprise cytokinins and / or lipoic acid. This culture medium may comprise, for example, water, basal salts, macronutrients, micronutrients and / or vitamins. In certain embodiments, the culture medium may comprise polyethylene glycol and / or an antimicrobial and / or 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 medium include 6-benzylaminopurine (BAP), thiadiazole (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino) purine (2iP) and 6-(3-hydroxybenzylamino) purine (topolin). In some embodiments, the concentration of the cytokinin in the bacterial culture medium can be between about 0.1 mg / L and about 50 mg / L, about 0.1 mg / L and about 45 mg / L, about 0.1 mg / L and about 40 mg / L, about 0.1 mg / L and about 35 mg / L, about 0.1 mg / L and about 30 mg / L, about 0.1 mg / L and about 25 mg / L, about 0.1 mg / L and about 20 mg / L, about 0.1 mg / L and about 15 ...5 mg / L, about 0.1 mg / L and about 20 mg / L, about 0.1 mg / L and about 15 mg / L, about 0.1 mg / L and about 15 mg / L, about 0.1 mg / L and about 25 mg / L, about 0.1 mg / L and about 25 mg / L, about 0.1 mg / L and about about 0.1 mg / L to about 10 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 0.1 mg / L to about 1 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 1 mg / L to about 5 mg / L, about 1 mg / L to about 10 mg / L. / 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, or a concentration of 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. g / 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 values ​​and ranges derivable therebetween.In certain embodiments, lipoic acid can be present in the bacterial culture medium at a concentration range of about 0.1 mg / L to about 500 mg / L, about 0.1 mg / L to about 400 mg / L, about 0.1 mg / L to about 300 mg / L, about 0.1 mg / L to about 200 mg / L, about 10 mg / L to about 200 mg / L, about 10 mg / L to about 180 mg / L, about 10 mg / L to about 160 mg / L, about 10 mg / L to about 140 mg / L, about 10 mg / L to about 120 mg / L, about 10 mg / L to about 100 mg / L, about 20 mg / L to about 80 mg / L, about 40 mg / L to about 60 mg / L, about 50 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, 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 11 0 mg / L, about 115 mg / L, about 120 mg / L, about 125 mg / L, about 130 mg / L, about 135 mg / L, about 140 mg / L, about 145 mg / L, about 150 mg / L, about 155 mg / L, about 160 mg / L, about 165 mg / L, about 170 mg / L, about 175 mg / L, about 180 mg / 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 mg / L, 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 ranges and values ​​derivable therebetween. In specific embodiments, including one or more cytokinins and / or lipoic acid in the bacterial culture medium can increase the developmental capacity, regeneration capacity and / or transformation / editing frequency following Rhizobium or Agrobacterium-mediated transformation. In certain embodiments, including lipoic acid in the bacterial culture medium reduces stress during Rhizobium- or Agrobacterium-mediated transformation.In some embodiments, the inclusion of one or more cytokinins and / or lipoic acid in the bacterial culture medium increases the developmental capacity, regeneration capacity, and / or transformation / editing frequency of dicot embryo explants. In a specific embodiment, the inclusion of one or more cytokinins and / or lipoic acid in the bacterial culture medium increases the developmental capacity, regeneration capacity, and / or transformation / editing frequency of soybean embryo explants.

[0063] In some embodiments, thidiazuron (TDZ) can be included in the bacterial culture medium at a concentration range 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 0.1 mg / L to about 1 mg / L, about 0.25 mg / L to about 1.75 mg / 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 From about 4 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 ranges and values ​​derivable therebetween. In some embodiments, cytokinins other than thidiazuron (TDZ), such as 6-benzylaminopurine (BAP), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (topolin) may be included in the bacterial culture medium, and their concentrations are adjusted and set according to their relative activities.For example, 6-benzylaminopurine (BAP), kinetin, zeatin, and / or 6-(3-hydroxybenzylamino)purine (topolin) can be present in the bacterial culture medium at 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. To about 8 mg / L, about 2.5 mg / L to about 7.5 mg / L, about 3 mg / L to 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 ranges and values ​​derivable therebetween, which can improve the developmental capacity, regeneration capacity and / or transformation / editing frequency of embryonic explants (such as dicotyledonous embryonic explants or soybean, cotton or rapeseed embryonic explants) after Rhizobium or Agrobacterium-mediated transformation. In some embodiments, 6-(γ,γ-dimethylallylamino)purine (2iP) can be present in the bacterial culture medium at a concentration 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 In some embodiments, the present invention provides a method for increasing the development capacity, regeneration capacity and / or transformation / editing frequency of soybean embryo explants after Rhizobium- or Agrobacterium-mediated transformation.

[0064] Embodiments of the present disclosure can include by introducing heterologous polynucleotide molecules jointly via rhizobium or agrobacterium-mediated conversion to carry out genetic transformation or genetic modification to embryo explant (at least one cell of each of at least two embryo explants as colony), and described conversion is generally related to making explant contact with Rhizobiales bacterium or Agrobacterium containing heterologous polynucleotide.This introducing step can include making the colony of explant or explant be present in the inoculation medium containing Rhizobiales bacterium or Agrobacterium, explant or explant's colony is added in the described inoculation medium and / or contacted with the described inoculation medium, and allow inoculation step to carry out for a period of time, from explant, remove Rhizobiales bacterium or Agrobacterium afterwards.Inoculation medium can comprise for example water, basal salts, macronutrients, micronutrients and / or vitamin.In certain embodiments, inoculation can comprise polyethylene glycol and / or antimicrobial and / or antifungal agent.Can measure and / or limit the concentration of Rhizobiales bacterium or Agrobacterium in inoculation medium according to optical density (OD). Higher Agrobacterium concentrations in the inoculation medium and / or co-culture medium can improve or increase the germination frequency and cultivation frequency, thereby improving or increasing the transformation frequency. The OD concentration of the Rhizobiales bacteria or Agrobacterium in the inoculation medium 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 0.25 to about 1.0, about 0.25 to about 0.5, 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, 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 ranges and values ​​derivable therein. According to some embodiments, transformation of some monocotyledonous plants or corn germplasm (including explants from male corn lines) can be improved by reducing the OD concentration of the Rhizobiales bacteria or Agrobacterium in the inoculation medium (e.g., in the range of about 0.25 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 ranges and values ​​derivable therebetween).

[0065] In certain embodiments, the inoculation medium may optionally include thiabendazole (TBZ) and / or nystatin, which can be used as antimicrobial or antifungal agents. The concentration of TBZ can 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 at a concentration of about 10 g / L, and / or the concentration of nystatin can 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 at a concentration of about 50 g / L. In certain embodiments, the inoculation medium may include the aforementioned Rhizobiales or Agrobacterium culture medium, which can be transferred in a volume to contact the explant. In certain embodiments, the inoculation medium may optionally contain or include cytokinins and / or lipoic acid. According to some embodiments, the explant (or a group or multiple explants) can be transferred or placed into a tube or container, and an amount of the Rhizobiales or Agrobacterium culture medium can be added to the tube or container. An amount of inoculum (or inoculation medium containing Rhizobia or Agrobacterium) can be added to the tube or container in an amount or volume sufficient to cover and / or submerge the explant.

[0066] According to some embodiments, can choose optionally the pipe or the container of the explant that is contained in the inoculation medium to carry out sonication, vortex or otherwise carry out physics or mechanical agitation.This type of agitation process can contribute to improving the conversion and / or sending or introducing of heterologous polynucleotide in the cell of explant, as by destroying and / or by increasing the infiltration or penetration in meristematic tissue or the explant tissue of Rhizobiales bacterium.According to some embodiments, can carry out sonication or agitation to explant, the time period scope that continues is approximately 1 second to approximately 10 minutes or approximately 5 seconds to approximately 5 minutes or approximately 10 seconds to approximately 4 minutes or approximately 10 seconds to approximately 3 minutes, or approximately 10 seconds, approximately 20 seconds, approximately 30 seconds, approximately 40 seconds, approximately 50 seconds, approximately 1 minute, approximately 1.5 minutes, approximately 2 minutes, approximately 2.5 minutes, approximately 3 minutes or approximately 4 minutes, comprise all ranges and the value that can be derived therebetween. As part of this inoculation step, the explant may be (subsequently) agitated, moved, or turned more gently (than the previous more vigorous agitation or disruption), such as by shaking or rocking, with or without a physical or mechanical agitation step, to improve transformation and / or delivery or introduction of the heterologous polynucleotide into the cells of the explant. This gentle movement may be for a period of time ranging from about 5 seconds to about 2 hours or from about 5 seconds to about 20 minutes or from about 10 seconds to about 15 minutes or from about 1 minute to about 15 minutes or from about 5 minutes to about 15 minutes or from about 7.5 minutes to about 12.5 minutes or from about 1 minute to about 2 hours or from about 5 minutes to about 2 hours or from about 10 minutes to about 2 hours or from about 20 minutes to about 1.5 hours or from about 30 minutes to about 1.5 hours or from about 45 minutes to about 1.25 hours or from about 1 minute to about 1 hour or from about 5 minutes to about 4 ... 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 ranges and values ​​derivable therein.

[0067] In some embodiments, the introducing or inoculating step can be performed under ambient light conditions. In some embodiments, such as for monocotyledonous embryonic explants, the introducing or inoculating step can include subjecting the population of embryonic explants to a force treatment, such as centrifugation and / or pressure treatment. According to some embodiments, the heterologous polynucleotide molecules, ribonucleoproteins, and / or site-specific nucleases are introduced into the population via particle bombardment.

[0068] According to the embodiment of the present disclosure, before or during inoculation, or before and during inoculation of the colony with Rhizobiales or Agrobacterium bacteria comprising heterologous polynucleotide molecules, the colony of the seed embryo explant is subjected to force treatment. In certain embodiments, during and / or after the rehydration of the seed embryo explant, force treatment is applied. In a specific embodiment, when the colony contacts with the inoculation medium, force treatment can be applied during the inoculation step. In one embodiment, the explant "contacting" with the "substratum" can be located in or on the substratum completely or in part. The non-limiting examples of the substratum that can contact the explant include liquid culture medium, solid culture medium and the matrix that comprises the substratum. When force treatment is applied, the colony can be immersed in a certain volume of inoculation medium. Alternatively, force treatment can be applied to the embryo explant of the colony after having removed excessive inoculation medium. For example, before force treatment is applied, the inoculation medium can be decantated, poured out, aspirated or blotted dry from the explant. If force treatment is applied during the inoculation step, the inoculation medium will not be completely free of contact with the explants even if a certain amount or volume of inoculation medium is removed from the explants of the population prior to force treatment.

[0069] As used herein, the term "heterologous polynucleotide molecule" refers to a polynucleotide molecule that is not naturally occurring or does not naturally occur in the same form or structure in a transformed or modified cell without human intervention. For example, a heterologous polynucleotide molecule may not be naturally occurring in a transformed or modified plant species, or may be expressed in a manner or genomic background that is different from the natural expression pattern or genomic background found in the transformed or modified species, such as in some embodiments, a heterologous polynucleotide molecule may be overexpressed. In specific embodiments, a heterologous polynucleotide molecule may be a combination of two or more polynucleotide molecules, wherein such a combination is not typically found in nature. In certain embodiments, the two polynucleotide molecules may be derived from different species, or may be derived from different genes, such as different genes from the same species or the same gene from different species. In some embodiments, a heterologous polynucleotide molecule may comprise two polynucleotide sequences that are not juxtaposed or operably linked in any naturally occurring polynucleotide molecule. In further embodiments, a heterologous polynucleotide molecule 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-stranded or double-stranded DNA or RNA polynucleotide molecule or sequence that can be derived from any source. For example, a polynucleotide molecule can comprise a polynucleotide sequence in which one or more nucleic acid sequences have been linked together in a functionally operable manner. As used herein, the term "nucleic acid sequence" refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence.

[0070] As used herein, the term "ribonucleoprotein" refers to a protein that can interact with nucleic acid or polynucleotide molecules.Ribonucleoprotein can be, for example, a site-specific nuclease associated with nucleic acid or polynucleotide molecules as known in the art. Non-limiting examples of site-specific nucleases that can be ribonucleoproteins include RNA-guided endonucleases, such as those of CRISPR / Cas systems (see, for example, U.S. Patents 8,697,359, 8,771,945, and 9,790,490 and U.S. Patent Application Publication No. 2014 / 0068797) and CRISPR-related transposases or CAST (see, for example, U.S. Patent Application Publication No. 2020 / 0190487), the entire contents of the above-mentioned patent documents and are disclosed herein are incorporated by reference. In some embodiments, polynucleotide molecules, heterologous polynucleotide molecules or ribonucleoproteins can be recombinant polynucleotide molecules or recombinant proteins. In some embodiments, polynucleotide molecules or heterologous polynucleotide molecules can be recombinant polynucleotide molecules. As used herein, the term "recombinant" when referring to polynucleotide (DNA or RNA) molecules, proteins, constructs, vectors, etc., refers to polynucleotide or protein molecules or sequences that are not naturally occurring or that are not naturally occurring in the same form and / or structure without human intervention. In specific embodiments, recombinant polynucleotide (DNA or RNA) molecules, proteins, constructs, vectors, etc. can include, for example, a combination of two or more polynucleotide or protein sequences that are not naturally occurring together in the same manner, such as a polynucleotide molecule, protein, construct, etc. that comprises at least two polynucleotide or protein sequences that are operably connected but are heterologous to each other. In other 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., plasmid, construct, vector, chromosome, protein, etc.), wherein such combination is artificial and is not typically found in nature. As used herein, the phrase "not typically found in nature" means not found in nature without human intervention. Recombinant polynucleotide or protein molecules, constructs, etc. can comprise polynucleotide or protein sequences that are (i) separated from other polynucleotide or protein sequences that exist near each other in nature, and / or (ii) adjacent to (or contiguous with) other polynucleotide or protein sequences that are not naturally near 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, recombinant polynucleotide molecules can include any engineered or artificial plasmids, vectors, etc., and can include linear or circular polynucleotide molecules.Such plasmids, vectors, etc. may contain various maintenance elements including, for example, a prokaryotic origin of replication and a selectable marker, and one or more transgenes or expression cassettes, possibly in addition to a plant selectable marker gene.

[0071] In order to improve the transformation or editing of the colony of explants, a variety of different force treatments can be used or applied to the colony before and / or during the inoculation step, such as centrifugal force treatment, gravity treatment, vacuum treatment, sonication, vortex treatment, shear treatment, mechanical force treatment, pressure treatment or any combination thereof. In some embodiments, force treatment may include pressure treatment and / or gravity (or centrifugal) force treatment. In specific embodiments, force treatment may include pressure treatment. In further embodiments, force treatment may include gravity (or centrifugal) force treatment. In certain embodiments, in addition to pressure treatment and / or gravity (or centrifugal) force treatment, the methods described herein may also include applying mechanical force treatment, vortex treatment, shaking or shear treatment, sonication and / or vacuum treatment. Without being bound by theory, applying force treatment before or during inoculation can improve transformation by increasing the contact of Rhizobiales bacteria with the explants of the colony and the attachment of Rhizobiales bacteria to the explants of the colony, by destroying the explants and / or by increasing the penetration or penetration of Rhizobiales bacteria into meristems or explant tissues.

[0072] In some embodiments, the force treatment can include applying a pressure or treatment to the population of embryonic explants in the range of about 100 pounds per square inch (psi) to about 20,000 psi, about 100 psi to about 18,000 psi, about 100 psi to about 16,000 psi, about 100 to about 14,000 psi, about 100 to about 12,000 psi, about 100 to about 10,000 psi, about 100 to about 8,000 psi, about 100 to about 6,000 psi, 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, such as 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 values ​​and ranges derivable therein. Other units of pressure are also known in the art. Methods for converting pressure from psi to other units are known in the art, such as standard atmospheres (atm) and Newtons (N) per square meter (N / m 2 The pressure in atm can be accurately calculated using the following formula: atm = pressure (psi) / 14.6959488, where 1 psi is equal to approximately 6894.76 N / m 2Thus, 100 psi is equal to about 6.80 atm, and 20,000 psi is equal to about 1360.9 atm. When the surface area is known or fixed, the pressure treatment can also be converted into an amount of force. For example, the French Press 40K pressure unit ( IEC, FA-032) has a piston / unit cavity surface area of ​​approximately 0.88 in 2 Therefore, 3,334 psi applied using a French Press 40K pressure unit is equivalent to approximately 13,000 N [(3,334 psi x 0.88 in 2 )] / [0.225 lbs / N]. In some embodiments, the pressure treatment can 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 values ​​and ranges derivable therein.

[0073] The methods described herein include applying gravity or centrifugal forces in the range of about 100 x g to about 10,000 x g, about 100 x g to about 5,000 x g, about 250 x g to about 5,000 x g, about 500 x g to about 5,000 x g, about 500 x g to about 3,000 x g, about 600 x g to about 2,700 x g, such as about 500 x g, about 550 x g, about 600 x g, about 650 x g, about 700 x g, about 750 x g, about 800 x g, about 850 x g, about 900 x g, about 950 x g, about 1000 x g, about 1500 x g, about 2000 x g, about 2500 x g, about 3000 x g, about 3500 x g, or about 4000 x g. g, including all values ​​and ranges derivable therein. Non-limiting examples of gravity treatments that can be applied to the population include centrifugal force or relative centrifugal force that can be applied using an appropriate 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. The relative centrifugal force can be calculated based on the rpm and known dimensions of the device using the following formula: rpm = √[RCF / (r×1.118)×1×10 5 ], where r = radius of rotation in centimeters. For the Sorvall gyroscope used in the examples described herein TM RC3BP centrifuge (ThermoFisher Scientific, Waltham, MA, USA) with a rotation radius of approximately 24.67 cm. Therefore, 2620 x g is equal to approximately 3,082 rpm [√[2620 / (24.67 x 1.118)] x 1 x 105 ]. Similarly, the centrifugal force in Newtons can be accurately estimated using the following formula: Force (N) = RCF x mass of the contents of the centrifuge tube (kg) x 9.82 m / s 2 In a particular embodiment, if the mass of the contents of the centrifuge tube is about 0.05 kg, then 2620 x g would be equal to about 1286 N [(2620 x g) x 0.05 kg x 9.82 m / s 2 In some embodiments, gravity or centrifugal force can be applied for a period of time ranging from 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, such as 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 values ​​and ranges derivable therein.

[0074] According to some embodiments, the methods described herein comprise subjecting the monocot seed embryo explant to a high gravity or centrifugal force or relative centrifugal force (RCF) ranging from about 2,500 x g to about 10,000 x g, about 2,500 x g to about 5,000 x g, about 2,500 x g to about 4,500 x g, about 2,800 x g to about 5,000 x g, about 2,800 x g to about 4,500 x g, about 3,000 x g to about 8,000 x g, about 3,000 x g to about 7,000 x g, about 3,000 x g to about 6,000 x g, about 3,000 x g to about 5,000 x g, about 3,500 x g to about 5,000 x g, or about 3,500 x g to about 5,000 x g. ,500xg to about 4,500xg, such as about 2,500xg, about 3,000xg, about 3,500xg, about 3,600xg, about 3,700xg, about 3,800xg, about 3,900xg, about 4,000xg, about 4,100xg, about 4,200xg, about 4,300xg, about 4,400xg, about 4,500xg, about 4,600xg, about 4,700xg, about 4,800xg, about 4,900xg, about 5,000xg, about 6,000xg, about 7,000xg, about 8,000xg, about 9,000xg or about 10,000xg, including all ranges and values ​​derivable therebetween. According to some embodiments, applying higher gravity or centrifugal force or relative centrifugal force (RCF) can improve the transformation and / or regeneration of monocot or corn embryo explants or genetically modified plants of certain monocot or corn lines (such as certain male germplasm corn lines) that are more resistant to efficient transformation and / or regeneration of genetically modified plants.

[0075] Force treatment, such as gravity (or centrifugal) and / or pressure treatment, can be applied at a temperature 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 values ​​and ranges derivable therein.

[0076] In one aspect of the method provided herein, force treatment may include applying pressure treatment and gravity treatment to the colony of the embryo explant. Pressure treatment and / or gravity treatment may be applied before, during, or before and during the colony is inoculated with the bacteria from the Rhizobium orders, wherein the Rhizobium orders bacteria comprise the heterologous polynucleotides of at least one plant cell of the explant of the colony for conversion, editing or genetic modification. In some embodiments, pressure treatment is applied before gravity treatment is applied. In other embodiments, gravity treatment is applied before pressure treatment. The order of applying pressure treatment and gravity treatment may preferably be based on the conversion or editing efficiency or frequency of improvement or based on the ease of treatment. In some embodiments, when the colony is subjected to a combination of pressure treatment and gravity treatment, pressure treatment may be applied before gravity treatment, which may be at least partially due to the fact that force treatment can be applied more evenly before the explant is granulated with gravity treatment or centrifugation. Alternatively, the explant of centrifugation or granulation may be resuspended before subsequent pressure treatment, or the explant of centrifugation or granulation may be subjected to pressure treatment without resuspending. In one aspect of the present disclosure, applying pressure treatment and gravity treatment before, during, or both before and during inoculation improves transformation or editing of plants compared to applying pressure treatment only or gravity treatment only.

[0077] On the other hand, method as herein described may also comprise that the colony of embryo explant is applied vacuum treatment.Vacuum treatment may comprise for example being immersed in the liquid inoculation medium that comprises Rhizobiales bacterium, and making the colony stand pressure reduction, then quickly or gradually repressurize.Perhaps, the colony that is not immersed in the liquid inoculation medium of embryo explant may be applied vacuum treatment.In some embodiments, can before applying force to handle, after applying force to handle, before applying gravity to handle, after applying gravity to handle, before applying pressure to handle and / or after applying pressure to handle, apply vacuum treatment.In specific embodiments, comprise applying pressure to handle and gravity to handle under the situation that force handles, can between applying pressure to handle and applying gravity to handle, apply vacuum treatment, no matter be first applying gravity to handle or first applying pressure to handle. In one embodiment, the population can 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 values ​​and ranges derivable therein.

[0078] Inoculate the colony of embryo explant with heterologous polynucleotide being incorporated into at least a cell of explant with rhizobium or agrobacterium, can before any subsequent transformation or culture step, remove the inoculum culture medium or most of described inoculum culture medium or at least excessive described inoculum culture medium that contains rhizobium or agrobacterium.Can remove inoculum culture medium by decantation (may use filter or net), filtration, suction, pipetting, vacuum treatment and / or blot or wick off any combination of inoculum culture medium.Blot or wick of inoculum culture medium can be finished by making substratum contact with filter paper or other absorbing material.The removal of inoculum culture medium can be carried out before the follow-up co-cultivation step.

[0079] C. Co-culture of Exosomes

[0080] In the colony of the embryo explant inoculated with the rhizobium or agrobacterium that comprise heterologous polynucleotide so that heterologous polynucleotide is incorporated into at least a cell of explant, and possibly after removing inoculation medium, explant can be contacted with co-culture medium and cultivate together.According to embodiments of the present invention, seed embryo explant can be transferred in the co-culture medium or make it to contact with co-culture medium, perhaps seed embryo explant can be transferred in one or more (cultivate together) plates that contain co-culture medium, or after explant is transferred, add co-culture medium wherein.Co-culture medium can comprise for example water, basal salts, macronutrients, micronutrients and / or vitamin.According to some embodiments, co-culture medium can not contain or comprise any plant hormone (as auxin and / or cytokinin) and / or any surfactant or wetting agent, perhaps co-culture medium can contain or comprise plant hormone (as auxin and / or cytokinin) and / or surfactant or wetting agent.In specific embodiments, co-culture medium comprises one or more cytokinins. Non-limiting examples of cytokinins that can be used in the co-culture medium include 6-benzylaminopurine (BAP), thiadiazole (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (topolin). According to some embodiments, the co-culture medium may optionally contain an antimicrobial or antifungal agent, such as nystatin and / or thiabendazole (TBZ), which may be present at the concentrations described above for bacterial culture and / or inoculation of the culture medium. In certain embodiments, the inclusion of one or more cytokinins and / or lipoic acid in the co-culture medium can increase the developmental capacity, regeneration capacity, and / or transformation / editing frequency following Rhizobium- or Agrobacterium-mediated transformation. In certain embodiments, the inclusion of lipoic acid in the co-culture medium reduces stress during Rhizobium- or Agrobacterium-mediated transformation (e.g., during the inoculation and co-cultivation steps).

[0081] The concentration of the cytokinin in the co-culture medium can be or within the concentrations described above for bacterial culture and / or inoculation medium. In some embodiments, the concentration of the cytokinin in the co-culture 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. , about 0.1 mg / L to about 10 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 0.1 mg / L to about 1 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 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, 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 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 values ​​and ranges derivable therebetween.

[0082] In certain embodiments, lipoic acid can be present in the co-culture medium at a concentration of about 0.1 mg / L to about 500 mg / L, about 0.1 mg / L to about 400 mg / L, about 0.1 mg / L to about 300 mg / L, about 0.1 mg / L to about 200 mg / L, about 10 mg / L to about 200 mg / L, about 10 mg / L to about 180 mg / L, about 10 mg / L to about 160 mg / L, about 10 mg / L to about 140 mg / L, about 10 mg / L to about 120 mg / L, about 10 mg / L to about 100 mg / L, about 20 mg / L to about 80 mg / L, about 4 0 mg / L to about 60 mg / L, about 50 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, 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 g / L, about 115 mg / L, about 120 mg / L, about 125 mg / L, about 130 mg / L, about 135 mg / L, about 140 mg / L, about 145 mg / L, about 150 mg / L, about 155 mg / L, about 160 mg / L, about 165 mg / L, about 170 mg / L, about 175 mg / L, about 180 mg / 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 mg / L, 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 ranges and values ​​derivable therebetween.

[0083] In some embodiments, thidiazuron (TDZ) can be included in the co-culture medium at a concentration range 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 0.1 mg / L to about 1 mg / L, about 0.25 mg / L to about 1.75 mg / 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 From about 4 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 ranges and values ​​derivable therebetween. Including thidiazuron (TDZ) in the co-culture medium can improve the developmental capacity, regeneration capacity and / or transformation / editing frequency after Rhizobium- or Agrobacterium-mediated transformation. In some embodiments, including one or more cytokinins and / or lipoic acid in the co-culture medium improves the developmental capacity, regeneration capacity and / or transformation / editing frequency of dicot embryo explants. In specific embodiments, including one or more cytokinins and / or lipoic acid in the co-culture medium improves the developmental capacity, regeneration capacity and / or transformation / editing frequency of soybean embryo explants, cotton embryo explants, or rapeseed embryo explants.

[0084] In some embodiments, cytokinins other than thidiazuron (TDZ) can be included in the co-culture medium, such as 6-benzylaminopurine (BAP), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino)purine (2iP) and 6-(3-hydroxybenzylamino)purine (topolin), and their concentrations are adjusted and set according to their relative activities. For example, 6-benzylaminopurine (BAP), kinetin, zeatin, and / or 6-(3-hydroxybenzylamino)purine (topolin) can be present in the bacterial culture medium at 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. To about 8 mg / L, about 2.5 mg / L to about 7.5 mg / L, about 3 mg / L to 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 ranges and values ​​derivable therebetween, which can improve the developmental capacity, regeneration capacity and / or transformation / editing frequency of embryonic explants (such as dicotyledonous embryonic explants or soybean, cotton or rapeseed embryonic explants) after Rhizobium or Agrobacterium-mediated transformation.

[0085] In some embodiments, 6-(γ,γ-dimethylallylamino)purine (2iP) can be present in the co-culture medium at a concentration 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 35 mg / L, about 22.5 mg / L to about 30 mg / L. / 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 ranges and values ​​derivable therein, which can improve the developmental capacity, regeneration capacity and / or transformation / editing frequency of embryonic explants (such as dicotyledonous embryonic explants or soybean, cotton or rapeseed embryonic explants) after Rhizobium or Agrobacterium-mediated transformation.

[0086] In certain embodiments, the co-culture medium may include a surfactant, which may be particularly suitable for transformation or editing of monocot embryonic explants and may include any suitable surfactant or combination of surfactants known in the art, such as detergents, wetting agents, emulsifiers, foaming agents, or dispersants. In some embodiments, the surfactant may be or similar surfactants.

[0087] According to some embodiments, method as herein described comprises that monocotyledonous plant seed embryo explant is transferred to co-cultivation plate or container, and the density of visible explant or total explant of each container or plate is lower.According to some embodiments, the density of lower visible explant or total explant of each co-cultivation plate can improve monocotyledonous plant or corn embryo explant or the genetically modified plant of some monocotyledonous plant or corn strain (such as some male germplasm corn strain) that are more resistant to effective transformation and / or regeneration of genetically modified plant.As described herein, compared with female corn strain or other monocotyledonous plant germplasm, the transformation of male corn strain and possible other monocotyledonous plant germplasm may be difficult or efficiency is lower usually, and this may be due to variable or lower germination rate, lower agrobacterium infection rate, cultivation and selection of crowding and tissue death (particularly under higher density) and / or minimal bud regeneration on culture medium. These differences may be owing to comparing with female corn strain or other germplasm, and male corn strain or other germplasm and from the heredity and characteristic of the embryo explant of the seed of male corn strain or other germplasm are different.Consider that the seed embryo explant of some male germplasm or strain or other monocotyledonous plant germplasm may have lower regeneration rate, this may reduce, reduce or decline relevant with developmental capacity and / or regeneration capacity, compare with female corn strain or other monocotyledonous plant germplasm with relatively high or regeneration rate (or developmental capacity and / or regeneration capacity that is higher or increase), may need more visible (or always) explant just can have the regenerable explant of similar number, and produce regeneration explant, plantlet or plant of similar number.Yet, if more visible (or always) explant is placed on the co-cultivation plate or the container of same number, then co-cultivation plate or container will become too crowded, and this may cause or further cause regeneration rate to reduce, reduce or decline (perhaps developmental capacity and / or regeneration capacity reduce, reduce or decline). In some embodiments, crowding can cause the drying of embryo explant to reduce, and this can cause the delivery of heterologous polynucleotide molecule to at least one cell of embryo explant to reduce.If the delivery of heterologous polynucleotide molecule reduces, then in some embodiments, if co-culture medium, bud inducing culture medium, prolongation bud inducing culture medium and / or regeneration culture medium comprise selection agent, this may cause regeneration rate to reduce, reduce or decline (perhaps developmental ability and / or regeneration ability reduce, reduce or decline) so.Therefore, according to the quantity of visible (or total) explant in cultivation plate or container and / or according to the quantity of the inoculation explant contacted with co-culture medium, increase the quantity of the cultivation plate of given size and / or reduce, reduce or reduce each plate or cultivate plate area together and can cause relatively higher or increased regeneration rate (perhaps developmental ability and / or regeneration ability that is higher or increases).In some embodiments, the quantity of visible explant can be defined as the sum of the explant that comprises reproducible and non-reproducible explant.In certain embodiments, the percentage of regenerable explants can be calculated as the number of regenerable explants per gram, or the number of viable explants capable of germination prior to transformation, divided by the total number of visible explants per gram, and multiplied by 100. According to some embodiments, a given number of visible (or total) monocot seed embryo explants (e.g., between about 500 and about 50,000, about 500 and about 25,000, about 500 and about 20,000, about 500 and about 15,000, about 500 and about 14,000, about 500 and about 13,000, about 500 and about 12,000, about 500 and about 11,000, about 500 and about 10,000, about 500 and about 9,000, about 500 and about 8,000, about 500 and about 7,000, about 500 and about 6,000, about 500 and about 5,000, about 500 and about 4,000) can be used to generate the regenerable explants. In some embodiments, the inoculated culture medium can be transferred from the inoculated culture medium to a relatively greater or larger number of co-cultivation plates or containers, such as 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more co-cultivation plates or containers.

[0088] According to some embodiments, a certain number of visible monocot seed embryo explants of a given germplasm or line (corresponding to a target number of regenerable seed embryo explants based on a known or theoretical regeneration rate of such germplasm or line) are added or transferred to a greater number of co-cultivation plates or containers or to a greater total surface area of ​​the co-cultivation plates or containers to achieve a lower density of visible (or total) explants per plate or container or per total surface area of ​​the co-cultivation plates or containers. According to some embodiments, the density of the number of visible (or total) monocot seed embryo explants transferred or added to the co-cultivation plates or containers can be less than or equal to about 800 seed embryo explants per plate, less than or equal to about 700 seed embryo explants per plate, or less than or equal to about 600 seed embryo explants per plate, or between about 50 and about 800 seed embryo explants per plate, about 50 and about 700 seed embryo explants per plate, or about 50 and about 600 seed embryo explants per plate. 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 per plate, about The number of seed embryo explants per plate is preferably about 150 to about 500, about 150 to about 400, about 200 to about 500, about 200 to about 400, about 300 to about 700, about 400 to about 700, or about 500 to about 700 seed embryo explants per plate, or about 100, about 150, about 175, about 200, about 200, about 300, about 700, or about 400 seed embryo explants per plate. 700, about 750, or about 800 seed embryo explants, including all ranges and values ​​derivable therein. For these density values ​​and ranges, the surface area of ​​each co-cultivation plate is approximately 11.9 square inches (in 2 ) or 76.8 square centimeters (cm 2Thus, all of the above density values ​​and ranges of seed embryo explants per plate can be readily converted to density values ​​and ranges of seed embryo explants per co-cultivation surface area (e.g., a density of 100 seed embryo explants per plate can be divided by the surface area of ​​each plate to yield a density of approximately 8.4 seed embryo explants per square inch (in2) of seed embryo explants per co-cultivation surface area). 2 ) or about 1.3 seed embryo explants / cm2 (cm 2 ), and similar conversions can be readily made for other density values ​​and ranges.) The density values ​​and ranges of seed embryo explants per co-cultivation surface area are a more general definition of the density of seed embryo explants in a variety of different co-cultivation plates or containers, which may each have a different surface area.

[0089] In some embodiments, the density of the number of visible (or total) monocot seed embryo explants transferred or added to the co-cultivation plate or container can be less than or equal to 1 cm2. 2) with a co-culture surface area of ​​about 11.0, about 10.9, about 10.8, about 10.7, about 10.6, about 10.5, about 10.4, about 10.3, about 10.2, about 10.1, about 10.0, about 9.9, about 9.8, about 9.7, about 9.6, about 9.5, about 9.4, about 9.3, about 9.2, about 9.1, about 9.0, about 8.9, about 8.8, about 8.7, about 8.6, about 8. 5, about 8.4, about 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, about 5.2, about 5.1, about 5.0, about 4.9, about 4.8, about 4.7, about 4.6, about 4.5, about 4.4, about 4.3, about 4.2, about 4.1, about 4.0, 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 embryo explants, including all ranges and values ​​derivable therein. In certain embodiments, the density range for the number of visible (or total) monocotyledonous seed embryo explants transferred or added to the co-cultivation plate or container can be per square centimeter (cm 2), a co-culture surface area of ​​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 5.5, 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 0.6 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 .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 .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 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 4.5 to about 7.5, about 4.5 to about 7.0, about 4.5 to about 6.5, about 4.5 to about 6.0, about 4.5 to about 5.5, about 4.5 to about 5.0 embryos; about 5.0 to about 11.0, about 5.0 to about 10.5, about 5.0 to about 10.0, about 5.0 to about 9.5, about 5.0 to about 9.0, about 5.0 to about 8.5, about 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, about 6.0 to about 7.0, about 6.0 to about 6.5, about 6.5 to about 11.0, about 6.5 to about 10.5, about 6.5 to about 10.0, about 6.5 to about 9.5, about 6.5 to about 9.0, about 6.5 to about 8.5, about 6.5 to about 8.0, about 6.5 to about 7.5, about 6.5 to about 7.0, about 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 9.5 or about 8.5 to about 9.0 explants, including all ranges and values ​​derivable therein.

[0090] In specific embodiments, the embryonic explants can be contacted with the co-culture medium at a temperature in the range of about 15°C to about 25°C, or about 17°C to about 23°C, or 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. In some embodiments, the explant can be contacted with the co-culture medium for a period of time ranging from 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, 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, such as 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 ranges and values ​​derivable therein. In further embodiments, the explant may be in contact with the co-culture medium for at least 5 days or at least 6 days.In embodiments of the invention, the co-culture medium in contact with the explant may be a solid, liquid or semi-solid culture medium.

[0091] According to some embodiments, monocot seed embryo explants can be contacted with the co-culture medium for a period of time 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, such as about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days, including all ranges and values ​​derivable therein. According to some embodiments, monocot seed embryo explants can be contacted with the co-culture medium for a longer period of time to increase transformation, germination, and / or regeneration frequency, which may be particularly useful for seed embryo explants of certain male germplasms or lines or other monocot germplasms with lower transformation, germination, and / or regeneration frequency. According to some embodiments, the monocot seed embryo explant can be contacted with the co-culture medium for a period of time ranging from about 5 days to about 10 days or about 5 days to about 9 days or about 5 days to about 8 days or about 5 days to about 7 days or about 5 days to about 6 days or about 6 days to about 7 days, such as about 5 days, about 6 days, about 7 days, about 8 days, about 9 days or about 10 days, including all ranges and values ​​derivable therein.

[0092] According to some embodiments, the co-culture medium may contain Rhizobiales bacteria or Agrobacterium that are capable of transforming at least one cell of the explant with a heterologous polynucleotide molecule. A higher concentration of Agrobacterium in the inoculation medium and / or co-culture medium may improve or increase the germination frequency and the cultivation frequency, thereby improving or increasing the transformation frequency. The OD of the Rhizobiales bacteria or Agrobacterium in the inoculation medium and / or co-culture medium may be greater than or equal to 0. 660 The concentration range can be 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 ranges and values ​​derivable therebetween. However, it may be that depending on the monocot germplasm or genetic line being transformed, in some cases, a lower OD 660 Concentrations (e.g., 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 ranges and values ​​derivable therein) can improve the developmental and / or regenerative capacity of the explant.

[0093] In certain embodiments, explant can be co-cultivated under the 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% (comprising all scopes and value that can be derived therebetween).

[0094] The co-cultivation step can also be performed under a variety of lighting conditions. While some degree of illumination is typically used, alternatively, all or part of the co-cultivation step can be performed in the dark. Light treatment can be quantified based on the light / dark cycle and light intensity, which can be expressed as photosynthetic photon flux density (PPFD) in μE / m 2 In some embodiments, the 2 ·s to about 200μE / m 2 ·s, 20μE / m 2 ·s to about 200μE / m2 ·s, 20μE / m 2 ·s to about 180μE / m 2 ·s、30μE / m 2 ·s to about 180μE / m 2 ·s、30μE / m 2 ·s to about 150μE / m 2 ·s、30μE / m 2 ·s to about 120μE / m 2 ·s, 60μE / m 2 ·s to about 120μE / m 2 ·s、70μE / m 2 ·s to about 110μE / m 2 ·s or 80μE / m 2 ·s to about 100μE / m 2 The co-cultivation step is performed under an average or set light intensity of photosynthetically active radiation (PAR) in the range of 10 μE / m s. In some embodiments, the co-cultivation step can be performed under an average or set light intensity of photosynthetically active radiation (PAR) in the range of 10 μE / m s. 2 ·s, about 10μE / m 2 ·s, about 20μE / m 2 ·s, about 30μE / m 2 ·s, about 40μE / m 2 ·s, about 50μE / m 2 ·s, about 60μE / m 2 ·s, about 70μE / m 2 ·s, about 80μE / m 2 ·s, about 90μE / m 2 ·s, about 100μE / m 2 ·s, about 110μE / m 2 ·s, about 120μE / m 2 ·s, about 130μE / m 2 ·s, about 140μE / m 2 ·s, about 150μE / m 2 ·s, about 160μE / m 2 ·s, about 170μE / m 2 ·s, about 180μE / m 2 ·s, about 190μE / m 2 ·s or about 200 μE / m 2The co-cultivation step is performed under an average or set light intensity of photosynthetically active radiation (PAR) of 1.5 s. In some embodiments, different amounts of light and dark cycles can be used during the co-cultivation step, which can include the presence of light for periods of between about 0 hours and about 24 hours of light, between about 2 hours and about 22 hours of light, between about 4 hours and about 20 hours of light, between about 8 hours and about 20 hours of light, between about 12 hours and about 20 hours of light, between about 16 hours and about 20 hours of light, each with a corresponding amount of relative darkness based on the corresponding period of a 24-hour day length. According to some embodiments, the amount of light and dark cycles during the co-cultivation step can be about 0 hours of light and about 24 hours of dark, about 1 hour of light and about 23 hours of dark, about 2 hours of light and about 22 hours of dark, about 3 hours of light and about 21 hours of dark, about 4 hours of light and about 20 hours of dark, about 5 hours of light and about 19 hours of dark, about 6 hours of light and about 18 hours of dark, about 7 hours of light and about 17 hours of dark, about 8 hours of light and about 16 hours of dark, about 9 hours of light and about 15 hours of dark, about 10 hours of light and about 14 hours of dark, about 11 hours of light and about 12 hours of dark. 3 hours of dark, about 12 hours of light and about 12 hours of dark, about 13 hours of light and about 11 hours of dark, about 14 hours of light and about 10 hours of dark, about 15 hours of light and about 9 hours of dark, about 16 hours of light and about 8 hours of dark, about 17 hours of light and about 7 hours of dark, about 18 hours of light and about 6 hours of dark, about 19 hours of light and about 5 hours of dark, about 20 hours of light and about 4 hours of dark, about 21 hours of light and about 3 hours of dark, about 22 hours of light and about 2 hours of dark, about 23 hours of light and about 1 hour of dark, about 24 hours of light and about 0 hours of dark.

[0095] In a specific embodiment, after introducing and / or inoculating step and any co-cultivating step, the embryo explant from the dicotyledonous plant species can be transferred to a regeneration medium to regenerate plant or its part. However, in some embodiments, the embryo explant from the monocotyledonous plant species can be transferred to a bud induction medium to carry out bud induction and possible extension bud induction steps after introducing and / or inoculating step and any co-cultivating step. After bud induction and possible extension bud induction steps, the monocotyledonous plant embryo explant can then be transferred to a regeneration medium to regenerate plant or its part, as further described below.

[0096] D. Bud Induction and Prolonged Bud Induction

[0097] According to an embodiment of the present invention, a population of monocotyledonous plant embryonic explants that have been transformed or edited by introducing a heterologous polynucleotide molecule into at least one cell of the embryonic explants can be contacted and cultured with at least a first bud induction medium comprising an auxin and a cytokinin. Prior to the bud induction step, the monocotyledonous plant embryonic explants can have been inoculated with an inoculation medium comprising Rhizobia or Agrobacterium containing the heterologous polynucleotide molecule, and the monocotyledonous plant seed embryonic explants can also have been contacted and co-cultured with a co-culture medium.

[0098] As provided herein, the monocotyledonous plant embryo explants can be further contacted with a second or extended bud induction medium comprising an auxin and a cytokinin, and then contacted with a regeneration medium to produce a genetically modified plant or plant part. In some embodiments, the methods described herein include contacting the monocotyledonous plant embryo explants with a bud induction medium (or a first bud induction medium) after the bud induction medium is contacted with the explants, and before the monocotyledonous plant embryo explants are contacted with the regeneration medium to regenerate or grow a genetically modified monocotyledonous plant or plant part. In further embodiments, the (first) bud induction medium and / or the second (or extended) bud induction medium can each comprise a high ratio of cytokinin to auxin.

[0099] In certain embodiments, bud inducing medium (or the first bud inducing medium) and the second bud inducing medium (or extending bud inducing medium) can each comprise a variety of standard culture media or solution ingredients or components, such as basal salts, macronutrients, micronutrients, sugars, antibiotics and / or vitamins. Bud inducing medium (or the first bud inducing medium) and the second bud inducing medium (or extending bud inducing medium) can each comprise auxin and cytokinin. Bud inducing medium (or the first bud inducing medium) and the second bud inducing medium (or extending bud inducing medium) can each comprise one or more selection agents, although according to many embodiments, in the first bud inducing medium, there is not selection agent. In the first bud inducing medium, there is not selection agent that can make the first bud inducing medium play the effect of delay culture medium. The identity of selection agent will depend on the selectable marker gene present in the heterologous polynucleotide molecule that is introduced into the colony of monocotyledonous plant embryo explant. Bud inducing medium (or the first bud inducing medium) and / or the second bud inducing medium (or extending bud inducing medium) can each be solid, semisolid or liquid culture medium, although each in these culture media can be solid culture medium usually. Solid media may comprise gelling or polymerizing agents or components, such as agarose or the like, which can solidify and form a solid medium.

[0100] As used herein, "high cytokinin to auxin ratio" generally refers to a situation in which the level of cytokinin activity is relatively high compared to the level of auxin activity present in the culture medium, which can generally be a cytokinin:auxin ratio of at least about 1:1 or higher on a weight / volume basis as provided. However, the exact cytokinin:auxin ratio will depend on the exact chemical identity of the auxin and cytokinin, as different auxins and cytokinins can have different activities and / or modes of action, as is known in the art. The levels of cytokinin and auxin in a culture medium with a high cytokinin to auxin ratio can, for example, be present in the culture medium (measured by weight / volume) at a ratio of 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 values ​​and ranges derivable therein.

[0101] The levels of cytokinin and auxin in a culture medium having a high cytokinin to auxin ratio can be, for example, greater than or equal to about 1:1 or at least about 1:1 or higher, greater than or equal to about 1.5:1 or at least about 1.5:1 or higher, greater than or equal to about 2:1 or at least about 2:1 or higher, greater than or equal to about 2.5:1 or at least about 2.5:1 or higher, greater than or equal to about 3:1 or at least about 3:1 or higher, greater than or equal to about 3.5:1 or at least about 3.5:1 or higher, greater than or equal to about 4:1 or at least about 4:1 or higher, greater than or equal to about 4.5:1 or at least about 4.5:1 or higher, greater than or equal to about 5:1 or at least about 5:1 or higher, greater than or equal to about 5.5:1 or at least about 5.5:1 or higher, greater than or equal to about 6:1 or at least about 6:1 or higher, greater than or equal to about 6:1 or at least about 6:1 or higher, greater than or equal to about 6:1 or at least about 6:1 or higher, or greater than about 6.5:1 or more, about 7:1 or more, about 7.5:1 or more, about 8:1 or more, about 8.5:1 or more, about 9:1 or more, about 9.5:1 or more, about 9.5:1 or more, about 10:1 or more, about 10.5:1 or more, about 11:1 or more, about 11.5:1 or more, about 11.5:1 or more, or about 12:1 or more, including all values ​​and ranges derivable therein.

[0102] The levels of cytokinin and auxin in a 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 ...1:1 to about 10:1, about 1:1 to about 10:1, about 1:1 to about 10:1, about 1:1 to about 10:1, 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 1:1 to about 10:1, about 1:1 to about 10:1, about 1:1 to about 10:1, about 1:1 to about 10:1, about 1:1 to about 10:1, about 1:1 to about 10:1, about 1:1 to about 10:1, about 1:1 to about 10:1, about 1:1 to about 10:1, about 1:1 to about 10:1, about 1:1 to about 10: Ranges between about 2:1 and about 8:1, about 4:1 and about 8:1, about 6:1 and about 8:1, about 1:1 and about 6:1, about 2:1 and about 6:1, about 4:1 and about 6:1, about 1:1 and about 5:1, about 2:1 and about 5:1, about 3:1 and about 5:1, about 1:1 and about 4:1, about 2:1 and about 4:1, about 3:1 and about 4:1, about 1:1 and about 3:1, or about 1:1 and about 2:1, including all values ​​and ranges derivable therebetween.

[0103] Non-limiting examples of cytokinins that can be used in accordance with the present disclosure can include, but are not limited to, 6-benzylaminopurine (BAP), thiadiazole (TDZ), N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino)purine (2iP), and 6-(3-hydroxybenzylamino)purine (topolin). Auxins that can be used according to the present disclosure may include, but are not limited to, 2,4-dichlorophenoxy-acetic acid (2,4-D), 4-amino-3,5,6-trichloro-picolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthylacetic 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-methoxy-benzoic acid (dicamba).

[0104] In embodiments of the present invention, the bud inducing medium (or the first bud inducing medium) may comprise an auxin that is the same or different from the second bud inducing medium (or extended bud inducing medium) and / or an cytokinin that is the same or different from the second bud inducing medium. The bud inducing medium (or the first bud inducing medium) may comprise a first auxin and a first cytokinin, and the second bud inducing medium (or extended bud inducing medium) may comprise a first auxin or a second auxin and a first cytokinin or a second cytokinin. In some embodiments, the second bud inducing medium (or extended bud inducing medium) may comprise an auxin that is the same as the bud inducing medium (or the first bud inducing medium) or a different auxin. In a further embodiment, the second bud inducing (or extended bud inducing medium) may comprise a cytokinin that is the same as the bud inducing medium (or the first bud inducing medium) or a different cytokinin.

[0105] In certain embodiments, the concentration of the cytokinin (or two or more cytokinins) or the total cytokinin concentration in the first bud inducing medium and / or the second (or extended) bud inducing medium is between about 0.1 mg / L and about 100.0 mg / L, 1 mg / L and about 90.0 mg / L, 1 mg / L and about 80.0 mg / L, 1 mg / L and about 75.0 mg / L, 2 mg / L and about 90.0 mg / L, 2 mg / L and about 80.0 mg / L, 2 mg / L and about 75.0 mg / L, 5 mg / L and about 90.0 mg / L, 5 mg / L and about 80.0 mg / L, 5 mg / L and about 75.0 mg / L, 10 mg / L and about 80.0 mg / L. / L to about 90.0 mg / L, 10 mg / L to about 80.0 mg / L, 10 mg / L to about 75.0 mg / L, 10 mg / L to about 70.0 mg / L, 15 mg / L to about 90.0 mg / L, 15 mg / L to about 80.0 mg / L, 15 mg / L to about 75.0 mg / L, 15 mg / L to about 70.0 mg / L, 20 mg / L to about 90.0 mg / L, 20 mg / L to about 80.0 mg / L, 20 mg / L to about 75.0 mg / L, 20 mg / L to about 70.0 mg / L, 20 mg / L to about 60.0 mg / L, 30 mg / L to about 90.0 mg / L, 30 mg / L to about 80.0 mg / L , 30 mg / L to about 75.0 mg / L, 30 mg / L to about 70.0 mg / L, 30 mg / L to about 60.0 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, 40 mg / L to about 60.0 mg / L, 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 0.5 mg / L to about 25.0 mg / L, about 0.5 mg / L to about 20.0 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 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, 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 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 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 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 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, or about 1.0 mg / L to about 3.0 mg / L, including all values ​​and ranges derivable therebetween. In some embodiments, the concentration of the cytokinin in the first bud induction medium or the second bud induction medium can be, 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, 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 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 values ​​and ranges derivable therebetween. The cytokinins in the first and second bud induction media may be the same or different, and each of these bud induction media may comprise one or more cytokinins.

[0106] In some embodiments, the concentration of the auxin (or two or more auxins) or the total auxin concentration in the first bud induction medium and / or the second (or extended) bud induction medium is between about 0.01 mg / L and about 25.0 mg / L, about 0.05 mg / L and about 25 mg / L, about 0.1 mg / L and about 25.0 mg / L, about 0.1 mg / L and about 20.0 mg / L, about 0.1 mg / L and about 15.0 mg / L, about 0.2 mg / L and about 25.0 mg / L, about 0.2 mg / L and about 20.0 mg / L, about 0.2 mg / L and about 15.0 mg / L, about 0.5 mg / L and about 25.0 mg / L, about 0.5 mg / L and about 25.0 mg / L. 0.0 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 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, 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, about 9.0 mg / L to about 11.0 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 7.0 mg / L, about 0.1 mg / L to about 6.0 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 0.2 mg / L to about 6.0 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 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 7.0 mg / L, about 1.0 mg / L to about 6.0 mg / L, about 2.0 mg / L to about 10.0 mg / L, about 2.0 mg / L to about 7.5 mg / L, about 2.0 mg / L to about 6.0 mg / L.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 to about 5.0 mg / L g / 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 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 2.0 mg / L, about 0.75 mg / L to about 1.5 mg / L, about 0.75 mg / L to about 1.25 mg / L, about 0.75 mg / L to about 1.2 mg / L, about 0.75 mg / L to about 1.1 mg / L, about 0.8 mg / L to about 2.0 mg / L, about 0.8 mg / L to about 1.5 mg / L, about 0.8 mg / L to about 1.25 mg / L, about 0.8 mg / L to about 1.2 mg / L, about 0.8 mg / L to about 1.1 mg / L, about 0.9 mg / L to about 2.0 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, about 0.9 mg / L to about 1.1 mg / L, about 0.1 mg / L to about 1.0 mg / L, about 0.1 mg / L to about 0.75 mg / L, about 0.1 mg / L to about 0.7 mg / L, About 0.1 mg / L to about 0.6 mg / L, about 0.2 mg / L to about 1.0 mg / L, about 0.2 mg / L to about 0.75 mg / L, about 0.2 mg / L to about 0.7 mg / L, about 0.2 mg / L to about 0.6 mg / L, about 0.3 mg / L to about 1.0 mg / L, about 0.3 mg / L to about 0.75 mg / L, about 0.3 mg / L to about 0.7 mg / L, about 0.3 mg / L to about In the range of about 0.6 mg / 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 values ​​and ranges derivable therebetween. In some embodiments, the concentration of the auxin in the first bud induction or second bud induction medium can be, 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, 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 values ​​and ranges derivable therein. The auxins in the first and second bud induction media may be the same or different, and each of these media may contain one or more auxins.

[0107] As used herein, the "total cytokinin concentration" of a culture medium is the total concentration of all cytokinins present in the culture medium. For example, if the concentration of TDZ in the culture medium is 1 mg / L, and the concentration of BAP in the culture medium is 2 mg / L, and no other cytokinins are present in the culture medium, the total cytokinin concentration in the culture medium is 3 mg / L. As used herein, the "total auxin concentration" of a culture medium is the total concentration of all auxins present in the culture medium. For example, if the concentration of 2,4-D in the culture medium is 0.5 mg / L, and the concentration of IAA in the culture medium is 1 mg / L, and no other auxins are present in the culture medium, the total auxin concentration in the culture medium is 1.5 mg / L. For clarity, if only one cytokinin is present in the culture medium, the total cytokinin concentration of the culture medium will be equal to the concentration of that one cytokinin in the culture medium, and if only one auxin is present in the culture medium, the total auxin concentration of the culture medium will be equal to the concentration of that one auxin in the culture medium.

[0108] As used herein, "co-cultivation surface area," "bud inducing surface area," "first bud inducing surface area," "extended bud inducing surface area," "second bud inducing surface area," and "regeneration surface area" each refer to the upper surface area of ​​a solid or semi-solid culture medium that serves as a co-cultivation medium, a bud inducing medium, a first bud inducing medium, an extended bud inducing medium, a second bud inducing medium, or a regeneration medium, respectively.

[0109] In certain embodiments, the first bud induction medium and / or the second (or extended) bud induction medium may comprise a cytokinin, wherein the cytokinin is 6-benzylaminopurine (BAP). In some embodiments, the concentration of BAP in the first bud induction medium and / or the second (or extended) bud induction medium may be from about 1.0 mg / L to about 25.0 mg / L, from about 1.0 mg / L to about 20.0 mg / L, from about 1.0 mg / L to about 15.0 mg / L, from about 1.0 mg / L to about 12.5 mg / L, from about 2.0 mg / L to about 25.0 mg / L, from about 2.0 mg / L to about 20.0 mg / L, from about 2.0 mg / L to about 15.0 mg / L, from about 2.0 mg / L to about 12.5 mg / L, from about 5.0 mg / L to about 6.0 mg / L, from about 1.0 mg / L to about 12.5 mg / L, from about 2.0 mg / L to about 25.0 mg / L, from about 2.0 mg / L to about 20.0 mg / L, from about 2.0 mg / L to about 15.0 mg / L, from about 2.0 mg / L to about 12.5 mg / L, from about 5.0 mg / L to about 6.0 mg / L. 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 values ​​and ranges derivable therebetween. In some embodiments, the concentration of BAP in the first bud induction medium and / or the second (or extended) bud induction medium can be 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 11.0 mg / L. / 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 values ​​and ranges derivable therebetween.

[0110] In a particular embodiment, the first bud induction medium and / or the second (or extended) bud induction medium may comprise a cytokinin, wherein the cytokinin is thidiazuron (TDZ). In some embodiments, the concentration of TDZ in the first bud induction medium and / or the second (or extended) bud induction medium can be between about 0.1 mg / L and about 10.0 mg / L, about 0.1 mg / L and about 7.5 mg / L, about 0.1 mg / L and about 7.0 mg / L, about 0.1 mg / L and about 6.0 mg / L, about 0.1 mg / L and about 5.0 mg / L, about 0.1 mg / L and about 4.0 mg / L, about 0.1 mg / L and about 3.0 mg / L, about 0.2 mg / L and about 10.0 mg / L, about 0.2 mg / L and about 7.5 mg / L, about 0.2 mg / L and about 6.0 mg / L. / 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 0.5 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 1.0 mg / L to about 10.0 mg / L, about 1.0 ... 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 values ​​and ranges derivable therebetween. In some embodiments, the concentration of TDZ in the first bud induction medium and / or the second (or extended) bud induction medium may 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 values ​​and ranges derivable therein.

[0111] In some embodiments, the first bud induction medium and / or the second (or extended) bud induction medium may comprise a cytokinin, wherein the cytokinin is N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU). In specific embodiments, the concentration of 4-CPPU in the first bud induction medium and / or the second (or extended) bud 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, 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 0.2 mg / L to about 10.0 mg / L, from about 0.2 mg / L to about 7.5 mg / L, from about 0.2 mg / L to about 7.0 mg / L, from about 0.2 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. 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 0.5 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 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 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 values ​​and ranges derivable therebetween. In further embodiments, the concentration of 4-CPPU in the first bud induction medium and / or the second (or extended) bud induction medium may 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 values ​​and ranges derivable therein.

[0112] In specific embodiments, the first bud inducing medium and / or the second (or extended) bud inducing medium may comprise a cytokinin, wherein the cytokinin is a kinetin. In some embodiments, the concentration of the kinetin in the first bud inducing medium and / or the second (or extended) bud inducing medium may be between about 1.0 mg / L and about 25.0 mg / L, about 1.0 mg / L and about 20.0 mg / L, about 1.0 mg / L and about 15.0 mg / L, about 1.0 mg / L and about 12.5 mg / L, about 2.0 mg / L and about 25.0 mg / L, about 2.0 mg / L and about 20.0 mg / L, about 2.0 mg / L and about 15.0 mg / L, about 2.0 mg / L and about 12.5 mg / L, about 5.0 mg / L and about 15.0 mg / L. 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 values ​​and ranges derivable therebetween. In further embodiments, the concentration of kinetin in the first bud induction medium and / or the second (or extended) bud induction medium can be 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 15.0 mg / L. g / 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 values ​​and ranges derivable therebetween.

[0113] In certain embodiments, the first bud inducing medium and / or the second (or extended) bud inducing medium may comprise a cytokinin, wherein the cytokinin is zeatin. In some embodiments, the concentration of zeatin in the first bud inducing medium and / or the second (or extended) bud inducing medium may be from about 1.0 mg / L to about 25.0 mg / L, from about 1.0 mg / L to about 20.0 mg / L, from about 1.0 mg / L to about 15.0 mg / L, from about 1.0 mg / L to about 12.5 mg / L, from about 2.0 mg / L to about 25.0 mg / L, from about 2.0 mg / L to about 20.0 mg / L, from about 2.0 mg / L to about 15.0 mg / L, from about 2.0 mg / L to about 12.5 mg / L, from about 5.0 mg / L to about 15.0 mg / L, from about 2.0 mg / L to about 12.5 mg / L, from about 5.0 mg / L to about 15.0 mg / L, from about 2.0 mg / L to about 12.5 mg / L, from about 2.0 mg / L to about 25.0 mg / L, from about 2.0 mg / L to about 20.0 mg / L, from about 2.0 mg / L to about 15.0 mg / L, from about 2.0 mg / L to about 12.5 mg / L, from about 5.0 mg / L to about 15.0 mg / L, from about 2.0 mg / L to about 12.5 mg / L, from about 2 ... 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 values ​​and ranges derivable therebetween. In further embodiments, the concentration of zeatin in the first bud induction medium and / or the second (or extended) bud induction medium can be 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 11.0 mg / L. g / 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 values ​​and ranges derivable therebetween.

[0114] In certain embodiments, the first bud induction medium and / or the second (or extended) bud induction medium may comprise a cytokinin, wherein the cytokinin is 6-(γ,γ-dimethylallylamino)purine (2iP). In some embodiments, the concentration of 2iP in the first bud induction medium and / or the second (or extended) bud induction medium may be between 5 mg / L and about 100.0 mg / L, 5 mg / L and about 90.0 mg / L, 5 mg / L and about 80.0 mg / L, 5 mg / L and about 75.0 mg / L, 5 mg / L and about 70.0 mg / L, 10 mg / L and about 100.0 mg / L, 10 mg / L and about 90.0 mg / L. / L, 10 mg / L to about 80.0 mg / L, 10 mg / L to about 75.0 mg / L, 10 mg / L to about 70.0 mg / L, 15 mg / L to about 100.0 mg / L, 15 mg / L to about 90.0 mg / L, 15 mg / L to about 80.0 mg / L, 15 mg / L to about 75.0 mg / L, 15 mg / L to about 70.0 mg / L, 20 mg / L to about 100.0 mg / L L, 20 mg / L to about 90.0 mg / L, 20 mg / L to about 80.0 mg / L, 20 mg / L to about 75.0 mg / L, 20 mg / L to about 70.0 mg / L, 20 mg / L to about 60.0 mg / L, 30 mg / L to about 100.0 mg / L, 30 mg / L to about 90.0 mg / L, 30 mg / L to about 80.0 mg / L, 30 mg / L to about 75.0 mg / L, In the range of 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, 40 mg / L to about 60.0 mg / L, including all values ​​and ranges derivable therebetween.In further embodiments, the concentration of 2iP in the first bud induction medium and / or the second (or extended) bud induction medium can be 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 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 values ​​and ranges derivable therebetween.

[0115] In certain embodiments, the first bud inducing medium and / or the second (or extended) bud inducing medium may comprise a cytokinin, wherein the cytokinin is 6-(3-hydroxybenzylamino)purine (intertopolin). In some embodiments, the concentration of the first bud inducing medium and / or the second (or extended) bud inducing medium intertopolin may be between about 1.0 mg / L and about 25.0 mg / L, about 1.0 mg / L and about 20.0 mg / L, about 1.0 mg / L and about 15.0 mg / L, about 1.0 mg / L and about 12.5 mg / L, about 2.0 mg / L and about 25.0 mg / L, about 2.0 mg / L and about 20.0 mg / L, about 2.0 mg / L and about 15.0 mg / L, about 2.0 mg / L and about 12.5 mg / L. / 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 values ​​and ranges derivable therebetween. In further embodiments, the concentration of the first bud induction medium and / or the second (or extended) bud induction medium intermediate topoisomer may be 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 15.0 mg / L. g / 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 values ​​and ranges derivable therebetween.

[0116] According to some embodiments, the second (or extended) bud induction medium may have a lower concentration of cytokinin to increase the frequency of transformation, germination and / or regeneration, which may be particularly useful for monocot seed embryo explants of certain male germplasms or lines or other monocot germplasms with lower frequencies of transformation, germination and / or regeneration. According to some embodiments, the cytokinin (or two or more cytokinins) may be present in the second (or extended) bud induction medium at a lower concentration, or the total cytokinin concentration in the second (or extended) bud induction medium may be lower, ranging from about 0.1 mg / L to about 20 mg / L, about 0.1 mg / L to about 15 mg / L, 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 0.1 mg / L to about 1 mg / L, about 0.2 mg / L to about 20 mg / L, about 0.2 mg / L to about 15 mg / L, about 0.2 mg / L to about 10 mg / L, about 0.2 mg / L to about 9 mg / L, about 0.2 mg / L to about 8 mg / L, about 0.2 mg / L to about 7 mg / L, about 0.2 mg / L to about 6 mg / L, about 0.2 mg / L to about 5 mg / L, about 0.2 mg / L to about 4 mg / L, about 0.2 mg / L to about 3 mg / L, about 0.2 mg / L to about 2 mg / L, about 0.2 mg / L to about 1 mg / L, about 0.5 mg / L to about 20 mg / L, about 0.5 mg / L to about 15 mg / L, about 0.5 mg / L L to 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 ranges and values ​​derivable therebetween.In some embodiments, the lower concentration of cytokinin in the second (or extended) bud inducing medium or the lower total cytokinin concentration in the second (or extended) bud inducing medium can be, 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 18.0 mg / L, about 19.0 mg / L or about 20.0 mg / L, including all ranges and values ​​derivable therebetween.

[0117] In some embodiments, the second (or extended) bud induction medium may comprise a lower concentration of a cytokinin, wherein the cytokinin is 6-benzylaminopurine (BAP), kinetin, zeatin, or 6-(3-hydroxybenzylamino)purine (intertopolin). In some embodiments, the lower concentration of BAP, kinetin, zeatin, or 6-(3-hydroxybenzylamino)purine (intertopolin) in the second (or extended) bud induction medium may be between about 0.1 mg / L and about 20 mg / L, about 0.1 mg / L and about 15 mg / L, about 0.1 mg / L and about 10 mg / L, about 0.1 mg / L and about 9 mg / L, about 0.1 mg / L and about 8 mg / L, about 0.1 mg / L and about 7 mg / L, about 0.1 mg / L and about 6 mg / L, about 0.1 mg / L and about 5 mg / L, about 0.1 mg / L and about 4 mg / L, about 0.1 mg / L and about 3 mg / L. , about 0.1 mg / L to about 2 mg / L, about 0.1 mg / L to about 1 mg / L, about 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 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, about 0.5 mg / L to about 1 mg / L g / L, about 1 mg / L to about 20 mg / L, about 1 mg / L to about 15 mg / 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 mg / L to about 4 mg / L, about 1 mg / L to about 3 mg / L, about 1 mg / L to about 2 mg / L, about 2 mg / L to about 20 mg / L, about 2 mg / L to about 15 mg / L, about 2 mg / L to about 10 mg / L, about 2 mg / L to about 9 mg / L, about 2 mg / L. In some embodiments, the present invention may be in the range of about 1 mg / L to about 8 mg / 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 at a concentration of 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 ranges and values ​​derivable therein.

[0118] In some embodiments, the second (or extended) bud induction medium may comprise a lower concentration of a cytokinin, wherein the cytokinin is thidiazuron (TDZ). In some embodiments, the lower concentration of TDZ in the second (or extended) bud induction medium may be between about 0.1 mg / L and about 10 mg / L, about 0.1 mg / L and about 9 mg / L, about 0.1 mg / L and about 8 mg / L, about 0.1 mg / L and about 7 mg / L, about 0.1 mg / L and about 6 mg / L, about 0.1 mg / L and about 5 mg / L, about 0.1 mg / L and about 4 mg / L, about 0.1 mg / L and about 3 mg / L, about 0.1 mg / L and about 2 mg / L. g / L, about 0.1 mg / L to about 1 mg / L, about 0.5 mg / L to 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, about 0.5 mg / L to about 1 mg / 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 mg / L to about 4 mg / L, about 1 mg / L to about 3 mg / L, about 1 mg / L to about 2 mg / L, about 2 mg / L to about 10 mg / L, about 2 mg / L to about 9 mg / L, about 2 mg / L to about 8 mg / 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 at a concentration of 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 ranges and values ​​derivable therebetween.

[0119] In some embodiments, the second (or extended) bud induction medium may comprise a lower concentration of a cytokinin, wherein the cytokinin is 6-(γ,γ-dimethylallylamino)purine (2iP). In some embodiments, the lower concentration of 2iP in the second (or extended) bud induction medium may be 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, about 1 mg / L to about 40 mg / L, 1 mg / L to about 30 mg / L, 1 mg / L to about 25 mg / L, 1 mg / L to about 20 mg / L, about 1 mg / L to about 15 mg / L, about 1 mg / L to about 10 mg / L, about 1 mg / L to about 5 mg / L, about 2 mg / L to about 40 mg / L, 2 mg / L to about 30 mg / L, 2 mg / L to about 25 mg / L, 2 mg / L to about 20 mg / L, about 2 mg / L to about 15 mg / L, about 2 mg / L to about About 10 mg / 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 2 The present invention also provides a concentration range of about 10 mg / L to about 15 mg / L, or about 10 mg / L to about 15 mg / L, or a concentration of 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 ranges and values ​​derivable therebetween.

[0120] In specific embodiments, the first shoot induction medium and / or the second (or extended) shoot induction medium may comprise an auxin, wherein the auxin is 2,4-dichlorophenoxy-acetic acid (2,4-D).In some embodiments, the concentration of 2,4-D in the first bud induction medium and / or the second (or extended) bud 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, 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, from about 0.1 mg / L to about 1.25 mg / L, from about 0.1 mg / L to about 1.2 mg / L, from about 0.1 mg / L to about 1. mg / L to about 1.1 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 0.2 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 3.0 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 10.0 mg / L 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 0.5 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, 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 2.0 mg / L, about 0.75 mg / L to about 1.5 mg / L, about 0.75 mg / L to about In the range of about 1.25 mg / L, about 0.75 mg / L to about 1.2 mg / L, about 0.75 mg / L to about 1.1 mg / L, about 0.8 mg / L to about 2.0 mg / L, about 0.8 mg / L to about 1.5 mg / L, about 0.8 mg / L to about 1.25 mg / L, about 0.8 mg / L to about 1.2 mg / L, about 0.8 mg / L to about 1.1 mg / L, about 0.9 mg / L to about 2.0 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, about 0.9 mg / L to about 1.1 mg / L, and all values ​​and ranges derivable therebetween.In further embodiments, the concentration of 2,4-D in the first bud induction medium and / or the second (or extended) bud induction medium may 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 values ​​and ranges derivable therein.

[0121] In some embodiments, the first shoot induction medium and / or the second (or extended) shoot induction medium may comprise 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 bud induction medium and / or the second (or extended) bud 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, 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, from about 0.1 mg / L to about 1.25 mg / L, from about 0.1 mg / L to about 1.2 mg / L, from about 0.1 mg / L to about 1. 0.1 mg / L to about 1.1 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 0.2 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 3.0 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 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, 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, 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 2.0 mg / L, about 0.75 mg / L to about 1.5 mg / L, about 0.75 mg / L In the range of about 1.25 mg / L, about 0.75 mg / L to about 1.2 mg / L, about 0.75 mg / L to about 1.1 mg / L, about 0.8 mg / L to about 2.0 mg / L, about 0.8 mg / L to about 1.5 mg / L, about 0.8 mg / L to about 1.25 mg / L, about 0.8 mg / L to about 1.2 mg / L, about 0.8 mg / L to about 1.1 mg / L, about 0.9 mg / L to about 2.0 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, about 0.9 mg / L to about 1.1 mg / L, and all values ​​and ranges derivable therebetween.In further embodiments, the concentration of 2,4,5-T in the first bud induction medium and / or the second (or extended) bud induction medium may 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 values ​​and ranges derivable therein.

[0122] In some embodiments, the first bud induction medium and / or the second (or extended) bud induction medium may comprise an auxin, wherein the auxin is 4-amino-3,5,6-trichloro-picolinic acid (picloram). In further embodiments, the concentration of picloram in the first bud induction medium and / or the second (or extended) bud induction medium may be between about 0.1 mg / L and about 10.0 mg / L, about 0.1 mg / L and about 7.5 mg / L, about 0.1 mg / L and about 7.0 mg / L, about 0.1 mg / L and about 6.0 mg / L, about 0.1 mg / L and about 5.0 mg / L, about 0.1 mg / L and about 4.0 mg / L. 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 0.2 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 3.0 mg / L, about 0.5 mg / L g / 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 0.5 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 1.0 mg / L to about 10.0 mg / L, about 1.0 ... 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 values ​​and ranges derivable therebetween. In specific embodiments, the concentration of picloram in the first bud induction medium and / or the second (or extended) bud induction medium may 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 values ​​and ranges derivable therein.

[0123] In certain embodiments, the first shoot induction medium and / or the second (or extended) shoot induction medium may comprise an auxin, wherein the auxin is indole-3-acetic acid (IAA). In some embodiments, the concentration of IAA in the first bud induction medium and / or the second (or extended) bud induction medium can be between about 0.1 mg / L and about 25.0 mg / L, about 0.1 mg / L and about 20.0 mg / L, about 0.1 mg / L and about 15.0 mg / L, about 0.2 mg / L and about 25.0 mg / L, about 0.2 mg / L and about 20.0 mg / L, about 0.2 mg / L and about 15.0 mg / L, about 0.5 mg / L and about 25.0 mg / L, about 0.5 mg / L and about 20.0 mg / L, about 0.5 mg / L and about 15.0 mg / L, about 0.5 mg / L and about 12.5 mg / L. From about 15.0 mg / L to about 12.5 mg / L, from about 2.0 mg / L to about 25.0 mg / L, from about 2.0 mg / L to about 20.0 mg / L, from about 2.0 mg / L to about 15.0 mg / L, from about 2.0 mg / L to about 12.5 mg / L, from about 5.0 mg / L to about 25.0 mg / L, from about 5.0 mg / L to about 20.0 mg / L, from about 5.0 mg / L to about 15.0 mg / L, from about 5.0 mg / L to about 12.5 mg / L, from about 7.5 mg / L to about 25.0 mg / L, from about 7.5 mg / L to about 20.0 mg / L, from about 7.5 mg / L to about 15.0 mg / L, from about 7.5 mg / L to about 12.5 mg / L, from about 8.0 mg / L to about 12.0 mg / L, or from about 9.0 mg / L to about 11.0 mg / L, including all ranges derivable therebetween.In further embodiments, the concentration of IAA in the first bud induction medium and / or the second (or extended) bud 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, 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 7.5 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 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 values ​​and ranges derivable therebetween.

[0124] In specific embodiments, the first shoot induction medium and / or the second (or extended) shoot induction medium may comprise an auxin, wherein the auxin is indole-3-butyric acid (IBA). In some embodiments, the concentration of IBA in the first bud induction medium and / or the second (or extended) bud induction medium can be from 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 0.2 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 0.1 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. / 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 0.5 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 1.0 mg / L to about 10.0 mg / L, about 1.0 ... 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 values ​​and ranges derivable therebetween. In further embodiments, the concentration of IBA in the first bud induction medium and / or the second (or extended) bud induction medium may 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 values ​​and ranges derivable therein.

[0125] In a specific embodiment, the first bud induction medium and / or the second (or extended) bud induction medium may comprise an auxin, wherein the auxin is naphthaleneacetic acid (NAA). In a further embodiment, the concentration of NAA in the first bud induction medium and / or the second (or extended) bud 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, from about 0.2 mg / L to about 15.0 mg / L, from about 0.1 mg / L to about 10.0 mg / L, 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 0.5 mg / L to about 25.0 mg / L, about 0.5 mg / L to about 20.0 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 to about 7.0 mg / L, about 0.5 mg / L to about 6.0 mg / L, 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 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 7.0 mg / L, about 1.0 mg / L to about 6.0 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, about 2.0 mg / L to about 10.0 mg / L, about 2.0 mg / L to about 7.5 mg / L, about 2.0 mg / L to about 7.0 mg / L, about 2.0 mg / L to about 6.0 mg / L, about 3.0 mg / L to about 25.0 mg / L, about 3.0 mg / L to about 20.0 mg / L, about 3.0 mg / L to about 15.0 mg / L, about 3.0 mg / L to about 12.5 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 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, about 4.0 mg / L to about 6.0 mg / L, and all values ​​and ranges derivable therebetween. In specific embodiments, the concentration of NAA in the first bud induction medium and / or the second (or extended) bud induction medium can be 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. g / 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 values ​​and ranges derivable therebetween.

[0126] In some embodiments, the first bud inducing medium and / or the second (or extended) bud inducing medium may comprise an auxin, wherein the auxin is 2,3,5-triiodobenzoic acid (TIBA). In further embodiments, the concentration of TIBA in the first bud inducing medium and / or the second (or extended) bud inducing 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, from about 0.2 mg / L to about 15.0 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 0.5 mg / L to about 25.0 mg / L, about 0.5 mg / L to about 20.0 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 to about 7.0 mg / L, about 0.5 mg / L to about 6.0 mg / L, 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 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 7.0 mg / L, about 1.0 mg / L to about 6.0 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, about 2.0 mg / L to about 10. 0 mg / L, about 2.0 mg / L to about 7.5 mg / L, about 2.0 mg / L to about 7.0 mg / L, about 2.0 mg / L to about 6.0 mg / L, about 3.0 mg / L to about 25.0 mg / L, about 3.0 mg / L to about 20.0 mg / L, about 3.0 mg / L to about 15.0 mg / L, about 3.0 mg / L to about 12.5 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 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, about 4.0 mg / L to about 6.0 mg / L, and all values ​​and ranges derivable therebetween. In other embodiments, the concentration of TIBA in the first bud induction medium and / or the second (or extended) bud induction medium can be 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, or about 20.0 mg / L, including all values ​​and ranges derivable therebetween.

[0127] In certain embodiments, the first shoot induction medium and / or the second (or extended) shoot induction medium may comprise an auxin, wherein the auxin is phenylacetic acid (PAA). In further embodiments, the concentration of PAA in the first bud induction medium and / or the second (or extended) bud induction medium can be between about 0.1 mg / L and about 25.0 mg / L, about 0.1 mg / L and about 20.0 mg / L, about 0.1 mg / L and about 15.0 mg / L, about 0.2 mg / L and about 25.0 mg / L, about 0.2 mg / L and about 20.0 mg / L, about 0.2 mg / L and about 15.0 mg / L, about 0.5 mg / L and about 25.0 mg / L, about 0.5 mg / L and about 20.0 mg / L, about 0.5 mg / L and about 15.0 mg / L, about 0.5 mg / L and about 12.5 mg / L, about 1.0 mg / L and about 25.0 mg / L, about 1.0 mg / L and about 20.0 mg / L, about 1.0 mg / L and about 15.0 mg / L. 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, 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 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 ranges derivable therebetween.In some embodiments, the concentration of PAA in the first bud induction medium and / or the second (or extended) bud 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, 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 or about 25.0 mg / L, including all values ​​and ranges derivable therebetween.

[0128] In certain embodiments, the first shoot induction medium and / or the second (or extended) shoot induction medium may comprise an auxin, wherein the auxin is 3,6-dichloro-2-methoxy-benzoic acid (dicamba). In further embodiments, the concentration of dicamba in the first bud induction medium and / or the second (or extended) bud induction medium may 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, 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 0.2 mg / L to about 10.0 mg / L, from about 0.2 mg / L to about 7.5 mg / L, from about 0.2 mg / L to about 7.0 mg / L, from about 0.2 mg / L to about 6.0 mg / L, g / 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 0.5 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 1.0 mg / L to about 10.0 mg / L, about 1.0 ... 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 values ​​and ranges derivable therebetween. In further embodiments, the concentration of dicamba in the first bud induction medium and / or the second (or extended) bud 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 values ​​and ranges derivable therein.

[0129] According to some embodiments, the first shoot induction medium comprises a first auxin and a first cytokinin, wherein the first auxin is 2,4-dichlorophenoxy-acetic 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 bud induction medium can be 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 1.1 mg / L, or about 0.1 mg / L to about 1.25 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 3.0 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 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, 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, 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 2.0 mg / L, about 0.75 mg / L to about 1.5 mg / L, about 0.75 mg / L to about 1.2 In some embodiments, the present invention relates to a method for preparing an aqueous solution of the present invention that is suitable for use in an aqueous solution of the present invention. The method comprises the following steps: providing a method for preparing an aqueous solution of the present invention wherein the aqueous solution contains at least one of the following: about 0.5 mg / L to about 1.5 mg / L, about 0.75 mg / L to about 1.2 mg / L, about 0.75 mg / L to about 1.1 mg / L, about 0.8 mg / L to about 2.0 mg / L, about 0.8 mg / L to about 1.5 mg / L, about 0.8 mg / L to about 1.25 mg / L, about 0.8 mg / L to about 1.2 mg / L, about 0.8 mg / L to about 1.1 mg / L, about 0.9 mg / L to about 2.0 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, about 0.9 mg / L to about 1.1 mg / L, and all values ​​and ranges derivable therebetween.According to these embodiments, the concentration of 6-benzylaminopurine (BAP) in the first bud induction medium may be between about 1.0 mg / L and about 25.0 mg / L, about 1.0 mg / L and about 20.0 mg / L, about 1.0 mg / L and about 15.0 mg / L, about 1.0 mg / L and about 12.5 mg / L, about 2.0 mg / L and about 25.0 mg / L, about 2.0 mg / L and about 20.0 mg / L, about 2.0 mg / L and about 15.0 mg / L, about 2.0 mg / L and about 12.5 mg / L, about 5.0 mg / L and about 15.0 mg / L. 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 ranges derivable therebetween.

[0130] According to certain embodiments, the second (or extended) shoot induction medium comprises a second auxin and a second cytokinin, wherein the second auxin is 4-amino-3,5,6-trichloro-picolinic acid (picloram) and the second cytokinin is thidiazuron (TDZ). According to these embodiments, the concentration of picloram in the second (or extended) shoot induction medium may be between about 0.1 mg / L and about 10.0 mg / L, about 0.1 mg / L and about 7.5 mg / L, about 0.1 mg / L and about 7.0 mg / L, about 0.1 mg / L and about 6.0 mg / L, about 0.1 mg / L and about 5.0 mg / L, about 0.1 mg / L and about 4.0 mg / L, about 0.1 mg / L and about 3.0 mg / L, about 0.2 mg / L and about 10.0 mg / L, about 0.2 mg / L and about 7.5 mg / L, about 0.2 mg / L and about 7.0 mg / L, about 0.1 mg / L and about 6.0 mg / L, about 0.1 mg / L and about 5.0 mg / L, about 0.1 mg / L and about 4.0 mg / L, about 0.1 mg / L and about 3.0 mg / L. 0.0 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, 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 g / L, about 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 values ​​and ranges derivable therebetween.According to these embodiments, the concentration of TDZ in the second (or extended) shoot induction medium may be between about 0.1 mg / L and about 10.0 mg / L, about 0.1 mg / L and about 7.5 mg / L, about 0.1 mg / L and about 7.0 mg / L, about 0.1 mg / L and about 6.0 mg / L, about 0.1 mg / L and about 5.0 mg / L, about 0.1 mg / L and about 4.0 mg / L, about 0.1 mg / L and about 3.0 mg / L, about 0.2 mg / L and about 10.0 mg / L, about 0.2 mg / L and about 7.5 mg / L, about 0.2 mg / L and about 6.0 mg / L, about 0.1 mg / L and about 5.0 mg / L, about 0.1 mg / L and about 4.0 mg / L, about 0.1 mg / L and about 3.0 mg / L. 0.0 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, 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 g / L, about 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 values ​​and ranges derivable therebetween.

[0131] According to some embodiments, method as herein described is included in the visible or total monocotyledonous plant seed embryo explant with lower density in the bud (or first bud) induction culture medium of each container or plate, this can be realized by transferring the monocotyledonous plant seed embryo explant of cultivation to the bud (or first bud) induction plate or container of relatively large number from the co-cultivation plate or container of given number.As described herein, compared with female corn strain or other monocotyledonous plant germplasm, the transformation of male corn strain and possible other monocotyledonous plant germplasm may be difficult or inefficient usually.Compared with the bud induction plate for female strain corn embryo explant, the bud induction plate for male strain corn embryo explant may show frequent pollution and tissue necrosis.According to some embodiments, the lower density of visible or total explant of each bud (or first bud) induction plate or container can improve monocotyledonous plant or corn embryo explant or to the effective transformation of genetically modified plant and / or regeneration more resistant some monocotyledonous plant or corn strain (as some male germplasm corn strain) genetically modified plant, budding and / or the regeneration frequency. According to some embodiments, monocot seed embryo explants can be transferred from one or more co-cultivation plates or containers to a relatively smaller or fewer number of shoot induction plates or containers, such as a 2:1 or 1:1 ratio of the number of co-cultivation plates or containers to the number of shoot induction plates or containers, but this may result in overcrowding of the number of explants per shoot induction plate.

[0132] According to some embodiments, monocot seed embryo explants can be transferred from one or more co-cultivation induction plates or containers to a relatively larger or greater number of shoot induction plates or containers, such as 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 the ratio of the number of co-cultivation plates or containers to the number of shoot induction plates or containers. According to some embodiments, a given number of visible (or total) monocotyledonous plant seed embryo explants (e.g., between about 10 and about 2,000, about 20 and about 2,000, about 30 and about 2,000, about 40 and about 2,000, about 50 and about 2,000, about 10 and about 1,500, about 10 and about 1,000, about 10 and about 1,500, about 10 and about 1,000, about 10 and about 750, about 10 and about 500, about 10 and about 600, about 10 and about 500, about 10 and about 400, about 10 and about 300, about 50 and about 200) can be added. , about 20 to about 2,000, about 20 to about 1,500, 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 60 0, 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 to about 400, about 50 to about 300, about 50 to about 200, including all ranges and values ​​derivable therein) from the co-cultivation plate or culture medium to a relatively larger or larger number of sprout inducing plates or containers, such as 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 sprout inducing plates or containers.

[0133] According to some embodiments, the density of the number of visible (or total) monocotyledonous plant seed embryo explants transferred or added to the bud induction plates or containers can be less than or equal to about 500 seed embryo explants per plate, less than or equal to about 400 seed embryo explants per plate, less than or equal to about 300 seed embryo explants per plate, less than or equal to about 200 seed embryo explants per plate, less than or equal to about 150 seed embryo explants per plate, less than or equal to about 100 seed embryo explants per plate, less than or equal to about 75 seed embryo explants per plate, less than or equal to about 10 ... The density ranges from 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 seed embryo explants per plate, about 25 per plate, or about 10 to about 200 seed embryo explants per plate. to about 300 seed embryo explants per plate, 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 150 seed embryo explants per plate The present invention provides a plurality of 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 ranges and values ​​derivable therein. For these density values ​​and ranges, the surface area of ​​each shoot induction plate is approximately 11.9 square inches (in 2 ) or 76.8 square centimeters (cm 2 Thus, all of the above density values ​​and ranges of seed embryo explants per plate can be readily converted to density values ​​and ranges of seed embryo explants per bud-inducing surface area (e.g., a density of 100 seed embryo explants per plate can be divided by the surface area of ​​each plate to yield a density of approximately 8.4 seed embryo explants per square inch (in) of seed embryo explants per bud-inducing surface area). 2) or about 1.3 seed embryo explants / cm2 (cm 2 ), and similar conversions can be readily made for other density values ​​and ranges.) The density values ​​and ranges of seed embryo explants per bud inducing surface area are a more general definition of the density of seed embryo explants in a variety of different bud inducing plates or containers, each of which may have a different surface area.

[0134] In some embodiments, the density of the number of visible (or total) monocot seed embryo explants transferred or added to the shoot induction plate or container can be 100% per square centimeter (cm 2 ) has a shoot inducing surface area of ​​less than or equal to 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 embryo explant, including all ranges and values ​​derivable therein. In certain embodiments, the density of the number of visible (or total) monocot seed embryo explants transferred or added to the shoot induction plates or containers may range from 1 to 2 cm2. 2) has a bud inducing surface area of ​​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 0.1 to about 1.0, about 0.1 to about 0.5, about 0.2 to about 6.0 .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 0.4 to about 5.5 .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 1.5, 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 embryonic explants, including all ranges and values ​​derivable therein.

[0135] According to embodiments of the present disclosure, a population of monocotyledonous plant embryo explants can be contacted and cultured with a first bud induction medium for about 2 days to about 14 days, about 4 days to about 12 days, about 5 days to about 10 days, or about 6 days to about 8 days, including all ranges derivable therein. In some embodiments, the monocotyledonous plant embryo explants are contacted and cultured with a first bud induction medium 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 ranges derivable therein. In further embodiments, the monocotyledonous plant embryo explants can be contacted and cultured with a first bud induction medium at a temperature in the range of 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 ranges derivable therein). According to specific embodiments, the monocotyledonous plant embryo explant can be contacted with the first bud induction medium and cultured at a temperature of 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 ranges derivable therein). According to one aspect of the present disclosure, the monocotyledonous plant embryo explants may be cultured in contact with the first shoot induction medium at an elevated temperature 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 ranges derivable therein. According to further embodiments, the monocot embryo explants can be contacted with a first bud induction medium 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 ranges derivable therein). The first bud induction medium typically may not contain a selective agent, but alternatively, the first bud induction medium may contain a selective agent.

[0136] On the other hand, culturing the monocot seed embryo explants in contact with the first shoot induction medium at an elevated temperature (e.g., a temperature in the range of about 30°C to about 40°C) for about one week during the first shoot induction step can improve transformation compared to culturing the explants in contact with the first shoot induction medium at a lower temperature (e.g., a temperature in the range of about 20°C to about 30°C).

[0137] The first bud induction step can also be performed under a variety of lighting conditions. Although some degree of light can generally be used, alternatively, all or part of the first bud induction step can be performed in the dark. According to some embodiments, the first bud induction step can be performed at about 0 μE / m 2 ·s to about 200μE / m 2 ·s, 20μE / m 2 ·s to about 200μE / m 2 ·s, 20μE / m 2 ·s to about 180μE / m 2 ·s、30μE / m 2 ·s to about 180μE / m 2 ·s、50μE / m 2 ·s to about 180μE / m 2 ·s、50μE / m 2 ·s to about 150μE / m 2 ·s, 60μE / m 2 ·s to about 150μE / m 2 ·s、70μE / m 2 ·s to about 140μE / m 2 s, 80μE / m 2 ·s to about 130μE / m 2 ·s or 90μE / m 2 ·s to about 120μE / m 2 The first bud induction step is carried out under an average or set light intensity of photosynthetically active radiation (PAR) in the range of 10 μE / m s. According to a further embodiment, the first bud induction step can be carried out under an average or set light intensity of photosynthetically active radiation (PAR) in the range of 10 μE / m s. 2 ·s, about 10μE / m 2 ·s, about 20μE / m 2 ·s, about 30μE / m 2 ·s, about 40μE / m 2 ·s, about 50μE / m 2 ·s, about 60μE / m 2 ·s, about 70μE / m 2 ·s, about 80μE / m 2 ·s, about 90μE / m 2 ·s, about 100μE / m 2 ·s, about 110μE / m 2 ·s, about 120μE / m 2 ·s, about 130μE / m 2 ·s, about 140μE / m 2 ·s, about 150μE / m 2 ·s, about 160μE / m 2 ·s, about 170μE / m2 ·s, about 180μE / m 2 ·s, about 190μE / m 2 ·s or about 200 μE / m 2 The first bud induction step is performed under an average or set light intensity of photosynthetically active radiation (PAR) of 1.5 s. In specific embodiments, different amounts of light and dark cycles can be used during the first bud induction step, which can include light being present for periods of between about 0 hours and about 24 hours of light, between about 2 hours and about 22 hours of light, between about 4 hours and about 20 hours of light, between about 8 hours and about 20 hours of light, between about 12 hours and about 20 hours of light, between about 16 hours and about 20 hours of light, each with a corresponding amount of relative darkness based on the corresponding period of a 24-hour day length.

[0138] According to some embodiments, the amount of light and dark cycle during the first bud induction step can be about 0 hours of light and about 24 hours of dark, about 1 hour of light and about 23 hours of dark, about 2 hours of light and about 22 hours of dark, about 3 hours of light and about 21 hours of dark, about 4 hours of light and about 20 hours of dark, about 5 hours of light and about 19 hours of dark, about 6 hours of light and about 18 hours of dark, about 7 hours of light and about 17 hours of dark, about 8 hours of light and about 16 hours of dark, about 9 hours of light and about 15 hours of dark, about 10 hours of light and about 14 hours of dark, about 11 hours of light and about 16 hours of dark. 13 hours of dark, about 12 hours of light and about 12 hours of dark, about 13 hours of light and about 11 hours of dark, about 14 hours of light and about 10 hours of dark, about 15 hours of light and about 9 hours of dark, about 16 hours of light and about 8 hours of dark, about 17 hours of light and about 7 hours of dark, about 18 hours of light and about 6 hours of dark, about 19 hours of light and about 5 hours of dark, about 20 hours of light and about 4 hours of dark, about 21 hours of light and about 3 hours of dark, about 22 hours of light and about 2 hours of dark, about 23 hours of light and about 1 hour of dark, about 24 hours of light and about 0 hours of dark.

[0139] According to embodiments of the present disclosure, the population of monocotyledonous plant embryo explants can be cultured in contact with the second (or extended) shoot induction medium 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 ranges derivable therein. In some embodiments, the monocot embryo explants can be cultured in contact with the second (or extended) shoot induction medium 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 ranges derivable therein. In further embodiments, the monocot seed embryo explants may be contacted and cultured with the second (or extended) shoot induction medium at a temperature in the range of 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 ranges derivable therebetween. According to certain embodiments, the monocot seed embryo explants may be contacted and cultured with the second (or extended) shoot induction medium at a temperature of 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 ranges derivable therebetween. In a specific embodiment, monocot seed embryo explants can be contacted and cultured with a first bud induction medium at a temperature in the range of about 20°C to about 30°C, or at an elevated temperature in the range of about 30°C to about 40°C, for a period of time in the range of about 2 days to about 14 days, and then subsequently contacted and cultured with a second (or extended) bud induction medium at a temperature in the range of about 20°C to about 32°C for a period of time in the range of about 4 days to about 28 days. The second (or extended) bud induction medium may also contain a selective agent. In a specific embodiment, contacting and culturing the explants with the second bud induction medium can improve transformation compared to contacting and culturing the explants with the first bud induction medium alone.

[0140] The second (or extended) bud induction step can also be performed under a variety of lighting conditions. A certain level of lighting can generally be used during the second (or extended) bud induction step. According to some embodiments, the buds can be grown at about 30 μE / m 2 ·s to about 200μE / m 2 ·s、30μE / m 2 ·s to about 180μE / m2 ·s、50μE / m 2 ·s to about 180μE / m 2 ·s、50μE / m 2 ·s to about 150μE / m 2 ·s, 60μE / m 2 ·s to about 150μE / m 2 ·s、70μE / m 2 ·s to about 140μE / m 2 s, 80μE / m 2 ·s to about 130μE / m 2 ·s or 90μE / m 2 ·s to about 120μE / m 2 The second (or extended) bud induction step is carried out under conditions of an average or set light intensity of photosynthetically active radiation (PAR) in the range of 10 μE / m s. According to a further embodiment, the bud induction step may be carried out under conditions of an average or set light intensity of photosynthetically active radiation (PAR) in the range of 10 μE / m s. 2 ·s, about 20μE / m 2 ·s, about 30μE / m 2 ·s, about 40μE / m 2 ·s, about 50μE / m 2 ·s, about 60μE / m 2 ·s, about 70μE / m 2 ·s, about 80μE / m 2 ·s, about 90μE / m 2 ·s, about 100μE / m 2 ·s, about 110μE / m 2 ·s, about 120μE / m 2 ·s, about 130μE / m 2 ·s, about 140μE / m 2 ·s, about 150μE / m 2 ·s, about 160μE / m 2 ·s, about 170μE / m 2 ·s, about 180μE / m 2 ·s, about 190μE / m 2 ·s or about 200 μE / m 2s of photosynthetically active radiation (PAR). In certain embodiments, different amounts of light and dark cycles can be used during the second (or extended) bud induction step, which can include light being present for durations between about 2 hours and about 24 hours of light, between about 2 hours and about 22 hours of light, between about 4 hours and about 20 hours of light, between about 8 hours and about 20 hours of light, between about 12 hours and about 20 hours of light, between about 16 hours and about 20 hours of light, each with a corresponding amount of relative darkness based on the corresponding duration of a 24-hour day length.

[0141] According to some embodiments, the amount of light and dark cycle during the second (or extended) bud induction step can be about 2 hours of light and about 22 hours of dark, about 3 hours of light and about 21 hours of dark, about 4 hours of light and about 20 hours of dark, about 5 hours of light and about 19 hours of dark, about 6 hours of light and about 18 hours of dark, about 7 hours of light and about 17 hours of dark, about 8 hours of light and about 16 hours of dark, about 9 hours of light and about 15 hours of dark, about 10 hours of light and about 14 hours of dark, about 11 hours of light and about 13 hours of dark, about 12 hours of light and about 14 hours of dark. light and about 12 hours of dark, about 13 hours of light and about 11 hours of dark, about 14 hours of light and about 10 hours of dark, about 15 hours of light and about 9 hours of dark, about 16 hours of light and about 8 hours of dark, about 17 hours of light and about 7 hours of dark, about 18 hours of light and about 6 hours of dark, about 19 hours of light and about 5 hours of dark, about 20 hours of light and about 4 hours of dark, about 21 hours of light and about 3 hours of dark, about 22 hours of light and about 2 hours of dark, about 23 hours of light and about 1 hour of dark, about 24 hours of light and about 0 hours of dark.

[0142] According to some embodiments, method as herein described is included in the visible or total monocotyledonous plant seed embryo explant with lower density in the second (or prolongation) bud induction culture medium of each container or plate, this can be realized by the monocotyledonous plant seed embryo explant of cultivation being transferred in the prolongation bud induction plate or the container of relatively large number from the bud induction plate or the container of given number.As described herein, compared with female corn strain or other monocotyledonous plant germplasm, the transformation of male corn strain and possible other monocotyledonous plant germplasm may be difficult or inefficient usually.Compared with the prolongation bud induction plate for female strain corn embryo explant, the prolongation bud induction plate for male strain corn embryo explant may show frequent pollution and tissue necrosis.According to some embodiments, the lower density of visible or total explant of each prolongation bud induction plate or container can improve monocotyledonous plant or corn embryo explant or to the effective transformation of genetically modified plant and / or regeneration more resistant some monocotyledonous plant or corn strain (as some male germplasm corn strain) genetically modified plant, budding and / or the regeneration frequency. According to some embodiments, monocot seed embryo explants can be transferred from one or more bud inducing plates or containers to a relatively smaller or fewer number of extended bud inducing plates or containers, such as a 2:1 or 1:1 ratio of the number of bud inducing plates or containers to the number of extended bud inducing plates or containers, but this may result in overcrowding of the number of explants per extended bud inducing plate.

[0143] According to some embodiments, monocot seed embryo explants can be transferred from one or more bud inducing plates or containers to a relatively larger or greater number of elongated bud inducing plates or containers, such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 in terms of the ratio of the number of bud inducing plates or containers to the number of elongated bud inducing plates or containers. According to some embodiments, a given number of visible (or total) monocotyledonous plant seed embryo explants (e.g., between 10 and about 2,000, about 20 and about 2,000, about 30 and about 2,000, about 40 and about 2,000, about 50 and about 2,000, about 10 and about 1,500, about 10 and about 1,000, about 10 and about 1,500, about 10 and about 1,000, about 10 and about 750, about 10 and about 500, about 10 and about 600, about 10 and about 500, about 10 and about 400, about 10 and about 300, about 50 and about 2 00, about 20 to about 2,000, about 20 to about 1,500, 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 3 0 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 5 0 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 to about 400, about 50 to about 300, about 50 to about 200) from the bud inducing plate or culture medium to a relatively larger or larger number of elongated bud inducing plates or containers, such as 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 elongated bud inducing plates or containers.

[0144] According to some embodiments, the density of the number of visible (or total) monocotyledonous plant seed embryo explants transferred or added to the extended bud induction plates or containers can be less than or equal to about 300 seed embryo explants per plate, less than or equal to about 200 seed embryo explants per plate, less than or equal to about 150 seed embryo explants per plate, less than or equal to about 100 seed embryo explants per plate, less than or equal to about 75 seed embryo explants per plate, less than or equal to about 50 seed embryo explants per plate, less than or equal to about 10 ... about 30 seed embryo explants per plate, or densities ranging from 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 250 seed embryo explants per plate, or ...00 seed embryo explants per plate. 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 150 seed embryo explants per plate, about The invention provides a plurality of seed embryo explants, wherein the plurality of seed embryo explants are arranged on a plate, wherein the plurality of seed embryo explants are arranged on a plate, wherein the plurality of seed embryo explants are arranged on a plate, wherein the plurality of seed embryo explants are arranged on a plate, wherein the plurality of seed embryo explants are arranged on a plate, wherein the plurality of seed embryo explants are arranged on a plate, wherein the plurality of seed embryo explants are arranged on a plate, wherein the plurality of seed embryo explants are arranged on a plate, wherein the plurality of seed embryo explants are arranged on a plate, wherein the plurality of seed embryo explants are arranged on a plate, wherein the plurality of seed embryo explants are arranged on a plate, wherein the plurality of seed embryo explants are arranged on a plate, wherein the plurality of seed embryo explants are arranged on a plate, 2 ) or 76.8 square centimeters (cm 2 Thus, all of the above density values ​​and ranges of seed embryo explants per plate can be readily converted to density values ​​and ranges of seed embryo explants per extended bud-inducing surface area (e.g., a density of 100 seed embryo explants per plate can be divided by the surface area of ​​each plate to yield a density of approximately 8.4 seed embryo explants per square inch (in) of seed embryo explants per extended bud-inducing surface area). 2 ) or about 1.3 seed embryo explants / cm2 (cm 2), and similar conversions can be readily performed for other density values ​​and ranges.) The density values ​​and ranges of seed embryo explants per elongated bud inducing surface area are more general definitions for the density of seed embryo explants in a variety of different elongated bud inducing plates or containers, which may each have a different surface area.

[0145] In some embodiments, the density of the number of visible (or total) monocot seed embryo explants transferred or added to the elongated shoot induction plates or containers may be 100% per square centimeter (cm 2 ) has an elongated shoot inducing surface area of ​​less than or equal to 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 embryo explant, including all ranges and values ​​derivable therein. In certain embodiments, the density of the number of visible (or total) monocot seed embryo explants transferred or added to the elongated shoot induction plates or containers can range from 1 to 2 cm2. 2) has an elongated bud inducing surface area of ​​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.or about 1.5 to about 2.0 embryonic explants, including all ranges and values ​​derivable therebetween.

[0146] Without being bound by theory, the bud induction step can cause the cell differentiation and / or proliferation of the explant to form a complex bud on the explant, and the complex bud can then be regenerated into a plant. According to some preferred embodiments, the first auxin and cytokinin are different from the second auxin and cytokinin, so as to affect the formation and development of the complex bud by slightly different activities and / or modes of action. Without being bound by theory, the first bud induction step can cause the cell differentiation of the explant to become a complex bud, and the second (or extended) bud induction step can promote the proliferation or expansion of the complex bud, to produce a more compact or solid complex bud explant for further cultivation and regeneration into a plant. A further benefit of including the second (or extended) bud induction step can be to reduce the mosaicism of the resulting genetically modified plant or plant part. According to certain embodiments, compared with culturing monocot seed embryo explants in the first bud induction medium before regeneration, but not in the second (or extended) bud induction medium, monocot embryo explants are cultivated in the first bud induction medium and then in the second (or extended) bud induction medium to reduce the mosaicism in the regenerated plant.

[0147] E. Regeneration of Transformed or Edited Plants

[0148] In another aspect of the present disclosure, a plurality of genetically modified plants or plant parts are regenerated from the colony of the embryo explant of the culture contacted with the regeneration medium. According to embodiments of the present invention, the regeneration medium can comprise a variety of standard culture media or solution ingredients or components, such as basal salts, macronutrients, micronutrients, sugars, antibiotics and / or vitamins. The regeneration medium can not comprise auxins or cytokinins, which may be particularly suitable for regenerating monocotyledonous plant embryo explants or by regeneration of monocotyledonous plant embryo explants, although alternatively, auxins and / or cytokinins may be present in the regeneration medium, depending on the dicotyledonous plant species, in some embodiments, which may be particularly suitable for regenerating dicotyledonous plant embryo explants or by regeneration of dicotyledonous plant embryo explants. The regeneration medium can generally comprise at least one selective agent, which may correspond to the selectable marker present in the heterologous polynucleotide molecule. The regeneration medium can be solid, semisolid or liquid culture medium, although the regeneration medium can generally be solid culture medium. The solid culture medium can comprise a gelling or polymerizing agent or component, such as agarose or the like, which can solidify and form a solid culture medium. As used herein, the term "regeneration" refers to the process of growing a plant or its part out of one or more plant cells or tissues of any progeny of an explant or its cell, and the term "regeneration medium" refers to a plant tissue culture medium prepared for regenerating a plant from an explant. In some embodiments, regeneration or regeneration step may refer to one or more regeneration steps that may involve contacting an explant or cultivated explant with two or more regeneration mediums. These two or more regeneration mediums may be identical or different regeneration mediums. In some embodiments, the explant passage culture of the colony may be transferred to a second regeneration medium from the first regeneration medium, and may be transferred to a third regeneration medium, etc.

[0149] According to some embodiments, method as herein described is included in the regeneration culture medium with less dense visible (or total) monocotyledonous plant seed embryo explant in each container or plate, this can be realized by the monocotyledonous plant seed embryo explant of cultivation from the prolongation bud induction plate or container of given number transferred in the regeneration plate or container of greater number.As described herein, compared with female corn strain or other monocotyledonous plant germplasm, the conversion of male corn strain and possible other monocotyledonous plant germplasm may be difficult or inefficient usually.Compared with the regeneration plate for female strain corn embryo explant, the regeneration plate for male strain corn embryo explant may show frequent pollution and tissue necrosis.According to some embodiments, the less dense visible or total explant of each regeneration plate or container can improve monocotyledonous plant or corn embryo explant or to the effective transformation of genetically modified plant and / or regeneration more resistant some monocotyledonous plant or corn strain (as some male germplasm corn strain) genetically modified plant, bud development and / or the regeneration frequency.

[0150] According to some embodiments, monocot seed embryo explants can be transferred from one or more extended bud induction plates or containers to a relatively small number of regeneration plates or containers, such as a 3:1 or 2:1 ratio of the number of extended bud induction plates or containers to the number of regeneration plates or containers, but this may result in overcrowding of the number of explants per regeneration plate. Transferring explants to solid regeneration medium after extended bud induction at a ratio of 1 regeneration plate for every 3 extended bud induction plates may result in increased contamination and tissue necrosis during regeneration, leading to loss of transformed tissue and regenerated plants.

[0151] According to some embodiments, monocot seed embryo explants can be transferred from one or more elongated bud induction plates or containers to a larger or greater number of regeneration plates or containers, such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 in terms of the ratio of the number of elongated bud induction plates or containers to the number of regeneration plates or containers. According to some embodiments, a given number of visible (or total) monocotyledonous plant 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 ranges and values ​​derivable therein) can be transferred from the elongated shoot induction plates or culture medium to regeneration plates or containers, such as 1 or more, 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 regeneration plates or containers.According to some embodiments, the number of visible (or total) monocotyledonous plant seed embryo explants transferred or added to the regeneration plates or containers can be at a density of less than or equal to about 300 seed embryo explants per plate, less than or equal to about 200 seed embryo explants per plate, less than or equal to about 150 seed embryo explants per plate, less than or equal to about 100 seed embryo explants per plate, less than or equal to about 75 seed embryo explants per plate, less than or equal to about 50 seed embryo explants per plate, less than or equal to about 300 seed embryo explants per plate, or less than or equal to about 100 seed embryo explants per plate. 0 seed embryo explants per plate, or a density ranging from 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 250 seed embryo explants per plate, 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 150 seed embryo explants per plate, The density of each plate is about 50 to about 100 seed embryo explants, 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 ranges and values ​​therebetween. For these density values ​​and scopes, the surface area of ​​each regeneration plate is about 11.9 square inches (in). 2 ) or 76.8 square centimeters (cm 2 Thus, all of the above density values ​​and ranges of seed embryo explants per plate can be readily converted to density values ​​and ranges of seed embryo explants per regeneration surface area (e.g., a density of 100 seed embryo explants per plate can be divided by the surface area of ​​each plate to yield a density of approximately 8.4 seed embryo explants per square inch (in2) of seed embryo explants per regeneration surface area). 2 ) or about 1.3 seed embryo explants / cm2 (cm 2), and similar conversions can be readily made for other density values ​​and ranges.) The density values ​​and ranges of seed embryo explants per regeneration surface area are a more general definition of the density of seed embryo explants in a variety of different regeneration plates or containers, each of which may have different surface areas.

[0152] In some embodiments, the density of the number of visible (or total) monocot seed embryo explants transferred or added to the regeneration plate or container can be 100% per square centimeter (cm 2 ) has a regeneration surface area of ​​less than or equal to 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 embryonic explant, including all ranges and values ​​derivable therein. In certain embodiments, the density of the number of visible (or total) monocot seed embryo explants transferred or added to the regeneration plate or container can range from 1 to 2 cm2. 2) has a regeneration surface area of ​​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 a...

Claims

1. A method for genetically modifying a population of plant embryo explants, the method comprising: The heterologous polynucleotide molecules are co-introduced into at least two embryonic explants of the population, each of the at least two plant embryonic explants comprising meristem tissue, wherein the population comprises embryonic explants of at least two different plant genotypes.

2. The method of claim 1, wherein the population is defined as a population of monocotyledonous plant embryonic explants.

3. The method of claim 2, wherein the population is defined as a population of maize, wheat, rice, barley, sorghum or turf grass embryo explants.

4. The method of claim 2 or 3, wherein the population is defined as a population of maize embryo explants.

5. The method of claim 2 or 3, wherein the population is defined as a population of wheat embryo explants.

6. The method of any one of claims 1 to 5, wherein the heterologous polynucleotide molecule comprises an expression cassette, and the expression cassette comprises a selectable marker gene, a screenable marker gene, a gene of interest, a nucleotide sequence encoding a site-specific nuclease, or a nucleotide sequence encoding a guide RNA.

7. The method of any one of claims 1-6, wherein the co-introduction comprises introducing the heterologous polynucleotide molecule into at least two explants of the population via bacteria-mediated transformation or Rhizobiales bacteria-mediated transformation.

8. The method of claim 7, wherein the Rhizobiales bacterium is selected from the group consisting of: a) bacteria of the families Rhizobiaceae, Leafbacteraceae, Brucellaceae, Bradyrhizobiaceae and Xanthomabacteraceae; or b) Bacteria of the genera Agrobacterium, Rhizobium, Sinorhizobium, Mesorhizobium, Leafbacterium, Ochrobacterium, Bradyrhizobium and Azotobium.

9. The method of any one of claims 1 to 8, wherein the co-introduction comprises introducing the heterologous polynucleotide molecule via Agrobacterium-mediated transformation, or wherein said co-introducing said heterologous polynucleotide molecule comprises inoculating said at least two embryo explants with an inoculation medium containing Rhizobiales bacteria capable of transforming said at least two embryo explants with said heterologous polynucleotide molecule.

10. The method of claim 9, wherein the force is applied to the population in contact with the inoculation medium, or wherein the force is applied prior to co-introducing the heterologous polynucleotide molecule.

11. The method of claim 9, wherein the embryo explants in contact with the inoculation medium are subjected to a force treatment, and wherein the force treatment comprises a gravity treatment in the 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.

12. The method of any one of claims 9 to 11, further comprising: The at least two embryo explants are co-cultivated with the Rhizobiales bacteria in contact with a co-culture medium.

13. The method of claim 12, wherein the population of embryo explants is a population of monocot seed embryo explants, and the method further comprises: a) per square centimeter (cm 2 ) contacting at least two embryonic explants of the population with the co-culture medium at a density of less than or equal to about 9.1 embryonic explants per co-culture surface area; or b) contacting at least two embryonic explants of the population with the co-culture medium for a period of time ranging from about 6 days to about 8 days.

14. The method of any one of claims 1-6, wherein said co-introducing comprises introducing said heterologous polynucleotide molecule into at least two explants of said population via particle bombardment.

15. The method of any one of claims 1 to 6 and 14, wherein: The co-introduction comprises introducing the site-specific nuclease into at least two embryonic explants of the population; or The heterologous polynucleotide molecule comprises a guide RNA molecule, wherein the co-introducing comprises introducing the site-specific nuclease into at least two embryonic explants of the population.

16. The method of claim 15, wherein the site-specific nuclease is a ribonucleoprotein, and wherein the ribonucleoprotein comprises the site-specific nuclease and the guide RNA molecule.

17. The method of any one of claims 1-16, wherein the co-introduction comprises introducing at least two heterologous polynucleotides into at least two explants of the population, wherein the at least two heterologous polynucleotides are different.

18. The method of claim 17, wherein: 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; The first expression cassette comprises 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 comprises a second selectable marker 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.

19. The method of any one of claims 1 to 16, wherein: The heterologous polynucleotide molecule comprises a first expression cassette and a second expression cassette; The first expression cassette comprises 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 comprises 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.

20. The method of any one of claims 6 to 19, further comprising: The at least two embryo explants are contacted and cultured with a first shoot induction medium comprising a first auxin and a first cytokinin.

21. The method of claim 20, wherein the first shoot induction medium comprises a high cytokinin to auxin ratio.

22. The method of claim 20 or 21, wherein the first auxin in the first bud induction medium is selected from the group consisting of: 2,4-dichlorophenoxy-acetic acid (2,4-D), 4-amino-3,5,6-trichloro-picolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthylacetic acid (NAA), 4-chlorophenoxyacetic acid or p-chloro-phenoxyacetic 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-methoxy-benzoic acid (dicamba), or wherein the first cytokinin in the first bud induction medium is selected from the group consisting of 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino)purine (2iP) and 6-(3-hydroxybenzylamino)purine (topolin).

23. The method of any one of claims 20-22, wherein the concentration of the first auxin in the first shoot induction medium is about 0.02 mg / L to about 25 mg / L or about 1 mg / L to about 2 mg / L.

24. The method of any one of claims 20-23, wherein the concentration of the first cytokinin in the first bud induction medium is in the range of about 0.1 mg / L to about 50 mg / L.

25. The method of any one of claims 20-24, wherein the population of embryo explants is a population of monocotyledonous plant seed embryo explants, and the method further comprises: 2 ) The at least two embryo explants are contacted and cultured with the first bud induction medium at a density of less than or equal to about 3.9 embryo explants.

26. The method of any one of claims 20-25, further comprising: The at least two embryo explants are contacted and cultured with a second shoot induction medium comprising the first auxin or the second auxin and the first cytokinin or the second cytokinin.

27. The method of claim 26, wherein the embryo explants are contacted with the second shoot induction medium at a temperature ranging from about 20°C to about 32°C, about 25°C to about 29°C, or about 27°C to about 28°C.

28. The method of claim 26 or 27, wherein the second shoot induction medium comprises: a) High cytokinin to auxin ratio; b) the first auxin and the first cytokinin; c) the first auxin and the second cytokinin; d) the second auxin and the first cytokinin; or e) the second auxin and the second cytokinin.

29. The method of any one of claims 26-28, wherein the population of embryo explants is a population of monocotyledonous plant seed embryo explants, and the method further comprises: 2 ) The at least two embryo explants are contacted and cultured with the second bud induction medium at a density of less than or equal to about 2.6 embryo explants.

30. The method of any one of claims 26-29, wherein the first auxin or the second auxin in the second shoot induction medium is selected from the group consisting of 2,4-dichlorophenoxy-acetic acid (2,4-D), 4-amino-3,5,6-trichloro-picolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthylacetic acid (NAA), 4-chlorophenoxyacetic acid or p-chloro-phenoxyacetic 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-methoxy-benzoic acid (dicamba).

31. The method of any one of claims 26-30, wherein the first cytokinin or the second cytokinin in the second bud induction medium is selected from the group consisting of 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino)purine (2iP) and 6-(3-hydroxybenzylamino)purine (topolin).

32. The method of any one of claims 26-31, wherein the concentration of the first cytokinin or the second cytokinin in the second bud 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.

33. The method of any one of claims 26-32, wherein the concentration of the first auxin or the second auxin in the second shoot induction 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.

34. The method of any one of claims 26-33, wherein said heterologous polynucleotide molecule comprises a selectable marker gene, wherein said second shoot induction medium comprises a selection agent, and wherein said selectable marker gene provides resistance to said selection agent in a plant.

35. The method of any one of claims 1 to 34, further comprising: A plurality of genetically modified plants or plant parts are regenerated or grown from contacting the at least two embryonic explants or any progeny of their cells with a regeneration medium.

36. The method of claim 35, wherein the population is a population of monocotyledonous plant seed embryo explants, and the method further comprises: 2 ) a density of about 2.6 embryo explants or less at a regeneration surface area of ​​less than or equal to about 2.6 embryo explants to regenerate or grow a plurality of genetically modified plants or plant parts from the at least two embryo explants in contact with the regeneration medium.

37. The method of claim 35, wherein the population is a population of monocot seed embryo explants, and the method further comprises: contacting a culture population of monocotyledonous plant seed embryo explants with a first regeneration medium, transferring the cultured population of monocotyledonous plant seed embryo explants or a subset of the cultured population of monocotyledonous plant seed embryo explants to a second regeneration medium, and Contacting with the second regeneration medium regenerates the plurality of genetically modified monocotyledonous plants or plant parts.

38. The method of any one of claims 35-37, wherein the regeneration medium, the first regeneration medium or the second regeneration medium has a low salt concentration.

39. The method of any one of claims 35-38, wherein the heterologous polynucleotide molecule comprises a selectable marker gene, wherein the regeneration medium, the first regeneration medium, or the second regeneration medium comprises a selection agent, and wherein the selectable marker gene provides resistance to the selection agent in the plant.

40. The method of any one of claims 35-39, wherein said plurality of genetically modified plants or plant parts comprises at least one genetic modification.

41. The method of claim 40, wherein: a) the at least one genetic modification comprises integration or insertion of the 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; b) the at least one genetic modification comprises editing introduced into the genome of the plurality of genetically modified plants or plant parts by genome editing technology using a site-specific nuclease or a guide RNA molecule; c) the heterologous polynucleotide molecule comprises at least one expression cassette, and the at least one expression cassette encodes a site-specific nuclease or a guide RNA molecule; or d) the heterologous polynucleotide molecule comprises at least two expression cassettes, the at least two expression cassettes comprising a first expression cassette encoding a site-specific nuclease and a second expression cassette encoding a guide RNA molecule.

42. The method of any one of claims 1-41, wherein the population comprises embryonic explants having an internal moisture content ranging from about 3% to about 25% prior to the introduction of the heterologous polynucleotide molecule.

43. The method of any one of claims 1-42, wherein the population comprises embryo explants comprising the apical portion of an embryonic axis lacking a radicle, or wherein the remainder of a seed from which the embryo explant was prepared has been substantially removed from the embryo explants.

44. The method of any one of claims 2-43, wherein the population comprising embryonic explants is prepared from a population of monocot seeds under conditions wherein the embryonic explants do not germinate and remain viable and genetically modifiable.

45. The method of any one of claims 1-44, wherein the population 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, at least 450, or at least 500 different or embryo explants of 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.

46. ​​The method of any one of claims 1-45, wherein at least two embryonic explants of the population comprise 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 more. 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.

47. The method of any one of claims 1-46, wherein the population 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, 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 about 2 to about 50,000, 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 20,000, about 5,000 to about 10,000, about 20 to about 30,000, about 5,000 to about 15,000, about 25 to about 30,000. or 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 embryo explants.

48. The method of any one of claims 1-47, wherein said population comprises at least one embryonic explant having at least one unconfirmed genotype prior to said co-introduction of said heterologous polynucleotide molecule.

49. The method of any one of claims 1-47, wherein at least two different genotypes of the embryonic explants are known.

50. The method of any one of claims 1-48, wherein each embryo explant of the population is an embryo explant of known genotype.

51. The method of any one of claims 35-50, wherein 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, 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 3500, at least 4000, at least 4500 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.

52. The method of any one of claims 1 to 51, further comprising: The genotype of at least one embryonic explant or at least one of said genetically modified plants or plant parts of said population is determined.

53. The method of claim 52, wherein the confirming the genotype comprises detecting at least one genetic marker in the at least one embryo explant or the at least one genetically modified plant or plant part, wherein the at least one genetic marker comprises a polynucleotide sequence characteristic of the genotype.

54. The method of claim 53, wherein the polynucleotide sequence is characteristic of the genotype.

55. as described in any one of the methods of claim 52-54, wherein said confirming described genotype 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 or at least 25 genetic markers in described at least one embryo explant or described at least one genetically modified plant or plant part, wherein said every kind of genetic marker comprises the polynucleotide sequence characteristic of described genotype.

56. The method of claim 55, wherein each of said genetic markers comprises a polynucleotide sequence that is uniquely characteristic of said genotype.

57. The method of any one of claims 52-56, wherein said confirming said genotype comprises: confirming the genotype before or after introducing the heterologous polynucleotide molecule into at least two embryonic explants of the population; confirming the genotype before or after co-culturing at least two embryonic explants of the population; confirming the genotype before or after contacting and culturing at least two embryo explants of the population with the first shoot induction medium; confirming the genotype before or after contacting and culturing at least two embryo explants of the population with the second shoot induction medium; or The genotype is confirmed before or after regenerating or growing the plurality of genetically modified plants or plant parts from the at least two embryonic explants of the population or any progeny of cells thereof.

58. The method of any one of claims 52-57, wherein said confirming said genotype comprises: performing genetic sequencing on a sample comprising polynucleotide molecules from or derived from said at least one embryonic explant or said 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 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 comprises a polynucleotide sequence characteristic of the genotype.

59. The method of any one of claims 52-57, wherein said confirming said genotype comprises: contacting a sample comprising polynucleotide molecules from or derived from said at least one embryonic explant or said at least one genetically modified plant or plant part with at least one polynucleotide probe, wherein said polynucleotide probe is specific for a genetic marker; subjecting the sample and the polynucleotide probe to stringent hybridization conditions; and detecting hybridization of the polynucleotide probe to at least one genetic 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 Wherein the genetic marker comprises a polynucleotide sequence characteristic of the genotype.

60. The method of any one of claims 52-59, wherein said confirming said genotype comprises: The genotypes of the plurality of said embryonic explants or the plurality of said genetically modified plants or plant parts are confirmed.

61. The method of any one of claims 52-59, wherein said confirming said genotype comprises: A plurality of genotypes of at least one of said embryonic explants or at least one of said genetically modified plants or plant parts of said population are identified.

62. The method of any one of claims 52-61, wherein said confirming said genotype comprises: The plurality of genotypes of the plurality of said embryonic explants or the plurality of said genetically modified plants or plant parts are confirmed.

63. The method of any one of claims 52-59, wherein said confirming said genotype comprises: At least two genetic markers are detected in at least two of said embryonic explants or at least two of said genetically modified plants or plant parts of said population.

64. A method as claimed in claim 63, wherein the at least two genetic markers include a first genetic marker and a second genetic marker, and wherein the first genetic marker comprises a first polynucleotide sequence characteristic of a first genotype, and the second genetic marker comprises a second polynucleotide sequence characteristic of a second genotype.

65. The method of claim 64, wherein the first polynucleotide sequence is characteristic exclusively of the first genotype, or the second polynucleotide sequence is characteristic exclusively of the second genotype.

66. The method of any one of claims 63-65, wherein the confirming 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 or at least 20 genetic markers are detected in the at least two embryonic explants or the at least two genetically modified plants or plant parts, wherein at least two of the genetic markers comprise a polynucleotide sequence characteristic of the first genotype and at least one of the genetic markers comprises a polynucleotide sequence characteristic of the second genotype.

67. The method of any one of claims 63-66, wherein the confirming comprises: genetically sequencing at least a first sample and a second sample, wherein the first sample comprises a first polynucleotide molecule from or derived from a first embryonic explant or a first genetically modified plant or plant part, and the second sample comprises a second polynucleotide molecule from or derived 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 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 Wherein each of the genetic markers comprises a polynucleotide sequence characteristic of the genotype, or wherein the genetic markers comprise a first genetic marker and a second genetic marker, the first genetic marker comprises a first polynucleotide sequence characteristic of a first genotype, and the second genetic marker comprises a second polynucleotide sequence characteristic of a second genotype.

68. The method of any one of claims 52-67, further comprising: confirming the presence of a genetic modification in at least one embryonic explant or at least one genetically modified plant or plant part of said population; and selecting embryonic explants or genetically modified plants or plant parts of said population comprising said genetic modification, wherein said selected embryonic explant or said selected genetically modified plant or plant part further comprises at least one genetic marker characteristic of said genotype, or wherein said selected embryonic explant or said selected genetically modified plant or plant part does not further comprise at least one genetic marker characteristic of said genotype.

69. The method of claim 68, further comprising: regenerating or growing a regenerated genetically modified plant or plant part from said selected embryonic explant or any progeny of its cells; or The selected genetically modified plant is crossed with itself or a different plant to obtain progeny plants or seeds.

70. The method of any one of claims 64-69, further comprising: confirming the genetic modification present in at least two embryonic explants of the population or in the at least two genetically modified plants or plant parts; and selecting a first embryonic explant or a first genetically modified plant or plant part of said population comprising said genetic modification and a second embryonic explant or a second genetically modified plant or plant part of said population comprising said genetic modification, wherein said first selected embryonic explant or said first selected genetically modified plant or plant part further comprises said first genetic marker or said second genetic marker, or wherein said first selected embryonic explant or said first selected genetically modified plant or plant part does not further comprise said first genetic marker or said second genetic marker, and wherein said second selected embryonic explant or said second selected genetically modified plant or plant part further comprises said first genetic marker or said second genetic marker, or wherein said second selected embryonic explant or said second selected genetically modified plant or plant part does not further comprise said first genetic marker or said second genetic marker.

71. The method of any one of claims 64-69, further comprising: confirming the presence of at least two genetic modifications in at least two embryonic explants or at least two genetically modified plants or plant parts of the population, the at least two genetic modifications comprising a first genetic modification and a second genetic modification; and selecting a first embryonic explant or a first genetically modified plant or plant part of said population comprising said first genetic modification and a second embryonic explant or a second genetically modified plant or plant part of said population comprising said second genetic modification, wherein said first selected embryonic explant or said first selected genetically modified plant or plant part further comprises said first genetic marker or said second genetic marker, or wherein said first selected embryonic explant or said first selected genetically modified plant or plant part does not further comprise said first genetic marker or said second genetic marker, and wherein said second selected embryonic explant or said second selected genetically modified plant or plant part further comprises said first genetic marker or said second genetic marker, or wherein said second selected embryonic explant or said second selected genetically modified plant or plant part does not further comprise said first genetic marker or said second genetic marker.

72. The method of claim 70 or 71, further comprising: A first regenerated genetically modified plant or plant part is regenerated or grown from any progeny of said first selected embryonic explant or a cell thereof, or a second regenerated genetically modified plant or plant part is regenerated or grown from any progeny of said second selected embryonic explant or a cell thereof.

73. The method of any one of claims 70-72, further comprising: observing culture characteristics of the first selected embryo explant and the second selected embryo explant; observing the phenotype of said first regenerated genetically modified plant or plant part and said second regenerated genetically modified plant or plant part; or The phenotype of said first selected genetically modified plant or plant part and said second selected genetically modified plant or plant part is observed.

74. The method of claim 73, further comprising: 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 phenotypes 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 The phenotypes of the first selected genetically modified plant or plant part and the second selected genetically modified plant or plant part are compared, and a determination is made 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.

75. The method of claim 73, further comprising: A first regenerated genetically modified plant or plant part is regenerated or grown from any progeny of the first selected embryonic explant or its cells, or a second regenerated genetically modified plant or plant part is regenerated or grown from any progeny of the second selected embryonic explant or its cells based on the culture characteristics of the first selected embryonic explant and the second selected embryonic explant.

76. The method of any one of claims 70-75, further comprising: 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; hybridizing the first regenerated genetically modified plant with itself or a different plant to obtain a third progeny plant or seed; or The second regenerated genetically modified plant is crossed with itself or a different plant to obtain a fourth progeny plant or seed.

77. The method of any one of claims 52-76, further comprising: A second heterologous polynucleotide molecule is introduced into at least one explant of a second population of embryonic explants, wherein the at least one explant of the second population has the same genotype or has a different genotype compared to: at least one embryonic explant of said population; said at least one genetically modified plant or plant part; said selected embryonic explant; or The selected genetically modified plant or plant part.

78. The method of any one of claims 1 to 77, further comprising: The genetic modification is detected in at least one of said embryonic explants or at least one of said genetically modified plants or plant parts of said population.

79. The method of any one of claims 35-78, wherein each of said plurality of genetically modified plants or plant parts comprises at least one genetic modification.

80. The method of claim 79, further comprising: selecting a genetically modified plant comprising said at least one genetic modification; and crossing the genetically modified plant with itself or a second plant to obtain progeny plants or seeds, wherein the second plant has the same genotype as the genetically modified plant or has a different genotype from the genetically modified plant.

81. The method of claim 80, wherein said selecting comprises identifying a genotype of said genetically modified plant and selecting said genetically modified plant comprising said genotype.

82. The method of claim 81, further comprising: 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; hybridizing the first genetically modified plant with itself or a first different plant to obtain a first progeny plant or seed; and The second genetically modified plant is crossed with itself, the first genetically modified plant, or a second different plant to obtain a second progeny plant or seed.

83. The method of claim 82, wherein the selecting comprises: confirming a first genotype of the first genetically modified plant, selecting the first genetically modified plant comprising the first genotype, and The second genotype of the second genetically modified plant is identified, and the second genetically modified plant comprising the second genotype is selected.

84. The method of any one of claims 52-83, further comprising: The genotype is correlated to at least one culture characteristic or at least one phenotype.

85. The method of claim 84, further comprising: Genetic markers or quantitative trait loci (QTLs) associated with the at least one culture characteristic or the at least one phenotype are identified.

86. The method of any one of claims 73-85, wherein the at least one culture characteristic is selected from the group consisting of: explant excision efficiency, regeneration efficiency, genetic modification efficiency, transformation efficiency, and the ability to regenerate into a genetically modified plant or plant part.

87. The method of any one of claims 73 to 85, wherein: The at least one phenotype is an observable plant trait, or the at least one phenotype results from expression of a selectable or screenable marker; or The at least one phenotype results from a genetic modification, wherein the genetic modification results from integration 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 gene of interest, a site-specific nuclease, or a guide RNA molecule.

88. The method of any one of claims 73-85, further comprising: introgressing a chromosome segment conferring said at least one culture characteristic or said at least one phenotype into a plant, said plant having a plant genotype lacking said culture characteristic or said phenotype in the absence of said chromosome segment; or A genetically modified plant comprising a chromosome segment that confers at least one culture characteristic or at least one phenotype is crossed with itself or a different plant to produce a progeny plant or seed comprising the chromosome segment.

89. like each described method in claim 1-88, the embryo explants of wherein said at least two different genotypes comprise the embryo explant of a first genotype and the embryo explant of a second genotype, and wherein the embryo explant of said first genotype and the embryo explant of said second genotype are present in said colony with a predetermined ratio.

90. The method of claim 89, wherein the predetermined ratio is determined based on at least one culture characteristic associated with the first genotype, with the second genotype, or with the first genotype and the second genotype.

91. The method of claim 90, wherein the at least one culture characteristic is selected from the group consisting of: explant excision efficiency, regeneration efficiency, shoot generation efficiency, genetic modification efficiency, transformation efficiency, and the ability to regenerate into a genetically modified plant or plant part.

92. as described in any one method among the claim 89-91, the described predetermined ratio of embryo explants of wherein said first genotype and said second genotype comprises embryo explants of said first genotype and said second genotype of approximately equal number.

93. as described in any one in claim 89-92 method, the described predetermined ratio of the embryo explant of wherein said first genotype and said second genotype causes the quantity of the regenerated genetically modified plant or plant part of said first genotype and said second genotype to be roughly equal.

94. The method of any one of claims 89 to 93, wherein: The first genotype is associated with a preferred culture characteristic relative to the second genotype, and the method comprises altering the predetermined ratio to include an increased number of embryonic explants of the second genotype compared to the first genotype in the population; or The second genotype is associated with the preferred culture characteristic relative to the first genotype, and the method includes altering the predetermined ratio to include an increased number of embryonic explants of the first genotype compared to the second genotype in the population.

95. The method of claim 94, wherein the predetermined ratio of embryonic explants 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.

96. The method of claim 95 or 96, wherein the preferred culture characteristics result in increased explant excision efficiency, regeneration efficiency, shoot generation efficiency, genetic modification efficiency, transformation efficiency, or the ability to regenerate into a genetically modified plant or part.

97. The method of any one of claims 1 to 96, further comprising: At least one culture characteristic or at least one phenotype of at least one of said embryonic explants or at least one of said genetically modified plants or plant parts of said population is observed.

98. The method of claim 97, wherein the at least one culture characteristic or the at least one phenotype is associated with a genetic modification of the at least one embryonic explant or the at least one genetically modified plant or plant part.

99. The method of any one of claims 1 to 98, further comprising: observing a first culture characteristic or a first phenotype of at least one embryonic explant of a first genotype or at least one genetically modified plant or plant part of said first genotype; and A second culture characteristic or a second phenotype is observed for at least one embryonic explant of a second genotype or at least one genetically modified plant or plant part of said second genotype.

100. The method of claim 99, wherein: The first culture characteristic or the first phenotype and the second culture characteristic or the second phenotype are the same; or The first culture characteristic or the first phenotype and the second culture characteristic or the phenotype are different.

101. The method of any one of claims 97-100, further comprising: The at least one embryonic explant or the at least one genetically modified plant or plant part of the first genotype and the at least one embryonic explant or the at least one genetically modified plant or plant part of the second genotype are evaluated by comparing the first culture characteristic or the first phenotype and the second culture characteristic or the second phenotype.

102. The method of any one of claims 1-101, wherein the co-introduction comprises site-directed integration of the heterologous polynucleotide or fragment thereof.

103. The method of any one of claims 1-102, wherein the heterologous polynucleotide molecule comprises or encodes a guide RNA.

104. The method of any one of claims 1-103, wherein said co-introduction comprises introducing said heterologous polynucleotide molecules into said population simultaneously.

105. The method of any one of claims 1, 6-19, and 35-104, wherein the population of embryo explants is a population of dicotyledonous embryo explants.

106. The method of claim 105, wherein the population of dicot embryo explants is a population of soybean, cotton or rapeseed embryo explants.

107. The method of claim 13, wherein the population of embryonic explants is a population of dicotyledonous embryonic explants, and wherein the co-culture medium comprises at least one cytokinin or lipoic acid.

108. The method of claim 107, wherein the at least one cytokinin is selected from the group consisting of 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino)purine (2iP) and 6-(3-hydroxybenzylamino)purine (topolin).

109. The method of claim 107 or 108, wherein the concentration of the cytokinin in the co-culture medium is about 0.1 mg / L to about 50 mg / L.

110. The method of claim 108 or 109, wherein 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.

111. The method of claim 108 or 109, wherein 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.

112. The method of claim 107, wherein the concentration of lipoic acid in the co-culture medium is about 0.1 mg / L to about 500 mg / L.

113. The method of claim 35, wherein the population of embryonic explants is a population of dicotyledonous embryonic explants.

114. The method of claim 113, wherein the heterologous polynucleotide molecule comprises a selectable marker gene, wherein the regeneration medium comprises a selection agent, and wherein the selectable marker gene provides resistance to the selection agent in the plant.

115. The method of claim 113 or 114, wherein the plurality of genetically modified plants or plant parts comprises at least one genetic modification.

116. The method of claim 115, wherein the at least one genetic modification comprises integrating or inserting the heterologous polynucleotide molecule or 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.

117. The method of claim 116, wherein the at least one genetic modification comprises an edit introduced into the genome of the plurality of genetically modified plants or plant parts using a site-specific nuclease or a guide RNA molecule by genome editing technology.

118. The method of claim 117, wherein the heterologous polynucleotide molecule comprises at least one expression cassette, and the at least one expression cassette encodes the site-specific nuclease or the guide RNA molecule, or the heterologous polynucleotide molecule comprises at least two expression cassettes, and the at least two expression cassettes include a first expression cassette encoding the site-specific nuclease and a second expression cassette encoding the guide RNA molecule.

119. The method of claim 113, wherein the regeneration medium comprises at least one cytokinin.

120. The method of claim 114, wherein the at least one cytokinin is selected from the group consisting of 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino)purine (2iP) and 6-(3-hydroxybenzylamino)purine (topolin).

121. The method of claim 119 or 120, wherein the concentration of the cytokinin in the regeneration medium is about 0.1 mg / L to about 50 mg / L.

122. The method of any one of claims 119-121, wherein 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 about 0.1 mg / L to about 15 mg / L.

123. The method of any one of claims 113-122, comprising regenerating or growing the plurality of genetically modified plants or plant parts in contact with the regeneration medium at about 15°C to about 40°C.

124. The method of claim 123, wherein the population of embryo explants is a population of cotton embryo explants, and the method comprises regenerating or growing the plurality of genetically modified plants or plant parts in contact with the regeneration medium at about 30°C to about 40°C for a first regeneration period.

125. The method of claim 124, wherein the first regeneration period is from about 1 hour to about 14 days.

126. The method of claim 125, comprising regenerating or growing the plurality of genetically modified cotton plants or cotton plant parts in contact with the regeneration medium at about 20°C to about 33°C for a second regeneration period.

127. The method of claim 126, wherein the second regeneration period is from about 7 days to about 56 days.

128. The method of any one of claims 113-124, comprising regenerating or growing the plurality of genetically modified plants or plant parts in contact with the regeneration medium for about 5 days to about 70 days or about 14 days to about 50 days.

129. The method of claim 128, wherein the population of embryo explants is a population of soybean embryo explants, and the method comprises regenerating or growing the plurality of genetically modified plants or plant parts in contact with the regeneration medium for about 5 days to about 70 days.

130. The method of claim 128, wherein the population of embryonic explants is a population of cotton embryonic explants, and the method comprises regenerating or growing the plurality of genetically modified plants or plant parts in contact with the regeneration medium for about 14 days to about 70 days.

131. The method of claim 128, wherein the population of embryo explants is a population of rapeseed embryo explants, and the method comprises regenerating or growing the plurality of genetically modified plants or plant parts in contact with the regeneration medium for about 14 days to about 70 days.

132. The method of any one of claims 113-131, further comprising regenerating or growing the plurality of genetically modified plants or plant parts by contacting with a second regeneration medium for an extended regeneration period.

133. The method of claim 132, wherein the second regeneration medium comprises at least one auxin, at least one cytokinin, or at least one auxin and at least one cytokinin.

134. The method of claim 133, wherein: The at least one auxin is selected from the group consisting of 2,4-dichlorophenoxy-acetic acid (2,4-D), 4-amino-3,5,6-trichloro-picolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthylacetic acid (NAA), 4-chlorophenoxyacetic acid or p-chloro-phenoxyacetic 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-methoxy-benzoic acid (dicamba); or The at least one cytokinin is selected from the group consisting of 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino)purine (2iP) and 6-(3-hydroxybenzylamino)purine (topolin).

135. The method of claim 133 or 134, wherein 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 wherein the concentration of the at least one cytokinin in the second regeneration medium is about 0.1 mg / L to about 50 mg / L.

136. The method of any one of claims 132-135, comprising regenerating or growing the plurality of genetically modified plants or plant parts in contact with a second regeneration medium at about 15°C to about 40°C.

137. The method of any one of claims 132-136, wherein the extended regeneration period is from about 7 days to about 56 days.

138. The method of any one of claims 132-137, wherein the population of embryo explants is a population of cotton embryo explants, and the method further comprises transferring selected portions of the multiple genetically modified cotton plants or cotton plant parts to the second regeneration medium before contacting the second regeneration medium to regenerate or grow the multiple genetically modified cotton plants or cotton plant parts.

139. The method of any one of claims 132-137, further comprising regenerating or growing the plurality of genetically modified plants or plant parts in contact with a first elongation medium for a first elongation period.

140. The method of claim 139, wherein the population of embryo explants is a population of rapeseed embryo explants.

141. The method of claim 139 or 140, wherein the first elongation medium comprises at least one auxin, at least one cytokinin, or at least one auxin and at least one cytokinin.

142. The method of claim 141, wherein: The at least one auxin is selected from the group consisting of 2,4-dichlorophenoxy-acetic acid (2,4-D), 4-amino-3,5,6-trichloro-picolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthylacetic acid (NAA), 4-chlorophenoxyacetic acid or p-chloro-phenoxyacetic 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-methoxy-benzoic acid (dicamba); or The at least one cytokinin is selected from the group consisting of 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino)purine (2iP) and 6-(3-hydroxybenzylamino)purine (topolin).

143. The method of claim 141 or 142, wherein 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 wherein the concentration of the at least one cytokinin in the first elongation medium is about 0.1 mg / L to about 50 mg / L.

144. The method of any one of claims 139-143, wherein the method comprises regenerating or growing the plurality of genetically modified plants or plant parts in contact with the first elongation medium at about 15°C to about 40°C; or wherein the first elongation period is about 7 days to about 56 days.

145. The method of any one of claims 139-144, further comprising regenerating or growing the plurality of genetically modified plants or plant parts for a second elongation period in contact with a second elongation medium.

146. The method of claim 145, wherein the second elongation medium comprises at least one auxin, at least one cytokinin, or at least one auxin and at least one cytokinin.

147. The method of claim 146, wherein: The at least one auxin is selected from the group consisting of 2,4-dichlorophenoxy-acetic acid (2,4-D), 4-amino-3,5,6-trichloro-picolinic acid (picloram), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthylacetic acid (NAA), 4-chlorophenoxyacetic acid or p-chloro-phenoxyacetic 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-methoxy-benzoic acid (dicamba); or The at least one cytokinin is selected from the group consisting of 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, zeatin, diphenylurea (DPU), 6-(γ,γ-dimethylallylamino)purine (2iP) and 6-(3-hydroxybenzylamino)purine (topolin).

148. The method of claim 146 or 147, wherein 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 wherein the concentration of the at least one cytokinin in the second elongation medium is about 0.1 mg / L to about 50 mg / L.

149. The method of any one of claims 145-148, comprising regenerating or growing the plurality of genetically modified plants or plant parts in contact with the second elongation medium at about 15°C to about 40°C.

150. The method of any one of claims 139-143, wherein the second extension period is from about 7 days to about 56 days.

151. The method of any one of claims 1-150, wherein said co-introducing comprises introducing said heterologous polynucleotide molecule into a population of said embryonic explants while said populations are present together in a single container.

152. The method of any one of claims 35-106 and 113-151, wherein the plant part comprises a shoot or a root.

153. The method of any one of claims 35-106 and 113-151, wherein the plant part comprises a seed.

154. The method of any one of claims 35-106 and 113-153, wherein said genetically modified plant or plant part is non-chimeric.

155. The method of any one of claims 35-106 and 113-154, wherein the genetically modified plant or plant part is cultivated or regenerated without producing a callus culture.

156. The method of any one of claims 1-155, wherein the population is a population of dry, dry, dry excised, wet excised or wet seed embryo explants.

157. The method of any one of claims 1-156, wherein the population is a population of mature or immature seed embryo explants.

158. A method for genetically modifying a population of plant embryo explants, the method comprising: The ribonucleoprotein or site-specific nuclease is co-introduced into at least two plant embryo explants of the population, each of the at least two plant embryo explants comprising a meristem, wherein the population comprises plant embryo explants of at least two different plant genotypes.

159. The method of claim 158, wherein the ribonucleoprotein comprises a site-specific nuclease and a guide RNA molecule.

160. The method of any one of claims 1 to 159, further comprising: The population of embryonic explants is excised from a population of plant seeds, wherein the excision is performed prior to the co-introduction of the heterologous polynucleotide molecule, the ribonucleoprotein or the site-specific nuclease, wherein the population of plant seeds comprises plant seeds of at least two different plant genotypes.

161. The method of claim 160, further comprising: The population of plant seeds is sorted into at least two batches of plant seeds based on plant seed size, plant seed shape, or a combination thereof prior to the excision.

162. The method of claim 161, wherein the at least two batches of plant seeds include a first batch of plant seeds and a second batch of plant seeds, wherein the population of plant embryo explants includes a first batch of embryo explants and a second batch of embryo explants, and the excision comprises: Using the same excision method to excise the first batch of embryo explants from the first batch of plant seeds, and to excise the second batch of embryo explants from the second batch of plant seeds; or The first batch of embryonic explants are excised from the first batch of plant seeds and the second batch of embryonic explants are excised from the second batch of plant seeds using different excision methods.

163. The method of claim 161 or 162, further comprising: 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 includes a first plant seed size, a first plant seed shape, or a combination thereof, and the second batch of plant seeds includes 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 a first batch of plant seeds and a second batch of plant seeds, wherein the first batch of plant seeds includes the first plant seed size, the first plant seed shape, or a combination thereof, and the second batch of plant seeds includes the second plant seed size, the second plant seed shape, or a combination thereof, The population of plant seeds comprises: a first predetermined ratio of plant seeds from said first batch of plant seeds of said first genotype and said second batch of plant seeds of said first genotype, and A second predetermined ratio of plant seeds from said first batch of plant seeds of said second genotype and said second batch of plant seeds of said second genotype.

164. The method of claim 163, wherein 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.

165. The method of claim 163 or 164, wherein 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 wherein 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.

166. The method of any one of claims 160-165, wherein the at least two different genotypes comprise a first genotype and a second genotype, and the excision results in the excision of approximately equal numbers of embryonic explants of the first genotype and the second genotype.

167. The method of any one of claims 160-166, wherein the at least two plant seeds of different genotypes include plant seeds of a first genotype and plant seeds of a second genotype, and 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.

168. The method of claim 167, wherein the predetermined ratio is determined based on at least one culture characteristic associated with the first genotype or the second genotype.

169. The method of claim 168, wherein the at least one culture characteristic is selected from the group consisting of: explant excision efficiency, regeneration efficiency, shoot generation efficiency, genetic modification efficiency, transformation efficiency, and the ability to regenerate into a genetically modified plant or plant part.

170. The method of any one of claims 163-169, 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.

171. The method of any one of claims 163 to 170, wherein: The first genotype is associated with a preferred cultivation characteristic relative to the second genotype, and the method comprises altering the predetermined ratio to include an increased number of plant seeds of the second genotype compared to the first genotype in the population; or the second genotype is associated with the preferred cultivation characteristic relative to the first genotype, and the method comprises altering the predetermined ratio to include an increased number of plant seeds of the first genotype compared to the second genotype in the population, wherein said predetermined ratio of plant seeds of said first genotype and said second genotype results in approximately equal numbers of regenerated genetically modified plants or plant parts of said first genotype and said second genotype.

172. The method of claim 171, wherein the preferred culture characteristics result in increased explant excision efficiency, regeneration efficiency, shoot generation efficiency, genetic modification efficiency, transformation efficiency, or the ability to regenerate into a genetically modified plant or part.

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