The effect of RdDM pathway mutants on MSH1 grafting results

Abstract

This invention provides a method for obtaining plants exhibiting useful traits by suppressing the expression of MSH1 and DRM2 genes in plant rootstock and grafting scions onto the rootstock. Methods for identifying gene loci that confer useful traits to plants, and plants produced using these loci, are also provided. Furthermore, plants exhibiting useful traits, plant parts such as seeds, plant products, and methods for using the plants are provided. Recombinant DNA vectors that induce the suppression of MSH1 and DRM2 genes and transgenic plants containing these vectors are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This international application claims the interests of U.S. Provisional Application No. 63 / 498,682, filed on 27 April 2023, which is incorporated herein by reference in its entirety.

[0002] Use of sequence listings This application includes a sequence listing submitted electronically in XML file format, the entire listing of which is incorporated herein by reference. The XML file, created on April 24, 2024, is named P13989WO00.xml and has a size of 78,790 bytes. [Background technology]

[0003] Evidence exists in both plants and animals supporting the link between environmental sensing and epigenetic changes (Bonasio et al., Science 330, 612, 2010). The intergenerational inheritance of these changes remains an active subject of research (Youngson et al. Annu. Rev. Genom. Human Genet. 9, 233, 2008). Previous studies have shown that modified methylation patterns are highly heritable across multiple generations and can be incorporated into quantitative analyses of mutational states (Vaughn et al. 2007; Zhang et al. 2008; Johannes et al. 2009). Early studies of methylation changes in Arabidopsis suggest that the epigenome can adapt to repeated selection, demonstrating the possibility of establishing new and stable epigenetic states. (F. Johannes et al. PLoS Genet. 5, e1000530 (2009); F. Roux et al. Genetics 188, 1015 (2011)). Manipulation of met1 and ddmt mutants of Arabidopsis thaliana has generated epigenetic recombinant inbred (epi-RIL) populations exhibiting novel methylation pattern inheritance and epiarelyl segregation, suggesting that epigenomic diversity likely influences plant adaptation (F. Roux et al. Genetics 188, 1015 (2011)). In natural populations, the majority of epiarelyl mutations detected in Arabidopsis thaliana are found as CpG methylation within gene-rich regions of the genome (C. Becker et al. Nature 480, 245 (2011), R. S. Schmitz et al. Science 334, 369 (2011)).

[0004] It has also been reported that repression of the MSH1 gene can induce traits exhibiting cytoplasmic inheritance (Redei Mutat.Res.18,149-162,1973; Sandhu et al. Proc Natl Acad Sci USA.104:1766-70,2007) or nuclear inheritance (WO2012 / 151254; Xu et al. Plant Physiol.Vol.159:711-720,2012). The plant msh1 system was first developed in the model plant Arabidopsis thaliana. In Arabidopsis thaliana, mutations or RNAi repression of msh1 result in a variety of plant phenotypic mutations associated with persistent stress responses (Xu et al.2012; Shao et al.2017) and epigenetic reprogramming of plants (Virdi et al.2015). Suppression of MSH1 expression induces a heritable and fully penetrating memory state in approximately 20% of the next generation of plants. This state similarly provides evidence of a sustained stress response and abiotic stress tolerance in plants (Yang et al. 2020; Kundariya et al. 2022). Plants obtained from grafting experiments using msh1 as the rootstock and wild-type plants with the same genetic background as the scion produce progeny with heritable enhanced growth vigor, resilience, and seed yield (Kundariya et al., 2020).

[0005] These manipulations of the epigenetic msh1 state have been successfully replicated in soybeans (Kechanmane Raju et al. 2018) and tomatoes (Yang et al. 2015; Kundariya et al. 2020), resulting in stress memory induced by MSH1 suppression and enhanced growth after grafting. However, the results of grafting experiments were varied; some grafted progeny performed better than the wild type, while others produced progeny with significantly less effect. These observations suggest that individual msh1 mutant rootstocks have different strengths of epigenetic "signals." Genetic experiments have demonstrated that the epigenetic phenomena of msh1 depend at least partially on the RNA-induced DNA methylation (RdDM) pathway (Yang et al. 2020; Kundariya et al. 2020). Therefore, the transmission of msh1-derived growth effects via grafting requires the production and transmission of sRNA by the rootstock (Kundariya et al. 2020; 2022). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Bonasio et al.,Science 330,612,2010 [Non-Patent Document 2] Youngson et al.Annu.Rev.Genom.Human Genet.9,233,2008 [Non-Patent Document 3] F.Johannes et al.PLoS Genet.5,e1000530(2009) [Non-Patent Document 4] F.Roux et al.Genetics 188,1015(2011) [Overview of the Initiative] [Means for solving the problem]

[0007] A grafted plant is provided, comprising a scion grafted onto a rootstock, wherein (i) the scion is derived from a wild-type plant, (ii) the expression of MSH1 and DRM2 genes is suppressed in the rootstock, and (iii) the rootstock imparts an improvement in yield or growth rate to the progeny of the grafted plant compared to a control plant, and the control plant comprises any of the following: (a) the progeny of a scion grafted onto a rootstock in which the expression of MSH1 and DRM2 genes has not been suppressed, (b) a complete plant in which no root grafting has been performed and the expression of MSH1 and DRM2 genes has not been suppressed, (c) a wild-type plant, or (d) the progeny of a plant having the same genetic background as the plant source of the scion of the grafted plant.

[0008] A selected population of progeny produced by a method comprising: (a) a step of obtaining a population of progeny from the aforementioned grafted plants, wherein the population of progeny is a first, second, or third generation of progeny obtained by self-propagating the grafted plants or by self-propagating the first or second generation of progeny; (b) a step of screening the population of progeny for improved yield or growth rate compared to a control population; and (c) a step of selecting the population of progeny for improvement in yield or growth rate compared to a control plant, wherein the selected population of progeny is A population of selected progeny plants is provided, which exhibits improvements in yield or growth rate and a nuclear chromosome DNA methylation pattern different from that of the control plant, wherein the control plant is cultivated under the same environmental conditions as the population of selected progeny plants and includes any of the following: (i) progeny of scions grafted onto rootstock in which the expression of MSH1 and DRM2 genes has not been suppressed; (ii) perfect plants in which no root grafting has been performed and the expression of MSH1 and DRM2 genes has not been suppressed; (iii) wild-type plants; or (iv) progeny of plants having the same genetic background as the plant source of the scion of the grafted plant.

[0009] A method for producing plants exhibiting useful traits is provided, comprising: (a) obtaining a population of progeny from a grafted plant including a scion grafted onto a rootstock, wherein the rootstock is obtained from a plant or its parent plant in which the expression of MSH1 and DRM2 genes has been suppressed; and (b) selecting one or more progeny from the population, wherein the selected progeny show improvement in the useful traits compared to a control plant, thereby producing plants exhibiting useful traits.

[0010] The accompanying drawings incorporated herein and constituting part thereof illustrate certain embodiments of the present invention. [Brief explanation of the drawing]

[0011] [Figure 1A] The graph shows the total leaf area of ​​grafted progeny (first generation) at different number of days after planting, and represents the average leaf area for Experiment 1 (January 22, 2022 to February 23, 2022). The growth of five independent Col-0 / msh1 (white triangles, dashed lines), six independent Col-0 / msh1,drm2 (white squares, thin dashed lines), and three independent Col-0 / Col-0 (black circles, solid lines) grafts was measured in a reach-in growth chamber divided into three shelves. [Figure 1B] The graph shows the total leaf area of ​​grafted offspring (first generation) at different number of days after planting, and the average leaf area for Experiment 2 (04 / 13 / 2022~05 / 05 / 2022). Five independent Col-0 / msh1 grafts (white triangles, dashed lines), six independent Col-0 / msh1,drm2 grafts (white squares, thin dashed lines), and three independent Col-0 / Col-0 grafts (black circles, solid lines) were screened in a reach-in chamber divided into three shelves. In the second experiment, measurements were limited to four time points due to a later infestation by gnats. Using ImageJ software, the leaf area of ​​7 to 18 plants taken from each graft was measured, and the average leaf area was calculated and displayed on the graph. [Figure 2]This figure shows the total leaf area of ​​grafted progeny (second generation) at different number of days after planting. The average leaf area of ​​the second generation progeny of six independent Col-0 / msh1 (white triangles, dashed lines), six independent Col-0 / msh1,drm2 (white squares, thin dashed lines), and three independent Col-0 / Col-0 (black circles, solid lines) grafts (shown in Figure 1) was measured for growth in a reach-in growth chamber divided into three shelves. Using ImageJ software, the leaf area of ​​7 to 18 plants taken from each graft was measured, and the average leaf area was calculated and displayed in a graph. [Figure 3] This figure shows the total leaf area of ​​the grafted progeny (third generation) 35 days after planting. The average leaf area of ​​the third generation progeny of six independent Col-0 / msh1, six independent Col-0 / msh1,drm2, and three independent Col-0 / Col-0 grafts (shown in Figure 1) was measured for growth in a reach-in growth chamber divided into three shelves. Using ImageJ software, the leaf area of ​​7 to 18 plants taken from each graft was measured, and the average leaf area was calculated and displayed in a graph. [Figure 4] This shows a rapeseed mutant plant derived from msh1CRISPR in the R016 genetic background. [Figure 5] This image shows rapeseed mutant plants derived from drm2CRISPR in the R016 genetic background. The larger plants in the upper row (two pots) are wild-type plants, and the smaller plants in the lower row (four pots) are drm2 mutant plants. [Modes for carrying out the invention]

[0012] As used herein, the term "and / or" is to be understood as a specific disclosure of each of two particular features or components, with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0013] As used herein, the term "chromosomal modification" refers to any of a) "a plurality of modified chromosomal loci" or "a modified chromosomal locus", b) "a plurality of chromosomal loci having mutations" or "a chromosomal locus having a mutation", "a plurality of chromosomal variants" or "a chromosomal variant", and c) a transgene.

[0014] As used herein, the expression "modified chromosomal locus" (plural) or "modified chromosomal locus" (singular) refers to a portion of a chromosome that has undergone a heritable and reversible epigenetic change as compared to the corresponding parental chromosomal locus. Heritable and reversible genetic changes at a modified chromosomal locus include, but are not limited to, methylation of chromosomal DNA, particularly methylation of cytosine residues to 5-methylcytosine residues, and / or post-translational modification of histone proteins, and particularly, histone modifications. Histone modifications include, but are not limited to, acetylation, methylation, ubiquitination, phosphorylation, and SUMOylation (covalent attachment of small ubiquitin-like modifier proteins). As used herein, "chromosomal locus" refers to a locus in a chromosome located in the nucleus of a cell.

[0015] As used herein, the term "clonal propagule" refers to a plant obtained from a plant cell or its progeny. Clonal propagules can be obtained by methods such as regenerating a whole plant from a plant cell, plant embryo, cutting, etc., but the method is not limited thereto. Various techniques used for such clonal propagation include, but are not limited to, meristem culture, somatic embryogenesis, thin cell layer culture, adventitious bud culture, callus culture, etc.

[0016] As used herein, the term "comprising" means "including but not limited to".

[0017] As used herein, the term "crop plant" includes, but is not limited to, cereal, seed, grain, fruit, and vegetable crop plants.

[0018] As used herein, the phrases "chromosomal locus having a mutation" (plural), "chromosomal locus having a mutation" (singular), "multiple chromosomal variants", and "chromosomal variant" refer to a portion of a chromosome that has undergone a heritable genetic change in its nucleotide sequence as compared to the nucleotide sequence of the corresponding parental chromosomal locus. A chromosomal locus having a mutation includes mutations including, but not limited to, inversions, insertions, deletions, substitutions, or combinations thereof in the nucleotide sequence. In certain embodiments, a chromosomal locus having a mutation can include reversible mutations. In this context, reversible mutations in a chromosome include, but are not limited to, insertion of a transposable element, deletion of a transposable element, and certain inversions. In certain embodiments, a chromosomal locus includes irreversible mutations. In this context, irreversible mutations in a chromosome include, but are not limited to, deletions.

[0019] As used herein, "discrete mutation" or "V" DThe term "male sterility" refers to distinct, heritable phenotypic variations that include traits such as male sterility, dwarfism, variegation, and / or delayed flowering, which can be observed individually or in any combination.

[0020] As used herein, the term “heterogeneous sequence” refers, when used in the context of an operablely linked promoter, to any sequence or sequence arrangement that differs from any sequence or sequence arrangement having a promoter as found in nature. Thus, an MSH1 promoter can be operablely linked to a heterogeneous sequence, which includes, but is not limited to, MSH1 sense, MSH1 antisense, combinations of MSH1 antisense and MSH1 sense, and other MSH1 sequences that differ from or are arranged differently from operablely linked sequences of MSH1 transcription units found in nature.

[0021] As used herein, the term “MSH-dr” refers to the variegated, cytoplasmic male sterility (CMS), reduced growth rate phenotype, delayed or non-flowering phenotype, increased plant tillering, reduced plant height, reduced internode elongation, plant tillering, and / or altered stomatal density observed in plants in which the MSH1 gene has been repressed. Genes that can be repressed to produce the MSH-dr phenotype include, but are not limited to, MSH1 and both MSH1 and DRM2.

[0022] As used herein, the term “heterotic group” refers to genetically related germplasm that, when crossed with genetically different germplasm belonging to another heterotic group, produces superior hybrids.

[0023] As used herein, the term “progeny” refers to any of the first, second, third, or subsequent generations obtained from a parent plant or plant cells.

[0024] Where used herein, “quantitative variation” or “V” QThe term "MSH1 suppression" refers to phenotypic variations observed in individual progeny lines obtained from cross-pollination of plants in which MSH1 expression is suppressed, and represents discrete variations that differ from those in other plants.

[0025] As used herein, the terms “microRNA” or “miRNA” refer to both miRNAs substantially similar to endogenous miRNAs present in plants and artificial miRNAs. In certain embodiments, by using transgenes, either miRNAs substantially similar to endogenous miRNAs occurring in plants or artificial miRNAs can be produced.

[0026] As used herein, the phrase "obtain nucleic acids associated with a modified chromosomal locus" refers to a method that enables the physical separation or concentration of nucleic acids associated with a modified chromosomal locus from unmodified covalent nucleic acids. In this context, the nucleic acids do not necessarily have to contain modifications (i.e., methylation, etc.), but they do contain at least one modified nucleotide base. Thus, nucleic acids associated with a modified chromosomal locus can be obtained by methods including, but are not limited to, molecular cloning, PCR, or direct synthesis based on sequence data.

[0027] As used herein, the term "operably linked" refers to the linking of nucleic acid sequences so that one sequence provides the necessary function for the linked sequence. In the context of promoters, "operably linked" means that a promoter is linked to a target sequence, and the transcription of that target sequence is controlled and regulated by that promoter. If the target sequence codes for a protein and the expression of that protein is desired, "operably linked" means that the promoter is linked to the sequence so that the resulting transcript is efficiently translated. If the linking of a promoter to a coding sequence is a transcriptional fusion and the expression of the encoded protein is desired, the linking is performed so that the first translation start codon in the resulting transcript is the start codon of the coding sequence. Alternatively, if the linking of a promoter to a coding sequence is a translational fusion and the expression of the encoded protein is desired, the linking is performed so that the linking of the first translation start codon in the 5' untranslated sequence associated with the promoter is in frame with the open reading frame (ORF) of the resulting translation product that codes for the desired protein. Nucleic acid sequences that can be manipulated and ligated include, but are not limited to, sequences that provide gene expression function (i.e., gene expression elements such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, polyadenylation sites, and / or transcription terminators), sequences that provide DNA transposition and / or integration function (i.e., site-directed recombinase recognition sites, integrase recognition sites), sequences that provide selection function (i.e., antibiotic resistance markers, biosynthetic genes), sequences that provide scoring marker function (i.e., reporter genes), sequences that facilitate in vitro or in vivo manipulation of sequences (i.e., polylinker sequences, site-directed recombination sequences, homologous recombination sequences), and sequences that provide replication function (i.e., bacterial origins of replication, autonomous replication sequences, centromere sequences).

[0028] As used herein, “suppression of MSH1 gene expression,” “suppression of MSH1 and DRM2 gene expression,” and similar phrases refer to any genetic modification that reduces the level of functional MSH1 and DRM2 activity in a plant or plant cell compared to the level of functional MSH1 and DRM2 activity that occurs in a plant or plant cell having the same genetic background as a plant or plant cell that has not undergone the genetic or environmental modification.

[0029] As used herein, in the context of chromosome modification, the term “transgene” refers to heterologous DNA incorporated into a chromosome that is stably maintained in a host cell. In this context, heterologous sources of DNA include, but are not limited to, DNA from an organism other than the host cell organism, DNA from a species other than the host cell species, DNA from different varieties within the same species or from the same variety, DNA that has been modified in vitro, recombinant DNA, and any combination thereof.

[0030] As used herein, the term “non-regenerative” refers to a part or cell of a plant that is incapable of producing a complete plant.

[0031] Heritable mutations resulting in plants exhibiting useful traits, as well as methods for introducing epigenetic and / or genetic mutations, are provided along with plants, plant seeds, plant parts, plant cells, and plant products obtained by these methods. In certain embodiments, the methods provided herein can be used to introduce epigenetic and / or genetic mutations into cultivar plants or nonhybrid plants, thereby obtaining useful plants or parts (seeds, plant cells, plant products, etc.) that exhibit, retain, or reflect the useful traits and the effects conferred by those useful traits. In other embodiments, the methods provided herein can be used to introduce epigenetic and / or genetic mutations into plants also suitable for hybridization.

[0032] In certain embodiments, a method for introducing heritable epigenetic or genetic mutations into a plant or its progeny may include the step of grafting a scion onto a rootstock obtained from a plant or its parent plant in which the expression of the MSH1 and DRM2 genes has been suppressed. In certain embodiments of any of the aforementioned methods, the heritable epigenetic mutation provides useful traits selected from the group consisting of increased yield, delayed flowering, non-flowering, enhanced tolerance to bioterrorism, enhanced tolerance to abiotic stress, improved lodging resistance, increased growth rate, increased biomass, increased tillering, increased branching, delayed flowering, delayed senescence, increased flower count, improved plant structure for high-density planting, improved photosynthesis, increased root mass, increased cell count, improved seedling vitality, improved seedling size, increased cell division rate, improved metabolic efficiency, and increased meristematic size, compared to a control plant. In certain embodiments, a plant, a plant progeny, or a scion contains one or more epigenetic changes in one or more nuclear chromosomes, which are not present in the nuclear chromosomes of a control plant or in the nuclear chromosomes of the plant from which the scion was obtained. In certain embodiments, rootstock subjected to plastid function disruption also contains epigenetic changes. In certain embodiments, epigenetic changes in plants, plant progeny, scions, or rootstocks are associated with the improvement of useful traits. In certain embodiments, epigenetic changes in plants, plant progeny, scions, or rootstocks induced by the repression of the MSH1 and DRM2 genes are associated with the improvement of useful traits. In certain embodiments, a plant, a plant progeny, a scion, or a rootstock contains one or more epigenetic changes in one or more nuclear chromosomes, which are not present in the nuclear chromosomes of rootstock obtained from plants that have not been subjected to plastid function disruption or in the nuclear chromosomes of the parent plants. In certain embodiments, plants, plant progeny, scions, and / or rootstocks exhibit CG hypermethylation in the region encompassing the MSH1 locus compared to control plants that have not undergone repression of the MSH1 and DRM2 genes.In certain embodiments, plants, plant progeny, scions, and / or rootstocks exhibit pericentromeric CHG hypermethylation compared to control plants that have not undergone repression of the MSH1 and DRM2 genes. In certain embodiments, plants, plant progeny, scions, and / or rootstocks exhibit CG hypermethylation and / or CHG hypermethylation at one or more nuclear chromosome loci compared to the corresponding nuclear chromosome loci of control plants that have not undergone repression of the MSH1 and DRM2 genes. In certain embodiments, plants are selected from the group consisting of crop plants, trees, shrubs, and climbing plants. In certain embodiments, crop plants are selected from the group consisting of maize, soybeans, cotton, canola, wheat, rice, tomatoes, tobacco, millet, potatoes, sugar beets, cassava, alfalfa, barley, oats, sugarcane, sunflowers, strawberries, and sorghum. In certain embodiments, the trees are selected from the group consisting of apple, apricot, grapefruit, orange, peach, pear, plum, lemon, coconut, poplar, eucalyptus, date palm, palm oil, pine, and olive trees. In certain embodiments, the shrubs are selected from the group consisting of blueberry, raspberry, and blackberry shrubs. In certain embodiments, the climbing plants are grapevines. Plants or their offspring obtained by any of the aforementioned methods are also provided. Plant parts obtained from plants or their offspring produced by any of the aforementioned methods are also provided.

[0033] Simply put, without being constrained by theory, DRM2 in Arabidopsis thaliana and other plants encodes a methyltransferase that functions in RdDM-targeted DNA methylation. Inactivation of DRM2 is thought to partially inactivate the RdDM pathway in the rootstock, preventing msh1-induced small RNA (sRNA) from being incorporated into the rootstock's epigenomic process, thereby enabling efficient transfer of sRNA to the scion. Since grafting is performed during the flowering period, source-sink conversion occurring within the plant is likely to facilitate the transfer of sRNA from rootstock to scion.

[0034] Furthermore, this specification describes grafted plants comprising scions grafted onto rootstock, wherein the rootstock is obtained from a plant or its parent plant in which the expression of the MSH1 and DRM2 genes has been suppressed, as well as progeny and clonal propagates obtained from such grafted plants. Such rootstocks can also be used to introduce epigenetic and / or genetic variation into cultivar or nonhybrid plants, thereby obtaining useful traits and useful plants or plant parts (such as seeds, plant cells, or processed plant products) that express, retain, or reflect the effects conferred by those useful traits. In other embodiments, such rootstocks can also be used to introduce epigenetic and / or genetic variation into plants suitable for hybridization.

[0035] Rootstocks useful for introducing epigenetic and / or genetic mutations into plants can be obtained from various rootstock plants in which the expression of the MSH1 and DRM2 genes has been suppressed. In certain embodiments, the rootstock plant is a plant that itself has undergone suppression of the expression of the MSH1 and DRM2 genes. In other embodiments, the rootstock plant is a progeny of a parent plant that itself has undergone suppression of the expression of the MSH1 and DRM2 genes. Various methods for producing rootstock plants by suppressing the expression of the MSH1 and DRM2 genes are described herein. Plants that can function as rootstock plants and methods for producing such plants are also disclosed in U.S. Patent Application Publication No. 20120284814, which is incorporated herein by reference in its entirety and expressly referenced elsewhere in this disclosure. In particular, it is provided that plants having useful traits and methods for producing such plants, disclosed in paragraphs

[0072] ,

[0085] , and

[0089] of U.S. Patent Application Publication No. 20120284814, may be used as rootstock sources, the contents of which are incorporated herein by reference in their entirety.

[0036] In a particular embodiment, where the rootstock source plant or its parent plant has undergone suppression of MSH1 and DRM2 gene expression, a population of progeny obtained from the grafted plant is screened, and individual progeny are selected for one or more useful traits. Such a population of progeny can be obtained by a method of self-pollinating or cross-pollinating the grafted plant, including the rootstock, to obtain seeds that produce a population. Such a population of progeny can be obtained by a method of growing a population of plants derived from independent clonal proliferators obtained from the grafted plant, including the rootstock, but is not limited to that method. Individual progeny thus selected that exhibit useful traits are propagated by sexual or asexual reproduction to produce a plant population exhibiting the useful traits, or a seed lot exhibiting or possessing the useful traits. Such sexual propagation can be achieved by self-pollinating or cross-pollinating the selected individual progeny exhibiting the useful traits.

[0037] In a particular embodiment, where the rootstock is a progeny of a parent plant in which the expression of the MSH1 and DRM2 genes has been suppressed, the rootstock itself can be a plant selected for one or more useful traits. By grafting a scion that does not exhibit the useful trait onto the rootstock of a plant selected for that trait, the trait can be conferred to the grafted plant or its progeny. The resulting grafted plant or its progeny exhibiting the useful trait can be propagated by sexual or asexual reproduction to obtain a population of plants exhibiting the useful trait or a seed lot exhibiting or possessing the useful trait.

[0038] In grafted plants or their progeny, the suppression of MSH1 and DRM2 gene expression in the rootstock can be persistent and continuous, or transient. Non-limiting and exemplary methods for achieving persistent and continuous suppression of MSH1 and DRM2 gene expression in the rootstock include suppressing MSH1 and DRM2 gene expression using loss-of-function mutations in endogenous genes and / or using transgenes that produce a product that suppresses the expression of endogenous genes. Alternatively, the suppression of MSH1 and DRM2 gene expression in the rootstock can be transient, or it may have occurred in the parent plant from which the rootstock was obtained but not in the rootstock used for grafting. Non-limiting and exemplary methods for achieving transient suppression of MSH1 and DRM2 gene function in rootstock include suppressing endogenous MSH1 and DRM2 gene expression by means of transgenes that enable inducible or repressive expression of products that suppress the expression of endogenous MSH1 and DRM2 genes, excisable transgenes, or heterozygous transgene insertions that are removed from the rootstock by isolation. Any of the methods for restoring plastid function after disturbance described herein can be used to generate rootstock used in a particular embodiment.

[0039] Grafting can be performed by any method that allows for the establishment of a vascular connection between the rootstock and the scion. Grafting methods that can be used to achieve a connection between the scion and the rootstock include, but are not limited to, apical grafting, lateral grafting, bark grafting, and root grafting. Such methods for grafting a scion onto a rootstock are disclosed in Chapter 12, "The Art of Grafting," of *Plant Propagation—Principles and Practices*, edited by Hartman, Kester, Davies, and Geneve, 7th edition. Methods for grafting a scion onto a rootstock in monocotyledonous plants, which can be used for the scions and rootstocks provided herein, are disclosed in Muzik and La Rue, *The Grafting of Large Monocotyledonous Plants*, Science 116, No. 3022: 589-591, 1952.

[0040] Rootstocks in which the MSH1 and DRM2 genes have been repressed, or rootstocks obtained from parent plants in which the MSH1 and DRM2 genes have been repressed, can exhibit modifications on one or more nuclear chromosomes. In certain embodiments, such rootstocks can exhibit characteristic DNA methylation and / or gene transcription patterns that occur in plants in which the MSH1 target gene has been repressed. Examples of such characteristic DNA methylation and / or gene transcription patterns that occur in plants or seeds in which the MSH1 target gene has been repressed include, but are not limited to, the patterns disclosed in U.S. Patent No. 1,0767,188, which is incorporated herein by example in its entirety. In certain embodiments, rootstocks of first-generation progeny from plants in which the MSH1 gene has been repressed exhibit CG differential methylation regions in various discrete chromosomal regions that include, but are not limited to, the MSH1 locus. In certain embodiments, the CG hypermethylation region including the MSH1 locus is about 5 to about 8 MBp (megabase pairs) in length. In certain embodiments, rootstocks of first-generation progeny from plants in which the MSH1 and DRM2 genes have been repressed also show alterations in the expression of plant defense and stress response genes. In certain embodiments, rootstocks, scions grafted thereon, and / or plant cells, seeds, progeny, plant populations, seed populations, and / or processed products derived therefrom, in which the MSH1 and DRM2 genes have been repressed, exhibit methylation repatterning similar to the methylation repatterning observed when MSH1 is repressed (Kundariya et al. 2020 and Kundariya et al. 2022). Such methylation repatterning can be evaluated by comparing the methylation status of samples from rootstocks, scions of plants grafted to rootstocks, plants or seeds in which the MSH1 and DRM2 genes have been repressed, or from progeny or seeds derived therefrom, with samples from control plants or seeds that have not been repressed with the MSH1 and DRM2 genes.In this context and in other specific contexts, such control plants include, but are not limited to, plants, grafted plants, their scions, and their rootstocks that have not undergone repression of the MSH1 and DRM2 genes. In certain embodiments, such aforementioned changes in methylation patterns, as shown by scions grafted onto rootstock, or as shown by plant cells, seeds, progeny, plant populations, seed populations, and / or products obtained from grafted plants, can be used to monitor the effectiveness of grafting in transmitting desired epigenetic changes, or to identify products obtained from plant cells, seeds, progeny, plant populations, seed populations, and / or products obtained from grafted plants.

[0041] Furthermore, this specification provides various methods for producing plants exhibiting useful traits, including crossing a grafted plant, which includes a scion grafted onto a rootstock subjected to plastid function disruption, with another plant, or crossing a progeny obtained from a grafted plant with another plant, and selecting one or more progeny from the resulting crosses that exhibit improved useful traits compared to a control plant. In certain embodiments, the second plant may also be a grafted plant, which includes a scion grafted onto a rootstock subjected to plastid function disruption, a progeny obtained from a grafted plant, which includes a scion grafted onto a rootstock subjected to plastid function disruption, any other ungrafted plant subjected to plastid function disruption, or any other ungrafted plant obtained from one or more parent plants subjected to plastid function disruption. Such a second plant is a plant selected for useful traits and may be a progeny of any plant or grafted plant subjected to plastid function disruption. Examples of control plants used as comparisons to identify the progeny of crosses showing improvements in useful traits include, but are not limited to, the progeny of crosses between plants that have not been grafted onto rootstock and plants having the same genetic background as the second plant, the self-pollinated progeny of plants that have not been grafted onto rootstock, and the self-pollinated progeny of the second plant; the progeny of crosses between plants having the same genetic background as the plant source of the scion of a grafted plant and plants having the same genetic background as the second plant; and the progeny of crosses between plants having the same genetic background as the plant source of the scion of a grafted plant and plants having the same genetic background as the plant source of the scion of the second plant when the second plant is a grafted plant. Methods are also provided in which at least the scion of the first plant originates from a different heterosis group than that of the second plant, or in which at least the scion of the first plant originates from the same heterosis group as that of the second plant.

[0042] Furthermore, this specification provides various methods for producing plants exhibiting useful traits, including self-pollinating grafted plants, which include scions grafted onto rootstock with disrupted plastid function, with other plants, or self-pollinating progeny obtained from grafted plants, and selecting one or more progeny obtained by self-pollination that exhibit improved useful traits compared to a control plant, in order to produce plants exhibiting useful traits. In a particular embodiment, the self-pollinated plant is a grafted plant in which the rootstock source plant is a progeny of a parent plant in which the expression of MSH1 and DRM2 genes has been suppressed, and the rootstock source plant itself has been selected for and exhibits one or more useful traits. Control plants used as comparisons to identify self-pollinated progeny showing improvements in useful traits include, but are not limited to, self-pollinated progeny of plants that have not been grafted onto rootstock, self-pollinated progeny of plants that have been grafted onto rootstocks in which the expression of MSH1 and DRM2 genes has not been suppressed, and self-pollinated progeny of plants that have the same genetic background as the plant source of the scion of the grafted plant.

[0043] In certain embodiments, the useful traits described herein can be more pronounced in subsequent generations of plants obtained from grafted plants, parent plants, or parent plant cells in which the expression of the MSH1 and DRM2 genes provided herein has been suppressed. Thus, a given initial plant obtained from a parent plant in which the expression of the MSH1 and DRM2 genes has been suppressed can be self-propagated to obtain first, second, third, or subsequent generations of progeny that exhibit the given useful traits more prominently than the initial plant or compared to a control plant. Thus, an initial grafted plant, including a scion grafted onto a rootstock in which the expression of the MSH1 and DRM2 genes has been suppressed, or a scion grafted onto a rootstock obtained from a parent plant in which the expression of the MSH1 and DRM2 genes has been suppressed, can be self-propagated to obtain first, second, third, or subsequent generations of progeny that exhibit the given useful traits more prominently than the grafted initial plant or compared to a control plant. In other embodiments, a given early plant obtained from parent plants in which the expression of MSH1 and DRM2 genes has been suppressed can be cross-pollinated to obtain F1, F2, F3, or subsequent generations of progeny that exhibit a given useful trait more prominently than the early plant or a control plant. In certain embodiments, a useful trait possessed by an early plant or early grafted plant is not shown or is shown to a lower degree in the early plant or early grafted plant. However, such a useful trait possessed by an early plant or early grafted plant is shown or is shown more prominently in progeny obtained by the early plant or by cross-pollinating the early grafted plant with another plant. Such a useful trait possessed by an early plant or early grafted plant is also shown or is shown more prominently in progeny obtained by self-pollinating the early plant or early grafted plant. In certain embodiments, the self-pollinated or cross-pollinated plant or grafted plant can be inbred. In certain embodiments, useful traits possessed by an inbred line are not exhibited in that line, or are exhibited to a lower degree.However, the useful traits of such inbred lines are shown, or more prominently shown, in progeny obtained by cross-pollinating the inbred lines with other plants. Thus, early grafted plants, including scions grafted onto rootstocks with suppressed MSH1 and DRM2 gene expression, or scions grafted onto rootstocks obtained from parent plants with suppressed MSH1 and DRM2 gene expression, can be cross-pollinated to obtain F1, F2, F3, or subsequent generations of progeny that more prominently exhibit a given useful trait compared to the early grafted plants or to control plants. Such cross-pollination of early or grafted plants can be performed with plants of the same genetic background or with genetically different plants. The methods provided herein allow for the selection of early or subsequent generations of progeny obtained from such self-pollination or crosses for useful traits. The methods provided herein also enable the identification of plants that possess various useful traits, but do not necessarily exhibit them to a complete degree.

[0044] Clonal propagation can be obtained by methods of regenerating a complete plant from plant cells, plant embryos, plant cells, plant embryos, cuttings, etc., obtained from scions or progeny of grafted plants provided herein, but is not limited to such methods. Various techniques used for such clonal propagation include, but are not limited to, meristematic tissue culture, somatic cell embryo development, thin cell layer culture, adventitious bud culture, and callus culture. In certain embodiments, clonal propagation is achieved by placing sterile plant cells, plant embryos, cuttings, etc., in a sterile plant culture medium containing suitable salts, sugars, and plant growth regulators to support the regeneration of the plant or plant part. Such techniques suitable for clonal propagation are often called "micropropagation." Typically, in culture materials, cytokinins are used to promote the formation of above-ground parts, and auxins are used to promote the formation of underground parts. Techniques available herein for the clonal propagation of potato plants include, but are not limited to, a method of growing sterile cuttings derived from tubers in a modified Murashige-Skoog medium to produce micropropagation plants, which are then transplanted into soil to produce microtubers that can function as seed potato tubers (Ahloowalia, Euphytica 75:163, 1994). Other methods available herein for the clonal propagation of potato plants include, but are not limited to, a method of culturing and propagating nodal tissue, meristematic tissue, or shoot apical tissue (Rosell, G. et al. Potato Research 30:111, 1987, and the references cited therein).Further methods that can be used for the clonal propagation of potato plants as provided herein include, but are not limited to, methods of culturing node segments in a bioreactor and then transplanting them into soil to produce a large number of microtubers that can function as seed potato tubers (Piao et al., Current Science 84(8):1129, 2003). Techniques that can be used for the clonal propagation of sugar beet plants as provided herein include, but are not limited to, propagation by petiole explants (Grieve, et al. Plant Growth Regulation 21:15, 1997), or propagation of leaf blades, apical meristems, stems, embryos, or hypocotyls (Mezei, S. et al. Biotechnology & Biotechnological Equipment, 20:1, 9-14, 2006).

[0045] In certain embodiments, the methods provided herein include suppressing the expression of MSH1 and DRM2 target genes, restoring the expression of functional MSH1 and DRM2 genes, and selecting progeny plants exhibiting one or more desirable traits. In certain embodiments, these desirable traits are associated with one or more modified chromosomal loci that have undergone heritable and reversible epigenetic changes.

[0046] In a particular embodiment, a method is provided for selectively repressing the expression of MSH1 and DRM2 target genes in a subpopulation of plant cells containing plastids referred herein as “sensing plastids.” Sensing plastids are plastids that arise in cells exhibiting preferential expression of at least the MSH1 promoter. In a particular embodiment, MSH1 and other promoters active in sensing plastids can thus be manipulably linked to heterologous sequences that disrupt plastid function in order to achieve selective gene repression in cells containing sensing plastids. Such cells containing sensing plastids are easily identifiable not only because they express MSH1, but also because their plastids are only about 30-40% the size of chloroplasts contained within mesophyll cells. Other promoters thought to be active in sensing plastids include, but are not limited to, the PPD3 gene promoter. Selective repression of MSH1 and DRM2 genes in cells containing sensing plastids can induce epigenetic changes that provide useful plant traits.

[0047] Table 1 shows examples of target genes for MSH1 and DRM2 in Arabidopsis thaliana, the accession numbers of the corresponding sequences in the Arabidopsis genome database (website address "Arabidopsis.org"), and orthologous MSH1 and DRM2 genes that can be targeted for suppression in other crop plants. Many orthologous genes in crop species can be obtained by BLAST comparisons of the protein sequences of the above Arabidopsis genes with genome databases (NCBI and publicly available genome databases for specific crop species) or by using the proper nomenclature of subunits.In particular, genomes, cDNAs, and EST sequences of apples, beans, barley, rapeseed, rice, cassava, coffee, eggplant, oranges, sorghum, tomatoes, cotton, grapes, lettuce, tobacco, papaya, pine, rye, soybeans, sunflowers, peaches, poplars, safflower beans, spruce, cocoa, cowpeas, corn, onions, peppers, potatoes, radishes, sugarcane, wheat, and other species are available at compbio.dfci.harvard.edu / tgi / plant.html, genomevolution.org / wiki / index.php / Sequenced_plant_genomes, ncbi.nlm.nih.gov / genomes / PLANTS / PlantList.html, plantgdb.org / , arabidopsis.org / portals / genAnnotation / other_genomesr, gramene.org / resources / , and genomenewsnetwor. It is available from the following internet or World Wide Web addresses: k.org / resources / sequenced_genomes / genome_guide_p1.shtml, jgi.doe.gov / programs / plants / index.jsr, chibba.agtec.uga.edu / duplication / , mips.helmholtz-muenchen.de / plant / genomes.jsp, science.co.il / biomedical / Plant-Genome-Databases.asp, jcvi.org / cms / index.php?id=16, rapdb.dna.affrc.go.jp / , ensembl.sorghumbase.org, genoscope.cns.fr, solgenomics.net, maizegdb.org, and phyto5.phytozome.net / Phytozome_resources.php.

[0048] [Table 1]

[0049] In general, the methods provided herein for introducing epigenetic and / or genetic mutations into plants only require suppressing the expression of MSH1 and DRM2 target genes for a sufficient amount of time to introduce mutations and / or in a suitable subset of cells (e.g., cells containing sensing plastids). Thus, a wide variety of methods for suppressing MSH1 and DRM2 genes can be employed to carry out the methods provided herein, and the methods are not limited to any particular suppression technique.

[0050] Sequences of MSH1 and DRM2 genes or fragments derived from Arabidopsis thaliana and various crop plants are shown herein (e.g., in Table 1). In certain embodiments, such genes may be used directly in either homologous or heterologous plant species to provide repression of endogenous MSH1 and DRM2 target genes in either homologous or heterologous plant species. A non-limiting example, in which an exemplary MSH1 gene from a certain species was shown to be effective in repressing both homologous and heterologous endogenous MSH1 genes, is provided by Sandhu et al. in 2007. There, it was shown that a transgene providing MSH1 inhibitory RNA (RNAi) containing the tomato MSH1 sequence inhibits endogenous MSH1 genes in both tomato and tobacco. In certain embodiments, endogenous MSH1 and DRM2 genes(s) in millet, sorghum, and maize can be inhibited by using a transgene providing MSH1 and / or DRM2 gene inhibitory RNA (RNAi) containing the maize MSH1 and / or DRM2 gene sequences. Furthermore, MSH1 and / or DRM2 genes(s) derived from other plants, including but not limited to cotton, canola, wheat, barley, flax, oats, rye, turfgrass, sugarcane, alfalfa, banana, broccoli, cabbage, carrot, cassava, cauliflower, celery, citrus fruits, cucurbits, eucalyptus, garlic, grapes, onions, lettuce, peas, peanuts, chili peppers, potatoes, poplar, pine, sunflower, safflower, soybeans, blackberries, blueberries, sugar beets, sweet potatoes, tobacco, strawberries, sugar beets, sweet potatoes, jatropha, camelina, and agave, can also be obtained by various techniques and used to suppress the expression of the corresponding MSH1 and DRM2 genes in these plants, or in separate plants.Methods for obtaining MSH1 and DRM2 genes from various plants include, but are not limited to, the following techniques: i) searching amino acid and / or nucleotide sequence databases containing plant species-derived sequences to identify MSH1 and DRM2 genes by sequence identity comparison; ii) cloning MSH1 and DRM2 genes by either PCR from genomic sequences or RT-PCR from expression RNA; iii) cloning MSH1 and DRM2 genes from genomic libraries or cDNA libraries using PCR and / or hybridization-based techniques; iv) cloning MSH1 and DRM2 genes from expression libraries where antibodies against MSH1 and DRM2 gene proteins are used to identify clones containing MSH1 and DRM2 genes; v) cloning MSH1 and DRM2 genes by complementation of MSH1 and DRM2 gene mutants or MSH1 and DRM2 gene-deficient plants; or vi) any combination of (i), (ii), (iii), (iv), and / or (v) above. The DNA sequence of the target gene can be obtained from the promoter or transcription region of the target gene by PCR isolation from genomic DNA, PCR of the cDNA of the transcription region, or synthesis of a commercially available DNA sequence. The RNA sequence can also be synthesized chemically, or more preferably by transcription of a suitable DNA template. Recovery of the MSH1 and DRM2 genes from plants can be easily determined or confirmed by constructing a plant transformation vector that enables gene repression, transforming the plants with the vector, and determining whether the plants transformed with the vector exhibit characteristic responses typically observed in various plant species when MSH1 expression is repressed (phenotypes such as leaf variegation, cytoplasmic male sterility (CMS), reduced growth rate, and / or delayed or non-flowering). The characteristic response to MSH1 repression has been previously described as developmental reprogramming or "MSH-dr1" (Xu et al. Plant Physiol. Vol.159:711-720, 2012).

[0051] In certain embodiments, the MSH1 and DRM2 genes or fragments used in the methods provided herein will have nucleotide sequences having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% nucleotide sequence identity with one or more of the MSH1 and DRM2 genes or fragments provided herein. The MSH1 and DRM2 genes or fragments include, but are not limited to, the genes and their orthologues found in various crop plants provided in Table 1. In certain embodiments, the MSH1 and DRM2 genes or fragments used in the methods provided herein will encode the MSH1 and DRM2 genes or parts thereof and will have amino acid sequences having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% amino acid sequence identity with one or more of the MSH1 and DRM2 gene proteins provided herein. MSH1 and DRM2 gene proteins include, but are not limited to, the MSH1 and DRM2 gene proteins encoded by the genes provided in Table 1.

[0052] Nucleic acid fragments of the MSH1 and DRM2 genes are expected to be usable to effectively suppress the endogenous MSH1 and DRM2 genes if they are 18-20 nucleotides, more preferably 21 nucleotides or longer. In certain embodiments, MSH1 and DRM2 gene nucleic acid fragments ranging from at least 18, 19, 20, or 21 nucleotides to about 50, 100, 200, 500, or more nucleotides can be used to achieve suppression of the endogenous MSH1 and DRM2 genes. For this purpose, regions of 20, 50, 100, 500, or more are preferred, and the length of the target gene sequence is preferably 100-300 bases, most preferably 300-500 bp or more. For use in hairpin-type or reverse repeat sequence knockdown designs, spacer regions having sequences unrelated to the target plant genome sequence can be used. A hairpin construct containing a portion of the target gene sequence of 300-500 bp or more in the antisense direction. A spacer region follows, but its sequence is not important and it may be an intron or non-intron. If the spacer is an intron, the castavean catalase intron (Tanaka, Mita et al. Nucleic Acids Res 18(23):6767-6770, 1990), which is effectively spliced ​​in both monocots and dicots, is well known to those skilled in the art and is useful in this embodiment. The same target gene sequence is located after the spacer in the sense direction, and after transcription of the transcription region, the antisense strand and sense strand can form a double-stranded RNA. A polyadenylated region follows the target gene sequence. Examples of 3' polyadenylated regions known to those skilled in the art that function in both monocots and dicots include, but are not limited to, the nopalin synthase (NOS) 3' region, the octopine synthase (OCS) 3' region, the cauliflower mosaic virus 35S 3' region, and the mannopinine synthase (MAS) 3' region. Additional 3' polyadenylated regions derived from monocotyledonous plant genes, such as those from rice, sorghum, wheat, and maize, are available to those skilled in the art and are available to provide similar polyadenylated regions and functions in the DNA constructs of this embodiment.In a particular embodiment, a transgene designed to repress a target gene in dicotyledonous plants is designed to have the following sequence: promoter / antisense to target gene / catalase intron / sense gene A / polyadenylated region. In embodiments where the gene is designed to repress a target gene in monocotyledonous plants, it may have the following sequence: promoter / intron for monocotyledonous plants / antisense to target gene / catalase intron / sense gene A / polyadenylated region.

[0053] Sequences providing repression of the MSH1 and DRM2 genes may include sequences complementary to any of the strands of the promoter, 5' or 3' untranslated region, intron, coding region, and / or any combination thereof. The target gene promoter region for gene repression may include the transcription start site, TATA box, and upstream region. The promoter region for gene silencing may be about 20, 50, 80, or 100 nucleotides in length, more preferably about 100 to 500 nucleotides in length. The promoter region used for such repression may be derived from different regions in the upstream promoter, preferably including a region at least about 500 nucleotides upstream from the transcription start site, and most preferably including a region at least about 500 nucleotides upstream from the translation start of the endogenous coding region of the endogenous gene. This also includes the UTR, which may or may not be part of the promoter. Various recombinant DNA constructs targeting the promoter region and / or adjacent regions of a target gene are described in U.S. Patent No. 8,293,975, which is incorporated herein by reference in its entirety.

[0054] In certain embodiments, the repression of the MSH1 and DRM2 genes in plants is achieved using transgenes. Transgenes that can be used to repress the expression of the MSH1 and DRM2 genes include, but are not limited to, transgenes that produce dominant-negative mutants of the MSH1 and DRM2 genes, small inhibitory RNAs (siRNAs), microRNAs (miRNAs), and co-repression sense RNAs and / or antisense RNAs that provide inhibition of endogenous MSH1 and DRM2 genes. U.S. patents describing the repression of endogenous plant genes by transgenes, which are incorporated herein by whole reference, include U.S. Patents 7,109,393, 5,231,020, and 5,283,184 (co-repression method), and U.S. Patents 5,107,065 and 5,759,829 (antisense method). In certain embodiments, transgenes specifically designed to produce double-stranded RNA (dsRNA) molecules homologous to the MSH1 and DRM2 genes can be used to reduce the expression of endogenous MSH1 and DRM2 genes. In such embodiments, the sense strand sequence of the dsRNA can be separated from the antisense sequence by a spacer sequence, preferably one that promotes the formation of the dsRNA (double-stranded RNA) molecule. Examples of such spacer sequences include, but are not limited to, those described in Wesley et al., Plant J., 27(6):581-90 (2001) and Hamilton et al., Plant J., 15:737-746 (1998). One exemplary and non-limiting vector shown to provide repression of MSH1 target genes in tobacco and tomato is described by Sandhu et al., 2007, in which an intron sequence separates the sense and antisense strands of the MSH1 target gene sequence.The design of recombinant DNA constructs for suppressing gene expression is also described in Helliwell, C. and P. Waterhouse (2003). “Constructs and methods for high-throughput gene silencing in plants.” Methods 30(4):289-295.

[0055] In certain embodiments, a transgene providing repression of the MSH1 and DRM2 genes may consist of a regulated promoter that provides either induction or downregulation of an inhibitory sequence of the MSH1 and DRM2 genes that is operablely linked. In this context, the MSH1 and DRM2 gene inhibitory sequences may include, but are not limited to, dominant-negative mutants of the MSH1 and DRM2 genes, small inhibitory RNAs (siRNAs), microRNAs (miRNAs), co-repression sense RNAs, and / or antisense RNAs that provide inhibition of the endogenous MSH1 and DRM2 genes in plants. Such promoters can provide repression of the MSH1 and DRM2 genes for a controlled period of time, depending on whether they provide an inducer or downregulator. Examples of inducible promoters include, but are not limited to, the PR-1a promoter (U.S. Patent Application Publication No. 20020062502) or the GST II promoter (WO1990 / 008826 A1). In other embodiments, both an induceable or repressible transcription factor and a promoter recognized by the transcription factor and manipulably linked to MSH1 and DRM2 gene inhibitory sequences are provided. Such transcription factor / promoter systems include, but are not limited to, i) an RF2a acidic domain-ecdysone receptor transcription factor / corresponding promoter that can be induced by methoxyphenozide, tebuphenozide, and other compounds (US Patent Application Publication No. 20070298499), and ii) a chimeric tetracycline presser transcription factor / corresponding chimeric promoter that can be repressed or depressed by tetracycline (Gatz, C., et al. (1992). Plant J. 2, 397-404).

[0056] In certain embodiments, a promoter is used that enables the selective expression of a heterologous sequence that represses the expression of a target gene in cells containing a sensor plastid. In certain embodiments, this promoter is the Msh1 or PPD3 promoter. In certain embodiments, this promoter is the Msh1 or PPD3 promoter, and the operablely linked heterologous sequence represses the expression of a target gene provided in U.S. Patent No. 1,0767188, which is incorporated herein by reference in its entirety. Examples of Msh1 promoters that can be used to express a heterologous sequence in cells containing a sensor plastid include, but are not limited to, the Arabidopsis, sorghum, tomato, and maize promoters provided herein or in U.S. Patent No. 1,0767188, which is incorporated herein by reference in its entirety, and their functional derivatives that similarly provide expression in cells containing a sensor plastid. In certain embodiments, deletion derivatives of the Msh1 promoter containing about 1500 Bp, about 1000 Bp, or about 750 Bp may also be used to express a heterologous sequence. PPD3 promoters that can be used to express heterologous sequences in cells containing sensor plastids include, but are not limited to, the Arabidopsis, rice, and tomato promoters provided in U.S. Patent No. 1,0767188, which is incorporated entirely herein by reference, as well as their functional derivatives that provide expression in cells containing sensor plastids. In certain embodiments, deletion derivatives of the Msh1 promoter containing a PPD3 promoter of about 800 Bp, about 600 Bp, or about 500 Bp can also be used to express heterologous sequences. In certain embodiments, the aforementioned PPD3 promoters and an additional 200, 500, or 1000 base pairs of endogenous 5' PPD3 promoter sequences can be used to express heterologous sequences. The additional 200, 500, or 1000 base pairs of endogenous 5' PPD3 promoter sequences can be obtained by methods including, but are not limited to, sequence retrieval from databases provided herein and recovery of adjacent promoter DNA by PCR amplification or direct synthesis of a genomic template sequence.In certain embodiments, a recombinant DNA construct for repressing a dicotyledonous plant target gene may include an MSH1 or PPD3 promoter derived from a dicotyledonous plant species such as Arabidopsis thaliana, soybean, or canola, and is bound to a hairpin construct containing 300-500 bp or more of the target gene sequence in the antisense direction, followed by a spacer region, the sequence of which is not important and may be intronic or non-intronic. The castavean catalase intron (Tanaka, Mita et al. Nucleic Acids Res 18(23):6767-6770, 1990) can be used as a spacer in certain embodiments. The same target gene sequence is present in the sense direction after the spacer, and after transcription of the transcription region, the antisense and sense strands can form double-stranded RNA. A polyadenylated region follows the target gene sequence. Various 3' polyadenylated regions known to function in monocots and dicots include, but are not limited to, the nopalin synthase (NOS) 3' region, the octopine synthase (OCS) 3' region, the cauliflower mosaic virus 35S 3' region, and the mannopinine synthase (MAS) 3' region. In certain embodiments, recombinant DNA constructs for repressing monocotyledonous target genes may include an MSH1 or PPD3 promoter derived from a monocotyledonous plant species such as rice, maize, sorghum, or wheat, and may be directly bound to a hairpin region or bound to a monocotyledonous intron prior to the hairpin region. In monocotyledonous plant introns that are beneficial for gene expression when located between the promoter and coding regions are the first intron of ubiquitin in maize (described in U.S. Patent No. 6,054,574, which is incorporated herein by reference in its entirety) and the first intron of rice actin 1 (McElroy, Zhang et al. Plant Cell 2(2):163-171, 1990).Additional introns that are beneficial to gene expression when located between the promoter and coding regions include the maize hsp70 intron (described in U.S. Patent No. 5,859,347, which is incorporated herein by reference in its entirety) and introns 2 and 6 of the maize alcohol dehydrogenase 1 gene (described in U.S. Patent No. 6,342,660, which is incorporated herein by reference in its entirety).

[0057] In yet another embodiment, a transgenic plant is provided in which a transgene enabling the repression of the MSH1 and DRM2 genes is flanked by sequences that provide removal of the transgene. Such sequences include, but are not limited to, transposable element sequences that are acted upon by the corresponding transposase. Non-exclusive examples of such systems that have been used in transgenic plants include the cre-lox system and the FLP-FRT system.

[0058] Suppression of the MSH1 and DRM2 genes can be readily identified or monitored by molecular biological techniques. In certain embodiments where the endogenous MSH1 and DRM2 genes are not impaired but their expression is inhibited, the production or accumulation of RNA encoding the MSH1 and DRM2 genes can be monitored. Molecular biological methods for monitoring the RNA expression levels of the MSH1 and DRM2 genes include, but are not limited to, the use of semi-quantitative or quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) techniques. The use of semi-quantitative PCR techniques to monitor MSH1 and DRM2 gene suppression resulting from RNAi-induced repression of the MSH1 and DRM2 genes has already been described (Sandhu et al. 2007). Various quantitative RT-PCR methods, including the TaqMan® reaction (Applied Biosystems, Foster City, Calif., US), the use of Scorpion® probes or Molecular Beacon® probes, or any of the methods described in Bustin, SA (Journal of Molecular Endocrinology (2002) 29, 23-39) can be used. Other RNA quantification techniques, such as Quantitative Nucleic Acid Sequence Based Amplification (Q-NASBA®) or Invader® technology (Third Wave Technologies, Madison, Wis.), can also be used.

[0059] In certain embodiments where the repression of the MSH1 and DRM2 genes is achieved by using loss-of-function mutations in the endogenous MSH1 and DRM2 genes of plants, the presence or absence of such loss-of-function mutations in the genomic DNA can be readily determined by various techniques. Furthermore, certain techniques can be used to identify mutations in the hemizygote state (i.e., one chromosome having the mutant MSH1 gene and the other chromosome having the wild-type MSH1 and DRM2 genes). These include insertions, deletions, nucleotide substitutions, and combinations thereof for MSH1 and DRM2. DNA sequence mutations are listed in US Patent Nos. 5,468,613, 5,217,863, 5,210,015, 5,876,930, 6,030,787, 6,004,744, 6,013,431, 5,595,890, 5,762,876, 5,945,283, 5,468,613, Mutations can be detected by a variety of effective methods, including those disclosed in U.S. Patents 6,090,558, 5,800,944, 5,616,464, 7,312,039, 7,238,476, 7,297,485, 7,282,355, 7,270,981, and 7,250,252. All of these documents are incorporated herein by reference in their entirety. For example, mutations can be detected by hybridization with allele-specific oligonucleotide (ASO) probes, as described in U.S. Patents 5,468,613 and 5,217,863. U.S. Patent Application 5,210,015 discloses the detection of annealed oligonucleotides in which an unannealed 5'-labeled nucleotide is released by 5'-3' exonuclease activity. U.S. Patent Application No. 6,004,744 discloses the detection of mutations in DNA by a DNA primer extension reaction. U.S. Patent No. 5,468,613 discloses allele-specific oligonucleotide hybridization.This method allows for the detection of single or multiple nucleotide mutations in a nucleic acid sequence by a process that involves amplifying the sequence containing the nucleotide mutation, immobilizing it on a support, and exposing it to a labeled sequence-specific oligonucleotide probe. Mutations can also be detected by the probe ligation method disclosed in U.S. Patent No. 5,800,944. In this method, the sequence of interest is amplified, hybridized with the probe, and then the labeled portion of the probe is detected by ligation. U.S. Patent Applications No. 6,613,509 and No. 6,503,710, and the references cited therein, provide methods for identifying mutations using mass spectrometry. These various mutation detection methods are intended to be illustrative and not limiting. The method of the present invention can be used in combination with any polymorphism typing method to identify the presence or absence of mutations in the MSH1 and DRM2 genes in genomic DNA samples. Furthermore, the genomic DNA samples used include, but are not limited to, genomic DNA isolated directly from plants, cloned genomic DNA, or amplified genomic DNA.

[0060] Loss-of-function mutations in endogenous plant MSH1 and DRM2 genes, including the MSH1 and DRM2 genes encoding cDNA shown in Table 1, can be obtained from various sources and by various techniques. Homologous substitution sequences containing one or more loss-of-function mutations in the MSH1 and DRM2 genes, along with homologous sequences at both ends of a double-strand break, enable homologous recombination, allowing the chromosomally constitutive wild-type MSH1 and / or DRM2 gene sequences to be replaced with MSH1 and / or DRM2 substitution sequences containing the loss-of-function mutation(s). Such loss-of-function mutations include, but are not limited to, insertions, deletions, and substitutions within the MSH1 and DRM2 genes, and can result in complete loss of function of the MSH1 and DRM2 genes, or loss of function of the MSH1 and DRM2 genes sufficient to induce changes at other chromosomal loci (i.e., heritable and reversible epigenetic changes) or mutations at other chromosomal loci. Loss-of-function mutations in the MSH1 and DRM2 genes include, but are not limited to, frameshift mutations, insertions of early translation termination codons, and deletions of one or more functional domains. In the case of MSH1, these functional domains include, but are not limited to, the DNA-binding domain (domain I), the ATPase domain (domain V), and / or the carboxyl-terminal GIY-YIG type endonuclease domain. This specification also describes mutations similar to the msh1 mutation in Arabidopsis thaliana, which were artificially introduced into the endogenous MSH1 plant gene to achieve similar effects. Methods of replacing endogenous chromosome sequences by homologous double-strand break repair have been reported in tobacco and maize (Wright et al., Plant J.44,693,2005; D'Halluin,et al., Plant Biotech.J.6:93,2008).Homologous substitution msh1 or drm2 sequences containing loss-of-function mutations (i.e., providing loss-of-function mutations in MSH1 or DRM2 target gene sequences) can also be introduced into target nuclease cleavage sites by non-homologous end joining or by a combination of non-homologous end joining and homologous recombination (review: Puchta, J.Exp.Bot. 56, 1, 2005; Wright et al., Plant J. 44, 693, 2005). In certain embodiments, meganucleases can introduce at least one site-specific double-strand break into endogenous MSH1 and DRM2 target genes. Genetic modification of meganucleases can create meganucleases that cleave within recognition sequences that either perfectly match or are closely related to specific endogenous target gene sequences (WO / 06097853A1, WO / 06097784A1, WO / 04067736A2, US20070117128A1). Methods for introducing loss-of-function mutations in MSH1 and DRM2 include the use of site-specific nucleases, which include meganucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and regularly spaced short palindromic repeat (CRISPR)-related Cas nucleases (e.g., Cas9, Cas12a, Cms1, Staphylococcus aureus Cas9 mutant, aCas9, nCas9 nickase, type V) used in combination with guide RNA. This includes, but is not limited to, Cas nucleases (Cas12a nuclease, nCas12a nickase, Cas12d (CasY), Cas12e (CasX), Cas12b (C2c1), Cas12c (C2c3), Cas12i, Cas12f, Cas12j, Cas14, or eSpCas9 nuclease). Methods using these site-specific nucleases, particularly Cas nucleases, and guide RNAs for MSH1 and DRM2, as part of the CRISPR / Cas system, are being considered.The Cpf1 or Csm1 nucleases described in U.S. Patent Application Publication 20180148735 (in its entirety by reference) can be used to obtain loss-of-function mutations in MSH1 and DRM2. The CRISPR-Cas systems disclosed in U.S. Patent Application Publications 20150344912, 20160138008, 20180179547, 20200172886, and 20220282244 (these documents are also incorporated herein by reference) can also be used to obtain loss-of-function mutations in MSH1 and DRM2. Thus, it is possible to select or design nucleases that cleave within the MSH1 or DRM2 gene sequence, which is expected to result in loss-of-function mutations through non-homologous end joining (NHEJ) mediated repair. In other embodiments, zinc finger nucleases can be used to introduce at least one site-specific double-strand break into the target sequence of endogenous MSH1 and / or DRM2 target genes. The use of artificially designed zinc finger nucleases to induce homologous recombination in plants has also been disclosed (WO03 / 080809, WO05 / 014791, WO07014275, WO08 / 021207). In yet another embodiment, mutations in the endogenous MSH1 and / or DRM2 target gene(s) can be identified using the TILLING technique (Targeting Induced Local Lesions in Genomes), as described by Henikoff et al., where a high-throughput screening is performed to identify plants containing point mutations or other mutations in the endogenous MSH1 and / or DRM2 target gene(s) following conventional chemical mutagenesis (Henikoff et al., Plant Physiol. 2004, 135:630-636). Restoration of mutations in the endogenous MSH1 and DRM2 genes is provided in particular herein.In certain embodiments (e.g., in certain crop plants provided in Table 1) in which the crop plant contains two or three MSH1 and DRM2 genes, loss-of-function mutations can be introduced into one, two, or possibly three MSH1 and / or DRM2 genes present in the crop plant. In certain embodiments (e.g., certain crop plants provided in Table 1) in which the crop plant has MSH1 and / or DRM2 genes that produce multiple transcripts (e.g., undergo alternative splicing), loss-of-function mutations, including deletions and / or frameshift mutations located near the 5' end of the MSH1 and / or DRM2 coding region, can be introduced into the MSH1 and / or DRM2 genes.

[0061] Any recombinant DNA construct provided herein can be introduced into the chromosomes of a host plant by methods such as Agrobacterium-mediated transformation, Rhizobium-mediated transformation, Sinorhizobium-mediated transformation, particle-mediated transformation, DNA transfection, DNA electroporation, or "whisker"-mediated transformation. Methods for introducing the aforementioned transgenes are well known to those skilled in the art and are described in U.S. Patent Publication No. 20050289673 (Agrobacterium-mediated transformation of maize), U.S. Patent No. 7,002,058 (Agrobacterium-mediated transformation of soybean), U.S. Patent No. 6,365,807 (Particle-mediated transformation of rice), and U.S. Patent No. 5,004,863 (Agrobacterium-mediated transformation of cotton), which are incorporated herein by reference in their entirety. Methods for transforming plants using bacteria such as Rhizobium and Sinorhizobium are described in Broothaerts, et al., Nature. 2005, 10;433(7026):629-33. It is further understood that recombinant DNA constructs may contain cis-site-specific recombination sites recognized by site-specific recombinases including Cre, Flp, Gin, Pin, Sre, pinD, Int-B13, and R. Subsequently, a method can be used to incorporate the DNA molecule into a specific location within the genome of the transgenic plant using site-specific recombinases (U.S. Patent No. 7,102,055). Those skilled in the art will further understand that any of these gene transfer techniques can be used to introduce recombinant DNA constructs into plant cells, plant tissues, or plant chromosomes.

[0062] A method for introducing plant minichromosomes, including plant centromeres, which provides maintenance of recombinant minichromosomes in transgenic plants, can also be used in carrying out the present invention (U.S. Patent No. 6,972,197 and U.S. Patent Application Publication 20120047609). In these embodiments of the present invention, the transgenic plant possesses minichromosomes as extrachromosomal elements that are not integrated into the host plant's chromosomes. Such minichromosomes are expected to be useful in providing variable transmission of commensal recombinant DNA constructs that suppress the expression of MSH1 and / or DRM2 target genes.

[0063] Methods for achieving repression of the MSH1 and DRM2 genes in cultured plant cells are also provided herein. In certain embodiments, repression of the MSH1 and DRM2 genes is achieved in cultured plant cells by introducing nucleic acids that enable such repression into the plant cells. Nucleic acids that can be used to provide repression of the MSH1 and DRM2 genes in cultured plant cells include, but are not limited to, transgenes that produce small inhibitory RNA (siRNA), microRNA (miRNA), co-repression sense RNA, and / or antisense RNA against the MSH1 and DRM2 genes. Nucleic acids that can be used to provide repression of the MSH1 and DRM2 genes include, but are not limited to, small inhibitory RNA (siRNA) or microRNA (miRNA) against the endogenous MSH1 and DRM2 genes. RNA molecules that inhibit the MSH1 and DRM2 genes can be introduced by electroporation. Introducing inhibitory RNA into cultured plant cells to inhibit a target gene can be achieved in certain embodiments as disclosed in Vanitharani et al. (Proc Natl Acad Sci USA., 2003, 100(16):9632-6), Qi et al. (Nucleic Acids Res. 2004 Dec. 15; 32(22):e179), or J. Cheon et al. (Microbiol. Biotechnol. (2009), 19(8), 781-786).

[0064] Methods for achieving repression of the MSH1 and DRM2 genes in vegetatively propagated or cloned plants are also provided herein. Such vegetatively propagated or cloned plants may include, but are not limited to, cuttings, cultured plants, and the like. In certain embodiments, the recovery of such plants or cloned plants with repressed MSH1 and DRM2 genes can be achieved by a method that enables transient repression of the MSH1 and DRM2 genes. In certain non-limiting examples, plants or cloned plants with repressed MSH1 and DRM2 genes are recovered by placing recombinant DNA constructs that repress the MSH1 and DRM2 genes in a vector that provides their excision or isolation. In certain embodiments, such excision can be facilitated by the use of a transposase-based system, or such isolation can be facilitated by the use of minichromosomes. In certain embodiments, such excision or isolation can be facilitated by ligating the transgenes that repress MSH1 and DRM2 in the recombinant DNA construct with a transgene that enables “conditionally lethal” counter-selection. Plants propagated vegetatively or clonally, in which the MSH1 and DRM2 genes have been repressed and which lack recombinant DNA constructs that repress the MSH1 and DRM2 genes, can then be screened and / or selected for useful traits. Methods are also provided for obtaining vegetatively propagated or clonal plants from plants obtained by self-pollination or cross-pollination, or from cultured plant cells, in which either the plant or plant cells have been repressed with the MSH1 and DRM2 genes. Such plants obtained by vegetative propagation or clonal propagation, from such plants obtained by self-pollination or cross-pollination, or from plant cells in which the MSH1 and DRM2 genes have been repressed, can also be screened and / or selected for useful traits. Furthermore, methods are provided for propagating sexually reproducing plants or plant populations containing useful traits by vegetative propagation or clonal propagation, and for producing seeds or seed lots using the plants or plant populations obtained thereby.

[0065] Suppression of the MSH1 and DRM2 genes can be readily identified or monitored by conventional methods of observing plant phenotypes. For example, suppression of the MSH1 and DRM2 genes can be identified or monitored by observing organic effects (phenotypic identification such as leaf variegation, cytoplasmic male sterility (CMS), reduced growth rate, delayed flowering, or failure to flower). Phenotypes showing MSH1 gene suppression in various plants are described in WO2012 / 151254, which is incorporated herein by reference in its entirety. These phenotypes associated with MSH1 gene suppression are referred herein as “discrete variations” (V DMSH1 gene repression is also known as MSH1-dr. Changes in the plant phenotype, including tillering, height, internode elongation, and stomatal density (hereinafter referred to as "MSH1-dr"), can also be used to identify or monitor MSH1 gene repression in plants, but are not limited to these changes. Other biochemical and molecular traits can also be used to identify or monitor MSH1 gene repression in plants. Such molecular-level traits may include changes in the expression of genes involved in cell cycle regulation, gibberellate catabolism, auxin biosynthesis, auxin receptor expression, and flowering and vernalization regulators (i.e., increased FLC expression and decreased SOC expression), and may also include increases in miR156 and decreases in miR172. Such biochemical traits include, but are not limited to, upregulation of most compounds in the TCA, NAD, and carbohydrate metabolic pathways, downregulation of amino acid biosynthesis, sucrose depletion in certain plants, increased sugars or sugar alcohols in certain plants, and increased levels of ascorbic acid, alpha-tocopherol, and stress-responsive flavones such as apigenin, and apigenin-7-glucoside, isovitexin, kaempferol 3-O-β-glucoside, luteolin-7-O-glucoside, and vitexin. In certain embodiments, elevated plastichromanol-8 levels in plant stems can serve as a biochemical marker that can be used to identify or monitor MSH1 gene repression. In particular, MSH1 and DRM2 target gene repression can be identified or monitored by comparing plastichromanol-8 levels in plant stems with MSH1 gene repression to levels in control plants without such repression. In certain embodiments, it is further intended that a combination of molecular, biochemical, and conventional methods may be used to identify or monitor the repression of MSH1 and DRM2 genes in plants.

[0066] Plants or rootstocks in which the MSH1 and DRM2 genes have been repressed, as well as scions grafted onto such rootstocks and their progeny, may exhibit a variety of nuclear chromosome DNA methylation patterns not seen in control plants, rootstocks, or scions that have not been repressed of the MSH1 and DRM2 genes. Such methylation patterns may include, but are not limited to, CG hypermethylation, pericentromeric CHG hypermethylation, and / or additional characteristic methylation patterns observed in plants or their progeny in which the expression of the MSH1 and DRM2 genes has been repressed. Such methylation patterns may also include, but are not limited to, changes in 5-hydroxymethylation, particularly 5-hydroxymethylcytosine (5-hmC). Changes in 5-hmC can be monitored by immunoassay (Quest 5-hmC® DNA ELISA Kit, Zymo Research Corp., Irvine, Calif., USA, or EpiSeeker® hydroxymethylated DNA Quantification Kit, Abeam, Inc., Cambridge, Mass.). The plants, plant parts, processed plant products, rootstocks, and scions provided herein, or the plants, plant parts, processed plant products, rootstocks, and scions produced by the methods provided herein, are expected to be identifiable by comparing the methylation patterns in the genomic DNA of such materials with the methylation patterns of control plants, plant parts, processed plant products, rootstocks, and scions.

[0067] In certain embodiments of the methods provided herein, progeny derived from plants in which the expression of MSH1 and DRM2 genes is suppressed, exhibiting male sterility, dwarfism, variegation, and / or delayed flowering, and expressing functional MSH1 and DRM2 genes, are obtained and maintained as independent breeding lines or as plant populations. Such phenotypes have been shown to be classifiable, and for example, it is possible to select cytoplasmic male-sterile plants that do not exhibit variegation at a normal growth rate, or male-fertile plants whose growth is inhibited and which exhibit a lot of variegation. Here, this phenomenon is referred to as discrete variation (V D This is called [a specific term]. An illustrative and non-limiting diagram of this phenomenon occurring in self-pollinating plant populations that have lost the transgene that inhibits the MSH1 gene through segregation is disclosed (WO2012 / 151254, the whole of which is incorporated herein by reference). Furthermore, discrete mutation (V D It is intended that such individual strains exhibiting the aforementioned genetic techniques, molecular genetic techniques, or a combination thereof can be obtained.

[0068] Among the individual lines obtained from plants in which the expression of MSH1 and DRM2 target genes was suppressed, discrete mutations (V D Individuals exhibiting ) are crossbred with other plants to introduce discrete variation (V D Progeny lacking phenotypes associated with (i.e., male sterility, dwarfism, variegation, and / or delayed flowering) can be obtained. In certain embodiments, such cross-pollinated progeny can be self-pollinated to obtain individual progeny lines exhibiting significant phenotypic variation. Such phenotypic variation observed in these individual progeny lines obtained from cross-pollination of plants in which the expression of MSH1 and DSM2 genes is suppressed, and which refers to discrete variation different from other plants, is referred to herein as "quantitative variation" (V Q) is referred to as. Certain individual progeny plant lines obtained from outcrossing a plant with suppressed expression of the MSH1 gene with other plants can exhibit useful phenotypic variations, where one or more traits are improved compared to either parental line and can be selected therefrom. Useful phenotypic variations that can be selected in such individual progeny lines include, but are not limited to, an increase in biomass of fresh weight and dry weight relative to either parental line. An exemplary and non-limiting figure of this phenomenon occurring in the F2 progeny of outcrossing plants showing discrete mutations with plants not showing discrete mutations is provided in WO2012 / 151254, which is hereby incorporated in its entirety by reference.

[0069] Among individual lines obtained from plants with suppressed expression of the MSH1 and DRM2 genes, individuals showing discrete mutations (V D ) can be selfed to obtain progeny plants lacking the phenotypes associated with the discrete mutations (V D ) (i.e., male sterility, dwarfism, variegation, and / or delayed flowering time). Recovery of such progeny plants lacking undesirable phenotypes can be facilitated, in certain embodiments, by removing the transgene or endogenous locus enabling suppression of the MSH1 and DRM2 genes. In certain embodiments, such selfed progeny can be used to obtain individual progeny lines or populations showing significant phenotypic variations. Certain individual progeny plant lines or populations obtained from selfing plants with suppressed expression of the MSH1 and DRM2 genes can exhibit useful phenotypic variations, where one or more traits are improved compared to the parental line that was not subjected to suppression of the MSH1 and DRM2 genes and can be selected therefrom. Useful phenotypic variations that can be selected in such individual progeny lines include, but are not limited to, an increase in biomass of fresh weight and dry weight relative to the parental line.

[0070] In certain embodiments, cross-pollination of individual lines exhibiting discrete variability can be performed on plants that have the same genetic background as the individual lines exhibiting discrete variability, but without the repression of the MSH1 and DRM2 genes. In certain exemplary embodiments, a line exhibiting discrete variability is obtained by repressing the MSH1 and DRM2 genes in a given germplasm and can be cross-pollinated on plants having the same germplasm without the repression of the MSH1 and DRM2 genes. In other embodiments, cross-pollination of individual lines exhibiting discrete variability can be performed on plants that do not have the same genetic background as the individual lines exhibiting discrete variability, but without the repression of the MSH1 and DRM2 genes. Thus, in certain embodiments, cross-pollination of individual lines exhibiting discrete variability can also be performed on plants containing one or more chromosomal polymorphisms that do not occur in the individual lines exhibiting discrete variability, plants derived from partially or entirely different germplasms, or plants of different heterosis groups (if such separate heterosis groups exist). Furthermore, it is understood that such cross-pollination can be performed in any direction. Therefore, in such cross-pollination, individual lines exhibiting discrete variability can be used as pollen donors or pollen recipients in plants that have not undergone repression of the MSH1 and DRM2 genes. In a particular embodiment, the cross-pollination progeny are then self-pollinated to establish individual lines that can be separately screened to identify lines with improved traits compared to the parent line. Individual lines exhibiting improved traits can then be selected and propagated by further self-pollination. Exemplary and non-exemplary examples of this procedure, in which F2 progeny of cross-pollination of plants exhibiting discrete variability and plants not exhibiting discrete variability are obtained, are provided in WO2012 / 151254, which is incorporated herein by reference in its entirety. Such F2 progeny lines are screened for desired trait improvements compared to the parent plant, and lines exhibiting such improvements are selected.

[0071] In certain embodiments, subpopulations of plants containing desirable traits and epigenetic changes induced by the repression of the MSH1 and DRM2 genes can be selected and bred as a population. Such populations can be further selected one or more times for desirable traits and / or epigenetic changes to obtain next-generation subpopulations of plants exhibiting desirable traits. Any of these subpopulations can also be used to generate seed lots. In an exemplary embodiment, plastid-disrupted plants exhibiting the Msh1-dr phenotype can be self-pollinated or cross-pollinated to obtain the F1 generation. Thus, batch selection in the F1, F2, and / or F3 generations can provide a population or seed lot of plants exhibiting desirable traits and / or epigenetic changes. In certain embodiments, it is also conceivable that a population of progeny plants or progeny seed lots containing a mixture of inbred germplasm and hybrid germplasm can be derived from a population containing hybrid germplasm (i.e., plants resulting from the cross of one inbred line with another inbred line). In certain embodiments, such subpopulations may include grafted plants comprising scions grafted onto rootstocks in which the MSH1 and DRM2 genes have been suppressed. Subpopulations of grafted plants may also be selected and bred as populations, using progeny of parent plants selected for one or more desirable traits, which have been suppressed for the MSH1 and DRM2 genes, as the rootstock source. Any of the aforementioned subpopulations may include 2 or more plants, 10 or more plants, 50 or more plants, 100 or more plants, 1000 or more plants, or 10000 or more plants. Seed lots obtained from these exemplary methods or other methods provided herein may contain seeds in which at least 25%, 50%, 60%, 70%, 80%, 90%, or 95% of the progeny grown from the seeds exhibit desirable traits. This selection will provide the most robust and vigorous populations for seed lot production. Seed lots thus produced may be used for breeding or commercial purposes. In a particular embodiment, a seed lot is obtained in which at least 25%, 50%, 60%, 70%, 80%, 90%, or 95% of the progeny plants grown from the seeds exhibit one or more useful traits associated with epigenetic changes.Here, the epigenetic changes are associated with CG hypermethylation and / or CHG hypermethylation at one or more nuclear chromosome loci compared to control plants that do not exhibit useful traits, and the seeds or progeny grown therefrom are epigenetically heterogeneous. Seed lots obtained by these methods may contain at least 100, 500, 1000, 5000, or 10000 seeds.

[0072] In a particular embodiment, a method for producing a seed lot is provided, comprising: (i) growing a population of plants, the population comprising two or more grafted plants consisting of scions and rootstocks obtained from plants that have been subjected to MSH1 and DRM2 gene suppression, or from parent plants that have been subjected to MSH1 and DRM2 gene suppression; and (ii) obtaining a seed lot from the population. A population of grafted plants whose rootstock source plants are progeny of parent plants that have been subjected to MSH1 and DRM2 gene suppression and selected for one or more useful traits may also be selected and bred as a population. Any of the aforementioned populations may include two or more plants, ten or more plants, fifty or more plants, 100 or more plants, 1000 or more plants, or 10,000 or more plants. Seed lots obtained by these exemplary methods or other methods provided herein may contain seeds in which at least 25%, 50%, 60%, 70%, 80%, 90%, or 95% of the progeny grown from the seeds exhibit a useful trait. This selection will provide the most robust and vigorous population for seed lot production. Seed lots thus produced may be used for breeding or commercial purposes. In a particular embodiment, a seed lot is obtained in which at least 25%, 50%, 60%, 70%, 80%, 90%, or 95% of the progeny grown from the seeds exhibit a useful trait associated with one or more epigenetic changes, where the epigenetic change is associated with CG hypermethylation and / or CHG hypermethylation at one or more nuclear chromosome loci compared to the corresponding nuclear chromosome loci of a control plant that does not exhibit the useful trait, and the seeds or progeny grown therefrom are epigenetically heterogeneous. Seed lots obtained by these methods may contain at least 100, 500, 1,000, 5,000, or 10,000 seeds.

[0073] Furthermore, by performing appropriate comparative analysis between test plants obtained from parent plants or plant cells in which the MSH1 and DRM2 genes have been suppressed and standard plants that do not exhibit useful traits, it is possible to identify and select modified chromosomal loci that can confer useful traits by obtaining either a modified locus or a plant containing a modified locus. It is expected that various standard plants and test plants will be used for such comparisons and selections. In a particular embodiment, a reference plant that does not exhibit a useful trait includes, but is not limited to, any of the following: a) a wild-type plant; b) a distinct subpopulation of plants within a given F2 population of a given plant lineage (where the F2 population is any applicable plant type or variety); c) an F1 population exhibiting a wild-type phenotype (where the F1 population is any applicable plant type or variety); and / or d) a plant having the same genetic background as the parent plant or plant cell prior to the repression of the MSH1 and DRM2 genes in the parent plant or plant cell of the test plant (i.e., the reference plant has the same genetic background as the plant or plant cell to which the MSH1 and DRM2 genes are subsequently repressed in order to obtain the test plant). In a particular embodiment, a test plant exhibiting a useful trait includes, but is not limited to, any: a) any non-transgenic isolate derived from a parent plant or plant cell that exhibits a useful trait and has been repressed via an transgene into MSH1 and DRM2 genes; b) a distinct subpopulation of plants within a given F2 population of a given plant line exhibiting a useful trait (where the F2 population is any applicable plant type or variety); c) any progeny obtained from the plant of (a) or (b) exhibiting a useful trait; or d) a plant or plant cell that has been repressed into MSH1 and DRM2 genes and exhibits a useful trait.

[0074] Generally, the purpose of these comparisons is to identify differences in small molecule RNA profiles and / or methylation of specific chromosomal DNA loci between test plants exhibiting desirable traits and control plants that do not. The modified loci thus identified can then be isolated or selected in plants to obtain plants exhibiting desirable traits.

[0075] In certain embodiments, modified chromosomal loci can be identified by identifying small RNA molecules that are upregulated or downregulated in the test plant (compared to the control plant). This method is partly based on identifying modified chromosomal loci where small interfering RNAs induce methylation of specific gene targets via RNA-induced DNA methylation (RdDM). The RNA-induced DNA methylation (RdDM) process has been described (Chinnusamy V et al. Sci China Ser C-Life Sci. (2009) 52(4):331-343). Any suitable technological platform can be used to compare small RNA molecules in the test plant and the control plant. These include, but are not limited to, microarray-based methods (Franco-Zorilla et al. Plant J. 2009 59(5):840-50) and deep sequencing-based methods (Wang et al. The Plant Cell 21:1053-1069 (2009)).

[0076] In certain embodiments, modified chromosomal loci can be identified by identifying histone proteins associated with the locus that are methylated or acylated in the test plant (compared to the control plant). Analysis of chromosomal loci associated with methylated or acylated histones can be achieved by enriching and sequencing those loci using antibodies that recognize methylated or acylated histones. Identification of chromosomal regions associated with methylation or acetylation of specific lysine residues of histone H3 using antibodies specific to H3K4me3, H3K9ac, H3K27me3, and H3K36me3 has been described (Li et al., Plant Cell 20:259-276, 2008; Wang et al. The Plant Cell 21:1053-1069 (2009)).

[0077] In certain embodiments, modified chromosomal loci can be identified by identifying chromosomal regions (genomic DNA) in which the methylation status is altered (compared to the control plant) in the test plant. The altered methylation status may include either the presence or absence of methylation at one or more chromosomal loci in the test plant compared to the control plant. Any applicable technical platform can be used to compare the methylation status of chromosomal loci in the test plant and the control plant. Applicable techniques for identifying chromosomal loci with altered methylation status include methods based on DNA immunoprecipitation using antibodies that recognize 5-methylcytidine, methods based on the use of methylation-dependent restriction endonucleases and PCR (such as McrBC-PCR) (Rabinowicz, et al. Genome Res. 13:2658-2664 2003; Li et al., Plant Cell 20:259-276, 2008), sequencing of bisulfite-treated DNA (Frommer et al. Proc. Natl. Acad. Sci. USA 89(5):1827-31; Tost et al. BioTechniques 35(1):152-156, 2003), and methylation-specific PCR analysis of bisulfite-treated DNA (Herman et al. Examples of such methods include, but are not limited to, al. Proc. Natl. Acad. Sci. USA 93(18):9821-6, 1996, deep sequencing-based methods (Wang et al. The Plant Cell 21:1053-1069 (2009)), methylation-sensitive single-strand nucleotide primer elongation (MsSnuPE; Gonzalgo and Jones Nucleic Acids Res. 25(12):2529-2531, 1997), fluorescence correlation spectroscopy (Umezu et al. Anal Biochem. 415(2):145-50, 2011), single-molecule real-time sequencing (Flusberg et al. Nature Methods 7, 461-465), and high-resolution melting analysis (Wojdacz and Dobrovic (2007) Nucleic Acids Res. 35(6):e41).

[0078] Methods for introducing various chromosomal modifications that can confer useful traits to plants, as well as plants, plant parts, and products of those plant parts, are also provided herein. Chromosomal alterations and / or mutations induced by the repression of the MSH1 and DRM2 genes can be identified as described herein. Once identified, the chromosomal modification (including, but not limited to, chromosomal alterations, mutations, or introduced genes that produce the same genetic effect as chromosomal alterations and / or mutations induced by the repression of the MSH1 and DRM2 genes) can be introduced into a host plant to obtain a plant exhibiting the desired trait. In this context, “same genetic effect” means that the introduced chromosomal modification allows for an increase and / or decrease in the expression of one or more endogenous plant genes, similar to that observed in plants that have undergone MSH1 and DRM2 gene repression and exhibit useful traits. In certain embodiments, chromosomal modifications are provided that, in plants that have undergone MSH1 and DRM2 gene repression, result in methylation of endogenous genes, exhibiting both decreased gene expression and useful traits, and similarly in other plants, result in decreased gene expression and useful traits. In certain embodiments, chromosomal modifications are provided that result in demethylation of endogenous genes in plants subjected to repression of the MSH1 and DRM2 genes, exhibiting both increased gene expression and a useful trait, and similarly in other plants.

[0079] In certain embodiments, the chromosomal modification introduced is a chromosomal alteration. Chromosomal alterations, including but not limited to differences in methylation states, can be introduced by crossing a plant containing the alteration with a plant without the alteration and selecting for the presence of the alteration in the F1, F2, or subsequent generations of the cross. In yet another embodiment, chromosomal modification in a specific target gene can be introduced by the expression of siRNA or hairpin RNA that targets that gene via RNA-induced DNA methylation (Chinnusamy V et al. Sci China Ser C-Life Sci. (2009) 52(4):331-343; Cigan et al. Plant J 43 929-940, 2005; Heilersig et al. (2006) Mol Genet Genomics 275 437-449; Miki and Shimamoto, Plant Journal 56(4):539-49; Okano et al. Plant Journal 53(1):65-77, 2008).

[0080] In certain embodiments, chromosomal modifications are chromosomal mutations. Chromosomal mutations that decrease or increase the expression of endogenous genes at a chromosomal locus may include, but are not limited to, insertions, deletions, and / or substitutions of nucleotide sequences within a gene. Chromosomal mutations can lead to decreased gene expression through various mechanisms, including, but are not limited to, the introduction of missense codons, frameshift mutations, premature translation arrest codons, promoter deletions, and mutations that disrupt mRNA processing. Chromosomal mutations that lead to increased gene expression may include, but are not limited to, promoter substitutions and the removal of negative regulators from a gene. Chromosomal mutations can be introduced into specific gene loci in plants using any applicable method. Applicable methods for introducing chromosomal mutations into endogenous chromosome loci in plants include homologous double-strand break repair (Wright et al., Plant J.44,693,2005; D'Halluin, et al., Plant Biotech.J.6:93,2008), non-homologous end joining, or a combination of non-homologous end joining and homologous recombination (Puchta, J.Exp.Bot.56,1,2005; Wright et al., Plant Examples include, but are not limited to, meganuclease-induced site-directed double-strand break repair (WO / 06097853A1, WO / 06097784A1, WO / 04067736A2, US20070117128A1), and homologous recombination mediated by zinc finger nucleases (WO03 / 080809, WO05 / 014791, WO07014275, WO08 / 021207). In further embodiments, desired mutations at endogenous chromosome loci in plants can be identified using the TILLING technique (Targeting Induced Local Lesions in Genomes), as described (Henikoff et al., Plant Physiol. 2004, 135:630-636).

[0081] In other embodiments, chromosomal modifications that produce the desired genetic effect may include transgenes. Transgenes that can reduce gene expression by various mechanisms include, but are not limited to, dominant-negative mutants, small inhibitory RNAs (siRNAs), microRNAs (miRNAs), co-repression sense RNAs, and / or antisense RNAs. U.S. patents describing the repression of endogenous plant genes by transgenes, which are incorporated herein by whole reference, include U.S. Patents 7,109,393, 5,231,020, and 5,283,184 (co-repression method), and U.S. Patents 5,107,065 and 5,759,829 (antisense method). In certain embodiments, a transgene specifically designed to produce a double-stranded RNA (dsRNA) molecule homologous to an endogenous gene at a chromosomal locus can be used to reduce the expression of that endogenous gene. In such embodiments, the sense strand sequence of dsRNA can be separated from the antisense sequence by a spacer sequence, preferably one that promotes the formation of a dsRNA (double-stranded RNA) molecule. Examples of such spacer sequences include, but are not limited to, those described in Wesley et al., Plant J., 27(6):581-90 (2001) and Hamilton et al., Plant J., 15:737-746 (1998). Vectors for inhibiting endogenous plant genes by expressing hairpin RNA via transgenes are disclosed in U.S. Patent Applications No. 20050164394, No. 20050160490 and No. 20040231016, each of which is incorporated herein by reference in its entirety.

[0082] Transgenes that result in increased expression of a gene at a chromosomal locus include, but are not limited to, recombinant genes fused with a heterologous promoter stronger than the endogenous promoter, recombinant genes containing elements such as heterologous introns, 5' untranslated regions, and 3' untranslated regions that result in increased expression, and combinations thereof. Such promoters, introns, 5' untranslated regions, 3' untranslated regions, and any necessary polyadenylation regions can be manipulably ligated to the target DNA in a recombinant DNA molecule containing a portion of the transgene useful for performing chromosomal modifications as provided herein.

[0083] Examples of promoters useful for transgene expression include, but are not limited to, enhanced or replicated versions of the viral CaMV35S and FMV35S promoters (U.S. Patent No. 5,378,619, which is incorporated herein by reference in its entirety), the cauliflower mosaic virus (CaMV) 19S promoter, the rice Act1 promoter, and the plantain mosaic virus (FMV) 35S promoter (U.S. Patent No. 5,463,175, which is incorporated herein by reference in its entirety). Exemplary introns useful for transgene expression include the maize hsp70 intron (U.S. Patent No. 5,424,412; the entire patent is incorporated herein by reference), the rice Act1 intron (McElroy et al., 1990, The Plant Cell, Vol. 2, 163-171), the CAT-1 intron (Cazzonnelli and Velten, Plant Molecular Biology Reporter 21:271-280, September 2003), the pKANNIBAL intron (Wesley et al., Plant J. 2001 27(6):581-90; Collier et al., 2005, Plant J 43:449-457), the PIV2 intron (Mankin et al. (1997) Plant Mol. Biol. Rep. 15(2):186-196), and Super Examples of polyadenylated sequences include, but are not limited to, the Ubiquitin intron (U.S. Patent No. 6,596,925, the entire patent is incorporated herein by reference; Collier et al., 2005, Plant J 43:449-457). Exemplary polyadenylated sequences include, but are not limited to, the polyadenylated sequences of the nopalin synthase (NOS) gene in the Agrobacterium tumor induction (Ti) plasmid and the ssRUBISCO E9 gene in pea.

[0084] Plant lines and populations obtained by the methods provided herein can be screened and selected for various useful traits using a wide range of techniques. In a particular embodiment provided herein, individual progeny plant lines or populations obtained from self-pollination of plants in which the expression of MSH1 and DRM2 genes is suppressed, or from cross-pollination to other plants, are screened and selected for desired useful traits.

[0085] In certain embodiments, the trait screened and selected is improved plant yield. In certain embodiments, such improved yield means that the yield of a plant line under non-stress conditions is improved compared to the yield of one or more parent lines. Non-stress conditions refer to conditions in which water, temperature, nutrients, minerals, and light are within the typical range for cultivation of the plant species. Such typical cultivation range includes conditions in which the amount or value of water, temperature, nutrients, minerals, and / or light is neither insufficient nor excessive. In certain embodiments, such improved yield means that the yield of a plant line under abiotic stress conditions is improved compared to the yield of one or more parent lines. Such abiotic stress conditions include, but are not limited to, conditions in which water, temperature, nutrients, minerals, and / or light are insufficient or excessive. Therefore, abiotic stress conditions may include, but are not limited to, drought stress, osmotic stress, nitrogen stress, phosphorus stress, mineral stress, heat stress, cold stress, and / or light stress. In this context, mineral stress includes, but is not limited to, stress caused by deficiencies or excesses of potassium, calcium, magnesium, iron, manganese, copper, zinc, boron, aluminum, and silicon. In this context, mineral stress includes, but is not limited to, stress caused by excess amounts of heavy metals such as cadmium, copper, nickel, zinc, lead, and chromium.

[0086] Yield improvements in plant lines obtained by the methods provided herein can be identified by direct measurement of wet or dry biomass, including (but not limited to) grains, lint, leaves, stems, or seeds. Yield improvements can also be evaluated by measuring yield-related traits, such as 100-grain weight, yield index, and seed weight. In certain embodiments, such yield improvements are improvements in the yield of the plant line compared to one or more parent lines, which can be easily determined by cultivating the plant lines obtained by the methods provided herein in parallel with the parent plants. In certain embodiments, field trials can be employed to determine yield differences, in which plots of test plants and control plants are replicated, randomized, and variability controlled (Giesbrecht FG and Gumpertz M L. 2004. Planning, Construction, and Statistical Analysis of Comparative Experiments. Wiley. New York; Mead, R. 1997. Design of plant breeding trials. In Statistical Methods for Plant Variety Evaluation. eds. Kempton and Fox. Chapman and Hall. London). Methods for setting the spacing between test plants (i.e., plants obtained by the method of the present invention) and control plants (parents or other controls) in order to obtain yield data suitable for comparison are described in references including, but not limited to, Cullis, B. et al. J. Agric. Biol. Env. Stat. 11:381-393 and Besag, J. and Kempton, R A. 1986. Biometrics 42:231-251.

[0087] In certain embodiments, the traits screened and selected are those that improve the plant's resistance to biological stress compared to the parent line. Biological stress on plants includes, but is not limited to, stress caused by plant fungal pathogens, plant bacterial pathogens, plant viral pathogens, insects, nematodes, and herbivores. In certain embodiments, a method is provided for screening and selecting plant lines that exhibit resistance to fungal pathogens, including but not limited to the genera Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diaporthe, Diplodia, Erysiphe, Fusarium, Gaeumanomyces, Helminthosporium, Macrophomina, Nectria, Peronospora, Phakopsora, Phialophora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, Puccinia, Podosphaera, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Sclerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, and Verticillium. In certain embodiments, a screening and selection of plant lines exhibiting resistance to bacterial pathogens including, but not limited to, the genera Erwinia, Pseudomonas, and Xanthamonas is provided. In certain embodiments, a screening and selection of plant lines exhibiting resistance to insects including, but not limited to, aphids, stinging / sap-sucking insects such as those of the genera Lygus, lepidopteran insects such as those of the genera Armigera sp., Helicoverpa, Heliothis, and Pseudoplusia, and coleopteran insects such as those of the genera Diabroticus is provided.In a particular embodiment, a method is provided for screening and selecting plant lines that exhibit resistance to nematodes, including but not limited to the genera Meloidogyne, Heterodera, Belonolaimus, Ditylenchus, Globodera, Naccobus, and Xipinema.

[0088] Other useful traits that can be obtained by the methods provided herein include, but are not limited to, traits relating to various seed qualities, including (but not limited to) improvements in the composition or quantity of oils, proteins, or starches in the seeds. Further useful traits that can be obtained by the methods provided herein include, but are not limited to, increased biomass, non-flowering, male sterility, digestibility, seed filling period, maturation period (either earlier or later as desired), reduced lodging, and plant height (either increased or decreased as desired). Further useful traits that can be obtained by the methods provided herein include, but are not limited to, delayed leaf senescence, increased flowering, improved plant structure for high-density planting, improved photosynthesis, increased root mass, increased cell count, improved seedling vitality, improved seedling size, increased cell division rate, improved metabolic efficiency, and increased meristematic size.

[0089] In addition to any of the traits described herein, particularly useful traits for sorghum that can be obtained by the methods provided herein include (but are not limited to): i) agricultural traits (flowering time, days to flowering, days to flowering under post-rainy season conditions, days to flowering under rainy season conditions); ii) fungal disease resistance (sorghum downy mildew resistance - greenhouse conditions, sorghum downy mildew resistance - field conditions, sorghum grain mold resistance, sorghum leaf blight resistance, sorghum rust resistance; iii) cereal-related traits (dry grain weight, number of grains, number of grains per square meter, grain weight per ear, seed color, seed luster, seed size); iv) traits related to growth and developmental stages (number of basal stalks, days to harvest, days to maturity, stalks, plant height, plant height - post-rainy season conditions); v) inflorescence anatomical and morphological traits (threshing ability); vi) pest resistance (sorghum stem and leaf fly resistance - post-rainy season conditions, sorghum stem and leaf fly resistance - rainy season conditions, sorghum stem borer resistance); vii) leaf-related traits (leaf color, leaf midrib color, leaf vein color, flag leaf weight, leaf weight, other leaf weight); viiii) mineral and ion content-related traits (aboveground potassium content, aboveground sodium content); ix) panicle x) traits related to the panicles (number of panicles, panicle density and shape, panicle protrusion, panicle yield index, panicle length, panicle weight, panicle weight excluding grain, panicle width); x) content of phytochemical compounds (plant pigments); xii) anatomical and morphological traits of the spikelets (glimmer color, glimmer coverage); xiii) stem-related traits (stem and leaf weight, stem weight); and xiv) other traits (stem and leaf-related traits, metabolic energy, nitrogen digestibility, organic matter digestibility, stem and leaf dry weight).

[0090] Embodiment This patent application includes, but is not limited to, the following embodiments.

[0091] 1. A grafted plant that includes a scion grafted onto a rootstock, (i) The scions are derived from wild-type plants, (ii) In the aforementioned rootstock, the expression of MSH1 and DRM2 genes is suppressed. (iii) The rootstock provides an improvement in yield or growth rate in the progeny of the grafted plant compared to a control plant, wherein the control plant comprises any of the following: (a) the progeny of a scion grafted onto a rootstock in which the expression of the MSH1 and DRM2 genes has not been suppressed; (b) a perfect plant in which no root grafting has been performed and the expression of the MSH1 and DRM2 genes has not been suppressed; (c) a wild-type plant; or (d) the progeny of a plant having the same genetic background as the plant source of the scion of the grafted plant.

[0092] 2. The grafted plant according to Embodiment 1, wherein the rootstock imparts an improvement in yield or growth rate to the grafted plant compared to a control plant.

[0093] 3. The grafted plant according to Embodiment 1 or 2, wherein the scion contains two or more epigenetic changes in one or more nuclear chromosomes, and the epigenetic changes are not present in the nuclear chromosomes of the control plant or in the nuclear chromosomes of the plant from which the scion was obtained.

[0094] 4. The grafted plant according to Embodiment 3, wherein the epigenetic changes (multiple changes) are also present in the rootstock.

[0095] 5. The grafted plant according to Embodiment 3, wherein the epigenetic changes are related to the improvement in the useful trait.

[0096] 6. The grafted plant according to Embodiment 3, wherein the rootstock contains one or more epigenetic changes in one or more nuclear chromosomes that are not present in the nuclear chromosomes of the rootstock obtained from a plant in which the expression of MSH1 and DRM2 genes was not suppressed, or in the nuclear chromosomes of the parent plant.

[0097] 7. A grafted plant according to any one of Embodiments 1 to 6, wherein the expression of the MSH1 and DRM2 genes in the rootstock is suppressed by loss-of-function mutations in the rootstock's endogenous MSH1 and / or DRM2 genes, and / or by small inhibitory RNA (siRNA), microRNA (miRNA), co-repression sense RNA, and / or by antisense RNA complementary to the endogenous MSH1 and / or DRM2 gene promoter, 5' or 3' untranslated region, intron, coding region, and / or any combination thereof, and optionally the grafted plant is a soybean, rapeseed, tomato, sorghum, rice, or maize plant, and the endogenous MSH1 and DRM2 genes (or more) encode the cDNA(or more) listed in Table 1.

[0098] 8. The grafted plant according to Embodiment 7, wherein loss-of-function mutations in the endogenous MSH1 and DRM2 genes (or more) suppress the expression of the MSH1 and DRM2 genes in the rootstock, and the grafted plant is optionally a soybean, rapeseed, tomato, sorghum, rice, or maize plant, and the MSH1 and DRM2 genes (or more) encode the cDNA (or more) listed in Table 1.

[0099] 9. The grafted plant according to Embodiment 7, wherein the expression of the MSH1 and / or DRM2 genes in the rootstock is suppressed by small inhibitory RNA (siRNA), microRNA (miRNA), co-repression sense RNA, and / or by antisense RNA complementary to the endogenous MSH1 and / or DRM2 gene promoter, 5' or 3' untranslated region, intron, coding region, and / or any combination thereof, and optionally the grafted plant is a soybean, rapeseed, tomato, sorghum, rice, or maize plant, and the MSH1 and / or DRM2 genes encode the cDNA(s) listed in Table 1.

[0100] 10. The grafted plant according to any one of Embodiments 1 to 9, wherein the plant is selected from the group consisting of crop plants, trees, shrubs, turfgrasses, pasture grasses, and climbing plants.

[0101] 11. The grafted plant according to Embodiment 10, wherein the crop plant is selected from the group consisting of corn, soybeans, cotton, canola, wheat, rice, tomato, tobacco, millet, potato, sugar beet, cassava, alfalfa, barley, oats, sugarcane, sunflower, strawberry, and sorghum.

[0102] 12. (a) A step of obtaining a population of progeny from a grafted plant according to Embodiment 1, wherein the population of progeny is a first-generation, second-generation, or third-generation progeny obtained by self-propagating the grafted plant or by self-propagating the first-generation or second-generation progeny, (b) A step of screening a population of progeny plants that have improved yield or growth rate compared to a control plant, (c) Progeny plants produced by a method comprising the step of selecting progeny plants from the population for improvement in yield or growth rate compared to a control plant, wherein the progeny plants exhibit improvement in yield or growth rate and exhibit a nuclear chromosome DNA methylation pattern different from that of the control plant, and the control plant is cultivated under the same environmental conditions as the selected progeny plants and includes any of the following: (i) progeny of scions grafted onto rootstock in which the expression of MSH1 and DRM2 genes has not been suppressed; (ii) perfect plants in which no root grafting has been performed and the expression of the MSH1 gene has not been suppressed; (iii) wild-type plants; or (iv) progeny of plants having the same genetic background as the plant source of the scion of the grafted plant.

[0103] 13. (a) A step of obtaining a population of progeny from a grafted plant according to Embodiment 1, wherein the population of progeny is a first-generation, second-generation, or third-generation progeny obtained by self-propagating the grafted plant or by self-propagating the first-generation or second-generation progeny, (b) A step of screening the progeny population for improved yield or growth rate compared to the control plant population, (c) A selected population of progeny plants produced by a method comprising the step of selecting a population of progeny plants for improvement in yield or growth rate compared to a control plant, wherein the selected population of progeny plants exhibits improvement in yield or growth rate and exhibits a nuclear chromosome DNA methylation pattern different from that of the control plant, and the control plant is cultivated under the same environmental conditions as the selected population of progeny plants, and includes any of the following: (i) progeny of scions grafted onto rootstocks in which the expression of MSH1 and DRM2 genes has not been suppressed; (ii) perfect plants in which no root grafting has been performed and the expression of MSH1 and DRM2 genes has not been suppressed; (iii) wild-type plants; or (iv) progeny of plants having the same genetic background as the plant source of the scion of the grafted plant.

[0104] 14. The method according to Embodiment 13, wherein loss-of-function mutations in the endogenous MSH1 and DRM2 genes (or more) of the rootstock suppress the expression of the MSH1 and DRM2 genes in the rootstock of the grafted plant, and optionally the grafted plant is a soybean, rapeseed, tomato, sorghum, rice, or maize plant, and the MSH1 and DRM2 genes (or more) encode the cDNA(s) listed in Table 1.

[0105] 15. A method for producing plants that exhibit useful traits, (a) A step of obtaining a population of progeny plants from a grafted plant including a scion grafted onto a rootstock, wherein the rootstock is obtained from a plant or its parent plant in which the expression of MSH1 and DRM2 genes has been suppressed, (b) A method comprising the step of selecting one or more progeny plants from the population, wherein the selected progeny plants show improvement in the useful traits compared to a control plant, thereby producing plants that exhibit the useful traits.

[0106] 16. The method according to Embodiment 15, wherein the population of progeny plants is obtained from the seeds of the grafted plant in step (a).

[0107] 17. The method according to Embodiment 15, wherein the population of progeny plants is obtained from the clonal propagates of the grafted plants of step (a).

[0108] 18. The method according to Embodiments 15, 16, or 17, wherein in step (a), the plastid function is restored in the rootstock grafted onto the scion.

[0109] 19. The method according to any one of embodiments 15 to 18, wherein the rootstock grafted onto the scion in step (a) is obtained from a plant derived from a parent plant that has been selected for the useful traits described above and has had the expression of the MSH1 and DRM2 genes suppressed.

[0110] 20. The method according to any one of Embodiments 15 to 19, wherein the useful traits are selected from the group consisting of increased yield, delayed flowering, non-flowering, enhanced tolerance to biological stress, enhanced tolerance to abiotic stress, improved lodging resistance, increased growth rate, increased biomass, increased tillering, increased branching, delayed flowering time, delayed senescence, increased number of flowers, improved plant structure for high-density planting, improved photosynthesis, increased root mass, increased cell number, improved seedling vitality, improved seedling size, increased cell division rate, improved metabolic efficiency, and increased meristematic size.

[0111] 21. The method according to any one of Embodiments 15 to 20, wherein the scion contains one or more epigenetic changes in one or more nuclear chromosomes, and the epigenetic changes are not present in the nuclear chromosomes of the control plant or are not present in the nuclear chromosomes of the plant from which the scion was obtained.

[0112] 22. The method according to Embodiment 21, wherein the epigenetic changes are also present in the rootstock whose plastid function has been disrupted.

[0113] 23. The method according to Embodiment 21 or 22, wherein the epigenetic change is related to the improvement of the useful trait.

[0114] 24. The method according to any one of embodiments 15 to 23, wherein the rootstock comprises one or more epigenetic changes in one or more nuclear chromosomes that are not present in the nuclear chromosomes of the rootstock obtained from a plant whose plastid function has not been disrupted, or in the nuclear chromosomes of the parent plant.

[0115] 25. The method according to any one of embodiments 15 to 24, wherein the plant is selected from the group consisting of crop plants, trees, shrubs, or climbing plants.

[0116] 26. The method according to Embodiment 25, wherein the crop plant is selected from the group consisting of corn, rapeseed, soybean, cotton, canola, wheat, rice, tomato, tobacco, millet, potato, sugar beet, cassava, alfalfa, barley, oat, sugarcane, sunflower, strawberry, and sorghum.

[0117] The order of steps described herein is illustrative and not intended to limit the scope of the inventive concept. For example, a step interpreted as being performed "first" may be performed as the first, second, third, or other step in the process. Similarly, a step described as "following" another step should not be interpreted as indicating an order unless explicitly indicated otherwise.

[0118] While exemplary embodiments of the concept of the present invention have been described with reference to specific times, temperatures, materials, and / or other measurements, the concept of the present invention should not be limited to these specific times, temperatures, materials, and / or other measurements. [Examples]

[0119] The following examples are included to illustrate preferred embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques that the inventors have found to function well in carrying out the present invention and can therefore be considered to constitute preferred embodiments for its implementation. However, those skilled in the art should understand that many modifications can be made in light of this disclosure to the specific embodiments disclosed, and similar or equivalent results can still be obtained without departing from the spirit and scope of the present invention.

[0120] Example 1: Utilization of msh1 and drm2 loss-of-function mutations in Arabidopsis thaliana rootstock and their effects on grafted progeny. Plants: Experiments were conducted using the accession Col-0 of Arabidopsis thaliana, including the msh1 T-DNA insertion mutant line SAIL_877_F01 and the drm2-2 mutant (CS16386; ABRC Stock Center). Washed seeds were sown in peat moss mixed medium in square pots (3.5 inches x 3.5 inches), stratified at 4°C for 2 days, and then transferred to a growth chamber (Percival AR-66L3; 22°C during the day, 20°C at night, 12 hours of sunlight, light intensity 120-150 μmol m-2s-1).

[0121] Method: Wedge grafting was performed using the main inflorescence meristem. The graft union was sealed with stretched Parafilm to prevent drying and further secured with silicone tubing to maintain close contact between the scion and rootstock. The grafted plants were kept under a clear plastic bag, which served as a mist chamber, until the scion began to grow. The plants were then slowly acclimatized to normal growing conditions. Other flower stalks were removed to encourage the growth of the main grafted flower stalk. Each grafted scion was harvested separately to produce the first generation of progeny.

[0122] Using progeny of the first generation, the total leaf area was measured at various time points. For this purpose, a Canon EOS Rebel XSi camera was mounted on a copier (RS-CS920) for image capture. The captured images were then analyzed using ImageJ software (version 1.52a; https: / / imagej.nih.gov / ij / index.html) to extract the total leaf area. The average leaf area for each population was calculated and plotted using the ggplot2 package (version 3.3.3) in RStudio (version 1.1.423).

[0123] Grafting was performed using inflorescence stems of Arabidopsis thaliana, with Col-0 used as the scion and Col-0, msh1 (Col-0 background) or msh1, drm2 double mutant (Col-0 background) used as the rootstock. The experiment was conducted twice, using three Col-0, five msh1, and six msh1, drm2 rootstocks, respectively. Seeds were collected from each graft and sown 18 seeds per tray in culture medium, and cultivated under controlled growing conditions. Each experiment was conducted in a single growth chamber using three shelves, and Col-0 / Col-0 grafts were incorporated into each shelf to compensate for variations between chambers. The growth (rosette area) of the first generation of grafted progeny was monitored, and the results are shown in Figure 1.

[0124] In each experiment, five out of six progeny populations derived from Col-0 / msh1,drm2 outperformed the wild-type (Col-0 / Col-0) control graft in terms of plant growth rate. In contrast, in Experiment 1, only one out of five Col-0 / msh1 grafted progeny outperformed the wild type, and in Experiment 2, three out of five Col-0 / msh1 grafted progeny showed improved growth rate. This data suggests that incorporating the drm2 mutation along with msh1 into the rootstock improved the frequency and degree of growth enhancement in grafted progeny compared to the effect of msh1 alone.

[0125] Variations in growth rate were observed on different shelves within the chamber, suggesting variations in light conditions. The second experiment was terminated earlier than the first due to insect damage occurring by day 24. However, since Experiment 1 showed that the data from day 22 strongly predicted the final outcome, the variability observed in the data from Experiment 2 is considered significant. The experiment focused more on plant growth rate than on seed yield. Neither we nor other researchers believe that Arabidopsis thaliana seed yield is useful in predicting yield results in other crops (Van Daele et al. 2012).

[0126] These data suggest that incorporating the drm2 mutation into msh1 improves rootstock performance and increases the frequency of resulting progeny growth enhancement. Furthermore, these results suggest that dual mutant rootstocks may exhibit even greater progeny growth enhancement.

[0127] Example 2: Growth-promoting effect in subsequent generations To investigate the sustainability of the growth promotion, first-generation plants (Figure 1) were self-pollinated to produce second-generation plants, and then the second-generation plants were self-pollinated to produce third-generation plants. The total leaf area of ​​the second and third-generation plants was analyzed in the same manner as the first generation described in Example 1, and the results are shown in Figures 2 and 3, respectively.

[0128] In the second generation, eight of the twelve progeny populations derived from Col-0 / msh1,drm2 showed superior plant growth rate compared to the wild-type (Col-0 / Col-0) control graft. On the other hand, ten of the twelve Col-0 / msh1 grafted progeny populations showed superior performance compared to the wild-type control graft. In the third generation, seven of the twelve progeny populations derived from Col-0 / msh1,drm2, and three of the twelve progeny populations derived from Col-0 / msh1 showed superior performance compared to the wild-type control graft.

[0129] These data suggest that the growth-enhancing effects observed in the first generation of Col-0 / msh1,drm2 and Col-0 / msh1 are inheritable up to at least the third generation. These results also suggest that the frequency of growth-enhancing effects may increase in the third generation of the double mutant rootstock.

[0130] Example 3: Creation of Brassica napus msh1, drm2 rootstocks To incorporate msh1 and drm2 rootstock grafts into crops, target mutations were induced in MSH1 and DRM2 in rapeseed (cv R016) using CRISPR-Cas9 technology. For this purpose, one target sequence for MSH1 (GCAGGCCACTGCACGTAGA, SEQ ID NO: 22) and two target sequences for DRM2 (Target 1: CGGTGGAACGTCGTACAAG, SEQ ID NO: 23; Target 2: ACAGAACGTTGATACCATA, SEQ ID NO: 24) were cloned into a pHSE401 vector (Xing et al 2014) containing gRNA and Cas9. The CRISPR constructs were introduced into rapeseed (cv R016) using the transformation protocol described by Cardoza et al. (2006). We successfully generated msh1 mutant plants exhibiting variegation and growth phenotypic diversity (Figure 4) and drm2 mutant plants exhibiting a growth-delay phenotype (Figure 5). To produce msh1,drm2 rootstocks for grafting, msh1 mutant plants and drm2 mutant plants were crossed, and msh1,drm2 double mutants were selected in the F2 generation. Subsequently, wild-type (WT) scions were grafted onto the Msh1,drm2 rootstocks as described in Example 1, and the progeny were field-tested to confirm improved yield.

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Claims

1. A grafted plant that includes a scion grafted onto a rootstock, (i) The scion is of wild type plant origin, (ii) In the aforementioned rootstock, the expression of the MSH1 and DRM2 genes is suppressed. (iii) The rootstock provides an improvement in yield or growth rate in the progeny of the grafted plant compared to a control plant, wherein the control plant comprises any of the following: (a) the progeny of a scion grafted onto a rootstock in which the expression of the MSH1 and DRM2 genes has not been suppressed; (b) a perfect plant in which no root grafting has been performed and the expression of the MSH1 and DRM2 genes has not been suppressed; (c) a wild-type plant; or (d) the progeny of a plant having the same genetic background as the plant source of the scion of the grafted plant.

2. The grafted plant according to claim 1, wherein the rootstock provides the grafted plant with an improvement in yield or growth rate compared to a control plant.

3. The grafted plant according to claim 1, wherein the scion contains one or more epigenetic changes in one or more nuclear chromosomes, and the epigenetic changes are not present in the nuclear chromosomes of the control plant or are not present in the nuclear chromosomes of the plant from which the scion was obtained.

4. The grafted plant according to claim 3, wherein the aforementioned epigenetic changes (or multiple changes) are also present in the rootstock.

5. The grafted plant according to claim 3, wherein the epigenetic changes are related to the improvement in the useful trait.

6. The grafted plant according to claim 3, wherein the rootstock contains one or more epigenetic changes in one or more nuclear chromosomes that are not present in the nuclear chromosomes of the rootstock obtained from a plant in which the expression of MSH1 and DRM2 genes was not suppressed, or in the nuclear chromosomes of the parent plant.

7. The grafted plant according to claim 1, wherein the expression of the MSH1 and DRM2 genes in the rootstock is suppressed by loss-of-function mutations in the rootstock's endogenous MSH1 and / or DRM2 genes, and / or by small inhibitory RNA (siRNA), microRNA (miRNA), co-repression sense RNA, and / or by antisense RNA complementary to the endogenous MSH1 and / or DRM2 gene promoter, 5' or 3' untranslated region, intron, coding region, and / or any combination thereof, and optionally the grafted plant is a soybean, rapeseed, tomato, sorghum, rice, or maize plant, and the endogenous MSH1 and DRM2 genes (or more) encode the cDNA(or more) listed in Table 1.

8. The grafted plant according to claim 7, wherein loss-of-function mutations in endogenous MSH1 and DRM2 genes (or more) suppress the expression of the MSH1 and DRM2 genes in the rootstock, and optionally the grafted plant is a soybean, rapeseed, tomato, sorghum, rice, or maize plant, and the MSH1 and DRM2 genes (or more) encode cDNA (or more) listed in Table 1.

9. The grafted plant according to claim 7, wherein the expression of the MSH1 and / or DRM2 genes in the rootstock is suppressed by small inhibitory RNA (siRNA), microRNA (miRNA), co-repression sense RNA, and / or by antisense RNA complementary to the endogenous MSH1 and / or DRM2 gene promoter, 5' or 3' untranslated region, intron, coding region, and / or any combination thereof, and optionally the grafted plant is a soybean, rapeseed, tomato, sorghum, rice, or maize plant, and the MSH1 and / or DRM2 genes encode the cDNA(s) listed in Table 1.

10. The grafted plant according to claim 1, wherein the plant is selected from the group consisting of crop plants, trees, shrubs, turfgrasses, pasture grasses, and climbing plants.

11. The grafted plant according to claim 10, wherein the crop plant is selected from the group consisting of corn, soybeans, cotton, canola, wheat, rice, tomato, tobacco, millet, potato, sugar beet, cassava, alfalfa, barley, oats, sugarcane, sunflower, strawberry, and sorghum.

12. (a) A step of obtaining a population of progeny from a grafted plant according to claim 1, wherein the population of progeny is a first-generation, second-generation, or third-generation progeny obtained by self-propagating the grafted plant or by self-propagating the first-generation or second-generation progeny; (b) A step of screening a population of progeny plants that have improved yield or growth rate compared to a control plant, (c) Progeny plants produced by a method comprising the step of selecting progeny plants from the population for improvement in yield or growth rate compared to a control plant, wherein the progeny plants exhibit improvement in yield or growth rate and exhibit a nuclear chromosome DNA methylation pattern different from that of the control plant, and the control plant is cultivated under the same environmental conditions as the selected progeny plants and includes any of the following: (i) progeny of scions grafted onto rootstock in which the expression of the MSH1 and DRM2 genes has not been suppressed; (ii) perfect plants in which no root grafting has been performed and the expression of the MSH1 gene has not been suppressed; (iii) wild-type plants; or (iv) progeny of plants having the same genetic background as the plant source of the scion of the grafted plant.

13. (a) A step of obtaining a population of progeny from a grafted plant according to claim 1, wherein the population of progeny is a first-generation, second-generation, or third-generation progeny obtained by self-propagating the grafted plant or by self-propagating the first-generation or second-generation progeny; (b) A step of screening the progeny population for improved yield or growth rate compared to the control plant population, (c) A selected population of progeny plants produced by a method comprising the step of selecting a population of progeny plants for improvement in yield or growth rate compared to a control plant, wherein the selected population of progeny plants exhibits improvement in yield or growth rate and exhibits a nuclear chromosome DNA methylation pattern different from that of the control plant, the control plant is cultivated under the same environmental conditions as the selected population of progeny plants, and the selected population of progeny plants includes any of the following: (i) progeny of scions grafted onto rootstock in which the expression of MSH1 and DRM2 genes has not been suppressed; (ii) perfect plants in which no root grafting has been performed and the expression of MSH1 and DRM2 genes has not been suppressed; (iii) wild-type plants; or (iv) progeny of plants having the same genetic background as the plant source of the scion of the grafted plant.

14. The method according to claim 13, wherein loss-of-function mutations in the endogenous MSH1 and DRM2 genes (or more) of the rootstock suppress the expression of the MSH1 and DRM2 genes in the rootstock of the grafted plant, and optionally the grafted plant is a soybean, rapeseed, tomato, sorghum, rice, or maize plant, and the MSH1 and DRM2 genes (or more) encode the cDNA (or more) listed in Table 1.

15. A method for producing plants that exhibit useful traits, (a) A step of obtaining a population of progeny from a grafted plant including a scion grafted onto a rootstock, wherein the rootstock is obtained from a plant or its parent plant in which the expression of the MSH1 and DRM2 genes has been suppressed, (b) A method comprising the step of selecting one or more progeny plants from the population, wherein the selected progeny plants show improvement in the useful traits compared to a control plant, thereby producing plants that exhibit the useful traits.

16. The method according to claim 15, wherein the population of progeny plants is obtained from the seeds of the grafted plant in step (a).

17. The method according to claim 15, wherein the population of progeny plants is obtained from clonal propagates of the grafted plants of step (a).

18. The method according to claim 15, wherein in step (a), the plastid function is restored in the rootstock grafted onto the scion.

19. The method according to claim 15, wherein the rootstock grafted onto the scion in step (a) is obtained from a plant derived from a parent plant that has been selected for the aforementioned useful traits and has had the expression of the MSH1 and DRM2 genes suppressed.

20. The method according to claim 15, wherein the useful traits are selected from the group consisting of increased yield, delayed flowering, non-flowering, enhanced tolerance to biological stress, enhanced tolerance to abiotic stress, improved lodging resistance, increased growth rate, increased biomass, increased tillering, increased branching, delayed flowering time, delayed senescence, increased number of flowers, improved plant structure for high-density planting, improved photosynthesis, increased root mass, increased cell number, improved seedling vitality, improved seedling size, increased cell division rate, improved metabolic efficiency, and increased meristematic size.

21. The method according to claim 20, wherein the scion contains one or more epigenetic changes in one or more nuclear chromosomes, and the epigenetic changes are not present in the nuclear chromosomes of the control plant or are not present in the nuclear chromosomes of the plant from which the scion was obtained.

22. The method according to claim 21, wherein the epigenetic changes are also present in the rootstock whose plastid function has been disrupted.

23. The method according to claim 21, wherein the epigenetic change is related to the improvement of the useful trait.

24. The method according to claim 21, wherein the rootstock comprises one or more epigenetic changes in one or more nuclear chromosomes that are not present in the nuclear chromosomes of the rootstock obtained from a plant whose plastid function has not been disrupted, or in the nuclear chromosomes of the parent plant.

25. The method according to claim 15, wherein the plant is selected from the group consisting of crop plants, trees, shrubs, or climbing plants.

26. The method according to claim 25, wherein the crop plant is selected from the group consisting of corn, rapeseed, soybean, cotton, canola, wheat, rice, tomato, tobacco, millet, potato, sugar beet, cassava, alfalfa, barley, oat, sugarcane, sunflower, strawberry, and sorghum.

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