Genetic transformation and genome engineering in legume species

Abstract

Methods and materials for generating leguminous plant tissue having one or more genetic modifications of interest are provided herein. For example, methods and materials that use developmental regulators (DRs) to improve the transformation process and efficiency in legumes are provided herein.

Description

[0001] Attorney Docket No. 09531-0530W01 2023-264

[0002] GENETIC TRANSFORMATION AND GENOME ENGINEERING

[0003] IN LEGUME SPECIES

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority from U.S. Provisional Application Serial No. 63 / 715,208, filed November 1, 2024. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

[0006] STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0007] This invention was made with government support under 2021-67013-34565 awarded by the National Institute of Food and Agriculture, and IOS2206920 awarded by the National Science Foundation. The government has certain rights in the invention.

[0008] TECHNICAL FIELD

[0009] This document relates to methods and materials for inducing genetic alterations in leguminous plants. For example, this document provides methods and materials that include using developmental regulators to improve transformation efficiency in legumes.

[0010] SEQUENCE LISTING

[0011] This application contains a Sequence Listing that has been submitted electronically as an XML file named “09531-0530W01_ST26.XML.” The XML file, created on October 31, 2025, is 81,894 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0012] BACKGROUND

[0013] Legumes are a large, diverse family of nitrogen fixing plants that include, for example, common bean, chickpea, pea, peanut, cowpea, pigeon pea, peanut ground nut, and many soybean varieties. Crop legumes are grown agriculturally, primarily for human consumption, livestock forage, and soil-enhancing green manure. Extensive efforts have been made to improve agronomically important traits in crop legumes through traditional breeding. Traditional breeding has certain limitations, but these limitations can be overcome Attorney Docket No. 09531-0530W01 2023-264 by genetic engineering and the use of advanced biotechnological tools. Genetic engineering requires efficient tissue culture and genetic transformation. Many legume crops, however, are recalcitrant to genetic transformation. Challenges facing current crop legume plant transformation and regeneration methods include the following: (1) explants vary among legume crops (e.g., cotyledon, shoot meristem, callus, embryonic axis) for efficient regeneration and transformation; (2) most soybean varieties and legume species exhibit poor regeneration and transformation efficiency, and in a genotype-dependent manner that requires prolonged tissue culture (with repeated subculture) timelines; and (3) every legume crop requires a different optimized plant growth medium and various growth regulators. The lack of efficient plant transformation methods has been a major limitation in applying biotechnology tools toward trait development in many crop legume species.

[0014] SUMMARY

[0015] This document provides methods and materials that can be used for robust, efficient, genotype-independent, and expedited genetic transformation and genome engineering in leguminous plants. The methods provided herein do not require time-consuming callus induction, shoot regeneration, or tissue culture selection steps. In general, the methods and materials provided herein can be used to directly induce shoot formation through the expression of developmental regulators (DRs) that promote axillary meristem initiation. This document also provides methods and materials that can be used for producing genetically modified and gene edited plants. The methods provided herein can include transforming germinated seeds of leguminous plants with nucleic acid encoding one or more DR polypeptides (e.g., WUS, IPT, STM, etc.) to induce shoot formation, and in some cases, generating genetically modified and gene edited leguminous plants without tissue culture or the use of plant hormones. The methods and materials provided herein can enable genotypeflexible genetic transformation and genome engineering in leguminous plants.

[0016] As demonstrated herein, T-DNA vectors containing sequences encoding WUS and IPT, as well as a reporter gene, can be transformed into Agrobacterium and transformed into germinating soybean embryos to yield plantlets having transgenic shoots. The transformation efficiency achieved using the methods described herein can be greater than 20%. In addition, co-delivery of a T-DNA encoding a CRISPR-Cas cassette to soybean germinating embryos Attorney Docket No. 09531-0530W01 2023-264 can yield CRISPR-mediated gene editing events. Thus, as demonstrated herein, DR-mediated transformation methods can effectively deliver gene editing reagents and achieve efficient gene editing in soybean.

[0017] In a first aspect, this document features a method for generating plant cells having one or more genetic modifications of interest. The method can include, or consist essentially of, (a) introducing, into a germinating embryo of a leguminous plant: (i) nucleic acid encoding one or more developmental regulators that, when expressed in cells of the germinating embryo, induce new meristem formation, and (ii) nucleic acid comprising one or more coding sequences that, when expressed in cells of the new meristem, edit DNA in cells of the new meristem to introduce one or more genetic modifications of interest, where the introducing includes infection of the germinating embryo with an Agrobacterium,' (b) incubating the germinating embryo with the Agrobacterium, such that the one or more developmental regulators and the one or more coding sequences within the nucleic acid of (ii) are expressed; (c) growing a plantlet developed from the germinating embryo of step (b) in a growth medium to induce shoot formation; and (d) identifying one or more shoots having the one or more genetic modifications of interest. The introducing can include incubating the germinating embryo with the Agrobacterium for about 3 days. The incubating can include incubating the germinating embryo with the Agrobacterium for about I day to 5 days. The growing can include growing the plantlets in a growth medium for about 1 week to about 8 weeks. The growing can include growing the plantlets in a growth medium for about 4 weeks. The method can further include, or consist of, introducing, into the germinating embryo, (iii) nucleic acid encoding a reporter. The reporter can include a RUBY polypeptide. The method can further include identifying plantlets in which the reporter is expressed. The growth medium can be a plant hormone-free growth medium. The one or more developmental regulators can include Wushel (WUS) and isopentenyl transferase (IPT). The one or more developmental regulators can further include one or more of Baby Boom, Irrepressible Variants of Monopteros, Shoot Meristemless, Leafy Cotyledon, WUS homeobox-containing, and APETALA2 / Ethylene Responsive Factor. The Agrobacterium can include Agrobacterium tumefaciens strain AGL-1, A. tumefaciens strain C58, A. tumefaciens strain GV3101, A. tumefaciens strain LBA4404, A. tumefaciens strain EHA105, or A. rhizogenes. The Agrobacterium can be A. tumefaciens strain AGL-1. The one or more Attorney Docket No. 09531-0530W01

[0018] 2023-264 sequences that, when expressed, edit the DNA in cells of the new meristem, can include a nucleotide sequence encoding a targeted endonuclease, and wherein the targeted endonuclease can include a Clustered Regularly-Interspaced Short Palindromic Repeats- associated nuclease, a Transcription Activator-Like Effector Nuclease, a meganuclease, or a zinc finger nuclease. The targeted endonuclease can be a Clustered Regularly -Interspaced Short Palindromic Repeats-associated (Cas) nuclease. The one or more sequences that, when expressed, edit the DNA in cells of the new meristem can include (1) a nucleotide sequence encoding a targeted endonuclease and (2) a repair template. The shoots can be meristematic and can be capable of deriving new plant tissue carrying the one or more genetic modifications of interest. The method can further include (e) culturing the shoots identified in step (d) in soil for about 5 days to 20 days to obtain modified plant tissue including the one or more genetic modifications of interest. The leguminous plant can be selected from the group including soybean, kidney bean, black bean, pinto bean, peanut, chickpea, lentil, pea, common bean, cowpea, pigeon pea, peanut ground nut, and faba bean. The leguminous plant can be soybean.

[0019] In another aspect, this document features a method for increasing transformation efficiency in a leguminous plant. The method can include, or consist essentially of, (a) introducing, into cells of a germinating embryo of a leguminous plant, (i) nucleic acid encoding one or more developmental regulators that, when expressed in cells of the germinating embryo, induce new meristem formation, wherein the introducing can include infection of the germinating embryo with an Agrobacterium., and (b) co-cultivating the germinating embryo anA Agrobacterium such that the one or more developmental regulators can be expressed. The method can have a transformation efficiency of about 15% to about 40%.

[0020] In another aspect, this document features a method for accelerating generation of a transgenic leguminous plant. The method can include, or consist essentially of, (a) introducing, into a germinating embryo of a leguminous plant, (i) nucleic acid encoding one or more developmental regulators that, when expressed in cells of the germinating embryo, induce new meristem formation, and (ii) nucleic acid comprising a transgene, wherein the introducing comprises infection of the germinating embryo with an Agrobacterium: (b) cocultivating the germinating embryo and the Agrobacterium such that the one or more Attorney Docket No. 09531-0530W01

[0021] 2023-264 developmental regulators can be expressed; and (c) growing a plantlet developed from the germinating embryo of step (b) in a growth medium to induce shoot formation, wherein at least one shoot can include genomic DNA that contains the transgene. The method can further include co-cultivating the germinating embryo and the Agrobacterhrm for about 3 days. The method can further include growing the plantlet in the growth medium for about 4 weeks. The growth medium can be a plant hormone-free growth medium. The developmental regulators can include one or more of WUS, IPT, Baby Boom, Irrepressible Variants of Monopteros, Shoot Meristemless, Leafy Cotyledon, WUS homeobox-containing, and APETALA2 / Ethylene Responsive Factor.

[0022] In another aspect, this document features a germinating leguminous plant embryo containing (i) exogenous nucleic acid encoding one or more developmental regulators that, when expressed in cells of the germinating embryo, induce new meristem formation, and (ii) exogenous nucleic acid comprising one or more coding sequences that, when expressed in cells of the new meristem, edit DNA in cells of the new meristem to introduce one or more genetic modifications of interest. The leguminous plant can be selected from the group comprising soybean, kidney bean, black bean, pinto bean, peanut, chickpea, lentil, pea, common bean, cowpea, pigeon pea, peanut ground nut, and faba bean. The germinating embryo can further contain (iii) exogenous nucleic acid encoding a reporter. The reporter can be a RUBY polypeptide. The germinating leguminous plant embryo can be in a plant hormone-free growth medium. The one or more developmental regulators can include WUS and IPT. The one or more developmental regulators can further include one or more of Baby Boom, Irrepressible Variants of Monopteros, Shoot Meristemless, Leafy Cotyledon, WUS homeobox-containing, and APETALA2 / Ethylene Responsive Factor. The one or more sequences that, when expressed, edit the DNA in cells of the new meristem, can include a nucleotide sequence encoding a targeted endonuclease, and the targeted endonuclease can be a Clustered Regularly-Interspaced Short Palindromic Repeats-associated nuclease, a Transcription Activator-Like Effector Nuclease, a meganuclease, or a zinc finger nuclease. The targeted endonuclease can be a Cas nuclease. The one or more sequences that, when expressed, edit the DNA in cells of the new meristem can include (1) a nucleotide sequence encoding a targeted endonuclease and (2) a repair template. Attorney Docket No. 09531-0530W01

[0023] 2023-264

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0025] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0026] DESCRIPTION OF DRAWINGS

[0027] FIG. 1 includes maps illustrating T-DNA constructs encoding WUS and IPT DRs. The WUS and IPT coding sequences were included between promoter and terminator sequences, and a RUBY reporter coding sequence also was included between the T-DNA border sequences.

[0028] FIG. 2 shows the steps of a representative method used for DR-mediated in-planta soybean transformation method. Soybean seeds were imbibed for 24 hours to initiate germination, and germinating embryos were carefully isolated from the imbibed seeds. The isolated embryos were sonicated twice for 30 seconds each to facilitate transformation. Following sonication, the embryos were infected with Agrobacterium containing T-DNA constructs encoding DRs for 30 minutes. The infected embryos then underwent a cocultivation period with the Agrobacterium for 3 days. Soybean plantlets were grown on a hormone-free growth medium for approximately 4 weeks, during which time multiple shoots regenerated due to the action of the DRs, and transgenic shoots were identified by their ruby color resulting from expression of the RUBY reporter. Transformed soybean shoots were then transferred to soil and acclimated for 10 days. Finally, the transformed soybean plants were grown to maturity over a period of 1-2 months.

[0029] FIGS. 3A-3D show the characterization of transformed soybean TO plants. FIG. 3A is a representative image showing a mature TO transgenic soybean plant grown in a Attorney Docket No. 09531-0530W01 2023-264 greenhouse. FIG. 3B is a representative image showing a close-up view of soybean leaves from TO transgenic plants, with ruby coloration indicating expression of the RUBY reporter gene. FIG. 3C is a representative image showing RUBY-colored flowers and pods from TO plants, indicated by the arrows. FIG. 3D is a representative image showing PCR results confirming of TO transgenic plants. M: DNA marker; WT: wild-type; N: negative control; P: T-DNA plasmid control. Upper band (-500 bp): Bar gene (herbicide resistance marker); lower band (-300 bp): endogenous Chllla gene (internal control).

[0030] FIG. 4 shows Sanger sequencing results confirming CRISPR-mediated mutations obtained via in-planta soybean transformation as described herein. CRISPR-induced gene editing events were identified in the trypsin inhibitor and P34 allergen genes of transformed soybean plants. The 20-bp CRISPR target sequences are indicated by underlining, and the 3- bp PAM (Protospacer Adjacent Motif) sequences are boxed. Mutations were identified based on Sanger sequencing results, which revealed either double peaks or deletions when compared to the wild-type (WT) sequences. WT trypsin inhibitor, SEQ ID NO: 1; mutated trypsin inhibitor, SEQ ID NO:2; WT P34 allergen, SEQ ID NO:3; mutated P34 allergen, SEQ ID NO:4.

[0031] FIGS. 5A-5C show nucleotide sequences for DRs cloned into T-DNA constructs. For Nos-GmWUS-OsT (FIG. 5A; SEQ ID NO: 17), lower case italicized indicates the Nos promoter sequence, upper case bold indicates the GmWUS sequence, and upper case underlined indicates the OCS terminator sequence. For CaMV35S-IPT-OcsT (FIG. 5B; SEQ ID NO:18), lower case italicized indicates the CaMV35S promoter sequence, upper case bold indicates the IPT sequence, and upper case underlined indicates the OCS terminator sequence. For CaMV35S-GmGRF4-GmGIFl-OcsT (FIG. 5C; SEQ ID NO: 19), lower case italicized indicates the CaMV35S promoter sequence, upper case bold indicates the GmGRF4 sequence, upper case italicized indicates the GIF sequence, and upper case underlined indicates the OCS terminator sequence.

[0032] FIGS. 6A-6B show comparisons of timelines between two widely used soybean transformation methods requiring extensive tissue culture and the DR-mediated approach described herein. FIG. 6A illustrates the timelines for methods compiled from studies described elsewhere (Paz et al., Plant Cell Rep., 25:206-213, 2006; Liu et al., Methods Mol. Biol., 1917:217-234, 2019; Paes de Melo et al., Front. Plant Set., doi: Attorney Docket No. 09531-0530W01 2023-264

[0033] 10.3389 / fpls.2020.01228, 2020; and Liang et al., Methods Mol. Biol., 2653:39-52, 2023) and from the studies described herein. The average total time required for each method is shown at the top. The Embryonic Axis method takes an average of 118 days, and the half cotyledon takes an average of 173 days, in contrast to the 70 day average for the methods provided herein. FIG. 6B includes images illustrating the trimming practice used in the DR-mediated transformation methods provided herein. The apical primary stem was trimmed after 2 weeks of growth on GM I medium to promote the emergence of RUBY-positive transformed shoots, which typically appeared within 4 weeks. Scale bar: 1 cm.

[0034] FIGS. 7A-7C show characterization of transgenic and gene-edited soybean plants. FIG. 7A includes representative images from phenotypic analysis of TO plants, showing transgenic versus non-transgenic tissues: independent transgenic shoots with varied ruby coloration (i-ii), flowers (iii), seed pods (iv), and seeds (v). A chimeric plant displaying both transgenic (left arrow) and non-transgenic (right arrow) shoots is shown in image (vi). Scale bar: 1 cm (i- v); 10 cm (vi). FIG. 7B shows the results of PCR genotyping of TO and T1 plants. Transgene integration was confirmed in nine TO plants (top) using T-DNA primers (Bar) with Chllla gene primers as an internal control, and in ten T1 progeny (bottom) using RUBY primers with the P34 gene primers as an internal control. M: 1 kb plus DNA ladder; N: no-DNA control; P: plasmid control; WT: non-transgenic wild type control; T0 / T1: individual plant samples. FIG. 7C shows Sanger sequencing results for the P3- / target site in plant T0-5 (SEQ ID NO: 16), and in two T0-5 progeny (Tl -5-5 and Tl-5-6) that exhibited heterozygous (Het) mutations with double peaks in sequencing chromatograms. The P34 target sequence at the top included a 20-bp CRISPR site (black bar), a 3 -bp PAM (white bar) and a 4-bp deletion region (boxed) found in T1 mutants.

[0035] FIGS. 8A-8B show construct maps for and results of DR-enabled soybean transformation. FIG. 8A is a schematic showing T-DNA construct maps including four modular components: a Cas9 coding sequence, a CRISPR gRNA coding sequence, a RUBY coding sequence, and DR cassettes. The DR components included GmWUS2, IPT, or GmGRF4-GIFl, as indicated. Black bars at the ends of each construct map indicate T-DNA borders. Arrowheads indicate the locations of transgene detection primers. FIG. 8B includes images showing the transformation workflow from germinating embryo isolation to transgenic / edited plant generation, with transformed plantlets displaying ruby-colored shoots Attorney Docket No. 09531-0530W01

[0036] 2023-264

[0037] (arrow). CC medium: Co-cultivation medium; GM I: Growth Medium I; GM II: Growth Medium II. The durations for each step are indicated. Scale bar: 1 cm.

[0038] FIGS. 9A-9B show multiple sequence alignments for DRs from several species. FIG. 9A shows a sequence alignment for WUS2 polypeptides from Arabidopsis thaliana (SEQ ID NO:20), Glycine max (SEQ ID NO: 13), and Zea mays (SEQ ID NO:21). FIG. 9B shows a sequence alignment for GRF4 polypeptides from thaliana (SEQ ID NO:22), Triticum aestivum, (SEQ ID NO:23), and G. max (SEQ ID NO:24).

[0039] FIGS. 10A-10B show PCT characterization of transgenic soybean plants. FIG. 10A is an image of a gel showing PCR genotyping of TO plants. Transgene integration of DRs was confirmed in nine TO plants using WUS and IPT primers. FIG. 10B is an image of a gel showing T-DNA segregation in ten T1 progeny using RUBY primers with P34 gene primers as internal control. M: 1 kb plus DNA ladder; N: no-DNA control; P: plasmid control; WT: non-transgenic wild type control; T0 / T1 : individual plant samples.

[0040] FIGS. 11A-11B show characterization of CRISPR-mediated gene editing in TO and T1 plants. Next-generation sequencing analysis was used to determine mutation rates at the P34 gene in nine TO plants (FIG. 11A) and two T1 plants (FIG. 11B). Pie charts generated by CRISPResso2 show the proportion of wild-type (unmodified) and edited (modified) sequences for each plant. FIG. 11B also includes sequence alignments of the T0-5 line and its T1 progenies with mutations: the target site is indicated in bold, the PAM sequence is underlined, and the deletion region is indicated.

[0041] FIG. 12 includes graphs plotting the results of analysis of IPT and WUS2 transgene expression. Expression levels of IPT (top) and WUS2 (Glyma.OlGl 66800,' bottom) were measured in individual samples at 3 and 6 days after transformation (DAT). The y-axis shows the number of sequencing reads (counts) from each RNA-seq dataset. Three biological replicates were analyzed for each transformation treatment.

[0042] FIGS. 13A-13D show the results of principal component analysis (PCA) analysis and hierarchical clustering of an RNA-seq dataset. FIG. 13A is a PCA plot of all 3 DAT and 6 DAT samples. As shown in the PCA plot of FIG. 13B, the 3 DAT samples showed clustering based on treatment, whereas at 6 DAT, IPT and WUS / IPT samples were clustered together distinct from EV and WUS (FIG. 13C). FIG. 13D is a hierarchical clustering and heat map of all RNA-seq samples based on gene expression similarity between samples. Attorney Docket No. 09531-0530W01

[0043] 2023-264

[0044] FIGS. 14A-14D show transcriptional dynamics of DR-transformed soybean shoots. FIG. 14A is a K-means clustering analysis heat map at 3 days after transformation (DAT), and FIG. 14B is a K-means clustering analysis heat map at 6 DAT, both with enriched GO terms and genes highlighted per cluster. FIG. 14C is a diagram showing gene pathway enrichment analysis of differentially expressed genes involved in plant hormone pathways. FIG. 14D is a schematic model showing the synergistic effects of WUS2 and IPT on de novo shoot formation through the inhibition of biotic / abiotic stress responses and promotion of growth hormone and stem cell activation pathways.

[0045] FIG. 15A illustrates the effects of DRs on biological processes through transformation, showing upregulated and downregulated genes. FIG. 15B is a PCA plot of all samples showing genes associated with key plant growth hormones. The distribution of samples appeared to be mainly based on the treatment effect. Sample distribution and scattering also suggested stronger effects of WUS and WUS / IPT on cytokinin and auxin pathways.

[0046] FIGS. 16A-16C show DR-mediated transgenic shoot formation in common bean (variety UI111). FIG. 16A shows a representative image of a TO transgenic common bean shoot (indicated by the arrow) grown in soil. The transgenic shoot exhibits green fluorescence under ultraviolet (UV) illumination. FIG. 16B shows a close-up view of leaves from TO wild type or transgenic plants displaying fluorescent signals, indicating expression of the green fluorescent reporter gene in the transgenic leaves, in contrast to non-fluorescent wild-type leaves. FIG. 16C shows the result of a PCR analysis that confirmed the presence of the transgene in TO plants. M, DNA marker; WT, wild type; N, negative control; LI and L2, leaf samples from independent transgenic lines showing positive PCR amplification (-500 bp) using primers specific to the T-DNA construct.

[0047] FIGS. 17A-17I show nucleic acid and amino acid sequences for Cas endonucleases. FIG. 17A: Streptococcus pyogenes Cas9 amino acid sequence (SEQ ID NO:36). FIG. 17B: S. pyogenes Cas9 coding sequence (SEQ ID NO:37). FIG 17C: Cas l amino acid sequence (SEQ ID NO:38). FIG. 17D: Cas l coding sequence (SEQ ID NO:39). FIG. 17E: Casl2f amino acid sequence (SEQ ID NO:40) and coding sequence (SEQ ID NO:41). FIG. 17F: Cas 2 amino acid sequence (SEQ ID NO:42). FIG. 17G: Casd>2 coding sequence (SEQ ID Attorney Docket No. 09531-0530W01 2023-264

[0048] NO:43). FIG. 17H CasO>3 amino acid sequence (SEQ ID NO:44). FIG. 171 Casd»3 coding sequence (SEQ ID NO:45).

[0049] FIG. 18 shows amino acid sequences for TnpB IsDra2 (SEQ ID NO:46) and TnpB IsYmul (SEQ ID NO:47).

[0050] FIG. 19 shows a nucleotide sequence (SEQ ID NO:9) encoding a RUBY reporter polypeptide.

[0051] FIG. 20 shows a nucleotide sequence (SEQ ID NO: 10) for a CRISPR-Cas9 cassette.

[0052] FIG. 21 shows a nucleotide sequence (SEQ ID NO:48) encoding an AmCyan reporter.

[0053] DETAILED DESCRIPTION

[0054] Provided herein are methods and materials that can be used to generate leguminous plant tissue having one or more genetic modifications of interest. For example, this document provides methods that can include introducing nucleic acids encoding one or more DRs into germinating embryos of leguminous plants, such that the DRs, when expressed, will induce meristem formation. The methods also can include introducing nucleic acids containing one or more nucleotide sequences that, when expressed, can edit the DNA of the germinating embryo to introduce one or more genetic modifications of interest. The methods and materials provided herein also can improve transformation efficiency in leguminous plants and accelerate the generation of transgenic leguminous plants.

[0055] The present disclosure provides methods for robust, efficient, and genotype-flexible genetic transformation of soybean and other legume crops, without relying on the use of plant growth hormones. The method described herein utilize DRs (e.g., WUS and IPT) to enhance transformation efficiency by promoting de novo in-planta shoot formation, with a transformation efficiency that can be greater than 20%. The methods described herein offer major advantages in genetic engineering and crop improvement efforts in legumes. By eliminating the need for plant growth hormones and utilizing DRs (e.g., WUS and IPT), the methods described herein overcome significant limitations of conventional transformation protocols. The methods described herein are applicable to a broad range of legume species, bypassing time-consuming plant-hormone based tissue culture techniques, and reducing dependency on genotyping. Genetically modified plants (e.g., legumes) can be produced Attorney Docket No. 09531-0530W01 2023-264 using the methods and materials described herein within 2-3 months, which is significantly faster than using conventional transformation techniques.

[0056] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0057] As used herein, the term “about,” when used herein in reference to a value, refers to a value that is ± 10% of the referenced value.

[0058] As used herein, a “developmental regulator” (DR) is an agent (e.g., a transcription factor, an enzyme, or a hormone) that can direct or influence plant development, and may guide the differentiation of plant cells, organs, or tissues. Non-limiting examples of developmental regulators that can be used in the methods provided herein include WUS, IPT, Baby Boom, Irrepressible Variants of Monopteros, Shoot Meristemless (STM), Leafy Cotyledons, WUS homeobox-containing, APETALA2 / Ethylene Responsive Factor, Wound Induced Dedifferentiation 1, Growth-regulating Factors (GRFs), GRF-interacting Factor (GIF), or any combination thereof. In some cases, the DRs used in the methods provided herein can be transcription factors (e.g., WUS, Baby Boom, Irrepressible Variants of Monopteros, or Shoot Meristemless) that can stimulate plant hormone biosynthesis or plant susceptibility to / sensing of hormones that affect plant development. In some cases, the DRs used in the methods provided herein can be enzymes (e.g., IPT) that lead to increased levels of plant hormones. IPT is in the cytokinin biosynthesis pathway. A DR can increase one or more cytokinins through ectopic application or through endogenous biogenesis, such as by increasing the expression of one or more enzymes involved in the synthesis of plant hormones. Other examples of enzymes that can lead to increased cytokinin levels and may be useful as DRs include, without limitation, tRNA-isopentenyl transferase, cytochrome P450 monooxygenase, LONELY GUY, adenosine kinase, and adenine phosphoribosyl transferase. In some cases, the DRs used in the methods provided herein can be combinations of transcription factors (e.g., WUS) and enzymes (e.g., IPT). In some cases, the DRs used in the methods provided herein further include any DR(s) described herein in addition to the combination of transcription factors (e.g., WUS) and enzymes (e.g., IPT).

[0059] In some cases, a nucleic acid encoding a DR (e.g., WUS or IPT) can be delivered to a germinating embryo in order to increase the level of the encoded DRs (e.g., WUS or IPT) in the germinating embryo. In some embodiments, a nucleic acid encoding one or more copies Attorney Docket No. 09531-0530W01

[0060] 2023-264

[0061] (e.g., one, two, three, four, or five copies) of WUS and a nucleic acid encoding one or more copies (e.g., one, two, three, four, or five copies) of IPT can be incorporated in a single DNA vector (e.g., T-DNA) for delivery into a germinating embryo. In some embodiments, a nucleic acid encoding one or more copies (e.g., one, two, three, four, or five copies) of WUS and a nucleic acid encoding one or more copies (e.g., one, two, three, four, or five copies) of IPT can be incorporated into separate DNA vectors (e.g., T-DNAs) for delivery into a germinating embryo.

[0062] The DR coding sequence(s) (e.g., WUS and / or IPT) can be operably linked to a promoter (e.g., NOS, 35 S, CmYLCV, AtUBQlO, AtUBQl, GmUBI, or any other suitable promoter) that can drive DR expression in plant cells. In some cases, expression of a DR (e.g., a DR that is a transcription factor) can lead to increased expression of genes downstream of the DR. In some embodiments, sequences encoding two different DRs (e.g., WUS and IPT) can be linked to different promoters. In some embodiments, sequences encoding two different DRs (e.g., WUS and IPT) can be linked to the same promoter. In some embodiments, a nucleotide sequence encoding a first DR (e.g., WUS) can be linked to a NOS promoter. In some embodiments, a nucleotide sequence encoding a second DR (e.g., IPT) can be linked to a 35S promoter.

[0063] Exemplary sequences for at least some of the above-referenced DRs and promoters are provided herein. It is to be noted, however, that homologs of these DRs exist in numerous plant species, and the methods provided herein are not limited to use of the listed DRs or to DRs having 100% identity to the provided sequences. In some cases, for example, a DR coding sequence can have at least 80% (e.g., at least 85%, at least 90%, or at least 95%) identity to the WUS sequence set forth in SEQ ID NO:5 or the IPT sequence set forth in SEQ ID NO:6. In some cases, a DR can have an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, or at least 95%) identical to the WUS sequence set forth in SEQ ID NO: 13 or the IPT sequence set forth in SEQ ID NO: 14.

[0064] The percent sequence identity between a particular amino acid or nucleic acid sequence and an amino acid or nucleic acid sequence referenced by a particular sequence identification number is determined as follows. First, an amino acid or nucleic acid sequence is compared to the sequence set forth in a particular sequence identification number using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing Attorney Docket No. 09531-0530W01 2023-264

[0065] BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from Fish & Richardson’s web site (e.g., www.fr.com / blast / ) or the U.S. government’s National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov). Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ. B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seql.txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output.txt); -q is set to -1; -r is set to 2; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\B12seq -i c:\seql.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q -1 -r 2. To compare two amino acid sequences, the options of B12seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seql.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\B12seq -i c:\seql .txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

[0066] Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. A matched position refers to a position in which an identical nucleotide or amino acid residue occurs at the same position in aligned sequences. The percent sequence identity is determined by dividing the number of matches by the length of the sequence set forth in the identified sequence (e.g., SEQ ID NO:5), followed by multiplying the resulting value by 100. For example, an amino acid sequence that has 880 matches when aligned with the sequence set Attorney Docket No. 09531-0530W01 2023-264 forth in SEQ ID NO:5 is 98.8 percent identical to the sequence set forth in SEQ ID NO:5 (i.e., 880 891 x 100 = 98.8). It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. It also is noted that the length value will always be an integer.

[0067] Non-limiting, representative sequences for at least some of the above-referenced promoters and DRs are provided below. It is to be noted, however, that homologs of these promoters and DRs exist in numerous plant species, and the methods provided herein are not limited to use of the listed promoters and DRs or to promoters and DRs having 100% identity to the provided sequences. Thus, in some cases, a promoter can have at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 98%, or at least 99%) sequence identity to the Nos promoter sequence set forth in SEQ ID NO:7 or to the 35S promoter sequence set forth in SEQ ID NO: 8. Further, in some cases, a DR coding sequence can have at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity to the WUS sequence set forth in SEQ ID NO:5, or the IPT sequence set forth in SEQ ID NO:6.

[0068] SEQ ID NO:5: Glycine max Gm WUS 1

[0069] ATGATGGAACCTCAACAACAACAACAACAAGCACAAGGGAGCCAACAAC AACAACAAAACGAGGATGGTGGCAGTGGAAAAGGGGGGTTTCTGAGCAGGCAA AGTAGTACACGGTGGACTCCAACAAACGACCAGATAAGAATATTGAAGGAACTT TACTACAACAATGGAATTAGATCCCCGAGTGCAGAGCAGATTCAGAGGATCTCT GCTAGGCTGAGGCAGTACGGTAAGATTGAAGGCAAGAATGTCTTTTATTGGTTCC AGAACCACAAAGCTCGAGAAAGGCAGAAGAAAAGGTTCACTTCTGATCATAATC ATAATAATGTCCCCATGCAAAGACCCCCAACTAATCCTTCTGCTGCTTGGAAACC TGATCTAGCTGATCCCATTCACACCACCAAGTATTGTAACATCTCTTCTACTGCA GGGATCTCTTCGGCATCATCTTCTGTTGAGATGGTTACTGTGGGACAGATGGGGA ATTATGGGTATGGTTCTGTGCCCATGGAGAAAAGTTTTAGGGACTGCTCGATATC AGCTGGGGGTAGCAGTGGCCATGTTGGATTAATAAACCACAACTTGGGGTGGGT TGGTGTGGACCCATATAATTCCTCAACCTATGCCAACTTCTTTGACAAAATAAGG CCAAGTGATCAAGAAACCCTTGAAGAAGAAGCAGAGAACATTGGTGCTACTAAG ATTGAAACCCTCCCTTTATTCCCTATGCACGGTGAGGACATCCATGGCTATTGCA ACCTCAAGTCTAATTCGTATAACTATGATGGAAACGGCTGGTATCATACTGAAGA Attorney Docket No. 09531-0530W01 2023-264

[0070] AGGGTTCAAGAATGCTTCTCGTGCTTCCTTGGAGCTCAGTCTCAACTCCTACACT CGCAGGTCTCCAGATTATGCTTAA

[0071] SEQ ID NO: 13: Glycine max GmWUSl

[0072] MMEPQQQQQQAQGSQQQQQNEDGGSGKGGFLSRQSSTRWTPTNDQIRILKE LYYNNGIRSPSAEQIQRISARLRQYGKIEGKNVFYWFQNHKARERQKKRFTSDHNHN NVPMQRPPTNPSAAWKPDLADPIHTTKYCNISSTAGISSASSSVEMVTVGQMGNYG YGSVPMEKSFRDCSISAGGSSGHVGLINHNLGWVGVDPYNSSTYANFFDKIRPSDQE TLEEEAENIGATKIETLPLFPMHGEDIHGYCNLKSNSYNYDGNGWYHTEEGFKNASR ASLELSLNSYTRRSPDYA

[0073] SEQ ID NO:6: Agrobacterium tumefaciens IPT

[0074] ATGGATCTGCGTCTAATTTTCGGTCCAACTTGCACAGGAAAGACGTCGAC CGCGATACGTCTTGCCCAGCAGACTGGCCTTCCAGTCCTTTCGCTCGATCGGGTC CAATGCTGTCCTCAACTGTCAACCGGAAGCGGACGACCAACAGTGGAAGAACTG AAAGGAACGACCCGTCTATACCTTGAAGATCGGCCTCTGGTGAAGGGTATCATC GCAGCCAAGCAAGCTCACGAAAGGCTGATCGGGGAAGTGTACAATTATGAGGCC CACGGCGGGCTTATTCTTGAGGGAGGATCTATCTCGTTGCTCAGGTGCATGGCGC AAAGCAGTTATTGGAGTACCGATTTTCGTTGGCATATTATTCGCCACAAGTTAGC AGACGAGGAGACATTCATGAACGCGGCCAAGGCCAGAGTTAGGCAGATGTTGCG CCCTGCTGTAGGCCCATCTATTATTCAAGAGTTGGTTCATCTTTGGAATGAGCCT CGGCTGAGGCCCATACTGAAAGAGATCGACGGATATCGATATGCCATGTTATTT GCTAGCCAGAACCAGATCACACCCGATATGCTATTGCAGCTTGACCCAGATATG GAGGGTGAGTTGATTCATGGAATCGCTCAGGAGTATCTCATCCATGCGCGCCGG CAGGAGCAGGAATTCCCTCCAGTGAGCGTGGTCGCTTTCGAAGGATTCGAAGGT CCACCGTTCGGAATGTGCTAG

[0075] SEQ ID NO: 14: A. tumifaciens IPT

[0076] MDLRLIFGPTCTGKTSTAIRLAQQTGLPVLSLDRVQCCPQLSTGSGRPTVEEL KGTTRLYLEDRPLVKGIIAAKQAHERLIGEVYNYEAHGGLILEGGSISLLRCMAQSSY WSTDFRWHIIRHKLADEETFMNAAKARVRQMLRPAVGPSIIQELVHLWNEPRLRPIL KEIDGYRYAMLFASQNQITPDMLLQLDPDMEGELIHGIAQEYLIHARRQEQEFPPVSV VAFEGFEGPPFGMC

[0077] SEQ ID NO: 7: A. tumefaciens Nos promoter Attorney Docket No. 09531-0530W01 2023-264

[0078] GATCATGAGCGGAGAATTAAGGGAGTCACGTTATGACCCCCGCCGATGAC GCGGGACAAGCCGTTTTACGTTTGGAACTGACAGAACCGCAACGTTGAAGGAGC CACTCAGCCGCGGGTTTCTGGAGTTTAATGAGCTAAGCACATACGTCAGAAACC ATTATTGCGCGTTCAAAAGTCGCCTAAGGTCACTATCAGCTAGCAAATATTTCTT GTCAAAAATGCTCCACTGACGTTCCATAAATTCCCCTCGGTATCCAATTAGAGTC TCATATTCACTCTCAATCCAAATAATCTGCACCGTA

[0079] SEQ ID NO:8: Cauliflower mosaic virus 35S promoter

[0080] AGATTTGCCTTTTCAATTTCAGAAAGAATGCTAACCCACAGATGGTTAGA GAGGCTTACGCAGCAGGTATCATCAAGACGATCTACCCGAGCAATAATCTCCAG GAAATCAAATACCTTCCCAAGAAGGTTAAAGATGCAGTCAAAAGATTCAGGACT AACTGCATCAAGAACACAGAGAAAGATATATTTCTCAAGATCAGAAGTACTATT CCAGTATGGACGATTCAAGGCTTGCTTCACAAACCAAGGCAAGTAATAGAGATT GGAGTCTCTAAAAAGGTAGTTCCCACTGAATCAAAGGCCATGGAGTCAAAGATT CAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGCGAACAGTTCATACAG AGTCTCTTACGACTCAATGACAAGAAGAAAATCTTCGTCAACATGGTGGAGCAC GACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGG GCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATT GCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTA CAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGA CAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAG ACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGT AAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGA

[0081] AGTTCATTTCATTTGGAGAGAACACGGGGGACT

[0082] In some embodiments of the methods described herein, introduction of an exogenous nucleic acid into a leguminous plant cell (e.g., a cell within a germinating embryo of a leguminous plant) can be achieved using a vector. Any appropriate vector can be used to introduce nucleic acid encoding one or more DRs and, optionally, nucleic acid encoding one or more DNA editing molecules, reporters, or other coding sequences into a leguminous plant cell. For example, a vector can be an expression vector that includes a promoter sequence operably linked to a nucleotide sequence encoding a DR to be expressed in the plant cell, and optionally a promoter operably linked to a nucleotide sequence encoding a Attorney Docket No. 09531-0530W01 2023-264 detectable marker to be expressed in the cell. Any appropriate reporter can be used (e.g., RUBY, 0-glucuronidase, fluorescent proteins such as GFP or RFP, luciferase, chloramphenicol acetyltransferase, anchocyanin, or another pigment-based reporter).

[0083] In some cases, a vector can be an expression vector that includes a promoter sequence operably linked to a nucleotide sequence encoding a polypeptide that, when expressed, can edit the DNA of the cell. The methods described herein can result in transient expression or stable integration of the delivered nucleic acid sequence(s). Non-limiting examples of vectors that can be used for plant cell transformation include Agrobacterium Ti plasmids (e.g., pBI121, pCAMBIA, pEarleyGate, or pGreen), viral vectors (e.g., tobacco mosaic virus-based vectors, geminivirus-based vectors such as Bean Yellow Dwarf virus (BeYDV) and Wheat Dwarf India Virus (WDIV) derived vectors), particle bombardment (biolistic) vectors (pUbi),and CRISPR / Cas9 vectors (e.g., pYLCRISPR vectors). Skilled practitioners will be capable of selecting suitable vectors for introducing any of the DRs and nucleic acid modifying / gene editing components described herein into leguminous plant cells. In some embodiments, a vector can include any of the exogenous nucleic acid molecules described herein.

[0084] This document provides methods of transformation that can have improved transformation efficiency in leguminous plant cells. In some cases, the transformation methods provided herein also can accelerate the transformation and generation of genomically edited leguminous plants. One or more DRs (e.g., WUS and / or IPT) can be delivered to cells within a germinating embryo, and cells expressing the one or more DRs (e.g., WUS and / or IPT), and their surrounding neighbors, can then be induced into a meristematic growth pattern that subsequently develops into plant tissues of interest (e.g., shoots).

[0085] Any appropriate method can be used to obtain germinating legume embryos. In some cases, legume seeds can be sterilized (e.g., using vapor-phase sterilization), and then incubated under conditions that result in germination. For example, sterilized seeds can be placed on sucrose agar plates and incubated in the dark (e.g., at room temperature for 4-10 hours), and then soaked in water for an appropriate length of time and under appropriate conditions (e.g., overnight at room temperature). Germinating embryos then can be isolated Attorney Docket No. 09531-0530W01 2023-264 by removing the seed coat and cotyledons, followed by removal of the two primary leaves with care to avoid damaging the meristematic region.

[0086] The methods disclosed herein can include using, for example, Agrobacterium, to introduce nucleic acid encoding one or more DRs into cells within a germinating embryo of a leguminous plant. In some cases, the methods provided herein can include introducing, into a germinating embryo of a leguminous plant, one or more nucleic acid molecules encoding WUS and IPT. In some cases, the methods provided herein also can include using, for example, Agrobacterium to introduce nucleic acid encoding a detectable marker, and / or nucleic acid encoding a polypeptide that, when expressed, can edit the DNA of the cells into which it was introduced. The methods provided herein also can include co-cultivation of a germinating embryo containing introduced nucleic acid with Agrobacterium (e.g., for about 12 hours to about 10 days, about 1 day to about 5 days, about 2 days to about 6 days, or about 3 days to about 7 days) to allow expression of the DRs and other polypeptides encoded by the introduced nucleic acid. . The methods provided herein also can include culturing plantlets that arise from the embryos in a growth medium for about 1 week to about 8 weeks (e g., about 1 to about 3 weeks, about 2 to about 4 weeks, about 3 to about 5 weeks, about 4 to about 6 weeks, about 5 to about 7 weeks, about 6 to about 8 weeks, or about 1, 2, 3, 4, 5, 6, 7, or 8 weeks) for shoot formation from the plantlets. When the nucleic acid introduced into the cells within a germinating embryo contains a nucleotide sequence encoding a detectable marker (e.g., RUBY or green fluorescent protein), shoots of the resulting plantlets can be assessed for expression of the marker (e.g., a ruby color in the case of a RUBY marker, or fluorescence in the case of a fluorescent marker), and can be identified as being transgenic shoots induced by the expression of the DRs.

[0087] Transformation efficiency is the percentage of successfully transformed cells or plant tissues (e.g., shoots) in relation to the total number of cells or plant tissues (e.g., embryos) exposed to the transformation process. Transformation efficiency can be expressed as the number of transformed cells or plant tissues (e.g., shoots) that have successfully incorporated a foreign nucleic acid sequence (e.g., a RUBY reporter coding sequence), divided by the total number of cells or tissues (e.g., embryos) that were subjected to the transformation procedure. In some embodiments, the transformation efficiency of the methods described herein can be about 15% to about 40% (e.g., about 1 %, about 16%, about 17%, about 18%, Attorney Docket No. 09531-0530W01 2023-264 about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40%). In some embodiments, the transformation efficiency of the methods described herein can be about 16% to about 30%. In some embodiments, the transformation efficiency of the methods described herein can be, on average, greater than 20% (e.g., about 22%, about 25%, or about 30%).

[0088] Transformation efficiency can indicate the effectiveness of a delivery system (e.g., an Agrobacterium strain and its encoded polypeptides). In some embodiments, the methods described herein can include Agrohacterium-me aie . transformation. Any appropriate Agrobacterium strain can be used. For example, Aie Agrobacterium strains used in the methods provided herein can include, without limitation, A. tumefaciens AGL-1, A. tumefaciens C58, A. tumefaciens GV3101, A. tumefaciens LBA4404, A. tumefaciens EHA105, and / or rhizogenes. In some embodiments, Ae Agrobacterium strain used in the methods provided herein can be A tumefaciens AGL-1.

[0089] The methods described herein also can include infecting and co-cultivating germinating embryos Agrobacterium X.o deliver nucleic acid encoding one or more DRs. In some embodiments, the infection of germinating embryos with Agrobacterium can be carried out for about 5 minutes to about 5 hours (e.g., about 5 to about 15 minutes, about 15 to about 30 minutes, about 30 to about 60 minutes, about 1 to about 2 hours, about 2 to about 3 hours, about 3 to about 4 hours, about 4 to about 5 hours, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, or about 5 hours). In some embodiments, the infection of germinating embryos with Agrobacterium can be carried out for about 30 minutes to about 2 hours. In some embodiments, the infection can be carried out for about 30 minutes. In some embodiments, the infection can be carried out for about 2 hours. In some embodiments, excess Agrobacterium in the infection medium can be removed after infection.

[0090] In some cases, co-cultivation of germinating embryos with Agrobacterium can be an incubation after an infection period, and can be carried out for about 1 day to about 5 days Attorney Docket No. 09531-0530W01

[0091] 2023-264

[0092] (e.g., about 1 to about 2 days, about 2 to about 3 days, about 3 to about 4 days, about 4 to about 5 days, about 1 to 3 days, about 2 to 4 days, about 3 to 5 days, about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days). A co-cultivation period can allow Agrobacterium o transfer T-DNA into plant cells. In some embodiments, co-cultivation can permit the introduction of nucleic acid (e.g., nucleic acid encoding one or more DRs or DNA editing reagents) into a plant cell’s genome. In some embodiments, co-cultivation can allow for expression of the one or more DRs (e.g., WUS and / or IPT) encoded by delivered nucleic acid. In some embodiments, the co-cultivation of germinating embryos with Agrobacterium can be carried out for about 3 days.

[0093] In some cases, the methods provided herein include delivering to cells of a germinating embryo of a leguminous plant, nucleic acid encoding one or more DRs and nucleic acid encoding a polypeptide that, when expressed, modifies (e.g., edits) the DNA of the cells. The nucleic acid can be delivered by any suitable method, including by Agrobacterium - in which case the DR(s) and the DNA editing sequence(s) can be delivered on the same T-DNA vector or on separate T-DNA vectors. In some cases, the nucleic acids can be delivered by direct injection, electroporation, biolistics (gene gun), nanoparticle delivery, particle bombardment, chemical transfection, viral infection, or any other useful method that can result in transient expression or stable integration of the delivered nucleic acid sequences. When two or more DRs (e.g., the combination of WUS and IPT) are delivered by Agrobacterium, they can be present on the same T-DNA vector or on separate T-DNA vectors. In some cases, different strains of Agrobacterium can be used to deliver the DR(s) and the gene editing component(s). In addition, it is to be noted that the T-DNA(s) used in the methods provided herein can include any suitable replicon. In some cases, for example, a T-DNA can include a viral replicon (e.g., a geminivirus replicon), which can include any appropriate virus component (e.g., RepA) to enable the generation of meristematic tissue.

[0094] In some cases, when a combination of two or more DRs (e.g., WUS and IPT) are delivered into cells of a germinating embryo, the two or more DRs (e.g., WUS and IPT) can be delivered into the cells simultaneously or sequentially. The two or more DRs can be delivered into the cells at any appropriate ratio. In some embodiments, the ratio of a first DR to a second DR (e.g., WUS : IPT) used for transformation refers to (the amount of nucleic Attorney Docket No. 09531-0530W01

[0095] 2023-264 acid encoding the first DR (e.g., WUS)) : (the amount of nucleic acid encoding the second DR (e.g., IPT)). Any appropriate ratio of a first DR to a second DR can be used. In some embodiments, the ratio of a first DR (e.g., WUS) to a second DR (e.g., IPT) used for transformation can be from about 9: 1 to about 1 :9 (e.g., about 9:1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3:1, about 2: 1, about 1 :1, about 1 :2, about 1 :3, about 1:4, about 1:5, about 1 :6, about 1 :7, about 1 :8, or about 1 :9).

[0096] In some cases, the methods provided herein can include delivering, to a germinating embryo of a leguminous plant, nucleic acid containing one or more transgenes to be integrated into the plant cell DNA. The nucleic acid containing one or more transgenes can be delivered in addition to the nucleic acid encoding the one or more DRs that promote new meristem formation and, optionally, the nucleic acid encoding a DNA editing polypeptide. In some cases, the transgene can encode a detectable reporter. Any appropriate reporter can be used. Non-limiting examples of suitable reporters include RUBY, P-glucuronidase, fluorescent proteins (e.g., GFP or RFP), luciferase, chloramphenicol acetyltransferase, anchocyanin, or other pigment-based reporters. In some embodiments, a reporter can be used for assessing transformation efficiency, promoter activity, gene expression, or tissue-specific gene expression. In some embodiments, when RUBY is used as a reporter, a plant or plant tissue (e.g., leaves or shoots) can be selected based on the presence of a pink or ruby color generated by the expression of RUBY reporter gene. In some cases, a transgene to be integrated into plant DNA or transiently expressed can encode one or more gene editing reagents that can make genetic alterations to the developing meristems, to create a desired genetic engineering (GE) event.

[0097] The methods and materials provided herein can be used with germinating embryos from any appropriate type of leguminous plant, including, but not limited to, soybean, kidney bean, black bean, pinto bean, peanut, chickpea, lentil, peas, common bean, cowpea, pigeon pea, peanut ground nut, faba bean, green beans, edamame, green lentils, green peas, green mung beans, sugar snap peas, and snow peas. The methods described herein can offer new and broadly applicable approaches to solve bottlenecks in delivery of GE reagents to leguminous plants, as well as the regeneration of leguminous plant tissues carrying GE events of interest. Since DRs are evolutionarily conserved, the methods provided herein are amenable to use across a variety of leguminous species. Finally, the methods may avoid Attorney Docket No. 09531-0530W01

[0098] 2023-264 regulatory hurdles in the development of agricultural crops, as there is potential for transient delivery of reagents and subsequent recovery of non-transgenic progeny carrying a GE event of interest.

[0099] In some cases, the methods provided herein can be used to obtain leguminous plants, leguminous plant tissues, leguminous plant parts, and leguminous plant cells having a desired trait, such as an agriculturally relevant trait. Agriculturally relevant traits can include, without limitation, herbicide tolerance, resistance to diseases and pests, growth rate, size, shape, color, yield, fatty acid composition, amino acid composition, reduced allergen, and flavor of harvested products. For example, the methods provided herein can be used to insert a transgene into the genomic sequence of a plant cell, where expression of the transgene yields an agriculturally relevant trait.

[0100] In some cases, expression (e.g., transient expression) of a transgene can produce one or more nucleic acid modifying agents that can modify / edit the plant DNA. The one or more nucleic acid modifying agents can include a polypeptide and / or an RNA that, when expressed within a plant cell, can edit nucleic acid of the plant cell to introduce one or more genetic or epigenetic modifications. Examples of such nucleic acid modifying agents include, without limitation, targeted rare-cutting endonucleases (e.g., meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) endonucleases, and RNA-guided endonucleases such as clustered regularly -interspaced short palindromic repeats (CRISPR) / CRISPR associated (Cas) endonucleases), as well as targeted cytosine or adenosine deaminases (e.g., apolipoprotein B mRNA editing enzyme, catalytic polypeptide- like (APOBEC)-CRISPR / Cas fusions such as BE3, and ABE), and prime editors. Methods for making and using such targeted DNA modifying enzymes include those described elsewhere. See, e.g., Sander et al., Nature Methods, 8:67-69, 2011; Jacoby et al., Nucl. Acids Res., 10.1093 / nar / gkrl303, 2012); Christian et al., Genetics, 186:757-761, 2010; U.S. Publication No. 2011 / 0145940; Cong et al., Science, 339:819-823, 2013; and Mali et al., Science, 339:823-826, 2013.

[0101] In some cases, a nucleic acid modifying agent can be selected to introduce a break (e.g., a single-strand break or a double-strand break) into a plant’s DNA (e.g., genomic DNA or epigenomic DNA). In general, a double-strand break at a target sequence to be modified can be repaired by one of two primary pathways: non-homologous end joining (NHEJ) or Attorney Docket No. 09531-0530W01 2023-264 homologous recombination (HR). In NHEJ, the ends of the broken chromosome are rejoined, sometimes imprecisely, which can introduce small insertions or deletions (indels) at the break site (Gorbunova and Levy, Nucl. Acids Res., 1997, 25:4650-4657). When indels occur in coding sequences, they may create frame shift mutations that disrupt gene function. In HR, or gene targeting (GT), the DNA break is repaired using a template with homology to the break site. The repair template can be the sister chromatid, a homologous or homeologous chromosome (in the case of polyploid species), or an exogenous template containing one or more specific sequence modifications to be incorporated into the break site.

[0102] Any appropriate nucleic acid modifying agent can be encoded by a transgene in a nucleic acid provided herein. Examples of nucleic acid modifying agents include, without limitation, targeted rare-cutting endonucleases such as meganucleases (Puchta et al., Nucl. Acids Res., 21 :5034-5040, 1993; Salomon and Puchta, EMBO J., 17:6086-6095, 1998; and Jacoby et al., Nucl. Acids Res., 10.1093 / nar / gkrl303, 2012), zinc-finger nucleases (ZFNs) (Kim et al., Proc. Natl. Acad. Sci. USA, 93: 1156-1160, 1996; Townsend et al., Nature, 459:442-445, 2009; and Sander et al., Nature Methods, 8:67-69, 2011), transcription activator-like effector (TALE) endonucleases (Christian et al., Genetics, 186:757-761, 2010; Bogdanove and Voytas, Science, 333: 1843-1846, 2011; and U.S. Publication No. 2011 / 0145940), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems, such as a CRISPR / Cas9 system (Hwang et al., Nat. Biotechnol. , 31 :227-229, 2013; Shan et al., Nat. Biotechnol, 31 :686-688, 2013; Cong et al., Science, 339:819-823, 2013; and Mali et al., Science, 339:823-826, 2013). CRISPR / Cas systems use RNA base pairing to direct DNA or RNA cleavage by a Cas endonuclease. CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) sequences direct the Cas enzyme to a specific target DNA sequence (Makarova et al., Nat. Rev. Microbiol., 9(6):467 -477 , 2011). The modification of a single targeting RNA can be sufficient to alter the nucleotide target of a Cas protein. In some cases, crRNA and tracrRNA can be engineered as a single cr / tracrRNA hybrid to direct Cas9 cleavage activity (Jinek et al., Science, 337(6096):816-821, 2012).

[0103] CRISPR / Cas systems use RNA base pairing to direct DNA or RNA cleavage by a Cas endonuclease. CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) sequences direct the Cas enzyme to a specific target DNA sequence (Makarova et al., Nat. Rev. Microbiol., 9(6):467-477, 2011). The modification of a single targeting RNA can be Attorney Docket No. 09531-0530W01 2023-264 sufficient to alter the nucleotide target of a Cas protein. In some cases, crRNA and tracrRNA can be engineered as a single cr / tracrRNA hybrid to direct Cas9 cleavage activity (Jinek et al., Science, 337(6096):816-821, 2012). Examples of CRISPR-Cas systems that can be used include, but are not limited to, CRISPR-Cas9, CRISPR-Cas 12a, CRISPR-Casl3, CRISPR- Cas3, CRISPR-Casl4, CRISPR-Cas , CRISPR-Cas9 with deaminase, or CRISPR-Cas9 with reverse transcriptase.

[0104] In some cases, a nucleic acid provided herein can contain sequences encoding a Cas endonuclease and a gRNA. The Cas-encoding sequence and the gRNA-encoding sequence can be independently and operably linked to any appropriate promoter(s) within the nucleic acid. The promoter(s) can be, for example, inducible, constitutive, cell specific, or tissue specific. Exemplary constitutive promoters include, without limitation, constitutive RNA pol II promoters such as the 35S, Nos-P, and ubiquitin promoters, and constitutive RNA pol III promoters such as the U6 promoter. Examples of inducible promoters include, without limitation, the virion-sense promoter from geminivirus, and the XVE promoter. In some embodiments, for example, a Cas coding sequence can be operably linked to an inducible XVE promoter, which can be activated by estradiol.

[0105] Any appropriate Cas endonuclease can be encoded by a nucleic acid provided herein. In some cases, the Cas endonuclease can be a Cas9 endonuclease. A Cas9 amino acid sequence from Streptococcus pyogenes is set forth in SEQ ID NO:36 (FIG. 17A), and a nucleotide sequence encoding an S. pyogenes Cas9 endonuclease is set forth in SEQ ID NO:37 (FIG. 17B). Other suitable Cas polypeptides include, without limitation, Casl2j (also referred to as CasO, including CasOl, Cas2, and Cas3) and Casl2f A representative Cas l amino acid sequence is set forth in SEQ ID NO:38 (FIG. 17C) and a representative nucleotide sequence encoding Cas l endonuclease is set forth in SEQ ID NO:39 (FIG. 17D). A representative Casl2f endonuclease amino acid sequence is set forth in SEQ ID NO:40, and a representative nucleotide sequence encoding a Casl2f endonuclease is set forth in SEQ ID NO:41 (both in FIG. 17E). A representative Cas 2 amino acid sequence is set forth in SEQ ID NO:42 (FIG. 17F) and a representative nucleotide sequence encoding Cas 2 endonuclease is set forth in SEQ ID NO:43 (FIG. 17G). A representative CasO3 amino acid sequence is set forth in SEQ ID NO:44 (FIG. 17H) and a representative nucleotide sequence encoding Cas3 endonuclease is set forth in SEQ ID NO:45 (FIG. 171). Attorney Docket No. 09531-0530W01 2023-264

[0106] Other examples of nucleic acid modifying agents that can be encoded by a nucleic acid provided herein include, without limitation, TnpB polypeptides, cytosine base editors (CBEs), adenine base editors (ABEs), and prime editing agents. TnpBs are Cas-related polypeptides that work with guide RNAs to modify DNA. TnpBs are relatively small in size - often less than about 500 amino acids. Representative examples of TnpB amino acid sequences are set forth in SEQ ID NOS:46 and 47 (FIG. 18). Base editing and prime editing technology can avoid the creation of double-stranded DNA breaks, and may provide enhanced editing efficiency and product purity. These technologies are described elsewhere (see, e.g., Kaya (2024), “Base Editing and Prime Editing” in: A Roadmap for Plant Genome Editing. Ricroch, Eriksson, Miladinovic, Sweet, Van Laere, and Wozniak-Gientka (eds), Springer, Cham.; doi.org / 10.1007 / 978-3-031-46150-7_2).

[0107] In some cases, a nucleic acid modifying agent can be a TALE endonuclease. TAL effectors of plant pathogenic bacteria in the genus Xanthomonas play important roles in disease and trigger defense by binding to host DNA and activating effector-specific host genes (see, e.g., Gu et al., Nature 435: 1122, 2005; Yang et al., Proc. Natl. Acad. Sci. USA 103: 10503, 2006; Kay et al., Science 318:648, 2007; Sugio et al., Proc. Natl. Acad. Sci. USA 104: 10720, 2007; and Romer et al., Science 318:645, 2007). Specificity depends on an effector-variable number of imperfect, typically 34 amino acid repeats (Schornack et al., J. Plant Physiol., 163:256, 2006). Polymorphisms are present primarily at repeat positions 12 and 13, which are referred to herein as the repeat variable-di residue (RVD). TALE nucleases contain (1) a DNA binding domain derived from a TAL effector, where the domain can be engineered to bind to a specific sequence based on the RVDs included in the repeats, and (2) an endonuclease domain, typically from a type II restriction endonuclease such as FokI (Kim et al., Proc. Natl. Acad. Sci. USA, 93: 1156-1160, 1996). Other useful endonucleases include, for example, Hhal, Hindll , Noll, BbvCI, EcoRI, BgH, and A / wl. The fact that some endonucleases (e.g., FokI) only function as dimers can be capitalized upon to enhance the target specificity of the TALE nuclease. For example, in some cases each FokI monomer can be fused to a TAL effector sequence that recognizes a different DNA target sequence, and only when the two recognition sites are in close proximity do the inactive monomers come together to create a functional enzyme. By requiring DNA binding to activate the nuclease, a highly site-specific restriction enzyme can be created. Thus, TALE nucleases can function as Attorney Docket No. 09531-0530W01

[0108] 2023-264 heterodimers, where each monomer of the pair is targeted to a selected target sequence, and when the monomers are bound to their targets, the nuclease dimerizes and cleaves the DNA at the target sequence between the monomer binding sites. See, e.g., U.S. Patent No. 8,586,363.

[0109] In some embodiments, a repair template also can be delivered to cells within a germinating embryo of a leguminous plant, along with nucleic acid encoding a targeted endonuclease. When the endonuclease cleaves the plant cell DNA, the repair template can become integrated into the plant cell’s genomic DNA, thus introducing a specific modification into the plant genome.

[0110] This document provides methods for generating leguminous plant cells, leguminous plant parts, leguminous plant tissues, and / or leguminous plants that contain one or more genetic modifications of interest. The methods provided herein can include introducing nucleic acid encoding one or more DRs and nucleic acid encoding one or more DNA editing polypeptides into cells within a germinating embryo of a leguminous plant, culturing the embryo to induce meristem development into shoots, and acclimating genetically transformed plantlets to obtain a whole plant or portion thereof with the genetic modification(s) of interest.

[0111] The introduced nucleic acid can (1) encode one or more (e.g., two, three, four, five or more) DRs such as WUS, IPT, Baby Boom, Irrepressible Variants of Monopteros, Shoot Meristemless, Leafy Cotyledon, WUS homeobox-containing, and APETALA2 / Ethylene Responsive Factor to induce de novo shoot formation, and (2) encode one or more polypeptides that, when expressed, act to modify / edit endogenous sequences within the cells of, or originating from, germinating embryos into which the nucleic acid was introduced, to result in a genetic modification of interest. As a result of introducing these nucleic acid sequences, de novo tissue subsequently derived from the plant can carry the genetic modification of interest. In some cases, the de novo tissue can be meristematic, and capable of deriving new tissue (e.g., branch, flower, or root tissue) carrying the genetic modification(s) of interest.

[0112] Germinating embryos into which nucleic acid(s) encoding one or more DRs and one or more DNA editing polypeptides have been introduced can be cultured in a growth medium for any appropriate length of time, to allow for shoot formation. For example, germinating Attorney Docket No. 09531-0530W01 2023-264 embryos into which nucleic acid(s) encoding one or more DRs and one or more DNA editing polypeptides have been introduced can be cultivated in a growth medium for about 1 week to about 8 weeks (e.g., about 1 to 2 weeks, about 2 to 4 weeks, about 4 to 6 weeks, about 6 to 8 weeks, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks) to induce shoot formation. In some embodiments, a germinating embryo, after transformation, can be grown in a growth medium that does not contain plant hormones. In some embodiments, the germinating embryos, after transformation, can be cultivated in a growth medium without plant hormones for about 4 weeks.

[0113] In some embodiments, this document provides methods in which plantlets arising from the transformed germinating embryos can be grown to a desired stage in either sterile or non-sterile conditions (e.g., soil). For example, plantlets having shoots displaying a detectable marker (e.g., ruby color, when RUBY is the marker) or identified as having one or more genetic modifications of interest, can be acclimated in soil to obtain modified whole plants or portions thereof carrying the one or more genetic modifications of interest. The acclimation in soil can be carried out for any appropriate length of time (e.g., about 5 days to about 20 days, about 5 to about 10 days, about 10 to about 15 days, about 7 to about 14 days, about 15 to about 20 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 20 days). In some embodiments, the acclimation in soil can be carried out for about 10 days. In some cases, before a plant is generated from a transformed germinating embryo, de novo derived tissue (e.g., one or more shoots) resulting from expression of the one or more DRs can be assessed to determine whether it contains a genetic modification of interest. For example, DNA from newly derived tissue can be isolated and assessed by restriction digest, hybridization methods (e.g., Southern blotting), or sequencing to determine whether a genetic modification has occurred at a target site. In some embodiments, the expression of a reporter (e.g., RUBY) delivered with nucleic acid encoding one or more DRs (e.g., WUS and / or IPT) and nucleic acid encoding a DNA editing polypeptide can first be detected, to identify tissues that are likely to carry the genetic modification. Attorney Docket No. 09531-0530W01 2023-264

[0114] This document also provides germinating embryos of leguminous plants that contain (i) exogenous nucleic acid encoding one or more DRs that can induce new meristem formation in cells of the germinating embryos, and (ii) exogenous nucleic acid containing coding sequences that, when expressed in cells of the new meristem, can edit DNA in cells of the new meristem to introduce one or more genetic modifications of interest.

[0115] Exemplary Embodiments

[0116] Embodiment 1 is 1 method for generating plant cells comprising one or more genetic modifications of interest, the method comprising: (a) introducing, into a germinating embryo of a leguminous plant: (i) nucleic acid encoding one or more developmental regulators that, when expressed in cells of said germinating embryo, induce new meristem formation, and (ii) nucleic acid comprising one or more coding sequences that, when expressed in cells of said new meristem, edit DNA in cells of said new meristem to introduce one or more genetic modifications of interest, wherein the introducing comprises infection of the germinating embryo with an Agrobacteriunr, (b) incubating said germinating embryo with said Agrobacterium, such that said one or more developmental regulators and said one or more coding sequences within the nucleic acid of (ii) are expressed; (c) growing a plantlet developed from the germinating embryo of step (b) in a growth medium to induce shoot formation; and (d) identifying one or more shoots having said one or more genetic modifications of interest.

[0117] Embodiment 2 is the method of embodiment 1, wherein the introducing comprises incubating said germinating embryo with said Agrobacterium for about 3 days.

[0118] Embodiment 3 is the method of embodiment 1 or embodiment 2, wherein the incubating comprising incubating said germinating embryo with said Agrobacterium for about 1 day to 5 days.

[0119] Embodiment 4 is the method of any one of embodiments 1-3, wherein the growing comprises growing said plantlets in a growth medium for about 1 week to about 8 weeks.

[0120] Embodiment 5 is the method of any one of embodiments 1-3, wherein the growing comprises growing said plantlets in a growth medium for about 4 weeks. Attorney Docket No. 09531-0530W01 2023-264

[0121] Embodiment 6 is the method of any one of embodiments 1-5, wherein the method further comprises introducing, into said germinating embryo, (iii) nucleic acid encoding a reporter.

[0122] Embodiment 7 is the method of embodiment 6, wherein said reporter comprises a RUBY polypeptide.

[0123] Embodiment 8 is the method of embodiment 6 or embodiment 7, wherein the method further comprises identifying plantlets in which said reporter is expressed.

[0124] Embodiment 9 is the method of any one of embodiments 1-8, wherein said growth medium is a plant hormone-free growth medium.

[0125] Embodiment 10 is the method of any one of embodiments 1-9, wherein said one or more developmental regulators comprise Wushel (WUS) and isopentenyl transferase (IPT).

[0126] Embodiment 11 is the method of embodiment 10, wherein said one or more developmental regulators further comprise one or more of Baby Boom, Irrepressible Variants of Monopteros, Shoot Meristemless, Leafy Cotyledon, WUS homeobox-containing, and APETALA2 / Ethylene Responsive Factor.

[0127] Embodiment 12 is the method of any one of embodiments 1-11, wherein said Agrobacterium comprises Agrobacterium tumefaciens strain AGL-1, A. tumefaciens strain C58, A. tumefaciens strain GV3101, A. tumefaciens strain LBA4404, A. tumefaciens strain EHA 105, or A. rhizogenes.

[0128] Embodiment 13 is the method of any one of embodiments 1 -12, wherein said Agrobacterium is A. tumefaciens strain AGL-1.

[0129] Embodiment 14 is the method of any one of embodiments 1-13, wherein said one or more sequences that, when expressed, edit the DNA in cells of said new meristem, comprise a nucleotide sequence encoding a targeted endonuclease, and wherein the targeted endonuclease comprises a Clustered Regularly-Interspaced Short Palindromic Repeats- associated nuclease, a Transcription Activator-Like Effector Nuclease, a meganuclease, or a zinc finger nuclease.

[0130] Embodiment 15 is the method of embodiment 14, wherein said targeted endonuclease is a Clustered Regularly-Interspaced Short Palindromic Repeats-associated (Cas) nuclease.

[0131] Embodiment 16 is the method of any one of embodiments 1-15, wherein said one or more sequences that, when expressed, edit the DNA in cells of said germinating embryo Attorney Docket No. 09531-0530W01

[0132] 2023-264 comprise (1) a nucleotide sequence encoding a targeted endonuclease and (2) a repair template.

[0133] Embodiment 17 is the method of any one of embodiments 1-16, wherein said shoots are meristematic and are capable of deriving new plant tissue carrying said one or more genetic modifications of interest.

[0134] Embodiment 18 is the method of any one of embodiments 1-17, further comprising (e) culturing the shoots identified in step (d) in soil for about 5 days to 20 days to obtain modified plant tissue comprising said one or more genetic modifications of interest.

[0135] Embodiment 19 is the method of any one of embodiments 1-18, wherein said leguminous plant is selected from the group comprising soybean, kidney bean, black bean, pinto bean, peanut, chickpea, lentil, pea, common bean, cowpea, pigeon pea, peanut ground nut, and faba bean.

[0136] Embodiment 20 is the method of embodiment 19, wherein said leguminous plant is soybean.

[0137] Embodiment 21 is a method for increasing transformation efficiency in a leguminous plant, the method comprising: (a) introducing, into cells of a germinating embryo of a leguminous plant, (i) nucleic acid encoding one or more developmental regulators that, when expressed in cells of the germinating embryo, induce new meristem formation, wherein said introducing comprises infection of said germinating embryo with an Agrobacterium, and (b) co-cultivating said germinating embryo and Agrobacterium such that said one or more developmental regulators are expressed.

[0138] Embodiment 22 is the method of embodiment 21, wherein said method has a transformation efficiency of about 15% to about 40%.

[0139] Embodiment 23 is a method for accelerating generation of a transgenic leguminous plant, the method comprising: (a) introducing, into a germinating embryo of a leguminous plant, (i) nucleic acid encoding one or more developmental regulators that, when expressed in cells of the germinating embryo, induce new meristem formation, and (ii) nucleic acid comprising a transgene, wherein the introducing comprises infection of the germinating embryo with an Agrobacterium, (b) co-cultivating said germinating embryo and said Agrobacterium such that said one or more developmental regulators are expressed; and (c) growing a pl anti et developed from the germinating embryo of step (b) in a growth medium to Attorney Docket No. 09531-0530W01

[0140] 2023-264 induce shoot formation, wherein at least one shoot comprises genomic DNA that comprises the transgene.

[0141] Embodiment 24 is the method of any one of embodiments 21-23, comprising cocultivating said germinating embryo and said Agrobacterium for about 3 days.

[0142] Embodiment 25 is the method of embodiment 23 or embodiment 24, comprising growing said plantlet in said growth medium for about 4 weeks.

[0143] Embodiment 26 is the method of any one of embodiments 23-25, wherein said growth medium is a plant hormone-free growth medium.

[0144] Embodiment 27 is the method of any one of embodiments 21-26, wherein said developmental regulators comprise one or more of Wushel (WUS), isopentenyl transferase (IPT), Baby Boom, Irrepressible Variants of Monopteros, Shoot Meristemless, Leafy Cotyledon, WUS homeobox-containing, and APETALA2 / Ethylene Responsive Factor.

[0145] Embodiment 28 is a germinating leguminous plant embryo comprising: (i) exogenous nucleic acid encoding one or more developmental regulators that, when expressed in cells of said germinating embryo, induce new meristem formation, and (ii) exogenous nucleic acid comprising one or more coding sequences that, when expressed in cells of said new meristem, edit DNA in cells of said new meristem to introduce one or more genetic modifications of interest.

[0146] Embodiment 29 is the germinating leguminous plant embryo of embodiment 28, wherein said leguminous plant is selected from the group comprising soybean, kidney bean, black bean, pinto bean, peanut, chickpea, lentil, pea, common bean, cowpea, pigeon pea, peanut ground nut, and faba bean.

[0147] Embodiment 30 is the germinating leguminous plant embryo of embodiment 28 or embodiment 29, wherein said germinating embryo further comprises (iii) exogenous nucleic acid encoding a reporter.

[0148] Embodiment 31 is the germinating leguminous plant embryo of embodiment 30, wherein said reporter comprises a RUBY polypeptide.

[0149] Embodiment 32 is the germinating leguminous plant embryo of any one of embodiments 28-31, wherein said germinating leguminous plant embryo is in a plant hormone-free growth medium. Attorney Docket No. 09531-0530W01

[0150] 2023-264

[0151] Embodiment 33 is the germinating leguminous plant embryo of any one of embodiments 28-32, wherein said one or more developmental regulators comprise Wushel (WUS) and isopentenyl transferase (IPT).

[0152] Embodiment 34 is the germinating leguminous plant embryo of embodiment 33, wherein said one or more developmental regulators further comprise one or more of Baby Boom, Irrepressible Variants of Monopteros, Shoot Meristemless, Leafy Cotyledon, WUS homeobox-containing, and APETALA2 / Ethylene Responsive Factor.

[0153] Embodiment 35 is the germinating leguminous plant embryo of any one of embodiments 28-34, wherein said one or more sequences that, when expressed, edit the DNA in cells of said new meristem, comprise a nucleotide sequence encoding a targeted endonuclease, and wherein the targeted endonuclease comprises a Clustered Regularly- Interspaced Short Palindromic Repeats-associated nuclease, a Transcription Activator-Like Effector Nuclease, a meganuclease, or a zinc finger nuclease.

[0154] Embodiment 36 is the germinating leguminous plant embryo of embodiment 35, wherein said targeted endonuclease is a Clustered Regularly-Interspaced Short Palindromic Repeats-associated (Cas) nuclease.

[0155] Embodiment 37 is the germinating leguminous plant embryo of any one of embodiments 28-36, wherein said one or more sequences that, when expressed, edit the DNA in cells of said new meristem comprise (1) a nucleotide sequence encoding a targeted endonuclease and (2) a repair template.

[0156] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0157] Attorney Docket No. 09531-0530W01

[0158] 2023-264

[0159] EXAMPLES

[0160] Example 1 - In-planta genetic transformation and genome engineering in legume species

[0161] MA TERIALS AND METHODS TABLE 1 : Media compositions

[0162] SOYBEAN EMBRYO AXIS TRANSFORMATION PROTOCOL

[0163] Fresh stocks of the following were prepared:

[0164] ■ L-Cysteine: 50 mg / mL in water, filter sterilized

[0165] ■ DTT (500mM): 231 mg / 3 mL in water, filter sterilized

[0166] ■ Acetosyringone: 39.24 mg / mL of DMSO, filter sterilized

[0167] ■ Cef: 200 mg / mL, filter sterilized

[0168] ■ Tim: 100 mg / mL, filter sterilized

[0169] ■ IBA: 1 mg / mL, filter sterilized Attorney Docket No. 09531-0530W01 2023-264

[0170] Agrobacterium cultures were prepared according to the following steps:

[0171] 1. Agrobacterium AGL-1 strain carrying T-DNA constructs from glycerol stock was struck on YEP agar plates with Rifampicin 25 mg / L and Kanamycin 50 mg / L antibiotics.

[0172] 2. Single colony with the proper T-DNA construct was inoculated into 20 ml of YEP medium supplemented with Rifampicin 10 mg / L and Kanamycin 50 mg / L antibiotics in 50 ml screw cap tubes and incubated at 28°C and 220 rpm overnight.

[0173] 3. The overnight cultures were centrifuged at 4000 rpm and 21 °C for 10 minutes. The pellets were resuspended in 10 ml of infection medium. Tubes were covered with foil and incubated at room temperature and 60 rpm for at least 2 hours before using the cultures for infection.

[0174] 4. The optical density (OD) value of the cultures was adjusted to 0.5 prior to infection. For co-transformation with individual DR-containing T-DNA construct, an equal volume of each culture was used.

[0175] Explants were prepared according to the following steps:

[0176] 5. Mature, dry seeds were surface sterilized for 16 hours using chlorine gas, produced by mixing 5 mL of 12N HC1 with 100 mL of commercial bleach.

[0177] 6. The disinfected seeds were imbibed on sucrose agar medium (TABLE 1; about 50 seeds in a 25 x 100 mm plate) at room temperature for 6-8 hours in the dark. The seeds were then soaked in sterile distilled water (around 10 seeds in a 25 x 100 mm plate, half filled with sterile water ) overnight at room temperature in the dark. Plates were stacked, taped on sides, covered with foil, and kept in a lab drawer.

[0178] 7. Intact embryonic axes were isolated using a scalpel and blade, then placed in 0.1 x liquid Gamborg’s B-5 medium (TABLE 1). The small two leaves were carefully broken off.

[0179] Infection was carried according to the following steps:

[0180] 8. Embryonic axes were placed in 25 x 100 mm deep Petri dishes with 15 ml of Agrobacterium suspension (OD 0.5) in infection medium. The plates were sealed with Attorney Docket No. 09531-0530W01 2023-264 parafilm and then sonicated (Sonicator Fisher Scientific model FS6) for 1 minute with 15-second intervals. Between sonications, the plates were gently shaken to mix the embryonic axes.

[0181] 9. After sonication, another 15 ml of Agrobacterium suspension was added to each plate, and plates were then wrapped with parafilm, covered with foil, and incubated for 30 minutes at room temperature and 60 rpm.

[0182] Co-cultivation was conducted as follows:

[0183] 10. After infection, excess bacterial suspension was removed from each plate and the embryonic axes were transferred to a single layer of autoclaved sterile filter paper in a 15 x 100 mm Petri dish. The embryonic axes were placed in a pile and the filter paper was moistened with 700 pl of infection medium (without Agrobacterium)' . The plates were sealed with parafilm and kept in a 21 °C incubator for 3 days under dim light (optionally covered with a single layer of paper).

[0184] Transfer was carried out as follows:

[0185] 11. After co-cultivation, the embryonic axes were lightly rinsed with 0.1 x Gamborg’s B-5 medium supplemented with Cef and Tim, and the base of each embryonic axis was embedded in growth medium I (TABLE 1) in a 25 x 100 mm Petri dish. The Petri dishes were wrapped with micropore tape and incubated in a growth chamber (at 24- 25°C with a 16:8 hour light-dark cycle)

[0186] 12. After 2 weeks, the plants were transferred to growth medium II (TABLE 1) and observed for the emergence of ruby color shoots.

[0187] DRs and Agrobacterium tumefaciens (A. tumefaciens) AGL-1 strain: Sequences encoding WUS (SEQ ID NO:5) and IPT (SEQ ID NO:6), driven by the NOS (SEQ ID NO:7) or 35S (SEQ ID NO:8) promoter, respectively, were cloned into a T-DNA vector containing a sequence encoding a RUBY reporter (FIG. 19; SEQ ID NO:9), allowing for the tracking of transgenic shoot formation (FIG. 1). The resulting constructs, including T-DNA encoding WUS, T-DNA encoding IPT, and T-DNA encoding both WUS and IPT, as well as a negative control T-DNA lacking any DR coding sequence, were subsequently transformed into the A. Attorney Docket No. 09531-0530W01 2023-264 tumefaciens AGL-1 strain. To prepare the bacterial culture for transformation, a single colony from each transformation was inoculated in 20 mL of YEP liquid medium supplemented with Rifampicin (10 mg / mL) and kanamycin (50 mg / tnL). The cultures were incubated at 28°C overnight and then pelleted by centrifugation at 4,000 rpm for 10 minutes at 21°C. The pellets were resuspended in infection medium, and the volume was adjusted to achieve an ODeoo of 0.5 for plant transformation.

[0188] DR-mediated soybean transformation via A. tumefaciens AGL-1 strain: As illustrated in FIG. 2, the soybean transformation process began with the sterilization of mature, dry soybean seeds by vapor-phase sterilization. These sterilized seeds were placed on sucrose agar plates and kept in the dark at room temperature for 6-7 hours. The seeds were then soaked in sterile water overnight at room temperature. Germinating embryos were isolated by removing the seed coat and cotyledons, followed by careful removal of the two primary leaves without damaging the meristematic region. The isolated embryos were then placed in Agrobacterium cultures, sonicated for 30 seconds twice, and then shaken for 30 minutes at 60 rpm. Post-infection, the embryos were transferred to a single layer of filter paper, wrapped with parafilm, and incubated at 21 °C for 3 days under low light conditions. Subsequently, they were planted in solid MS medium plates and incubated at 25°C under an 18-hour light photoperiod. After approximately 4 weeks, transgenic shoots began to emerge from plantlets transformed with the DRs (FIG. 2).

[0189] A RUBY reporter coding sequence was included in the T-DNA constructs to facilitate the identification of transgenic shoots. Positive transgenic shoots were readily identified by the ruby color in the leaves (FIGS. 3A and 3B). Transgenic shoots were grown to maturity and successfully produced ruby-colored flowers and pods (FIG. 3C). The transformation efficiency varied depending on the DRs used. The combination of WUS and IPT provided the highest transformation efficiency at an average of 22.0%, while WUS or IPT genes alone resulted in lower transformation efficiencies of 3.3% and 6.4%, respectively (TABLE 2). The transgenic nature of the ruby-colored shoots was confirmed by Polymerase Chain Reaction (PCR) using primer sequences for a T-DNA backbone sequence (“Bar,” FIG. 3D).

[0190] TABLE 2: Transformation efficiency mediated by IPT alone, WUS alone, and WUS and IPT in combination. Transformation with T-DNA without DRs was used as a control. Attorney Docket No. 09531-0530W01 2023-264

[0191] Gene editing via DR-mediated soybean transformation: To evaluate the efficacy of gene editing using DR-mediated transformation method, a CRISPR-Cas9 cassette (FIG. 20; SEQ ID NO: 10) was incorporated into T-DNA constructs containing the WUS and IPT coding sequences.

[0192] The CRISPR construct was designed to encode two guide RNAs (gRNAs). The first gRNA targeted the soybean trypsin inhibitor gene and was encoded by the sequence GTTGTGGAGGATCTACCAGAgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaa gtggcaccgagtcggtgc (SEQ ID NO: 11). The second gRNA targeted the P34 allergen gene and was encoded by the sequence GGCCAACAAGAAAATGAAGAgttttagagctagaaatagcaagttaa aataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc (SEQ ID NO: 12). These constructs were applied to transform germinating embryos using DR-mediated transformation methods described herein. Subsequently, leaf samples were collected from the resulting ruby-colored shoots and genomic DNA was extracted. PCR amplification was performed across each CRISPR target site using individual DNA samples, followed by Sanger sequencing of the amplified regions. Analysis of 16 ruby-colored plants revealed that one plant exhibited CRISPR-mediated gene editing events in both target genes (FIG. 4). This result Attorney Docket No. 09531-0530W01 2023-264 demonstrated that the DR-mediated transformation methods described herein effectively delivered gene editing reagents, achieving efficient gene editing in soybean.

[0193] Example 2 - Developmental regulators enable rapid and efficient soybean transformation and CRISPR-mediated genome editing

[0194] The results in this Example re-present and expand on at least some of the results provided in Example 1.

[0195] Materials and Methods

[0196] Construction of T-DNA vectors: All vectors were cloned using the Golden Gate assembly approach, which was based on modules A, B, C’ and D (Cermak et al., Plant Cell, 29: 1196-1217, 2017). Module A carried the Cas9 coding sequence driven by the GmUbi promoter, while Module B contained a sequence encoding a CRISPR guide RNA (GGCCAACAAGAAAATGAAGAA; SEQ ID NO: 15) that was targeted to the soybean P34 gene and was driven by the CmYLCV promoter. The coding sequences of each developmental regulator gene, including GmWUSCHEL2 (WUS2) driven by the Nos promoter, as well as IPT and GmGRP'4-GI 1 driven by the CaMl '35S promoter, were cloned into module C’(FIGS. 5A-5C). NRUBY visual reporter gene was cloned under the d / Ubi l O constitutive promoter in module D. These modules were then assembled into a destination vector, pTRANS_231 d and pTRANS_230, following the Golden Gate assembly method (Cermak et al., supra).

[0197] Seed sterilization and explant preparation: Mature dry seeds of soybean (cv. Williams 82 and Bert) were surface sterilized for 16 hours using chlorine gas, produced by mixing 5 mL of hydrochloric Acid (HC1, TraceMetal™ Grade, Thermo Fisher) with 100 mb of commercial bleach. Sterilized seeds were placed on germination medium plates (TABLE 3) in the dark at room temperature for 6 to 7 hours. Seeds were then removed from the germination plates and soaked in sterile water overnight at room temperature. Intact embryonic axes were isolated and undeveloped leaves were carefully broken off without damaging the meristematic region. All isolated embryonic axes were maintained in Cocultivation medium (TABLE 3) before Agrobacterium transformation. Attorney Docket No. 09531-0530W01 2023-264

[0198] Preparation of Agrobacterium suspension: The T-DNA vectors were transformed into the AGL1 strain of A. tumefaciens using the freeze-thaw method and plated onto YEP agar containing 10 pg / mL rifampicin and 50 pg / mL kanamycin. A single colony was inoculated in 20 mL of YEP liquid medium (rifampicin 10 pg / mL and kanamycin 50 pg / mL) and cultured at 28°C overnight with shaking at 220 rpm. The bacterial culture was centrifuged at 4000 rpm for 10 minutes at 21 °C, and the pellet was resuspended in the cocultivation medium (TABLE 3). The Agrobacterium suspension was then incubated at room temperature in an orbital shaker at 60 rpm for about 2 hours in the dark, and then OD600 of the cultures was adjusted to 0.5 before infection. For co-transformation with two constructs, equal volumes with equal OD600 of each culture were mixed before transformation.

[0199] Agrobacterium mediated transformation of germinating embryos: Isolated embryonic axes (EAs) were mixed with 15 L Agrobacterium suspension in a petri dish and sealed with parafdm. The sealed plate was subjected to sonication in water (Sonicator, Fisher Scientific Model-FS6) for 1 minute. After sonication, another 15 mL (A Agrobacterium suspension was added to each plate in a laminar flow hood. The plate was covered with foil and kept on a shaker at 60 rpm for 2 hours at room temperature. After inoculation, excess bacterial suspension was removed and EAs were transferred to a single layer of autoclaved sterile filter paper in a 15 * 100 mm petri dish. The filter paper was moistened with 700 pL of co-cultivation (CC) medium. Plates were sealed with parafilm and kept in a 22°C transparent incubator for 3 days under low light (10 pmol m-2 s— 1 ). After 3 days of cocultivation, EAs were rinsed with co-cultivation medium containing cefotaxime (200 pg / mL) and timentin (100 pg / mL), and the base of each embryonic axis was embedded in Growth medium I (GM I) (TABLE 3) at 24°C with a 16 / 8-hour light / dark cycle. After two weeks, the apical primary stem was trimmed from each explant and transferred to Growth medium II (GM II) (FIG. 6B; TABLE 3) to facilitate the emergence of transformed pink shoots.

[0200] TABLE 3: Media compositions for soybean transformation Attorney Docket No. 09531-0530W01 2023-264

[0201] ^Growth media I and II were modified based on Cho et al., Plant Biotechnol. J., 20(5):977- 990, 2022.

[0202] Hardening acclimatization and plant growth condition: Soybean plantlets with transgenic shoots were removed from plant tissue culture containers and gently washed to remove agar gel without damaging the roots. Plants were transplanted to 4-inch biodegradable peat pots containing potting mix soil and covered with transparent plastic covers to maintain humidity. After 10-14 days, the covers were gradually removed. After 15 days, the 4-inch pots were transferred into 5-gallon pots. The plants were grown in the growth chamber with temperature maintained at 26°C, 55% relative humidity, and a 16 / 8- hour light / dark cycle until maturity. Plants were continuously watered and fertilized at one- week intervals.

[0203] DNA extraction, PCR genotyping, and gene editing mutation detection: DNA was extracted from the TO and T1 plants using the bead grinder and cetyltrimethylammonium bromide (CTAB) method (Allen et al., Nat. Protoc., 1 :2320-2325, 2006). Polymerase chain reaction (PCR) was performed from the genomic DNA with the oligonucleotides listed in TABLE 4 using Q5 high fidelity DNA Polymerase as per the manufacturer’s protocol (New England Biolabs; Ipswich, MA). For TO plants, PCR amplicons of the P34 gene target site were sequenced using Illumina paired-end read sequencing (Genewiz Inc.; South Plainfield, New Jersey, USA). Sequencing reads and mutation rates were analyzed by CRISPResso2 Attorney Docket No. 09531-0530W01

[0204] 2023-264 using the default setting for CRISPR-Cas9 analysis (Clement et al., Nat. Biolechnol.. 37:224- 226, 2019). To detect mutations in T1 plants, the PCR product from the P34 gene of each plant was subjected to Sanger sequencing. The samples showing double peaks near the target site were further confirmed by Nanopore-sequencing (Plasmidsaurus, OR) and analyzed by CRISPResso2 (Clement et al., supra). The same P34 PCR primers also were used as the endogenous gene control for T1 plant screening, as the CRISPR is unlikely to disrupt the primer binding sites of this gene, based on the mutation profile from the TO parent line (FIG. 7B).

[0205] TABLE 4: List of oligo sequences.

[0206] Sample collection and RNA-seq: Explants transformed with vectors encoding WUS2, IPT, or WUS2UPT, as well as an Empty Vector (EV), were subjected to sampling. Infection, co-cultivation and transfer of embryonic axes were carried out as described above. At zero days, three days, or six days after transformation, shoot regions (4-6 mm) were excised from corresponding explants and immediately frozen in liquid nitrogen for RNA Attorney Docket No. 09531-0530W01 2023-264 extraction. For each time point, five excised shoot samples were pooled for one biological replicate, and three biological replicates were collected for each treatment. Total RNA was extracted using a RNeasy Plant Mini Kit (Qiagen; Redwood City, CA) according to the manufacturer’s protocol, including an on-column DNase digestion. Library preparation and Illumina sequencing were carried out at Novogene using the NovaSeq 6000 PE150 (Novogene; Sacramento, CA).

[0207] RNAseq data analysis: Raw reads were processed using fastp (0.23.2) with the “- detect_adapter_for_pe”, “-trim_poly_x”, and “-trim_poly_g” parameters to remove adapters and filter bad reads. Filtered reads were aligned to the Glycine max reference genome (Wm82.a4.vl) downloaded from phytozome-next.jgi.doe.gov using kallisto (0.48.0). Kallisto abundance files were used to perform differential analysis in R using DESeq2 (1.44.0) with a threshold of p.adj < 0.05. Variance stabilized transformation (VST) values for gene expression were used for sample-wise PCA, distance matrix plotting, and hormonal PCA. Z- scaled VST expression values were used for k-means clustering and visualization and venn diagrams. Heatmaps were generated using the “pheatmap” function in ComplexHeatmap (2.20.0). Hormonal PCA plot was generated using the “fviz_pca_biplof ’ function in factoextra (1.0.7).

[0208] Data availability: The gene IDs or accession numbers included A / WUS2 (AT2G17950.1), GmWUS2 (Glyma.01G166800.1), AtGRF4 (AT3G52910), GmGRF4 (Glyma.l2G014700. 1), TaGRF4 (TraesCS6A01G269600), and the soybean P34 gene (Glyma.08Gl 16300). Sequence information for the DRs is available in FIGS. 5A-5C. Sequence information for the oligos is available in TABLE 4. Next-Generation sequencing data generated in these studies were deposited in the NCBI Sequence Read Archive (SRA) under accession code PRJNA1219159. RNA-seq data were deposited under accession code PRJNA1218798. Processed CRISPResso2 output files for each sample are available from github . com / ZhangLab-UMN / SoybeanDR_NGS .

[0209] Results

[0210] Synergistic effect of WUS2 and IPT induces de novo shoot formation in soybean: To deliver DR genes into soybean, a series of T-DNA vectors were constructed, each containing three distinct functional modules: a DR expression cassette, a CRISPR-Cas9 Attorney Docket No. 09531-0530W01 2023-264 system, and a visual marker gene cassette (FIG. 8A). Within each T-DNA, one of three DR coding sequences was individually cloned: WUS2, IPT, or GRF4-GIF1. For WUS2, the maize WUS2 sequence was used to search against the soybean genome and identify the homologous gene, GmWUS2 (FIG. 9A). For GRF4-GIF1, the reported sequence from wheat was used to search against the soybean genome and identify the homologous genes, GmGRF4 and GmGIFl (Kong et al., Front. Plant Set., 11 : 572319, 2020; FIG. 9B). For IPT, sequences reported elsewhere (Maher et al., Nat. Biotechnol., 38:84-89, 2020) were used. The CRISPR- Cas9 module included guide RNAs targeting the soybean major allergen gene P34 (Glyma.08Gl 16300) (Joseph et al., Crop Set., 46: 1755-1763, 2006). For non-invasive, realtime monitoring of transgenic events, the RUBY reporter gene was selected as a visual marker (He et al., Hortic. Res., 7: 1-6, 2020; FIG. 8A). The T-DNA constructs were transformed either individually or in combinations as listed in TABLE 5.

[0211] While various explant types have been used for soybean transformation, including cotyledonary nodes from immature and mature seeds, half-seeds, hypocotyls, primary nodes, immature embryos, and mature embryonic axis (see, e.g., Xu et al., Front. Plant Set., 13 :900318, 2022), 2-day germinating embryos (embryonic axis or EA) were chosen for these studies based on two key considerations: (1) during primary shoot development, the DRs could promote de novo axillary shoot formation on the explants (Maher et al., supra), and (2) the transformed embryos could continue to grow into plantlets without exogenous growth hormones. The transformation protocol involved isolating germinating embryos from imbibed mature seeds, followed by transformation using Ayrobacterium strain AGL1 carrying the T-DNA constructs (FIG. 8B). After a three-day co-cultivation period, the treated embryos were cultured on Growth medium I and II (TABLE 3) without growth hormones or selection reagents. The transformed embryos germinated and produced multiple shoots, averaging 6-8 shoots per explant. A subset of these explants exhibited 1-2 emerging RUBY- colored shoots (FIG. 8B). The plantlets with RUBY-colored shoots were then transplanted to soil and grown as TO plants to maturity, resulting in T1 seed production (FIG. 8B). These transgenic shoots were readily identified by their RUBY coloration throughout development. This hormone-free approach shortened the transformation timeline to 9-11 weeks from initial embryo isolation to T1 seed production, making it about 25-45% faster than current methods that require extensive tissue culture (FIG. 6A). Attorney Docket No. 09531-0530W01

[0212] 2023-264

[0213] The transgenic shoot formation frequency with DR genes in the soybean variety Williams 82 was evaluated. The frequency was calculated as the percentage of explants developing RUBY -colored shoots relative to the total number of treated explants. In single-DR treatment groups, the transformation frequencies were 3.3% with WUS2, 6.4% with IPT, and 0% with GRF4-GIF, demonstrating that individual DRs - particularly IPT - could induce de novo shoot formation (TABLE 5). However, the dual-DR combination of WUS2 and IPT exhibited substantially higher transformation frequency, averaging 22.3%, while the WUS2 and GRF4-GIF1 combination showed 5.8% frequency. These results indicated that DR combinations including WUS2 significantly enhanced transgenic shoot formation, with the WUS2HPT combination substantially outperforming all other DR groups (TABLE 5). To validate this finding across genotypes, the WUS2HPT combination was appled to another soybean variety, Bert. Bert was chosen as a Group I maturity soybean line mature with improved disease resistance, yield potential, and agronomic traits (Orf and Kennedy, Crop Sci., 32(3):830, 1992). Bert has been used in studies that investigated the mechanism and genomic impacts of Agrobacterhim-based transformation of soybean (Olhoft et al., Vitro Cell Dev. Biol. - Plant, 43:536-549, 2007; Michno et al., BMC Biotechnol. , 20: 10, 2020; and Liu et al., Plant Genome, 18: e70056, 2025), making it an excellent genotype to compare with Williams 82 for testing the methods described herein. These studies demonstrated a slightly lower but comparable transformation frequency of 14.6% when using Bert.

[0214] Among the transgenic shoots, varying intensities of RUBY coloration were observed in leaves, flower petals, seed pods, and seed coats (FIG. 7A). To confirm that the RUBY- colored shoots were genuine transformation events, nine TO plants were sampled and the presence of transgenes was verified using polymerase chain reaction (PCR) (FIG. 7B). Additionally, using DR gene-specific primers, it was found that all nine TO plants contained WUS2 and IPT transgene sequences (FIG. 10A). All RUBY-positive shoots from these TO plants produced seeds. However, the RUBY-colored seed pods and seeds were noticeably smaller compared to those from non-transgenic sibling shoots (FIG. 7A). To assess transgene inheritance, 10 T1 seeds collected from two independent RUBY-positive TO plants were grown, and PCR-based genotyping was performed. These studies revealed differential transgene transmission rates, with 8 of 10 T1 plants from one TO parent and 3 of 10 T1 plants from the other TO parent showing successful transgene inheritance (FIGS. 7B and 10B). Attorney Docket No. 09531-0530W01 2023-264

[0215] TABLE 5: DR-mediated soybean transformation frequency

[0216] *S.D.: Standard deviations were calculated separately for each transformation group. Heritable CRISPR-Cas9 mutations generated from DR-induced shoots: In addition to evaluating DR gene inheritance, the efficiency of CRISPR-mediated genome editing was assessed, as each DR-expressing T-DNA construct contained a CRISPR-Cas9 cassette targeting the P34 allergen gene. A 20-bp CRISPR guide RNA sequence (GGCCAACAAGAAAATGAAGAA; SEQ ID NO: 15) was used, which was validated to target exon 1 of the single copy P34 gene (Glyma.08Gl 16300) as described elsewhere (Liu et al., supra). The target regions were PCR amplified from nine individual TO plants and analyzed using Next Generation Sequencing. Sequence analysis revealed that 6 of 9 TO Attorney Docket No. 09531-0530W01 2023-264 plants carried editing events, with efficiencies ranging from 0.1% to 34.5% (FTG. 11A). To examine mutation heritability, 10 T1 plants derived from event T0-5 (which showed a 26.3% mutagenesis rate) were screened using PCR and Sanger sequencing (FIG. 7C). Among these T1 plants, two heterozygous mutants were identified with a 4-bp deletion at the target site (FIGS. 7C and 11B) These results demonstrated that the transformation method effectively delivered CRISPR-Cas9 and induced heritable mutations in soybean. Together, these findings demonstrated that combining WUS and IPT dramatically enhanceed soybean transformation frequency through promoting de novo shoot formation. By simplifying the transformation process while maintaining high editing efficiency, this method promises to accelerate soybean genetic engineering efforts.

[0217] Transcriptional dynamics of WUS2 / IPT synergy during de novo shoot formation: The synergistic effect of WUS2 and IPT on de novo shoot formation efficiency led to further studies to investigate the transcriptional dynamics induced by these DR genes. Bulk RNA sequencing was performed at 0, 3, and 6 days after transformation (DAT) using four treatments: empty no-DR vector control (EV), WUS2 alone, IPT alone, and WUS2HPT combination. To confirm the expression of both TFT and WUS2 transgenes (FIG. 12), their transcript levels in individual samples were analyzed. Strong IPT transcript accumulation was detected in both IPT and WUS2UPT samples at 3 and 6 DAT, with negligible expression in the empty vector and riTAS'2-only samples. For WUS2, which corresponds to the endogenous soybean locus Glyma.01G 166800 , expression was elevated in both WUS2 and WUS2HPT treatments at 3 and 6 DAT, while only low basal expression was observed for the empty vector and / F7-only samples. These results confirmed transgene expression and activity in this system.

[0218] Principal component analysis (PCA) revealed distinct temporal separation of transcriptional profiles at 3 and 6 DAT (FIG. 13A). At 3 DAT, samples clustered primarily by treatment with high concordance between replicates (FIG. 13B). By 6 DAT, samples were segregated into two distinct clusters: one comprising EV and WUS2 treatments, and another containing IPT and WUS2UPT treatments (FIG. 13C). This clustering pattern was further supported by distance matrix analysis (FIG. 13D). To delve into these expression patterns, K-means clustering of differentially expressed genes (DEGs) was performed, revealing five distinct clusters at 3 DAT and two at 6 DAT (FIGS. 14A and 14B). At 3 Attorney Docket No. 09531-0530W01 2023-264

[0219] DAT, synergistic expression of genes regulating distinct yet interconnected pathways associated with early DR response was identified. Gene Ontology (GO) enrichment analysis revealed that genes involved in cell cycle progression, hormone response, cell wall remodeling, and meristematic tissue establishment were highly upregulated with WUS2IIPT treatment (Clusters 3 and 4; FIG. 14A), suggesting initiation of somatic cell reprogramming toward a regenerative state. Specifically, genes involved in DNA replication and cytokinin pathway activation were significantly upregulated with WUS2UPT treatment. In contrast, stress response, defense, and light-responsive genes were highly upregulated with EV, IPT, and WUS2 treatments (Clusters 1 and 2; FIG. 14A). Notably, coordinated expression of WUS2UPT not only suppressed stress and defense response genes, but also promoted cell cycle activation and hormone response (FIG. 15A).

[0220] The transcriptional landscape evolved further at 6 DAT, where two major clusters emerged, with ZPZ and WUS2HPT showing similar DEG patterns that were distinct from the patterns for WUS2 and EV (FIG. 14B). This was consistent with the observation that IPT played a dominant role in activating genes involved in cytokinin signaling and meristem regulation (Nguyen et al., Plant Biotechnol. J., 19: 1297-1313, 2021). Key genes involved in stem cell activation, including RGI5, ATHB-15, and cytokinin targets such as ARR9, ARR2, and KNAT, were upregulated with WUS2UPT treatment (Baima et al., Plant Physiol., 126:643-655, 2001; Dai et al., J. Integr. Plant Biol., 59:747-758, 2017; and Fernandez et al., Nat. Plants, 6:533-543, 2020). Additionally, negative regulators of gibberellin (GA) response, including DELLA subfamily GRAS regulatory genes (GAI and RGLT), were upregulated with both WUS2HPT and IPT treatments. Interestingly, brassinosteroid- associated genes (FER, BAK1, EXO, and THE!) were downregulated with WUS2HPT treatment compared to EV at both timepoints (FIG. 14B) (Hamant et al., Plant Physiol., 130:657-665, 2002).

[0221] To further understand the role of hormone signaling in this process, a targeted PC A of hormone-responsive genes was conducted (FIGS. 14C and 15B). This analysis revealed a clear temporal transition in hormone profiles. At 3 DAT, samples clustered closely and showed strong associations with stress-related hormones, including jasmonic acid (J A), abscisic acid (ABA), and ethylene, suggesting an initial biotic stress response to Agrobacterinm co-cultivation (FIGS. 14C and 15B). By 6 DAT, a marked shift occurred: Attorney Docket No. 09531-0530W01 2023-264

[0222] WUS2UPT and IPT treatments showed stronger induction of auxin and cytokinin pathways that are known to be associated with de novo shoot development (FIGS. 14C and 15B). This temporal shift from stress response to regenerative processes highlighted the dynamic role of DRs in orchestrating both initial stress adaptation and subsequent developmental reprogramming. Notably, the distinct hormone profdes between WUS2UPT and other treatments at 6 DAT aligned with their different regeneration efficiencies, highlighting the importance of hormone balance in shoot formation. Together, the transcriptional analyses revealed that WUS2IIPT expression initiates a complex developmental program: it initially prepares explants for tissue dedifferentiation by modulating stress responses and cell fate genes, and then promotes de novo shoot formation through coordinated regulation of meristem activity and growth hormone signaling networks (FIG. 14D).

[0223] Example 3 - DR-mediated transgenic shoot formation in common bean The methods used as described in the Examples above for soybean were used to transform common bean (variety UI111) with Agrobacterium containing T-DNA constructs encoding gmWUS and IPT and an AmCyan reporter (SEQ ID NO:48; FIG. 21). These studies resulted in TO transgenic common bean shoots (e.g., as indicated by the arrow in FIG. 16A), which exhibited green fluorescence under UV illumination. A close-up view of leaves from a TO wild type plant and a TO transgenic plant is shown in FIG. 16B. The leaf from the transgenic plant displayed a green fluorescent signal, indicating expression of the AmCyan reporter gene in the transgenic leaves, in contrast to the non-fluorescent wild-type leaf. PCR analysis using primers specific to the T-DNA construct confirmed the presence of the transgene in TO plants (FIG. 16C).

[0224] OTHER EMBODIMENTS

[0225] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Attorney Docket No. 09531-0530W01 2023-264WHAT TS CLAIMED IS:

1. A method for generating plant cells comprising one or more genetic modifications of interest, the method comprising:(a) introducing, into a germinating embryo of a leguminous plant: (i) nucleic acid encoding one or more developmental regulators that, when expressed in cells of said germinating embryo, induce new meristem formation, and (ii) nucleic acid comprising one or more coding sequences that, when expressed in cells of said new meristem, edit DNA in cells of said new meristem to introduce one or more genetic modifications of interest, wherein the introducing comprises infection of the germinating embryo with an Agrobacterium.,(b) incubating said germinating embryo with said Agrobacterium, such that said one or more developmental regulators and said one or more coding sequences within the nucleic acid of (ii) are expressed;(c) growing a plantlet developed from the germinating embryo of step (b) in a growth medium to induce shoot formation; and(d) identifying one or more shoots having said one or more genetic modifications of interest.

2. The method of claim 1, wherein the introducing comprises incubating said germinating embryo with s A Agrobacterium for about 3 days.

3. The method of claim 1, wherein the incubating comprising incubating said germinating embryo with said Agrobacterium for about 1 day to 5 days.

4. The method of claim 1, wherein the growing comprises growing said plantlets in a growth medium for about 1 week to about 8 weeks.

5. The method of claim 1, wherein the growing comprises growing said plantlets in a growth medium for about 4 weeks.

6. The method of claim 1, wherein the method further comprises introducing, into said germinating embryo, (iii) nucleic acid encoding a reporter.

7. The method of claim 6, wherein said reporter comprises a RUBY polypeptide.Attorney Docket No. 09531-0530W01 2023-2648. The method of claim 6 or claim 7, wherein the method further comprises identifying plantlets in which said reporter is expressed.

9. The method of claim 1, wherein said growth medium is a plant hormone-free growth medium.

10. The method of claim 1, wherein said one or more developmental regulators comprise Wushel (WUS) and isopentenyl transferase (IPT).

11. The method of claim 10, wherein said one or more developmental regulators further comprise one or more of Baby Boom, Irrepressible Variants of Monopteros, Shoot Meristemless, Leafy Cotyledon, WUS homeobox-containing, and APETALA2 / Ethylene Responsive Factor.

12. The method of claim 1, wherein said Agrobacterium comprises Agrobacterium tumefaciens strain AGL-1, A. tumefaciens strain C58, A. tumefaciens strain GV3101, A. tumefaciens strain LBA4404, A. tumefaciens strain EHA 105, or A rhizogenes.

13. The method of claim 1, wherein said Agrobacterium is tumefaciens strain AGL-1.

14. The method of claim 1, wherein said one or more sequences that, when expressed, edit the DNA in cells of said new meristem, comprise a nucleotide sequence encoding a targeted endonuclease, and wherein the targeted endonuclease comprises a Clustered Regularly-Interspaced Short Palindromic Repeats-associated nuclease, a Transcription Activator-Like Effector Nuclease, a meganuclease, or a zinc finger nuclease.

15. The method of claim 14, wherein said targeted endonuclease is a Clustered Regularly-Interspaced Short Palindromic Repeats-associated (Cas) nuclease.

16. The method of claim 1, wherein said one or more sequences that, when expressed, edit the DNA in cells of said germinating embryo comprise (1) a nucleotide sequence encoding a targeted endonuclease and (2) a repair template.

17. The method of claim 1, wherein said shoots are meristematic and are capable of deriving new plant tissue carrying said one or more genetic modifications of interest.Attorney Docket No. 09531-0530W01 2023-26418. The method of claim 1, further comprising (e) culturing the shoots identified in step (d) in soil for about 5 days to 20 days to obtain modified plant tissue comprising said one or more genetic modifications of interest.

19. The method of claim 1, wherein said leguminous plant is selected from the group comprising soybean, kidney bean, black bean, pinto bean, peanut, chickpea, lentil, pea, common bean, cowpea, pigeon pea, peanut ground nut, and faba bean.

20. The method of claim 19, wherein said leguminous plant is soybean.

21. A method for increasing transformation efficiency in a leguminous plant, the method comprising:(a) introducing, into cells of a germinating embryo of a leguminous plant, (i) nucleic acid encoding one or more developmental regulators that, when expressed in cells of the germinating embryo, induce new meristem formation, wherein said introducing comprises infection of said germinating embryo with a Agrobacterium, and(b) co-cultivating said germinating embryo and Agrobacterium such that said one or more developmental regulators are expressed.

22. The method of claim 21, wherein said method has a transformation efficiency of about 15% to about 40%.

23. A method for accelerating generation of a transgenic leguminous plant, the method comprising:(a) introducing, into a germinating embryo of a leguminous plant, (i) nucleic acid encoding one or more developmental regulators that, when expressed in cells of the germinating embryo, induce new meristem formation, and (ii) nucleic acid comprising a transgene, wherein the introducing comprises infection of the germinating embryo with an Agrobacterium,(b) co-cultivating said germinating embryo and said Agrobacterium such that said one or more developmental regulators are expressed; andAttorney Docket No. 09531-0530W01 2023-264(c) growing a plantlet developed from the germinating embryo of step (b) in a growth medium to induce shoot formation, wherein at least one shoot comprises genomic DNA that comprises the transgene.

24. The method of any one of claims 21-23, comprising co-cultivating said germinating embryo and said Agrobacterium for about 3 days.

25. The method of claim 23, comprising growing said plantlet in said growth medium for about 4 weeks.

26. The method of claim 23, wherein said growth medium is a plant hormone-free growth medium.

27. The method of any one of claims 21-23, wherein said developmental regulators comprise one or more of Wushel (WUS), isopentenyl transferase (IPT), Baby Boom, Irrepressible Variants of Monopteros, Shoot Meristemless, Leafy Cotyledon, WUS homeobox-containing, and APETALA2 / Ethylene Responsive Factor.

28. A germinating leguminous plant embryo comprising:(i) exogenous nucleic acid encoding one or more developmental regulators that, when expressed in cells of said germinating embryo, induce new meristem formation, and(ii) exogenous nucleic acid comprising one or more coding sequences that, when expressed in cells of said new meristem, edit DNA in cells of said new meristem to introduce one or more genetic modifications of interest.

29. The germinating leguminous plant embryo of claim 28, wherein said leguminous plant is selected from the group comprising soybean, kidney bean, black bean, pinto bean, peanut, chickpea, lentil, pea, common bean, cowpea, pigeon pea, peanut ground nut, and faba bean.

30. The germinating leguminous plant embryo of claim 28, wherein said germinating embryo further comprises (iii) exogenous nucleic acid encoding a reporter.

31. The germinating leguminous plant embryo of claim 30, wherein said reporter comprises a RUBY polypeptide.Attorney Docket No. 09531-0530W01 2023-26432. The germinating leguminous plant embryo of claim 28, wherein said germinating leguminous plant embryo is in a plant hormone-free growth medium.

33. The germinating leguminous plant embryo of claim 28, wherein said one or more developmental regulators comprise Wushel (WUS) and isopentenyl transferase (IPT).

34. The germinating leguminous plant embryo of claim 33, wherein said one or more developmental regulators further comprise one or more of Baby Boom, Irrepressible Variants of Monopteros, Shoot Meristemless, Leafy Cotyledon, WUS homeobox-containing, and APETALA2 / Ethylene Responsive Factor.

35. The germinating leguminous plant embryo of claim 28, wherein said one or more sequences that, when expressed, edit the DNA in cells of said new meristem, comprise a nucleotide sequence encoding a targeted endonuclease, and wherein the targeted endonuclease comprises a Clustered Regularly-Interspaced Short Palindromic Repeats- associated nuclease, a Transcription Activator-Like Effector Nuclease, a meganuclease, or a zinc finger nuclease.

36. The germinating leguminous plant embryo of claim 35, wherein said targeted endonuclease is a Clustered Regularly-Interspaced Short Palindromic Repeats-associated (Cas) nuclease.

37. The germinating leguminous plant embryo of claim 28, wherein said one or more sequences that, when expressed, edit the DNA in cells of said new meristem comprise (1) a nucleotide sequence encoding a targeted endonuclease and (2) a repair template.

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