WO2012021494A1 - Method for gene delivery into grain legumes and in vitro regeneration of same - Google Patents

Abstract

The disclosure generally relates to methods for genetically transforming a grain legume, in particular methods that are independent of the selected grain legume species and/or genotype. The methods include processes for both transgene delivery as well as in vitro regeneration of transgenic plant material. The genotype-independent genetic transformation of grain legumes such as common beans and soybeans can include (a) the integration of selected transgene(s) into a specific cell layer of an apical shoot meristem explant of the grain legume, (b) release of apical dominance in the transgenically modified explant in favor of meristem branching, and/or (c) and effective chemical selection and proliferate rooting of multiplied shootlets. The disclosure is further directed to the resulting genetically transformed grain legume plants or components thereof, such as plant seeds.

Description

METHOD FOR GENE DELIVERY INTO GRAIN LEGUMES

AND IN VITRO REGENERATION OF SAME

CROSS-REFERENCE TO RELATED APPLICATION

[0001] Priority is claimed to U.S. Provisional Application No. 61/401 ,335, filed August 1 1 , 2010, the disclosure of which is incorporated herein by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED IN A COMPUTER READABLE FORMAT

[0002] The application contains nucleotide sequences which are identified with SEQ ID NOs. The sequence listing is provided in computer readable form and is incorporated herein by reference in its entirety. The information recorded on the form is identical to the written sequence listing provided with the application.

BACKGROUND OF THE DISCLOSURE

Field of the Disclosure

[0003] The present disclosure is directed to methods for genetically transforming a grain legume as well as the resulting transgenic plant material.

Brief Description of Related Technology

[0004] Grain legumes supplement the human diet and supply dietary protein. Grain legumes typically are rich in lysine but poor in methionine content, thereby complementing the reverse amino acid pattern found in cereal grains. Grain legumes, as nitrogen-fixers, also generally reduce the cost of nitrogen inputs for agricultural production (Hymowitz, 1990).

[0005] Common beans as a type of grain legume are a very important source of vegetable protein, especially in those regions of the world in which animal and fish protein is scarce. Common beans satisfy 22 % of the total protein requirement worldwide and account for over 50 % of all legumes consumed globally. Conventional breeding has contributed singularly to the improvement of cultivated common beans. Whilst plant breeding has contributed to the much-needed genetic variation necessary for trait improvement, certain genes that can add to the value of agronomic traits in common beans do not exist naturally in its gene pool. Due to this limitation of plant breeding, new trait improvement approaches such as interspecific horizontal gene transfer via genetic engineering can complement the limitations encountered by conventional breeding (Aragao et al. 1996, 1998, 2002).

[0006] Soybean as a food, feed, biofuel and bioindustrial crop is planted in the United States in 72.7 million acres (the same acreage used for planting corn) with 12.5 billion dollar sales. Certain elite inbred lines of soybean are very difficult to genetically transform. Like other mono-cultured crops, soybean plants are susceptible to a large set of biotic and abiotic stresses and are subjected to genetic improvements. However, traditional methods of soybean transformation are very genotype dependent and do not target undifferentiated meristem cells in the apical shoot meristem primordium. For example, when using differentiated apical shoot meristem explants, all transgenic soybean plants developed were chimerically transformed or certain parts of transgenic plants were not transgenic (Christou 1992).

[0007] Abiotic stresses, including drought, salinity and high temperatures, pose a major obstacle for crop yield and production, with more than 90% of arable land experiencing one or more of these stresses. In an effort to overcome or reduce these stress factors, plants have evolved to adapt by synthesizing low molecular weight osmolytes. Drought and salt stresses share a similar pathway. When drought occurs or high salt content is present, ionic and osmotic homeostasis of cells becomes unbalanced. As a result, plants lose cellular turgidity, followed by the aggregation and misfolding of proteins.

[0008] Late embryogenesis abundant (LEA) proteins are a class of Heat shock proteins (Hsp) that are extremely hydrophilic and resilient towards heat, such that they do not coagulate at boiling temperatures (e.g., the HVA1 gene from barley that encodes a type III LEA protein). These proteins may play a role in water binding, ion sequestration and macromolecule and membrane stabilization.

[0009] Sclerotinia sclerotiorum is the most devastating necrotrophic soil borne pathogen in the temperate region. Species of this pathogen cause both southern stem rot and white mold which result in the rotting of both seedling and pod in most grain legumes, especially in common bean, soybean, sunflower, lentils and peanut. This pathogen accounts for annual agricultural losses in the United States alone of more than $200 million.

[0010] Oxalic acid (ethanedioic acid), has been implicated as the main pathogenicity factor of S. sclerotiorum. During the early stages of pathogenesis, oxalic acid accumulates in host plant infected tissue. As the oxalic acid concentration increases, it lowers the pH to 4 or 5 (Bolton et al. 2006). It is this low pH produced by oxalic acid that allows S. sclerotiorum to escape the inhibitory action of innate plant defense system mediated by cell-wall- associated glycoproteins such as polygalacturose-inhibiting protein (PGIPs) (Favaron et al. 2004, Zuppini et al. 2005). In addition, oxalic acid chelates, calcium and pectic material allows polygalacturonase to hydrolyze pectates and disrupt the integrity of host cell walls. As a consequence, calcium-dependent plant defense response, production of polyphenol oxidases, and the oxidative burst are compromised due to the action of oxalic acid (Cessna et al. 2000, Bolton et al. 2006).

[0011] Germin is an oxalate oxidase (G-OXO) which degrades oxalic acid into C0₂ and H₂0₂ (Schmitt 1991 ). H₂0₂ promotes localized hypersensitive response (HR) cell death, but most importantly, H₂0₂ is toxic to the oxalate producing pathogens such as S. sclerotiorum. H₂0₂ also promotes lignification and cross linking of cell walls, which provides a barrier against invading fungal pathogens. Germin-OXO is able to liberate chelated Ca²⁺ bound by oxalic acid (Apostol et al. 1989). As a consequence, the germin gene helps to promote plant defense against fungal pathogens such as S. sclerotiorum. Isoforms of the germin gene are present in all cereals, such as the wheat isoform of G-OXO (gf2.8). This is why most cereals are not susceptible to oxalate producing fungi such as S. sclerotiorum.

SUMMARY

[0012] The disclosure relates to a method for genetically transforming a grain legume, the method comprising: (a) providing a shoot apical meristem primordial of a grain legume; and (b) delivering one or more transgenes into the shoot apical meristem primordial of the grain legume, thereby forming a transgene-modified shoot apical meristem primordial. In an embodiment, the shoot apical meristem primordial can comprise undifferentiated

subepidermal meristem tissue to which the one or more transgenes are delivered in part (b). The one or more transgenes can be delivered only undifferentiated stem cell tissue in the subepidermal meristem tissue. The shoot apical meristem primordial to which the one or more transgenes are delivered in part (b) can exclude differentiated cells.

[0013] Various refinements and embodiments of the disclosed methods are possible. For example, the grain legume is selected from the group consisting of beans, peanuts, lentils, peas, and chickpeas. In various embodiments, the grain legume is a common bean (e.g., having a genotype selected from the group consisting of red beans, navy beans, white kidney beans, black beans, pink beans, red kidney beans, pinto beans, and great northern beans) or a soybean. In another embodiment, the one or more transgenes are selected to impart one or more transgenic traits selected from the group consisting of herbicide resistance, pesticide resistance, fungal resistance, microbial resistance, salt tolerance, drought tolerance, temperature tolerance, and combinations thereof. In another

embodiment, the one or more transgenes are selected from the group consisting of a gus (β- glucuronidase) gene, a bar gene, an HVA1 (barley Hordeum vulgare LEA3) gene, a g†2.8 (wheat germin) gene, and combinations thereof. In another embodiment, the delivered transgene is stably integrated into the transgene-modified shoot apical meristem primordial. In various embodiments, delivering the one or more transgenes can comprise performing a gene bombardment process (e.g., having a transformation efficiency of at least 2%) or an Agrobacterium- mediated transformation process. In a refinement, providing the shoot apical meristem primordial in part (a) comprises: (i) sterilizing seeds of a grain legume plant; (ii) dissecting the seeds and excising seed embryos therefrom; (iii) excising the shoot apical meristem primordial from the excised seed embryos; and (iv) optionally regenerating in vitro the shoot apical meristem primordial (e.g., by incubating/culturing the shoot apical meristem primordial in a culture medium comprising an anti-oxidant and optionally one or more plant hormones).

[0014] In one aspect, an extension of the disclosed methods further comprises (c) regenerating in vitro the transgene-modified shoot apical meristem primordial of part (b). The in vitro regeneration of the transgene-modified shoot apical meristem primordial can comprise releasing apical dominance in the transgene-modified shoot apical meristem primordial and growing multiple shoots of a transgene-modified grain legume from the transgene-modified shoot apical meristem primordial, for example such that growing multiple shoots provides one or more non-chimeric transgene-modified grain legume shoots. In an extension, the method further comprises (d) growing the transgene-modified grain legume shoots to provide a rooted transgenic grain legume plant comprising a DNA encoding the one or more transgenes, wherein the transgenic grain legume plant is capable of expressing the one or more transgenes, for example such that the transgenic grain legume plant is non- chimeric and/or the DNA encoding the one or more transgenes is stably integrated into the chromosomes of the transgenic grain legume plant. In an embodiment, in vitro regeneration of the transgene-modified shoot apical meristem primordial comprises incubating the transgene-modified shoot apical meristem primordial in a culture medium comprising an antioxidant. The anti-oxidant can be selected from the group consisting of silver nitrate, ascorbic acid, activated charcoal, and glutathione. The culture medium can further comprise a cytokinin and an auxin. In an embodiment, the in vitro regeneration produces between 6 and 20 shoots per transgene-modified shoot apical meristem primordial explant.

[0015] In another aspect, the disclosure relates to transgenic grain legume or component thereof comprising: (a) a DNA encoding a transgene, wherein the transgenic grain legume or a transgenic grain legume formed from the transgenic grain legume component is capable of expressing the transgene. In an embodiment, the transgenic grain legume or component thereof is a transgenic grain legume plant. In another embodiment, the transgenic grain legume or component thereof is a transgenic seed of the transgenic plant, the transgenic seed comprising a transgene-modified shoot apical meristem primordial comprising the DNA encoding the transgene. In another embodiment, the transgenic grain legume or component thereof is formed according to the foregoing methods in any of their various embodiments. The transgenic grain legume or component thereof can be non-chimeric. In an embodiment, the DNA encoding a transgene is stably integrated into the chromosomes of the transgenic grain legume.

[0016] All patents, patent applications, government publications, government regulations, and literature references cited in this specification are hereby incorporated herein by reference in their entirety. In case of conflict, the present description, including definitions, will control.

[0017] Additional features of the disclosure may become apparent to those skilled in the art from a review of the following detailed description, taken in conjunction with the examples, drawings, and appended claims, with the understanding that the disclosure is intended to be illustrative, and is not intended to limit the claims to the specific embodiments described and illustrated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] For a more complete understanding of the disclosure, reference should be made to the following detailed description and accompanying drawing wherein:

[0019] Figure 1 illustrates an apical shoot meristem.

[0020] Figure 2 is a linear map of the pACT1 F cassette (not drawn to scale) used as a selectable marker for transformation of β-glucuronidase (gus) into common beans: rice actin promoter (Act), gus gene (UidA), and nopaline synthase terminator (Tnos).

[0021] Figure 3 is a linear map of the pBY520 cassette (not drawn to scale) used for the transformation of common bean with the HVA1 gene conferring drought and salt stress tolerance as well as the bar gene as a selectable marker: rice actin promoter (Act1 ), barley or Hordeum vulgare (HVA1 ) LEA3 gene, Cauliflower Mosaic Virus 35S promoter (35S), bar gene (bar), and nopaline synthase terminator (Nos).

[0022] Figure 4 is a circular map of the 6.9 Kb of pBKSbar/gf2.8 (not drawn to scale) used for the transformation of common bean with the gf2.8 gene conferring white mold resistance and the bar gene as a selectable marker: ampicilin resistant marker (Amp), herbicide selectable marker (bar), origin of replication of the pUC 18 plasmid vector (pUC ori). [0023] Figure 5 is a linear map of the pCAMBIA3301 T-DNA cassette (not to scale) used for Agrobacterium transformation of common bean with the gus and bar genes as selectable markers (a variant of this construct was also used with the SLPI gene in place of the bar gene): left/right T-DNA border sequences (LB/RB), CaMV 35S promoter/terminator

(P35S/T35S), coding region of the phosphinothricin resistance gene (bar), nopaline synthase terminator (Tnos), gusA gene coding region with intron sequence (gus-intron).

[0024] Figure 6 illustrates in vitro regeneration methods according to the disclosure.

[0025] Figure 7 illustrates the effect of co-cultivation period (1 , 5, 10 and 15 d) on the transient transformation frequency for the gus gene of two genotypes of common bean (Matterhorn and Sedona) using three different strains of A. tumefaciens (EHA105, GV3301 and LBA4404).

[0026] Figure 8 illustrates the effect of using different strains of A. tumefaciens (EHA105, GV3301 and LBA4404) with two common bean genotypes (Matterhorn and Sedona) on the relative stable transformation frequency for the gus gene of ΤΊ (second generation) plants after 15 days of co-cultivation.

[0027] Figure 9 illustrates PCR results of T₃ transgenic plants Montcalm (Mon), Condor (Con), Sedona (Sed), and Matterhorn (Mat) in comparison to wild type (Wt) plants, where the expected band size is 670 bp for the HVA1 transgene.

[0028] Figure 10 is a Southern blot showing integration of the HVA1 gene in Condor (Co), Montcalm (Mo), Sedona (Se), Matterhorn (Ma), and wild type (Wt) plants digested with BamH1 .

[0029] Figure 1 1 illustrates the Northern blot expression of the HVA1 gene from T₃ transgenic plants subjected to drought stress: Montcalm (Mon), Condor (Con), Sedona (Sed), and Matterhorn (Mat) in comparison to wild type (Wt) plants.

[0030] Figure 12 illustrates the relative rate of infection and development spread of fungal pathogen as measured by lesion size on the leaf surface of T₂ Matterhorn, Sedona, Olathe, Condor, and wild type plants.

[0031] While the disclosed methods, compositions, and resulting transgenic plant materials are susceptible of embodiments in various forms, specific embodiments of the disclosure are illustrated in the drawings (and will hereafter be described) with the understanding that the disclosure is intended to be illustrative, and is not intended to limit the claims to the specific embodiments described and illustrated herein. DETAILED DESCRIPTION

[0032] The disclosure is generally directed to methods for genetically transforming a grain legume, in particular methods that are independent of the selected grain legume species and/or genotype thereof (e.g., in terms of the ability of the method to efficiently transform the selected grain legume). The methods include processes for both transgene delivery as well as in vitro regeneration of transgenic plant material. More specifically, the genotype- independent genetic transformation of grain legumes such as common beans and soybeans can include (a) the integration of selected transgene(s) into a specific cell layer of an apical shoot meristem explant of the grain legume, (b) release of apical dominance in the transgenically modified explant in favor of meristem branching (e.g., resulting in

multiplication to remove transgene chimerism), and/or (c) and effective chemical selection and proliferate rooting of multiplied shootlets. The disclosure is further directed to the resulting genetically transformed grain legume plants (or components thereof, such as plant seeds).

Grain Legume Transformation

[0033] The disclosed methods utilize the shoot apical meristem primordial of a grain legume as the target explant tissue for genetic modification. One or more transgenes are then delivered into the shoot apical meristem primordial of the grain legume to form a transgene-modified shoot apical meristem primordial (e.g., transgenes not normally present in wild-type members of the particular grain legume and/or genes not otherwise obtainable by conventional breeding of non-transgenic members of the particular grain legume).

[0034] Figure 1 illustrates a general shoot apical meristem 10 of a plant such as a grain legume. In plants, the shoot apical meristem 10 includes completely undifferentiated meristematic tissue in a small and relatively round bud shape. This round meristem 10 includes multiple different layers, including an epidermal cell layer 12 with cells 12A, a subepidermal cell layer 14 with cells 14A, and inner cell layer 16 (or corpus tissue) with cells 16A. The subepidermal cell layer 14 forms meristems from which eventually the gametes are derived. Both the epidermal and subepidermal cell layers 12, 14 (also collectively termed the tunica) are able to horizontally produce new cells in both directions (e.g., generally laterally relative to a longitudinal direction of meristem growth as illustrated by the arrows in Figure 1 ), thereby maintaining the distinctness of the cell layers. The cells 14A in the subepidermal cell layer 14 layer cells are called stem cells or primordium cells because they can divide indefinitely and reproduce into gametes resulting in fertile plants (i.e., undifferentiated cells capable of differentiation into whole plants). The cells 12A in the epidermal cell layer 12 are undifferentiated cells but are not stem cells, and are outermost layer cells of plant shoot apical meristem. The cells 16A in the inner cell layer 16 grow vertically and horizontally (e.g., in the longitudinal direction of meristem growth and lateral thereto as illustrated by the arrows in Figure 1 ), and its cells are not considered stem cells because these cells are not capable of producing whole plants.

[0035] The grain legume stem cells are targeted for genetic transformation in the disclosed methods, not only for genotype independency, but also because the stem cells multiply fast (i.e., which facilitates the elimination transgene chimerism through removal of non-transgenic tissues). Thus, the shoot apical meristem primordial explant to which the transgenes are delivered suitably includes undifferentiated (e.g., completely undifferentiated) subepidermal meristem cells/tissue. The apical shoot meristem primordial explant can be obtained from a grain legume by any suitable method, such as by excising seed embryos from sterilized seeds of the desired grain legume, followed by excising the apical shoot meristem primordial from the excised seed embryo. The transgenes can be targeted only to the sub-epidermal cells (e.g., where the transgenes are directed only to undifferentiated tissue in a shoot apical meristem explant), for example excluding entry of transgenes into the epidermal cell layer and/or the corpus tissue (e.g., either or both of which can be present in the explant but not the target of transgene delivery). In an embodiment, the explant can exclude differentiated and/or non-stem cells/tissue, for example excluding the epidermal cell layer and/or the corpus tissue from the explant (e.g., where one or both of the epidermal layer and the corpus tissue from a general apical shoot meristem have been removed prior to transgene delivery to promote delivery to the desired subepidermal tissue).

[0036] Any of a variety of grain legumes can be transformed using the disclosed method due to its broad applicability and genotype independence. Example grain legumes include beans, peanuts, lentils, peas, and chickpeas. Various suitable genera (and species) of grain legumes include Vicia (e.g., Faba or broad bean), Vigna (e.g., Aconitifolia or Moth bean, Angularis or azuki bean, mungo or urad bean, radiata or mung bean, umbellatta or ricebean, unguiculata or cowpea), Cicer (e.g., arietinum or chickpea (garbanzo bean)), Pisum (e.g., sativum or pea), Lathyrus (e.g., sativus or Indian pea, tuberosus or tuberous pea), Lens (e.g., culinaris or lentil), Lablab (e.g., purpureus or hyacinth bean), Phaseolus (e.g., acutifolius or tepary bean, coccineus or runner bean, lunatus or lima bean, vulgaris or common bean), Glycine (e.g., max or soybean), Psophocarpus (e.g., tetragonolobus or winged bean), Cajanus (e.g., cajan or pigeon pea), Stizolobium (e.g., spp or velvet bean), Cyamopsis (e.g., tetragonoloba or guar), Canavalia (e.g., ensiformis or jack bean, gladiata or sword bean), Macrotyloma (e.g., uniflorum or horse gram), Lupinus or Lupin (e.g., mutabilis or tarwi, albus or lupini bean), Erythrina (e.g., herbacea or Coral bean).

[0037] Any desired genotype for a given grain legume can be targeted for transgene delivery. For example, representative genotypes for common beans (P. vulgaris) include: (1 ) Merlot, which is a small red bean from middle American gene pool belonging to the race Durango, (2) Seahawk, which is a navy bean from middle American gene pool belonging to the race Mesoamerica, (3) Beluga, which is a white kidney bean from the Andean gene pool belonging to the race Nueva Granada, (4) Condor, which is a black bean from the middle American gene pool belonging to the race Mesoamerica, (5) Sedona, which is a pink bean from the middle American gene pool belonging to the race Durango, (6) Red Hawk, which is a red kidney bean from the Andean gene pool belonging to the race Nueva Granada, (7) Olathe, which is a pinto bean from the middle American gene pool belonging to the race Durango, (8) Matherhorn, which is the great northern bean from the Middle American gene pool belonging to the Race Durango, (9) Montcalm, which is a red kidney bean from the Andean gene pool belonging to the race Nueva Granada, and (10) Jaguar, which is a black bean from the middle American gene pool belonging to the race Mesoamerica.

Transqenes

[0038] The particular transgenes delivered to the shoot apical meristem primordial are not particularly limited; one or more transgenes can be selected for genetic modification to impart any desired trait to the transgenic plant, for example genetic traits not normally present in wild-type members of the particular grain legume and/or not otherwise obtainable by conventional breeding of non-transgenic members of the particular grain legume.

General examples of genetic traits include (i) traits improving the ability of the transgenic plant to survive when subjected to adverse environmental factors (natural or artificial), (ii) traits serving as a genetic marker or indicator that a particular plant is transgenic, (iii) traits providing additional functionality to the transgenic plant, (iv) traits improving the biotic and abiotic resistance of the transgenic plants, (v) traits improving the nutritional quality of the transgenic plants, and/or (vi) traits improving the production of biobased matter in grain legumes. Example transgenic traits providing improved resistance or tolerance to environmental factors include those providing herbicide resistance (e.g., herbicides directed to the control of weed or other plant pests), pesticide resistance (e.g., pesticides directed to the control of insect, rodent, or other animal pests), fungal resistance, microbial resistance, salt tolerance, drought tolerance, and/or temperature tolerance. Example transgenic traits providing additional functionality include transgenes that express one or more lignocellulolytic enzymes (e.g., which can be used to degrade transgenic plant matter not usable as a food source (i.e., non-edible portions of the grain legume) into fermentable sugars such as for use in a biofuel (ethanol) formation process). An example transgenic trait increasing the production of biobased matter includes a late/delayed flowering gene (e.g., FLC) to increase the biomass of the transgenic grain legume.

[0039] An example of a suitable marker for identifying transgenic plant material is the gus (β-glucuronidase) gene from Escherichia co// (e.g., to provide visibly colored transgenic products). Examples of markers that additionally provide resistance to herbicides include the tar gene from Streptomyces hygroscopicus encoding phosphinothricin acetylase (PAT), which confers resistance to the herbicide glufosinate; mutant genes which encode resistance to imidazalinone or sulfonylurea such as genes encoding mutant a form of the ALS and AHAS enzyme (e.g., U.S. Patent 5,773,702); genes which confer resistance to

glycophosphate such as mutant forms of EPSP synthase and aroA; resistance to L- phosphinothricin such as the glutamine synthetase genes; resistance to glufosinate such as the phosphinothricin acetyl transferase (PAT and bar) gene; and resistance to phenoxy propionic acids and cyclohexones such as the ACCAse inhibitor-encoding genes.

[0040] Examples of genes which confer resistance to pests or disease include: genes encoding an oxalate oxidase (G-OXO) wheat isoform of G-OXO (gf2.8) to promote plant defense against fungal pathogens such as Sclerotinia sclerotiorum; genes encoding a Bacillus thuringiensis protein such as the delta-endotoxin (e.g., U.S. Patent 6,100,456); genes encoding lectins; genes encoding vitamin-binding proteins such as avidin and avidin homologs which can be used as larvicides against insect pests; genes encoding protease or amylase inhibitors, such as the rice cysteine proteinase inhibitor and the tobacco proteinase inhibitor; genes encoding insect-specific hormones or pheromones such as ecdysteroid and juvenile hormone, and variants thereof, mimetics based thereon, or an antagonists or agonists thereof; genes encoding insect-specific peptides or neuropeptides which, upon expression, disrupts the physiology of the pest; genes encoding insect-specific venom such as that produced by a wasp, snake, etc.; genes encoding enzymes responsible for the accumulation of monoterpenes. sesquiterpenes, asteroid, hydroxamic acid, phenylpropanoid derivative or other non-protein molecule with insecticidal activity; genes encoding enzymes involved in the modification of a biologically active molecule (U.S. Patent 5,539,095); genes encoding peptides which stimulate signal transduction; genes encoding hydrophobic moment peptides such as derivatives of Tachyplesin which inhibit fungal pathogens; genes encoding a membrane permease, a channel former or channel blocker; genes encoding a viral invasive protein or complex toxin derived therefrom; genes encoding an insect-specific antibody or antitoxin or a virus-specific antibody; and genes encoding a developmental- arrestive protein produced by a plant, pathogen or parasite which prevents disease.

[0041] Examples of genes which confer resistance to environmental stress include, but are not limited to, mtld and HVA 1 (barley Hordeum vulgare LEA3), which are genes that confer resistance to environmental stress factors such as drought and/or salt; rd29A and rdlPB, which are genes of Arabidopsis thaliana that encode hydrophilic proteins which are induced in response to dehydration, low temperature, salt stress, or exposure to abscisic acid and enable the plant to tolerate the stress. Other suitable genes can be found in U.S. Patents 5,296,462 and 5,356,816.

[0042] The transgenic grain legumes can include transgenes for the expression of one or more lignocellulolytic enzymes such as cellulases, hemicellulases, and ligninases.

Cellulases generally include endoglucanases (e.g., E1 beta-1 ,4-endoglucanase precursor gene (e1 ) of Acidothermus cellulolyticus), exoglucanases (e.g., cellobiohydrolase gene (cbhl ) of Trichoderma reesei; dextranase gene of Streptococcus salivarius encoding the 1 ,6- alpha-glucanhydrolase gene) and β-glucosidases (e.g., β-glucosidase gene from Butyrivibrio fibrisolvens or Actinomyces naeslundi). Hemicellulases include enzymes that degrade any type of hemicellulose such as xylan, glucuronoxylan, arabinoxylan, glucomannan and xyloglucan. Ligninases include enzymes which degrade lignins such as lignin peroxidases (e.g., lignin peroxidase gene of Phanerochaete chrysosporium), manganese-dependent peroxidases, hybrid lignin and manganese-dependent peroxidases, and laccases

Transgene Delivery Constructs

[0043] Delivery of a desired transgene into the shoot apical meristem primordial explant can be performed by methods generally know in the art. Nucleic acid constructs (e.g., polynucleotides or oligonucleotides comprising nucleic acid sequences not normally associated in nature and/or in the targeted grain legume; including a deoxyribonucleotide (DNA) or ribonucleotide (RNA) polymer either in single- or double- stranded form) for transgene delivery generally include gene expression cassettes for expression of the desired gene product in the transgenic grain legume. The gene expression cassette generally includes 5' and 3' regulatory sequences as well as a gene nucleotide sequence encoding a gene product, which sequences can be operably linked (e.g., expression of one of the nucleic acid sequences is controlled by, regulated by or modulated by the other nucleic acid sequence, for example where two operably linked sequences are covalently linked, either directly or indirectly, to each other). [0044] A gene includes a discrete nucleic acid sequence responsible for a discrete cellular product and/or performing one or more intracellular or extracellular functions, for example a nucleic acid that includes a portion encoding a protein and optionally encompasses regulatory sequences, such as promoters, enhancers, terminators, and the like, which are involved in the regulation of expression of the protein encoded by the gene of interest. The gene and regulatory sequences may be derived from the same natural source, or may be heterologous to one another. Genes additionally can provide for transcription of functional RNA molecules such as tRNAs, rRNAs, etc. or can define a genomic location for a particular event/function, such as the binding of proteins and/or nucleic acids. A transgene includes an exogenous gene which is expressed by a host cell upon introduction therein and is integrated into the cell's DNA such that the trait or traits produced by the expression of the transgene is inherited by the progeny of the transformed cell. A transgene may be partly or entirely heterologous (i.e., foreign to the cell into which it is introduced) or homologous to an endogenous gene of the host cell (e.g., designed to be inserted (or is inserted) into the cell's genome in such a way as to alter the genome of the cell). A transgene can include one or more transcriptional regulatory sequences and other nucleic acids, such as introns.

[0045] Gene expression includes the conversion of a gene's information into a gene product (e.g., a direct transcriptional product of the gene (such as mRNA, tRNA, rRNA, antisense RNA, ribozyme structural RNA, or any other type of RNA) or a protein produced by translation of an mRNA).

[0046] Techniques used to isolate or clone a gene encoding a desired gene product (e.g., a gene from a different organism encoding a desired enzyme or other protein) are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. Cloning of a gene from genomic DNA, can be performed using a polymerase chain reaction (PCR) process, antibody screening, or expression libraries to detect cloned DNA fragments with shared structural features. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used.

[0047] The gene expression cassette can include (in the 5'-3' direction of transcription) a transcriptional and translational initiation region, a coding sequence for the desired gene product, and a transcriptional and translational termination region functional in plants. The transcriptional initiation region (promoter) can be native or analogous (i.e., found in the native plant) or foreign or heterologous (i.e., not found in the native plant) to the grain legume host. Additionally, the promoter can be the natural sequence or alternatively a synthetic sequence. The transcriptional and translational termination region can be native with the transcription initiation region, can be native with the operably linked polynucleotide sequence of interest, or can be derived from another source. Convenient termination regions are available from the TYplasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. Other sequences can be included in the gene expression cassette to enhance gene expression such as intron sequences and leader sequences. Examples of non-translated leader sequences include leader sequences from Tobacco Mosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AIMV).

[0048] A promoter (or plant promoter) generally includes a polynucleotide that regulates expression of a selected polynucleotide sequence operably linked to the promoter, and which effects expression of the selected polynucleotide sequence in the transgenic plant cells. Constitutive promoters as well as tissue-specific promoters (e.g., directing expression of the gene product to a plant part such as leaf, stem, bean, pod, flower, and/or seed) and/or cell-specific promoters (e.g., directing expression of the gene product to a specific cellular compartment or organelle such as vacuole, vesicle, cytosol, apoplast, nucleus, endoplasmic reticulum, peroxisome, and/or plastid) can be used (e.g., U.S. Publication 2007/0192900, International Publication WO 2007/100897). Examples of plant promoters include the 35S cauliflower mosaic virus (CaMV) promoter, a promoter of nopaline synthase from

Agrobacterium tumefaciens, and a promoter of octopine synthase. Examples of other constitutive promoters used in plants are the 19S promoter and promoters from genes encoding actin and ubiquitin. Promoters may be obtained from genomic DNA by using polymerase chain reaction (PCR), and then cloned into the nucleic acid construct.

[0049] Nucleic acid constructs according to the disclosure may be cloned into an expression vector (e.g., a plasmid). Vectors suitable for transforming plant cells include, but are not limited to, plasmids from Agrobacterium tumefaciens, a plasmid containing a β- glucuronidase gene and a cauliflower mosaic virus (CaMV) promoter plus a leader sequence from alfalfa mosaic virus, or a plasmid containing a bar gene cloned downstream from a CaMV 35S promoter and a tobacco mosaic virus (TMV) leader. Other plasmids may additionally contain introns, such as that derived from alcohol dehydrogenase (Adhl), or other DNA sequences. For constructs intended to be used in Agrobacterium-me0\ate0 transformation, the plasmid may contain an origin of replication that allows it to replicate in Agrobacterium and a high copy number origin of replication functional in E. colHo permit facile production and testing of transgenes in E. coli prior to transfer to Agrobacterium for subsequent introduction in plants. [0050] The above gene expression cassettes can be constructed using methods generally known in the art, for example conventional molecular biology cloning methods. In a particularly convenient method, PCR is used to produce the nucleotide fragments for constructing the gene expression cassettes. By using the appropriate PCR primers, the precise nucleotide regions of the above DNAs can be amplified to produce nucleotide fragments for cloning. By further including in the PCR primers restriction enzyme cleavage sites which are most convenient for assembling the heterogenous gene expression cassettes (e.g., restriction enzyme sites that are not in the nucleotide fragments to be cloned), the amplified nucleotide fragments are flanked with the convenient restriction enzyme cleavage sites for assembling the nucleotide fragments into heterogenous gene expression cassettes. The amplified nucleotide fragments are assembled into the heterogeneous gene expression cassettes using conventional molecular biology methods. Based upon the nucleotide sequences provided herein, how to construct the heterogenous gene expression cassettes using conventional molecular biology methods with or without PCR would be readily apparent to one skilled in the art.

[0051] Figures 2-5 illustrate embodiments of various gene cassettes for use in the transgene delivery methods of the present disclosure, for example in particle bombardment or Agrobacterium transformation methods as described in more detail below in the examples. Figure 2 is a linear map of the pACT1 F cassette (not drawn to scale) used as a selectable marker for transformation of β-glucuronidase (gus) into common beans: rice actin promoter (Act), gus gene (UidA), and nopaline synthase terminator (Tnos). Figure 3 is a linear map of the pBY520 cassette (not drawn to scale) used for the transformation of common bean with the HVA1 gene conferring drought and salt stress tolerance as well as the bar gene as a selectable marker: rice actin promoter (Act1 ), barley or Hordeum vulgare (HVA1 ) LEA3 gene, Cauliflower Mosaic Virus 35S promoter (35S), bar gene (bar), and nopaline synthase terminator (Nos). Figure 4 is a circular map of the 6.9 Kb of

pBKSbar/gf2.8 (not drawn to scale) used for the transformation of common bean with the gf2.8 gene conferring white mold resistance and the bar gene as a selectable marker:

ampicilin resistant marker (Amp), herbicide selectable marker (bar), origin of replication of the pUC 18 plasmid vector (pUC oh). Figure 5 is a linear map of the pCAMBIA3301 T-DNA cassette (not to scale) used for Agrobacterium transformation of common bean with the gus and bar genes as selectable markers (a variant of this construct was also used with the SLPI gene in place of the bar gene): left/right T-DNA border sequences (LB/RB), CaMV 35S promoter/terminator (P35S/T35S), coding region of the phosphinothricin resistance gene (bar), nopaline synthase terminator (Tnos), gusA gene coding region with intron sequence (gus-intron).

Transformation Methods

[0052] Transformation generally relates to the process by which the desired transgene is delivered to a target grain legume explant (e.g., introduction of an expression vector including the desired exogenous nucleic acid construct into a recipient cell, callus or protoplast). The transgene may or may not be integrated into (i.e., covalently linked to) chromosomal DNA making up the genome of the host cell, callus or protoplast (e.g., the exogenous polynucleotide may be maintained on an episomal element, such as a plasmid or it may become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication). Stable transformation of the grain legume (or stable integration of the transgene into the grain legume) relates to state in which an inserted exogenous nucleic acid construct is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. Stability is demonstrated by the ability of the transformed cells to establish cell lines or clones comprised of a population of daughter cells containing the transgene.

[0053] Delivery of the transgene into the apical shoot meristem primordial and the resulting grain legume transformation can be accomplished by any method generally known to the art, for example including Agrobacterium- mediated transformation, particle bombardment, electroporation, and virus-mediated transformation. The method of transformation is not particularly limited and suitably incorporates the nucleic acid construct containing the gene product encoding region and any desired regulatory sequences into the plant host. Using the various transformation methods, genetically modified plants, plant cells, plant tissue, and/or seeds of the transformed grain legume can be obtained. In general, transformation of the grain legume includes first introducing a nucleic acid construct including the desired transgene(s) into the apical shoot meristem primordial tissue of a grain legume, and then optionally regenerating a whole grain legume plant from the transformed apical shoot meristem primordial tissue, in particular where the selected transgene is stably integrated into the apical shoot meristem primordial tissue and/or resulting plant.

[0054] Microprojectile bombardment (e.g., biolistic or gun bombardment) can be used to make the disclosed transgenic grain legumes by transforming the apical shoot meristem primordial tissue with plasmids, each containing a particular gene expression cassette including the nucleic acid construct for the delivered transgene (e.g., U.S. Patents

5,036,006; 5,302,523; 5,322,783, 5,538,880; 5,550,318; 5,563,055, 5,610,042, 5,736,369, and 5,767,368; WO 94/09699 and WO 95/06128). In this method, nucleic acid constructs are delivered to living subepidermal meristem cells in the explant tissue by coating or precipitating the gene expression cassette onto a particle or microprojectile (for example tungsten, platinum or gold particles), and propelling the coated microprojectile into the living cells. Each gene expression cassette is thus introduced into the explant tissue, and the transformed tissue is then regenerated to produce a transgenic plant that contains the particular gene expression cassette. The result is a transgenic plant containing the gene expression cassette expressing the gene product of the desired transgene.

[0055] Microprojectile bombardment is an effective method for transforming the apical shoot meristem primordial tissue of grain legumes according to the disclosure. The bombardment method suitably results in a transformation efficiency (e.g., stable

transformation efficiency) of at least 2% (i.e., expressed as the number of explants successfully transformed relative to the number of explants subjected to transformation). More generally, the transformation efficiency can be at least 2%, 3%, 4%, 5%, 6%, 8% and/or up to 10%, 12%, 15%, 20%, 30%, 40%, 50%, 70%, 90%. Suitable operating conditions for the bombardment method include (i) a bombardment pressure of at least 800 or 1000 psi and/or up to 1200 or 1500 psi, (ii) a bombardment frequency of 1 , 2, or 3 (e.g., the total number of times an explant tissue is subjected to bombardment, such as at 24 hr intervals), and/or (iii) a plasmid DNA concentration of 1 μg to 4 μg per bombardment.

[0056] Alternatively, transformation of the grain legume can be achieved using bacterial- mediated transformation using a bacterium such as Agrobacterium tumefaciens to mediate the transformation of the meristem primordia. Agrobacterium-mediated transformation of plant cells is generally known (e.g., U.S. Patents 5,563,055, 5,591 ,616). Generally, the steps of cloning and DNA modifications are performed in E. coli, and then the plasmid containing the gene construct of interest is transferred by heat shock treatment into an Agrobacterium strain, and the resulting Agrobacterium strain is used to transform plant cells (e.g., by co- cultivation of the strain with apical shoot meristem primordial tissue).

[0057] Transformed grain legumes containing multiple transgenic traits can be formed using the above methods. For example, a single gene expression cassette can include nucleic acid sequences for multiple transgenes (e.g., as shown in Figure 3 for the pBY520 cassette with the HVA1 gene and the bar gene). Additionally, multiple gene expression cassettes including different nucleic acid sequences can be delivered simultaneously to the same apical shoot meristem primordial tissue (e.g., as described in Example 1 where two plasmid vectors pACT1 F and pBY520 are mixed and then coated onto tungsten particles used for bombardment of the explant tissue). Alternatively, separate explant tissues can be transformed with different gene expression cassettes (e.g., a first explant tissue transformed with a first gene expression cassette and a second explant tissue transformed with a second gene expression cassette) so that first generation transgenic plant progeny from each explant tissue can be crossbred by sexual fertilization to produce second generation transgenic plants including multiple transgenes from the different cassettes. In another embodiment, an explant tissue is transformed with a first gene expression cassette and regenerated to from a first generation transgenic plant progeny whose transgenic apical shoot meristem primordial tissue including the first transgene can then be transformed with a second gene expression cassette to provide second generation progeny additionally containing the second transgene. It will be readily apparent to one skilled in the art that transgenic grain legumes (or components thereof) containing any combination of transgenes can be obtained by the above methods.

Regeneration

[0058] Sticklen and Oraby (2005) describe suitable possibilities for recovering transgenic plants via transfer of DNA into the shoot apical meristem. One possibility is to genetically transform the meristem subepidermal germline cells (stem cells or layer 14 as in Figure 1 ) followed by the development of a partially transgenic reproductive organ such as a seed or kernel from in vitro transgenic stem cells. In this case, the primary seed transformants will always be chimeric, and must be multiplied several generations in order to remove their chimerism. A second possibility is to transfer transgenes into the subepidermal or stem cell layer and then release the meristem apical dominancy via a combination of growth conditions and light regimes in favor of meristem primordium differentiation and branching or multiplication of transgenic apical meristems. In this case, the chance for transgene chimerism of transgenic plants is almost zero percent. In vitro selection can be used in both cases, each after multiplication. In the event of chemical selection of transformants prior to multiplication, chimeric transformants will most probably not survive the selection, especially when the transgenic plantlets are highly chimeric.

[0059] In vitro regeneration of the apical shoot meristem primodial (e.g., either untransformed tissue or transgene-modified tissue) generally involves the incubation of the explant tissue with a suitable culture medium (e.g., a gelled/solidified culture medium in a petri plate). Callus tissue is formed and shoots may be induced from callus and

subsequently roots. Alternatively, somatic embryo formation can be induced in the callus tissue, and the somatic embryos germinate as natural embryos to form plants. In either case, the resulting plant can be a transgenic plant if derived from transgene-modified explant tissue. Primary (transgenic) plants may then be grown using any suitable conventional method for plant cultivation known in the art. The plants can be grown in soil, or alternatively can be grown hydroponically. Primary transgenic plants may be either pollinated with the same transformed strain or with a different strain, and the resulting hybrid having the desired phenotypic characteristics can be identified and selected. Two or more generations may be grown to ensure that the desired phenotypic characteristics are stably maintained and inherited, and then seeds are harvested to ensure that the desired phenotype or other property has been achieved.

[0060] In vitro regeneration of the transgene-modified shoot apical meristem primordial suitably involves releasing apical dominance in the transgene-modified shoot apical meristem primordial so that multiple shoots of a transgene-modified grain legume are then grown from the transgene-modified shoot apical meristem primordial (e.g., multiple shoots per transformed explant are grown in the culture medium). Apical dominance release to facilitate branching can be effected by culture/regeneration conditions such as cytokinin selection (e.g., type and/or amount can influence the expression of certain genes favoring apical shoot meristem branching) and other factors such as light and growth regulator therapy. The in vitro regeneration of grain legume explants according to the disclosure suitably produces between 6 and 20 shoots per transgene-modified shoot apical meristem primordial explant (e.g., at least 6, 8, or 10 and/or up to 12, 15, or 20 shoots per explants; such as where the value represents an average multiplication or branching factor over multiple explants). The growing of multiple shoots per explant in the regeneration process provides a convenient, rapid means to one or more non-chimeric transgene-modified grain legume shoots (e.g., which can be grown into non-chimeric transgene-modified grain legume plants). The transgene-modified grain legume shoots (e.g., non-chimeric, stably

transformed) can be grown to provide a rooted transgenic grain legume plant including a DNA encoding the delivered transgenes so that the resulting plant is capable of expressing the delivered transgenes (e.g., the gene product thereof).

[0061] The culture medium generally contains various conventional ingredients, such as nutrients (e.g., various nitrate, sulfate, chloride, and/or phosphate salts), amino acids (e.g., glutamic acid, proline), sugars (e.g., sucrose), gelling agents (e.g., agar, gellan gum) , and/or plant hormones (e.g., auxins, cytokinins). A suitable base culture medium is that of

Murashige and Skoog (1962) ("MS medium"). The cytokinin plant hormone (or a synthetic plant hormone analog) is suitably included in the medium at levels of at least 0.5, 1 , or 2.5 mg/l and/or up to 2.5, 5, or 10 mg/l, such as about 2.5 mg/L to 5 mg/L. Specific cytokinins can include adenine-type cytokinins (e.g., kinetin, zeatin, and 6-benzylaminopurine (BAP)), and phenylurea-type cytokinins (e.g., diphenylurea, thidiazuron (TDZ)). The auxin plant hormone (or a synthetic plant hormone analog) is suitably included in the medium at levels of at least 0.02, 0.05, or 0.1 mg/l and/or up to 0.2, 0.5, or 1 mg/l auxin. Specific auxins can include naturally occurring (endogenous) auxins (e.g., indole-3-acetic acid(IAA), 4- chloroindole-3-acetic acid (4-CI-IAA), phenylacetic acid (PAA), and indole-3-butyric acid (IBA)) and synthetic auxin analogs (e.g., 1 -naphthaleneacetic acid (NAA), 2,4- dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2-methoxy- 3,6-dichlorobenzoic acid, and 4-amino-3,5,6-trichloropicolinic acid).

[0062] In an embodiment, the culture medium further includes an antioxidant. Growing grain legume explant tissue in culture can be characterized by substantial excretion of phenolic compounds. The inclusion of the antioxidant is effective in preventing or reducing the oxidation of the phenolic compounds into toxic oxides in the culture that kill tissue growing in vitro. The antioxidant is suitably included in the medium at levels of at least 1 , 2, 5, or 10 mg/l and/or up to 20, 30, 50, or 100 mg/l in culture. Specific suitable antioxidants include silver nitrate, ascorbic acid, activated charcoal, and glutathione. Inclusion of the antioxidant can enhance the regeneration frequency of cultured explants, for example with an increase of at least 5%, 10%, or 15% and/or up to 20% or 30% relative to a comparable culture medium without the antioxidant.

[0063] Figure 6 illustrates various pathways for the in vitro regeneration of (transgenic) plant material according to the disclosure. Different letters (A-E) along the pathways represent different culture media that can be used to effect the growth along the indicated path after a sufficient culturing period. Example media formulations suitable for regeneration of common bean plant tissue include are A: 4.4 mg/L MS medium ; B: 4.4 mg/L MS medium, 2.5 mg/L BAP, and 0.1 mg/L IAA or NAA; C: 4.4 mg/L MS medium, 1 mg/L TDZ, and 0.5 mg/L IAA or NAA; D: 4.4 mg/L MS medium, 1 mg/L TDZ or BAP, and 1 mg/L IAA or NAA; and E: 4.4 mg/L MS medium and 0.1 mg/L IAA or NAA. The foregoing media can be further supplemented with an antioxidant as described above to suppress the inhibitory effects of phenolic growth byproducts.

Transgenic Plants

[0064] The disclosure additionally relates transgenic grain legume plants or plant material, for example as formed by the transgene delivery and/or regeneration methods described above. A transgenic grain legume can include a whole plant or plant components such as plant parts (e.g., cuttings, tubers, pollen), plant organs (e.g., leaves, stems, flowers, roots, fruits, branches, etc.), individual plant cells, groups of plant cells (e.g., cultured plant cells), protoplasts, plant extracts, seeds, and progeny thereof.

[0065] The transgenic grain legume includes a DNA encoding a transgene, such that cells in the transgenic grain legume are capable of expressing the transgene (e.g., capable of producing the gene product of the transgene not normally found in native, non-transgenic plants of the same strain). More generally, the transgenic grain legume includes transgenic cells whose DNA contains an exogenous nucleic acid not originally present in non- transgenic cells of the grain legume. A transgenic cell may be derived or regenerated from a transformed cell (e.g., as described above) or derived from another transgenic cell.

Exemplary transgenic cells include plant calli derived from a stably transformed plant cell and particular cells (such as leaf, root, stem, or reproductive cells) obtained from a transgenic plant. The transgenic grain legume plant includes plant in which one or more of the cells of the plant contain the delivered transgenes. The transgenic grain legume can include the progeny from the transgenic grain legume plant or from crosses involving the transgenic plant in the form of plants, seeds, tissue cultures and isolated tissue and cells, which carry at least part of the modification originally introduced by genetic engineering.

[0066] In an embodiment, the DNA encoding the transgene is stably integrated into the chromosomes of the transgenic grain legume. In another embodiment, the transgenic grain legume is non-chimeric (e.g., contains only transformed/transgenic tissue; as opposed to chimeric plants having cells with two or more different genotypes coexisting in a meristem such that tissues transformed with exogenous genes could contain both transformed cells and non-transformed cells).

Examples

[0067] The following examples illustrate various methods and compositions according to the disclosure for forming a transgene-modified shoot apical meristem primordial as well as related transgenic plant materials therefrom, but are not intended to limit the scope of the claims appended hereto. In particular, several genetically distant common bean (Phaseolus vulgaris) varieties were transformed via direct DNA transfer in a genotype-independent manner. The subepidermal cells of the apical shoot meristem were targeted for genetic transformation and then the apical dormancy was chemically released in favor of meristem branching (multiple apical shoot meristem production) to remove transgene chimerism in regenerated plantlets.

[0068] Plant Material: Five genotypes of common bean ("Condor," "Matterhorn," "Sedona," "Olathe," and "Montcalm") were used. In vitro regeneration of the common beans was performed as described above and in Kwapata et al. (2010) (incorporated herein by reference in its entirety). As described in the examples below, transformation of these genotypes was performed either through gene gun bombardment or via Agrobacterium tumefaciens-me0\ate0 transformation of the apical shoot meristem primordial explant.

[0069] Explant Preparation: In a suitable method for obtaining apical shoot meristem primordia, seeds are rinsed twice with sterile distilled water then immersed in 75% ethanol for 3 min, rinsed thrice with sterile distilled water and immersed for 20 min in a solution of 25% commercial bleach (CLOROX), 5m l/l polysorbate surfactant (TWEEN20), and 10m l/l of 0.02% HgCI₂. Following sterilization, the seeds are rinsed five times in sterile distilled water and soaked overnight for 20 hours. After the soaking period, the seeds are dissected and the embryos are excised. The hypocotyl and cotyledons are removed, leaving the epicotyl with the shoot apical meristem primordial. The obtained shoot apical meristem primordial can be subjected to a genetic transformation method (e.g., for as yet untransformed plant material) or to an in vitro regeneration method (e.g., for untransformed or transformed plant material). The excised epicotyl with shoot apical meristem primordial can be incubated in vitro for 5 days at 25^QC with a 16 h photoperiod and light intensity of 45-70 μιηοΙ/ιη²/8βο in the culture media described below.

[0070] In Vitro Multiple Shoot Regeneration Media: A suitable regeneration culture medium contains 4.43 g/L MS medium (Murashige and Skoog 1962), 3% sucrose, 100 mg/L casein hydrolysate, 2.5 g/L GELRITE (gellan gum), 2.5 mg/L 6-benzylaminopurine (BAP; or benzyladenine), and 0.1 mg/L indole-3-acetic acid (IAA). Silver nitrate 30 mg/L is added as an anti-oxidant to eliminate of the phenolic compounds produced during regeneration. After 3 weeks of visible shoot primordial growth, the explants are transferred to shoot

development medium containing the above ingredients excluding silver nitrate, and adjusting BAP and IAA to 1 mg/L each. Explants are kept on this medium for 7 wk before being transferred to rooting media which contains all ingredients of the shoot development media excluding BAP and adjusting IAA to 0.1 mg/L supplemented with 4 mg/L of glufosinate of ammonium for selection. Shootlets are kept on this medium for 5 wk until firm roots develop. Growth regulators are added after autoclaving the media for 25 min at 120 °C and 100 psi. The final medium combinations are then poured into 100 ^χ 25 mm petri dishes and solidified under a laminar flow hood. The in vitro cultures are incubated at 25 °C with 16 h photoperiod and light intensity of 45-70 μιηοΙ/ιη²/8.

[0071] Genetic Transformation Vectors: Four different plasmid vectors were used:

pACT1 F harboring the gus gene (Figure 2), pBY520 harboring the HVA1 and bar gene which confers drought tolerance and herbicide (phosphinothricin) resistance respectively (Figure 3), pBKSbar/gf2.8 harboring the germin (gf2.8) gene that confers resistance towards white mold fungus (Figure 4), and the binary vector pCAMBIA3301 for Agrobacterium tumefaciens-me0\ate0 transformation harboring the bar and gus-intron gene (Figure 5).

[0072] Polymerase Chain Reaction (PCR) Analysis: Polymerase chain reaction (PCR) analysis for the detection of HVA 1, gf.2.8 and bar genes was conducted on T₀-T₃ plants. Genomic DNA was obtained from leaf disks with diameters the size of the lid of a 1 .5 ml Eppendorf tube. Extraction of DNA was done using REDExtract-N-Amp Plant PCR Kit (Sigma-Aldrich, St. Louis, MO, Cat No. XNA-P), as per manufacturer's instruction. The primers used were; bar F, 5^{^}-ATG AGC CCA GAA CGA CG-3^' (forward primer; SEQ ID NO: 1 ); tar R, 5^{^}-TCA CCT CCA ACC AGA ACC AG-3^' (reverse primer; SEQ ID NO: 2); and HVA 1 F, 5^'-TGG CCT CCA ACC AGA ACC AG-3^' (forward primer; SEQ ID NO: 3); HVA 1 R, S^{^}-ACG ACT AAA GGA ACG GAA AT-3^' (reverse primer; SEQ ID NO: 4); g†2.8 F, 5^{^}-ATG GGG TAC TCC AAA ACC CTA G-3^' (forward primer; SEQ ID NO: 5); g†2.8 R, 5^{^}-CTA GAA ATT AAA ACC CAG CG-3^' (reverse primer; SEQ ID NO: 6). Optimized PCR conditions were 94 °C for 3 min for initial denaturation; 35 cycles of 50 s at 94 °C; 50 s at 56 °C, 1 min at 72 °C and a final 10 min extension at 72 °C. The PCR product was loaded onto a 1 % (w/v) agarose gel stained with 2 μΙ ethidium bromide and visualized under UV light.

[0073] Southern Blot Hybridization Analysis: The Southern blot hybridization analysis was conducted to determine the stability of the transgenic event and determine the gene copy numbers of HVA 1, gf2.8 and bar gene. The DIG High Prime DNA Labeling and Detection Starter Kit (Roche Co., Cat. No. 1 585 614) was used as per manufacturer's instructions. Transgenic and non-transgenic genomic DNA was isolated using the methods described by Saghai-Maroof et al. (1984). Hind III or BamHI restriction enzymes were used to digest 20 μg of genomic DNA, which was electrophoresed at 70 v on 1 % agarose gel and transferred to a Hybond-N+ membrane (Amersham-Pharmacia Biotech) and fixed with a UV crosslinker (Stratalinker UV Crosslinker 1800, Stratagene, CA) at an energy level of 2,000 J. The DIG- labeled probes that were used for bar, HVA 1 and g/2.8 were synthesized using the primers for the specific genes as described above for the PCR analysis.

[0074] Northern Blot Analysis: Northern blot analysis was conducted using the DIG- labeled Northern Starter Kit (Roche Co., Cat. No. 12039672910). Total RNA from the leaves of transgenic and non-transgenic plants was isolated using TRI reagent (Sigma-Aldrich, St. Louis, MO) as per manufacturer's instructions. A total of 30 μg of RNA per sample was loaded onto a 1 .2% (m/v) agarose-formaldehyde denaturing gel as described by Sambrook et al. (1989) and transferred to a Hybond-N+ membrane (Amersham-Pharmacia Biotech) and fixed with a UV crosslinker (Stratalinker UV Crosslinker 1800, Stratagene, CA) at an energy level of 200 J. An RNA or DNA DIG-labeled probe, containing the coding region of the gene of interest, was used for detection of transcripts.

[0075] Histochemical gus Assay: Gus activity was tested on transgenic and non- transgenic seeds and embryos using histochemical staining with 5-bromo-4-chloro-3-indoyl- β-D-glucuronicacid salt (X-gluc). Plant samples were dipped into gus substrate buffer, according to Jefferson et al. (1987), and incubated at 37°C for 24 hours. The tissue samples were washed with 100 percent ethanol to remove all coloration.

Example 1 Bombardment Transformation of Common Beans

[0076] Apical shoot meristem primordia of mature common bean embryos were excised and then bombarded with the helium particle delivery system (gene gun), model PDS-1000 (DuPont, Wilmington, DE). The plasmid DNA was coated onto 50 μg/L of 10 μιη tungsten particles with 2.5 M calcium chloride and 0.1 M spermidine suspended in a solution of 1 :1 (v/v) of 75% ethanol and 50% glycerol. Three levels of pressure were applied (500, 1000 and 1 100 psi) to assess the most effective pressure. The concentration of plasmid DNA per bombardment was varied at 1 .5 μg and 3.0 μg in order to see which concentration was most favorable. Three levels of bombardment frequency (1 , 2, and 3 total bombardment events at 24-hour intervals) were used, and plant tissues were kept for 24 hours before re-bombarding tissue. In one set of experiments, the plasmid vector pACT1 F (Figure 2) containing the gus marker gene was mixed in a ratio of 1 :1 (v/v) with the plasmid vector pBY520 (Figure 3) containing the bar herbicide resistant selection marker gene and the HVA1 drought resistance gene. In another set of experiments, the plasmid vector pBKSbar/gf2.8 (Figure 4) for white mold resistance and bar selection marker was transformed independently.

[0077] The transformed apical shoot meristem primordia were then regenerated in vitro as described above to provide a T₀ generation rooted common bean plant, from which subsequent plant generations to T₃ were grown. Plant material from the T₀ to T₃ generations was tested to confirm the level of transgene integration and expression. Table 1 below summarizes the mean stable transformation efficiency as a function of various bombardment conditions (PCR data for gus transgene integration averaged across the T₀ generation for the five genotypes). The results suggest that bombarding the plant twice, using a pressure setting of 1 100 psi with a concentration of 1 .5 μg of plasmid DNA per bombardment yielded the highest transformation efficiency of 8.4%. The gene gun pressure was an important factor for successful integration of a transgene. Low pressures yielded very low and poor transformation efficiencies, while increased pressure and/or frequency of bombardment could result in damaged explants. The transformation efficiency that was obtained was higher than those that have been reported by other researchers who have bombarded explants only once or used different psi pressure of the gene gun (Somers et al., 2003, Popelka et al., 2004).

Table 1 : Stable transformation efficiencies for varying bombardment conditions

Bombardment Concentration of Bombardment Mean Transformation Pressure (psi) plasmid DNA (ug) Frequency (%)

500 1 .5 1 0.1 ±0.04

500 1 .5 2 0.2±0.1 0

500 1 .5 3 0.4±0.30

500 3 1 0.1 ±0.04

500 3 2 0.6±0.32

500 3 3 0.7±0.32

1000 1 .5 1 2.9±0.67

1000 1 .5 2 3.9±1 .4

1000 1 .5 3 5.1 ±1 .2

1000 3 1 5.6±1 .0

1000 3 2 8.1 ±0.3

1000 3 3 7.4±1 .0

1 1 00 1 .5 1 7.2±0.70

1 1 00 1 .5 2 8.4±0.74

1 1 00 1 .5 3 8.2±0.50

1 1 00 3 1 7.5±0.69

1 1 00 3 2 4.8±0.93

1 1 00 3 3 3.3±0.92

Examole 2 Aarobacterium Transformation of Common Beans

[0078] Three strains of Agrobacterium tumefaciens (EHA105, GV3301 , and LBA4404) were used. These were transformed with the pCAMBIA-3301 binary vector (Figure 5) containing the gus gene driven by the 35S promoter with or without the bar gene. These were cultured in 50 ml LB medium (Luria-Bertani medium or Lysogeny broth) in the dark at 37 °C in a rotator at 280 rpm for 48 hours; OD₆₀₀ = 1 . The strains used were co-cultivated with the apical shoot meristem primordia explants for 1 , 5, 10 and 15 days. The

regeneration media described above (2.5 mg/L BAP, 0.1 mg/L IAA, and 30 mg/L silver nitrate) was supplemented with 500 mg/L of timentin to kill the A. tumefaciens after the appropriate co-cultivation period to yield the transformed apical shoot meristem primordia. [0079] Similar to Example 1 , the transformed apical shoot meristem primordia were then regenerated in vitro as described above to provide a T₀ generation rooted common bean plant, from which subsequent plant generations to T₃ were grown (i.e., as in Kwapata et al. (2010)). Plant material from the T₀ to T₃ generations was tested to confirm the level of transgene integration and expression.

[0080] Figure 7 illustrates the effect of co-cultivation period (1 , 5, 10 and 15 d) on the transient transformation frequency for the gus gene of two genotypes of common bean (Matterhorn and Sedona) using three different strains of A. tumefaciens (EHA105, GV3301 and LBA4404). Figure 8 similarly illustrates the relative stable transformation frequency for the gus gene of ΤΊ (second generation) plants after 15 days of co-cultivation. The results indicate that for both transient and stable expression of the gus gene, Sedona was more amenable to Agrobacterium transformation than Matterhorn. For both transient and stable expression of gus gene, the Agrobacterium strain GV3301 was the most effective when compared to EHA105 or LBA4404 strains. The most favorable co-cultivation period for high transformation frequency was 15 days. It was noted that there was a significant discrepancy between transformation efficiencies of tissues that were transiently being expressed as compared to those with stable transformation (Figure 7 and Figure 8). With a co-cultivation period of 15 days, using GV3301 , transient expression efficiencies of gus were 51 % with Matterhorn and 81 % with Sedona (Figure 7). Using the same co-cultivation period and with the strain EHA105, transient expression efficiencies for gus were 66% and 69% for

Matterhorn and Sedona, respectively (Figure 7). Under the same conditions using LBA4404, 18% and 50% transient expression efficiencies were achieved for Matterhorn and Sedona, respectively (Figure 7). Stable expression of the gus transgene also was observed in T₃ seeds and zygotic embryos.

Example 3 Herbicidal Resistance of Transformed Common Beans

[0081] The herbicide LIBERTY (Aventis, Strasboug, France), with the active ingredient ammonium glufosinate, was used in both multiple shoot and rooting media, and applied to determine which plants were transgenic as well as to score the segregation ratios of the transgenic progeny. Plants were sprayed at different stages of growth and development ranging from three-week-old young seedlings to two and three-month-old plants. Different foliar application rates of the herbicide were assessed ranging from 50, 100, 250, and 350 mg/L of the herbicide.

[0082] Integration of the bar gene integration was demonstrated using PCR in the Condor, Sedona, Montcalm and Matterhorn genotypes. However the chi-square test of T₂ and T₃ plants revealed that the segregation of the bar gene does not follow Mendelian inheritance. Southern blot analysis of T₂ plants bombarded with a construct containing the bar gene showed integration of four different transgenic Condor plants with a single gene copy insert. Two Matterhorn plants showed successful integration of the bar gene with a single and four copy numbers of the bar transgene. Sedona and Montcalm showed three and two copy number of bar transgene respectively. Northern blot analysis of T₂ and T₃ plants confirmed the transcription of bar transgene. The expression levels are not very high except for Matterhorn and Sedona which shows a higher expression level than the others.

[0083] The LIBERTY herbicide resistance test of two-months-old T₂ plants showed that they were still chimeric, because certain portions of the leaves got scotched by the herbicide three days after being sprayed with 150 mg/L of Liberty herbicide (although they were still performing better as compared to the wild type plants). T₃ plants also were tested to see if their level of resistance towards the herbicide had improved. The foliar application of the herbicide was increased to 250 mg/ L, and it was noted that the transgenic plants were still chimeric because some of the leaves got scotched by the herbicide.

Example 4 Drought Resistance of Transformed Common Beans

[0084] Seedlings were raised in the growth chamber for three weeks or until trifoliate leaves appeared. They were then transferred to the greenhouse into 15 cm diameter clay pots containing BACCTO High Porosity Professional Planting Mix (Michigan Peat Company, Houston, TX). The plants were watered daily for three weeks, after which moisture was withheld for 21 days. Observations were recorded on plant survival, degree of leaf wilting, root length, plant growth and height. After the 21 days, moisture was applied to the plants continuously for 14 days and the percentage of plants recovered was recorded.

[0085] Confirmation of HVA1 transgene integration also was confirmed in the plants using PCR, Southern blot, and Northern blot analyses. Figure 9 illustrates PCR results of T₃ transgenic plants Montcalm (Mon), Condor (Con), Sedona (Sed), and Matterhorn (Mat) and confirms the stability of HVA1 transgene integration in all four genotypes. Figure 10 is a Southern blot showing integration of the HVA1 gene in Condor (Co), Montcalm (Mo), Sedona (Se), Matterhorn (Ma), and wild type (Wt) plants digested with BamH1 . The results indicate that there is a double gene integration in all genotypes except Montcalm, which has a single copy number, while the wild type shows no transgene integration. 9 illustrates the Northern blot expression of the HVA1 gene from T₃ transgenic plants subjected to drought stress: Montcalm (Mon), Condor (Con), Sedona (Sed), and Matterhorn (Mat) in comparison to wild type (Wt) plants. Matterhorn and Sedona showed some expression, while the wild type, Montcalm, and Condor plants showed no expression at all.

[0086] When water was withheld for 21 days continuously, all the Condor plants regardless whether they were transgenic or non-transgenic wild types died within 12 days of treatment. No differences could be distinguished between the transgenic and the wild types. Similar results were also obtained for Montcalm plants that died within 16 days of treatment with no clear distinction between transgenic plants and wild types. On the other hand, Sedona and Matterhorn transgenic plants persisted for 21 days without water. They showed symptoms of drought stress but soon recovered after three days when moisture application resumed. The wild types showed more severe symptoms of drought stress with most of the leaves wilted and dehisced. Out of 30 plants that were planted for each genotype in the experiment, 15 were wild types and the other 15 were transgenic. Survival of wild type Sedona plants was only 13.3%, and for transgenic plants it was 33.3%. Survival of

Matterhorn wild type plants was 20%, and for transgenic plants it was 53.3%.

[0087] The percent leaf abscission was used as an indirect measure of the degree of plant wilting. Wilting was defined as the difference of ratios between the number of leaves on plant before 21 days of moisture withdrawal and the number of green leaves on plant remaining after 21 days of moisture withdrawal. The percent leaf abscission for transgenic Sedona plants was 78% compared to 91 % for wild type; for Matterhorn it was 72% and 88% for transgenic and wild type, respectively. It appears that Matterhorn possesses a genotypic advantage over Sedona in terms of tolerating drought as indicated by the results of the performance of their wild types.

[0088] The mean height or growth rate of transgenic versus non-transgenic plants did not differ significantly. For example, before the experiment was conducted, plants of uniform height (20 cm) were selected. After the treatment period height measurement was taken again. The results showed that the mean height for Sedona transgenic plants was 23 cm and that for wild type plants was 22 cm. The mean height for Matterhorn transgenic plants was 24 cm and for wild type plants it was 23 cm. In contrast, the control normal-watered plants grew to a height of 33 cm and had a net growth of 13 cm. This is an average of threefold increase in growth compared to the plants under drought stress.

[0089] The rooting ability was also examined and showed that the root growth of transgenic plants was more robust than wild type plants under stress but less developed than wild type plants under normal moisture regime. The average root length measured after 21 days of treatment for Sedona transgenic plants was 15 cm and for wild type plants was 1 1 cm. For Matterhorn, the average root length measured after the same treatment application was 17 cm for transgenic plants and 13 cm for wild type plants. In contrast, for control plants under normal irrigation the average root length was 28 cm. From the results of this experiment it was shown that transgenic plants engineered with HVA1 utilize their energy in developing and growing their root system as opposed to the above ground stem and canopy which exhibited little growth under drought stress conditions and showed no significant phenotypic difference between transgenic plants and wild types.

[0090] The drought stress test results are summarized in Table 2 below for transgenic (Tr) as compared to wild type (Wt) plants.

Table 2: Drought test results

Number of Plants Plant growth in Root growth in Surviving per 15 Percentage of (cm) after 21 days (cm) After 21 days plants Leaf Abscission of drought of drought

Genotype Tr Wt Tr Wt Tr Wt Tr Wt

Matterhorn 8 3 72 88 24 23 1 7 13

Sedona 5 2 78 91 23 22 15 1 1

Condor 0 0 100 100 21 21 8 7

Montcalm 0 0 100 100 22 22 9 9

Example 5 Fungal Resistance of Transformed Common Beans

[0091] PCR analysis showed about 6.9% of the bombarded plant material contained the gf2.8 insert in the T₀ generation. PCR tests also were positive for J and T₂ plants, and three plants (of 2000 separately bombarded explants) showed positive integration of the transgene using Southern blot analysis. This therefore means that the other plants were chimerically transgenic or that the transformed plasmid is resident in the cytoplasm and not on the chromosome in the nucleus. In Southern blot, the number of integrated transgenes ranged from two to four copies . PCR positives for these plants was demonstrated in the T₃ population along with other plants that did not show Southern blot positive.

[0092] A modified protocol from Livingstone et al. (2005) was used in analyzing the effectiveness of transgenic plants against the white mold fungus. Trifoliate leaves were detached and inoculated with S. sclerotiorum mycelia by placing a 6 mm diameter agar plug with inoculum onto the center of the detached leaf. The inoculation was conducted in a glass tray covered with a plastic paper containing wet paper towel placed at the bottom to keep the leaves and the fungus moist during the infection process. [0093] The level of resistance of different independent transgenic lines that were inoculated with the fungal pathogen was compared to the non transgenic wild type plants that were used as controls. The transformed plant leaves showed some resistance against the pathogen when compared to the wild-type, non-transgenic leaves. As shown in Figure 12, the best performed plant that delayed the onset of lesions was Matterhorn, followed by Sedona, then Olathe, and finally Condor.

[0094] Summary: The foregoing examples illustrate that the most promising strains for Agrobacterium-me0\a[e0 transformation are EHA105 and GV3301 , both of which showed better results for gene delivery than LBA4404. In addition to the type of strains used in the studies, it was also shown that the co-cultivation period significantly affects the efficiency of the Agrobacterium-me0\ate0 transformation systems. The results also show that gene bombardment of multiple shoots offered a relatively better way of delivering transgene than Agrobacterium. The potential of using the H VA 7 transgene to alleviate symptoms of drought in common bean has been demonstrated. The potential of using the germin gene (G-OXO) which expresses an oxalate oxidase conferring resistance to white mold also was demonstrated.

[0095] Because other modifications and changes varied to fit particular operating requirements and environments will be apparent to those skilled in the art, the disclosure is not considered limited to the examples chosen for purposes of illustration, and covers all changes and modifications which do not constitute departures from the true spirit and scope of this disclosure. For example, it is expressly intended that all combinations of those elements and/or method steps which perform substantially the same function in substantially the same way to achieve the same results are within the scope of the disclosure. Moreover, it should be recognized that structures and/or elements and/or method steps shown and/or described in connection with any disclosed form or embodiment of the disclosure may be incorporated in any other disclosed or described or suggested form or embodiment as a general matter of design choice. It is the intention, therefore, to be limited only as indicated by the scope of the claims appended hereto.

[0096] Accordingly, the foregoing description is given for clarity of understanding only, and no unnecessary limitations should be understood therefrom, as modifications within the scope of the disclosure may be apparent to those having ordinary skill in the art.

[0097] Throughout the specification, where the compositions, kits, processes, or apparatus are described as including components, steps, or materials, it is contemplated that the compositions, processes, or apparatus can also comprise, consist essentially of, or consist of, any combination of the recited components or materials, unless described otherwise. Component concentrations expressed as a percent are weight-percent (% w/w), unless otherwise noted. Numerical values and ranges can represent the value/range as stated or an approximate value/range (e.g., modified by the term "about"). Combinations of components are contemplated to include homogeneous and/or heterogeneous mixtures, as would be understood by a person of ordinary skill in the art in view of the foregoing disclosure.

References

1 . Apostol I, Heinstein PF, Low PS (1989) Rapid stimulation of an oxidative burst during elicitation of cultured plant cells: role in defense and signal transduction. Plant Physiol 90: 109-1 16

2. Aragao FJL, Barros LMG, Brasileiro ACM (1996) Inheritance of foreign genes in

transgenic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment. Theor Appl Genet 93: (1 -2) 142-150

3. Aragao FJL, Ribeiro SG, Barros LMG (1998) Transgenic beans (Phaseolus vulgaris L.) engineered to express viral antisense RNAs show delayed and attenuated symptoms to bean golden mosaic geminivirus. Mol Breeding 4: (6) 491 -499

4. Aragao FJL, Vianna GR, Albino MMC, Rech EL (2002) Transgenic dry bean tolerant to the herbicide glufosinate ammonium. Crop Science 42: (4) 1298-1302

5. Bolton M D, Bart P, Thomma H J, Berlin D N (2006) Sclerotinia sclerotiorum (Lib.) de Bary: Biology and molecular traits of a cosmopolitan pathogens. Mol. Plant Pathology 7: 1 -16

6. Cessna AS, Sears VE, Dickman MB, Low PS (2000) Oxalic Acid, a Pathogenicity Factor for Sclerotinia sclerotiorum, Suppresses the Oxidative Burst of the Host Plant." The Plant Cell 12: 2191 -2199

7. Christou P. (1992) Genetic transformation of crop plants using microprojectile

bombardment. The Plant Journal. 2(3): 275-281

8. Favaron F, Sella L, D'Ovidio R (2004) Relationships among endopolygalacturonase, oxalate, pH, and plant polygalacturonaseinhibiting protein (PGIP) in the interaction between Sclerotinia sclerotiorum and soybean. Mol. Plant-Microbe Interact. 17:1402- 1409

9. Kwapata K, Sabzikar R, Sticklen MB, Kelly JD (2009) In vitro regeneration and

morphogenesis studies in common bean. Plant Cell Tiss Organ Cult. 100:97-105

10. Hymowitz, T. (1990) Grain legumes, p. 54-57. In: J. Janick and J.E. Simon (eds.),

Advances in new crops. Timber Press, Portland, OR.

1 1 . Murashige T., Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant. 15: 473-497. Livingstone DM, Hampton JL, Phipps PM, Grabau EA (2005) Enhancing resistance to Sclerotinia minor in peanut by expressing a barley oxalate oxidase gene. Plant Physiol. 137:1354-1362.

Popelka J.C, N. Terryn and T.J.V. Higgins (2004) Gene technology for grain legumes: can it contribute to the food challenge in developing countries?, Plant Sci 167, pp. 195— 206.

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: a laboratory manual. 2nd ed. N.Y., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, pp. 1659 . ISBN 0-87969-309-6.

Somers D. A, D.A. Samac, P.M. Olhoft (2003) Recent advances in legume

transformation Plant Physiol. 131 892-899.

Sticklen M and Oraby H (2005) Shoot apical meristem: A sustainable explant for genetic engineering of cereal crops. In Vitro Cellular & Developmental-PLANT 41 : 187-200. Zuppini A, Navazio L, Sella L, Castiglioni C, Favaron F, Mariani P (2005) An

endopolygalacturonase from Sclerotinia sclerotiorum induces calcium-mediated signaling and programmed cell death in soybean cells. Mol. Plant-Microbe Interact. 18: 849-855

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Claims

What is claimed is:

1. A method for genetically transforming a grain legume, the method comprising:

(a) providing a shoot apical meristem primordial of a grain legume; and

(b) delivering one or more transgenes into the shoot apical meristem primordial of the grain legume, thereby forming a transgene-modified shoot apical meristem primordial.

2. The method of claim 1 , wherein the shoot apical meristem primordial comprises undifferentiated subepidermal meristem tissue to which the one or more transgenes are delivered in part (b).

3. The method of claim 2, wherein the one or more transgenes are delivered only undifferentiated stem cell tissue in the subepidermal meristem tissue.

4. The method of claim 1 , wherein the grain legume is selected from the group consisting of beans, peanuts, lentils, peas, and chickpeas.

5. The method of claim 1 , wherein the grain legume is a common bean.

6. The method of claim 5, wherein the common bean has a genotype selected from the group consisting of red beans, navy beans, white kidney beans, black beans, pink beans, red kidney beans, pinto beans, and great northern beans.

7. The method of claim 1 , wherein the grain legume is a soybean.

8. The method of claim 1 , wherein the one or more transgenes are selected from the group consisting of a gus (β-glucuronidase) gene, a bar gene, an HVA1 (barley Hordeum vulgare LEA3) gene, a gf2.8 (wheat germin) gene, and combinations thereof.

9. The method of claim 1 , wherein the one or more transgenes are selected to impart one or more transgenic traits selected from the group consisting of herbicide resistance, pesticide resistance, fungal resistance, microbial resistance, salt tolerance, drought tolerance, temperature tolerance, and combinations thereof.

10. The method of claim 1 , wherein the delivered transgene is stably integrated into the transgene-modified shoot apical meristem primordial.

11. The method of claim 1 , wherein delivering the one or more transgenes comprises performing a gene bombardment process.

12. The method of claim 1 1 , wherein the bombardment process has a transformation efficiency of at least 2%.

13. The method of claim 1 , wherein delivering the one or more transgenes comprises performing an Agrobacterium-me0\ate0 transformation process.

14. The method of claim 1 , further comprising:

(c) regenerating in vitro the transgene-modified shoot apical meristem primordial of part (b).

15. The method of claim 14, wherein in vitro regeneration of the transgene-modified shoot apical meristem primordial comprises releasing apical dominance in the transgene- modified shoot apical meristem primordial and growing multiple shoots of a transgene- modified grain legume from the transgene-modified shoot apical meristem primordial.

16. The method of claim 15, wherein growing multiple shoots provides one or more non-chimeric transgene-modified grain legume shoots.

17. The method of claim 15, further comprising:

(d) growing the transgene-modified grain legume shoots to provide a rooted transgenic grain legume plant comprising a DNA encoding the one or more transgenes, wherein the transgenic grain legume plant is capable of expressing the one or more transgenes.

18. The method of claim 17, wherein the transgenic grain legume plant is non- chimeric.

19. The method of claim 17, wherein the DNA encoding the one or more transgenes is stably integrated into the chromosomes of the transgenic grain legume plant.

20. The method of claim 14, wherein in vitro regeneration of the transgene-modified shoot apical meristem primordial comprises incubating the transgene-modified shoot apical meristem primordial in a culture medium comprising an anti-oxidant.

21. The method of claim 20, wherein the anti-oxidant is selected from the group consisting of silver nitrate, ascorbic acid, activated charcoal, and glutathione.

22. The method of claim 20, wherein the culture medium further comprises a cytokinin and an auxin.

23. The method of claim 14, wherein in vitro regeneration produces between 6 and 20 shoots per transgene-modified shoot apical meristem primordial explant.

24. The method of claim 1 , wherein providing the shoot apical meristem primordial in part (a) comprises:

(i) sterilizing seeds of a grain legume plant;

(ii) dissecting the seeds and excising seed embryos therefrom ;

(iii) excising the shoot apical meristem primordial from the excised seed embryos; and

(iv) optionally regenerating in vitro the shoot apical meristem primordial.

25. The method of claim 24, wherein in vitro regeneration of the shoot apical meristem primordial comprises incubating the shoot apical meristem primordial in a culture medium comprising an anti-oxidant and optionally one or more plant hormones.

26. A transgenic grain legume or component thereof comprising:

(a) a DNA encoding a transgene, wherein the transgenic grain legume or a transgenic grain legume formed from the transgenic grain legume component is capable of expressing the transgene.

27. The transgenic grain legume or component thereof of claim 26, wherein the grain legume or component thereof is a transgenic grain legume plant.

28. The transgenic grain legume or component thereof of claim 26, wherein the grain legume or component thereof is a transgenic seed of the transgenic plant, the transgenic seed comprising a transgene-modified shoot apical meristem primordial comprising the DNA encoding the transgene.

29. The transgenic grain legume or component thereof of claim 26, wherein the plant or component thereof is formed according to the method of claim 17.

30. The transgenic grain legume or component thereof of claim 26, wherein the transgenic grain legume or component thereof is non-chimeric.

31. The transgenic grain legume or component thereof of claim 26, wherein the DNA encoding a transgene is stably integrated into the chromosomes of the transgenic grain legume.

32. The transgenic grain legume or component thereof of claim 26, wherein the grain legume is selected from the group consisting of beans, peanuts, lentils, peas, and chickpeas.

33. The transgenic grain legume or component thereof of claim 26, wherein the grain legume is a common bean.

34. The transgenic grain legume or component thereof of claim 33, wherein the common bean has a genotype selected from the group consisting of red beans, navy beans, white kidney beans, black beans, pink beans, red kidney beans, pinto beans, and great northern beans.

35. The transgenic grain legume or component thereof of claim 26, wherein the grain legume is a soybean.

36. The transgenic grain legume or component thereof of claim 26, wherein the transgene is selected from the group consisting of a gus (β-glucuronidase) gene, a bar gene, an HVA1 (barley Hordeum vulgare LEA3) gene, a gf2.8 (wheat germin) gene, and combinations thereof.

37. The transgenic grain legume or component thereof of claim 26, wherein transgene is selected to impart one or more transgenic traits selected from the group consisting of herbicide resistance, pesticide resistance, fungal resistance, microbial resistance, salt tolerance, drought tolerance, temperature tolerance, and combinations thereof.