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WO2024221290A1 - Procédés de transformation du soja et produits/compositions associés - Google Patents

Procédés de transformation du soja et produits/compositions associés Download PDF

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Publication number
WO2024221290A1
WO2024221290A1 PCT/CN2023/090935 CN2023090935W WO2024221290A1 WO 2024221290 A1 WO2024221290 A1 WO 2024221290A1 CN 2023090935 W CN2023090935 W CN 2023090935W WO 2024221290 A1 WO2024221290 A1 WO 2024221290A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
seq
plant
acid sequence
growth regulating
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PCT/CN2023/090935
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English (en)
Inventor
Heng Zhong
Lizhao GENG
Qiudeng Que
Yinghui Dan
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Syngenta Crop Protection Ag
Syngenta Biotechnology China Co., Ltd.
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Publication date
Application filed by Syngenta Crop Protection Ag, Syngenta Biotechnology China Co., Ltd. filed Critical Syngenta Crop Protection Ag
Priority to PCT/CN2023/090935 priority Critical patent/WO2024221290A1/fr
Priority to PCT/US2024/025924 priority patent/WO2024226554A2/fr
Publication of WO2024221290A1 publication Critical patent/WO2024221290A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development

Definitions

  • the disclosure relates to the field of transforming plants, in particular to a plant-growth regulator mediated transformation of soybean via somatic embryogenesis.
  • Soybean is one of the most important crops in the world. However, to increase/maintain production of soybean at levels necessary to sustain global demand, new traits are constantly needed. Unfortunately, current methods of soybean transformation primarily rely on transforming soybean during organogenesis resulting in a high risk of chimeric plants. The chimeras result due to the multicellular nature of organogenesis. These chimeras impair the ability to effectively develop new traits. An internal analysis of 3974 T1 transgenic plants from 74 T0 editing transgenic soybean events showed that only 34.7%of the 3974 edited plants were not chimeric, and the rest (65.3%) were chimeric and not useful.
  • One embodiment of the disclosure is directed to methods for producing somatic embryos and directly regenerating plantlets from somatic embryos of a Glycine max plant comprising: introducing into immature cotyledons from the Glycine max plant a nucleic acid encoding a growth regulating factor and/or a morphogenic gene; and culturing immature cotyledons with a culture medium supplemented with a greatly reduced level of auxin under conditions allowing for expression of the nucleic acid encoding a growth regulating factor and/or a morphogenic gene.
  • the introduction of the nucleic acid encoding a growth regulating factor and/or morphogenic gene comprises bombardment.
  • the introduction of the nucleic acid encoding a growth regulating factor and/or morphogenic gene comprises Agrobacterium-mediated delivery. In some embodiments, two or more, three or more, and four or more different growth regulating factors and/or a morphogenic genes are introduced into the Glycine max plant.
  • the nucleic acid encodes a growth regulating factor (overexpression) , such as e.g., growth regulating factor 4.
  • the growth regulating factor 4 comprises SEQ ID NO: 3, a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 7, a nucleic acid that is 85 %identical to SEQ ID NO: 3, or a nucleic acid that is 85%identical to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 7.
  • the nucleic acid may also further encode a GRF-interacting factor (GIF) , such GIF1 including a GIF1 comprising SEQ ID NO: 4, a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 8, a nucleic acid that is 85%identical to SEQ ID NO: 4, or a nucleic acid that is 85%identical to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 8.
  • GIF GRF-interacting factor
  • the nucleic acid encodes a chimeric growth regulating factor and GRF-interacting factor.
  • the chimeric growth regulating factor and GRF-interacting factor comprises GRF4 and GIF1.
  • the chimeric growth regulating factor and GRF-interacting factor comprises: SEQ ID NO: 2; or SEQ ID NO: 3 and SEQ ID NO: 4.
  • the methods culturing the immature cotyledons with less than 40 mg/L of the auxin. In certain embodiments, the methods culturing the immature cotyledons with less than 20 mg/L of the auxin.
  • the auxin is 2, 4-dichlorophenoxyacetic acid (2, 4-D) . In another embodiment, the method comprises culturing with about 4 mg/L of 2, 4-D.
  • the methods may include other steps before the introducing or after the culturing step.
  • the method further comprises isolating the immature of cotyledons from the Glycine max plant prior to introducing the nucleic acid encoding a growth regulating factor and/or a morphogenic gene.
  • the methods further include transforming the immature cotyledon tissues of the Glycine max plant to express a nucleic acid, such as e.g., a gene of interest.
  • the methods after culturing, the methods further include transforming the induced callus of the Glycine max plant to express a nucleic acid, such as e.g., a gene of interest.
  • the methods after culturing, the methods further include transforming the induced somatic embryos of the Glycine max plant to express a nucleic acid, such as e.g., a gene of interest.
  • the somatic embryos are transformed by bombardment.
  • the somatic embryos are transformed by Agrobacterium-mediated transformation.
  • the somatic embryos are transformed using nanoparticle-mediated transformation.
  • the methods also include further steps after transformation to express the nucleic acid, such as e.g., a gene of interest.
  • the method in some embodiments, includes increasing the amount of carbohydrate in the culture medium during embryo maturing.
  • the methods may also include lowering the amount of carbohydrate in the culture medium during embryo maturation.
  • the methods may include determining the presence of the nucleic acid of interest in the transgenic plant.
  • the methods also include constructing a vector comprising the nucleic acid encoding a growth regulating factor and/or a morphogenic gene.
  • the vector comprises a nucleic acid encoding a GRF, such as GFRF4, and/or a nucleic acid encoding a GIF, such as e.g., GIF1.
  • the GRF4 comprises SEQ ID NO: 3, a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 7, a nucleic acid that is 85 %identical to SEQ ID NO: 3, or a nucleic acid that is 85%identical to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 7.
  • the GIF1 comprises SEQ ID NO: 4, a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 8, a nucleic acid that is 85%identical to SEQ ID NO: 4, or a nucleic acid that is 85%identical to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 8.
  • the vector comprises a chimeric growth regulating factor and GRF-interacting factor.
  • the chimeric growth regulating factor and GRF-interacting factor comprises GRF4 and GIF1.
  • the chimeric growth regulating factor and GRF-interacting factor comprises: the nucleic acid sequence of SEQ ID NO: 2; a nucleic acid encoding the amino acid sequence of SEQ ID NO: 6; a nucleic acid that is 85%identical to nucleic acid sequence of SEQ ID NO: 2; a nucleic acid that is 85%identical to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 6; the nucleic acid sequence of SEQ ID NO: 3 and the nucleic acid sequence of SEQ ID NO: 4; a nucleic acid encoding the amino acid sequence of SEQ ID NO: 7 and a nucleic acid encoding the amino acid sequence of SEQ ID NO: 8; a nucleic acid that is 85%identical to the nucleic acid sequence of SEQ ID NO:
  • the morphogenic factor comprises a protein selected from SEQ ID NO: 12 to SEQ ID NO: 29. In another embodiments, the morphogenic factor comprises a protein with more than 85%sequence identity to SEQ ID NO: 12 to SEQ ID NO: 29, in some embodiments, more than 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity, or even 100%sequence identity.
  • the vector comprises the prUBQ3 promoter, such as e.g., a prUBQ3 promoter comprising SEQ ID NO: 1.
  • the vector also includes a terminator tMt12344 from a Medicago truncatula gene.
  • the terminator tMt12344 from a Medicago truncatula gene comprises SEQ ID NO: 5.
  • the promoter driving expression of morphogenic factor gene expression is inducible.
  • the inducible promoter driving expression of morphogenic factor gene expression is AtHSP18.2 as in SEQ ID NO. 30.
  • the vector comprises an inducible auto-excisable morphogenic factor gene expression cassette flanked by site-specific recombinase recognition sites as shown in Figure 2.
  • the inducible promoter driving expression of site-specific recombinase gene expression is AtHSP18.2 as in SEQ ID NO. 30 and Figure 2.
  • the methods include additional steps prior to introducing the nucleic acid.
  • the methods also include storing Glycine max seed pods at a temperature of about 4°C for about 4-6 days, alternatively about 4 days, and then culturing the immature cotyledons for about 3 days prior to introducing into immature cotyledons a nucleic acid encoding a growth regulating factor and/or a morphogenic gene.
  • the culturing comprises culturing in a medium supplemented with an auxin.
  • the methods may be used to transform a variety of different types of soybeans.
  • the Glycine max plant is an elite Glycine max plant.
  • the disclosure also includes: plants or plant parts produced by the methods of the disclosure; and a derivative or a commodity product produced or obtained from the plant or plant part.
  • the disclosure includes a progeny seed produced by crossing such a plant with a second plant or by selfing the plant.
  • FIG. 1 is a schematic map of transformation vector pBSC26550 expressing vGRF4-GIF1 fusion protein.
  • FIG. 2 is a schematic map of transformation vector pHSP-BnBBM expressing BnBBM gene under inducible heat shock inducible promoter.
  • FIG. 3 shows differentiation and formation of somatic embryos at four developmental stages in the presence of the GRF-GIF genes of grape and auxin using immature cotyledon explants in soybean (genotype NE802) .
  • FIG. 3, panel A shows somatic embryos at the globular stage.
  • FIG. 3, panel B shows somatic embryos at the heart stage.
  • FIG. 3, panel C shows somatic embryos at the torpedo stage.
  • FIG. 3, panel D shows somatic embryos at the cotyledon stage.
  • FIG. 4 shows spectinomycin resistant somatic embryos formed directly from immature cotyledons 3 weeks after bombardment in the presence of GRF-GIF genes and auxin.
  • FIG. 5 shows multiple spectinomycin resistant somatic embryos formed from immature cotyledons 4 weeks after bombardment in the presence of GRF-GIF genes and auxin.
  • FIG. 6 shows spectinomycin resistant embryo-like structure formed from embryo axis derived from mature seeds 2 weeks after bombardment in the presence of GRF-GIF genes and auxin.
  • FIG. 7 shows germinated seedling from somatic embryo in the presence of GRF-GIF genes and auxin.
  • transformation technologies via organogenesis in soybean have a major disadvantage of high frequencies of chimeric transgenic and edited soybean plants.
  • transformation technologies via somatic embryogenesis as disclosed, herein are ideal for eliminating the risk of generating chimeras due to unicellular origin of somatic embryos.
  • This disclosure is based on the discovery that soybean transformation can be improved if the soybeans are modified to, at least transiently, express a growth regulating factor and/or morphogenic gene.
  • This disclosure provides for methods of transformation of soybean via somatic embryogenesis that rely on Growth Regulating Factor (GRF) polypeptide and/or a GRF-interacting Factor (GIF) polypeptide, or chimeric GRF-GIF genes and proteins, and/or morphogenic factor genes or proteins including BBM, WUS, WOX, STM, LEC1, LEC2, MYB115, MYB118 and their family members (SEQ ID NO 6 to SEQ ID NO 29) .
  • GRF Growth Regulating Factor
  • GRF GRF-interacting Factor
  • this disclosure provides methods and compositions for producing somatic embryos of soybean and other Glycine species using immature cotyledons, cultured on media containing low concentration of auxin, such as 2, 4-dichlorophenoxyacetic acid (2, 4-D) , in the presence of growth regulating factors and morphogenic genes including GFR, GIF or chimeric GFR-GIF, BBM, WUS, WOX, STM, LEC1, LEC2, MYB115, MYB118 and their family members (SEQ ID NO 6 to SEQ ID NO 29) .
  • the disclosure also provides methods for inducible excision of morphogenic factor gene expression cassette to allow high efficiency regeneration and recovery of transgenic or edited plants without abnormal phenotypes.
  • Another aspect of the disclosure includes a further method for such somatic embryogenesis and includes culture medium containing lower concentrations of auxin, such as e.g., 2, 4-D, for embryo proliferation, higher carbohydrate for embryo maturation, and lowered carbohydrate for embryo germination.
  • the method includes storage of immature cotyledon explants and seed pods.
  • the methods also include regeneration of transgenic plants from transformed somatic embryos and construction of transformation vectors containing the genes.
  • references to “a cell” include a plurality of such cells
  • references to “the protein” include references to one or more proteins and their equivalents known to those skilled in the art, and so on.
  • all technical and scientific terms used herein have the same meanings generally understood by those of ordinary skill in the art to which the present invention belongs.
  • Explant refers to tissue, a piece of tissue, or pieces of tissue derived from a plant or a plant part, such as a seed.
  • An explant can be a part of a plant, such as immature embryos, leaves meristems, or can be derived from a portion of the shoot, leaves, immature embryos or any other tissue of a plant or seed.
  • the word “or” refers to any member of a particular list and also comprises any combination of members of the list.
  • the term “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ( “or” ) .
  • Arange may be expressed in the present invention as being from “about” one specific value and/or to “about” another specific value.
  • other aspects include from one specific value and/or to other specific values.
  • the value is expressed as an approximation, it should be understood that by using the antecedent “about, ” the specific value forms another aspect.
  • the endpoints of each of the ranges are both significantly related to and independent of the other endpoint.
  • each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.
  • consists essentially of (and grammatical variants thereof) , as applied to a polynucleotide sequence of this invention, means a polynucleotide sequence that consists of both the recited sequence (e.g., SEQ ID NO 2 to 4) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides on the 5’ and/or 3’ ends of the recited sequence such that the function of the polynucleotide is not materially altered.
  • the total of ten or less additional nucleotides includes the total number of additional nucleotides on both ends added together.
  • polynucleotides of the invention refers to an increase or decrease in ability to express the polynucleotide sequence of at least about 50%or more as compared to the expression level of a polynucleotide sequence consisting of the recited sequence.
  • introgression means accomplished by any manner including but not limited to; introgression, transgenic, Clustered Regularly Interspaced Short Palindromic Repeats modification (CRISPR) , Transcription activator-like effector nucleases (TALENs) (Feng et al. 2013, Joung &Sander 2013) , meganucleases, or zinc finger nucleases (ZFNs) .
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats modification
  • TALENs Transcription activator-like effector nucleases
  • ZFNs zinc finger nucleases
  • soybean refers to soybean and any plant variety bred or cultured using soybean including “wild glycine” plants.
  • wild glycine refers to a perennial Glycine max plant, for example any one of G. canescens, G. argyrea, G. clandestine, G. latrobeana, G. albicans, G. aphyonota, G. arenaria, G. curvata, G. cyrtoloba, G. dolichocarpa, G. falcate, G. gracei, G. hirticaulis, G. lactovirens, G. latifolia, G. microphylla, G. montis-douglas, G. peratosa, G. pescadrensis, G. pindanica, G. pullenii, G. rubiginosa, G. stenophita, G. syndetika, or G. tomentella.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in single-stranded or double-stranded form and, unless otherwise limited, encompasses known analogues (e.g., peptide nucleic acids) that have the basic properties of natural nucleotides in the following aspects: it hybridizes to single-stranded nucleic acids in a manner similar to that of naturally occurring nucleotides.
  • analogues e.g., peptide nucleic acids
  • variants comprise deletion and/or addition of one or more nucleotides at one or more sites in the native nucleic acid molecule, and/or substitution of one or more nucleotides at one or more sites in the native nucleic acid molecule.
  • protein refers to a polymer of amino acid residues.
  • the term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of corresponding natural amino acids, and to natural amino acid polymers.
  • nucleic acid molecule or protein comprises a naturally occurring nucleotide sequence or an amino acid sequence, respectively.
  • nucleic acid comprises the desired information, which is specified by the use of codons to direct the translation of nucleotide sequences (for example, leguminous sequences) into specific proteins.
  • a nucleic acid coding a protein may comprise an untranslated sequence (e.g., an intron) within the translation region of the nucleic acid or may lack such an intermediate untranslated sequence (e.g., as in cDNA) .
  • allele refers to one of two or more different nucleotides or nucleotide sequences that occur at a specific locus.
  • Amarker is “associated with” a trait when it is linked to it and when the presence of the marker is an indicator of whether and/or to what extent the desired trait or trait form will occur in a plant/germplasm comprising the marker.
  • a marker is “associated with” an allele when it is linked to it and when the presence of the marker is an indicator of whether the allele is present in a plant/germplasm comprising the marker.
  • a marker associated with enhanced pathogen resistance refers to a marker whose presence or absence can be used to predict whether and/or to what extent a plant will display a pathogen resistant phenotype.
  • backcross and “backcrossing” refer to the process whereby a progeny plant is repeatedly crossed back to one of its parents.
  • the “donor” parent refers to the parental plant with the desired gene or locus to be introgressed.
  • the “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. For example, see Ragot, M. et al. Marker-assisted Backcrossing: A Practical Example, in TECHNIQUES ET UTILISATIONS DES MARQUEURS MOLECULAIRES LES COLLOQUES, Vol. 72, pp.
  • Acentimorgan ( “cM” ) is a unit of measure of recombination frequency.
  • One cM is equal to a 1%chance that a marker at one genetic locus will be separated from a marker at a second locus due to crossing over in a single generation.
  • chromosomal interval defined by and including, ” used in reference to particular loci and/or alleles refers to a chromosomal interval delimited by and encompassing the stated loci/alleles.
  • cross refers to the fusion of gametes via pollination to produce progeny (e.g., cells, seeds, or plants) .
  • progeny e.g., cells, seeds, or plants
  • the term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, e.g., when the pollen and ovule are from the same plant) .
  • crossing refers to the act of fusing gametes via pollination to produce progeny.
  • cultivar and “variety” refer to a group of similar plants that by structural or genetic features and/or performance can be distinguished from other varieties within the same species.
  • the terms “desired allele” , “favorable allele” and “allele of interest” are used interchangeably to refer to an allele associated with a desired trait (e.g., ASR resistance) .
  • the terms “inhibit, ” “reduce, ” etc., and grammatical variations thereof refer to any reduction in the expression or function of a target gene product, including any relative reduction in the expression or function up to and including complete elimination of the expression or function of the target gene product.
  • the term “enhance” and grammatical variations thereof refer to improvement, increase, amplification, reproduction, rise and/or elevation to reduce one or more disease symptoms.
  • the terms “increase, ” “enhance” etc., and grammatical variations thereof are used to refer to any promotion or gain or increase in the expression, function, or activity of a product of a target gene (for example, a resistance gene) as compared to a susceptible plant, thereby providing increased resistance to one or more pathogens (for example, Phakopsora) or diseases (for example, rust) .
  • a target gene for example, a resistance gene
  • pathogens for example, Phakopsora
  • diseases for example, rust
  • the term “cause” or “increase” and grammatical variations thereof may refer to a higher expression of a target gene product such that the level is increased by 10%or more, 50%or more, or 100%, relative to a cell or plant lacking the target gene or protein disclosed herein.
  • immunoscopically visible disease symptoms refer to the absence of any macroscopically visible disease symptoms.
  • partial resistance is used in the present invention to refer to the presence of macroscopically visible lesions without or with limited spore formation and/or a reduction in the scope or degree of any disease symptoms and/or a delay in the progression of any disease symptoms, and may, for example, manifest a reduction in the number of lesions or lesions with reduced spore formation.
  • the term “susceptibility” or the phrase “lack of resistance” in terms of rust refers to the occurrence of a lesion in the case where the spore formation level is equal to or higher than the spore formation level observed in a reference standard, such as, for example, the variety Williams or Peking.
  • resistance is used herein to refer to the absence or reduction of one or more disease symptoms caused by plant pathogens in plants. Resistance may mean that disease symptoms, such as the number of diseased plants, defoliation, and associated yield loss, are reduced, minimized, or decreased when compared to plants susceptible to the diseases or plants that do not comprise effective resistance genes that reduce one or more disease symptoms. In addition, resistance may include prevention or delay of pathogen proliferation. Generally speaking, the term “resistance” includes immunity and partial resistance as defined above.
  • the terms “enhanced pathogen resistance” , “enhanced plant pathogen resistance” , or “enhanced disease resistance” refers to an improvement, enhancement, or increase in a plant’s ability to endure and/or thrive despite being infected with a disease (e.g., Asian soybean rust) as compared to one or more control plants (e.g., one or both of the parents, or a plant lacking a marker associated with enhanced pathogen resistance to respective pathogen/disease) .
  • Enhanced disease resistance includes any mechanism (other than whole-plant immunity or resistance) that reduces the expression of symptoms indicative of infection for a respective disease such as Asian soybean rust, soybean cyst nematode, Pytophthora, etc.
  • a plant pathogen and grammatical variations thereof can be used herein to refer to, for example, a fungal pathogen of the genus Phakopsora of the class Basidiomycetes (including Phakopsora pachyrhizi and Phakopsora meibomiae) .
  • the plant diseases or the diseases of leguminous crops may be, for example, rust.
  • disease resistance gene or “resistance gene” is used in the present invention to refer to a gene encoding a protein capable of enhancing or improving the defense or immune system response in plants.
  • orthologue and grammatical variations thereof refer to genes derived from common ancestral genes and present in different species due to speciation.
  • An “elite line” or “elite strain” is an agronomically superior line that has resulted from many cycles of breeding and selection for superior agronomic performance. Numerous elite lines are available and known to those of skill in the art of soybean breeding. An “elite population” is an assortment of elite individuals or lines that can be used to represent the state of the art in terms of agronomically superior genotypes of a given crop species, such as soybean. Similarly, an “elite germplasm” or elite strain of germplasm is an agronomically superior germplasm, typically derived from and/or capable of giving rise to a plant with superior agronomic performance, such as an existing or newly developed elite line of soybean.
  • An “elite” plant is any plant from an elite line, such that an elite plant is a representative plant from an elite variety.
  • elite soybean varieties that are commercially available to farmers or soybean breeders include: AG00802, A0868, AG0902, A1923, AG2403, A2824, A3704, A4324, A5404, AG5903, AG6202 AG0934; AG1435; AG2031; AG2035; AG2433; AG2733; AG2933; AG3334; AG3832; AG4135; AG4632; AG4934; AG5831; AG6534; and AG7231 (Asgrow Seeds, Des Moines, Iowa, USA) ; BPR0144RR, BPR 4077NRR and BPR 4390NRR (Bio Plant Research, Camp Point, Ill., USA) ; DKB17-51 and DKB37-51 (DeKalb Genetics, DeKalb, Ill., USA) ; DP 4546 RR, and DP 7870 RR (Delta
  • agronomically elite means a genotype that has a culmination of many distinguishable traits such as emergence, vigor, vegetative vigor, disease resistance, seed set, standability, yield and threshability which allows a producer to harvest a product of commercial significance.
  • commercially significant yield or “agronomically acceptable yield” refers to a grain yield of at least 100%of a commercial check variety such as AG2703 or DKB23-51.
  • exotic, ” “exotic line” and “exotic germplasm” refer to any plant, line or germplasm that is not elite. In general, exotic plants/germplasms are not derived from any known elite plant or germplasm, but rather are selected to introduce one or more desired genetic elements into a breeding program (e.g., to introduce novel alleles into a breeding program) .
  • Germplasm is used in the present invention to refer to genetic material derived from an individual (e.g., a plant) , a group of individuals (e.g., a plant germline, variety, or family) , or a clone derived from a strain, variety, species, or culture. Germplasm can be part of an organism or a cell, or can be isolated from an organism or a cell. Germplasm provides genetic material having a specific molecular composition that provides the physical basis for some or all of the genetic properties of an organism or cell culture
  • a “genetic map” is a description of genetic linkage relationships among loci on one or more chromosomes within a given species, generally depicted in a diagrammatic or tabular form. For each genetic map, distances between loci are measured by the recombination frequencies between them. Recombinations between loci can be detected using a variety of markers.
  • a genetic map is a product of the mapping population, types of markers used, and the polymorphic potential of each marker between different populations. The order and genetic distances between loci can differ from one genetic map to another.
  • genotype refers to the genetic constitution of an individual (or group of individuals) at one or more genetic loci, as contrasted with the observable and/or detectable and/or manifested trait (the phenotype) .
  • Genotype is defined by the allele (s) of one or more known loci that the individual has inherited from its parents.
  • genotype can be used to refer to an individual’s genetic constitution at a single locus, at multiple loci, or more generally, the term genotype can be used to refer to an individual’s genetic make-up for all the genes in its genome. Genotypes can be indirectly characterized, e.g., using markers and/or directly characterized by nucleic acid sequencing.
  • germplasm refers to genetic material of or from an individual (e.g., a plant) , a group of individuals (e.g., a plant line, variety, or family) , or a clone derived from a line, variety, species, or culture.
  • the germplasm can be part of an organism or cell, or can be separate from the organism or cell.
  • germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture.
  • germplasm may refer to seeds, cells (including protoplasts and calli) or tissues from which new plants may be grown, as well as plant parts that can be cultured into a whole plant (e.g., stems, buds, roots, leaves, etc. ) .
  • haplotype is the genotype of an individual at a plurality of genetic loci, i.e., a combination of alleles. Typically, the genetic loci that define a haplotype are physically and genetically linked, i.e., on the same chromosome segment.
  • haplotype can refer to polymorphisms at a particular locus, such as a single marker locus, or polymorphisms at multiple loci along a chromosomal segment.
  • heterozygous refers to a genetic status wherein different alleles reside at corresponding loci on homologous chromosomes.
  • homozygous refers to a genetic status wherein identical alleles reside at corresponding loci on homologous chromosomes.
  • hybrid refers to a seed and/or plant produced when at least two genetically dissimilar parents are crossed.
  • the term “inbred” refers to a substantially homozygous plant or variety.
  • the term may refer to a plant or variety that is substantially homozygous throughout the entire genome or that is substantially homozygous with respect to a portion of the genome that is of particular interest.
  • the term “indel” refers to an insertion or deletion in a pair of nucleotide sequences, wherein a first sequence may be referred to as having an insertion relative to a second sequence or the second sequence may be referred to as having a deletion relative to the first sequence.
  • a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome.
  • transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome.
  • the desired allele may be a selected allele of a marker, a QTL, a transgene, or the like.
  • Offspring comprising the desired allele can be repeatedly backcrossed to a line having a desired genetic background and selected for the desired allele, with the result being that the desired allele becomes fixed in the desired genetic background.
  • a marker associated with enhanced ASR tolerance may be introgressed from a donor into a recurrent parent that is not disease resistant. The resulting offspring could then be repeatedly backcrossed and selected until the progeny possess the ASR tolerance allele (s) in the recurrent parent background.
  • operably linked, ” “operatively linked, ” “operatively associated” or “in operative association” and the like mean that elements of a nucleic acid construct such as an expression cassette or nucleic acid molecule are configured so as to perform their usual function.
  • regulatory or control sequences e.g., promoters
  • operatively associated with a nucleotide sequence are capable of effecting expression of the nucleotide sequence.
  • a promoter is operably linked with a coding sequence or functional RNA when it is capable of affecting the expression of that coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter) .
  • Coding sequences in sense or antisense orientation can be operably-linked to regulatory sequences.
  • the control sequences need not be contiguous with the nucleotide sequence of interest, as long as they function to direct the expression thereof.
  • intervening untranslated, yet transcribed, sequences can be present between a promoter and a coding sequence, and the promoter sequence can still be considered “operably linked” to the coding sequence.
  • plant refers to any plant, particularly to agronomically useful plants (e.g., seed plants)
  • plant cell is a structural and physiological unit of the plant, which comprises a cell wall but may also refer to a protoplast.
  • the plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized units such as for example, a plant tissue, or a plant organ differentiated into a structure that is present at any stage of a plant’ s development.
  • a plant may be a monocotyledonous or dicotyledonous plant species.
  • the term “expression cassette” refers to a nucleotide capable of directing expression of a particular nucleic acid sequence in a host cell.
  • the expression cassette comprises, consists essentially of or consists of one or more promoter sequences (e.g., one or more constitutive/inducible promoter sequences, one or more tissue-and/or organ-specific promoter sequences and/or one or more developmental stage-specific promoter sequences) operably linked to a nucleic acid of interest, which is operably linked to a termination sequence.
  • promoter sequences e.g., one or more constitutive/inducible promoter sequences, one or more tissue-and/or organ-specific promoter sequences and/or one or more developmental stage-specific promoter sequences
  • Expression cassettes often comprise sequences required for proper translation of the nucleic acid sequence of interest in the host cell.
  • the expression cassette may be chimeric in that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may be one that is naturally occurring but that has been obtained in a recombinant form useful for heterologous expression.
  • the expression cassette is heterologous with respect to the host (i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event) .
  • genome editing agent refers to an agent that is capable of inducing a deletion, insertion, indel, or other modification in the genome of a cell, e.g., by creating a single or double-stranded break in the genome.
  • genome editing agents include CRISPR/Cas agents (e.g., Cas proteins and guide RNAs) , transcription activator-like effector nucleases (TALENs) , DNA-guided nucleases, meganucleases, recombinases, and zinc finger nucleases.
  • Cas proteins include Cas9, Casl2a (also known as Cpfl) , C2cl, C2c2, and C2c3, and functional variants thereof.
  • Example Cas9 and Casl2a proteins include Streptococcus pyogenes Cas9 (SpCas9) , Streptococcus thermophilus Cas9 (StCas9) , Streptococcus pasteurianus (SpaCas9) , Campylobacter jejuni Cas9 (CjCas9) , Staphylococcus aureus (SaCas9) , Francisella novicida Cas9 (FnCas9) , Neisseria cinerea Cas9 (NcCas9) , Neisseria meningitis Cas9 (NmCas9) , Francisella novicida Cpfl (FnCpfl) , Acidaminococcus sp.
  • SpCas9 Streptococcus pyogenes Cas9
  • StCas9 Streptococcus thermophilus Cas9
  • Streptococcus pasteurianus Spa
  • a “variant” of a Cas protein refers to a protein or polypeptide derivative of a Cas protein, e.g., a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof.
  • the Cas variant is a functional variant which substantially retains the nuclease activity of or has better nuclease activity than the wild-type Cas protein.
  • Example guide RNAs include single guide RNAs and dual guide RNAs.
  • heterologous refers to a polynucleotide/polypeptide at least a part of which originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a nucleotide sequence derived from an organism or species different from that of the cell into which the nucleotide sequence is introduced is heterologous with respect to that cell and the cell's descendants.
  • a heterologous nucleotide sequence includes a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g., present in a different copy number, and/or under the control of different regulatory sequences than that found in the native state of the nucleic acid molecule.
  • a nucleic acid sequence can also be heterologous to other nucleic acid sequences with which it may be associated, for example in a nucleic acid construct, such as e.g., an expression vector.
  • a promoter may be present in a nucleic acid construct in combination with one or more regulatory element and/or coding sequences that do not naturally occur in association with that particular promoter, i.e., they are heterologous to the promoter.
  • plant part indicates a part of a plant, including single cells and cell tissues such as plant cells that are intact in plants, cell clumps and tissue cultures from which plants can be regenerated.
  • plant parts include, but are not limited to, single cells and tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, and seeds; as well as pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, scions, rootstocks, seeds, protoplasts, calli, and the like.
  • plant part also includes explants.
  • stably introducing or “stably introduced” in the context of a polynucleotide introduced into a cell is intended the introduced polynucleotide is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide.
  • “Stable transformation” or “stably transformed” as used herein means that a nucleic acid is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations.
  • “Genome” as used herein also includes the nuclear, mitochondrial and the plastid genome, and therefore includes integration of the nucleic acid into, for example, the chloroplast genome.
  • Stable transformation as used herein can also refer to a transgene that is maintained extrachromasomally, for example, as a minichromosome.
  • Selection agent refers to an agent (e.g., a chemical) that interacts with a selectable marker to give a plant cell a selective advantage.
  • agent e.g., a chemical
  • Example selection agents are known in the art and described herein, such as glyphosate, glufosinate, spectinomycin, bensulfuron-methyl, and kanamycin.
  • a “selectable marker” or “selectable marker gene” refers to a gene whose expression in a plant cell gives the cell a selective advantage. “Positive selection” refers to a transformed cell acquiring the ability to metabolize a substrate that it previously could not use or could not use efficiently, typically by being transformed with and expressing a positive selectable marker gene. This transformed cell thereby grows out of the mass of nontransformed tissue.
  • Positive selection can be of many types from inactive forms of plant growth regulators that are then converted to active forms by the transferred enzyme to alternative carbohydrate sources that are not utilized efficiently by the nontransformed cells, for example mannose, which then become available upon transformation with an enzyme, for example phosphomannose isomerase (PMI) , that allows them to be metabolized.
  • Nontransformed cells either grow slowly in comparison to transformed cells or not at all.
  • Other types of selection may be due to the cells transformed with the selectable marker gene gaining the ability to grow in presence of a negative selection agent, such as an antibiotic or an herbicide, compared to the ability to grow of non-transformed cells.
  • a selective advantage possessed by a transformed cell may also be due to the loss of a previously possessed gene in what is called “negative selection” . In this, a compound is added that is toxic only to cells that did not lose a specific gene (a negative selectable marker gene) present in the parent cell (typically a transgene) .
  • transformation refers to the transfer of a nucleic acid into a host cell, which includes integration into a chromosome, heritable extrachromosomal events and transient transfer.
  • the introduction into a plant, plant part and/or plant cell is via bacterial-mediated transformation, particle bombardment transformation (also called biolistic particle transformation) , calcium-phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, liposome-mediated transformation, nanoparticle-mediated transformation, polymer-mediated transformation, virus -mediated nucleic acid delivery, whisker-mediated nucleic acid delivery, microinjection, sonication, infiltration, polyethylene glycol-mediated transformation, protoplast transformation, or any other electrical, chemical, physical and/or biological mechanism that results in the introduction of a nucleic acid into the plant, plant part and/or cell thereof, or a combination thereof.
  • transgenic refers to any plant, plant cell, callus, plant tissue, or plant part that contains all or part of at least one heterologous polynucleotide.
  • all or part of the heterologous polynucleotide is stably integrated into a chromosome or stable extra-chromosomal element, so that it is passed on to successive generations.
  • SEQ ID NO: 1 the nucleotide sequence of Arabidopsis thaliana promoter prAtUBQ3.
  • SEQ ID NO: 2 the nucleotide sequence encoding rGRF4-GIF1.
  • SEQ ID NO: 3 the nucleotide sequence of rGFR4.
  • SEQ ID NO: 4 the nucleotide sequence of rGIF1.
  • SEQ ID NO: 5 the nucleotide sequence of Medicago truncatula terminator tMt1233.
  • SEQ ID NO: 6 the protein sequence of rGRF4-GIF1.
  • SEQ ID NO: 7 the protein sequence of rGRF4.
  • SEQ ID NO: 8 the protein sequence of rGIF1.
  • SEQ ID NO: 9 the protein sequence of Arabidopsis thaliana AtGRF4-GIF1 fusion (AtGRF4-AtGIF1) .
  • SEQ ID NO: 10 the protein sequence of Arabidopsis thaliana GRF4 (AtGRF4) .
  • SEQ ID NO: 11 the protein sequence of Arabidopsis thaliana GIF1 (AtGIF1) .
  • SEQ ID NO: 12 the protein sequence of Brassica napus BBM (BnBBM) .
  • SEQ ID NO: 13 the protein sequence of Arabidopsis thaliana EMBRYMAKER (AtEMB) , aka. AINTEGUMENTA-like 5 (AIL5) .
  • SEQ ID NO: 14 the protein sequence of Arabidopsis thaliana PLETHORA1 (AtPLT1) .
  • SEQ ID NO: 15 the protein sequence of Setaria italica BBM (SiBBM) .
  • SEQ ID NO: 16 the protein sequence of Brachypodium distachyon BBM (BdBBM) .
  • SEQ ID NO: 17 the protein sequence of Arabidopsis thaliana Wuschel (AtWUS) .
  • SEQ ID NO: 18 the protein sequence of Arabidopsis thaliana Wuschel-related homeobox protein 1 (AtWOX1) .
  • SEQ ID NO: 19 the protein sequence of maize (Zea mays) Wuschel 2 (ZmWUS2) .
  • SEQ ID NO: 20 the protein sequence of Brachypodium distachyon Wox5 (BdWOX5) .
  • SEQ ID NO: 21 the protein sequence of Arabidopsis thaliana AGL15 (AtAGL15) .
  • SEQ ID NO: 22 the protein sequence of Arabidopsis thaliana GRF5 (AtGRF5) .
  • SEQ ID NO: 23 the protein sequence of Arabidopsis thaliana MYB115 (AtMYB115) .
  • SEQ ID NO: 24 the protein sequence of Arabidopsis thaliana MYB118 (AtMYB118) .
  • SEQ ID NO: 25 the protein sequence of Arabidopsis thaliana Lec1 (AtLEC1) .
  • SEQ ID NO: 26 the protein sequence of Arabidopsis thaliana Lec2 (AtLEC2) .
  • SEQ ID NO: 27 the protein sequence of Medicago truncatula Somatic Embryo-Related Factor 1 (MtSERF1) .
  • SEQ ID NO: 28 the protein sequence of Arabidopsis thaliana Somatic Embryo Receptor-like Kinase 1 (AtSERK1) .
  • SEQ ID NO: 29 the protein sequence of Arabidopsis thaliana SHOOT MERITEMLESS (AtSTM)
  • SEQ ID NO: 30 the nucleotide sequence of Arabidopsis thaliana heat shock inducible promoter prAtHSP18.2.
  • SEQ ID NO: 31 the nucleotide sequence of heat inducible BnBBM gene expression cassette comprising of AtHSP18.2 promoter, BnBBM coding sequence and AtUbq3 terminator) .
  • SEQ ID NO: 32 the nucleotide sequence of codon-optimized Cre recombinase gene with an Arabidopsis thaliana intron (iAtRPS5A) .
  • SEQ ID NO: 33 the nucleotide sequence of heat inducible Cre gene expression cassette comprising of AtHSP18.2 promoter, Cre with AtRPS5A intron and NOS terminator
  • SEQ ID NO: 34 the nucleotide sequence of loxP recognition sequence for Cre recombinase
  • SEQ ID NO: 35 the nucleotide sequence of binary vector pHSPCre-BnBBM.
  • SEQ ID Nos: 1-35 are provided in Table A, below:
  • the disclosure provides a variety of different methods that may be used alone or together.
  • One aspect of the disclosure is directed an efficient method for rapid generation of transformed somatic embryos at globular, heart, torpedo and cotyledon stages in soybean using growth regulating factors and/or morphogenic genes, and auxins and/or cytokinins as regulators for the genes.
  • Another aspect of the disclosure is directed to methods for storage of immature cotyledon explants and seed pods.
  • Yet another aspect of the disclosure is directed to methods for regeneration of transgenic plants from transformed somatic embryos.
  • Yet another aspect of the disclosure is directed to methods for constructing transformation constructs using the genes and for controlling appropriate expression of the genes using inducible expression, developmentally regulated expression, and transient expression in soybean transformation.
  • One aspect of the disclosure is directed to efficient methods for rapid generation of transformed somatic embryos at globular, heart, torpedo and cotyledon stages of Glycine max plants using growth regulating factor and morphogenic genes, and auxins and/or cytokinins.
  • the methods include producing somatic embryos of a Glycine max plant that have been transformed to express a (at least one) nucleic acid encoding a growth regulating factor and/or a morphogenic gene.
  • the Glycine max plant has been transformed to express at least 2, at least 3, at least 4, at least 5, at least 5, at least 6, or at least 8 different nucleic acids encoding growth regulating factors and/or morphogenic genes.
  • the methods include transforming such an embryo to express a heterologous polynucleotide, e.g., a heterologous gene of interest.
  • the methods include both producing the somatic embryo and transforming the somatic embryo to express the protein of interest. The methods may use explants of somatic embryos at globular, heart, torpedo, and cotyledon stages of Glycine max plants.
  • One embodiment of the disclosure is directed to a method for producing somatic embryos of a Glycine max plant including at least the steps of introducing into immature cotyledons (isolated) from the Glycine max plant a nucleic acid encoding a growth regulating factor and/or a morphogenic gene; and culturing immature cotyledons with a culture medium supplemented with an auxin under conditions allowing for expression of the nucleic acid encoding a growth regulating factor and/or a morphogenic gene.
  • the methods provide for somatic Glycine max embryos that have improved capability to be further transformed to express a heterologous polynucleotide, e.g., a heterologous gene of interest.
  • the method includes introducing into somatic embryos at globular, heart, and/or torpedo stages of Glycine max plants a nucleic acid encoding a growth regulating factor and/or a morphogenic gene; and culturing immature cotyledons with a culture medium supplemented with an auxin under conditions allowing for expression of the nucleic acid encoding a growth regulating factor and/or a morphogenic gene.
  • the nucleic acid may be introduced into the somatic embryos using conventional methods known at the time of the invention. In one embodiment, the nucleic acid is introduced using bombardment. In other embodiments, the nucleic acid is introduce using Agrobacterium. In certain embodiments, the nucleic acid may be part of a vector, such as e.g. a plasmid.
  • the nucleic acid introduced into the somatic embryo is a growth regulating factor and/or a morphogenic gene.
  • the nucleic acid encodes a growth regulating factor.
  • the nucleic acid encodes a morphogenic gene.
  • the nucleic acid encodes both a growth regulating factor and a morphogenic gene.
  • the nucleic acid encodes a chimeric construct of both a growth regulating factor and a morphogenic gene.
  • growth regulating factors include but are not limited to Glycine max growth regulating factors (GmGRFs) , in particular GmGRF1-22, which are for example disclosed in Chen et al., BMC Plant Biology Volume 19, Article number 269 (2019) .
  • GmGRFs Glycine max growth regulating factors
  • the disclosure also includes nucleic acids encoding GmGRF1-12 polypeptide or fragments thereof having growth regulating activities.
  • the nucleic acid encodes growth regulating factor 4.
  • the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 3, a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, a nucleic acid encoding the amino acid sequence of SEQ ID NO: 7, or a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 7.
  • the nucleic acid encodes a morphogenic factors.
  • morphogenic factors include BBM (SEQ ID NO: 12) , WUS (SEQ ID NO: 17) , AGAMOUS-LIKE15 (SEQ ID NO: 21) and MYB118 (SEQ ID NO: 24) .
  • BBM SEQ ID NO: 12
  • WUS SEQ ID NO: 17
  • AGAMOUS-LIKE15 SEQ ID NO: 21
  • MYB118 SEQ ID NO: 24
  • suitable morphogenic genes are disclosed in Gordon-Kamm B et al., Plants (Basel) . 2019; 8(2) : 38, and Nalapalli et al, 2021, Methods Mol. Biol. 2238: 37-61, the disclosure of which is incorporated herein as it pertains to morphogenic genes.
  • the embryo may be transformed to express more than one growth regulating factor and/or a morphogenic gene.
  • the embryo can, for example, be transformed express both Growth Regulating Factor (GRF) polypeptide and a GRF-interacting Factor (GIF) polypeptide.
  • GRF Growth Regulating Factor
  • the GIF is GIF1 such as e.g., a GIF1 encoded by the nucleic acid sequence of SEQ ID NO: 4, a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 4, a nucleic acid encoding the amino acid sequence of SEQ ID NO: 8, or a nucleic acid that is or a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 8.
  • GIF1 such as e.g., a GIF1 encoded by the nucleic acid sequence of SEQ ID NO: 4, a nucleic acid that is 85%, 86%, 87%, 8
  • the embryo has been transformed with a nucleic acid encoding a GRF and a nucleic acid encoding GIF, such as GIF1.
  • the nucleic encodes a GRF (GmFRF) , such as GFR-4 (e.g., GFR-4 of SEQ ID NO: 3) , and a GIF, such as GIF1, a nucleic acid comprising SEQ ID NO: 4, a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 4, a nucleic acid encoding the amino acid sequence of SEQ ID NO: 8, or a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleic acid
  • the nucleic acid is a chimeric nucleic acid encoding a GRF (GmFRF) , such as GFR-4 (e.g., GFR-4 of SEQ ID NO: 3) , and a GIF, such as GIF1, a nucleic acid comprising SEQ ID NO: 4, a nucleic acid comprising SEQ ID NO: 4, a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 4, a nucleic acid encoding the amino acid sequence of SEQ ID NO: 8, or a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 8.
  • GRF
  • the nucleic acid is a chimeric nucleotide encoding GRF-4 and GIF1.
  • the chimeric nucleotide comprises: (1) a GRF-4 nucleic acid comprising SEQ ID NO: 3, a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, a nucleic acid encoding the amino acid sequence of SEQ ID NO: 7, or a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 7; and a GIF-1 nucleic acid a nucleic acid comprising SEQ ID NO: 4, a nucleic acid that is 85%, 86%, 8
  • the chimeric nucleotide comprising GRF-4 and GIF1 comprises: SEQ ID NO: 2; a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 2; a nucleic acid encoding the amnio acid sequence of SEQ ID NO: 6; or a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 6.
  • GRF Growth Regulating Factor
  • GRF-interacting Factor GRF
  • WO 2021/007284 Further examples of Growth Regulating Factor (GRF) polypeptides and/or GRF-interacting Factor (GIF) polypeptides, or chimeric GRF-GIF genes and proteins are disclosed in WO 2021/007284.
  • the methods include culturing with a culture medium supplemented with an auxin under conditions allowing for expression of the nucleic acid encoding a growth regulating factor and/or a morphogenic gene.
  • auxins may be used.
  • the auxin is a naturally occurring auxin such as e.g., indole-3-acetic acid, 4-chloroindole-3-acetic acid, phenylacetic acid, indole-3-butyric acid, and indole-3-propionic acid.
  • synthetic analogs of auxins such as 2, 4-Dichlorophenoxyacetic acid (2, 4-D) , are used.
  • the methods use less than 40 mg/ml of the auxin.
  • the medium is supplemented with about 4 mg/ml, alternatively about 3-40 mg/ml, about 4-40 mg/ml, alternatively about 4-35 mg/ml, alternatively about 4-30 mg/ml, alternatively about 4-25 mg/ml of auxin.
  • the auxin is 2, 4-Dichlorophenoxyacetic acid (2, 4-D) .
  • the method comprises culturing with about 4 mg/ml, alternatively less than 40 mg/ml, alternatively about 3-40 mg/ml, about 4-40 mg/ml, alternatively about 4-35 mg/ml, alternatively about 4-30 mg/ml, alternatively about 4-25 mg/ml of 2, 4-D.
  • the methods also include isolating the somatic embryos.
  • the method includes isolating the immature of cotyledons from the Glycine max plant prior to introducing the nucleic acid encoding a growth regulating factor and/or a morphogenic gene.
  • somatic embryos of a Glycine max plant are transformed to express a nucleic acid encoding a growth regulating factor and/or a morphogenic gene using the methods described above, they can be further transformed to express a heterologous polynucleotide, e.g., a heterologous gene of interest.
  • a heterologous polynucleotide e.g., a heterologous gene of interest.
  • the transformation of these somatic embryos of the Glycine max plant is carried out sequentially after transforming the Glycine max embryo to express a nucleic acid encoding a growth regulating factor and/or a morphogenic gene using the methods described above.
  • the transformation is carried out using Glycine max embryos that were previously transformed to express a nucleic acid encoding a growth regulating factor and/or a morphogenic gene using the methods described above.
  • the embryos may optionally be stored prior to being transformed to express the heterologous polynucleotide, e.g., a gene of interest.
  • the somatic embryos are transformed by bombardment to express the nucleic acid, e.g., a gene of interest.
  • the somatic embryos are transformed using Agrobacterium-mediated transformation to express a heterologous polynucleotide.
  • plants, plant parts, and plant cells transformed with a heterologous polynucleotide using the methods of the disclosure can be selected, e.g., using selectable markers present in the heterologous polynucleotide.
  • the plants, plant parts and plant cells transformed with a heterologous polynucleotide are selected using one or more selection steps or selection agents described in the Examples.
  • selectable markers include, but are not limited to, genes that provide resistance or tolerance to antibiotics such as kanamycin (Dekeyser et al. 1989, Plant Phys 90: 217-23) , spectinomycin (Svab and Maliga 1993, Plant Mol Biol 14: 197-205) , streptomycin (Maliga et al. 1988, Mol Gen Genet 214: 456-459) , hygromycin B (Waldron et al. 1985, Plant Mol Biol 5: 103-108) , bleomycin (Hille et al. 1986, Plant Mol Biol 7: 171-176) , sulphonamides (Guerineau et al.
  • antibiotics such as kanamycin (Dekeyser et al. 1989, Plant Phys 90: 217-23) , spectinomycin (Svab and Maliga 1993, Plant Mol Biol 14: 197-205) , streptomycin (Maliga et al. 1988, Mol Gen Genet
  • selectable markers include genes that provide resistance or tolerance to herbicides, such as the S4 and/or Hra mutations of acetolactate synthase (ALS) that confer resistance to herbicides including sulfonylureas, imidazolinones, triazolopyrimidines, and pyrimidinyl thiobenzoates; 5-enol-pyrovyl-shikimate-3-phosphate-synthase (EPSPS) genes, including but not limited to those described in U.S. Patent. Nos.
  • ALS acetolactate synthase
  • EPSPS 5-enol-pyrovyl-shikimate-3-phosphate-synthase
  • PPO Sprotophorphyrinogen oxidase mutants and variants, which confer resistance to peroxidizing herbicides including fomesafen, acifluorfen-sodium, oxyfluorfen, lactofen, fluthiacet-methyl, saflufenacil, flumioxazin, flumiclorac-pentyl, carfentrazone-ethyl, sulfentrazone) ; and genes conferring resistance to dicamba, such as dicamba monoxygenase (Herman et al. 2005, J Biol Chem 280: 24759-24767 and U.S. Patent No. 7,812,224 and related applications and patents) .
  • selectable markers can be found in Sundar and Sakthivel (2008, J Plant Physiology 165: 1698-1716) .
  • Additional selectable markers for use in the disclosure are known in the art such as Phosphinothricin N-acetyl transferase (PAT) and Aminoglycoside 3’-adenylyiltransferase (ad) (see, e.g., Rosellini (2012) Selectable Markers and Reporter Genes: A Well Furnished Toolbox for Plant Science and Genetic Engineering, Critical Reviews in Plant Sciences, 31: 5, 401-453) .
  • Phosphinothricin N-acetyl transferase PAT
  • Aminoglycoside 3’-adenylyiltransferase (see, e.g., Rosellini (2012) Selectable Markers and Reporter Genes: A Well Furnished Toolbox for Plant Science and Genetic Engineering, Critical Reviews in Plant Sciences, 31: 5, 401-453) .
  • selection systems include using drugs, metabolite analogs, metabolic intermediates, and enzymes for positive selection or conditional positive selection of transgenic plants. Examples include, but are not limited to, a gene encoding phosphomannose isomerase (PMI) where mannose is the selection agent, or a gene encoding xylose isomerase where D-xylose is the selection agent (Haldrup et al. 1998, Plant Mol Biol 37: 287-96) . Finally, other selection systems may use hormone-free medium as the selection agent.
  • PMI phosphomannose isomerase
  • xylose isomerase where D-xylose is the selection agent
  • other selection systems may use hormone-free medium as the selection agent.
  • the maize homeobox gene knl whose ectopic expression results in a 3-fold increase in transformation efficiency (Luo et al. 2006, Plant Cell Rep 25: 403-409) . Examples of various selectable markers and genes encoding them are disclosed in Miki and McHugh (J Biotechn
  • the selectable marker may be plant derived.
  • An example of a selectable marker which can be plant derived includes, but is not limited to, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) .
  • EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
  • the enzyme 5-enolpyruvylshikimate-3 -phosphate synthase (EPSPS) catalyzes an essential step in the shikimate pathway common to aromatic amino acid biosynthesis in plants.
  • the herbicide glyphosate inhibits EPSPS, thereby killing the plant.
  • Transgenic glyphosate-tolerant plants can be created by the introduction of a modified EPSPS transgene which is not affected by glyphosate (for example, U.S. Patent No. 6,040,497) .
  • Other sources of EPSPS which are not plant derived and can be used to confer glyphosate tolerance include but are not limited to an EPSPS P101S mutant from Salmonella typhimurium (Comai et al. 1985, Nature 317: 741-744) and a mutated version of CP4 EPSPS from Agrobacterium sp.
  • EPSPS is synthesized as a preprotein containing a transit peptide, and the precursor is then transported into the chloroplast stroma and proteolytically processed to yield the mature enzyme (della-Cioppa et al. 1986, PNAS 83: 6873-6877) . Therefore, to create a transgenic plant which has tolerance to glyphosate, a suitably mutated version of EPSPS which correctly translocates to the chloroplast could be introduced.
  • transgenic plant then has a native, genomic EPSPS gene as well as the mutated EPSPS transgene. Glyphosate could then be used as a selection agent during the transformation and regeneration process, whereby only those plants or plant tissue that are successfully transformed with the mutated EPSPS transgene survive.
  • the heterologous polynucleotide comprises a selectable marker and the method further comprises contacting the plant with a selection agent to eliminate or reduce untransformed tissue.
  • the selection agent is an herbicide, an antibiotic, or a non-metabolizable sugar.
  • the selection agent is glyphosate, glufosinate, spectinomycin, bensulfuron-methyl, imazapyr, D-xylose, mannose, or kanamycin.
  • the selectable marker is EPSPS, and the selection agent is glyphosate.
  • the contacting with the selection agent comprises adding the selection agent to a medium (e.g., soil or hydroponics) in which the plant is growing (e.g., by watering or applying to the soil or other medium a composition comprising the selection agent, such as between l ⁇ M to 1 M of a selection agent, e.g., 100 ⁇ M to 500 ⁇ M of glyphosate) , spraying the plant with the selection agent (e.g., with a sprayable composition comprising the selection agent, such as l ⁇ M to 1 M of a selection agent, e.g., between 10 ⁇ M to 50 mM glyphosate) , or applying the selection agent (such as between 1 ⁇ M to 1 M of a selection agent, e.g., 100 ⁇ M to 200 ⁇ M glyphosate or l0 ⁇ M to 100 ⁇ M Bensulfuron-methyl) to the regenerated axillary meristem (e.g., using a medium (
  • the contacting with the selection agent occurs for at least one day, at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, or longer. In some embodiments, the contacting with the selection agent occurs for between 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, or 5-6 weeks. In some embodiments, the contacting with the selection agent occurs for between 1 day to 6 weeks. In some embodiments, the contacting with the selection agent occurs for between 3-6 weeks.
  • the method further comprises performing an assay on a sample of the regenerated axillary meristem to assess the presence or absence of transformed cells in the sample and/or to assess the number of transformed cells in the sample.
  • Example assays include fluorescent protein detection, qPCR, real-time PCR, immunoassays, and the like.
  • the method further comprises growing the plant to produce a seed (e.g., one seed, two seeds, ten seeds, twenty seeds, fifty seeds or more) optionally comprising at least part of the heterologous polynucleotide and harvesting the seed.
  • all seeds produced by the plant comprise at least part of the heterologous polynucleotide.
  • at least one seed, or more seeds e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%
  • the method further comprises growing the seed (s) to produce a progeny plant (s) , optionally comprising at least part of the heterologous polynucleotide.
  • the heterologous polynucleotide encodes a genome editing agent, e.g., a CRISPR/Cas’s agent, a TALEN, a DNA-guided nuclease, a mega nuclease, a recombinase, or a zinc finger nuclease.
  • the heterologous protein comprises a genome editing agent, e.g., a Cas protein, a TALEN, a DNA-guided nuclease, a meganuclease, a recombinase, or a zinc finger nuclease.
  • the heterologous polynucleotide comprises one or more polynucleotides encoding a Cas protein and/or a guide RNA. In some embodiments, the heterologous polynucleotide comprises one or more guide RNAs, optionally wherein the heterologous polynucleotide is comprised within a ribonucleoprotein (RNP) with a Cas protein.
  • RNP ribonucleoprotein
  • the Cas protein is Cas9 or Casl2a, or a functional variant thereof.
  • the heterologous polynucleotide comprises an expression cassette comprising a coding sequence.
  • the coding sequence encodes a protein or non-coding RNA of interest.
  • the protein or non-coding RNA of interest confers one or more desired traits on a plant, such as enhanced growth, enhanced yield, drought tolerance, salt tolerance, herbicide tolerance, insect resistance, pest resistance, disease resistance, temperature tolerance, enhanced nitrogen utilization and the like.
  • the coding sequence encodes a genome editing agent, such as a Cas protein and/or a guide RNA.
  • the heterologous polynucleotide comprises a coding sequence encoding a protein or non-coding RNA of interest and a coding sequence a selection marker.
  • the expression cassette further comprises a promoter operably linked to the coding sequence (s) .
  • the promoter may be, e.g., a constitutive promoter, a tissue-specific promoter, or an inducible promoter.
  • the step of introducing the nucleic acid encoding a growth regulating factor and/or a morphogenic gene and/or the step of transforming are performed with Agrobacterium, viral particles, particles such as microparticles or nanoparticles (e.g., gold or tungsten microparticles or nanoparticles) , cell membrane penetrating peptides, aerosol beam, chemicals, electroporation, or pressure (e.g., vacuum) .
  • the step of introducing the nucleic acid encoding a growth regulating factor and/or a morphogenic gene and/or the step of transforming are performed with Agrobacterium.
  • the step of introducing the nucleic acid encoding a growth regulating factor and/or a morphogenic gene and/or the step of transforming are performed with viral particles. In some embodiments, the step of introducing the nucleic acid encoding a growth regulating factor and/or a morphogenic gene and/or the step of transforming are performed with gold or tungsten particles, such as microparticles or nanoparticles. In some embodiments, the step of introducing the nucleic acid encoding a growth regulating factor and/or a morphogenic gene and/or the step of transforming are performed with cell membrane penetrating peptides.
  • the step of introducing the nucleic acid encoding a growth regulating factor and/or a morphogenic gene and/or the step of transforming are performed with an aerosol beam. In some embodiments, the step of introducing the nucleic acid encoding a growth regulating factor and/or a morphogenic gene and/or the step of transforming are performed with chemicals. In some embodiments, the step of introducing the nucleic acid encoding a growth regulating factor and/or a morphogenic gene and/or the step of transforming are performed with electroporation. In some embodiments, the step of introducing the nucleic acid encoding a growth regulating factor and/or a morphogenic gene and/or the step of transforming are performed with pressure (e.g., vacuum) .
  • pressure e.g., vacuum
  • the step of introducing the nucleic acid encoding a growth regulating factor and/or a morphogenic gene and/or the step of transforming are performed with Agrobacterium or viral particles and the step comprises an infection step and an incubation step.
  • the infection step is performed for at least 30 minutes, e.g., 30 minutes to 24 hours, such as 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, 11-12, 1-11, 2-11, 3-11, 4-11, 5-11, 6-11, 7-11, 8-11, 9-11, 10-11, 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 1-9, 2-9, 3-9, 4-9, 5-9, 6-9, 7-9, 8-9, 1-8, 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 1-6, 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, 4-5, 1-4, 2-4, 3-4, 1-3, 2-3, or 1-2 hours, and the incubation step is performed in darkness or in light or in a light/dark cycle for
  • the infection step comprises contacting the wounded axillary meristem (s) with a solution, gel, absorbable material, or other material that contains the Agrobacterium or viral particles.
  • the infection step occurs for 5-12 hours.
  • the incubation step is performed in darkness for 3-7 days.
  • antibiotics e.g., Timentin, Cefotaxime and/or Vancomycin
  • Agrobacterium-mediated transformation is a method used for transforming plants.
  • Agrobacterium-mediated transformation typically involves transfer of a binary vector carrying the foreign DNA of interest to an appropriate Agrobacterium strain that may depend on the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (see, e.g., Uknes et al. 1993, Plant Cell 5: 159-169) .
  • the transfer of the recombinant binary vector to Agrobacterium can be accomplished, e.g., by a tri-parental mating procedure using Escherichia coli carrying the recombinant binary vector, a helper E.
  • the recombinant binary vector can be transferred to Agrobacterium by nucleic acid transformation (see, e.g., Hofgen and Willmitzer 1988, Nucleic Acids Res 16: 9877) .
  • Another embodiment of the disclosure is directed to methods of storing immature cotyledon explants and seed pods.
  • the immature cotyledon explants and seed pods have been transformed to express a heterologous polynucleotide, e.g., a heterologous gene of interest.
  • the immature cotyledon explants and seed pods have been transformed to express a heterologous polynucleotide, e.g., a heterologous gene of interest using the methods described herein.
  • the immature cotyledon explants and seed pods are grown and stored using these methods prior to being transformed using for example the transformation methods described above.
  • the method includes storing immature cotyledon by first storing immature seed pods at 4°C for 5 days and then pre-culturing immature cotyledon on a Murashige and Skoog (MS) basal medium for 3 days. Using this method, the success of transformation was increased.
  • MS Murashige and Skoog
  • one embodiment is directed to a storage method comprising storing seed pods at a temperature of about 4°C for about 5 days, alternatively about 4-6 days and culturing the somatic embryos at globular, heart, torpedo, and cotyledon stages of Glycine max plants for about 3 days, alternatively about 3-5 days on a suitable medium prior to transformation.
  • the suitable medium is supplemented with an auxin, such as the auxins described above in the amounts described above.
  • the storage method comprising storing seed pods at a temperature of about 4°C for about 5 days, alternatively about 4-6 days and culturing the immature cotyledon explants of Glycine max plants for about 3 days, alternatively about 3-5 days on a suitable medium prior to transformation.
  • the suitable medium is supplemented with an auxin, such as the auxins described above in the amounts described above.
  • the suitable medium is MS basal medium.
  • Another aspect of the disclosure is directed to methods for regeneration of transgenic plants from transformed somatic embryos.
  • the transformed embryos having been generated using the methods described herein.
  • the methods for storage of immature plant explants and seed pods were used prior to generating the transformed embryos using e.g., the methods described herein.
  • somatic embryos were proliferated on a medium with lower auxin medium followed by embryo maturation in a medium with higher carbohydrate and germination in a medium with lower carbohydrate to produce transgenic plants.
  • the method of regenerating transgenic plants comprises: culturing transformed somatic embryo on a medium supplemented with an auxin, such as an auxin described above, using a concentration of auxin less than the concentration described above; embryo maturation in a medium with carbohydrate concentration higher than during the culturing; and germination in a medium with lower carbohydrate concentration lower than during the embryo maturation to produce transgenic plants.
  • an auxin such as an auxin described above
  • the disclosure also includes methods of generating transformed constructs encoding the growth regulating factors and/or morphogenic genes introduced into Glycine max using the methods of the disclosure.
  • the methods include controlling appropriate expression of the genes using inducible expression, developmentally regulated expression, and transient expression in soybean transformation.
  • one embodiment of the disclosure is directed to a method of constructing a vector encoding the growth regulating factors and/or morphogenic genes introduced into Glycine max using the methods of the disclosure.
  • the method is directed to constructing a chimeric vector encoding a growth regulating factor, such as e.g., GRF4, and its cofactor GFR interacting factor, such as e.g., GIF1.
  • the methods comprise synthesizing a chimeric GRF-GIF nucleotide.
  • the chimeric nucleotide comprises GRF-4 and GIF1.
  • the chimeric nucleotide acid comprises: (1) a GRF-4 nucleic acid having SEQ ID NO: 3, a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, a nucleic acid encoding the amino acid sequence of SEQ ID NO: 7, or a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 7; and (2) a GIF-1 nucleic acid a nucleic acid
  • the chimeric nucleotide comprising GRF-4 and GIF1 and comprises: SEQ ID NO: 2; a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 2; a nucleic acid encoding the amnio acid sequence of SEQ ID NO: 6; or a nucleic acid that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 6.
  • the nucleotide comprising the GRF and GIF contain a terminator.
  • the nucleotide comprising a chimeric nucleotide comprising GFR4 and GIF1 comprises a terminator such as e.g., tMt12344 from a Medicago truncatula gene (GeneChip ID Mtr. 12344.1. S1_at) .
  • the terminator comprises SEQ ID NO: 5.
  • the nucleotide is under the control of Arabidopsis Ubiquitin3 (AT5G03240) promoter prUBQ3, such as e.g., a promoter of SEQ ID NO: 1 or a functional fragment thereof.
  • the chimeric nucleotide comprising GRF and GIF (such as e.g., GRF4 and GIF1) , a terminator such as e.g., t12344, and a promoter, such as e.g., prUBQ3, for an expression cassette that may be inserted into a plasmid.
  • the expression cassette is inserted into a plasmid, it may further include a restriction enzyme cleavage site.
  • the expression cassette insert contains a restriction enzyme cleavage site at both the 5’ and the 3’ end of the cassette.
  • the expression cassette includes a AsiSI at the 5’ prime end and BamHI at the 3’ prime end.
  • aspects of the disclosure relates to a plant or plant part produced by any of the methods described above or elsewhere herein, including in the Examples.
  • Other aspects of the disclosure relate to progeny seed produced by crossing the plant produced by any of the methods described above or elsewhere herein with a second plant or by selfing the plant.
  • Other aspects of the disclosure relate to a derivative or a commodity product produced or obtained from the plant or plant part produced by any of the methods described above or elsewhere herein.
  • the commodity product is selected from the group consisting of whole or processed seeds, flour, protein isolates, concentrates, liquids, syrups, pastes, sauces or other food or product produced from the plant or plant part.
  • Example 1 DNA constructs for expression of morphogenic factors and Agrobacterium strains
  • pBSC26550 containing an expression cassette for the chimeric rGRF-GIF fusion was constructed as shown in the schematic map of FIG. 1.
  • pBSC26550 T-DNA region contains four expression cassettes: 1) The grape GRF4-GIF1 fusion protein coding sequence (Debernardi et al 2020) with prAtUbq3 promoter and tMt12344 terminator, 2) SpecR (aadA) gene with prGmEF promoter and tPsE9 terminator, 3) LbCas12a (D156R) coding sequence with prAtEF1A promoter and tNOS terminator, and 4) gRNA (crRNA direct repeat and guide/spacer sequence) flanked by Hammerhead (HH) and hepatitis delta virus (HDV) ribozymes controlled by prGmUbi1 promoter and tNOS terminator.
  • HH Hammerhead
  • HDV hepatitis delta virus
  • the DNA fragment comprising the Vitis rGRF-GIF gene fusion was prepared as described in Debernardi JM, et al., (2020) A GRF–GIF chimeric protein improves the regeneration efficiency of transgenic plants, Nature Biotech 38: 1274–1279.
  • the expression cassette is controlled by Arabidopsis Ubiquitin3 gene (AT5G03240) promoter and contains a tMt12344 terminator from a Medicago truncatula gene (GeneChip ID Mtr. 12344.1. S1_at) .
  • the sequences used for this study are provided in SEQ ID NOs: 1-5.
  • the morphogenic factor genes are preferably placed under the control of an inducible promoter. For example, binary transformation vector pHSP-BnBBM (FIG.
  • Syngenta soybean varieties Var1 and Var 2 were grown in a greenhouse with 14 hours of light and 10 hours dark at 24 °C. Immature soybean pods were harvested from the plants. The pods were sterilized in a container with about 1 liter of 20%Clorox solution and 5 drops of Tween-20; then agitated on a platform shaker for about 20 minutes in a sealed container. The pods were thoroughly rinsed with sterile distilled water to remove all traces of Clorox and Tween 20.
  • Immature seeds with zygotic embryos that were no more than 5 mm long in size were excised from seed pods.
  • the seed coat was carefully removed from the immature seeds.
  • the young cotyledons were dissected and the end with the embryonic axis was discarded.
  • the excised cotyledons were placed on a callus induction medium or basal MS medium (4.3g/L basal salt mixture 1 x B5 vitamins, 20g/L sucrose, 7 g/L purified agar) with the FLAT side up.
  • the excised immature cotyledon explants were then incubated at 25+1 degree C with 16/8hour light/dark, respectively, photoperiods for 0-5 days (pre-culturew) before use for bombardment. (Table 1.
  • immature cotyledon explants were selected and arranged by placing them in the center of Petri dish containing osmoticum medium (MS-12%sucrose: 4.3 g/L basal salt mixture 1 x B5 vitamins, 2, 7 g/L purified agar, 12%sucrose) .
  • MS-12%sucrose 4.3 g/L basal salt mixture 1 x B5 vitamins, 2, 7 g/L purified agar, 12%sucrose
  • Example 3 Biolistic delivery of DNA to immature embryo explants.
  • the plasmid DNA of Example 1 (pBSC26550 or pHSP-BnBBM) was precipitated onto 0.6 ⁇ m gold particle as described in the Bio-Rad Biolistics TM Manual. Bombardment was performed using a Biolistic PDS-1000 Particle Delivery System (Bio-Rad) according to the instruction manual. The immature cotyledon soybean explants were placed with the flat side upward in petri dishes containing MS medium immediately before the bombardment. Genes were delivered to the target tissue cells using a PDS-1000He Biolistics TM device. The settings on the device were as follows: 8 mm (3/8 inch) between the rupture disk and the macrocarrier, 10 mm between the microcarrier and the stopping screen and 7 cm between the stopping screen and the target.
  • the immature cotyledon target plates were shot twice with 0.08 pmole of DNA per shot using 1100-psi rupture disks.
  • a stainless steel mesh with 200 openings per lineal inch horizontally and vertically was placed between the stopping screen and the target tissue.
  • the bombarded explants were transferred to fresh MSD4 medium (4.3g/L basal salt mixture 1 x B5 vitamins, 4mg/L 2, 4-D, 7 g/L purified agar) for recovery and incubated at 24°C for 1-3 days in the dark, and then transferred to MSD4 medium containing selection agent.
  • Example 4 GRF-GIF gene and auxin formation of somatic embryos using immature cotyledon explants in soybean.
  • Example 5 Storage of immature cotyledon explants and seed pods
  • MSD4 4.30g/L MS Basal Salt mixture, 1x B5 Vitamins, 4mg/L 2, 4-D 7g/L purified agar
  • MSM 64.3 g/L MS Basal Salt mixture 1x B5 Vitamins, 60g/L Maltose, 6g/L purified agar
  • MSO MS 4.3g/L Basal Salt mixture, 1x B5 Vitamins, 15g/L Sucrose, 6g/L purified agar
  • Germinated seedlings from transformed somatic embryos started to appear 2 to 3 months after bombardment (FIG. 7) .
  • Example 7 Agrobacterium-mediated soybean immature cotyledon transformation facilitated by Growth Regulating Factor (GRF) or Morphogenic Factors (BnBBM)
  • GEF Growth Regulating Factor
  • BnBBM Morphogenic Factors
  • Agrobacterium cells containing transformation vector pBSC26550 (FIG. 1) or pHSPCre-BnBBM (FIG. 2) were collected from the YP culture plate using a disposable plastic inoculation loop and resuspended in liquid infection medium (SoyInf, 2.15 g/L MS Basal Salt Mixture, 1x B5 Vitamins, 20 g/L sucrose, 10 g/L glucose, 5 mg/L 2, 4-D, 4 g/L MES, pH5.4) , in a sterile disposable 50 ml centrifuge tube. The tube was mixed until Agrobacterium cells were uniformly dispersed in the suspension.
  • SoyInf 2.15 g/L MS Basal Salt Mixture, 1x B5 Vitamins, 20 g/L sucrose, 10 g/L glucose, 5 mg/L 2, 4-D, 4 g/L MES, pH5.4
  • Immature cotyledon explants were prepared as described in Example 2. The young cotyledons were cut with the end with the embryonic axis discarded. The excised cotyledon explants were prepared by then immediately mixing the isolated explants with the Agrobacterium suspension. Optionally, the explants were treated with heat shock at 42 °C for 30 sec to 5 minutes before mixing with Agrobacterium cell suspension. The explants were incubated with Agrobacterium suspension for 30 minutes at room temperature with occasional shaking (preferably in the dark) .
  • the Agrobacterium suspension was then removed and the infected immature cotyledon explants were placed in cocultivation medium (CCM, 2.15 g/L MS Basal Salt Mixture, 1x B5 Vitamins, 20 g/L sucrose, 10 g/L glucose, 4 g/L MES, 40 mg/L acetosyringone, 5 mg/L 2, 4-D, 6 g/L purified agar, pH5.4) at 24+1°C with 16/8 hr light/dark photoperiod for 0-5 days (pre-culture) for 2-5 days.
  • CCM cocultivation medium
  • CCM 2.15 g/L MS Basal Salt Mixture
  • 1x B5 Vitamins 20 g/L sucrose, 10 g/L glucose, 4 g/L MES, 40 mg/L acetosyringone, 5 mg/L 2, 4-D, 6 g/L purified agar, pH5.4
  • Explants were incubated in the Agrobacterium suspension for at least 30 minutes and up to overnight at room temperature in the dark (preferably 1 hour) . In case of overnight incubation, the mixture was covered by black plastic wrap or placed in the dark at room temperature (23 °C) .
  • the explants were removed from the Agrobacterium suspension and transferred to Petri dishes with CIM (2.15 g/L MS Basal Salt Mixture, 1x B5 Vitamins, 20 g/L sucrose, 10 g/L glucose, 4 g/L MES, 40 mg/L acetosyringone, 5 mg/L 2, 4-D, 7 g/L purified agar pH5.4, 150 mg/L Timentin, 75 mg/L Cefotaxime, 50 mg/L vancomycin) with explants placed adaxial (flat) side up for co-cultivation.
  • the plates were placed in a plastic Flambeau container and incubated for 7-14 days (preferably 4 to 5 days) at 22+1 °C in the dark to induce callus and somatic embryo formation.
  • MSD5 medium containing appropriate selection , e.g. MSD5Spec (4.3 g/L MS Basal Salt Mixture, 1 x B5 vitamins, 5 mg/L 2, 4-D, 50 mg/L spectinomycin, 7 g/L purified agar) for pBSC26550 (FIG. 1) or other vectors with SpecR/aadA selectable marker) or MSD5BSU (4.3 g/L MS Basal Salt Mixture, 1 x B5 vitamins, 5 mg/L 2, 4-D, 2 ⁇ M bensulfuron-methyl, 7 g/L purified agar, pH5.8) for pHSPCre-BnBBM (FIG. 2) or other vectors ALS selectable marker) .
  • MSD5Spec 4.3 g/L MS Basal Salt Mixture, 1 x B5 vitamins, 5 mg/L 2, 4-D, 50 mg/L spectinomycin, 7 g/L purified agar
  • MSD5BSU 4.3 g/L MS Bas
  • MSD5 (4.30 g/L MS Basal Salt Mixture, 1x B5 Vitamins, 5 mg/L 2, 4-D, pH5.8, 7 g/L purified agar) followed by embryo maturation in a medium with higher carbohydrate (MSM6, 4.30 g/L MS Basal Salt Mixture, 1x B5 Vitamins, 60 g/L Maltose, pH5.8, 7 g/L purified agar) .
  • MSM6 4.30 g/L MS Basal Salt Mixture, 1x B5 Vitamins, 60 g/L Maltose, pH5.8, 7 g/L purified agar
  • Somatic embryos were subjected heat shock treatments (42 to 45 °C for 2-3 hours each for 1-3 times over the period of 2 weeks) for inducing auto-excision of BnBBM and Cre cassettes when pHSPCre-BnBBM vector was used for transformation.
  • Mature somatic embryos were germinated by transferring to a medium with lower carbohydrate (MS0: MS 4.3 g/L Basal Salt Mixture, 1x B5 Vitamins 15.0 g/L Sucrose, pH5.8, 7 g/L Purified agar) to produce transgenic plants.
  • Germinated seedlings were transplanted to soil and sampled for transgene copy number analysis by Taqman qPCR method and grown to maturity for seed production as described (Que et al., WO2008112267, Transformation of Immature Soybean Seeds through Organogenesis; Zhong and Li, WO2021108337, Methods of Transformation) .
  • spectinomycin resistant somatic embryos appeared as early as 10 days (FIG. 3) after culture compared with approx. 30 to 50 days when using existing methods (Channareddy et al., 2018) . Under the described conditions 36.5%of cotyledons produced spectinomycin resistant somatic embryos 4 weeks after bombardment (Table 1) . Germinating somatic embryos appeared approx. 2 to 3 months after bombardment (FIG. 6) . Certain specific advantages include, but are not limited to:

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Abstract

La présente invention concerne des procédés et des compositions pour produire des embryons somatiques de soja et d'autres espèces de glycine à l'aide de cotylédons immatures, cultivés sur des milieux contenant une faible concentration d'auxine, tels que l'acide 2, 4-dichlorophénoxyacétique (2, 4-D) en présence de facteurs de régulation de croissance et de gènes morphogéniques tels que GFR, GIF ou GFR-GIF chimérique. L'invention concerne également un autre procédé pour une telle embryogenèse somatique à l'aide d'un milieu de culture contenant des concentrations inférieures d'auxine pour la prolifération d'embryons, un glucide supérieur pour la maturation d'embryons, et un glucide abaissé pour la germination d'embryons. Dans certains modes de réalisation, le procédé comprend le stockage d'explants de cotylédon immatures et de capsules de graines. Dans d'autres modes de réalisation, les procédés comprennent également la régénération de plantes transgéniques à partir d'embryons somatiques transformés et la construction de vecteurs de transformation contenant les gènes.
PCT/CN2023/090935 2023-04-26 2023-04-26 Procédés de transformation du soja et produits/compositions associés WO2024221290A1 (fr)

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