Genotype-independent media regimen for the initiation, maintenance, and transformation of soybean callus and suspension cultures

Abstract

The disclosure relates to methods of producing soybean cell suspension cultures. Induction media, proliferation media, and suspension media that effectively induce and maintain soybean callus and cell suspension cultures independent of genotype are provided.

Description

Agent Ref.: P15010WO00TITLE : GENOTYPE-INDEPENDENT MEDIA REGIMEN FOR THE INITIATION, MAINTENANCE, AND TRANSFORMATION OF SOYBEAN CALLUS AND SUSPENSION CULTURESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to European patent application No. 25386006.8, filed January 24, 2025, which is hereby incorporated herein by reference in its entirety.BACKGROUND

[0002] Recent advances in genome editing technologies have provided opportunities for precise modification of the genome in many types of organisms, including plants and animals. For example, technologies based on genome editing proteins, such as zinc finger nucleases, TALENs, and CRISPR systems are advancing rapidly and it is now possible to target genetic changes to specific DNA sequences in the genome. Efficient soybean cell culture systems are essential for advancements in genome editing, yet current practices rely on media regimens tailored to specific genotypes. This specificity limits the broader applicability of these regimens across diverse soybean genotypes.SUMMARY

[0003] Genotype-independent methods of producing a soybean cell suspension culture comprising (i) inducing callus from a tissue of a soybean plant on an induction medium; (ii) proliferating the induced callus obtained in step (i) on a proliferation medium; and (iii) suspending the proliferated callus obtained in step (ii) in a suspension medium, thereby producing the soybean cell suspension culture are provided.DETAILED DESCRIPTION

[0004] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0005] As used herein, the term “calli” or “callus” refers to a group of structurally undifferentiated cells derived from any plant parts of plants.Agent Ref.: P15010WO00

[0006] As used herein, “consisting essentially of’ means that the medium composition does not include any other component in an amount that would materially affect the ability of the medium composition to effectively induce and / or proliferate callus.

[0007] As used herein, “cotyledon” refers to an embryonic leaf or “primary leaf’ of the embryo of a seed plant. A cotyledon is also referred to in the art as a “seed leaf.” Dicotyledonous species, such as soybean, have two cotyledons.

[0008] As used herein, the term “cytokinin” refers to a class of plant growth regulators that enhance cell division, cell differentiation, and axillary bud growth, and inhibit apical dominance. There are two types of cytokinins based on their chemical structures: adenine-type cytokinins (e.g., kinetin, zeatin, 6-benzylaminopurine) and phenylurea-type cytokinins (e.g., diphenylurea, thidiazuron).

[0009] As used herein, the terms “include,” “includes,” and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

[0010] As used herein, the term “introduced” means providing a nucleic acid (e.g, expression construct or gene editing molecules) or protein into a cell. Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell and includes reference to the transient provision of a nucleic acid or protein to the cell. Introduced includes reference to stable or transient transformation methods. Thus, “introduced” in the context of inserting a nucleic acid fragment (e.g, a recombinant DNA construct / expression construct) into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., nuclear chromosome, plasmid, plastid, chloroplast, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA). The term “introduced” can also apply to introduction of gene editing molecules that can cause a deletion, base substitution, and / or insertion in the genome of the cell.

[0011] As used herein, the term “plant” includes a whole plant and any descendant, cell, tissue, or part of a plant. The term “plant parts” include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; or a plant organ (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, and explants). A plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit.Agent Ref.: P15010WO00

[0012] As used herein, the term “protoplast” refers to a plant cell that had its cell wall completely or partially removed, with the lipid bilayer membrane thereof naked.

[0013] As used herein, the term “subculturing” refers to conditions that typically involve harvesting (withdrawing) cells, diluting or splitting the cells with fresh cell suspension media, and cultivating the diluted or split cell culture.

[0014] As used herein, the terms “suspension cell” or “suspension culture” refer to a specialized population of homogeneous, undifferentiated plant cells grown in liquid nutrient or culture media. The cells are typically suspended within the media as opposed to adhering to a surface.

[0015] To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.

[0016] The present disclosure provides genotype-independent methods for producing soybean cell suspension cultures. The methods comprise (i) inducing callus from a tissue of a soybean plant on an induction medium; (ii) proliferating the induced callus obtained in step (i) on a proliferation medium; and (iii) suspending the proliferated callus obtained in step (ii) in a suspension medium, thereby producing the soybean cell suspension culture.

[0017] Methods of the disclosure include the step of inducing callus from a tissue of a soybean plant on an induction medium. In certain embodiments, the induction medium comprises inorganic salts, vitamins, a carbon source, a nitrogen source, L-proline, a plant growth regulator, and optionally a gelling agent.

[0018] In certain embodiments, the induction medium comprises inorganic salts and / or vitamins. A variety of inorganic salts and vitamins are known that support plant tissue growth and development. These inorganic salts and vitamins can either be purchased as a commercial preparation or custom prepared and modified by those of skill in the art. Examples of inorganic salts for use in the induction medium include, but are not limited to, Murashige and Skoog (MS) basal salts (see, e.g., Murashige and Skoog (1962) Physiol. Plant., 15: 473-497), Gamborg B5 basal salts (see, e.g., Gamborg et al. (1968) Exp. Cell Res., 50: 151-158), Nitsch and Nitsch basal salts, Chu N6 basal salts, and Schenk and Hildebrandt basal salts, or derivations of these basal salt mixtures. In certain embodiments, the induction medium comprises MS basal salts. Examples of vitamins for use in the induction medium include, but are not limited to, MS vitamins, Gamborg B5 vitamins, Nitsch and Nitsch vitamins, and Chu N6 vitamins. In certain embodiments, the induction medium comprises Gamborg B5 vitamins. In certain embodiments, the induction medium comprises MS basal salts and Gamborg B5 vitamins.Agent Ref.: P15010WO00

[0019] In certain embodiments, the induction medium comprises a carbon source, such as a carbohydrate. Examples of a carbon source for use in the induction medium include, but are not limited to, glucose, sucrose, maltose, mannose, fructose, lactose, galactose, and dextrose. In certain embodiments, the induction medium comprises at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 25 g / L, at least about 30 g / L, at least about 35 g / L, or at least about 40 g / L of a carbon source. In certain embodiments, the induction medium comprises from about 5 g / L, about 10 g / L, about 15 g / L, about 20 g / L, or about 25 g / L to about 35 g / L, about 40 g / L, about 45 g / L, or about 50 g / L of a carbon source. In certain embodiments, the induction medium comprises sucrose as the carbon source. In certain embodiments, the induction medium comprises at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 25 g / L, at least about 30 g / L, at least about 35 g / L, or at least about 40 g / L of sucrose. In certain embodiments, the induction medium comprises from about 5 g / L, about 10 g / L, about 15 g / L, about 20 g / L, or about 25 g / L to about 35 g / L, about 40 g / L, about 45 g / L, or about 50 g / L of sucrose. In certain embodiments, the induction medium comprises about 30 g / L of sucrose.

[0020] In certain embodiments, the induction medium comprises a nitrogen source. Examples of a nitrogen source include, but are not limited to, casein hydrolysate, yeast extract, malt extract, and corn steep liquor. In certain embodiments, the induction medium comprises at least about 0.05 g / L, at least about 0.10 g / L, at least about 0.15 g / L, at least about 0.20 g / L, at least about 0.25 g / L, or at least about 0.30 g / L of a nitrogen source. In certain embodiments, the induction medium comprises from about 0.05 g / L, about 0.10 g / L, about 0.15 g / L, about 0.20 g / L to about 0.25 g / L, about 0.3 g / L, about 0.35 g / L, or about 0.40 g / L of a nitrogen source. In certain embodiments, the induction medium comprises casein hydrolysate as the nitrogen source. In certain embodiments, the induction medium comprises at least about 0.05 g / L, at least about 0.10 g / L, at least about 0.15 g / L, at least about 0.20 g / L, at least about 0.25 g / L, or at least about 0.30 g / L of casein hydrolysate. In certain embodiments, the induction medium comprises from about 0.05 g / L, about 0.10 g / L, about 0.15 g / L, about 0.20 g / L to about 0.25 g / L, about 0.3 g / L, about 0.35 g / L, or about 0.40 g / L of casein hydrolysate. In certain embodiments, the induction medium comprises about 0.23 g / L of casein hydrolysate.

[0021] In certain embodiments, the induction medium comprises L-proline. In certain embodiments, the induction medium comprises at least about 0.1 g / L, at least about 0.2 g / L, at least about 0.3 g / L, at least about 0.4 g / L, or at least about 0.5 g / L of L-proline. In certain embodiments, the induction medium comprises from about 0.2 g / L, about 0.3 g / L, or about 0.4Agent Ref.: P15010WO00g / L to about 0.6 g / L, about 0.7 g / L, or about 0.8 g / L of L-proline. In certain embodiments, the induction medium comprises about 0.5 g / L of L-proline.

[0022] In certain embodiments, the induction medium comprises a plant growth regulator. The term plant growth regulator refers to naturally occurring plant hormones and to synthetic chemical analogs of naturally occurring plant hormones that are applied to mimic the effects of plant hormones. Naturally occurring plant hormones generally fall under one of five classes: auxin, cytokinin, gibberellin, ethylene, and abscisic acid. In certain embodiments, the induction medium comprises an auxin. Auxins include naturally occurring and synthetic auxins. Naturally occurring auxin is indole-3 -acetic acid (IAA). In certain embodiments, the induction media does not comprise IAA. In certain embodiments, the induction medium comprises a synthetic auxin. Other auxins include, but are not limited to, di chlorophenoxy acetic acid (2,4-D), 4-chlorophenoxyacetic acid (4-CPA), 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB), tris[2-(2,4-dichlorophenoxy)ethyl]phosphite (2,4-DEP), 2-(2,4-Dichlorophenoxy)propionic acid (dicloroprop), (RS)-2-(2,4,5-trichlorophenoxy)propionic acid (fenoprop), naphthaleneacetamide, a-naphthaleneacetic acid (NAA), 1 -naphthol, naphthoxyacetic acid, potassium naphthenate, (2,4,5-trichlorophenoxy)acetic acid (2,4, 5-T), indole-3 -acetic acid, indole-3 -butyric acid (IBA), 4-amino-3,5,6-trichloropyridine-2-carboxylic acid (picloram), 3,6-dichloro-o-anisic acid (dicamba), indole-3 -propionic acid (IP A), phenyl acetic acid (PAA), benzofuran-3 -acetic acid (BFA), and phenyl butyric acid (PBA). In certain embodiments, the induction medium comprises 2,4-D. In certain embodiments, the induction medium comprises at least about 0.5 mg / L, at least about 1 mg / L, at least about 1.5 mg / L, or at least about 2 mg / L, or at least about 2.5 mg / L of 2,4-D. In certain embodiments, the induction medium comprises from about 1 mg / L, about 1.5 mg / L, about 2 mg / L, or about 2.3 mg / L to about 2.8 mg / L, about 3 mg / L, about 3.5 mg / L, or about 4 mg / L of 2,4-D. In certain embodiments, the induction medium comprises about 2.5 mg / L of 2,4-D. In certain embodiments, the induction medium does not comprise a cytokinin or does not comprise a cytokinin at a concentration sufficient to exert a cytokinin effect on cultured soybean cells. Examples of cytokinins include, but are not limited to, 6-benzylaminopurine (BAP), meta-topolin, zeatin, kinetin, thiadiazuron (TDZ), 6-(y,y-Dimethylallylamino)purine (2iP), 1,3 -Diphenylurea (DPU), adenine hemisulfate, N-Benzyl-9-(2-tetrahydropyranyl)adenine (BP A), and N-(2-Chloro-4-pyridyl)-N'-phenylurea (4-CPPU). In certain embodiments, the induction medium does not comprise BAP or does not comprise BAP at a concentration sufficient to exert a cytokinin effect on cultured soybean cells.

[0023] The induction medium can be formulated as solid or semi-solid media, or as a liquid. In certain embodiments, the induction medium is a solid or semi-solid medium. In theseAgent Ref.: P15010WO00embodiments, a gelling agent can be included using standard media formulation procedures. Suitable gelling agents for use in the induction media include, but are not limited to, agar and gellan gum (e.g., GELRITE™, PHYTAGEL™).

[0024] In certain embodiments, the callus is induced on the induction medium at a temperature of at least about 20 °C, at least about 21 °C, at least about 22 °C, at least about 23 °C, at least about 24 °C, or at least about 25 °C. In certain embodiments, the callus is induced on the induction medium at a temperature from about 20 °C, about 21 °C, about 22 °C, about 23 °C, or about 24 °C to about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C. In certain embodiments, the callus is induced on the induction medium at a temperature of about 25 °C. In certain embodiments, the callus is induced on the induction medium for at least about 7 days (e.g., at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days). In certain embodiments, the callus is induced on the induction medium for about 7 days to about 28 days (e.g., at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, or 27 days and less than 28 days, 27 days, 26 days, 25 days, 24 days, 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, or 8 days). In certain embodiments, the callus is induced on the induction medium under a 24-hour dark photoperiod.

[0025] Methods of the disclosure include the step of proliferating the induced callus on a proliferation medium. In certain embodiments, the proliferation medium comprises inorganic salts, vitamins, a carbon source, a plant growth regulator, and optionally a gelling agent.

[0026] In certain embodiments, the proliferation medium comprises inorganic salts and / or vitamins. Examples of inorganic salts for use in the proliferation medium include, but are not limited to, MS basal salts, Gamborg B5 basal salts, Nitsch and Nitsch basal salts, Chu N6 basal salts, and Schenk and Hildebrandt basal salts, or derivations of these basal salt mixtures. In certain embodiments, the proliferation medium comprises MS basal salts. Examples of vitamins for use in the proliferation medium include, but are not limited to, MS vitamins, Gamborg B5 vitamins, Nitsch and Nitsch vitamins, and Chu N6 vitamins. In certain embodiments, the proliferation medium comprises MS vitamins and / or Gamborg B5 vitamins. In certain embodiments, the MS vitamins are modified MS vitamins supplemented with additional thiamine. In certain embodiments, the proliferation medium comprises MS basal salts, MS vitamins, and Gamborg B5 vitamins.

[0027] In certain embodiments, the proliferation medium comprises a carbon source, such as a carbohydrate. Examples of a carbon source for use in the proliferation medium include, but areAgent Ref.: P15010WO00not limited to, glucose, sucrose, maltose, mannose, fructose, lactose, galactose, and dextrose. In certain embodiments, the proliferation medium comprises at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 25 g / L, at least about 30 g / L, at least about 35 g / L, or at least about 40 g / L of a carbon source. In certain embodiments, the proliferation medium comprises from about 5 g / L, about 10 g / L, about 15 g / L, about 20 g / L, or about 25 g / L to about 35 g / L, about 40 g / L, about 45 g / L, or about 50 g / L of a carbon source. In certain embodiments, the proliferation medium comprises sucrose as the carbon source. In certain embodiments, the proliferation medium comprises at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 25 g / L, at least about 30 g / L, at least about 35 g / L, or at least about 40 g / L of sucrose. In certain embodiments, the proliferation medium comprises from about 5 g / L, about 10 g / L, about 15 g / L, about 20 g / L, or about 25 g / L to about 35 g / L, about 40 g / L, about 45 g / L, or about 50 g / L of sucrose. In certain embodiments, the proliferation medium comprises about 30 g / L of sucrose.

[0028] In certain embodiments, the proliferation medium comprises a plant growth regulator. In certain embodiments, the proliferation medium comprises an auxin. In certain embodiments, the proliferation media does not comprise IAA. In certain embodiments, the proliferation medium comprises a synthetic auxin. In certain embodiments, the proliferation medium comprises 2,4-D. In certain embodiments, the proliferation medium comprises at least about 0.2 mg / L, at least about 0.3 mg / L, at least about 0.4 mg / L, at least about 0.5 mg / L, at least about 0.6 mg / L, at least about 0.7 mg / L, at least about 0.8 mg / L, at least about 0.9 mg / L, or at least about 1 mg / L of 2,4-D. In certain embodiments, the proliferation medium comprises from about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, or about 1 mg / L to about 1.1 mg / L, about 1.2 mg / L, about 1.3 mg / L, about 1.4 mg / L, about 1.5 mg / L, about 1.6 mg / L, about 1.7 mg / L, about 1.8 mg / L, about 1.9 mg / L, about 2 mg / L, about 2.5 mg / L, or about 3 mg / L of 2,4-D. In certain embodiments, the proliferation medium comprises about 1 mg / L of 2,4-D. In certain embodiments, the proliferation medium does not comprise a cytokinin or does not comprise a cytokinin at a concentration sufficient to exert a cytokinin effect on cultured soybean cells. In certain embodiments, the proliferation medium does not comprise BAP or does not comprise BAP at a concentration sufficient to exert a cytokinin effect on cultured soybean cells.

[0029] The proliferation medium can be formulated as solid or semi-solid media, or as a liquid. In certain embodiments, the proliferation medium is a solid or semi-solid medium. In these embodiments, a gelling agent can be included using standard media formulation procedures.Agent Ref.: P15010WO00Suitable gelling agents for use in the proliferation media include, but are not limited to, agar and gellan gum (e.g., GELRITE™, PHYTAGEL™).

[0030] In certain embodiments, the callus is proliferated on the proliferation medium at a temperature of at least about 20 °C, at least about 21 °C, at least about 22 °C, at least about 23 °C, at least about 24 °C, or at least about 25 °C. In certain embodiments, the callus is proliferated on the proliferation medium at a temperature from about 20 °C, about 21 °C, about 22 °C, about 23 °C, or about 24 °C to about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C. In certain embodiments, the callus is proliferated on the proliferation medium at a temperature of about 25 °C. In certain embodiments, the callus is proliferated on the proliferation medium at least about 7 days (e.g., at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days). In certain embodiments, the callus is proliferated on the proliferation medium for about 7 days to about 28 days (e.g., at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, or 27 days and less than 28 days, 27 days, 26 days, 25 days, 24 days, 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, or 8 days). In certain embodiments, the callus is proliferated on the proliferation medium under a 24-hour dark photoperiod.

[0031] Methods of the disclosure include the step of suspending the proliferated callus in a suspension medium. In certain embodiments, the suspension medium comprises inorganic salts, vitamins, a carbon source, and a plant growth regulator.

[0032] In certain embodiments, the suspension medium comprises inorganic salts and / or vitamins. Examples of inorganic salts for use in the suspension medium include, but are not limited to, MS basal salts, Gamborg B5 basal salts, Nitsch and Nitsch basal salts, Chu N6 basal salts, and Schenk and Hildebrandt basal salts, or derivations of these basal salt mixtures. In certain embodiments, the suspension medium comprises MS basal salts. Examples of vitamins for use in the suspension medium include, but are not limited to, MS vitamins, Gamborg B5 vitamins, Nitsch and Nitsch vitamins, and Chu N6 vitamins. In certain embodiments, the suspension medium comprises MS vitamins and / or Gamborg B5 vitamins. In certain embodiments, the MS vitamins are modified MS vitamins supplemented with additional thiamine. In certain embodiments, the suspension medium comprises MS basal salts, MS vitamins, and Gamborg B5 vitamins.

[0033] In certain embodiments, the suspension medium comprises a carbon source, such as a carbohydrate. Examples of a carbon source for use in the suspension medium include, but areAgent Ref.: P15010WO00not limited to, glucose, sucrose, maltose, mannose, fructose, lactose, galactose, and dextrose. In certain embodiments, the suspension medium comprises at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 25 g / L, at least about 30 g / L, at least about 35 g / L, or at least about 40 g / L of a carbon source. In certain embodiments, the suspension medium comprises from about 5 g / L, about 10 g / L, about 15 g / L, about 20 g / L, or about 25 g / L to about 35 g / L, about 40 g / L, about 45 g / L, or about 50 g / L of a carbon source. In certain embodiments, the suspension medium comprises sucrose as the carbon source. In certain embodiments, the suspension medium comprises at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 25 g / L, at least about 30 g / L, at least about 35 g / L, or at least about 40 g / L of sucrose. In certain embodiments, the suspension medium comprises from about 5 g / L, about 10 g / L, about 15 g / L, about 20 g / L, or about 25 g / L to about 35 g / L, about 40 g / L, about 45 g / L, or about 50 g / L of sucrose. In certain embodiments, the suspension medium comprises about 30 g / L of sucrose.

[0034] In certain embodiments, the suspension medium comprises a plant growth regulator. In certain embodiments, the suspension medium comprises an auxin. In certain embodiments, the suspension medium does not comprise IAA. In certain embodiments, the suspension medium comprises a synthetic auxin. In certain embodiments, the suspension medium comprises 2,4-D. In certain embodiments, the suspension medium comprises at least about 0.2 mg / L, at least about 0.3 mg / L, at least about 0.4 mg / L, at least about 0.5 mg / L, at least about 0.6 mg / L, at least about 0.7 mg / L, at least about 0.8 mg / L, at least about 0.9 mg / L, or at least about 1 mg / L of 2,4-D. In certain embodiments, the suspension medium comprises from about 0.2 mg / L, about 0.3 mg / L, about 0.4 mg / L, about 0.5 mg / L, about 0.6 mg / L, about 0.7 mg / L, about 0.8 mg / L, about 0.9 mg / L, or about 1 mg / L to about 1.1 mg / L, about 1.2 mg / L, about 1.3 mg / L, about 1.4 mg / L, about 1.5 mg / L, about 1.6 mg / L, about 1.7 mg / L, about 1.8 mg / L, about 1.9 mg / L, about 2 mg / L, about 2.5 mg / L, or about 3 mg / L of 2,4-D. In certain embodiments, the suspension medium comprises about 1 mg / L of 2,4-D. In certain embodiments, the suspension medium does not comprise a cytokinin or does not comprise a cytokinin at a concentration sufficient to exert a cytokinin effect on cultured soybean cells. In certain embodiments, the suspension medium does not comprise BAP or does not comprise BAP at a concentration sufficient to exert a cytokinin effect on cultured soybean cells.

[0035] The suspension medium can be formulated as solid or semi-solid media, or as a liquid. In certain embodiments, the suspension medium is a liquid medium.

[0036] In certain embodiments, the suspension culture is maintained at a temperature of at least about 20 °C, at least about 21 °C, at least about 22 °C, at least about 23 °C, at least about 24 °C,Agent Ref.: P15010WO00or at least about 25 °C. In certain embodiments, the suspension culture is maintained at a temperature from about 20 °C, about 21 °C, about 22 °C, about 23 °C, or about 24 °C to about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C. In certain embodiments, the suspension culture is maintained at a temperature of about 25 °C. In certain embodiments, the suspension culture is maintained for at least about 7 days (e.g., at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days). In certain embodiments, the suspension culture is maintained for about 7 days to about 28 days (e.g, at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, or 27 days and less than 28 days, 27 days, 26 days, 25 days, 24 days, 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, or 8 days). In certain embodiments, the suspension culture is maintained under a 24-hour dark photoperiod.

[0037] In certain embodiments, the methods further comprise subculturing the suspension culture. In certain embodiments, the suspension culture is subcultured at least about every 5 days (e.g, at least every 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days). In certain embodiments, the suspension culture is subcultured about every 5 days to about every 15 days (e.g., at least every 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days and less than every 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, or 6 days). In certain embodiments, the suspension culture is subcultured at least one time (e.g., at least two, three, four, five, six, seven, eight, nine, or ten times) prior to use of the soybean cells from the suspension culture in a downstream application (e.g, obtaining at least one soybean protoplast from the suspension culture, introducing one or more biological molecules into at least one soybean cell or protoplast from the suspension culture.

[0038] The methods of the disclosure can further include the step of recovering at least one soybean cell from the suspension culture. As used herein, the term “recovering” refers to the isolation of the soybean cell such that it is substantially free of any media component.

[0039] The methods of the disclosure can further include the step of obtaining at least one soybean protoplast from the suspension culture. In certain embodiments, the protoplasts are produced by subjecting soybean cells from the suspension culture to enzymatic breakdown of the cell walls. Suitable cell wall degrading enzymes include cellulases, hemicellulases, pectinases, and lignin-modifying enzymes.Agent Ref.: P15010WO00

[0040] The methods of the disclosure can further include the step of introducing one or more biological molecules (e.g., comprising a polynucleotide, polypeptide or combination thereof) into at least one soybean cell or protoplast from the suspension culture.

[0041] In certain embodiments, the biological molecules include gene editing molecules. Gene editing molecules of use in the methods provided herein include molecules capable of introducing a double-strand break (“DSB”) or single-strand break (“SSB”) at a specific site or sequence in a double-stranded DNA, such as in genomic DNA or in a target gene located within the genomic DNA as well as accompanying guide RNA or donor or other DNA template polynucleotides. Examples of such gene editing molecules include: (a) a nuclease comprising an RNA-guided nuclease, an RNA-guided DNA endonuclease or RNA directed DNA endonuclease (RdDe), a class 1 CRISPR type nuclease system, a type II Cas nuclease, a Cas9, a nCas9 nickase, a type V Cas nuclease, a Cas 12a nuclease, a nCasl2a nickase, a Cas 12d (CasY), a Casl2e (CasX), a Casl2b (C2cl), a Casl2c (C2c3), a Casl2i, a Casl2j, a Casl2L, a Casl4, an engineered nuclease, a codon-optimized nuclease, a zinc-finger nuclease (ZFN) or nickase, a transcription activator-like effector nuclease (TAL-effector nuclease or TALEN) or nickase (TALE-nickase), an Argonaute, and a meganuclease or engineered meganuclease; (b) a polynucleotide encoding one or more nucleases capable of effectuating site-specific alteration (including introduction of a DSB or SSB) of a target nucleotide sequence; (c) a guide RNA (gRNA) for use with an RNA-guided nuclease, or a DNA encoding a gRNA for use with an RNA-guided nuclease; (d) donor DNA template polynucleotides suitable for insertion at a break in genomic DNA by homology-directed repair (HDR) or microhomology-mediated end joining (MMEJ); and (e) other DNA templates (e.g., dsDNA, ssDNA, or combinations thereof) suitable for insertion at a break in genomic DNA (e.g., by non-homologous end joining (NHEJ).

[0042] CRISPR elements, e.g., gene editing molecules comprising CRISPR endonucleases and CRISPR guide RNAs including single guide RNAs or guide RNAs in combination with tracrRNAs or scoutRNA, or polynucleotides encoding the same, are useful in effectuating genome editing without remnants of the CRISPR elements or selective genetic markers occurring in progeny. In certain embodiments, the CRISPR elements are provided directly to the soybean cell or protoplast as isolated molecules, as isolated or semi-purified products of a cell free synthetic process e.g., in vitro translation), or as isolated or semi-purified products of a cell-based synthetic process (e.g., such as in a bacterial or other cell lysate). In certain embodiments, soybean cells or protoplasts used in the methods provided herein can comprise a transgene that expresses a CRISPR endonuclease (e.g., a Type II (e.g., Cas9), a Type V (e.g., Casl2a, Casl2b, Casl2c, Casl2f, Casl2i, or Casl2j) or other CRISPR endonuclease). In certainAgent Ref.: P15010WO00embodiments, one or more CRISPR endonucleases with unique PAM recognition sites can be used. Guide RNAs (sgRNAs or crRNAs and a tracrRNA) to form an RNA-guided endonuclease / guide RNA complex which can specifically bind sequences in the gDNA target site that are adjacent to a protospacer adjacent motif (PAM) sequence. The type of RNA-guided endonuclease typically informs the location of suitable PAM sites and design of crRNAs or sgRNAs. G-rich PAM sites, e.g., 5’-NGG are typically targeted for design of crRNAs or sgRNAs used with Cas9 proteins. Examples of PAM sequences include 5’-NGG (Streptococcus pyogenes), 5’-NNAGAA (Streptococcus thermophilus CRISPR1), 5’-NGGNG (Streptococcus thermophilus CRISPR3), 5’-NNGRRT or 5’-NNGRR (Staphylococcus aureus Cas9, SaCas9), and 5’-NNNGATT (Neisseria meningitidis). T-rich PAM sites (e.g., 5’-TTN or 5’-TTTV, where "V" is A, C, or G) are typically targeted for design of crRNAs or sgRNAs used with Casl2a proteins. In some instances, Casl2a can also recognize a 5’-CTA PAM motif. Other examples of potential Casl2aPAM sequences include TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN, ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN, GCCN, and CCGN (wherein N is defined as any nucleotide). In certain embodiments provided herein, a miniature Type V Cas endonuclease, which include Casl2f, Casl2i, Casl2j, and variants thereof, is used. Miniature Type V Cas endonucleases are disclosed in Nguyen et al., doi.org / 10.1016 / j.sbi.2022.102466 as well as in US20210395784A1, US20210254038 Al, WO2024091775, and US11649444B1, each of which are incorporated herein by reference in their entireties.

[0043] In certain embodiments, zinc finger nucleases or zinc finger nickases can also be used in the methods provided herein. Zinc-finger nucleases are site-specific endonucleases comprising two protein domains: a DNA-binding domain, comprising a plurality of individual zinc finger repeats that each recognize between 9 and 18 base pairs, and a DNA-cleavage domain that comprises a nuclease domain (typically FokI). The cleavage domain dimerizes in order to cleave DNA; therefore, a pair of ZFNs are required to target non-palindromic target polynucleotides. The zinc finger binding domains of the zinc finger nuclease or nickase provide specificity and can be engineered to specifically recognize any desired target DNA sequence. The zinc finger DNA binding domains are derived from the DNA-binding domain of a large class of eukaryotic transcription factors called zinc finger proteins (ZFPs). The DNA-binding domain of ZFPs typically contains a tandem array of at least three zinc “fingers” each recognizing a specific triplet of DNA. A number of strategies can be used to design the binding specificity of the zinc finger binding domain. One approach, termed “modular assembly”, relies on the functional autonomy of individual zinc fingers with DNA. In this approach, a given sequence is targeted byAgent Ref.: P15010WO00identifying zinc fingers for each component triplet in the sequence and linking them into a multifmger peptide. Several alternative strategies for designing zinc finger DNA binding domains have also been developed. These methods are designed to accommodate the ability of zinc fingers to contact neighboring fingers as well as nucleotide bases outside their target triplet. Typically, the engineered zinc finger DNA binding domain has a novel binding specificity, compared to a naturally-occurring zinc finger protein. Engineering methods include, for example, rational design and various types of selection. Rational design includes, for example, the use of databases of triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. Exemplary selection methods (e.g., phage display and yeast two-hybrid systems) can be adapted for use in the methods described herein. In addition, individual zinc finger domains may be linked together using any suitable linker sequences. The nucleic acid cleavage domain is non-specific and is typically a restriction endonuclease, such as Fokl. This endonuclease must dimerize to cleave DNA. Thus, cleavage by Fokl as part of a ZFN requires two adjacent and independent binding events, which must occur in both the correct orientation and with appropriate spacing to permit dimer formation. The requirement for two DNA binding events enables more specific targeting of long and potentially unique recognition sites.

[0044] Transcription activator like effectors (TALEs) are proteins secreted by certain Xanthomonas species to modulate gene expression in host plants and to facilitate the colonization by and survival of the bacterium. TALEs act as transcription factors and modulate expression of resistance genes in the plants. Recent studies of TALEs have revealed the code linking the repetitive region of TALEs with their target DNA-binding sites. TALEs comprise a highly conserved and repetitive region consisting of tandem repeats of mostly 33 or 34 amino acid segments. The repeat monomers differ from each other mainly at amino acid positions 12 and 13. A strong correlation between unique pairs of amino acids at positions 12 and 13 and the corresponding nucleotide in the TALE-binding site has been found. The simple relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for the design of DNA binding domains of any desired specificity. TALEs can be linked to a nonspecific DNA cleavage domain to prepare genome editing proteins, referred to as TAL-effector nucleases or TALENs. As in the case of ZFNs, a restriction endonuclease, such as Fokl, can be conveniently used.

[0045] In certain embodiments, one or more treatments is employed to deliver the gene editing or other biological molecules (e.g., comprising a polynucleotide, polypeptide or combinationAgent Ref.: P15010WO00thereof) into a soybean cell or protoplast, e.g., through barriers such as a cell wall, a plasma membrane, a nuclear envelope, and / or other lipid bilayer. In certain embodiments, a polynucleotide-, polypeptide-, or RNP (ribonucleoprotein)-containing composition comprising the molecules are delivered directly, for example by direct contact of the composition with a soybean cell or protoplast. Aforementioned compositions can be provided in the form of a liquid, a solution, a suspension, an emulsion, a reverse emulsion, a colloid, a dispersion, a gel, liposomes, micelles, an injectable material, an aerosol, a solid, a powder, a particulate, a nanoparticle, or a combination thereof can be applied directly to a soybean cell or protoplast (e.g., through abrasion or puncture or otherwise disruption of the cell wall or cell membrane, by spraying or dipping or soaking or otherwise directly contacting, by microinjection). For example, a soybean cell or protoplast is soaked in a liquid genome editing molecule-containing composition. In certain embodiments, the composition is delivered using negative or positive pressure, for example, using vacuum infiltration or application of hydrodynamic or fluid pressure. In certain embodiments, the composition is introduced into a soybean cell or protoplast, e.g., by microinjection or by disruption or deformation of the cell wall or cell membrane, for example by physical treatments such as by application of negative or positive pressure, shear forces, or treatment with a chemical or physical delivery agent such as surfactants, liposomes, or nanoparticles. Other techniques useful for delivering the composition to a soybean cell or protoplast include: ultrasound or sonication; vibration, friction, shear stress, vortexing, cavitation; centrifugation or application of mechanical force; mechanical cell wall or cell membrane deformation or breakage; enzymatic cell wall or cell membrane breakage or permeabilization; abrasion or mechanical scarification (e.g., abrasion with carborundum or other particulate abrasive or scarification with a file or sandpaper) or chemical scarification (e.g., treatment with an acid or caustic agent); and electroporation. In certain embodiments, the composition is provided by bacterially mediated (e.g. , Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.) transfection of the plant cell or plant protoplast with a polynucleotide encoding the genome editing molecules (e.g., RNA dependent DNA endonuclease, RNA dependent DNA binding protein, RNA dependent nickase, ABE, or CBE, and / or guide RNA). Any of these techniques or a combination thereof are alternatively employed on the soybean cells or protoplasts.

[0046] The biological molecules (e.g., gene editing molecules) used in the methods provided herein can include a nucleotide sequence encoding a selectable marker which can be used to select a soybean cell expressing the biological molecules. Examples of selectable markers include, but are not limited to, DNA segments that comprise restriction enzyme sites; DNAAgent Ref.: P15010WO00segments that encode products which provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT)); DNA segments that encode products which are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA segments that encode products which can be readily identified (e.g., phenotypic markers such as P-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), yellow-green (mNeonGreen), red (RFP; mScarlet), and cell surface proteins); the generation of new primer sites for PCR (e.g., the juxtaposition of two DNA sequence not previously juxtaposed), the inclusion of DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; and, the inclusion of DNA sequences required for a specific modification (e.g., methylation) that allows its identification. Additional selectable markers include genes that confer resistance to herbicidal compounds, such as glyphosate, glufosinate, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).

[0047] Various selection procedures for the cells based on the selectable marker can be used, depending on the nature of the marker gene. In certain embodiments, use is made of a selectable marker, i.e., a marker which allows a direct selection of the cells based on the expression of the marker. A selectable marker can confer positive or negative selection and is conditional or nonconditional on the presence of external substrates. Most commonly, antibiotic or herbicide resistance genes are used as a marker, whereby selection is performed by growing the transformed plant material on media containing an inhibitory amount of the antibiotic or herbicide to which the marker gene confers resistance. Examples of such genes are genes that confer resistance to antibiotics, such as hygromycin (hpt) and kanamycin (nptll), and genes that confer resistance to herbicides, such as phosphinothricin (bar), chlorsulfuron (als), aroA, glyphosate acetyl transferase (GAT) genes, phosphinothricin acetyl transferase (PAT) genes from Streptomyces species, and ACCase inhibitor-encoding genes. Detoxifying genes can also be used as a marker, with examples including an enzyme encoding a phosphinothricin acetyltransferase and hydroxyphenylpyruyate dioxygenase (HPPD) inhibitors. Transformed plants and plant cells may also be identified by screening for the activities of a visible marker, typically an enzyme capable of processing a colored substrate (e.g., the P-glucuronidase, luciferase, B or CI genes).

[0048] In certain embodiments, the method provides a soybean cell containing at least one genetic alteration that is absent in the source tissue (or the source plant from which the source tissue was obtained). The genetic alteration can be variously characterized as transientAgent Ref.: P15010WO00transformation, stable genomic changes, gene editing (genome editing), base editing, and single or multiplexed genetic changes. In certain embodiments, a soybean cell contains in its genome one or more “genome edits” such as deletion of one or more nucleotides, insertion of one or more nucleotides, insertion of a nucleotide sequence encoded by a donor polynucleotide, allele substitution or replacement, and combinations of such genomic changes.

[0049] The soybean cells of the disclosure (optionally comprising one or more biological molecules and / or at least one genetic alteration) can be cryopreserved. As used herein, the term “cry opreserving” or “cry opreservation” refers to the storage of biological material, e.g., cells, tissues or organs, at temperatures below 4°C. Generally, the intention of the cryopreservation is to maintain the cells in a preserved or dormant state, after which time the cells are returned to a temperature above 4°C for subsequent use. In certain embodiments, the cryopreserving temperature is below 0°C. For example, the cry opreserving temperature may be below 0°C, -5°C, -10°C, -20°C, -60°C, or -80°C. Such temperatures can be reached by exposing the soybean cells to liquid nitrogen, liquid helium, carbon dioxide (‘dry-ice’), or slurries of carbon dioxide with other solvents. In certain embodiments, the cryopreserving temperature is about -20°C to about -80°C. In certain embodiments, the cryopreserved soybean cells further comprise a cryopreservation agent or cryoprotectant. As used herein, the terms “cryopreservation agent” or “cryoprotectant” refer to a substance that is used to protect plant cells from freezing damage. Further, the cryopreservation agent or cryoprotectant may protect the soybean cells from cold and heat shock, dehydration, and cryo-toxicity during cryopreservation. The cryopreservation agent or cryoprotectant may be cell penetrating or nonpenetrating. Non-limiting examples of cryoprotectants include glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, acetamide, and methanol.

[0050] The following numbered embodiments also form part of the present disclosure:

[0051] 1. A method of producing a soybean cell suspension culture, the method comprising: (i) inducing callus from a tissue of a soybean plant on an induction medium; (ii) proliferating the induced callus obtained in step (i) on a proliferation medium; and (iii) suspending the proliferated callus obtained in step (ii) in a suspension medium, thereby producing the soybean cell suspension culture.

[0052] 2. The method of embodiment 1, wherein the tissue comprises a cotyledon, a leaf, an embryo axis, or a root.

[0053] 3. The method of embodiment 1 or embodiment 2, wherein the induction medium comprises Murashige and Skoog (MS) basal salts, Gamborg B5 vitamins, a carbon source, a nitrogen source, L-proline, and 2,4-dichlorophenoxyacetic acid (2,4-D).Agent Ref.: P15010WO00

[0054] 4. The method of any one of embodiments 1-3, wherein the induction medium comprises sucrose as the carbon source, optionally wherein the induction medium comprises from about 25 g / L to about 35 g / L of sucrose.

[0055] 5. The method of any one of embodiments 1-4, wherein the induction medium comprises casein hydrolysate as the nitrogen source, optionally wherein the induction medium comprises from about 0.20 g / L to about 0.25 g / L of casein hydrolysate.

[0056] 6. The method of any one of embodiments 1-5, wherein the induction medium comprises from about 0.4 g / L to about 0.6 g / mL of L-proline, optionally wherein the induction medium comprises about 0.5 g / L of L-proline.

[0057] 7. The method of any one of embodiments 1-6, wherein the induction medium comprises from about 2 mg / L to about 3 mg / L of 2,4-D, optionally wherein the induction medium comprises about 2.5 mg / L of 2,4-D.

[0058] 8. The method of any one of embodiments 1-7, wherein the induction medium does not comprise 1 -naphthaleneacetic acid (NAA) or 6-benzylaminopurine (BAP).

[0059] 9. The method of any one of embodiments 1-8, wherein the induction medium does not comprise a cytokinin.

[0060] 10. The method of any one of embodiments 1-9, wherein the induction medium consists of or consists essentially of MS basal salts, Gamborg B5 vitamins, sucrose as the carbon source, casein hydrolysate as the nitrogen source, L-proline, 2,4-D, and a gelling agent.

[0061] 11. The method of any one of embodiments 1-10, wherein the callus is induced under a 24-hour dark photoperiod.

[0062] 12. The method of any one of embodiments 1-11, wherein the callus is induced at a temperature from about 20 °C to about 30 °C, optionally wherein the callus is induced at a temperature of about 25 °C.

[0063] 13. The method of any one of embodiments 1-12, wherein the callus is induced on the induction medium for about 7 days to about 28 days, optionally wherein the callus is induced on the induction medium for about 14 days.

[0064] 14. The method of any one of embodiments 1-13, wherein the proliferation medium comprises MS basal salts, Gamborg B5 vitamins, MS vitamins, a carbon source, and 2,4-D.

[0065] 15. The method of any one of embodiments 1-14, wherein the proliferation medium comprises sucrose as the carbon source, optionally wherein the proliferation medium comprises from about 25 g / L to about 35 g / L of sucrose.Agent Ref.: P15010WO00

[0066] 16. The method of any one of embodiments 1-15, wherein the proliferation medium comprises from about 0.5 mg / L to about 2 mg / L of 2,4-D, optionally wherein the proliferation medium comprises about 1 mg / L of 2,4-D.

[0067] 17. The method of any one of embodiments 1-16, wherein the proliferation medium does not comprise NAA or BAP.

[0068] 18. The method of any one of embodiments 1-17, wherein the proliferation medium does not comprise a cytokinin.

[0069] 19. The method of any one of embodiments 1-18, wherein the proliferation medium consists of or consists essentially of MS basal salts, Gamborg B5 vitamins, MS vitamins, sucrose as the carbon source, 2,4-D, and a gelling agent.

[0070] 20. The method of any one of embodiments 1-19, wherein the callus is proliferated under a 24-hour dark photoperiod.

[0071] 21. The method of any one of embodiments 1-20, wherein the callus is proliferated at a temperature from about 20 °C to about 30 °C, optionally wherein the callus is proliferated at a temperature of about 25 °C.

[0072] 22. The method of any one of embodiments 1-21, wherein the callus is proliferated on the proliferation medium for about 7 days to about 28 days, optionally wherein the callus is proliferated on the proliferation medium for about 14 days.

[0073] 23. The method of any one of embodiments 1-22, wherein the suspension medium comprises MS basal salts, Gamborg B5 vitamins, MS vitamins, a carbon source, and 2,4-D.

[0074] 24. The method of any one of embodiments 1-23, wherein the suspension medium comprises sucrose as the carbon source, optionally wherein the suspension medium comprises from about 25 g / L to about 35 g / L of sucrose.

[0075] 25. The method of any one of embodiments 1-24, wherein the suspension medium comprises from about 0.5 mg / L to about 2 mg / L of 2,4-D, optionally wherein the suspension medium comprises about 1 mg / L of 2,4-D.

[0076] 26. The method of any one of embodiments 1-25, wherein the suspension medium does not comprise NAA or BAP.

[0077] 27. The method of any one of embodiments 1-26, wherein the suspension medium does not comprise a cytokinin.

[0078] 28. The method of any one of embodiments 1-27, wherein the suspension medium consists of or consists essentially of MS basal salts, Gamborg B5 vitamins, MS vitamins, sucrose as the carbon source, and 2,4-D.Agent Ref.: P15010WO00

[0079] 29. The method of any one of embodiments 1-28, wherein the suspension culture is maintained under a 24-hour dark photoperiod.

[0080] 30. The method of any one of embodiments 1-29, wherein the suspension culture is maintained at a temperature from about 20 °C to about 30 °C, optionally wherein the suspension culture is maintained at a temperature of about 25 °C.

[0081] 31. The method of any one of embodiments 1-30, further comprising subculturing the cells of the suspension culture.

[0082] 32. The method of any one of embodiments 1-31, further comprising obtaining at least one protoplast from the suspension culture.

[0083] 33. The method of any one of embodiments 1-32, further comprising introducing one or more biological molecules comprising a polynucleotide and / or a polypeptide into at least one soybean cell of the suspension culture.

[0084] 34. The method of embodiment 33, wherein the biological molecule comprises a guide RNA and / or a RNA-guided nuclease, a ribonucleoprotein complex comprising a guide RNA and a RNA-guided nuclease, or a polynucleotide encoding a guide RNA and / or a RNA-guided nuclease; and optionally a donor polynucleotide.

[0085] 35. The method of embodiment 33 or embodiment 34, wherein the biological molecule comprises a transcription activator-like effector nuclease (TALEN) or a polynucleotide encoding a TALEN, a meganuclease or a polynucleotide encoding a meganuclease, or an artificial zinc finger nuclease or a polynucleotide encoding an artificial zinc finger nuclease; and optionally a donor polynucleotide.

[0086] 36. The method of any one of embodiments 33-35, wherein the polynucleotide is introduced by Agrobacterium-mediated transformation, bombardment-mediated transformation, microinjection, electroporation, or polyethylene glycol (PEG)-mediated transformation.

[0087] 37. A soybean cell suspension culture produced according to the method of any one of embodiments 1-31.EXAMPLESExample 1: Callus induction and proliferation

[0088] This example describes methods of inducing and maintaining friable callus from leaf and cotyledon tissue for the eventual purpose of initiating a suspension culture.Leaf callus induction

[0089] Sterilized seeds were germinated on half strength MS media for 20-24 days in a growth chamber at 26°C and with a 16 / 8 photoperiod to provide leaf explant material. A midsized leafAgent Ref.: P15010WO00(approx. 1 cm across) was removed from the second or third layer from the top of the plant using microscissors. The leaf was transferred to a petri dish and placed upside down, with the underside of the leaf facing up. Using a scalpel, the center vein was excised from the leaf as tightly as possible. Continuing with the scalpel, the remaining leaf tissue was cut into 5-10 mm squares, ensuring every edge of the square was created with a cut ( / .< ., not using the natural edge of the leaf as an edge of a square). Each square was poked repeatedly with the tip of the scalpel to produce more wound sites for callus formation.

[0090] Each square, still upside down, was placed on a plate of induction media (Table 1). A maximum of 8 squares were placed on each plate. Once the plate was full, the edge was wrapped with Micropore tape to seal. The plates were placed in a dark growth chamber set to 25°C for two weeks for callus induction.TABLE 1Cotyledon callus induction

[0091] Seed was sterilized with chlorine gas in a chemical fume hood. The desired number of sterile seeds (usually about 20-30 seeds) were obtained 24 hours in advance of induction and placed in a petri dish with water. The plate was wrapped in foil and stored overnight for seeds to imbibe. On the day of induction, a seed was removed using forceps and placed on a petri dish. The seed coat was gently split using a scalpel to reveal the natural line of separation between the cotyledons. The cotyledons were split, and the embryonic axis present on one or both cotyledons was completely removed. The primary leaves were also removed using a scalpel. The cotyledon was wounded all along the edges and the adaxial surface (flat side) using the scalpel. TheAgent Ref.: P15010WO00cotyledon was placed adaxial side down (rounded side facing up) onto a plate of induction media (Table 1). A maximum of 6 cotyledons were placed on each plate. Once the plate was full, the edge was wrapped with Micropore tape to seal. The plates were placed in a dark growth chamber set to 25°C for two weeks for callus induction. In addition to the cotyledons, the removed embryo axis was cultured in a similar manner. Callus was produced and suspension cultures were created. Likewise, cotyledons could be allowed to proliferate on media where roots were formed. Such roots were utilized to make callus cultures and suspension cultures were created.Callus proliferation

[0092] Bits of friable callus were removed and placed onto a fresh proliferation plate (Table 2) in an approximately 0.8 cm square, ensuring the callus was not spread too thin but was placed in full patches. Where present, any mucinous material as well as non-friable callus was not transferred. Once the plate was full of callus, the edge was wrapped with Micropore tape to seal. The plates were placed in a dark growth chamber set to 25°C for two weeks for callus proliferation. Proliferation was repeated every two weeks for a maximum of 24 weeks.TABLE 2Example 2: Suspension culture initiation and subculturing

[0093] This example describes methods for initiating and subculturing soybean leaf and cotyledon suspension cultures.

[0094] Proliferation plates to use in a suspension culture were chosen based on the presence of white-beige callus chunks on the plate that were full and visibly friable to the naked eye. Each 250 mL flask was filled with 80 mL suspension media (Table 3), and 4 g of friable callus wasAgent Ref.: P15010WO00added using forceps. The callus was gently broken up using the forceps. The flask was gently swirled for several seconds to mix the callus with the media and release single cells. The color of the culture was a creamy, slightly translucent white with defined chunks but also visible single cells. The flasks were placed in a fully dark shaking incubator set to 110 rpm for cotyledon-derived cultures and 150 rpm for leaf-derived cultures. The temperature was set to 25 °C with a humidity range of between 40-65%. Suspension cultures were subcultured every 7 days by adding 35-40 mL suspension culture to 45-40 mL fresh media using a wide bore pipette.TABLE 3Example 3: Validation of media regimen for soybean callus and suspension

[0095] The purpose of this example was to validate whether the callus and suspension media regimen was able to facilitate callus induction, callus proliferation, and suspension culture maintenance using the soybean genotype NING1295. Callus was induced and proliferated as described in Example 1, and suspension cultures were initiated as described in Example 2.Callus and suspension cultures were successfully generated from cotyledon, leaf, embryonic axis, and root tissue of NING1295 using the media regimen.Example 4: Genotype-independent media regimen for soybean callus and suspension

[0096] The purpose of this example was to determine whether the callus and suspension media regimen was able to facilitate callus induction, callus proliferation, and suspension culture maintenance of diverse soybean genotypes. Callus was induced and proliferated as described in Example 1, and suspension cultures were initiated as described in Example 2. The genotypes used in this example were as follows: GINE9231, GINE9252, GINE9523, GINF8201, GINF9812, and GINF9543.Agent Ref.: P15010WO00

[0097] Callus was successfully induced from cotyledon tissue from each of the six ex-PVP genotypes and cultured through the callus proliferation and suspension culture process. All six ex-PVP genotypes were able to grow and survive in suspension culture.Example 5: Agrobacterium-mediated transformation of suspension cultured cells

[0098] Cultures for transformation were freshly subcultured the day before use. Agrobacterium containing a T-DNA with a selectable (glyphosate tolerance) and screenable marker (green fluorescent protein) was utilized for transformation. Agrobacterium \\!zs grown overnight in Yeast Extract Peptone (YEP) media with appropriate antibiotics. Agrobacterium was centrifuged for 15 minutes at 4000 g before use and media was replaced with infection media to an optical density of 0.3. Infection media was the same as Table 3 but with the addition of MES (3.9 mg / L) and Acetosyringone (40 mg / L).

[0099] For each transformation, an amount of culture volume that would contain 1-1.5 mL of settled suspension cultured cells was centrifuged at 100g for 2-5 minutes and the culture media was removed. The cells were resuspended using 12-15 mL of prepared Agrobacterium culture, transferred to a 125 mL flask and placed on a rotary shaker at 100 rpm for 40 minutes at 25 °C for infection. The shaker was stopped for the last 5 minutes of the infection. Following infection, cells were centrifuged at 100g for 2-5 minutes and washed twice with a total of 35 mL of cocultivation media and transferred to a flask for coculture on a rotary shaker at 100 rpm for up to 5 days. Cocultivation media was the same as Table 3 but with the addition of L-cysteine (400 mg / L), DTT (154 mg / L) and Acetosyringone (40 mg / L).

[0100] Following co-cultivation, cells were centrifuged at 150g for 3 minutes and supernatant was removed. Following a rinse, the cells were resuspended in resting media and returned to the shaker overnight. Recovery media was as Table 3 with the exception being that 2,4-D was replaced by NAA at 0.1 mg / L and BAP at 1 mg / L. The antibiotic timentin was also added at 100 mg / L to control Agrobacterium. Timentin was added in all subsequent media.

[0101] Following overnight resting, cells were washed as above with selection media. Selection media contained appropriate selection agent (glyphosate) at a level that was determined to inhibit multiplication of non-transgenic cells (7.5 mg / L). Transgenic calli was determined by visualizing actively growing GFP clusters in selective media. Further growth, selection and development is supported in liquid culture or in some cases in solidified medium. Selection media was as described in Table 3 but without 2,4-D which was replaced by BAP (1 mg / L) as well as selection agent and antibiotic.Agent Ref.: P15010WO00Example 6: Protoplast isolation and transfection of suspension culture cells

[0102] In this example, suspension cultured cells initiated and established from leaf and cotyledon explants were utilized as a source for isolation of viable protoplast useful for transfection experiments. 10 mL of suspension cultured cells were filtered through a sterile, 1000 pM mesh filter. Approximately 1 g of filtered suspension culture cells was utilized for each isolation. Cells were mixed with 10 mL of filter sterilized Enzyme Solution (ES: 50 mL contained Cellulase R10 (0.75 g), Pectolyase Y23 (0.1 g), Kao and Michayluk (Kao) basal salt (0.183 g), Mannitol (6.5 g)) for 3 hours with gentle shaking (40 rpm) at 25 °C. Digested cells were filtered through a 40 pM filter and washed twice with a solution containing Kao basal salts, Kao vitamins, NAA (1 mg / L), Zeatin (0.5 mg / L), 2,4-D (2 mg / L), mannitol (1,125 g / L) and sucrose (68.4 g / L) and resuspended in the same solution at a density of approximately 2-8 x 105cells / mL. Protoplasts were tested for viability with fluorescein diacetate and for complete removal of cell walls with calcofluor white. Experimental samples showed up to 92.9% viability of isolated protoplasts.

[0103] Protoplasts are transformed using methods such as polyethylene glycol coprecipitation of DNA, RNA, Protein or mixtures of both nucleic acids and proteins. Transfection efficiencies are determined by utilizing marker genes or proteins. Guide RNA efficiency and editing enzyme efficiencies are determined by sequencing methods.

[0104] The breadth and scope of the present disclosure should not be limited by any of the above-described examples.

Claims

Agent Ref.: P15010WO00What is claimed is:

1. A method of producing a soybean cell suspension culture, the method comprising:(i) inducing callus from a tissue of a soybean plant on an induction medium comprising Murashige and Skoog (MS) basal salts, Gamborg B5 vitamins, a carbon source, a nitrogen source, L-proline, and 2,4-dichlorophenoxyacetic acid (2,4-D);(ii) proliferating the induced callus obtained in step (i) on a proliferation medium; and(iii) suspending the proliferated callus obtained in step (ii) in a suspension medium, thereby producing the soybean cell suspension culture.2 The method of claim 1, wherein the tissue comprises a cotyledon, a leaf, an embryo axis, or a root.3 The method of claim 1, wherein the induction medium comprises sucrose as the carbon source, optionally wherein the induction medium comprises from about 25 g / L to about 35 g / L of sucrose.4 The method of claim 1, wherein the induction medium comprises casein hydrolysate as the nitrogen source, optionally wherein the induction medium comprises from about 0.20 g / L to about 0.25 g / L of casein hydrolysate.5 The method of claim 1, wherein the induction medium comprises from about 0.4 g / L to about 0.6 g / mL of L-proline, optionally wherein the induction medium comprises about 0.5 g / L of L-proline.6 The method of claim 1, wherein the induction medium comprises from about 2 mg / L to about 3 mg / L of 2,4-D, optionally wherein the induction medium comprises about 2.5 mg / L of 24-D.7 The method of claim 1, wherein the induction medium does not comprise 1-naphthaleneacetic acid (NAA) or 6-benzylaminopurine (BAP).8 The method of claim 1, wherein the induction medium does not comprise a cytokinin.Agent Ref.: P15010WO009. The method of claim 1, wherein the induction medium consists of or consists essentially of MS basal salts, Gamborg B5 vitamins, sucrose as the carbon source, casein hydrolysate as the nitrogen source, L-proline, 2,4-D, and a gelling agent.

10. The method of claim 1, wherein the callus is induced under a 24-hour dark photoperiod.

11. The method of claim 1, wherein the callus is induced at a temperature from about 20 °C to about 30 °C, optionally wherein the callus is induced at a temperature of about 25 °C.

12. The method of claim 1, wherein the callus is induced on the induction medium for about 7 days to about 28 days, optionally wherein the callus is induced on the induction medium for about 14 days.

13. The method of claim 1, wherein the proliferation medium comprises MS basal salts, Gamborg B5 vitamins, MS vitamins, a carbon source, and 2,4-D.

14. The method of claim 1, wherein the proliferation medium comprises sucrose as the carbon source, optionally wherein the proliferation medium comprises from about 25 g / L to about 35 g / L of sucrose.

15. The method of claim 1, wherein the proliferation medium comprises from about 0.5 mg / L to about 2 mg / L of 2,4-D, optionally wherein the proliferation medium comprises about 1 mg / L of 2,4-D.

16. The method of claim 1, wherein the proliferation medium does not comprise NAA or BAP.

17. The method of claim 1, wherein the proliferation medium does not comprise a cytokinin.

18. The method of claim 1, wherein the proliferation medium consists of or consists essentially of MS basal salts, Gamborg B5 vitamins, MS vitamins, sucrose as the carbon source, 24-D, and a gelling agent.Agent Ref.: P15010WO0019. The method of claim 1, wherein the callus is proliferated under a 24-hour dark photoperiod.

20. The method of claim 1, wherein the callus is proliferated at a temperature from about 20 °C to about 30 °C, optionally wherein the callus is proliferated at a temperature of about 25 °C.

21. The method of claim 1, wherein the callus is proliferated on the proliferation medium for about 7 days to about 28 days, optionally wherein the callus is proliferated on the proliferation medium for about 14 days.

22. The method of claim 1, wherein the suspension medium comprises MS basal salts, Gamborg B5 vitamins, MS vitamins, a carbon source, and 2,4-D.

23. The method of claim 1, wherein the suspension medium comprises sucrose as the carbon source, optionally wherein the suspension medium comprises from about 25 g / L to about 35 g / L of sucrose.

24. The method of claim 1, wherein the suspension medium comprises from about 0.5 mg / L to about 2 mg / L of 2,4-D, optionally wherein the suspension medium comprises about 1 mg / L of 24-D.25 The method of claim 1, wherein the suspension medium does not comprise NAA or BAP.26 The method of claim 1, wherein the suspension medium does not comprise a cytokinin.27 The method of claim 1, wherein the suspension medium consists of or consists essentially of MS basal salts, Gamborg B5 vitamins, MS vitamins, sucrose as the carbon source, and 2,4-D.28 The method of claim 1, wherein the suspension culture is maintained under a 24-hour dark photoperiod.Agent Ref.: P15010WO0029. The method of claim 1, wherein the suspension culture is maintained at a temperature from about 20 °C to about 30 °C, optionally wherein the suspension culture is maintained at a temperature of about 25 °C.

30. The method of claim 1, further comprising subculturing the cells of the suspension culture.

31. A soybean cell suspension culture produced according to the method of any one of claims 1-30.

32. The method of claim 1, further comprising obtaining at least one protoplast from the suspension culture.

33. The method of claim 1, further comprising introducing one or more biological molecules comprising a polynucleotide and / or a polypeptide into at least one soybean cell of the suspension culture.

34. The method of claim 33, wherein the biological molecule comprises a guide RNA and / or a RNA-guided nuclease, a ribonucleoprotein complex comprising a guide RNA and a RNA-guided nuclease, or a polynucleotide encoding a guide RNA and / or a RNA-guided nuclease; and optionally a donor polynucleotide.

35. The method of claim 33, wherein the biological molecule comprises a transcription activator-like effector nuclease (TALEN) or a polynucleotide encoding a TALEN, a meganuclease or a polynucleotide encoding a meganuclease, or an artificial zinc finger nuclease or a polynucleotide encoding an artificial zinc finger nuclease; and optionally a donor polynucleotide.

36. The method of claim 33, wherein the polynucleotide is introduced by Agrobacterium-mediated transformation, bombardment-mediated transformation, microinjection, electroporation, or polyethylene glycol (PEG)-mediated transformation.

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