METHODS FOR TRANSFORMATION OF DICOT PLANT CELLS

Abstract

This disclosure relates to methods of transformation of dicots, such as tomato and watermelon. In some aspects, the methods comprise use of dicot explants obtained by dividing seeds and soaking the dicot explants in various media. In some aspects, transformed cells of a dicot plant are provided as well as dicot plants comprising transformed cells.

Claims

1. A method of transforming a dicot plant cell, comprising: (a) obtaining a dicot plant seed; (b) soaking the dicot plant seed in a suitable seed-soak medium; (c) dividing the soaked dicot plant seed of step (b) into a plurality of dicot explants; (d) inoculating the plurality of dicot explants of step (c) with Agrobacterium comprising a nucleic acid of interest, thereby obtaining Agrobacterium-inoculated dicot explants; and (e) co-cultivating the Agrobacterium-inoculated dicot explants to produce a transformed dicot plant cell.

2. A method of transforming a dicot plant cell, comprising: (a) obtaining a dicot plant seed; (b) soaking the dicot plant seed in a suitable seed-soak medium; (c) dividing the soaked dicot plant seed of step (b) into a plurality of dicot explants; and (d) delivering a nucleic acid of interest into the plurality of dicot explants using a biolistic particle deliver system to produce a transformed dicot plant cell.

3. The method of claim 1 wherein the dicot plant seed is a mature seed, optionally a dry mature seed.

4. The method of claim 1 wherein the dicot plant seed is a fresh seed harvested from fresh fruit or a vigorous seed harvested from a dried fruit, optionally a fresh maturing seed harvested from fresh fruit or a vigorous maturing seed harvested from a dried fruit.

5. The method of claim 1, wherein the co-cultivation of step (e) occurs in darkness over a multi-day period.

6. The method of claim 5, wherein the multi-day period is 1 to 5 days, and preferably 2 days, at a temperature of approximately 22° C.

7. The method of claim 1, further comprising: (f) removing the dicot explants from the co-culture of step (e) or the delivery of step (d) and allowing said explants to recover; and (g) incubating the recovered dicot explants from step (f) on at least one selection medium, wherein the at least one selection medium permits survival of transformed dicot explants.

8. The method of claim 7, wherein the recovering step of step (f) occurs in darkness over an at least one day period in a recovery medium at approximately 25° C., optionally wherein the recovery medium comprises MS salts and vitamins, 0.01 mg/L IAA, 1 mg/L Zeatin, 150 mg/L timentin, and 150 mg/L carbenicillin.

9. The method of claim 7, further comprising placing the selected transformed dicot explants of step (g) onto growth medium.

10. The method of claim 8, wherein the at least one day period is one to seven days.

11. The method of claim 1, wherein the suitable seed-soak medium comprises a medium selected from the group consisting of water, B5, Woody, and MS.

12. The method of claim 1, wherein a seed coat of the dicot plant seed is removed or damaged before the inoculation or delivery of step (d).

13. The method of claim 12, wherein the seed coat is removed or damaged by a manual means or a mechanical means.

14. The method of claim 1, wherein the soaking step of step (b) is within the range of approximately 1 hour to approximately 72 hours, with or without light.

15. The method of claim 9, further comprising growing the selected transformed dicot explants into seedlings, and optionally sampling said seedlings for molecular analysis.

16. The method of any one of the preceding claims, wherein the dividing in step (c) comprises cutting or crushing the soaked dicot plant seed of step (b).

17. The method of claim 16, wherein the cutting or crushing in step (c) comprises use of a razor blade, a scalpel, a blender, a burr grinder, a press, a series of blades, a mortar and pestle, a microtome, a knife, or a combination thereof.

18. The method of any one of the preceding claims, wherein the transformed dicot plant cell is selected from the group consisting of a tomato cell, a watermelon cell, a spinach cell, a soybean cell, a peanut cell, a sunflower cell, a Brassica species cell, a Cucurbit species cell, an alfalfa cell, a thale cress (Arabidopsis) cell, and a Solanaceae species cell, optionally wherein the transformed dicot plant cell a tomato cell or a watermelon cell.

19. The method of any one of the preceding claims, wherein the plurality of dicot explants is at least 2, 3, or more discrete explants, but preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 discrete explants.

20. The method of any one of the preceding claims, wherein the plurality of dicot explants is 14, 15, 16, 17, or 18 discrete explants.

21. The method of any one of the preceding claims, wherein the discrete explants are approximately 0.2 mm-8.0 mm in size.

22. The method of claim 7, wherein the at least one selection medium comprises an antibiotic selection agent, a metabolic selection agent, or herbicide selection agent.

23. The method of claim 22, wherein the antibiotic selection agent is selected from the group consisting of spectinomycin, kanamycin, ampicillin, streptomycin, tetracycline, and the like.

24. The method of claim 22, wherein the metabolic selection agent is a non-metabolizable sugar.

25. The method of claim 22, wherein the herbicide selection agent is glyphosate, glufosinate, bialaphose, ALS, phosphinothricin (PPT).

26. The method of claim 24, wherein the non-metabolizable sugar is selected from the group consisting of mannose-6-phosphate, palatinose, and turanose.

27. The method of claim 1, wherein the method produces less than 15% aberrant tetraploid transformants.

28. A method comprising the steps of a process described any of the Examples.

29. A transformed dicot plant cell produced or obtainable by a method of claim 1.

30. A seedling produced or obtainable by the method of claim 15.

31. A method comprising, (a) growing the seedling of claim 15 into a plant comprising transformed cells; and (b) crossing the plant comprising transformed cells with another plant to produce a progeny plant, optionally wherein the another plant does not comprise transformed cells.

32. A progeny plant produced or obtainable by the method of claim 31.

33. A method of regenerating a dicot plant, comprising: (a) obtaining a dicot plant seed; (b) imbibing the dicot plant seed in a suitable imbibition medium; (c) dividing the whole imbibed dicot plant seed of step (b) to create a plurality of dicot plant explants; (d) incubating the plurality of dicot explants from step (c) on regeneration medium, wherein the regeneration medium allows survival of dicot explants, optionally wherein the dicot plant seed is a mature dicot plant seed.

34. The method of claim 33, wherein the method comprises one or more of the steps of a process described any of the Examples.

Description

DETAILED DESCRIPTION

[0047] The disclosure relates to methods of transformation of dicot plants and plant cells, such as tomato and watermelon plants and plant cells. In some embodiments, the methods described herein result in improved transformation efficiency, reduced aneuploidy in transformed plants, capability for high-throughput output, lower cost, a faster transformation process, and/or genotype-independent transformation.

[0048] In some embodiments, the method of transforming a dicot plant or plant cell comprises one or more steps as described herein, e.g., one or more steps described in the Examples, such one or more or all of the steps in any of Examples 1-9.

[0049] In some embodiments, the method of transforming a dicot plant or plant cell comprises obtaining a dicot plant seed; soaking the dicot plant seed in a suitable seed-soak medium; dividing the soaked dicot plant seed into a plurality of dicot explants; and introducing into at least some of the plurality of dicot explants a nucleic acid of interest. In some embodiments, the dicot plant seed is a mature dicot plant seed. The dividing can be into equal-sized explants or into unequal-sized explants. In some embodiments, the explants are of about the same size. In some embodiments, the suitable seed-soak medium is selected from the group consisting of water, B5, Woody, and MS. B5, Woody, and MS can be obtained from any source known in the art, such as from PhytoTechnology Laboratories (e.g., Cat Nos. M524, G398+G219, or L449).

[0050] In some embodiments, the introducing into at least some of the plurality of dicot explants is via bacterial-mediated transformation (e.g., Agrobacterium transformation), particle bombardment 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, polyethyleneglycol-mediated transformation, any other electrical, chemical, physical and/or biological mechanism that results in the introduction of nucleic acid into at least some of the plurality of dicot explants, or a combination of any of the foregoing means. Preferably, the introducing into at least some of the plurality of dicot explants is via bacterial-mediated transformation (e.g., Agrobacterium transformation) or particle bombardment transformation.

[0051] Agrobacterium-mediated transformation is a commonly used method for transforming plants because of its relatively high efficiency and increased throughput of transformation and because of its broad utility with many different species. 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. coli strain that carries a plasmid that is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by nucleic acid transformation (see, e.g., Höfgen and Willmitzer 1988, Nucleic Acids Res 16:9877).

[0052] Transformation of a plant by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant. Transformed tissue is typically regenerated on selection medium carrying an antibiotic or herbicide resistance marker between the binary plasmid T-DNA borders. In some embodiments, the co-cultivation comprises one or more of the steps described in the Examples, such one or more of the steps in any of Examples 1-9.

[0053] Another method for transforming plants, plant parts and plant cells involves propelling inert or biologically active particles at plant tissues and cells, also referred to as biolistic particle bombardment. See, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006 and 5,100,792. Generally, this method involves propelling inert or biologically active particles at the plant cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the nucleic acid of interest. Alternatively, a cell or cells can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing one or more nucleic acids sought to be introduced) also can be propelled into plant tissue.

[0054] In some embodiments, plants, plant parts and plant cells transformed with a nucleic acid of interest can be selected, e.g., using selectable markers present in the nucleic acid of interest. In some embodiments, the plants, plant parts and plant cells transformed with a nucleic acid of interest are selected using one or more selection steps described in the Examples. In some embodiments, the selectable marker is a selectable marker used in one or more of the Examples, such one or more of the selectable markers in any of Examples 1-9.

[0055] Examples of 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. 1990, Plant Mol Biol 15: 127-136), streptothricin (Jelenska et al. 2000, Plant Cell Rep 19: 298-303), or chloramphenicol (De Block et al. 1984, EMBO J 3: 1681-1689). Other 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. Pat. Nos. 4,940,935, 5,188,642, 5,633,435, 6,566,587, 7,674,598 (as well as all related applications) and the glyphosate N-acetyltransferase (GAT) which confers resistance to glyphosate (Castle et al. 2004, Science 304:1151-1154, and U.S. Patent Application Publication Nos. 20070004912, 20050246798, and 20050060767); BAR which confers resistance to glufosinate (see e.g., U.S. Pat. No. 5,561,236); aryloxy alkanoate dioxygenase or AAD-1, AAD-12, or AAD-13 which confer resistance to 2,4-D; genes such as Pseudomonas HPPD which confer HPPD resistance; Sprotophorphyrinogen oxidase (PPO) 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. Pat. No. 7,812,224 and related applications and patents). Other examples of selectable markers can be found in Sundar and Sakthivel (2008, J Plant Physiology 165: 1698-1716), herein incorporated by reference.

[0056] Other 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. One non-limiting example 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 Biotechnol, 2004. 107: 193-232; incorporated by reference).

[0057] In some embodiments of the disclosure, 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). 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. Pat. No. 6,040,497; incorporated by reference). Other examples of a modified plant EPSPS which can be used as a selectable marker in the presence of glyphosate includes a P106L mutant of rice EPSPS (Zhou et al 2006, Plant Physiol 140: 184-195) and a P106S mutation in goosegrass EPSPS (Baerson et al 2002, Plant Physiol 129: 1265-1275). 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. Strain CP4 (Funke et al 2006, PNAS 103: 13010-13015). Although the plant EPSPS gene is nuclear, the mature enzyme is localized in the chloroplast (Mousdale and Coggins 1985, Planta 163:241-249). 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. Such a 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.

[0058] In some embodiments, the method further comprises permitting the dicot explant to recover after transformation. In some embodiments, the recovering step occurs prior to one or more selection steps. In some embodiments, the recovering step occurs after one or more selection steps. In some embodiments, the recovering step occurs both before and after one or more selection steps. In some embodiments, the recovering occurs for at least one day (e.g., at least 1, 2, 3, 4, 5, 6, 7 or more days) and in darkness. In some embodiments, the recovering occurs in a recovery medium, such as a medium described in the Examples, such one or more of the recovery media in any of Examples 1-9.

[0059] In some embodiments, after obtaining or selecting the transformed dicot explant, the method further comprises placing the selected or obtained transformed dicot explant onto growth medium. In some embodiments, the growth medium is a medium described in the Examples, such one or more of the growth media in any of Examples 1-9.

[0060] Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are illustrative of the present invention, and the scope of the present invention is not limited by the following examples.

EXAMPLES

Example 1: Method of Transformation in Tomatoes

[0061] Below is an example method for transforming a dicot. The method is exemplified with tomatoes. The method was altered in certain examples as indicated in the relevant example.

[0062] (1) Sterilization of Seeds

[0063] a. Dry mature tomato seeds at least 14 days post pollination were soaked in 10% Clorox with 0.04% silwet-77 for 20 min. The seeds were then rinsed with sterilized water at least 3 times.

[0064] (2) Seeds Imbibition

[0065] a. The sterilized tomato seeds were transferred to Germination medium (Gamborg B5 medium with 20 g/L sucrose, 8 g/L agar, pH5.6), or a sterilized filter paper soaked with sterilized water. The seeds were then incubated at 25° C. in dark for 1 to 3 days (before the cotyledons emerged from the seed coat). One day of incubation was preferred.

[0066] (3) Inoculum Preparation

[0067] The Agrobacterium strain EHA101 containing a vector of interest stored at −80° C. was streaked on solid LB (Lennox) agar (Caisson Laboratories, Cat No. LBP04) with appropriate antibiotics at least 3 days before inoculation, and re-streaked once on a new solid LB (Lennox) medium 1 day before inoculation. The Agrobacterium were collected and re-suspended in liquid infection medium (MS medium with 0.5 mg/L BAP, 20 g/L sucrose, 10 g/L glucose, 4 g/L MES, pH5.5) with 400 μM Acetosyringone (AS) and 1 mM Dithiothreitol (DTT). The Agrobacterium suspension was adjusted OD660 to 0.5.

[0068] (4) Explants Preparation and Infection

[0069] There were two ways used to prepare the explants:

[0070] a. The imbibed seeds were cut into small pieces with a scalpel to be used as explants for transformation. The seed coat was removed either before or after the cutting. The size of the explants was generally between 0.1 mm to 2 mm, with about 0.5 mm being preferred. The explants were immersed into Agrobacterium suspension for inoculation for about 2 hours, but it is also expected that between 5 minutes to 6 hours inoculation will be sufficient. OR

[0071] b. The imbibed seeds were cut into small pieces to be used as explants using a blender (BAMIX® GASTRO 350). The container and knife of the blender were sterilized with 75% alcohol. The imbibed seeds and some water were placed in the container. The seed coat was optionally removed before blending. The imbibed seeds were cut using the blender at the speed of 18,000 rpm. The explants with appropriate size were isolated with a sieve. The explants were immersed into Agrobacterium suspension for inoculation for about 2 hours, but it is also expected that between 5 minutes to 6 hours inoculation will be sufficient.

[0072] (5) Co-Culture

[0073] a. After inoculation, the Agrobacterium suspension was removed from the explants with a dropper or pipette. The explants were transferred to a co-culture medium plate (½ MS medium with 0.5 mg/L BAP, 20 g/L sucrose, 10 g/L glucose, 4 g/L MES, 200 μM, 1 mg/L Silver Nitrate, 7 g/L Agar, pH 5.4) with a piece of filter paper on top of the medium, or a filter paper soaked with liquid co-culture medium (¼ MS medium with 0.25 mg/L BAP, 10 g/L sucrose, 5 g/L glucose, 2 g/L MES, 100 μM Acetosyringone, 0.5 mg/L Silver Nitrate, pH 5.4). The explants were dispersed evenly on the medium. The plate was sealed with parafilm and incubated at 22° C. in the dark for 2-3 days.

[0074] (6) Recovery

[0075] a. The explants were transferred to recovery medium (MS medium with 20 g/L sucrose, 10 g/L glucose, 1 mg/L Zeatin, 0.01 mg/L IAA, 150 mg/L Timentin, 150 mg/L Carbenicillin, 10 g/L Agar, pH 5.8). The explants were cultured at 25° C. in the dark for 4 days.

[0076] (7) Selection 1

[0077] a. The explants were transferred to selection medium 1 (MS medium with 1× B5 vitamins, 30 g/L sucrose, 1 mg/L Zeatin, 0.01 mg/L IAA, 150 mg/L Timentin, 150 mg/L Carbenicillin, 10 g/L Agar, pH 5.8, with selection agent corresponding to the selectable marker, for example, spectinomycin 100 mg/L or glyphosate). The explants were cultured at 25° C. 16 hours/8 hours (light/dark) for 3 weeks.

[0078] (8) Selection 2

[0079] a. Explants with green buds were transferred to selection medium 2 (MS medium with 1× B5 vitamins, 30 g/L sucrose, 0.2 mg/L Zeatin, 150 mg/L Timentin, 150 mg/L Carbenicillin, 10 g/L Agar, pH 5.8, with selection agent corresponding to the selectable marker, for example 100 mg/L spectinomycin or glyphosate). The explants were kept at 25° C. 16 hours/8 hours (light/dark). The explants were sub-cultured to new selection medium 2 every 3 weeks, and continue sub-culture if needed

[0080] (9) Rooting

[0081] a. The selected green shoots were transferred to rooting medium (MS medium with 0.1 g/L Inositol, 1 mg/L thiamine, 0.5 mg/L pyridoxine, 0.5 mg/L nicotinic acid, 1 mg/L glycine, 10 g/L sucrose, 150 mg/L Timentin, 150 mg/L Carbenicillin, 8 g/L Agar, pH 5.8). The shoots were kept at 24-25° C. 16 hours/8 hours (light/dark) to promote rooting.

[0082] The vectors used in the below examples are as follows:

TABLE-US-00001 Vector ID Selectable marker Selection agent and pressure 19741 EPSPS Glyphosate 15 μM 22810 PAT Glufosinate 2 mg/L 24133 aadA Spectinomycin 100 mg/L 24134 NPT II Kanamycin 100 mg/L 24416 aadA Spectinomycin 100 mg/L

[0083] The following protocol describes the collection and analysis of the plant material for the below examples:

Plant Leaf Material and Genomic DNA Extraction

[0084] Two leaf discs per plant were collected and placed into a 96-well block. Genomic DNA was extracted following the Magbeads Plant genome extraction protocol from Promega.

TAQMAN® Assays Design and qPCR

[0085] TAQMAN® assay was designed using Primer Express 3.0 (Life Technologies, Inc.) to the coding sequence of the selectable marker present in the vector to detect the presence of vector sequence in the extracted genomic DNA. The amplicon was blasted with the tomato genome database to ensure assay specificity. Primers and probes were purchased from Life Technologies.

[0086] Each 25 μl qPCR reaction contained 12.5 μl 2× Sigma JumpStart Master Mix (Sigma-Aldrich Corporation, P2893), 5 μl DNA, 0.5 μl TAQMAN® assay (final concentration: 300 nM for primers and 100 nM for probe) and 6.5 μl water. qPCR was performed in an ABI 7900HT real-time PCR system under conditions: 95° C. for 5 min; 40 cycles of 95° C. for 5 sec followed by 60° C. for 30 sec.

Data Analysis

[0087] Data were analyzed using the SDS 2.4 software. The Cycle threshold values (Ct) were generated by selecting a threshold line that was placed in the region of exponential amplification across all of the amplification plots, and clearly above the background fluorescence and above the level where splitting or fork effects between replicates can be observed. The baseline was set at a cycle number three to five cycles earlier than the cycle number at which the threshold line crosses the first amplification curve (e.g. earliest Ct=24, set the baseline crossing at Ct=24−3=21).

Example 2: Transformation of Explants from 1-Day Imbibed Seeds and 7-Day Imbibed Seedlings from Tomatoes

[0088] Variety used: Moneymaker

[0089] Vector used: 24416

[0090] The procedure of transformation of explants from 1-day imbibed seeds at least 14 days post pollination was as described in Example 1.

[0091] The procedure of transformation of explants from 7-day seedlings:

[0092] 1. Sterilization of seed: as described in Example 1.

[0093] 2. Seeds imbibition: The sterilized seeds were transferred to a growth container with appropriate amount of germination medium (0.5×MS medium with 10 g/L sucrose, 10 g/L Phytagel, pH5.8). The seeds were kept at 25° C. 16 hours/8 hours (light/dark), for 7 days.

[0094] 3. Agrobacterium: Agrobacterium suspension were prepared as described in Example 1.

[0095] 4. Explants preparation and infection: The hypocotyl and cotyledon were cut into segments of about 5 mm in length. The segments were inoculated in Agrobacterium suspension for about 5 minutes.

[0096] 5. Co-culture: as described in Example 1.

[0097] 6. Recovery: as described in Example 1.

[0098] 7. Selection 1: as described in Example 1.

[0099] 8. Selection 2: as described in Example 1.

[0100] 9. Rooting: as described in Example 1.

[0101] The results from the transformation are shown in the below tables. The Transformation Frequency (TF) is equal to the number of confirmed positive plants divided by the total explants initiated for transformation, which is multiplied by 100 to obtain the percentage.

TABLE-US-00002 TABLE 1 Transformation Efficiency No. No. TaqMan Transformation Explant Source of No. Regenerate aadA Positive Frequency type explant Explants shoots events (TF) Hypocotyl 7-day imbibed 131 26 23 17.56% seedlings Cotyledon 7-day imbibed 121 1 1 0.83% seedlings Hypocotyl 1-day imbibed 58 2 2 3.45% seeds Cotyledon 1-day imbibed 102 12 12 11.76% seeds

TABLE-US-00003 TABLE 2 Large % of abnormal ploidy plants recovery from 7 day seedlings explants Source of No. of plants for No. of plants with expected explants ploidy assay normal ploidy (2n) ploidy rate 1-day imbibed 11 11  100% seeds 7-day imbibed 11 3 27.3% seedlings

TABLE-US-00004 TABLE 3 Comparison between explants from 1-day imbibed seeds in water and on solid germination medium No. No. No. TaqMan Germ Explants Regenerate Positive Explants Medium number shoots events TF Hypocotyl medium 58 2 2 3.45% Cotyledon medium 102 12 12 11.76% Hypocotyl Water 70 0 0 0.00% Cotyledon Water 68 6 6 8.82%

Example 3: Transformation of Different Explant Types

[0102] Variety used: Syngenta Variety 1

[0103] Vector used: 24133

[0104] Source of explant: 1-day imbibed tomato seeds at least 14 days post pollination

[0105] The process of transformation was performed as described in Example 1.

TABLE-US-00005 TABLE 4 Results No. No. TaqMan Explant No. Regenerate aadA Positive type Explants shoots events TF Apical meristem 15 1 1 6.7% Cotyledonary 36 2 2 5.6% node Hypocotyl 37 2 2 5.4% Cotyledon 108 5 5 4.6%

Example 4: Transformation of Explants from Different Sources

[0106] Variety used: Syngenta Variety 1

[0107] Vector used: 24133

[0108] The process of transformation was performed as described in Example 1.

TABLE-US-00006 TABLE 5 Results No. No. TaqMan Explant Source of No. Regenerate aadA Positive types explant Explants shoots events TF Cotyledon 1-day 108 5 5 4.6% imbibed tomato seeds Cotyledon 3-day 82 3 3 3.7% imbibed tomato seeds

Example 5: Transformation Using Different Selection Agents

[0109] Source of explant: 1-day imbibed tomato seeds at least 14 days post pollination

[0110] The process of transformation was performed as described in Example 1.

TABLE-US-00007 TABLE 6 Results No. selective No. marker Vector Selective Explant No. Regenerate Positive Variety ID marker type Explants shoots events TF Syngenta Variety 1 19741 EPSPS Cotyledon 90 2 2 2.2% Syngenta Variety 2 22810 PAT Cotyledon 66 22 4 6.1% Moneymaker 24134 NPT II Mix 60 1 1 1.7%

Example 6: Transformation of Multiple Varieties Having Diverse Genetic Backgrounds

[0111] Vector used: 24133

[0112] Source of explant: 1-day imbibed tomato seeds at least 14 days post pollination

[0113] The process of transformation was performed as described in Example 1. The results in Table 7 show that the transformation method works in several varieties with diverse genetic backgrounds.

TABLE-US-00008 TABLE 7 Results No. of No. of Rate of plants plants with plants No. of sent to normal with Syngenta variety No. of Positive ploidy ploidy level expected name or public name Explants events TF assay (2n) ploidy level Syngenta Variety 3 124 5 4.03% 0 n/a n/a Syngenta Variety 4 91 7 7.69% 8  8 100.00% Syngenta Variety 5 111 5 4.50% 0 n/a n/a Syngenta Variety 6 214 22 10.28% 12 12 100.00% Syngenta Variety 7 146 10 6.85% 0 n/a n/a Syngenta Variety 1 117 4 3.42% 0 n/a n/a Syngenta Variety 2 117 8 6.84% 0 n/a n/a Syngenta Variety 8 107 11 10.28% 8  8 100.00% Syngenta Variety 9 124 9 7.26% 0 n/a n/a Syngenta Variety 10 324 46 14.20% 35 30  85.71% Syngenta Variety 11 111 10 9.01% 0 n/a n/a Syngenta Variety 12 119 6 5.04% 0 n/a n/a Syngenta Variety 13 128 18 14.06% 7  6  85.71% Syngenta Variety 14 63 9 14.29% 0 n/a n/a Syngenta Variety 15 146 9 6.16% 0 n/a n/a Syngenta Variety 16 313 7 2.24% 0 n/a n/a Syngenta Variety 17 190 11 5.79% 0 n/a n/a Alisacraig 104 16 15.38% 13 12  92.31% Moneymaker 67 18 26.87% 13 13 100.00%

Example 7: Watermelon Transformation with 1-Day Imbibed Seeds

[0114] Source of explant: 1-day imbibed watermelon seeds at least 14 days post pollination

[0115] Below is an example transformation process which was used for watermelon.

[0116] 1. Sterilization of seeds: Removed the seed coat. Sterilized with 15% Clorox+0.1% ul of Tween 20 for 15 min, then washed with sterilized water for >3 times.

[0117] 2. Seeds imbibition: as described in Example 1 for 1 day.

[0118] 3. Agrobacterium: as described in Example 1.

[0119] 4. Explants preparation and infection: As described in Example 1 except as follows. Separated the embryo axis and cotyledon. The cotyledon was inoculated in Agrobacterium suspension (OD=0.5) for at least 2 hours.

[0120] 5. Co-culture: As described in Example 1 except as follows. After inoculation, transferred the explants to co-culture medium, and sealed the petri dish with parafilm. The co-culture medium was an empty petri dish with a filter paper, and added 1 ml liquid coculture medium. Kept at 22° C. in the dark for 3 days.

[0121] 6. Recovery: After co-culture, transferred the explants to recovery medium (MS medium with 1× B5 vitamins, 1× MS lion, 20 g/L sucrose, 10 g/L glucose, 0.01 mg/L IAA, 2 mg/L Zeatin, 1 mg/L BAP, 150 mg/L Timentin, 150 mg/L Carbenicillin, 10 g/L Agar, pH=5.8). Kept at 25° C. in dark for 4 days

[0122] 7. Selection 1: After Recovery, transferred to selection medium (MS medium with 1× B5 vitamins, 1×Ms Iron, 20 g/L sucrose, 10 g/L glucose, 0.01 mg/L IAA, 2 mg/L Zeatin, 1 mg/L BAP, 150 mg/L Timentin, 150 mg/L Carbenicillin, 150 mg/L Spectinomycin, 10 g/L Agar, pH=5.8). Kept at 25° C. in 16 hours/8 hours (light/dark) for about 14 days. Then subcultured to new selection medium and kept at 25° C. in 16 hours/8 hours (light/dark) for about 21 days.

[0123] 8. Selection 2: After Selection 1, transferred to selection medium (MS medium with 1× B5 vitamins, 1× Ms Iron, 30 g/L sucrose, 0.2 mg/L kinetin, 150 mg/L Timentin, 150 mg/L Carbenicillin, 150 mg/L Spectinomycin, 10 g/L Agar, pH=5.8) and kept at 25° C. in 16 hours/8 hours light/dark for about 14 days.

[0124] 9. Rooting: transferred the regenerated shoots to new rooting medium (MS basal salts and vitamins, plus 1× B5 vitamins, plus 1× Ms Iron, 30 g/L sucrose, 0.2 mg/L kinetin, 0.2 mg/L NAA, 150 mg/L Timentin, 150 mg/L Carbenicillin, 150 mg/L Spectinomycin, 10 g/L Agar, pH=5.8) in pot for rooting.

TABLE-US-00009 TABLE 8 Results No. No. of Transformation Explant No. of Regenerate Positive frequency type Explants shoots events (TF) Variety Vector Cotyledonary 20 14 14 70.00% 97103 24133 node

Example 7: Biolistic Transformation of Tomato

[0125] Variety used: Ailsa Craig

[0126] Vector used: linearized fragment contains selectable marker and reporter GUS cassette from 24133

[0127] Source of explant: 1-day imbibed seeds

[0128] Below is an example biolistic transformation for tomato.

[0129] 1. Sterilization of seeds: as described in Example 1.

[0130] 2. Seeds imbibition: as described in Example 1, in germination medium

[0131] 3. Explants preparation: Split the seeds and isolated the halved embryo. Cut the embryo into pieces to be used as explants. Transferred the explants to Osmotic medium (MS medium with 1× Ms Iron, 30 g/L sucrose, 60 g/L Mannitol, 10 g/L Agar, pH=5.8). Each Osmotic medium plate contains explants from 20 seeds. Rearranged the explants to the center of the Osmotic medium plate, and do not overlap them. Kept the explants at 25° C. in the dark for 4-8 hours.

[0132] 4. Gold particle preparation: the method of coating DNA to gold particle followed by Sanford et al (1992). Two mg 0.6 μm gold particle was washed by 1 ml absolute ethanol by sonication and replaced by ddH2O and sonication again in a test tube. After pellet the goal and completely remove the water, the gold was suspended in 50 μl H2O. 0.6 pMol DNA, 250 μl CaCl2 (2.5 M) and 50 μl Spermidine (0.1 M) was added into the gold suspension and adjust the volume with water to final 500 μl. Mixed gently and keep on ice for >30 min. Pellet the gold by centrifugation and remove the supernatant, wash the DNA coated gold twice with 1 ml ethanol. The gold was resuspended in 60 μl ethanol. Resuspended the gold particle, and transferred the suspension to micro carrier for biolistic transformation

[0133] 5. Biolistic: The type of biolistic particle delivery system used: BIO-RAD, PDS-1000. The distance between Stop screen and explants was set to 6 cm, pressure set to 1100 psi, vacuum set to 28 inches Hg. One shot for each plate.

[0134] 6. After biolistic: The explants were kept on Osmotic medium overnight. Then washed in 1 ml liquid ¼ MS medium (¼ MS medium with 1 mg/L Zeatin, 10 g/L sucrose, 5 g/L glucose, 2 g/L MES, 20 mg/L Acetosyringone, 0.5 mg/L Silver Nitrate). Transfer the explants to recovery medium (MS medium with 1× Ms Iron, 20 g/L sucrose, 10 g/L glucose, 10 g/L Polyvinyl Pyrrolidone, 1 mg/L Zeatin, 0.01 mg/L IAA, 150 mg/L Timentin, 150 mg/L Carbenicillin, 10 g/L Agar). Kept the explants at 25° C. in dark for 4 days

[0135] 7. Selection 1: as described in Example 1.

[0136] 8. Selection 2: as described in Example 1.

[0137] 9. Rooting: as described in Example 1.

TABLE-US-00010 TABLE 9 Results Imbibed Estimated No. No. of days of Explants Regenerate Positive Estimated explants Seeds number shoots events TF 1 20 200 1 1 0.50%

Example 8: Mechanically Prepared Explants for Transformation

[0138] Variety used: Ailsa Craig

[0139] Explant type: mixed

[0140] Vector used: 24416

[0141] The process of transformation was as described in Example 1 except for the explant preparation method, which is described below.

TABLE-US-00011 TABLE 10 Cut method and results No. of Explants Explants Positive Seeds number Cut method per seeds events TF 20 139 First cut with 6.95 12 8.6% scalpel to split the seeds, then immerge the halved embryo in water and blender at low speed (18000 rpm) for about 3 seconds, collect large pieces of explants and blender again until all explants could pass through the sieve. Count the alive explants when transfer from recovery to Selection 1 10 165 First cut with 16.5 20 12.1% scalpel to split the seeds, then cut perpendicularly, inoculate the cut seeds, then remove the seed coat 10 170 Cut the seeds 17 20 11.8% into slices with scalpel, inoculate the cut seeds, then remove the seed coat

Example 9: Use of Fresh Seeds Harvested from Mature Tomato Fruits as Source of Explants

[0142] Variety: Syngenta Variety 1

[0143] Explant type: mixed

[0144] Vector used: 24133

[0145] A process of transformation of explants from fresh seeds is described:

[0146] 1. Ripened fruits were harvested from plants grown in a greenhouse.

[0147] 2. The surface of ripened fruits were sterilized with high-temperature metal tools. Specifically, a spoon was placed into dry sterilizer (250° C.). After 1-2 minutes, the hot spoon was pressed on the surface of the tomato fruit. This was repeated several times.

[0148] 3. The sterilized surface was cut and fresh seeds were retrieved with a spoon.

[0149] 4. The fresh seeds were cut into small explants, and the seed coat was removed for inoculation.

[0150] 5. Inoculated the explants in Agrobacterium suspension for 0.5-1 hour, and completely remove the Agrobacterium suspension.

[0151] 6. Added a sterilized filter paper to solid co-culture plate, and transferred the explants to the plate and co-cultured on the filter paper at 22° C. in dark for 2 days.

[0152] 7. The other steps are the same as described in Example 1.

TABLE-US-00012 TABLE 11 Results No. No. of Explant No. of Regenerate Positive source Explants shoots events TF Fresh 165 9 9 5.45% Seeds

REFERENCES

[0153] U.S. Pat. No. 5,422,259A [0154] U.S. Pat. No. 5,986,181A [0155] WO0113707A1 [0156] US2002157139 A1 [0157] Ellul, P., Garcia-Sogo, B., Pineda, B., Rios, G., Roig, L. A. and Moreno, V. (2003) The ploidy level of transgenic plants in Agrobacterium-mediated transformation of tomato cotyledons (Lycopersicon esculentum Mill.) is genotype and procedure dependent [corrected]. Theor Appl Genet, 106, 231-238. [0158] Sigareva, M., Spivey, R., Willits, M. G., Kramer, C. M. and Chang, Y. F. (2004) An efficient mannose selection protocol for tomato that has no adverse effect on the ploidy level of transgenic plants. Plant Cell Rep, 23, 236-245.