AND AGROB ACTERIUM STRAINS FOR TRANSFORMATION OF PLANTS

BACKGROUND OF THE INVENTION

[0001] The present invention relates to the field of the production of transgenic plants through Agrobacierium-mediaxzd transformation of cells of somatic embryogenic calli or embryogemc suspension cultures and regeneration of the transformed cells into fruit-setting plants. In particular, the present invention relates to the production of transgenic plants in the Euphorbiaceae family. The present invention further relates to media compositions _. , selection methods and engineered Agrobacterium tumefaciens strains that improve Agrobacterium- mediated transformation efficiency.

[0002] The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the Bibliography.

[0003] The world is facing dwindling supply is fossil fuel and worsening Green House Effect. There is an urgent demand to increase production and consumption of renewable energy. Biofueis have been recognized as a national priority for many countries in their search for alternative sources to meet their energy security needs and at the same time help reduce CO ₂ emissions that cause the Green House Effect. The demand for biofuel has put increasing pressure on food production. For example, to satisfy the biofuel need for Germany in 2017 as mandated by the German government the entire farm land of this country would have to be used for growing bioenergy crops with no land left for food production. To ease this competition for land and to satisfy our need for renewable fuels, there is a strong need to utilize marginal iand for bio-energy production.

[0004] Euphorbiaceae family includes valuable trees species such as rubber (Hevea brasiliensis ) and Jatropha curcas and castoT oil piant [Ricinus communis). Several unique characteristics of Jatropha curcas make it an ideal piant for biodiesel production. These characteristics include the ability to grow on marginal land; low requirement for water; a nonfood crop status; fast oil production in 0.5-2 years after planting compared to more than 3.5 years for oil palm. Accordingly, the Indonesian government has announced that they will dedicate about 3 million hectares of land for jatropha planting in the next 5 years. Many other Asian countries are adopting similarly bold plans. Amongst the various countries, India is the most advanced in terms of establishment of Jatropha plantations. However, the seed yield of an Indian Jatropha plantation remains low, ranging from 0.4 to 12 MT/Ha (compared with about 19Mt Ha for palm fruit). Similarly, the reported kernel oil contents varied wildly from 40.4 - 85.3% (Sun, W.B., Jatropha International Congress 2008, Singapore). Castor oil is an important source of lubricant and is also a potential source of biodiesel in the future.

[0005] This difference is at least in pan attributed to the lack of research in breeding and farm management in Jatropha curcas. Genetic engineering is recognized as a fast method for crop improvement. Plant transformation is essentially a two step process, i.e., delivery of genes into a host cell followed by regeneration of the transformed cell into a plant. Somatic embryogenic calli or somatic embryogenic suspension cultures is generally regarded as the most efficient method of regeneration as most of the transformed cells have already acquired the embryogenic potential that will drive them to develop into a somatic embryo quite spontaneously. An efficient method for initiation of Jatropha somatic embryos and maintenance of embryogenic suspension cultures have been described (International patent application No. PCT/SG2009/000015; US Provisional Patent: 61/025,430).

[0006] Plant transformation methods may be broadly classified into two groups: Direct DNA transfer, such as eiectroporation and particle bombardment, and Agrobacterium-meaiatsd transformation. To date, the later has become method of choice in transformation of many plants. In fact, most commercially available transgenic varieties of cotton and rapeseed derived from this method (Cerdeira and Duke, 2006; Dunwell, 2000). Previous work in maize (Dunwell, 1999; Vega et al., 2008; Wang and Frame, 2009), rice (Christou, 1997; Hayashimoto et al., 1990: Lee et al, 1991 ; Zhang et al., 1997) and soybean (Christou et al., 1988; Christou et al, 1987; Olhoft et al., 2003) demonstrate different examples of different methods for introduction of foreign DNA into crop plants. Transgenic plants may be obtained following Agrobacterium- mediated transformation of the. This method is characterized by a low frequency of transgenic production and by the formation of chimeras (Gould et al., 1991). Transgenic plants may also be obtained following Agrobacterium-med ^' iated transformation of immature embryos. Immature embryos have been the choicest explant to date since there is usually a very high frequency of callus induction and plant regeneration from immature embryos. Following transformation, immature embryos, regardless of the method of DNA delivery, are very hard to regenerate into fertile plants (Cheng et al., 1997). In addition, it is usually very difficult to obtain immature embryos throughout the year, and their suitable stage for culture is also strictly time dependent, thus limiting their use. While the frequency of transformation with immature embryos ranges from about 0.14 to about 4.3%, the number of transgenics recovered is a small fraction of the number of embryos transformed (Cheng et al., 1997). Somatic embryos are suitable for transformation via Agrobacterium t mefaciens and particle bombardment (Christou, 1997; Sidorov and Duncan, 2009). Somatic embryogenic calli can be maintained conveniently as a liquid culture and fast-amplified for production of clonal populations. Thus, once a somatic embryogenic culture of an elite variety of established, it will save years of time developing genetically pure lines that are stacked with transgenic traits.

[0007] Many different explants can be co-cultured with an Agrobacterium strain harboring a T-DNA vector to produce a transgenic cell, which can be regenerated into a normal plant through somatic embryogenesis or organogenesis pathway. Research on regeneration and transformation of Jatropha curcas has been very limited.

[0008] Jha et al. (2007) reported production of somatic embryos of Jatropha curcas using leaf tissues. (Li et al., 2008) report transformation of Jatropha curcas by co-culturing leaf disc with Agrobacterium and regenerate via organogenesis pathway. However, both methods are not easily repeatable in the Jatropha research community (Z. Mao et al., Temasek Life Sciences Laboratories, Singapore, peronsonal communication; A. Suwanto, Bogor Agricultural University, Indonesia; personal communication).

[0009] Mao et al. (U.S. provisional patent application No. 61/122,454) discloses production of transgenic Jatropha curcas plants by Agrob cterium-mediated transformation of leaf explants and regeneration via somatic embryogenesis pathway. The invention also used grafting technique to overcome rooting difficulties of the transgenic plantlets.

[0010] International Publication No. WO 2008/012832 discloses an efficient process for in vitro propagation of Jatropha through direct regeneration of leaf disc without any intermediary callus phase. Genetic transformation based on this regeneration method has not been reported.

[0011] Thus, a reliable and efficient method for transformation of Jatropha is yet to be developed, improvements that would lead to shorter regeneration time, higher transformation rate and high throughput transformation work are particularly welcome.

[0012] Delivery of T-DNA by Agrobacterium cells into plant host cells depends on pre- induction of virulence genes. Known inducers that are naturally produced in wounded plant tissues include a variety of phenolic compounds such as acetosyringone, sinapinic acid (3,5 dimethoxy-4-hydroxycinnamic acid), syringic acid (4-hydroxy-3,5 dimethoxybenzoic acid), ferulic acid (4-hydroxy-3-methoxycinnamic acid), catechol (1,2-dihydroxybenzene), p- hydroxybenzoic acid (4-hydroxybenzoic acid), β- resorcylic acid (2,4 dihydroxybenzoic acid), protocatechuic acid (3,4-dihydroxybenzoic acid), pyrrogallic acid (2,3,4-trihydroxybenzoic acid), gallic acid (3,4,5-trihydroxybenzoic acid) and vanillin (3-methoxy- - hydroxybsnzaldehyde) (U.S. Patent No. 6,483,013).

[0013] A constitutively expressed virG mutant protein (virGN54D) has been found to enhance Agrobacterium virulence and transformation efficiency (Hansen et al., 1994). Similarly, supplementation of virulence inducers, e.g., acetosyringone or nopaline. increased transformation efficiency of cotton shoot tips (Veluthambi et al., 1989) and somatic embryogenic callus that was propagated on solid media (U.S. Patent No. 6,483,013). Acetosyringone is indispensable for transformation of a large number of fungi, which are unable to secrete Agrobacterium virulence inducing compounds (Bundock et al., 1995; de Groot et al., 1998).

[0014] U.S. Patent Nos. 6,162,965 and 6,310,273 disclose that Agrobacterium tamefaciens induces necrosis during co-culture with plant cells, particularly of the Gramineae. Compounds that inhibit necrosis. e.g., 2,5-norbomadiene, norbornene, silver thiosulfate, aminoethoxyvinylglycine, cobalt salts, acetyl salicylic acid, or salicylic acid, may be used to improve transformation efficiency. Similarly, a nucleotide sequence that encodes a cell death suppressor protein (p35, iap or dad-1) may be included in the transformation vector to achieve the same goal.

[0015] Similarly, wounding and A. tumefaciens infection of soybean cotyledonary node explants result in extensive enzymatic browning and cell death in the tissue. Thiol compounds, such as L-cysteine, dithiothreitol (DTT), and sodium thiosulfate, improves T-DNA delivery by inhibiting the activity of plant pathogen and wound-response enzymes, such as peroxidases (PODs) and polyphenol oxidases (PPOs). Additive effect was observed with the thiol compounds. The most effective combination for soybean is 1 mM Na-thiolsulfate + 8.8 mM L- cysteine + 1 mM DTT (Olhoft et al., 2003).

[0016] Nitric oxide (NO) has been implicated in defense responses and its control or elimination may increase pathogen infection such as that of Agrobacterium during plant transformation (U.S. Patent No. 7,388,126). NO modulators include of NG-monomethyl-L- arginine, monoacetate salt (N-Me-L-Arg, AcOH; L-NMMA), NG-monomethyl-L- homoarginine, monoacetate salt (NMMHA, AcOH), NG-monoethyl-L-arginine, monoacetate salt (NMEA, AcOH), NG-monomethyl-L-arginine, di-p-hydroxyazobenzene-p"-sulfonate salt (N-Me-L-Arg, diHABS; L-NMMA), and clinorotation.

[0017] Activation of plasma membrane NADPH oxidase is associated with the incompatible interaction of plants with microbes, leading to production of superoxide, which can be converted into H2O2 by superoxide dismutases. NADPH oxidase can be effectively inhibited by diphenylene iodonium (Sagi and Fluhr. 2001). To date, there is no evidence to suggest that NADPH oxidase plays any role in compatible plant-microbe interactions, such as jatropha- agrobacterium interaction during transformation process. Neither exist any report on the use of NADPH oxidase inhibitors, such as diphenylene iodonium, to improve Agrobacterium-memated plant transformation efficiency.

[0018] Overgrowth of Agrobaeterium jeopardizes the survival of the transformed plant cells and also has an effect on the T-DNA transfer process. Insertion of multiple copies of a gene of interest into a plant cell is influenced by the frequency of T-DNA transfer into the cell. Agrobacterium-msdiated transformation protocols strive to attain transformation events with a limited number of copies of DNA entering into any one cell. The presence of multiple inserts can lead to gene silencing or reduce expression levels of transformed genes, which is caused by several mechanisms including recombination between the multiple copies.

[0019] International Publication No. WO 2001/09302 and U.S. Patent Application Publication 2003/0204875 discloses control of Agrobaeterium growth during the transformation process in order to improve transformation efficiency. The use of inhibiting agents during inoculation and co-culture of Agrobaeterium with a transformable plant cell results, according to the disclosure, in increased transformation efficiencies and a low copy number of an introduced genetic component in several plant systems. Preferred growth-inhibiting agents are compounds containing heavy metals such as silver nitrate or silver thiosulfate, antibiotics such as carbenicillin, and a combination of antibiotics and a clavulanic acid such as augmentin or timentin.

[0020] U.S. Patent No. 6,323,396 claims a process to produce a dicotyledonous transgenic plant by systemically infecting auxotrophic Agrobaeterium strain with explant tissue, protoplast or microspore and regeneration on a plant regeneration medium, in the absence of a compound with a bacteriocidal or bacteriostatic effect on the auxotrophic Agrobaeterium strain. The Agrobaeterium strain is LBA4404metHV (LMG P-18486) or ATHV ade, his, (LMG P-18485). International Publication No. WO/2005/103271 discloses improved method for Agrobaeterium mediated transformation of embryogenic suspension culture which is achieved by the use of solid support for co-culturing of the embryogenic cells and a set of media for co-culture and selection of transformants. The method is particularly suitable for transformation of cotton embryogenic calli. SUMMARY OF THE INVENTION

[0021] The present invention relates to the field of the production of transgenic plants through Agrobacterium-m diaxed transformation of cells of somatic embryogenic calli or embryogenic suspension cultures and regeneration of the transformed cells into fruit-setting plants. In particular, the present invention relates to the production of transgenic plants in the Euphorbiaceae family. The present invention further relates to media compositions, selection methods and engineered Agrobacterium tumefaciens strains that improve Agrobacterium- mediated transformation efficiency.

[0022] In particular this invention relates to method for preparation of somatic embryogenic calli that are highly competent for T-DNA conjugation by agrobacterial cells; medium compositions and co-culture conditions for Agrob cterium-mediatsd transformation and cell line maintenance; method for selection for transgenic cells that are prone to killing by Agrobacterium due to over-growth and antibiotic resistance of the agrobacterial cells, and regeneration of the transformed cells into fruit-setting plants.

[0023] Thus in a first aspect, the present invention provides method for the transformation of plants of the Euphorbiaceae family. The method, comprises the steps of:

[0024] (a) establishing and maintaining cells of embryogenic calli;

[0025] (b) co-culturing the cells with a culture of Agrobacterium harboring a T-DNA vector of interest containing a polynucleotide of interest to produce transformed cells having the polynucleotide of interest stably incorporated into their genome in a co-culture medium;

[0026] (c) selecting the transformed cells on a selection medium;

[0027] (d) developing the selected transformed cells into transgenic somatic embryos on a development medium;

[0028] (e) maturing the transgenic somatic embryos on a maturation medium;

[0029] (f) germinating the somatic embryos into plantlets on a germination medium; and

[0030] (g) transferring the plantlets to soil to further develop into flowering plants.

[0031] In one embodiment, the present invention relates to method for preparation of somatic embryogenic calli that are highly competent for T-DNA conjugation by Agrobacterial cells. In one embodiment, the embryogenic calli are derived from somatic embryos. In another embodiment, the embryogenic calli are derived from zygotic embryos. In one embodiment, the calli are subcultured regularly, preferably about every week, by transferring part of the cell mass to about 20 ml to about 100 ml liquid medium in a suitable container. Freshly subcultured calli that are about 2 days to about 7 days after subculture are preferred for transformation. In one embodiment, a preferred liquid medium contains modified MS salts (MS salts without NH4NO ₃ ), MS vitamins, about 10 g/1 to about 30 g/1 sucrose, about 10 g/1 to about 80 g/1 polyethylene glycol (PEG) 8000, about 0.01 mg/l to about 2 mg l 2,4-dichiorophenoxyacetic acid (2,4-D), about 0.1 g/1 to about 1 g/1 glutamine and about 0.1 g/1 to about 1 g/1 asparagine. Altematively, solidified media or filter-supported medium containing the media components may be used to propagate embryogenic calli.

[0032] In another embodiment, the present invention relates to a medium composition for efficient delivery of T-DNA into embryogenic calli. In one embodiment, the medium contains an inhibitor of NADPH oxidase, such as diphenylene iodonium (DPI) in the range between about 1 μΜ and about 50 μΜ. In one embodiment, the medium is preferably supplemented with an inducer of the Agro bacterium virulence, such as acetylsyringone (AS) in the range between about 5 μΜ ando about 500 μΜ. In another embodiment, the medium contains the Agrobacterium MinAB salts (Chilton et al., 1974). In a further embodiment, the medium contains low concentration of glucose, in the range between about 5 mM and about 50 mM. In an additional embodiment, the medium contains glycerol, in the range between about 0.1% and about 2%. In a further embodiment, the medium optionally contains a compound, such as histidine, that supports normal growth of Agrobacterial cells.

[0033] In another embodiment, the present invention relates to use of a preferred Agobacterium strain (AGL2). in accordance with this embodiment the Agrobacterium strain contains a constitutively active virA gene. In one embodiment, the strain needs no chemical inducers for activation of virulence. In another embodiment the virA gene is integrated into the genome or Ti plasmid for improved stability. In a further embodiment, the virA gene is derived from a super-virulent strain. In an additional embodiment, the virA gene is derived from AGL1. In one embodiment, the strain does not contain a wild type allele of virA. In another embodiment, the use of the strain improves transformation efficiency.

[0034] In another embodiment, the present invention relates to use of a preferred Agrobacterium strain (AGL3). In one embodiment, the strain further contains a mutation that suppresses growth of Agrobacterium cells in selection media. In another embodiment, the strain contains a mutation affecting amino acid utilization, which preferably does not affect utilization of glutamine or asparagine. In a further embodiment, the mutation is in the a background with a constitutively active virA allele. In another embodiment, the use of the strain improves transformation efficiency. [0035] In another embodiment, the present invention relates to use of a medium-over-la)' technique for selection of transformants. In one embodiment, the technique improves control of over-growth of AgTobacterium. In another embodiment the medium contains a compound that inhibits growth of Agrobacterium cells. In a further embodiment, the said medium further contains a selection agent that inhibits growth of non-transformed plant cells.

[0036] In another embodiment, the present invention relates to an improved method for selection of transformed cells and regeneration of the transformed cells into normal plants. In one embodiment, the method includes a liquid stage. In another embodiment, the liquid stage shortens the regeneration time. In a further embodiment, the liquid stage is immediately or shortly after co-culture with an Agrobacterium strain. In an additional embodiment, the strain is preferably one which is not able to utilize at least one amino acid. In another embodiment, the liquid medium contains an inhibitor of Agrobacterium growth, such as cefotaxime, carbenicillin, timentin and the like, and a selecting agent for transformed plants cells, such as a plant active antibiotic or herbicide.

BRIEF DESCRIPTION OF THE FIGURES

[0037] Figure 1 shows suspension culture that has been transformed by AGL2(pC1305.1). Embryos were stained with x-gluc (5-bromo-4-chloro-3-indolyl beia-D-glucuronide) 2 months after co-culture.

[0038] Figure 2 shows embryogenic calli that were co-cultured with either AGL2(pCambia 1305.1) or AGL3(pCambiaI305.1). 0.3 mg/l histidine was added in all media before or during the co-culture stage. Thoroughly washed calli were transferred in small clumps on solid Bd2 medium supplemented with 7 mg/l hygromycin and 300 mg/l cefotaxime and cultured under the same condition. The plate was photographed 4 weeks after the first culture on solid selection medium.

[0039] Figure 3 shows embryogenic calli that were co-cultured for 53 hours with either AGLl(pCambtal305.1bar) or AGL2(pCambial305.1bar) in medium with (200 uM) or without AS as described in Example 2. The calli were immediately stained with X-gluc and photographed.

[0040] Figure 4 shows somatic embryogenic calli from 4 different cell lines that were co- cultured with AGL2(pCambial 305.1) and selection was done by embedding the calli in LMP Bd2 medium. The co-culture medium JIM2 was supplemented with either 1 μΜ diphenyl iodonium (DPI), 5 μΜ DPI or a mixture of antioxidants with a final concentration of 1 mM Na- tfaiosulfate, 8.8 mM L-cysteine and 1 mM dithiothxeitol (DTT). Hygromycin resistant somatic embryos were scored after two months selection. The number of somatic embryos derived from co-culture with DPi-free JIM2 (CK) was set at 100 in each cell line and the relative number of somatic embryos in other co-culture conditions was calculated by dividing the actual number by that of CK. The results derived from the average of four cell lines, each cell line consisting of 6 selection plates. "AOC" indicates culture medium with 1 mM Na-thiosulfate, 8.8 mM L-cysteine. and 1 mM DTT.

[0041] Figure 5 shows somatic embryos vigorously developing in selection medium that were selected about 2 months after co-culture and small pieces of the leaf tissues were removed and placed in a well in a microtitre plats filled with 200 μΐ phosphate buffer (50 mM) with 1 mM X-gluc. After vacuum infiltration, the tissue was incubated at 37°C overnight and the tissue was cleared with 70% ethanol.

[0042] Figures 6A-6C show hygromycin resistant plantlets 4 months after co-culture. Fig. 6A: Somatic embryogenic calli were co-cultured with AGL2(pCambial305.1 ) and selection was done in embedded in LMP Bd2 medium as described in Example 2. Fig. 6B: One of the plants of Fig. 6A was stained with X-gluc. Fig. 6C: Examples of transgenic plants in soil pots 7 months after co-culture.

[0043] Figure 7 shows an example of flowering transgenic plant with developing fruits.

[0044] Figure 8 shows Southern blot analysis of 17 randomly selected transgenic plants. Ck lane is DNA of wild type Jcurapha curcas plant.

[0045] Figures 9A-9C show Jatropha curcas somatic embryogenic calli. Fig. 9A: Jatropha curcas somatic embryogenic calli was co-cultured with AGL3(pCambial305J ) for 2.5 day and selection was ^' done in J20P medium with 7 mg/1 hygromycin and 300 mg 1 cefotaxime for two weeks. Subculture was done in a similar medium with reduced sucrose (lOg/1) for another 2 weeks. Fig. 9B: The same co-cultured calli selected on solid B2d medium with 7mg/l hygromycin and 300 mg/1 cefotaxime for 4 weeks. Fig. 9C: The same co-cultured calli with selection done in embedded LMP B2d medium for 4 weeks.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention relates to the field of the production of transgenic plants through Agrobacteriu -medm ^' icd transformation of cells of somatic embryogenic calli or embryogenic suspension cultures and regeneration of the transformed cells into fruit-setting plants. In particular, the present invention relates to the production of transgenic plants in the Euphorbiaceae family. The present invention further relates to media compositions, seiection methods and engineered Agro bacteriu turnefaciem strains that improve Agro bacterium- mediated transformation efficiency.

[0047] Thus in a first aspect, the present invention provides method for the transformation of plants of the Euphorbiaceae family. The method comprises the steps of:

[0048] (a) establishing and subculturing cells of embryogenic calli;

[0049] (b) co-culturing freshly subcultured cells with a culture of Agrobacterium harboring a T-DNA vector of interest containing a polynucleotide of interest to produce transformed cells having the polynucleotide of interest stably incorporated into their genome in a co-culture medium;

[0050] (c) selecting the transformed cells on a selection medium;

[0051] (d) developing the selected transformed cells into transgenic somatic embryos on a development medium;

[0052] (e) maturing the transgenic somatic embryos on a maturation medium;

[0053] (f) germinating the somatic embryos into plantlets on a germination medium; and

[0054] (g) transferring the plantlets to soil to further develop into flowering plants.

[0055] In one embodiment, the cells for co-culturing are derived from the Euphorbiaceae family. In another embodiment, the cells are derived from the Jatropha genera. In one embodiment, the embryogenic calli are derived from somatic embryos. In another embodiment, the embryogenic calli are derived from zygotic embryos. In a preferred embodiment, the cells are derived from somatic embryogenic calli. In one embodiment, these cells are cultured on a solid medium. In another embodiment, the cells are cultured in a liquid medium. Any medium that is suitable for maintaining cells with high embryogenecity can be used. It has been found that a medium that contains modified MS salts, MS vitamins, a carbohydrate source, such as sucrose, polytheylene glycol (PEG), an auxin, such as 2.4-dichlorophenoxyacetic acid (2,4-D), glutamine and asparagnine is particularly suitable. In one embodiment, the medium comprises modified MS salts (MS salts without NH4NO3), MS vitamins, about 10 g/1 to about 30 g/1 sucrose, about 10 g/1 to about 80 g/1 PEG 8000, about 0.01 mg 1 to about 2 mg/I 2,4-D, about 0.1 g/1 to about 1 g/1 glutamine and about 0.1 g/1 to about 1 g/1 asparagine is particularly suitable and is a preferred medium. The cells are subcultured regularly, preferably about every week. In one embodiment, the cells are cultured in a condition with 2000-3000 lux lighting with 16/8 day/night cycles, 26°C - 30° C and gentle shaking at 50-100 rpm. [0056] In one embodiment, a method is provided for preparing somatic embryo genie calli that are highly competent for T-DNA conjugation by Agrobacterial cells, in one embodiment, the embryogenic calli are subcultured regularly, preferably about every week, by transferring part of the cell mass to about 20 ml to about 100 ml liquid medium in a suitable container. As a variation, solidified media or filter-supported medium containing the media components may be used as the cell culture medium. Freshly subcultured calli that are about 2 days to about 7 days after subculture are preferred for transformation.

[0057] In another embodiment, the present invention relates to a co-culture medium composition for efficient delivery of T-DNA into embryogenic calli. In one embodiment the medium is one that favors induction of virulence of Agrobacterial cells, such as a medium that contains the Agrobacterium MinAB salts (Chilton et al., 1974). In one embodiment, the medium also contains vitamins, such as B5 vitamins. In another embodiment, the medium also contains an inhibitor of NADPH oxidase, such as diphenylsne iodonium (DPI) in the range between about 1 μΜ and about 50 uM. The use of an inhibitor of NAPDH oxidase leads to an improved plant transformation efficiency. In one embodiment, the medium is preferably supplemented with an inducer of the Agrobacterium virulence, such as acetylsyringone (AS) in the range between about 5 μΜ and about 500 μΜ. Other known inducers include sinapinic acid (3,5 dimethoxy-4-hydroxycinnamic acid), syringic acid (4-hydroxy-3,5 dimethoxybenzoic acid), ferulic acid (4-hydroxy-3-methoxycinnamic acid), catechol (l,2-dihydroxybenzene), p- hydroxybenzoic acid (4-hydroxybenzoic acid), β- resorcylic acid (2,4 dihydroxybenzoic acid), protocatechuic acid (3,4-dihydroxybenzoic acid), pyrrogallic acid (2,3,4-trihydroxybenzoic acid), gallic acid (3,4,5-trihydroxybenzoic acid) and vanillin (3-methoxy-4- hydroxybenzaldehyde). In a further embodiment, the medium optionally contains a compound, such as histidine, that supports normal growth of Agrobacterial cells. In one embodiment, the medium contains a low concentration of glucose, in the range between about 5 mM and about 50 mM. In an additional embodiment, the medium contains glycerol, in the range betwee about 0.1% and about 2% and a buffering agent, such as 2-[N-morphoiino]ethanesulfonic acid (MES), in a range between about 1 mM to about 20 mM and adjusted about pH 5.7.

[0058} In one embodiment, Agrobacterium that are pre-induced are used in the co-culture step. In another embodiment, the Agrobacterium is a supervirulent strain, such as Agrobacterium tumefaciens strain AGL1. In a further embodiment, the Agrobacterium contains a mutant virA protein that needs no added inducer for acquisition of virulence. In one embodiment, the Agrobacterium containing a mutant virA protein is A. tumefaciens strain AGL2. In another embodiment, the Agrobacterium is an auxotrophic strain, such as A. tumefaciens strain AGL3. in one embodiment, the auxotrophic strain is not auxotrophic to giutamine and asparagine.

[0059] The cells with high somatic embryogenesis potential are co-cultured with Agrobacterium containing a T-DNA vector containing a polynucleotide of interest to produce transformed cells having the polynucleotide of interest stably integrated into their genome. The Agrobacterium typically also contains a selectable marker gene, such as described herein. In one embodiment, the co-culturing is performed on as solid medium. In another embodiment, the co-culturing is preferably performed on a porous support such as a porous membrane or filter paper. The porous support may be soaked in liquid, medium, may be overlaid on a solid medium or may be overlaid on a liquid medium. Co-culture is performed in an environment with about 1000 lux constant lighting, 24° C - 25° C for 2-4 days.

[0060] The transformed cells are selected by culturing them on a selection medium, hi one embodiment, the selection is performed on a solid medium. In a preferred embodiment, the selection is performed by embedding the co-cultured plant cells in a solid medium, in another preferred embodiment, the selection is performed in a liquid medium. Any suitable medium may be used for selecting transformed cells. In one embodiment, the selection medium comprises Gamborg B5 salts and B5 vitamins. The selection medium further contains a carbohydrate source, such as sucrose, an auxin, such as 2,4-D. In one embodiment, the medium comprises Gamborg B5 salts and B5 vitamins, about 10 g/1 to about 30 g/1 sucrose, and about 0.1 mg/1 to about 10 mg/1 2,4-D, preferably about 2 mg/1 2,4-D. The medium may be solidified using typical solidifying agents such as phytagel, agar and low melting point (LMP) agarose. The selection medium also contains a compound for selection of transformed cells (selection agent) and is dependent on the selectable marker gene that is included in the Agrobacterium. Examples of compounds that can be used include plant active antibiotics or herbicides. The selection medium also preferably contains a compound that suppresses the growth of agrobacterial cells. Such compounds are well known in the art and include cefotaxime, carbenicillin, and timentin.

[0061] In one embodiment, the selection of transformed cells is done in condition with 25° C - 30° C temperature, preferably about 26° C and with 16 hours lighting per day and under one 36W (about 2000 lux) warm-light tube. Subculturing can be done in the same fresh medium every 2-3 weeks. Calli resistant to the selecting agent are transferred to a hormone-free medium supplemented with giutamine and asparagine and further supplemented with a selection agent, such as hygromycin, and an Agrobacterium inhibiting compound, such as cefotaxime. An example of such a medium is the DGA medium described herein, which contains NFLNOrfree MS salt, Gamborg B5 vitamins, 30 g/1 sucrose, 2.4 g/1 phytagel, 1 g/1 glutamine, 0.5 g/1 asparagine. pH 5.8. Once green cotyledonary stage embryos are produced, they can be transferred to an embryo maturation medium for 2-4 weeks. An example of a suitable embryo maturation medium is the M9-2 medium described herein, which contains NH _t NOs-free MS salts, Gamborg B5 vitamins, 20 g/1 sucrose, 20 g/1 maltose, 1 g/i glutamine, 0.5 g 1 asparagine, 2.4 g/1 phytagel, 0.1 g/1 casein hydrolysate, pH 5.8, and supplemented with a selection agent and a. Agrobacterium inhibiting agent. Germination of fully developed embryos is well known in the art. An example of a suitable germination medium is the G35-2 medium described herein, which contains MS salts, vitamins, 20 g/1 sucrose, 5.5 g/1 agar. 0.1 g/I casein hydrolysate, 2 mg/1 gibberellic acid (GA ₃ ), pH 5.8. Germinated embryos with roots are further developed in a medium with low concentration of auxin, such as a-naphthaleneacetic acid (NAA) at about O.OlmgA. An example of such a medium is the W9 medium described herein, which contains MS vitamins, 20 g/1 sucrose, 1 mg 1 FeS0 ₄ , 0, 1 g/1 casein hydrolysate, 0.01 mg/1 NAA, 5.5 g/1 agar, pH 5.8. Activated charcoal at about Q.l g 1 to about 0.5 g may be included in the medium to improve growth of small plants. Another example of such a medium is the W15 medium described herein, which contains ½ MS salts, ½ MS vitamins, 15 g/1 sucrose, 1 mg/1 FeS0 ₄ , 0.1 g 1 activated charcoal, 0.1 g/1 casein hydrolysate, 0.01 mg/1 NAA, 5.5 g/1 agar, pH 5.8.

[0062] Selection for transformed cells by the medium embedding method is performed by spreading thoroughly washed co-cultured somatic embryogenic calli, at about 0.1 g per 90 mm Petri dish, on a solid medium, such as Bd2 medium described herein that contains Gamborg B5 salts, B5 vitamins, 30 g/1 sucrose, 2.2 g/1 phytagel. 2 mg/1 2,4-D sodium salt, pH5.8, and supplemented with selection agent and a Agrobacterium inhibiting agent. About 10 ml low melting point (LMP) agarose (0.6%) containing Bd2 medium (after cooled down to about 40° C) supplemented with the same selection agent was over-laid the calli. Subculture was done by carefully picking up resistant calli, placing on a fresh Bd2 medium and overlayed with LMP Bd2 medium. Selection in the LMP medium is done for 8-10 weeks until cotyledonary stage embryos are produced. Resistant embryos at around torpedo to cotylendonary stages are transferred to DGA medium supplemented with a selection agent, in about 3-4 weeks, small green cotyledonary stage embryos are transferred to M9-2 medium supplemented with a selection agent. In about 3-4 weeks, the embryos are transferred to G35-2 medium as described herein containing a selection agent. At this stage, up to four 36W light tubes may be used. Lighting remains 16 hr per day. In about 3 weeks, germinated embryos are transferred to W9 medium containing a selection agent. In about 2-3 weeks, the plantlets are transferred to W15 medium (without hygromycin). Rooted plantlets are ready for planting in soil in 2-3 weeks.

[0063] Selection of transformed cells in liquid medium can be done in any suitable medium for a period of time sufficient to produce transgenic torpedo or cotyledonary stage somatic embryos. In one embodiment, the liquid selection medium comprises Gamborg B5 salts and B5 vitamins. The selection medium further contain a carbohydrate source, such as sucrose, an auxin, such as 2,4-D. In one embodiment, the medium comprises Gamborg B5 salts and B5 vitamins, about 10 g/1 to about 20 g/1 sucrose, and about 0.1 mg 1 to about 10 mg/1 2,4-D, preferably about 2 mg/1 2,4-D. In one embodiment, the amount of the carbohydrate source, such as sucrose, is reduced in each subculture from a high of about 20 g/1 to a low of about 10 g 1. The selection medium also contains a compound for selection of transformed cells and is dependent on the selectable marker gene that is included in the Agrobacterium. Examples of compounds that can be used include plant active antibiotics or herbicides. The selection medium also preferably contains a compound that suppresses the growth of Agrobacterial cells. Such compounds are well known in the art and include cefotaxime, carbenicillin, and timentin. In one embodiment, the selection is performed in a small growth vessel, such as a multi-well plate. In one embodiment, the cells are cultured in a condition with 2000-3000 lux lighting with 16/8 hours day/night cycles, 26° C - 30° C temperature and gentle shaking at 50-100 rpm. Somatic embryos thus produced are matured and germinated as those from solid medium. A desiccation step after the liquid stage may facilitate embryos germination. Alternatively, chemical agents, such as abscisic acid (ABA), that mimic the desiccation effect may be used.

[0064] The germinated transgenic plants are then transferred to soil to develop into flowering plants. The transgenic plants can be used as a parent in conventional breeding programs.

[0065] In another aspect, the present invention relates to a novel Agrobacterium strain which improves transformation efficiency. In one embodiment, the novel Agrobacterium strain contains a constitutively active virA gens. The strain needs no added chemicals for virulence induction. The virA gene is integrated into the chromosomal genome or Ti piasmid for improved stability. The novel Agrobacterium strain does not contain a wild type allele of virA. The virA gene is expressed in a super-virulent background, such as in a pTiBo542 background, and is derived from pTiBo542. In one embodiment, the novel Agrobacterium strain is AGL2. A culture of Agrobacterium strain AGL2 has been deposited under the Budapest Treaty on 28 October 2009 with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia, 20110-2209 USA under ATCC® Patent Deposit Designation PTA-10447. The deposit has been tested for viability on 4 November 2009 by the ATCC and the culture was viable.

[0066] In another aspect, the present invention relates to a novel Agrobacterium strain which improves regeneration efficiency. In one embodiment, the improved regeneration efficiency occurs at post-co-cuiture steps. In one embodiment, the novel Agrobacterium strain contains a mutation that leads to auxotrophy for growth. The novel Agrobacterium strain may further contain a mutant gene encoding a constitutively active virA protein. The virA protein is expressed in a super-virulent background, such as in a pTiBo542 background. In one embodiment, the novel Agrobacterium strain is AGL3. A culture of Agrobacterium strain AGL3 has been deposited under the Budapest Treaty on 28 October 2009 with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia, 20110-2209 USA under ATCC® Patent Deposit Designation PTA-10448. The deposit has been tested for viability on 4 November 2009 by the ATCC and the culture was viable.

[0067] In another aspect, the present invention provides a method for improved plant transformation efficiency. In this method, the embryogenic calli are co-cultured with an Agrobacterium strain in a medium that contains an inhibitor of NADPH oxidase. In one embodiment, the inhibitor of NADPH oxidase is diphenylene iodonium (DPI) in the range between about 1 μΜ and about 50 μΜ, preferably between about 1 μΜ and about 10 μΜ.

[0068] Techniques for co-culture of embryogenic calii with an Agrobacterium are well known in the art. Any strain harboring a DNA construct that contains a gene or nucleic acid of interest can be used. A preferred method for co-culture is the method that is disclosed in International Publication No. WO 2005/103271.

[0069] The polynucleotide that is inserted (the polynucleotide of interest) into plants in accordance with the present invention is not critical to the transformation process. Generally the polynucleotide of interest that is introduced into a plant is part of a construct. The polynucleotide of interest may be a gene of interest, e.g., a coding sequence for a protein, or it may be a sequence that is capable of regulating expression of a gene, such as an antisense sequence, a sense suppression sequence or a miRNA sequence. The construct typically includes regulator;,' regions operatively linked to the 5' side of the DNA of interest and/or to the 3' side of the DNA of interest. A cassette containing all of these elements is also referred to herein as an expression cassette. The expression cassettes may additionally contain 5' leader sequences in the expression cassette construct. The regulator}' regions (i.e., promoters, transcriptional regulator}' regions, and translational termination regions) and/or the polynucleotide encoding a signal anchor may be native/anaiogous to the host cell or to each other. Alternatively, the regulatory regions and/or the polynucleotide encoding a signal anchor may be heterologous to the host cell or to each other. See, U.S. Patent No. 7,205.453 and U.S. Patent Application Publication Nos. 2006/0218670 and 2006/0248616. The expression cassette may additionally contain selectable marker genes. See, U.S. Patent No. 7.205,453 and U.S. Patent Application Publication Nos. 2006/0218670 and 2006/0248616.

[0070] Generally, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Usually, the plant selectable marker gene will encode antibiotic resistance, with suitable genes including at least one set of genes coding for resistance to the antibiotic spectinomycin, the streptomycin phosphotransferase (spt) gene coding for streptomycin resistance, the neomycin phosphotransferase (nptll) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (hpt or aphiv) gene encoding resistance to hygromycin, acetolactate synthase (als) genes. Alternatively, the plant selectable marker gene will encode herbicide resistance such as resistance to the sulfonylurea-rype herbicides, glufosinate, glyphosate, ammonium, bromoxynil, imidazolinones, and 2,4- dichlorophenoxyacetate (2,4-D), including genes coding for resistance to herbicides which act to inhibit the action of glutamine synthase such as phosphinothricin or basta (e.g., the bar gene). See generally, International Publication No. WO 02/36782, U.S. Patent No. 7,205,453 and U.S. Patent Application Publication Nos. 2006/0248616 and 2007/0143880, and those references cited therein. This list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used.

[0071] A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. That is, the nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in the host cell of interest. Such constitutive promoters include, for example, the core promoter of the Rsyn7 (International

Publication No. WO 99/48338 and U.S. Patent No. 6,072,050); the core CaMV ^{35 S} promoter (Odell etal., 1985); rice actin (McElroy et at, 1990); ubiquitin (Christensen and Quail, 1989 and Christensen et al., 1992); pEMU (Last et al., 1991); MAS (Velten et al, 1984); ALS promoter (U.S. Patent No. 5,659,026), and the like. Other constitutive promoters include, for example, those disclosed in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. Jatropha promoters include those disclosed in U.S. provisional patent application No. 61/184,416.

[0072] Other promoters include inducible promoters, particularly from a pathogen-inducibie promoter. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen: e.g., PR proteins, SAR proteins, beta- 1 ,3- glucanase, chitinase, etc. Other promoters include those that are expressed locally at or near the site of pathogen infection. In further embodiments, the promoter may be a wound-inducible promoter. In other embodiments, chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. The promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. In addition, tissue-preferred promoters can be utilized to target enhanced expression of a polynucleotide of interest within a particular plant tissue. Each of these promoters are described in U.S. Patent Nos. 6,506,962, 6,575,814, 6,972,349 and 7,301,069 and in U.S. Patent Application Publication Nos. 2007/0061917 and 2007/0143880. Tissue preferred Jatropha promoters include those disclosed in U.S. provisional patent application No. 61/184,416.

[0073] Where appropriate, the polynucleotide of interest may be optimized for increased expression in the transformed plant. That is, the coding sequences can be synthesized using plant-preferred codons for improved expression. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, 5,436,391, and 7,205,453 and U.S. Patent Application Publication Nos. 2006/0218670 and 2006/0248616.

[0074] The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, ceil biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et ai, 1982, Molecular Cloning (Cold Spring Harbor Laborator}' Press, Cold Spring Harbor, New York); Sambrook et al, 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York): Ausubel et ai, 1992), Current Protocols in Molecular Biology (John Wiley & Sons, including periodic updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Russell, 1984, Molecular biology of plants: a laborator)' course manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press. New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor. New York); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. I. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B . Perbal, A Practical Guide To Molecular Cloning (1984); the treatise. Methods In Enzymology (Academic Press, Inc., N.Y.); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Fir et al., UNA Interference Technology: From Basic Science to Drug Development, Cambridge University Press, Cambridge, 2005; Schepers, RNA Interference in Practice, Wiley-VCH, 2005; Engelke, RNA Interference (RNAi): The Nuts & Bolts of siRNA Technology, DNA Press, 2003 ; Gott, RNA Interference, Editing, and Modification: Methods and Protocols (Methods in Molecular Biology), Human Press, Totowa, NJ, 2004; Sohail, Gene Silencing by RNA Interference: Technology and Application, CRC, 2004.

EXAMPLES

[0075] The present invention is described by reference to the following Examples, which is offered by way of illustration and is not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were utilized.

EXAMPLE 1

Media Formulations

[0076] J20P: MLJ^-free MS salts, MS vitamins, 20 g/1 sucrose, 50 g/1 PEG 8000, 0.5 g/1 glutamine, 0.25 g/1 asparagine, 0.2 mg 1 2,4-D sodium salt, pH 5.8

[0077] IM (Chilton et al., 1974; de Groot et al, 1998): 2.05 g/1 K ₂ HP0 ₄ , 1.45 g/1 KH ₂ P0 ₄ , 0.6 g/1 MgS0 ₄ -7H ₂ 0, 0.5 g/1 (N¾) ₂ S0 ₄ , 0.5 g/1 ΝΉ ₄ ΝΟ3, 0.01 g/1 CaCl _2; 0.5 mg/1 ZnS0 ₄ - 7¾0, 0.5 mg/1 CuS0 ₄ -5H ₂ 0, 0.5 mg 1 H3BO3, 0.5 mg 1 MnS0 ₄ -H ₂ 0, 0.5 mg/1 NaMb0 - 2¾0, 1 mg/l FeS0 ₄ , 0.5% glycerol, 0.5g/l 2-[N-morphoIino]ethanesulfonic acid (MES), 0-100 μΜ acetosyringone, 2 g/1 glucose, pH 5.7-5.8 [0078] JIM2: IM medium plus: B5 vitamins, 2 mg/1 2,4-D, 1-50 μΜ diphenylene iodonium (DPI).

[0079] JIM2 solid: JIM2 with 2g/l phytagel.

[0080] JIM2H: JIM2 medium plus 0.3g/L histidine.

[0081] JIM2H solid (for co-culture): J1M2H with 2g/l phytagel.

[0082] Bd2: Gamborg B5 salts, B5 vitamins, 30 g/1 sucrose, 2.2 g/1 phytagel, 2 mg/1 2,4-D sodium salt, pH5.8.

[0083] DGA: NHiNOs-free MS salt, Gamborg B5 vitamins, 30 g/1 sucrose, 2.4 g/1 phytagel, 1 g/1 glutamine, 0.5 g/1 asparagine, pH 5.8.

[0084] M9-2: NT^NCVfree MS salts, Gamborg B5 vitamins, 20 g/1 sucrose, 20 g 1 maltose, 1 gA glutamine, 0.5 g/1 asparagine, 2.4 g/1 phytagel, 0.1 g/1 casein hydrolysate, pH 5.8.

[0085] G35-2: MS salts, vitamins, 20 g/1 sucrose, 5 g/1 agar, 0.1 A casein hydrolysate, 2 mg/1 gibberellic acid (GA ₃ ), pH 5.8.

[0086] W9: MS salts, MS vitamins, 20 g 1 sucrose, 1 mg/1 FeS0 ₄ , 0.1 g/1 casein hydrolysate, 0.01 mg/1 a-naphthaleneacetic acid ( AA), 5.5 g/1 agar, pH 5.8.

[008η W15: ½ MS salts, ½ MS vitamins, 15 g/l sucrose, 1 mg/1 FeS0 , 0.1 g/1 activated charcoal, 0.1 g 1 casein hydrolysate, 0.01 mg/1 NAA, 5 g/1 agar, pH 5.8.

EXAMPLE 2

Co-culture of Embryogenic Calli with Agrobacterium

[0088] Embryogenic cell lines, for example, JS4 cell line, which was derived from a zygotic embryo of an Indonesian Jatropha curcas collection, were subcultured every week by transferring about 3 g of the cell mass to 20 ml J20P medium. 3-5 days after subculture, the embryogenic calli cell mass was collected.

Co-culture plates were made by pouring about 30ml JFM2H solid medium to each 90 mm Petri dish. Alternatively, 3-4 sheets of cellulose filter paper can be soaked with JIM2H liquid medium before placing into a 90 mm Petri dish. One sheet of 82 mm Hybond N membrane was over-laid to the medium-soaked cellulose filter paper or solid JIM2H medium. About 0.75 g embryogenic calli was mixed with 200 ul Agrobacterium cells on the membrane. After one hour incubation at room temperature, excess liquid medium containing the Agrobacterium cells was removed. The plates were capped, sealed with 3M tape and incubated at 24° C with dim light for 2-3 days. EXAMPLE 3

Preparation of Agrobacterium Cultures

[0089] Fresh colonies of AGL2 or AGL3 harboring pCambial305.1 were inoculated into 30 ml LB medium that had been supplemented with 25 mg 1 rifampicm and 50 mg/1 kanamycrn. Cells were cultured at 28° C, 200-230 RPM until OD600 reached 0.8-2.0. The culture was diluted to 0.3 OD600 units with LB medium with appropriate antibiotics and incubated in the same condition for about 2-3 hr at 28° C until about 0.6 OD600 unit. 25 ml of the Agrobacterium culture were transferred to a 50 ml Falcon tube and the cells were spun down at 3500 RCF, 15° C for 20min in a benchtop centrifuge with a swing-out rotor. The LB medium was removed and the pellet was resuspended in 25ml JTM2H medium. The cells were allowed to induced at room temperature with no shaking for about 2 hours and the Agrobacterium preparation was adjusted to about 0.5 OD600 using the same medium.

[0090] For transformants of wt AGL1 strain, the cells were cultured in the Agrobacterium MinAB medium and pre-induced in induction medium (IM) supplemented with AS for about 6 hours before being used for co-culture with plants cells.

EXAMPLE 4

Selection and Regeneration of Transformants

[0091] The co-cultured somatic embryogenic calli were washed for three times on the membrane with Bd2 medium supplemented with 5-10 mg/1 hygromycin and 300 mg/1 cefotaxime. The calli were transferred to solid Bd2 medium supplemented with 5-10 mg/1 hygromycm and 300 mg/1 cefotaxime. The plates were placed in a 26° C tissue culture room with 16 hr lighting per day on shelves with one 36W Phillips Lifemax light tube (warm light). Subculture was done in the same fresh medium after 2-3 weeks. In another 4-6 weeks, resistant calli were transferred to DGA medium supplemented with 7mg l hygromycin and 300rng/L cefotaxime, in about 3-4 weeks, small green cotyledonary stage embryo appeared. They were transferred to M9-2 medium supplemented with 7 mg/l hygromycin and 300 mg/1 cefotaxime.

[0092] In about 3 weeks, the embryos were transferred to G35-2 medium with 10 mg 1 hygromycin. At this stage, up to four 36W light tubes may be used. Lighting remains 16 hr per day. In about 3-4 weeks, germinated embryos were transferred to W9 medium with 7 mg/1 hygromycin. In about 2-3 weeks, the plantlets were transferred to W15 medium (without hygromycin). Rooted plantlets were ready for planting in soil in 2-3 weeks. [0093] Selection for transformants can also be done with herbicides, such as glyfosinate, and other antibiotics, such as kanamycin.

[0094] Selection on solid medium was always associated with heavy Agrobacterium overgrowth, which kills the majority of transgenic calli/somatic embryos. Table ] summarizes the numerous examples of selection using this approach.

TABLE 1

Selection of Transgenic Jatropha Plants on Various Media

Code Date Strain Plasmid Calli Source Selection Results

Method

TE1 10/6/07- AGLl pST2eGFP solid SE on soiid no transformants due to 10/30/08 medium SE medium heavy Agro contamination

TE2 10/30/07- AGL1 pST2eGFP liquid SE on solid no transformants due to 10/30/08 medium SE medium heavy Agro contamination

ΊΈ3 11/5/07- AGLl pST2eGFP liquid SE on soiid no transformants due to 10/30/08 medium SE medium heavy Agro contamination

TE4 1 1/20/07- AGLl pST2eGFP liquid SE on solid no transformants due to 3/6/08 medium SE medium with heavy Agro contamination

Kan-LMP

coating

ΊΈ5 11/27/07- AGLl pPZP 11135SGFP liquid SE on Kan no transformants due to 3/6/08 medium SE solid medium heavy Agro contamination with Kan-LMP

coating

TE6 1/8/08- AGLl pC1305.1 liquid Hyg 0, 1.0, 20, no transformants due to 2/12/08 medium SE 30 and 50 mg 1 heavy Agro contamination

SE on solid

medium

high selection pressure

TE7 1/14/08- ' AGLl pST2eGFP liquid Kan 0, 25, 50 50-80 mg/1 OK, heavy 2/19/08 medium SE and 80 mg/1 Agro contamination

SE on soiid DPI caused no damage on medium expiants

TE8 1/15/08- AGLl pC1305.1 liquid Hyg O, 10, 20, Agro contamination, NA 2/18/08 medium SE 30 and 50 mg/1 did not kill agrobacteria) cells

SE on soiid

medium too high selection pressure

TE10 1/30/08- AGLl pCI305.1 liquid Hyg O, 10, 20, NA able to inhibit. AGLl 3/4/08 medium SE 30 and 50 mg 1 at 80 mg/i yet ineffective

SE on solid no effect on plant cells medium

TEH 2/3/08- AGLl DC1305.1 liquid Hyg 0, 2, 4, 5, no transformants due to 3/4/08 medium SE 6 and 8 mg/1 heavy Agro contamination

1 SE on solid medium

TE14 3/3/08- AGL1 j pC1305.I bar liquid GLF 0, 2, 5 no transformants due to 10/30/08 medium SE and lO mg 1 heavy Agro contamination

SE on solid

medium

TE19 3/23/08- LBA4404 pC 1305.1 bar liquid GLF 0, 5, 8 no transformants due to 12/23/08 medium SE and 10 mg/i heavy Agro contamination

SE on solid

medium

TE26 5/9/08- LBA4404 pC1305.1 bar liquid GLF 3 and 5 no transformants due to 1/23/09 medium SE mg/i heavy Agro contamination

SE on solid

medium

TE29 6/23/08- LBA4404 pC 1305.1 bar liquid GLF 3, 5 and 8 no transformants due to 1/23/09 medium SE mg/1 heavy Agro contamination

MSO 1 μΜ

SE on solid

medium

TE32 7/21/08- LBA4404 pC1305.1 bar liquid GLF 10 and 5 no transformants due to 1/23/09 medium SE then 10 mg/1 heavy Agro contamination and SEC

SEC/SE on

solid medium

3/18/09 9,12 cm

TE34 7/25/08- LBA4404 pC1305.1 bar liquid GLF 5, 10 no transformants due to 1/23/09 medium mg/1 heavy Agro contamination

SEC

SEC

embedded in

LMP medium

TE37 8/1/08- AGL2 pC 1305.1 liquid Hyg O. 1, 2, 5. transgenic plant generated 6/17/09 medium mg/1

Southern blot positive,

SEC

SEC potted to soil embedded in

LMP medium

TE40 8/8/08- AGL2 pC1305.1 bar liquid GLF 3 and 5 after intense rescue effort 6/3/09 medium mg/1 from contamination, one

SEC resistant plant obtained.

SEC GUS staining positive embedded in

LMP medium

TE4 8/15/08- AGL1 pC1305.1 bar liquid GLF 0, 3, 5 1 resistant plant by AGL1.

6/3/09 medium and 10 mg/1 number too small to draw

AGL2

SEC conclusion. GUS staining

SEC

positive

embedded in

LMP medium

TE45 8/19/08- AGL1 pC1305.1 bar liquid GLF 0, 3, 5 . 1 resistant plant by AGL1.

5/21/09 medium and 10 mg/1 number too small to draw

AGL2

SEC conclusion. GUS staining

SEC

positive

embedded in

LMP medium TE46 8/19/08- LBA4404 pC1305.1 bar liquid GLF 0, 3, 5 no resistant plant 4/18/09 medium and 10 mg 1

SEC

SEC

embedded in

LMP medium

TE48 8/26/08- AGL2 pCl 305.1 bar liquid GLF 0, 5 and no resistant plant

4/9/09 medium .10 mg/1

SEC

SEC

embedded in

LMP medium

TE49 8/25/08- AGL2 pC1305.1 bar - liquid GLF 0, 5 and 1 resistant plant, potted 5/6/09 medium 10 mg/1

SEC

SEC

embedded in

LMP medium

TE50 9 7/08- LBA4404 pC1305.1 bar iiquid GLF 0 and 5 no resistant plant

4/9/09 medium mg/1

SEC

SEC

embedded in

LMP medium

TE51 9/9/0S- AGL2 pC1305.1 bar liquid GLF 0 and 10 no resistant plant

4/9/09 medium mg/1

heavy contamination

SEC

SEC

embedded in

LMP medium

TE52 9/12/08- AGL1 pC1305.1 bar iiquid GLF 0, 5 and no resistant plant

medium 10 mg/1

AGL2 heavy contamination

SEC

SEC

embedded in

LMP medium

TE53 9/19/08- AGL2 pC1305.1 iiquid Hyg 0, 5, 6, 8 got resistant plants. GUS 6/30/09 medium and 10 mg 1 staining positive

SEC

SEC optimal Hyg 6-8mg/l embedded in

LMP medium

TE54 10/3/08- AGL2 pCI305.1 bar . liquid GLF 0, 5, 10, no resistant plant

2/12/09 medium 20, 30 and 50

SEC mg 1

SEC

embedded in

LMP medium

TE55 10/9/08- AGL2 DC1305.1 liquid Hyg 0, 8 mg 1 heavy agro contarmination 1/30/09 medium

SEC

SEC

embedded in

LMP medium

TE56 1 1/4/08- AGL2 pC1305. I iiquid Hyg 0, 2, 3, 5 transgenic plant generated, 7/18/09 medium mg/1 then 7 Southern positive, potted

SEC mg/1 to soil

SEC embedded in

LMP medium

TE57 11/21/08- AGL2 pC1305.1 bar liquid MSO 0, L 2, no resistant plant

2/3/09 medium 5, 10 and 20

SEC μΜ

SEC

embedded in

LMP medium

TE58 11/21/08- AGL2 pC1305.1 bar liquid GLF 5 mg 1 no resistant plant

3/3/09 medium then 30 mg 1

SEC

SEC

embedded in

LMP medium

TE59 12/2/08- AGL2 pC1305.1 iiquid Hyg 0 and 7 cell line 1 : 9 trasngenic medium mg/1 lines from 5 co-cultur

SEC

SEC cell line 2: none from 2 co- embedded in culture

LMP medium

cell line 3: 1 transgenic line from 2 co-culture cell line 4: none from 3 co- culture

TE60 12/5/08- AGL2 pC1305.1 liquid Hyg 0 and 8 cell line 1, 5 transgenic medium mg/1 lines from 3 co-culture

SEC

SEC cell line 2: 1 transgenic embedded in line from 3 co-culture LMP medium

cell line 3: 3 transgenic lines from 3 co-culture cell line 4: 2 transgenic lines from 2 co-culture

TE61 1/8/09- AGL2 pC1305.1 iiquid Hyg 0, 2, 5, 7 90 byg resisistant plants, medium and 10 mg 1 48 GUS positive plants

SEC then shifted to from 6 co-culture

7 mg/1

30% GUS negative plants

SEC contained transgene embedded in

46% dish discarded due to LMP medium

agrobacterium overgrowth

Notes: IM with AS: Agrobacterium induction medium with 200μΜ acetosyrin one. Kan: kanarnycin. Hyg: hygromycin. GLF: glyfosinate. SE: somatic embryos. SEC somatic embryogenic calli. MSO: methionine sulfoximine

[0095] Alternatively, co-cultured somatic embryogenic calli were washed for three times on the membrane with Bd2 medium supplemented with 5-10 mg/I hygromycin and 300 mg/1 cefotaxime. About 0.1 g co-cultured calli was spread at low density on solid Bd2 medium supplemented with 5-10 mg/1 hygromycin and 300 mg/1 cefotaxime. Ten ml low melting point (LMP) agarose (0.6%) containing Bd2 medium supplemented with 5-10 mg/1 hygromycin and 300 mg/1 cefotaxime was over-laid to a Bd2 solid medium plate that contains the same selection agents. The plates were placed in a 26° C tissue culture room with 16 hr lighting per day on shelves with one 36W Phillips Liiemax light tube (warm light). Calii embedded in LMP agarose medium at low density reduced plate contamination rate from > 90% to about 50% (TE 61 ).

[0096] Selection in the LMP medium was done for 8-10 weeks until cotyledonary stage embryos were produced. Subculture was done by carefully picking up resistant calli, placing on a fresh Bd2 medium and overlayed with LMP Bd2 medium. Resistant embryos at torpedo to cotylendonary stages were transferred to DGA medium supplemented with 7mg/l hygromycin and 300mg/L cefotaxime. In about 3-4 weeks, small green cotyledonary stage embryo appeared. They were transferred to M9-2 medium supplemented with 10 mg/1 hygromycin and 300 mg 1 cefotaxime. In about 3 weeks, the embryos were transferred to G35-2 medium with lOmg/1 hygromycm. At this stage, up to four 36 light tubes may be used. Lighting remains 16 hr per day. In about 3 weeks, germinated embryos were transferred to W9 medium with lOmg/1 hygromycin. In about 2-3 weeks, the plantlets were transferred to W15 medium (without hygromycin). Rooted plantlets were ready for planting in soil in 2-3 weeks.

[0097] Numerous hygromycin resistant somatic embryos were observed with embryogenic calli co-cultured with Agrobacterium. strains harboring pCambial 305.1 when selection was done by the medium embedding method (Figure 1).

EXAMPLE 5

Construction of Agrobacterium tumefaciens Mutants AGL2 and AGL3

[0098] AGLl-AvirA strain: An approximately 10-kb fragment that contains the AGL1 virA gene in pUC18-S/?I was isolated from a genomic library of AGL1 using C5S virA gene as a probe. A 4.6-kb fragment containing entire AGL1 virA gene was made by digestion with Ascl and Kpnl and subcloned into pUC18-S//I to generate pUC18-virA. A 1.4-kb hygromycin cassette with flanking with loxP sites was cloned into the pUC18-virA that was cut with Nhel and BgUl to produce pUC18-AvirA-Hyg, from which the entire AvirA cassette was released by digestion with Sfil and inserted into pUT-mini-Tn5-sp, creating pTn5-AvirA-Hyg.

[0099] Two plasmids pPZP200-virB::LacZ (spectinomycin reisistance) and pRil5A- Tacr.RecA (kanamycin resistance, containing the RecA gene from A. tumefaciens C58) were co- introduced into supervirulent A. tumefaciens strain AGL1 (pTiBo542 in C58 chromosomal background) (Lazo et al.. 1991) by electroporation. the transferred strain was named AGLl(LacZ RecA). The virB::LacZ fusion gene was used as a reporter gene for monitoring the functionality of virA gene of AGL1 while the RecA gene restores the recombintion function AGL1 so that knockout of virA could be achieved. The suicide vector pTnS-AvirA-Hyg was delivered from the donor strain E. coli SI 7-1 (λρίτ) to the recipient strain AGLl(LacZ RecA) by bi-parentai mating at 28° C. Conjugants were selected on MiniAB agar containing hygromycin (100 μg/ml) and rifampicin (50 μg/ml) as well as 5-bromo-4-chloro-3-indolyl-galactoside (X- Gal; 40 μ l) . Nine randomly selected colorless colonies were transferred to liquid MiniAB or 2YT with the same antibiotics. The Southern analysis confirmed deletion of the virA (not shown).

[0100] The two plasmids in AGLl-AvirA were removed by growing the cells in liquid MiniAB without antibiotics at 36° C. consecutively for 6 days with 3 subcultures in between. After streaking on 2YT medium, 84 colonies were tested for growth in medium with spectinomycin (50 ug/ml), kanamycin (25 g/ ^' ml) or hygromycin (100 μg/ml) respectively. Only one colony (named 8D12) was found to have lost both spectinomycin and kanamycin resistance, but it has maintained hygromycin resistance.

[0101] To remove hygromycin resistance from strain 8D12, plasmid pRil A-Tac::Cre which contains the site-specific D A recombinase gene Cre was introduced into the strain 8D12 by electroporation. The transformants were incubated in 2YT supplemented with kanamycin (25 /ml) and rifampicin (50 \iglm\) at 28° C for 2 days. Colonies that had lost resistance to hygromycin were selected. Plasmid pRil 5A-Tac::Cre was cured according to the above mentioned method. One such colony, ΔΑ4-4, which was sensitive to spectinomycin, kanamycin and hygromycin was selected as the AGLl-AvirA strain.

[0102] AGL2 strain: virA protein containing mutation G665D has been shown to strongly activate vir gene expression in the absence of acetosyringone and monosaccharides (McLean et aL 1994). The work was done in non-supervirulent octopine strain and the functionality and efficacy of virA at the corresponding site was unknown.

[0103] To re-introduce mutated virA gene into AGLl-AvirA, a ~3.5-kb fragment containing the entire VirA gene promoter, coding sequence containing G665D mutation and 3 ' flanking sequence (including vir] promoter) was created by fusion of two PCR fragments over-lapping at the G665D residue, which as made by primer-directed mutagenesis. The fused PCR product was cloned into plasmid pUC18-S ?I to product pUC18-virA-on. Subsequently, a 1.4-kb hygromycin cassette with flanking loxP sites was inserted into downstream of VirA-on gene to generate pUC18-virA-on-Hyg. Meanwhile, plasmid pRil5A.-Tac::RecA was re-introduced into A. tumejaciens strain ΔΑ4-4 by electroporation to make strain AA4-4R. pUC18-virA-on-Hyg was introduced to &A4-4R ^' and transformants containing the constitutive virA mutant were selected on MiniAB agar supplemented with hygromycin (100 i lm\) and rifampicin (50 μ£πι1). One hygromycin and rifampicin-resistant colony was picked and presence of the constitutive virA gene was confirmed by X-gal staining after introducing pPZP200-virB::LacZ.

[0104] After curing of pRil5A-Tac::RecA, pRi!5A-Tac::Cre was introduced back into the strain so as to excise the hygromycin resistance gene cassette. After curing of the hygromycin sensitive strains, AGL2 strain was created. Molecular analysis showed that the virA-on gene was integrated at the loxP site next to the deleted virA.

[0105] To confirm that the AGL2 retains the full supervirlence and independence of AS for vir gene induction, pPZP200-virB::LacZ was introduced into the strain and its lacZ activity in AS-free media was compared to that of wt AGL1 induced with 100 μΜ AS. Results showed that AGL2 expressed comparable LacZ protein in AS-free IM media to that of wt AGLl with 100 μΜ AS. (not shown).

[0106] Several verions of virA mutants were made in AGL1 background, eg., C58 virA G665D mutant and AGL1 virA G665D mutant linked to the strong E. coli tac promoter or AGLl virG promoter. All were several folds weaker than AGL2.

[0107] To further confirm that AGL2 was able to deliver T-DNA into host cell, pEX2 was tested for transformation efficiency by this strain in Ustilago maydis. Surprisingly, AGL2 outperformed AGLl in producing transgenic cells (Table 2). Similar enhancing effect was seen in transformation of U. scitaminae and Aspergillus niger. Southern blot analyses showed no significant differences from the wild type AGLl with regard to T-DNA transfer fidelity and copy number.

TABLE 2

Transformation of Ustilago maydis Without vir Inducer

Note: Agrobacterium cells transformed with pEX2 (100 ul, OD600=0.3) were cultured in IM medium (de Groot et aL, 1998) for 6 hour before co-cultured with U. maydis sporidia (100 μΐ, OD600=0.7) for 48 hr. Selection for transformants was done by placing the membranes on YPD medium with 150 um/ml hygromycin B and 300 μg/ml cefotaxime.

[0108] AGLl was further tested for AS-independence in plant transformation. Cotton somatic embryogenic calli were co-cultured with AGL2(pST2-EGFP) or AGLl(pST2-EGFP) in the presence or absence of 100 μΜ AS. Selection for transformants was done by transferring the calli clumps to solid medium as described in PCT application WO/2005/103271. About 60-70% calli gave rise to kanamycin resistant somatic embryos when AGL2(pST2-EGFP) was used for co-culture with no AS added. This is essentially the same as that achieved with AGLl that was supplemented with 100 μΜ AS.

[0109] Auxotrophic strains of AGLl: Transposon plasmid pTn5-Hyg was constructed by replacing spectinomycin resistance gene in pUC-miniTn5-sp-eGFP with a 1.4-kb hygromycin cassette flanked with loxP sites. pRil5A-Tac::RecA was introduced into AGL2 and transposon Tn5-Hyg was transferred from E. coli SI 7-1 (Apir) to the Ag obacteriums strain by bi-parental conjugation at 28°C. Conjugants were selected on 2YT agar containing hygromycin (100 and chloramphenicol (20 μg/ml). Resistant colonies were transferred in duplicates to both 2YT medium and MS medium. Colonies that failed to grow in MS medium were inoculated in 2ml 3YT medium with hygromycin and chloramphenicol. The overnight cultures were pelleted, washed with MS medium and re-inpcuiated in MS medium supplemented with various amino acids, such as arginine (R), asparagine (N), glutamic acid (E), glutamine (Q), histidine (H), leucine (L), lysine (K) and proline (P). From 5000 transposon conjugants, 5 auxotrophic strains were isolated. Among them strain No.14 and No.22 required histidine for growth in MS medium. Strain 14 was cured of pRi l 5A-Tac::RecA and the hygromycin resistance cassette was removed by the Cre recombinase as described previously and the hygromycin sensitive derivative was named AGL3.

EXAMPLE 6

Effective Control of Agrobacterium Over-growth During Selection Stage by the Use of Auxotropic Agrobacterium Strains

[0110] Embryogenic calli were co-cultured with either AGL2(pCambial305.1) or

AGL3(pCambial305.1) as described in Example 2. 0.3 mg/1 histidine was added in all media before or during co-culture stage. Thoroughly washed calli were transferred in small clumps on solid Bd2 medium supplemented with 7 mg/l hygromycin and 300 mg/1 cefotaxime and cultured under the same condition for 4 weeks. Only 1 in 8 calli sector was free of Agrobacterium cells while the rest was covered with thick layer of Agrobacterium cells. In contract, ali 10 calli sectors co-cultured with AGL3 showed no sign of Agrobacterium contamination and the calli showed healtliy growth (greenish color) under the selection medium, indicating generation of transformed somatic embryos (Figure 2).

Example 7

Improved Transformation Efficiency of Constitutive virA Strain

[0111] Embryogenic calli were co-cultured for 53 hours with either AGLl(pCambial305.1bar) or AGL2(pCambial 305.1 bar) in medium with or without AS (200 μΜ) as described in Example 2. The calli were immediately stained with X-gluc. Stronger blue staining was observed in all calli co-cultured with AGL2 and AS further boosted transformation efficiency in AGL2 (Figure 3).

Example 8

Improved Transformation Efficiency by the Use of DPI

[0112] Somatic embryogenic calli from 4 different cell lines were co-cultured with AGL2(pCambial 305.1) and selection was done by the LMP Bd2 embedding method. Tne co- culture medium JIM2 was supplemented with either 1 μΜ DPI, 5 μΜ DPI or a mixture of antioxidants with a final concentration of 1 mM Na-thiosulfate, 8.8 mM L-cysteine and I mM DTT. Hygromycin resistant somatic embryos were scored after two months selection. In transformation using each cell line, the number of somatic embryos derived from co-culture with DPI- free JIM2 (CK) was set at 100 and relative number of somatic embryos in other co-culture conditions was calculated by dividing the actual number by that of CK. The results (Figure 4) clearly demonstrate that DPI at about 10 μΜ improved transformation efficiency by more than 2 folds whereas the combination of 1 mM Na-thiosulfate, 8.8 mM L-cysteine, 1 mM DTT led to little improvement.

[0113] The transformation enhancing effect of DPI was also tested in soybean. Soybean embryogenic suspension culture (derived Peking 18) was co-cultured for 2.5 day at 24° C with pre-induced AGLl(pCambial3Q5.1) on nylon membrane, which was placed on top of solid medium (IM with 20 mg/1 2,4-D and B5 vitamins) that was supplemented with 1 μΜ DPI or 1 mM Na-thiosulfate, 8.8 mM L-cysteine and 1 mM DTT. After selection with lOmg/1 hygromycin, the calli clumps were stained with X-gluc. GUS staining was observed in about 40% calli clumps that was co-cultured the control medium (with 20 mg/S 2,4-D and B5 vitamins). In contrast, more than 80% calli clumps showed GUS staining when 1 μΜ DPI was added in the co-culture medium. Surprisingly, combination of 1 mM Na-thiosulfate, 8.8 mM L- cysteine and 1 mM DTT did not improve transformation rate in our co-culture system.

Example 9

Analyses of Transgenic Plants

[0114] Somatic embryos vigorously developing in selection medium were selected about 2 months after co-culture and small pieces of the leaf tissues were removed and placed in a well in a microtitre plate filled with 200 μΐ phosphate buffer (50 mM) with 1 mM X-gluc (5-bromo-4- chloro-3-indolyl beta-D-glucuronide). After vacuum infiltration, the tissue was incubated at 37° C overnight and the tissue was cleared with 70% ethanol. Usually, 50-60% somatic embryos were positive for the staining (Figure 5). More than 140 hygromycin resistant plants were produced from co-culture of about 1.5 g embryogenic calli.

[0115] About 4 months after co-culture, fully developed plamlet were ready for transplantation in soil (Figure 6A). Chimera was not observed in 10 plantiets stained. (Figure 6B). The plants show no sign of developmental abnormality (Figure 6C) and normal flowers and fruits were produced (Figure 7). As expected, Tl seeds segregate into GUS positive and GUS negative off-springs with no sign of chimera for the transgene.

[0116] Total DNA was extracted from the hygromycin resistant plants and Southern blot was performed using the hpt gene as a probe. Only 1 in 17 plants did not contain the transgene (Figure 8).

Example 10

Selection in Liquid Medium.

[0117] Jatropha curcas somatic embryogenic calli were co-cultured with AGL3(pCambia 1305.1) for 2.5 days and selection was dons in J20P medium with 7 mg/1 hygromycin and 300 mg/1 cefotaxime for three weeks. Subculture was done in a similar medium with reduced sucrose (10 g/1) for another 2 weeks. Resistant cotyledonary stage somatic embryos starting to emerge along with earlier stage embryos (Figure 9A). This was about 3-4 weeks faster than those from selection on solid Bd2 medium (Figure 9B) or embedded in LMP Bd2 (Figure 9C). [0118] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10-15 is disclosed, then 11 , 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "suc as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.

[0119] It will be appreciated that the methods and compositions of the instant invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary' skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0120] The present invention has several embodiments and relies on patents, patent applications and other references for details known to those of the art. Therefore, when a patent, patent application, or other reference is cited or repeated herein, it should be understood that it is incorporated by reference in its entirety for all purposes as well as for the proposition that is recited. BIBLIOGRAPHY

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