The present invention relates to methods for the regeneration and Agrobacterium-mediated transformation of plants in the genera of Jatropha, more specifically, in Jatropha curcas.

Current U.S. Class:	435/430; 800/298; 800/306
Current CPC Class:	C12N 15/8205 (20130101)
Current International Class:	C12N 5/00 (20060101); A01H 5/00 (20060101)
Field of Search:	;435/430,410,420 ;800/298,306,295

Rothwell, Figg, Ernst & Manbeck P.C.

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Parent Case Text

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CROSS-REFERENCE TO RELATED APPLICATION

The present application is a national stage filing under 35 U.S.C. .sctn.371 of PCT/SG2009/000479, filed on 15 Dec. 2009, and claims the benefit of priority to U.S. provisional patent application Ser. No. 61/122,454 filed 15 Dec. 2008, each application is incorporated herein by reference in its entirety.

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Claims

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What is claimed is:

1. A method for regenerating plants of Jatropha curcas comprising: (a) culturing cotyledon explants on a solid callus formation medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose, 6-benzylaminopurine (6-BA) and 1-naphthaleneacetic acid (NAA) for a period of time to produce explants with callus tissue; (b) culturing the callus tissue on a solid first shoot regeneration medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose, adenine, 6-BA and 3-indolebutyric acid (IBA) for a period of time to produce callus tissue with shoots and callus tissue; (c) culturing the shoots on a solid second shoot regeneration medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose, 6-BA, IBA and gibberellic acid (GA.sub.3) for a period of time to produce shoots; (d) culturing shoots from step (c) on a solid shoot elongation medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose, 6-BA and GA.sub.3 for a period of time to produce elongated shoots; and (e) rooting or grafting the elongated shoots, wherein rooting is performed by culturing the elongated shoots on a solid rooting medium which comprises MS mineral salts, B5 vitamins, sucrose and IBA for a period of time to produce plantlets or wherein grafting is performed by grafting the elongated shoots to J. curcas rootstock.

2. The method of claim 1, which further comprising culturing the callus tissue from step (b) on a solid third shoot regeneration medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose, 6-BA and IBA for a period of time to produce callus tissue with shoots which is then cultured in step (c).

3. The method of claim 1, wherein the concentration of media components are: (a) citric acid: about 10 mg/L to about 30 mg/L, preferably about 10 mg/L; glutamine: about 150 mg/L to about 200 mg/L, preferably about 150 mg/L; casein hydrolysate: about 100 mg/L; sucrose: about 3% w/v; 6-BA: about 1.5 mg/L; and NAA: is about 0.05 mg/L to about 0.1 mg/L, preferably about 0.05 mg/L in the callus formation medium; (b) citric acid: about 10 mg/L to about 30 mg/L, preferably about 10 mg/L; glutamine: about 150 mg/L to about 200 mg/L, preferably about 150 mg/L; casein hydrolysate: about 100 mg/L; sucrose: about 3% w/v; adenine: about 2 mg/L to about 4 mg/L, preferably about 2 mg/L; 6-BA: about 1.5 mg/L; and IBA: about 0.05 mg/L in the first shoot regeneration medium; (c) citric acid: about 10 mg/L to about 30 mg/L, preferably about 10 mg/L; glutamine: about 150 mg/L to about 200 mg/L, preferably about 150 mg/L; casein hydrolysate: about 100 mg/L; sucrose: about 3% w/v; 6-BA: about 1.5 mg/L; IBA: about 0.05 mg/L and GA.sub.3: about 0.05 mg/L to about 0.5 mg/L, preferably about 0.5 mg/L in the second shoot regeneration medium; (d) citric acid: about 10 mg/L to about 30 mg/L, preferably about 10 mg/L; glutamine: about 150 mg/L to about 200 mg/L, preferably about 150 mg/L; casein hydrolysate: about 100 mg/L; sucrose: about 3% w/v; 6-BA: about 0.3 mg/L; and GA.sub.3: about 0.1 mg/L to about 0.5 mg/L, preferably about 0.1 mg/L in the shoot elongation medium; and (e) sucrose: about 3% w/v and IBA: is about 0.07 g/L in the rooting medium.

4. The method of claim 2, wherein the concentration of the media components in the third shoot regeneration medium are citric acid: about 10 mg/L to about 30 mg/L, preferably about 10 mg/L; glutamine: about 150 mg/L to about 200 mg/L, preferably about 150 mg/L; casein hydrolysate: about 100 mg/L; sucrose: about 3% w/v; 6-BA: about 1.5 mg/L; and IBA: about 0.05 mg/L.

5. The method of claim 1, wherein periods of time for the culturing are: (a) about two weeks to about three weeks on the callus formation; (b) about three weeks to about four weeks on the first shoot regeneration medium; (c) about three weeks to about four weeks on the second shoot regeneration medium; (d) about two weeks to about three weeks on the shoot elongation medium; and (e) about three weeks to about four weeks on the rooting medium.

6. The method of claim 2, wherein the period of time for culturing on the third shoot regeneration medium is about four weeks to about five weeks, preferably about four weeks.

7. The method of claim 5, wherein the periods of time for the culturing are: (a) about three weeks on the callus formation; (b) about three weeks on the first shoot regeneration medium; (c) about four weeks on the second shoot regeneration medium; (d) about two weeks on the shoot elongation medium; and (e) about four weeks on the rooting medium.

8. The method of claim 4, wherein the period of time for culturing on the third shoot regeneration medium is about four weeks to about five weeks, preferably about four weeks.

9. A method for Agrobacterium-mediated transformation of plants of Jatropha curcas comprising: (a) co-culturing cotyledon explants with Agrobacterium on a solid medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose, acetospringone (AS), 6-benzylaminopurine (6-BA) and 1-naphthaleneacetic acid (NAA) for a period of time to produce transformed cotyledon explants (b) culturing transformed cotyledon explants on a solid callus formation medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose, 6-BA, NAA, a selective agent and an Agrobacterium eradicant in the dark for a period of time to produce explants with transformed callus tissue; (c) culturing the transformed callus tissue on a solid first shoot regeneration medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose, adenine, 6-BA, 3-indolebutyric acid (IBA), a selective agent and an Agrobacterium eradicant for a period of time to produce transformed callus tissue with transformed shoots and transformed callus tissue; (d) culturing the transformed shoots on a solid second shoot regeneration medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose, 6-BA, IBA, gibberellic acid (GA.sub.3), a selective agent and an Agrobacterium eradicant for a period of time to produce transformed shoots; (e) culturing transformed shoots from step (c) on a solid shoot elongation medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose, 6-BA and GA.sub.3 for a period of time to produce elongated transformed shoots; and (f) rooting or grafting the elongated transformed shoots, wherein rooting is performed by culturing the elongated transformed shoots on a solid rooting medium which comprises MS mineral salts, B5 vitamins, sucrose and IBA for a period of time to produce transformed plantlets or wherein grafting is performed by grafting the elongated transformed shoots to J. curcas rootstock.

10. The method of claim 9, which further comprising culturing the transformed callus tissue from step (c) on a solid third shoot regeneration medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose, 6-BA, IBA a selective agent and an Agrobacterium eradicant for a period of time to produce callus tissue with shoots which is then cultured in step (d).

11. The method of claim 9, wherein the concentration of media components are: (a) citric acid: about 10 mg/L to about 30 mg/L, preferably about 10 mg/L; glutamine: about 150 mg/L to about 200 mg/L, preferably about 150 mg/L; casein hydrolysate: about 100 mg/L; sucrose: about 3% w/v; AS: about 20 mg/L; 6-BA: 1.5 mg/L; and NAA: is about 0.05 mg/L to about 0.1 mg/L, preferably about 0.05 mg/L in the co-culturing medium; (b) citric acid: about 10 mg/L to about 30 mg/L, preferably about 10 mg/L; glutamine: about 150 mg/L to about 200 mg/L, preferably about 150 mg/L; casein hydrolysate: about 100 mg/L; sucrose: about 3% w/v; 6-BA: about 1.5 mg/L; and NAA: is about 0.05 mg/L to about 0.1 mg/L, preferably about 0.05 mg/L in the callus formation medium; (c) citric acid: about 10 mg/L to about 30 mg/L, preferably about 10 mg/L; glutamine: about 150 mg/L to about 200 mg/L, preferably about 150 mg/L; casein hydrolysate: about 100 mg/L; sucrose: about 3% w/v; adenine: about 2 mg/L to about 4 mg/L, preferably about 2 mg/L; 6-BA: about 1.5 mg/L; and IBA: about 0.05 mg/L in the first shoot regeneration medium; (d) citric acid: about 10 mg/L to about 30 mg/L, preferably about 10 mg/L; glutamine: about 150 mg/L to about 200 mg/L, preferably about 150 mg/L; casein hydrolysate: about 100 mg/L; sucrose: about 3% w/v; 6-BA: about 1.5 mg/L; IBA: about 0.05 mg/L and GA.sub.3: about 0.05 mg/L to about 0.5 mg/L, preferably about 0.5 mg/L in the second shoot regeneration medium; (e) citric acid: about 10 mg/L to about 30 mg/L, preferably about 10 mg/L; glutamine: about 150 mg/L to about 200 mg/L, preferably about 150 mg/L; casein hydrolysate: about 100 mg/L; sucrose: about 3% w/v; 6-BA: about 0.3 mg/L; and GA.sub.3: about 0.1 mg/L to about 0.5 mg/L, preferably about 0.1 mg/L in the shoot elongation medium; and (f) sucrose: about 3% w/v and IBA: is about 0.07 g/L in the rooting medium.

12. The method of claim 10, wherein the concentration of the media components in the third shoot regeneration medium are citric acid: about 10 mg/L to about 30 mg/L, preferably about 10 mg/L; glutamine: about 150 mg/L to about 200 mg/L, preferably about 150 mg/L; casein hydrolysate: about 100 mg/L; sucrose: about 3% w/v; 6-BA: about 1.5 mg/L; and IBA: about 0.05 mg/L.

13. The method of claim 9, wherein the periods of time for the co-culturing or culturing are: (a) about 2-3 days; (b) about two weeks to about three weeks on the callus formation; (c) about three weeks to about four weeks on the first shoot regeneration medium; (d) about three weeks to about four weeks on the second shoot regeneration medium; (e) about two weeks to about three weekson the shoot elongation medium; and (f) on the rooting medium.

14. The method of claim 10, wherein the period of time for culturing on the third shoot regeneration medium is about four weeks to about five weeks, preferably about four weeks.

15. The method of claim 13, wherein the periods of time for the co-culturing or culturing are: (a) about 2-3 days; (b) about three weeks on the callus formation; (c) about three weeks on the first shoot regeneration medium; (d) about four weeks on the second shoot regeneration medium; (e) about two weeks on the shoot elongation medium; and (f) about four weeks on the rooting medium.

16. The method of claim 12, wherein the period of time for culturing on the third shoot regeneration medium is about four weeks to about five weeks, preferably about four weeks.

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Description

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BACKGROUND OF THE INVENTION

The present invention relates to the field of plant regeneration and transformation, particularly to methods for the regeneration and transformation of Jatropha. More specifically, the present invention relates to a method and media compositions for regeneration and transformation of plants of Jatropha curcas.

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.

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. Biofuels 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.sub.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 land for bio-energy production.

Jatropha curcas is a small woody plant belonging to the Euphorbiaceae family. Several unique characters of Jatropha curcas make it an ideal plant for biodiesel production. These include its rapid growth, easy propagation, low cost of seeds, high oil content, short gestation period, wide adaptability, drought tolerance and the ability to thrive on degraded soils. Moreover, its plant size renders convenient collection of seed (Jones, 1991; Sujatha et al., 2008).

However, Jatropha suffers from several shortcomings that may limit its wide adoption. The productivity of the plant is constrained by the unfavourable male to female flower ratio and its oil content has not been optimized by breeding. This plant is also sensitive to biotic stresses such as viral (Narayanna et al., 2007), fungal and bacterium pathogens and abiotic stresses, especially cold and drought (http colon www dot jatropha dot org). The presence of several toxic components (e.g. the protein toxin, curcin, and the cancer-causing agent phorbol esters) in seeds and leaves of the plant possess health hazards for farmers and bioprocess workers in the Jatropha industry.

The traditional way to improve on quality traits of plants is by breeding for superior genotypes. However, an assessment of genetic diversity using molecular markers disclosed low inter-accessional variability amongst local J. curcas germplasm (Sujatha at al., 2008). Therefore, alternative genetic manipulation tools, such as genetic transformation methods, are urgently required to provide additional strategies for genetic improvement of this crop. Agrobacterium-mediated genetic transformation has become the principal choice for generating transgenic plants. However, very few reports have appeared on the use of Agrobacterium-mediated transformation of plants belonging to the Euphorbiaceae family. The only one reported transformation protocol for Jatropha (Li et al., 2008) is not reproducible in our hands.

Thus, there is a need for methods of transforming J. curcas to provide means for genetic improvement in this crop species.

SUMMARY OF THE INVENTION

The present invention relates to methods for the regeneration and Agrobacterium-mediated transformation of plants in the genera of Jatropha, more specifically, in Jatropha curcas.

Thus, in one aspect the present invention provides an efficient and reproducible plant regeneration protocol for J. curcas by optimizing tissue culture and shoot regeneration conditions. This regeneration protocol has been used in combination with Agrobacterium-mediated transformation to produce T.sub.0 transgenic Jatropha shoots/plants. The present invention also provides the use of a grafting step using T.sub.0 transgenic shoots as scions and non-transgenic plants as root stocks. This grafting step obviates the need for regenerated plants to produce roots in tissue culture and considerably shortens the time for transgenic shoots to flower and produce T.sub.1 seeds.

In one embodiment, the present invention provides a method of regenerating J. curcas plants. According to this embodiment, explants are obtained from cotyledons from 5-7 day old seedlings. The explants are cultured on callus formation medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose and 6-benzylaminopurine (6-BA) and 1-naphthaleneacetic acid (NAA) as plant hormones. Callus tissue is then transferred to a first shoot regeneration medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, adenine, sucrose and 6-BA and 3-indolebutyric acid (IBA) as plant hormones. Any shoots that regenerated from the callus tissue are transferred to a second shoot regeneration medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose and 6-BA, IBA and gibberellic acid (GA.sub.3) as plant hormones. Callus tissue with no regenerates shoots are transferred to a third shoot regeneration medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose and 6-BA and IBA as plant hormones for further regeneration of shoots. The shoots that have regenerated are transferred to a shoot elongation medium which comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose and 6-BA and GA.sub.3 as plant hormones for elongation and bud multiplication. Elongated shoots are transferred to a rooting medium which comprises MS mineral salts, B5 vitamins, sucrose and IBA. After rooting, the plantlets are transferred to soil. Alternatively, the elongated shoots can be grafted to J. curcas root stock.

In a second embodiment, the present invention provides a method for Agrobacterium-mediated transformation of J. curcas plants. According to this embodiment, the Agrobacterium-mediated transformation of J. curcas utilizes the same basic scheme as described above for the regeneration of J. curcas. For transformation, the explants are first co-cultured with Agrobacterium cells prior to transfer to the callus formation medium with subsequent transfers to the shoot regeneration media, shoot elongation medium and rooting medium as described above. The co-culturing medium comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose, glucose, acetosyringone and 6-BA and NAA as plant hormones. The callus formation medium is the same as for regeneration except that it further comprises a selective agent and an Agrobacterium eradicant. Similarly, the shoot regeneration media further comprise a selective agent and an Agrobacterium eradicant. For transformation, culturing on the callus formation medium is performed in the dark. Conventional selective agents can be used for the Agrobacterium-mediated transformation of J. curcas plants. Examples of selective agents include, but are not limited to, the herbicide BASTA, hygromycin and the like.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an Agrobacterium-mediated Jatrohpa transformation method in accordance with the present invention. The time scale listed in the left is to use rooting protocol while the left is to use grafting protocol.

FIG. 2 illustrates Agrobacterium transformation vectors utilized to demonstrate the transformation method of the present invention.

FIGS. 3A-3K show the transformation, regeneration, flowering and seedling of J. curcas. FIG. 3A: J. curcas MD 5 day's seedling suitable for transformation. FIG. 3B: Callus formation and shoot generation. Left, cotyledons inoculated with Agrobacterium without carrying any vector. Right, cotyledons inoculated with Agrobacterium carrying a vector with a trait gene. Note shoot regeneration from explants. FIG. 3C: an enlarged view of hygromycin-resistant callus and shoot-like organs on the surface of brownish cotyledons. FIG. 3D: Regeneration of hygromycin-resistant shoots of J. curcas. FIG. 3E: Shoot elongation. FIG. 3F: Rooting of transgenic shoots. FIG. 3G: High rooting efficacy for transgenic J. curcas. FIG. 3H: Transgenic J. curcas grown on soil. FIG. 3I and FIG. 3J: Transgenic J. curcas shoots grafted onto non-transgenic rootstock. White arrow indicates the grafting site. FIG. 3K: Transgenic J. curcas flowering and seeding. The scale bars indicate 10 mm.

FIG. 4 shows PCR analyse of hyg-resistant ubi:GFP J. curcas plants. Lane -: wildtype Jatropha control; Lane +: plasmid DNA of p1300-GFP; Lanes #1-#10 from hygromycin-resistant Jatropha shoot leaves.

FIGS. 5A-5P show the expression of GFP in T.sub.0 plant root (FIG. 5B, FIG. 5D), male flower (FIG. 5F, FIG. 5H), and T.sub.1 seeds 3 week after fertilization (FIG. 5J, FIG. 5 K, FIG. 5L, FIG. 5N, FIG. 5O, FIG. 5P). FIG. 5A, FIG. 5C, FIG. 5E, FIG. 5G, FIG. 5I and FIG. 5M are wildtype controls for every plant organ. The scale bars indicate 2 mm.

FIG. 6 shows PCR analyses of BASTA-resistant 35S:JcWRI1 J. curcas plants. Lane M, DNA ladder; Lane #1-#7 from BASTA-resistant Jatropha shoot leaves; lane -, wildtype control; lane +, plasmid DNA of pBA002-MYC-JcWRI1.

FIG. 7 shows Western blot analysis of RcFAH12 and JcDGAT1 levels in leaves of transgenic Jatropha plants expressing 35S:RcFAH12 and 35S:JcDGAT1 using anti-HA antibody. Bottom panel: Coomassie Bright Blue staining of the RUBL (the large subunit of RUBISCO) which serves as a loading control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for the regeneration and Agrobacterium-mediated transformation of plants in the genera of Jatropha, more specifically, in Jatropha curcas.

In one aspect, the present invention provides a method of regenerating J. curcas plants. According to this embodiment, explants are obtained from cotyledons from about 5 day to about 12 day old seedlings, preferably about 5-7 day old seedlings. The culturing is performed in the light at 25.degree. C..+-.2.degree. C. in a 16 h light (100 .mu.mol/m.sup.2S)/8 h dark cycle. The seedlings are grown in tissue culture. Seed kernels of J. curcas are surface sterilized using conventional techniques and immersed in sterile water overnight at 28.degree. C. in the dark. The endosperm-free embryos are germinated on hormone free germination medium with the radicals in contact with the medium. The germination medium comprises 1/2 strength MS mineral salts, B5 vitamins and sucrose. The concentration of sucrose is about 5% (w/v). The germination medium may further comprise a buffer. In one embodiment, the buffer is 2-(4-morpholino)ethanesulfonic acid (MES) at about 0.5 g/L at a pH of about 5.6. The germination medium is solidified with agar or phytogel. The culturing is performed in the light at 25.degree. C..+-.1.degree. C. in a 16 h light (100 .mu.mol/m.sup.2S)/8 h dark cycle.

The explants are cultured on callus formation medium in the dark for about 2 weeks to about 3 weeks, preferably about three weeks. The callus formation medium comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose and 6-benzylaminopurine (6-BA) and 1-naphthaleneacetic acid (NAA) as plant hormones. The concentration of citric acid is about 10 mg/L to about 30 mg/L, preferably about 10 mg/L. The concentration of glutamine is about 150 mg/L to about 200 mg/L, preferably about 150 mg/L. The concentration of casein hydrolysate is about 100 mg/L. The concentration of sucrose is about 3%. The concentration of 6-BA is about 1.5 mg/L. The concentration of NAA is about 0.05 mg/L. The callus formation medium preferably further comprises MgCl.sub.2 at a concentration of about 0.5 g/L to about 0.95 g/L, preferably 0.5 g/L. The callus formation medium has a pH of about 5.8 to about 6.0. The callus formation medium is solidified with agar or phytagel, preferably phytagel at a concentration of about 2.5 g/L to about 3 g/L, preferably 2.5 g/L.

Callus tissue is then transferred to a first shoot regeneration medium and cultured in the light for about 2 weeks to about 3 weeks, preferably about three weeks. The first shoot regeneration medium comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, adenine, sucrose and 6-BA and 3-indolebutyric acid (IBA) as plant hormones. The concentrations of the citric acid, glutamine, casein hydrolysate and 6-BA are the same as in the callus formation medium. The concentration of adenine is about 2 mg/L to about 4 mg/L, preferably about 2 mg/L. The concentration of IBA is about 0.05 mg/L. The first shoot regeneration medium preferably further comprises MgCl.sub.2 at a concentration of about 0.5 g/L to about 0.95 g/L, preferably 0.5 g/L. The first shoot regeneration medium has a pH of about 5.8 to about 6.0. The first shoot regeneration medium is solidified with agar or phytagel, preferably phytagel at a concentration of about 2.5 g/L to about 3.0 g/L, preferably 2.5 g/L.

Any shoots that regenerated from the callus tissue are transferred to a second shoot regeneration medium and cultured in the light for about 3 weeks to about 4 weeks, preferably about four weeks. The second shoot regeneration medium comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose and 6-BA, IBA and gibberellic acid (GA.sub.3) as plant hormones. The concentrations of the citric acid, glutamine, casein hydrolysate, 6-BA and IBA are the same as in the first shoot regeneration medium. The concentration of GA.sub.3 is about 0.05 mg/L to about 0.5 mg/L, preferably about 0.5 mg/L. The second shoot regeneration medium preferably further comprises MgCl.sub.2 at a concentration of about 0.5 g/L. The second shoot regeneration medium has a pH of about 5.8 to about 6.0. The second shoot regeneration medium is solidified with agar or phytagel, preferably agar at a concentration of about 6.5 g/L to about 7 g/L, preferably 7 g/L.

Callus tissue with no regenerates shoots are transferred to a third shoot regeneration medium and cultured in the light for about 4 weeks to about 5 weeks, preferably about 4 weeks. The third shoot regeneration medium comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose and 6-BA and IBA as plant hormones for further regeneration of shoots. The concentration of the citric acid, glutamine, casein hydrolysate, 6-BA and IBA are the same as in the first shoot regeneration medium. The third shoot regeneration medium preferably further comprises MgCl.sub.2 at a concentration of about 0.5 g/L to about 0.95 g/L, preferably 0.5 g/L. The third shoot regeneration medium has a pH of about 5.8 to about 6.0. The third shoot regeneration medium is solidified with agar or phytagel, preferably phytagel at a concentration of about 2.5 g/L to about 3 g/L, preferably 2.5 g/L.

The shoots that have regenerated on the second shoot regeneration medium are transferred to a shoot elongation medium and cultured in the light for about 2 weeks to about 3 weeks, preferably about two weeks. The shoot elongation medium comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose and 6-BA and GA.sub.3 as plant hormones for elongation and bud multiplication. The concentrations of the citric acid, glutamine and casein hydrolysate are the same as in the first shoot regeneration medium. The concentration of 6-BA is about 0.3 mg/L. The concentration of GA.sub.3 is about 0.1 mg/L to about 0.5 mg/L, preferably about 0.1 mg/L. The shoot elongation medium has a pH of about 5.8 to about 6.0. The shoot elongation medium is solidified with agar or phytagel, preferably agar at a concentration of about 6.5 g/L to about 7 g/L, preferably 7 g/L.

Elongated shoots are transferred to a rooting medium and cultured in the light for about 3 weeks to about 4 weeks, preferably about four weeks. The rooting medium comprises MS mineral salts, B5 vitamins, sucrose and IBA. The concentration of sucrose is about 3%. The concentration of IBA is about 0.07 mg/L. The rooting medium has a pH of about 5.6. The rooting medium is solidified with agar or phytagel, preferably phytagel at a concentration of about 2.2 g/L. After rooting, the plantlets are transferred to soil. Alternatively, the elongated shoots can be grafted to J. curcas root stock using conventional techniques instead of being transferred to the rooting medium.

In a second aspect, the present invention provides a method for Agrobacterium-mediated transformation of J. curcas plants. According to this embodiment, the Agrobacterium-mediated transformation of J. curcas utilizes the same basic scheme as described above for the regeneration of J. curcas. Vectors containing DNA of interest are introduced into Agrobacterium using conventional techniques, such as electroporation. Transformed Agrobacterium cells are cultured prior to use using conventional techniques. In accordance with one such technique, Agrobacterium cells are inoculated into LB medium supplemented with kanamycin and carbicillin. The concentration of kanamycin is about 25 mg/L to about 100 mg/L, preferably about 50 mg/L. The concentration of carbicillin is about 50 mg/L to about 100 mg/L, preferably about 100 mg/L. The Agrobacterium cells are grown overnight at 28.degree. C., 250 rpm. The Agrobacterium cells are collected by centrifugation and re-suspended in liquid MS medium supplemented with sucrose, glucose, acetosyringone (AS), 6-BA and NAA. The concentration of sucrose is about 30 g/L. The concentration of glucose is about 10 g/L. The concentration of AS is about 20 mg/L. The concentration of 6-BA is about 1.5 mg/L. The concentration of NAA is about 0.05 mg/L to about 0.1 mg/L, preferably about 0.1 mg/L.

For transformation, the explants are first co-cultured with Agrobacterium cells prior to transfer to the callus formation medium with subsequent transfers to the shoot regeneration media, shoot elongation medium and rooting medium as described above. The co-culturing is performed in the dark for about 2-3 days. The co-culturing medium comprises MS mineral salts, B5 vitamins, citric acid, glutamine, casein hydrolysate, sucrose, AS and 6-BA and NAA as plant hormones. The concentrations of citric acid, glutamine, casein hydrolysate and sucrose are the same as in the callus formation medium. The concentration of AS is about 20 mg/L. The concentration of 6-BA is about 1.5 mg/L. The concentration of NAA is about 0.05 mg/L to about 0.1 mg/L, preferably about 0.05 mg/L. The co-culturing medium may further comprise a suitable buffer. In one embodiment, the buffer is MES. The concentration of MES is about 0.5 g/L at a pH of about 5.0 to about 5.2.

The callus formation medium used for the Agrobacterium-mediated transformation of J. curcas is the same as that used for regeneration except that it further comprises a selective agent and an Agrobacterium eradicant. The selective agent may be any selective agent for which a marker gene, such as described below, has been included in the transformed Agrobacterium. In one embodiment, the selective agent is hygromycin at a concentration of about 3 mg/L to about 5 mg/L, preferably 3.5 mg/L. In another embodiment, the selective agent is glufosinate ammonium at a concentration of about 1 mg/L. The Agrobacterium eradicant may be any conventional eradicant, such as cefotaxinme, and the like. In one embodiment, the Agrobacterium eradicant is cefotaxinme at a concentration of about 100 mg/L to about 150 mg/L, preferably 100 mg/L. Culturing on the callus formation medium for Agrobacterium-mediated transformation is performed in the dark for about 2 weeks to about 3 weeks, preferably about 3 weeks.

The callus tissue is then treated as described above with respect to the regeneration of J. curcas with transfers and culturing in the light as described above to the first shoot regeneration medium, second shoot regeneration medium, third shoot regeneration medium, shoot elongation medium, rooting medium or grafting. The first shoot regeneration medium used for the Agrobacterium-mediated transformation of J. curcas is the same as that used for regeneration except that it further comprises a selective agent and an Agrobacterium eradicant. The selective agent may be any selective agent for which a marker gene, such as described below, has been included in the transformed Agrobacterium. In one embodiment, the selective agent is hygromycin at a concentration of about 3 mg/L to about 5 mg/L, preferably 3.5 mg/L. In another embodiment, the selective agent is glufosinate ammonium at a concentration of about 1 mg/L. The Agrobacterium eradicant may be any conventional eradicant, such as cefotaxinme. In one embodiment, the Agrobacterium eradicant is cefotaxinme at a concentration of about 100 mg/L to about 150 mg/L, preferably 100 mg/L.

The second shoot regeneration medium used for the Agrobacterium-mediated transformation of J. curcas is the same as that used for regeneration except that it further comprises a selective agent and an Agrobacterium eradicant. The selective agent may be any selective agent for which a marker gene, such as described below, has been included in the transformed Agrobacterium. In one embodiment, the selective agent is hygromycin at a concentration of about 4 mg/L to about 5 mg/L, preferably 4 mg/L. In another embodiment, the selective agent is glufosinate ammonium at a concentration of about 1 mg/L. The Agrobacterium eradicant may be any conventional eradicant, such as cefotaxinme. In one embodiment, the Agrobacterium eradicant is cefotaxinme at a concentration of about 100 mg/L to about 150 mg/L, preferably 100 mg/L.

The third shoot regeneration medium used for the Agrobacterium-mediated transformation of J. curcas is the same as that used for regeneration except that it further comprises a selective agent and an Agrobacterium eradicant. The selective agent may be any selective agent for which a marker gene, such as described below, has been included in the transformed Agrobacterium. In one embodiment, the selective agent is hygromycin at a concentration of about 3 mg/L to about 5 mg/L, preferably 3.5 mg/L. In another embodiment, the selective agent is glufosinate ammonium at a concentration of about 1 mg/L. The Agrobacterium eradicant may be any conventional eradicant, such as cefotaxinme. In one embodiment, the Agrobacterium eradicant is cefotaxinme at a concentration of about 100 mg/L to about 150 mg/L, preferably 100 mg/L.

The shoot elongation medium and the rooting medium used for the Agrobacterium-mediated transformation of J. curcas are the same as that used for regeneration.

The DNA that is inserted (the DNA of interest) into plants of the genera Jatropha is not critical to the transformation process. Generally the DNA that is introduced into a plant is part of a construct. The DNA 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 regulatory 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 regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the polynucleotide encoding a signal anchor may be native/analogous 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. Pat. 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. Pat. No. 7,205,453 and U.S. Patent Application Publication Nos. 2006/0218670 and 2006/0248616.

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 (nptII) 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-type 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, WO 02/36782, U.S. Pat. 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.

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 (WO 99/48338 and U.S. Pat. No. 6,072,050); the core CaMV.sup.35S promoter (Odell et al., 1985); rice actin (McElroy et al., 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. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, those disclosed in U.S. Pat. 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.

Other promoters include inducible promoters, particularly from a pathogen-inducible 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. Pat. 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.

Where appropriate, the DNA 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. Pat. Nos. 5,380,831, 5,436,391, and 7,205,453 and U.S. Patent Application Publication Nos. 2006/0218670 and 2006/0248616.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ausubel et al., 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 laboratory 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, N.Y.); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. 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; Fire et al., RNA 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, N.J., 2004; Sohail, Gene Silencing by RNA Interference: Technology and Application, CRC, 2004.

EXAMPLES

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

Materials and Methods

Plant Materials and Culture Methods:

Jatropha curcas (L.) MD seeds were obtained from Indonesia. After removing the outer seed coat, seeds kernel were surface sterilized for 60 seconds with 75% (v/v) ethanol, following immersed in 10% (v/v) H.sub.2O.sub.2 for 1 h, then rinsed with sterile water for two times, finally immersed in sterile water overnight at 28.degree. C. in darkness. The endosperm-free embryos were germinated on hormone-free half-strength Murashige and Skoog salt (1/2 MS) medium (Murashige and Skoog, 1962) containing B5 Vitamins (Gamborg et al., 1968), 5 g/L sucrose, 0.5 g/L 2-(4-morpholino)ethanesulfonic acid (MES) and 2.2 g/L phytagel (Sigma), pH 5.6, with the radicals in contact with the medium, and cultured in a tissue culture room, at 25.degree. C..+-.2.degree. C. in a 16 h light (100 .mu.mol/m.sup.2S)/8 h dark cycle.

Media:

The media used in the present invention are as follows.

Medium I (Basal Medium):

MS major salts, MS minor salts and B5 vitamins, 10 mg/L citric acid, 150 mg/L glutamine, 100 mg/L casein enzymatic hydrolysate, 3% (w/v) sucrose, 0.5 g/L MgCl.sub.2 (only used in phytagel-containing medium) in combination with plant growth regulators was used. Medium I was adjusted to pH 5.8-6.0 with 1 N KOH, solidified with 2.5 g/L phytagel and autoclaved at 121.degree. C. for 20 min. All plant growth regulators were filter sterilized before being added to autoclaved medium.

Co-Cultivation Medium:

basal medium plus 20 mg/L acetosyringone (AS), 0.5 g/L MES, 1.5 mg/L 6-benzylaminopurine (6-BA) and 0.05 mg/L 1-naphthaleneacetic acid (NAA), pH 5.0-5.2.

Callus Formation Medium:

basal medium plus 1.5 mg/L 6-BA, 0.05 mg/L NAA, 3.5 mg/L hygromycin (hyg, A.G scientific, SanDiego, Calif.) as selective agent for plant transformation or 1 mg/L glufosinate ammonium (BASTA, Crescent Chemical, NY) and 100 mg/L cefotaxinme (Cef) for elimination of Agrobacteria cells.

Shoot Regeneration Medium I:

basal medium plus 1.5 mg/L 6-BA, 0.05 mg/L 3-indolebutyric acid (IBA), 2 mg/L adenine (adenine hemisulfate salt, SIGMA), 3.5 mg/L Hyg or 1 mg/L glufosinate ammonium and Cef 100 mg/L.

Shoot Regeneration Medium II:

basal medium plus 1.5 mg/L 6-BA, 0.05 mg/L IBA, 0.5 mg/L gibberellic acid (GA.sub.3), 4 mg/L Hyg or 1 mg/L glufosinate ammonium and 100 mg/L Cef 100, change phytagel to 7 g/L agar.

Shoot Regeneration Medium III:

basal medium plus 1.5 mg/L 6-BA, 0.05 mg/L IBA, 3.5 mg/L Hyg or 1 mg/L glufosinate ammonium and 100 mg/L Cef.

Shoot Elongation Medium:

basal medium plus 0.3 mg/L 6-BA, 0.1 mg/L GA.sub.3, change phytogel to 7 g/L agar.

Rooting Medium:

MS major salts, MS minor salts and B5 vitamins, 3% sucrose, 0.5 g/L MES, 0.07 mg/L IBA, 2.2 g/L phytagel, pH5.6.

Medium II:

liquid MS medium supplemented with 10 g/L glucose, 0.5 g/L MES, 20 mg/L AS, 1.5 mg/L 6-BA, 0.1 mg/L NAA, pH 5.0-5.2.

RNA Extraction and Analysis:

Fresh leaf or seed tissue (100 mg) was ground in liquid nitrogen and extracted with plant RNA purification reagent (Invitrogen). RNA concentration was measured by Nanodrop (Thermo, USA). DNase treatment and reverse transcription (RT) reaction were performed as described (Qu et al., 2007).

Agrobacterium Strain and Vectors:

J. curcas WRINKLE1 (JcWRI1) and DGAT1 sequences were identified by sequencing a Jatropha seed cDNA library. The JcWRI1 full-length cDNA was amplified from J. curcas seed first stranded cDNA product with two primers 5'-AATCGGATCCTAATGAAGAGGTCTTCTGCT-3' (SEQ ID NO:1) and 5'-TCATGTTAATT AATCAAACAGAATAGTTACAAGAAA-3' (SEQ ID NO:2) (underlined nts are enzyme recognition sites). The PCR product was further inserted into the pBA002-MYC vector treated with the BamHI and PacI to form pBA002-MYC-JcWRI1. The JcDGAT1 full-length cDNA was amplified from J. curcas seed first stranded cDNA product with two primers 5'-CAATATCTAGACCATGACGATTTTGGAGACCACT-3' (SEQ ID NO:3) and 5'-TATTAGATCTGGTCTTAATTCAGCATTGCC-3' (SEQ ID NO:4) (underlined nts are enzyme recognition sites). The PCR product was further inserted into the pBA002-HA vector treated with XbaI and BamHI to form pBA002-JcDGAT1-HA. The RcFAH12 full-length cDNA was amplified from castor bean seed first stranded cDNA product with two primers: 5'-CAATATCTAGACCATGGGAGGTGGTGGTC-3' (SEQ ID NO:5) and 5'-TGTAGGATCCGGATACTTGTTCCGGTACCAG-3' (SEQ ID NO:6) (underlined nts are enzyme recognition sites). The PCR product was further inserted into the pBA002-HA vector treated with XbaI and BamHI to form pBA002-RcFAH12-HA. Vectors were introduced into Agrobacterium strain AGL1 by electroporation (BIO-RAD, CA, USA). Transformed Agrobacterium cells were used to inoculate liquid LB medium supplemented with 50 mg/L kanamycin (for pCAMBIA 1300-GFP) or 50 mg/L spectimycin (for pBA002-MYC-WRI1, pBA002-JcDGAT1-HA, pBA002-RcFAH12-HA) and 100 mg/L carbicillin and were grown overnight at 28.degree. C., 250 rpm to a final OD.sub.595=0.7-1. Agrobacterium cells were collected by centrifugation at 4200 rpm for 10 min at 20.degree. C. The cell pellet was re-suspended with Medium II and adjusted to an OD.sub.595 of 0.25-0.35 (only Agrobacterium AGL1) prior to co-cultivation.

Isolation of DNA from J. curcas Leaves and Genotype Analysis:

Fifty mg of fresh J. curcas leaves were disrupted in liquid nitrogen and incubated at 65.degree. C. for one hour after addition of 400 .mu.L CTAB extraction buffer (100 mM Tris, pH 8.0; 1.4 M NaCl; 20 mM EDTA; 2% cetyl trimethylammonium bromide (CTAB)). After two times extraction with chilled-chloroform, DNA was precipitated with isoprepanol and collected by centrifugation. For hygromycin gene genotyping, the primers used were hyg5: 5'-CGATGTAGGAGGGCGTGG-3' (SEQ ID NO:7), hyg3: 5'-ACTTCTACACAGCCATCGGT CC-3' (SEQ ID NO:8). For bar gene genotyping, the primers used were bar5: 5'-GTCTGCAC CATCGTCAACC-3' (SEQ ID NO:9), bar3: 5'-GAAGTCCAGCTGCCAGAAAC-3' (SEQ ID NO:10).

Antibodies and Protein Gel Blot Analysis:

Curcin protein antibody was prepared by Dr. Yin Zhongcao's lab. Western blot analysis was performed as previously described (Qu et al., 2007). Total plant proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ECL peroxidase conjugated donkey anti-rabbit immunoglobulin G was used as a secondary antibody. Immunoreactive bands were visualized using ECL Western blotting Detection Reagents (GE healthcare).

Example 2

J. curcas Cotyledon Explant Transformation

FIG. 1 illustrates the Agrobacterium-mediated Jatropha transformation method as set forth in further detail in this Example. FIG. 2 illustrates the Agrobacterium transformation vectors that were used in this Example.

Co-Cultivation:

Cotyledons from 5-7 day old seedlings (Example 1; FIG. 3A) were cut into small pieces (5.times.5 mm) and incubated with Agrobacterium cells (Example 1) harboring the target expression cassette in 20 ml of medium II for 10-20 min at 25.degree. C. Explants were then transferred to the co-cultivation medium for 2-3 days at 22.degree. C. in the dark. Following co-cultivation, explants were rinsed several times with sterile water, following one wash with 300 mg/L cefotaxine. Cotyledon tissues were blotted dry by putting them on a pad of sterilized paper to remove excess surface water.

Selection of Hygromycin-Resistant or Glufosinate Ammonium-Resistant Calli:

After co-cultivation, the explants were plated onto the callus formation medium plate and transferred to darkness at 25.degree. C..+-.1.degree. C. for three weeks. The nontransformed and transformed explants form callus tissue (FIG. 3B) and some form callus upon culturing (FIG. 3B, right panel; FIG. 3C). Nontransformed explants normally will turn brown when cultured in the dark.

Shoot Regeneration:

Explants with newly emerged hygromycin-resistant or glufosinate ammonium-resistant callus were transferred onto the shoot regeneration medium I for 3 weeks at 25.degree. C. with 16 h light (100 .mu.mol/m.sup.2S.sup.1)/8 h dark cycles. The methods described here are based on direct shoot induction from transformed callus by adding adenine. While the term "regeneration" is used here to describe the re-creation of a whole plant from such transformed calli. Although 6-BA (6-benzyladenine) has similar effect on shoot regeneration, it can not be used in this special step during the methods described here. Furthermore, higher or lower concentration, early adding or later adding adenine will make the shoot regeneration more difficult or unnormal shooting. In an alternative embodiment, the method for obtaining shoot regeneration involves adding 2 mg/L plus normal 6-BA or other adenine derivative such as 2-isopentenyl adenine. During this period, any shoots regenerated from calli (about 40-50%) were transferred to the shoot regeneration medium II (FIG. 3D). Calli with no regenerated shoots were transferred to the shoot regeneration medium III for further culturing and regeneration of shoots.

Shoot Elongation:

After 4 weeks, regenerated shoots were transferred onto shoot elongation medium for elongation and bud multiplication (FIG. 3E).

Rooting:

The elongated shoots about 2.5 cm in length were rooted in rooting medium (FIG. 3F). Normally it takes more than one month to get roots such as shown in FIG. 3F. Our rooting protocol can provide high rooting efficacy about 45% (FIG. 3G) and one main root length longer than 10 mm can be successfully transferred into soil and get more than 90% live (FIG. 3H).

Grafting:

Elongated, transgenic shoots can also be used as scions for grafting onto non-transgenic root stocks. Healthy and vigorously growing J. curcas plants were chosen to be rootstocks. Both scions and rootstocks were cut into the cambium region so that phloem tissues from both will connect after joining. The graft joint was wrapped with parafilm and secured by a tape. Grafted J. curcas plants were maintained under low light intensity (28.degree. C. with 16 h light (50 .mu.mol/m.sup.2S.sup.1)/8 h dark cycles) and 85% humidity for 7 days. Transgenic J. curcas shoots grafted onto non-transgenic root stock are shown in FIGS. 3I and 3J. Transgenic J. curcas plant showed normal flowering and seeding in greenhouse (FIG. 3K).

Example 3

Transformation and Analysis of Transgenic J. curcas

Examples of Jatropha transformation and regeneration of BASTA or hygmycin plants from the transformed cells using the method of the present invention are detailed below. Briefly, the method requires that a heterologous DNA construct comprising a plant promoter, a DNA sequence encoding a protein that confers a selective advantage, such as BASTA or hygromycin tolerance, and a 3' untranslated transcriptional terminator region be provided. The DNA constructs comprise a plant promoter operably connected to a DNA coding region encoding a protein that confers BASTA or hygromycin tolerance, and a 3' termination signal. Preferably, the DNA construct encodes an additional gene of interest. For example, the DNA construct may include a gene the expression of which results in increased yields or altered fatty acid content in transformed plants.

In the example below, hygromycin tolerant Jatropha plants expressing green fluorescent protein (GFP) were obtained from tissue that was transformed with DNA constructs that included a GFP gene. This GFP gene and other genes such as GUS, luciferase gene, which can serve as easily screenable markers, were used in some of the examples described below, simply because their phenotypes can be readily detected in the transformed plants. It is reasonable to expect that by using DNA constructs created by standard molecular biological techniques, the present invention may be employed to obtain a Jatropha plants expressing virtually any other gene. In an alternative embodiment, the method for obtaining transformed Jatropha plants involves the cotransformation of two DNA constructs, one of which comprises a selectable marker, such as a BASTA or hygromycin tolerance marker, and the other of which comprises a gene of interest.

The transformation and shoot regeneration of hygromycin resistant putative GFP transgenic Jatropha plants was accomplished according to the method described in Example 2. Genomic DNA of hygromycin resistant shoots was extracted with the method described in Example 1. Genotyping was performed with the hygromycin gene primer pair (SEQ ID NO:7 and SEQ ID NO:8). Nine of 10 events were PCR positive, while the non-transformation control shows no band in the CK lane (FIG. 4). GFP expression was fast screened when transgenic Jatropha roots were excited with ultraviolet light (FIG. 3B). The fluorescence indicated that this new introduced GFP expression cassette was expressed in T.sub.0 Jatropha plants. After ubi:GFP transgenic Jatropha flowering, we checked the GFP expression in the inflorescence. The male flower, especially the pollen has some weak green fluoresce (FIG. 5H). We also checked GFP expression in the 3 week after fertilization seeds. Strong GFP expression can be seen in the whole transgenic T.sub.1 seed both from outside (FIG. 5N, FIG. 5O) or inside (FIG. 5P). This indicates GFP also express well in the progeny seeds in transgenic Jatropha.

Triacylglycerols (TAG) is the main energy storage form after the plant converts solar energy into chemical energy. But the standard biochemical route for its synthesis was thought to be quite wasteful when plants use a variation of glycolysis as an intermediate. WRINKLED1 (WRI1), a transcription factor of the AP2/EREB family, has an impact on more specific aspects of the seed storage process especially transcriptional control conversion of sugar variants into TAG, and therefore, shows a very important role in control seed oil content. Expression of the Arabidopsis WRI1 cDNA under the control of the cauliflower mosaic virus 35S-promoter led to increased 10-20% seed oil content. Moreover, the ectopic expression of the WRINKLED1 cDNA caused the accumulation of triacylglycerols in developing seedlings (Cernac and Benning, 2004). We proposed that ectopic expression of Jatropha WRI1 gene in Jatropha would lead to higher oil content. In addition, the transgenic seedling can develop into embryos or embryo-like organ producing oil when fed with sugars, just like a lipid reactor that can be supplied with sugar-containing liquid substrate for the constitutive CaMV 35S promoter-driven to WRI1 strong expression in vegetative organs.

We cloned the full-length cDNA of Jatropha WRI1 (JcWRI1) PCR amplified from Jatropha seed RT-PCR products using PCR primers (SEQ ID NO:1 and SEQ ID NO:2) for the JcWRI1 clone sequence derived from the Jatropha seeds cDNA library sequencing. The full length JcWRI1 cDNA sequence is set forth in SEQ ID NO:11. The overexpression vector (pBA002-MYC-JcWRI1) having the JcWRI1 cDNA under the control of CaMV 35S promoter was constructed and transformed into the Agrobacterium AGL1 strain. The proposed 6.times.MYC tag fusion WRI1 could be detected with MYC tag antibody. Transformation and shoot regeneration of BASTA resistant putative JcWRI1 overexpression transgenic Jatropha plants was accomplished according to the method described in Example 2. Genomic DNA of hygmycin resistant shoots was extracted with the method described in Example 1. Genotyping was performed with the BASTA gene primer pair (SEQ ID NO:9 and SEQ ID NO:10). All events we tested were PCR positive, while the non-transformation control showed no band in the CK (FIG. 6).

Plant and animal diacylglycerol acyltransferases (DGAT) are responsible for packaging of nascent fatty acids into TAGs, which subsequently accumulate in oil bodies that bud off from the endoplasmic reticulum. Plant type 1 DGAT (DGAT1) genes have been shown to contribute significantly to seed oil content, both by overexpression and through mutational downregulation studies (Zou et al., 2999; Jako et al., 2001). We proposed that ectopic expression of Jatropha DGAT1 gene in Jatropha would leads to higher level oil content.

We cloned the full-length Jatropha DGAT1 cDNA from Jatropha seed RT-PCR products using PCR primers (SEQ ID NO:3 and SEQ ID NO:4) according to the DGAT1 clone sequence. The full length JcDGAT1 cDNA sequence is set forth in SEQ ID NO:13. The overexpression vector (pBA002-JcDGAT1-HA) having the JcDGAT1 cDNA under the control of CaMV 35S promoter was constructed and transformed into the Agrobacterium AGL1 strain. The proposed 3.times.HA tag fusion DGAT1 could be detected with HA tag antibody. Transformation and shoot regeneration of BASTA resistant putative JcDGAT1 transgenic Jatropha plants was accomplished according to the method described in Example 2. 35S-JcDGAT1 expression was proved by Western blot based on HA-antibody with the method describing in Example 1 (FIG. 7). HA-specific band can be seen in two lanes out of three transgenic Jatropha lines.

Plant oils (and their derivatives) can be used in numerous cases and applications for plant-derived industrial feedstocks. When compared with non-renewable petroleum, the renewable nature making them especially attractive for many industrial applications for total loss applications where environmental concerns are an issue. Castor (Ricinus communis) oil has numerous applications in transportation, cosmetics and pharmaceuticals, and manufacturing industries. Castor oil contains more than 90% ricinoleic acid, which is a monounsaturated, 18-carbon fatty acid. It is unusual in that it has a hydroxyl functional group on the twelfth carbon. This functional group causes ricinoleic acid (and castor oil) to be unusually polar (http colon en dot wikipedia dot org slash wiki slash Castor_oil). One specific enzyme: fatty acid hydroxylase 12 (FAH12) is responsible for adding the hydroxyl group instead of normal FAD2 function to introduce unsaturated band on the twelfth carbon (van de Loo et al., 1995). Compared to other seed oils which lack the hydroxyl group, castor oil demands a higher price. Despite a widespread demand for castor oil, however, cultivation of this crop is restricted due to the presence of a toxin (ricin) and allergenic proteins, and thus the cost of castor oil is relatively high. Transgenic exogenous FAH12 can produce hydroxyl-castor oil in Arabidopsis seeds (Lu et al., 2006). We proposed that ectopic expression of castor bean FAH12 gene in Jatropha would lead to the production of castor oil.

We cloned the full-length castor bean FAH12 cDNA (RcFAH12) from castor bean seed RT-PCR products using PCR primers (SEQ ID NO:5 and SEQ ID NO:6) according to the FAH12 CDS sequence. The full length RcFAH12 cDNA sequence is set forth in SEQ ID NO:15. The overexpression vector (pBA002-RcFAH12-HA) having the RcFAH12 cDNA under the control of CaMV 35S promoter was constructed and transformed into the Agrobacterium AGL1 strain. The proposed 3.times.HA tag fusion RcFAH12 could be detected with HA tag antibody. Transformation and shoot regeneration of BASTA resistant putative RcFAH12 transgenic Jatropha plants was accomplished according to the method described in Example 2. 35S-RcFAH12 expression was proved by Western blot based on HA-antibody with the method describing in Example 1. HA-specific band can be seen in 5 lanes out of 7 transgenic Jatropha lines (FIG. 7). Two lines, #2 and #5, have very high FAH12-HA fusion expression protein level.

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., "such 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.

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.

BIBLIOGRAPHY

Cernac, A. and Benning, C. (2004). WRINKLED1 encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis. Plant J 40:575-585. Gamborg, O. L. et al. (1968). Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151-158. Jako, C. et al. (2001). Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. Plant Physiol 126:861-874. Jones, N. M. J. (1991). Jatropha curcas--a multipurpose species for problematic sites. Land Resources Series 1:1-12. Li, M. L. H. et al. (2008). Establishment of an Agrobacterium-mediated cotyledon disc transformation method for Jatropha curcas. Plant Cell Tiss Org Cult 92:173-181. Lu, C. et al. (2006). A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis. Plant J 45:847-856. Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473-497. Narayana, D. S. A et al. (2007). Distinct Begomoviruses Closely Related to Cassava Mosaic Viruses cause Indian Jatropha Mosaic Disease. Int'l J Virol 3:1-11. Qu, J. et al. (2007). Artificial microRNA-mediated virus resistance in plants. J Virol 81:6690-6699. Sujatha, M. et al. (2008). Role of biotechnological interventions in the improvement of castor (Ricinus communis L.) and Jatropha curcas L. Biotechnol Adv 26:424-435. van de Loo, F. J. et al. (1995). An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog. Proc Natl Acad Sci USA 92:6743-6747. Zou, J. et al. (1999). The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene. Plant J 19:645-653.

SEQUENCE LISTINGS

1

16130DNAJatropha curcas 1aatcggatcc taatgaagag gtcttctgct 30236DNAJatropha curcas 2tcatgttaat taatcaaaca gaatagttac aagaaa 36334DNAJatropha curcas 3caatatctag accatgacga ttttggagac cact 34430DNAJatropha curcas 4tattagatct ggtcttaatt cagcattgcc 30529DNARicinus communis 5caatatctag accatgggag gtggtggtc 29631DNARicinus communis 6tgtaggatcc ggatacttgt tccggtacca g 31718DNAEscherichia coli 7cgatgtagga gggcgtgg 18822DNAEscherichia coli 8acttctacac agccatcggt cc 22919DNAStreptomyces hygroscopicus 9gtctgcacca tcgtcaacc 191020DNAStreptomyces hygroscopicus 10gaagtccagc tgccagaaac 20111251DNAJatropha curcasCDS(1)..(1251)misc_feature(518)..(518)n is a, c, g, or t 11atg aag agg tct tct gct tca tct tgc tct tct tct tct tct tct tct 48Met Lys Arg Ser Ser Ala Ser Ser Cys Ser Ser Ser Ser Ser Ser Ser1 5 10 15tct tct cca tcc tct tct tcg tct tct gct tgt tct gct tcg tct tct 96Ser Ser Pro Ser Ser Ser Ser Ser Ser Ala Cys Ser Ala Ser Ser Ser 20 25 30tgc tta gat tca gta tct cct cct aat cac cat caa tta cga tca gag 144Cys Leu Asp Ser Val Ser Pro Pro Asn His His Gln Leu Arg Ser Glu 35 40 45aaa tca aaa tcc aaa cgc att cga aaa att caa acc aag caa gat aaa 192Lys Ser Lys Ser Lys Arg Ile Arg Lys Ile Gln Thr Lys Gln Asp Lys 50 55 60tgt cag act aca gct act acc acc agt cca agc ggc ggc ggt agg aga 240Cys Gln Thr Thr Ala Thr Thr Thr Ser Pro Ser Gly Gly Gly Arg Arg65 70 75 80agc tcc att tac aga gga gtc acc cgg cat aga tgg act gga agg ttt 288Ser Ser Ile Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg Phe 85 90 95gaa gct cat ctt tgg gat aag agt tct tgg aat aac att caa aac aag 336Glu Ala His Leu Trp Asp Lys Ser Ser Trp Asn Asn Ile Gln Asn Lys 100 105 110aaa gga agg caa gtt tat ttg ggg gct tac gac aat gag gaa gca gct 384Lys Gly Arg Gln Val Tyr Leu Gly Ala Tyr Asp Asn Glu Glu Ala Ala 115 120 125gct cat acc tat gat ctt gct gct ctc aag tac tgg gga caa gac acc 432Ala His Thr Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly Gln Asp Thr 130 135 140act ttg aat ttt ccg ata gag aca tac tca aag gag ctt gaa gag atg 480Thr Leu Asn Phe Pro Ile Glu Thr Tyr Ser Lys Glu Leu Glu Glu Met145 150 155 160caa aag atg agc aag gaa gag tac tta gca tct ctt cna cgg aga agc 528Gln Lys Met Ser Lys Glu Glu Tyr Leu Ala Ser Leu Xaa Arg Arg Ser 165 170 175agt gga ttt tca aga gga gtt tct aag tac cgg gga gta gct agg cat 576Ser Gly Phe Ser Arg Gly Val Ser Lys Tyr Arg Gly Val Ala Arg His 180 185 190cat cac aat ggc cgg tgg gaa gct cga att ggc cgg gtt ttt ggc aat 624His His Asn Gly Arg Trp Glu Ala Arg Ile Gly Arg Val Phe Gly Asn 195 200 205aag tat ctc tac ctc gga act tac aat aca caa gaa gag gca gca gca 672Lys Tyr Leu Tyr Leu Gly Thr Tyr Asn Thr Gln Glu Glu Ala Ala Ala 210 215 220tat gat atg gca gca ata gag tac aga gga gca aat gca gta acc aat 720Tyr Asp Met Ala Ala Ile Glu Tyr Arg Gly Ala Asn Ala Val Thr Asn225 230 235 240ttt gat gtc agc cat tac ata gac cgt ttg aag aag aaa ggc att cct 768Phe Asp Val Ser His Tyr Ile Asp Arg Leu Lys Lys Lys Gly Ile Pro 245 250 255tta gat aaa atc cta cca gaa acn ctt tct aaa ggc tca aaa gag tca 816Leu Asp Lys Ile Leu Pro Glu Thr Leu Ser Lys Gly Ser Lys Glu Ser 260 265 270gaa gaa atc gag cga acc tca ccc tta ccg ttg cca tca cca cca tca 864Glu Glu Ile Glu Arg Thr Ser Pro Leu Pro Leu Pro Ser Pro Pro Ser 275 280 285cca tca ata aca cca tta cac gaa gaa ata gtc tca cca cag ctg ctt 912Pro Ser Ile Thr Pro Leu His Glu Glu Ile Val Ser Pro Gln Leu Leu 290 295 300gaa act gaa tgc cca caa cat cct cca tgt atg gat act tgt act atg 960Glu Thr Glu Cys Pro Gln His Pro Pro Cys Met Asp Thr Cys Thr Met305 310 315 320atc gtt atg gac cct ata gaa gag cac gag ctt act tgg agc ttc tgt 1008Ile Val Met Asp Pro Ile Glu Glu His Glu Leu Thr Trp Ser Phe Cys 325 330 335ctc gat tcg ggg tta gtt ccg ctc cct gtg cct gac cta cca cta gca 1056Leu Asp Ser Gly Leu Val Pro Leu Pro Val Pro Asp Leu Pro Leu Ala 340 345 350aat ggc tgt gag tta cca gac ttg ttg gat gac aca ggc ttt gaa gac 1104Asn Gly Cys Glu Leu Pro Asp Leu Leu Asp Asp Thr Gly Phe Glu Asp 355 360 365aat att gac ttg ata ttt gat gct tgt tgc ttc gga aat gat gcc aac 1152Asn Ile Asp Leu Ile Phe Asp Ala Cys Cys Phe Gly Asn Asp Ala Asn 370 375 380cct gca gat gag aat ggg aaa gag agg ttg tct tcc gct tca act tct 1200Pro Ala Asp Glu Asn Gly Lys Glu Arg Leu Ser Ser Ala Ser Thr Ser385 390 395 400cca tct tgt tcc aca aca tta act tct gtt tct tgt aac tat tct gtt 1248Pro Ser Cys Ser Thr Thr Leu Thr Ser Val Ser Cys Asn Tyr Ser Val 405 410 415tga 125112416PRTJatropha curcasmisc_feature(173)..(173)The 'Xaa' at location 173 stands for Gln, Arg, Pro, or Leu. 12Met Lys Arg Ser Ser Ala Ser Ser Cys Ser Ser Ser Ser Ser Ser Ser1 5 10 15Ser Ser Pro Ser Ser Ser Ser Ser Ser Ala Cys Ser Ala Ser Ser Ser 20 25 30Cys Leu Asp Ser Val Ser Pro Pro Asn His His Gln Leu Arg Ser Glu 35 40 45Lys Ser Lys Ser Lys Arg Ile Arg Lys Ile Gln Thr Lys Gln Asp Lys 50 55 60Cys Gln Thr Thr Ala Thr Thr Thr Ser Pro Ser Gly Gly Gly Arg Arg65 70 75 80Ser Ser Ile Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg Phe 85 90 95Glu Ala His Leu Trp Asp Lys Ser Ser Trp Asn Asn Ile Gln Asn Lys 100 105 110Lys Gly Arg Gln Val Tyr Leu Gly Ala Tyr Asp Asn Glu Glu Ala Ala 115 120 125Ala His Thr Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly Gln Asp Thr 130 135 140Thr Leu Asn Phe Pro Ile Glu Thr Tyr Ser Lys Glu Leu Glu Glu Met145 150 155 160Gln Lys Met Ser Lys Glu Glu Tyr Leu Ala Ser Leu Xaa Arg Arg Ser 165 170 175Ser Gly Phe Ser Arg Gly Val Ser Lys Tyr Arg Gly Val Ala Arg His 180 185 190His His Asn Gly Arg Trp Glu Ala Arg Ile Gly Arg Val Phe Gly Asn 195 200 205Lys Tyr Leu Tyr Leu Gly Thr Tyr Asn Thr Gln Glu Glu Ala Ala Ala 210 215 220Tyr Asp Met Ala Ala Ile Glu Tyr Arg Gly Ala Asn Ala Val Thr Asn225 230 235 240Phe Asp Val Ser His Tyr Ile Asp Arg Leu Lys Lys Lys Gly Ile Pro 245 250 255Leu Asp Lys Ile Leu Pro Glu Thr Leu Ser Lys Gly Ser Lys Glu Ser 260 265 270Glu Glu Ile Glu Arg Thr Ser Pro Leu Pro Leu Pro Ser Pro Pro Ser 275 280 285Pro Ser Ile Thr Pro Leu His Glu Glu Ile Val Ser Pro Gln Leu Leu 290 295 300Glu Thr Glu Cys Pro Gln His Pro Pro Cys Met Asp Thr Cys Thr Met305 310 315 320Ile Val Met Asp Pro Ile Glu Glu His Glu Leu Thr Trp Ser Phe Cys 325 330 335Leu Asp Ser Gly Leu Val Pro Leu Pro Val Pro Asp Leu Pro Leu Ala 340 345 350Asn Gly Cys Glu Leu Pro Asp Leu Leu Asp Asp Thr Gly Phe Glu Asp 355 360 365Asn Ile Asp Leu Ile Phe Asp Ala Cys Cys Phe Gly Asn Asp Ala Asn 370 375 380Pro Ala Asp Glu Asn Gly Lys Glu Arg Leu Ser Ser Ala Ser Thr Ser385 390 395 400Pro Ser Cys Ser Thr Thr Leu Thr Ser Val Ser Cys Asn Tyr Ser Val 405 410 415131566DNAJatropha curcasCDS(1)..(1566) 13atg acg att ttg gag acc act act agc gga ggt gat ggt gtt gct gag 48Met Thr Ile Leu Glu Thr Thr Thr Ser Gly Gly Asp Gly Val Ala Glu1 5 10 15tcg tct tcc gat ctt aac gta tcg ctt cga cgg aga cgg aaa ggc acc 96Ser Ser Ser Asp Leu Asn Val Ser Leu Arg Arg Arg Arg Lys Gly Thr 20 25 30agc tcg gat gga gct ttg ccg gaa ttg act tcg aat att gtt gaa ttg 144Ser Ser Asp Gly Ala Leu Pro Glu Leu Thr Ser Asn Ile Val Glu Leu 35 40 45gaa tct gaa agc ggt ggc cag gtg atg atg gat cca ggt gtg gtg acg 192Glu Ser Glu Ser Gly Gly Gln Val Met Met Asp Pro Gly Val Val Thr 50 55 60gaa ccg gag aca gag aaa att aat gga aaa gat tgc ggc ggt gac aag 240Glu Pro Glu Thr Glu Lys Ile Asn Gly Lys Asp Cys Gly Gly Asp Lys65 70 75 80gat aag att gac aat cgc gag aat cgt ggg agg tcg gat att aaa ttc 288Asp Lys Ile Asp Asn Arg Glu Asn Arg Gly Arg Ser Asp Ile Lys Phe 85 90 95acg tac cgg cca tcg gtg ccg gct cat cga gcg ctc agg gag agt ccg 336Thr Tyr Arg Pro Ser Val Pro Ala His Arg Ala Leu Arg Glu Ser Pro 100 105 110ctt agc tct gat gct ata ttt aaa caa agt cat gca ggt ctg ttc aac 384Leu Ser Ser Asp Ala Ile Phe Lys Gln Ser His Ala Gly Leu Phe Asn 115 120 125ctc tgt ata gta gtg ctt gtt gct gtt aac agc agg ctt atc att gaa 432Leu Cys Ile Val Val Leu Val Ala Val Asn Ser Arg Leu Ile Ile Glu 130 135 140aat cta atg aag tac ggt tgg tta att aaa acg ggg ttt tgg ttt agt 480Asn Leu Met Lys Tyr Gly Trp Leu Ile Lys Thr Gly Phe Trp Phe Ser145 150 155 160tca aga tcg ttg aga gat tgg ccc ctt ctt atg tgc tgt ctt acc ctc 528Ser Arg Ser Leu Arg Asp Trp Pro Leu Leu Met Cys Cys Leu Thr Leu 165 170 175cct ata ttc tct ctt gcc gcc tat cta gtt gag aag ttg gca tat cga 576Pro Ile Phe Ser Leu Ala Ala Tyr Leu Val Glu Lys Leu Ala Tyr Arg 180 185 190aaa tat ata tct gca cct att gtt att ttc ttt cat atg ctc att acc 624Lys Tyr Ile Ser Ala Pro Ile Val Ile Phe Phe His Met Leu Ile Thr 195 200 205aca aca gca gtt ttg tac cca gtt tct gtg att ctc agt tgt ggg tct 672Thr Thr Ala Val Leu Tyr Pro Val Ser Val Ile Leu Ser Cys Gly Ser 210 215 220gct gtt ctg tct ggt gtt gca ttg atg ctc ttt gct tgt atc gtg tgg 720Ala Val Leu Ser Gly Val Ala Leu Met Leu Phe Ala Cys Ile Val Trp225 230 235 240ttg aaa tta gta tct tat gca cat aca aac tat gac atg aga gcc att 768Leu Lys Leu Val Ser Tyr Ala His Thr Asn Tyr Asp Met Arg Ala Ile 245 250 255gcc aac tca gct gac aag gga gat gca cta tcc gat act tca ggt gca 816Ala Asn Ser Ala Asp Lys Gly Asp Ala Leu Ser Asp Thr Ser Gly Ala 260 265 270gat tct tca cgt gat gtt agc ttc aag agt ttg gtc tac ttc atg gtt 864Asp Ser Ser Arg Asp Val Ser Phe Lys Ser Leu Val Tyr Phe Met Val 275 280 285gct cct acg cta tgt tac cag cca agt tat cct cga aca gat tca gtt 912Ala Pro Thr Leu Cys Tyr Gln Pro Ser Tyr Pro Arg Thr Asp Ser Val 290 295 300aga aag ggt tgg gtg gtt cgt caa ttt gtc aag tta ata ata ttt aca 960Arg Lys Gly Trp Val Val Arg Gln Phe Val Lys Leu Ile Ile Phe Thr305 310 315 320gga ttc atg gga ttt atc ata gaa caa tat atc aat cct att gtc cag 1008Gly Phe Met Gly Phe Ile Ile Glu Gln Tyr Ile Asn Pro Ile Val Gln 325 330 335aat tca caa cat ccc tta aag ggg gat cta tta tat gcc att gaa agg 1056Asn Ser Gln His Pro Leu Lys Gly Asp Leu Leu Tyr Ala Ile Glu Arg 340 345 350gtt ttg aag ctc tca gtt cca aac tta tat gtg tgg ctt tgc atg ttc 1104Val Leu Lys Leu Ser Val Pro Asn Leu Tyr Val Trp Leu Cys Met Phe 355 360 365tac tgc ttt ttt cat cta tgg tta aat ata ctt gct gag ctc ctt cgg 1152Tyr Cys Phe Phe His Leu Trp Leu Asn Ile Leu Ala Glu Leu Leu Arg 370 375 380ttt ggt gac aga gag ttc tat aaa gat tgg tgg aat gca agg acc gtt 1200Phe Gly Asp Arg Glu Phe Tyr Lys Asp Trp Trp Asn Ala Arg Thr Val385 390 395 400gag gag tac tgg aga atg tgg aat atg cct gtt cat aag tgg atg gtt 1248Glu Glu Tyr Trp Arg Met Trp Asn Met Pro Val His Lys Trp Met Val 405 410 415cgc cat atc tac ttt cca tgc ttg cgg cat aaa ata cca agg ggg gta 1296Arg His Ile Tyr Phe Pro Cys Leu Arg His Lys Ile Pro Arg Gly Val 420 425 430gcc ttg tta att gct ttc ttc gtt tca gct gta ttt cat gag ttg tgc 1344Ala Leu Leu Ile Ala Phe Phe Val Ser Ala Val Phe His Glu Leu Cys 435 440 445att gct gtt cct tgc cac atg ttc aag ctc tgg gct ttt att gga att 1392Ile Ala Val Pro Cys His Met Phe Lys Leu Trp Ala Phe Ile Gly Ile 450 455 460atg ttt cag att cca ttg gtc ggg atc act aat tac ctc cag aac aag 1440Met Phe Gln Ile Pro Leu Val Gly Ile Thr Asn Tyr Leu Gln Asn Lys465 470 475 480ttc aga agc tcc atg gtg gga aat atg atc ttt tgg ttc att ttc tgc 1488Phe Arg Ser Ser Met Val Gly Asn Met Ile Phe Trp Phe Ile Phe Cys 485 490 495att ctt ggt caa ccc atg tgt gtg cta ttg tat tat cat gac cta atg 1536Ile Leu Gly Gln Pro Met Cys Val Leu Leu Tyr Tyr His Asp Leu Met 500 505 510aat cgg aaa ggc aat gct gaa tta aga tga 1566Asn Arg Lys Gly Asn Ala Glu Leu Arg 515 52014521PRTJatropha curcas 14Met Thr Ile Leu Glu Thr Thr Thr Ser Gly Gly Asp Gly Val Ala Glu1 5 10 15Ser Ser Ser Asp Leu Asn Val Ser Leu Arg Arg Arg Arg Lys Gly Thr 20 25 30Ser Ser Asp Gly Ala Leu Pro Glu Leu Thr Ser Asn Ile Val Glu Leu 35 40 45Glu Ser Glu Ser Gly Gly Gln Val Met Met Asp Pro Gly Val Val Thr 50 55 60Glu Pro Glu Thr Glu Lys Ile Asn Gly Lys Asp Cys Gly Gly Asp Lys65 70 75 80Asp Lys Ile Asp Asn Arg Glu Asn Arg Gly Arg Ser Asp Ile Lys Phe 85 90 95Thr Tyr Arg Pro Ser Val Pro Ala His Arg Ala Leu Arg Glu Ser Pro 100 105 110Leu Ser Ser Asp Ala Ile Phe Lys Gln Ser His Ala Gly Leu Phe Asn 115 120 125Leu Cys Ile Val Val Leu Val Ala Val Asn Ser Arg Leu Ile Ile Glu 130 135 140Asn Leu Met Lys Tyr Gly Trp Leu Ile Lys Thr Gly Phe Trp Phe Ser145 150 155 160Ser Arg Ser Leu Arg Asp Trp Pro Leu Leu Met Cys Cys Leu Thr Leu 165 170 175Pro Ile Phe Ser Leu Ala Ala Tyr Leu Val Glu Lys Leu Ala Tyr Arg 180 185 190Lys Tyr Ile Ser Ala Pro Ile Val Ile Phe Phe His Met Leu Ile Thr 195 200 205Thr Thr Ala Val Leu Tyr Pro Val Ser Val Ile Leu Ser Cys Gly Ser 210 215 220Ala Val Leu Ser Gly Val Ala Leu Met Leu Phe Ala Cys Ile Val Trp225 230 235 240Leu Lys Leu Val Ser Tyr Ala His Thr Asn Tyr Asp Met Arg Ala Ile 245 250 255Ala Asn Ser Ala Asp Lys Gly Asp Ala Leu Ser Asp Thr Ser Gly Ala 260 265 270Asp Ser Ser Arg Asp Val Ser Phe Lys Ser Leu Val Tyr Phe Met Val 275 280 285Ala Pro Thr Leu Cys Tyr Gln Pro Ser Tyr Pro Arg Thr Asp Ser Val 290 295 300Arg Lys Gly Trp Val Val Arg Gln Phe Val Lys Leu Ile Ile Phe Thr305 310 315 320Gly Phe Met Gly Phe Ile Ile Glu Gln Tyr Ile Asn Pro Ile Val Gln 325 330 335Asn Ser Gln His Pro Leu Lys Gly Asp Leu Leu Tyr Ala Ile Glu Arg 340 345 350Val Leu Lys Leu Ser Val Pro Asn Leu Tyr Val Trp Leu Cys Met Phe 355 360

365Tyr Cys Phe Phe His Leu Trp Leu Asn Ile Leu Ala Glu Leu Leu Arg 370 375 380Phe Gly Asp Arg Glu Phe Tyr Lys Asp Trp Trp Asn Ala Arg Thr Val385 390 395 400Glu Glu Tyr Trp Arg Met Trp Asn Met Pro Val His Lys Trp Met Val 405 410 415Arg His Ile Tyr Phe Pro Cys Leu Arg His Lys Ile Pro Arg Gly Val 420 425 430Ala Leu Leu Ile Ala Phe Phe Val Ser Ala Val Phe His Glu Leu Cys 435 440 445Ile Ala Val Pro Cys His Met Phe Lys Leu Trp Ala Phe Ile Gly Ile 450 455 460Met Phe Gln Ile Pro Leu Val Gly Ile Thr Asn Tyr Leu Gln Asn Lys465 470 475 480Phe Arg Ser Ser Met Val Gly Asn Met Ile Phe Trp Phe Ile Phe Cys 485 490 495Ile Leu Gly Gln Pro Met Cys Val Leu Leu Tyr Tyr His Asp Leu Met 500 505 510Asn Arg Lys Gly Asn Ala Glu Leu Arg 515 520151164DNARicinus communisCDS(1)..(1164) 15atg gga ggt ggt ggt cgc atg tct act gtc ata acc agc aac aac agt 48Met Gly Gly Gly Gly Arg Met Ser Thr Val Ile Thr Ser Asn Asn Ser1 5 10 15gag aag aaa gga gga agc agc cac ctt aag cga gcg ccg cac acg aag 96Glu Lys Lys Gly Gly Ser Ser His Leu Lys Arg Ala Pro His Thr Lys 20 25 30cct cct ttc aca ctt ggt gac ctc aag aga gcc atc cca ccc cat tgc 144Pro Pro Phe Thr Leu Gly Asp Leu Lys Arg Ala Ile Pro Pro His Cys 35 40 45ttt gaa cgc tct ttt gtg cgc tca ttc tcc tat gtt gcc tat gat gtc 192Phe Glu Arg Ser Phe Val Arg Ser Phe Ser Tyr Val Ala Tyr Asp Val 50 55 60tgc tta agt ttt ctt ttc tac tcg atc gcc acc aac ttc ttc cct tac 240Cys Leu Ser Phe Leu Phe Tyr Ser Ile Ala Thr Asn Phe Phe Pro Tyr65 70 75 80atc tct tct ccg ctc tcg tat gtc gct tgg ctg gtt tac tgg ctc ttc 288Ile Ser Ser Pro Leu Ser Tyr Val Ala Trp Leu Val Tyr Trp Leu Phe 85 90 95caa ggc tgc att ctc act ggt ctt tgg gtc atc ggc cat gaa tgt ggc 336Gln Gly Cys Ile Leu Thr Gly Leu Trp Val Ile Gly His Glu Cys Gly 100 105 110cat cat gct ttt agt gag tat cag ctg gct gat gac att gtt ggc cta 384His His Ala Phe Ser Glu Tyr Gln Leu Ala Asp Asp Ile Val Gly Leu 115 120 125att gtc cat tct gca ctt ctg gtt cca tat ttt tca tgg aaa tat agc 432Ile Val His Ser Ala Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser 130 135 140cat cgc cgc cac cat tct aac ata gga tct ctc gag cga gac gaa gtg 480His Arg Arg His His Ser Asn Ile Gly Ser Leu Glu Arg Asp Glu Val145 150 155 160ttc gtc ccg aaa tca aag tcg aaa att tca tgg tat tct aag tac tca 528Phe Val Pro Lys Ser Lys Ser Lys Ile Ser Trp Tyr Ser Lys Tyr Ser 165 170 175aac aac ccg cca ggt cga gtt ttg aca ctt gct gcc acg ctc ctc ctt 576Asn Asn Pro Pro Gly Arg Val Leu Thr Leu Ala Ala Thr Leu Leu Leu 180 185 190ggc tgg cct tta tac tta gct ttc aat gtc tct ggt aga cct tac gat 624Gly Trp Pro Leu Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp 195 200 205cgc ttt gct tgc cat tat gat ccc tat ggc cca ata ttt tcc gaa aga 672Arg Phe Ala Cys His Tyr Asp Pro Tyr Gly Pro Ile Phe Ser Glu Arg 210 215 220gaa agg ctt cag att tac att gct gac ctc gga atc ttt gcc aca acg 720Glu Arg Leu Gln Ile Tyr Ile Ala Asp Leu Gly Ile Phe Ala Thr Thr225 230 235 240ttt gtg ctt tat cag gct aca atg gca aaa ggg ttg gct tgg gta atg 768Phe Val Leu Tyr Gln Ala Thr Met Ala Lys Gly Leu Ala Trp Val Met 245 250 255cgt atc tat ggg gtg cca ttg ctt att gtt aac tgt ttc ctt gtt atg 816Arg Ile Tyr Gly Val Pro Leu Leu Ile Val Asn Cys Phe Leu Val Met 260 265 270atc aca tac ttg cag cac act cac cca gct att cca cgc tat ggc tca 864Ile Thr Tyr Leu Gln His Thr His Pro Ala Ile Pro Arg Tyr Gly Ser 275 280 285tcg gaa tgg gat tgg ctc cgg gga gca atg gtg act gtc gat aga gat 912Ser Glu Trp Asp Trp Leu Arg Gly Ala Met Val Thr Val Asp Arg Asp 290 295 300tat ggg gtg ttg aat aaa gta ttc cat aac att gca gac act cat gta 960Tyr Gly Val Leu Asn Lys Val Phe His Asn Ile Ala Asp Thr His Val305 310 315 320gct cat cat ctc ttt gct aca gtg cca cat tac cat gca atg gag gcc 1008Ala His His Leu Phe Ala Thr Val Pro His Tyr His Ala Met Glu Ala 325 330 335act aaa gca atc aag cct ata atg ggt gag tat tac cgg tat gat ggt 1056Thr Lys Ala Ile Lys Pro Ile Met Gly Glu Tyr Tyr Arg Tyr Asp Gly 340 345 350acc cca ttt tac aag gca ttg tgg agg gag gca aag gag tgc ttg ttc 1104Thr Pro Phe Tyr Lys Ala Leu Trp Arg Glu Ala Lys Glu Cys Leu Phe 355 360 365gtc gag cca gat gaa gga gct cct aca caa ggc gtt ttc tgg tac cgg 1152Val Glu Pro Asp Glu Gly Ala Pro Thr Gln Gly Val Phe Trp Tyr Arg 370 375 380aac aag tat taa 1164Asn Lys Tyr38516387PRTRicinus communis 16Met Gly Gly Gly Gly Arg Met Ser Thr Val Ile Thr Ser Asn Asn Ser1 5 10 15Glu Lys Lys Gly Gly Ser Ser His Leu Lys Arg Ala Pro His Thr Lys 20 25 30Pro Pro Phe Thr Leu Gly Asp Leu Lys Arg Ala Ile Pro Pro His Cys 35 40 45Phe Glu Arg Ser Phe Val Arg Ser Phe Ser Tyr Val Ala Tyr Asp Val 50 55 60Cys Leu Ser Phe Leu Phe Tyr Ser Ile Ala Thr Asn Phe Phe Pro Tyr65 70 75 80Ile Ser Ser Pro Leu Ser Tyr Val Ala Trp Leu Val Tyr Trp Leu Phe 85 90 95Gln Gly Cys Ile Leu Thr Gly Leu Trp Val Ile Gly His Glu Cys Gly 100 105 110His His Ala Phe Ser Glu Tyr Gln Leu Ala Asp Asp Ile Val Gly Leu 115 120 125Ile Val His Ser Ala Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser 130 135 140His Arg Arg His His Ser Asn Ile Gly Ser Leu Glu Arg Asp Glu Val145 150 155 160Phe Val Pro Lys Ser Lys Ser Lys Ile Ser Trp Tyr Ser Lys Tyr Ser 165 170 175Asn Asn Pro Pro Gly Arg Val Leu Thr Leu Ala Ala Thr Leu Leu Leu 180 185 190Gly Trp Pro Leu Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp 195 200 205Arg Phe Ala Cys His Tyr Asp Pro Tyr Gly Pro Ile Phe Ser Glu Arg 210 215 220Glu Arg Leu Gln Ile Tyr Ile Ala Asp Leu Gly Ile Phe Ala Thr Thr225 230 235 240Phe Val Leu Tyr Gln Ala Thr Met Ala Lys Gly Leu Ala Trp Val Met 245 250 255Arg Ile Tyr Gly Val Pro Leu Leu Ile Val Asn Cys Phe Leu Val Met 260 265 270Ile Thr Tyr Leu Gln His Thr His Pro Ala Ile Pro Arg Tyr Gly Ser 275 280 285Ser Glu Trp Asp Trp Leu Arg Gly Ala Met Val Thr Val Asp Arg Asp 290 295 300Tyr Gly Val Leu Asn Lys Val Phe His Asn Ile Ala Asp Thr His Val305 310 315 320Ala His His Leu Phe Ala Thr Val Pro His Tyr His Ala Met Glu Ala 325 330 335Thr Lys Ala Ile Lys Pro Ile Met Gly Glu Tyr Tyr Arg Tyr Asp Gly 340 345 350Thr Pro Phe Tyr Lys Ala Leu Trp Arg Glu Ala Lys Glu Cys Leu Phe 355 360 365Val Glu Pro Asp Glu Gly Ala Pro Thr Gln Gly Val Phe Trp Tyr Arg 370 375 380Asn Lys Tyr385

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