The Genetic Transformation of Jatropha curcas
Aurellia Whitmore Southern University at New Orleans & The Pennsylvania State University-Harrisburg The Phylogeny and Advantages of Jatropha Curcas Habitat: tropical & subtropical SEEDSAdvantagesare considered: anthelemintic in • GrowsBrazil. on marginal land LATEX(Pennsylvaniacontains has aan number alkaloid of abandonedknown as “Jatrophine” (having anti- mind landscancerous) properties.) BARK•Droughtyields resistance a dark blue dye used in the •Non foodPhilippines crop for coloring cloth, finishing nets and lines. Ranjan Parajuli “Jatropha Curcas and Its •30-40 % oil content in seed Potential Applications; A Compilation Paper on LEAVES are for fumigating houses Plantation and Application of Jatropha Curcas” •USED AS BIODIESEL http://www.environmental-http://gas2.org/2008/10/02/jatropha-from- against bed bugs, in Ghana. expert.com/Files/0/articles/73213/Jatropha.pdfhaitian-voodoo-to-biodiesel-holy-grail/ What is transformation and why is it important and helpful?
A desired (single) gene of interest Combines multiple genes Introduce foreign genes simultaneously Rapidly generate plants with improved Mendelian Genetics (same species) varieties Enhance market value of the plant
Challenge: Jatropha is susceptible to cold.
Transformation with CBF3 can make it cold resistant. 1 Proof of concept for cold and drought tolerance induced by
CBF3 demonstrated in Petunia and corn (Sairam et al, 2008)
Plant 1: After exposure to -6° C ( 22°F) Plant 2: 2 Watered to saturation No watering for 15 days 24 hours after watering
CBF NT Sairam R.V., Al-Abed, D., Johnson, J., Muzynski, M., Raab, M., Reddy, T.V. and Goldman, S.L. Maize. (2008) In: Kole, C., Hall, T.C. (eds) A Compendium of Transgenic Crop Plants 49-82. What are GUS, GFP, & CBF3 genes?
CBF3 A. Agrobacterium Cell Rd29 CBF3 A176 CBF3-A354 CBF3-350 B. Agrobacterium DNA CBF3A478 C. Ti Plasmid CBF3 A-648 CBF3 A779 D. Plant Cell CBF3 Second Exon CBF3A-952 E. Plant Mitochondria CBF3 A1099 F. Plant Chloroplast CBF3A-1256 CBF3 A1403 G. Plant Nucleus CBF3 A-1400 a. T-DNA CBF3 A-1551 CBF3 A-1701 b. Vir genes c. Replication origin
http://en.wikipedia.org/wiki/File:Transf ection_by_Agrobacterium.svg Procedures for Transformation
In vitro germinated Embryos leaves
Biolistic Agrobacterium-mediated transformation transformation Immerse in GUS, GFP and Coat gold with Plasmid DNA CBF3 cultures 20 mins. Load explants on Transfer explants to co- regeneration media. cultivation media. http://www.ag.ndsu.edu/pu Prepare gene gun. bs/plantsci/crops/a1219w.ht m Dark, 25° C, 72 hrs. FIRE! Procedure for GUS staining solution Transformation with GUS gene
• Explants: Embryo & Leaf
Explants from JAT: 1st picture- 6/17 Jat Embryo. 2nd picture- 7/22 Jat leaf invitro. Transformation with GFP EXPLANT: Leaf 7/22 Transformation with CBF3 Explant: Embryo
Explants from 6/17/11 JPB exp. PCR Results
Jatropha Jatropha Agrobacterium- Particle mediated BombardmentTransformation (GUS) (GFP)
• Gradient PCR with different concentrations into different wells.
Summary & Future Research
SUMMARY .GUS and GFP expression was positive in PCR results.
FUTURE RESEARCH .To perform molecular analysis for CBF3 .Develop Jatropha to be resistant to other abiotic and biotic stress. Industrial use of Jatropha Oil
On January 7, 2009 Continental Airlines successfully completed a test flight from Houston, Texas using a 50/50 mixture of algae/Jatropha oil-derived biofuel & http://phx.corporate-ir.net/phoenix.zhtml? traditional Jet fuel in one of the 2 engines. c=85779&p=irol-newsArticle&ID=1241576 Acknowledgements
• National Science Foundation (NSF) for their fellowship
• The Pennsylvania State- Harrisburg
• Ben Tabatabai (Mentor, tissue culture, transformation and procedures), Deepkamal Karelia(Biolistic Transformation)Mathew Ritzel (Molecular Analysis)
• Dr. Puthiyaparambil Josekutty, Dr. Sairam Rudrabhatla
• NSF-REU staff and colleagues
Work Cited
Continental Airlines Flight Demonstrates Use of Sustainable Biofuels as Energy Source for Jet Travelhttp://phx.corporate-ir.net/phoenix.zhtml?c=85779&p=irol-newsArticle&ID=1241576
Plant Transformation: Problems and strategies for Practical Application. R.G. Birch Department of Botany. The University of Queensland, Brisbane,4072, Australia
Sairam R.V., Al-Abed, D., Johnson, J., Muzynski, M., Raab, M., Reddy, T.V. and Goldman, S.L. Maize. (2008) In: Kole, C., Hall, T.C. (eds) A Compendium of Transgenic Crop Plants 49-82.
Agrobacterium-mediated transformation diagramhttp://en.wikipedia.org/wiki/File:Transfection_by_Agrobacterium.svg(Image)
Bayer Crop Science, Jatropha: From Hatain vodoo biodiesal holy grain. http://gas2.org/2008/10/02/jatropha-from-haitian-voodoo-to-biodiesel-holy- grail/ (Images) http://www.biodiesel.org/pdf_files/fuelfactsheets/emissions.pdf http://www.environmental-expert.com/Files/0/articles/73213/Jatropha.pdf