The Genetic Transformation

of Jatropha curcas

Aurellia WhitmoreSouthern University at New Orleans

&The Pennsylvania State University-Harrisburg

The Phylogeny and

Advantages of Jatropha Curcas

Habitat: tropical & subtropical Advantages:• Grows on marginal land (Pennsylvania has a number of abandoned

mind lands)•Drought resistance•Non food crop•30-40 % oil content in seed•USED AS BIODIESEL

http://gas2.org/2008/10/02/jatropha-from-haitian-voodoo-to-biodiesel-holy-grail/

SEEDS are considered anthelemintic in Brazil.

LATEX contains an alkaloid known as “Jatrophine” (having anti-cancerous properties.)

BARK yields a dark blue dye used in the Philippines for coloring cloth, finishing nets and lines.

LEAVES are for fumigating houses against bed bugs, in Ghana.

Ranjan Parajuli “Jatropha Curcas and Its Potential Applications; A Compilation Paper on Plantation and Application of Jatropha Curcas” http://www.environmental-expert.com/Files/0/articles/73213/Jatropha.pdf

What is transformation and why is it

important and helpful?

Challenge: Jatropha is susceptible to cold.

Transformation with CBF3 can make it cold resistant.

A desired (single) gene of interest

Introduce foreign genes

Rapidly generate plants with improved varieties

Enhance market value of the plant

Combines multiple genes simultaneously

Mendelian Genetics (same species)

Plant 1: After exposure to -6° C ( 22°F)Plant 2:Watered to saturationNo watering for 15 days24 hours after watering

NT CBF

1

2

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.

Proof of concept for cold and drought tolerance induced by CBF3 demonstrated in Petunia and corn (Sairam et al, 2008)

What are GUS, GFP, & CBF3 genes?

A. Agrobacterium Cell B. Agrobacterium DNA C. Ti Plasmid D. Plant CellE. Plant MitochondriaF. Plant ChloroplastG. Plant Nucleusa. T-DNAb. Vir genesc. Replication origin

http://en.wikipedia.org/wiki/File:Transfection_by_Agrobacterium.svg

Rd29

CBF3

CBF3 A176CBF3-A354CBF3-350

CBF3A478CBF3 A-648

CBF3 A779CBF3 Second ExonCBF3A-952CBF3 A1099

CBF3A-1256CBF3 A1403

CBF3 A-1400

CBF3 A-1551CBF3 A-1701

Procedures for Transformation

Immerse in GUS, GFP and CBF3 cultures 20 mins.

Transfer explants to co-cultivation media.

Dark, 25° C, 72 hrs.

Coat gold with Plasmid DNA

Load explants on regeneration media.

Prepare gene gun.

FIRE!

Agrobacterium-mediated transformation

Biolistic transformation

EmbryosIn vitro germinated leaves

http://www.ag.ndsu.edu/pubs/plantsci/crops/a1219w.htm

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 GFPEXPLANT: Leaf 7/22

Transformation with CBF3Explant: Embryo

Explants from 6/17/11 JPB exp.

PCR Results Jatropha Particle Bombardment (GUS)

Jatropha Agrobacterium- mediated Transformation (GFP)

• Gradient PCR with different concentrations into different wells.

SUMMARYGUS and GFP expression was positive in PCR results.

FUTURE RESEARCHTo perform molecular analysis for CBF3Develop Jatropha to be resistant to other abiotic and biotic stress.

Summary & Future Research

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 & traditional Jet fuel in one of the 2 engines.

http://phx.corporate-ir.net/phoenix.zhtml?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 CitedContinental 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