IMPROVING STRESS TOLERANCE IN CAMELINA SATIVA, A SECOND GENERATION BIOFUEL CROP. Krysta Haggins NSF-REU Summer Internship2011

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1 IMPROVING STRESS TOLERANCE IN CAMELINA SATIVA, A SECOND GENERATION BIOFUEL CROP Krysta Haggins NSF-REU Summer Internship2011

2 Camelina sativa inter annual (adapted to cooler climates) asic advantages Cold tolerant (adapted to late fall/ early winter cropping) Less water and fertilizer input than other oilseed crops biotic Stress forming disadvantages Drought accompanied by heat in summer Increase in salinity Objective for working with Camelina Introduce ysamdc into Camelina (via Agrobacterium-mediated transformation) Enhance stress tolerance (Ehrensing, D, & Guy, S., 2008)

3 Agrobacterium tumefaciens Naturally occurring gram-negative soil bacterium Natural Genetic Engineer Causes crown gall tumors in plants T-DNA, a part of the bacterial tumor-inducing (Ti) plasmid, is transferred from the bacterium to the plant cell with the aid of a number of Virulence (Vir) proteins encoded by the Ti-plasmid The gene of interest or the T-DNA would be cloned initially into Agrobacterium to facilitate plant transformation (Keefer, J.,2002)

4 Yeast S-adenosyl methionine decarboxylase SAMdC is derived from yeast Saccharomyces cerevisiae verexpression of ysamdc results in the improved tolerance to abiotic stress (Cheng L, Zou Y, Ding S, Zhang J, Yu X, Cao J, Lu G, 2009)

5 Seed Surface Sterilization and Explant Preparation

6 Seed Surface Sterilization and Germination Shoot regeneration Washing of explants and subculture Genetic Transformation of Camelina Preparation of explants (cotyledons and young leaves) Co-cultivation Agrobacterium-mediated transformation

7 Agrobacterium-mediated transformation reat explants with Agrobacterium o-cultivation of plant explants on solid nutrient medium grobacterium cleanup of explants using antibiotics election and Regeneration of Explants allus Induction, Shooting, Rooting (Keefer, J., 2002)

8 Regeneration of transformed Camelina explants

9 Results

10 Expression of Glucouronidase (GUS) in Camelina 50µm 50µm

11 Expression of Green Fluorescent Protein (egfp) in Camelina 50µm

12 Conclusion us and egfp illustrate successful transformation oung leaves showed better and faster results than cotyledons urther analysis for presence of gene of interest

13 Future Work ontinue the process of regeneration egenerated plants will transferred into Green House xtraction of genomic DNA from putative transgenic plants to test for GUS, egfp, or ysamdc

14 Knowledge gained from this NSF-REU Penn State Harrisburg experience Personal Work ethics Continued from College Experience -Being Committed -Being Compassionate -Being Competent Science is full of -Acknowledging mistakes and make modifications -Proper preparation -Study

15 ACKNOWLEDGEMENTS *National Science Foundation- Research Experience Undergraduate Program *Penn State Harrisburg Dr. Shobha Devi Potlakayala Dr. R. V. Sairam Dr. Puthiyaparambil Josekutty Diego Morales Imran Hussain Matt Reitzel Mike Chennavasin Nasie Constantino Joe Meisenbach Ben Tabatabi Deepkamal Karelia Aneel Maine Julianne Dauber Alison Shuler NSF-REU Students

16 REFERENCES CHENG L, ZOU Y, DING S, ZHANG J, YU X, CAO J, LU G (2009). POLYAMINE ACCUMULATION IN TRANSGENIC TOMATO ENHANCES THE TOLERANCE TO HIGH TEMPERATURE STRESS. J. INTEGR. PLANT BIOL /J X. EHRENSING, D, & GUY, S. (2008). CAMELINA. UNPUBLISHED MANUSCRIPT, EXTENSION SERVICE, OREGON STATE UNIVERSITY, RETRIEVED FROM PDF/EM/EM8953-E.PDF KEEFER, J. (2002, JUNE 5). GENETIC MODIFICATION IN NATURE. RETRIEVED FROM ATURAL.HTML PUTNAM, D.H., J.T. BUDIN, L.A. FIELD, AND W.M. BREENE CAMELINA: A PROMISING LOW-INPUT OILSEED. P IN: J. JANICK AND J.E. SIMON (EDS.), NEW CROPS. WILEY, NEW YORK.

17 IMPROVING STRESS TOLERANCE IN CAMELINA SATIVA, A SECOND GENERATION BIOFUEL CROP Krysta Haggins NSF-REU Summer Internship2011