NSF-REU Program Summer 2011 Nasie Constantino

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1 NSF-REU Program Summer 2011 Nasie Constantino

2 Oil Producing Crops in US Land Crop Yields based on US average 2006 Crop Avg Harvest (lbs) Peanut Canola Soybean Sunflower Camelina Safflower Corn Cottonseed Ethrensing et a1, Oil content% (avg) Gal/acre(approx.)

3 Camelina sativa Introduction Camelina sativa is a member of mustard family Native to central Asia and Mediterranean region Second generation biofuel crop Matures in 85 to 100 days and will grow 1 to 3 feet tall Yields 1,800 to 2,000 pounds of seeds per acre in areas with 16 to 18 inches of rainfall New Crop to US Example: 50,000 acres planted in Montana 2007

4 My NSF-REU Objective To successfully transform Camelina sativa with AtNPR1 using Agrobacterium-mediated transformation AtNPR1: Arabidopsis thaliana Non-expressor of Pathogenesis Related gene Key and positive regulator of disease resistance Antimicrobial Activity Broad range of disease resistance: against viruses, bacteria and fungi Has been overexpressed in rice and has Frederick et al lead to antimicrobial activity and enhanced tolerance to pathogens 2008 Native American Rehabilitation Association

5 Surface Sterilization

6 Preparation of Explants

7 Agrobacterium mediated Transformation

8 Summary of Transformations Transformation number Explant used (#) Gene Number of calli Number of shoot regenerated NCT1 Cotyledons NPR1, GUS 33 2 NCT2 Cotyledons SAMdc, GUS 33 1 NCT3 Cotyledons NPR1, egfp 46 0 NCT4 Young leaves and cotyledons NPR1, egfp, GUS 49 9 NCT5 Young leaves and cotyledons NPR1, egfp, GUS 12 6 NCT6 Young leaves and cotyledons NPR1, egfp, GUS 17 8 Total NCT1 = Nasie Camelina Transformation # 1 GUS = B-Glucouronidase egfp = Green Fluorescent protein

9 GUS expression in Camelina

10 egfp expression in Camelina

11 Camelina Regeneration

12 Research Summary Preparation of Camelina explants (cotyledons and young leaves) Agrobacterium-mediated transformation Camelina regeneration

13 Future Work All explants will be taken through the process of regeneration, including rooting. Successfully regenerated plants will be acclimatized in the green house. Molecular analyses: Genomic DNA will be extracted from putative transgenic plants and will be tested for either GUS, egfp or AtNPR1 using Polymerase chain reaction (PCR)

14 Techniques that I learned this summer Sterile technique with plant tissue cultures Preparation of overnight Agrobacterium cultures for plants transformation Preparation of plant tissue culture media Measuring optical densities of bacterial cultures Agrobacterium-mediated transformation that included: Preparing of Agrobacterium glycerol stocks -Seed sterilization and plating Plasmid purification -Explant preparation DNA quantification using Nanovue -Agrobacterium mediated transformation Transformation of plasmid DNA into Agrobacterium competent -Subculturing cells using electroporation -Washing Agrobacterium overgrowth Resuspension of primers and aliquottingfrom explants -Taking observations Plant genomic DNA isolation PCR -Taking pictures of cultures in the hood and under a GFP microscope Gel Electrophoresis -GUS staining Protein extraction from plant leaf tissue

15 What I ve learned from this Experience Planning ahead Calculations: Molarity, %, conversions That ph measurement is very crucial for plant tissue culture media Being very very careful with sub culturing in the hood Patience is an absolute must in Science Dedication An extra mile takes you a long way

16 Acknowledgments National Science Foundation; for the fellowship Penn State Harrisburg for facilitating this research experience through the brand new Biotechnology Laboratory. Dr Shobha Potlakayala teaching me new techniques and making this a wonderful experience Dr Sairam Rudrabhatla Dr Josekutty for teaching us how to make plant tissue culture media Matt Reitzel, Mike Chennavasin and Imran Hussain for being my student mentors Ben Tabatabai and Diego Morales Joe Meisenbach and Krysta Haggins who also worked with Dr Shobha; for all the help with some new techniques Aneel Maini for assisting me All students from Dr Sairam s lab for helping us all the time Alison Shuler and Julie Dauber for helping with coordination of the program

17 Sources Biofuel Variety Trials Fact Sheet, USDA-ARS and WSU, Prosser, wa, Camelina sativa by Agrobacterium-mediated transformation. Plant Cell Rep Feb;27(2):273-8 Després, C., Chubak. C., Rochon, A., Clark, R., Bethune, T., Desveaux, D., and Fobert, P. R. (2003). The Arabidopsis NPR1 Disease Resistance Protein Is a Novel Cofactor That Confers Redox Regulation of DNA Binding Activity to the Basic Domain/Leucine Zipper Transcription Factor TGA1. Plant Cell,15(9): Ethrensing, D. T., and S. O. Guy. (2008). Camelina. Oregon State University, 1-7. Gelvin, S. B. (2003). Agrobacterium-Mediated Plant Transformation: the Biology behind the Gene-Jockeying Tool. Microbiology and Molecular Biology Reviews, 67(1): Gelvin, S. B. (2010). Plant Proteins Involved in Agrobacterium-Mediated Genetic Transformation. Annual Review of Phytopathology, 48: