1. Charlene Carr Department of Plant and Environmental Science New Mexico State University Faculty Advisor Dr. Champa Sengupta-Gopalan Development of an Efficient Transformation and Regeneration System for Chile (Capsicum annuum)

2. Road Map I. Plant Genetic Engineering Background a) Regeneration b) Transformation II. Previous Modified Plants vs Chili III. Research Objectives IV. Materials and Methods V. Results

3. Regeneration

4. Plant Regeneration Technology  Whole plants from single cells.  Involves developing media and other growth conditions.  Unique culturing conditions have to be developed for each plant. Collaborative effort from CSG lab.

5. Plant Tissues Used (Ochoa-Alejo, et. al 2001) Modified by Charlene Carr. By Suman Bagga

6. Plant Tissue Culture  Types of Regeneration Organogenesis (direct plantlet formation) Callus-induced (indirect plantlet)  When exposed to specific plant hormones un-differentiated growth (callusing) plant embryogenesis Collaborative effort from Chile Team CSG lab.

7. Previous Regeneration Studies  Identification of plant growth Murashige and Skoog media (MS media) (1962)  Complimentary growth regulators (plant hormones) Essential to the regeneration efficiency Promotes callus, embryo, root, shoot and plantlet formation Callusing Multiple Embryo Root Development Whole Plant Formation Collaborative effort from Chile Team CSG lab.

8. Previous Growth Regulator Studies  Complimentary growth regulators (plant hormones) BAP (benzylamino purine) at 5mg/L – a synthetic cytokinin (shoot) IAA (indole acetic acid) at 1mg/L – is an auxin (cell division) GA (giberrillic acid) at 2mg/L - (Arous S et. al 2001)  Once the regeneration system is standardized, it can be integrated with the transformation system.

9. Transformation

10.  Recombinant DNA delivery technologies (transformation)  The concept of using Agrobacterium tumefaciens soil bacterium responsible for crown gall disease a vector to create transgenic plants

11. Plant Transformation

12. Agrobacterium tumefaciens www.bio.davidson.eduwww.bio.davidson.edu. 2003

13. pCAMBIA Vector of Interest

14. -glucuronidase -GUS Reporter gene  Chemical assay with X-Gluc as the substrate  When cells are stained with substrate Transformed plant cells that express the gene appears blue Confirms presences of GUS gene Arabidopsis thaliana (www.zmbp.uni-tuebingen.de. 2007)(www.zmbp.uni-tuebingen.de Tobacco(www.nature.com.2006)

15. Previously GMO Crops tomato Flavr Savr® herbicide resistant soybean and insect-resistant corn and Bt cotton high methionine protein in alfalfa foliage vitamin A produced in golden rice (http://www.ucsusa.org(http://www.ucsusa.org. 2006)

16. Previous GMO vs. Chili  Previous GMOs have been improved with respect to rotting, herbicide, insect resistance  Any plant tissue can be used in tissue culture  Previous GMO crops have high regeneration capabilities  Solanaceae - tobacco and tomato  Many economically important crop species such as chili lies many challenges  Low to produce whole plants from cells in tissue culture  Only colyledons and hypocotyledons  Protocols not repeatable  Other reports are not complete

17. Objectives 1. Develop methodologies for in vitro regeneration of chile 2. Optimize conditions for Agrobacterium- mediated transformation of chile. a. Optimize DNA delivery to cells b. Standardize whole plant transformation

18. Methods and Materials 1. Regeneration a. Plant Materials b. MS Media c. Sterilization and Germination d. Tissue Culture 2. Transformation a. Preparation of Cultures b. Infiltration Studies c. Vacuum Infiltration d. GUS Assay

19. Regeneration

20. Plant Materials Chili Cultivars: NM-S Subicho CM-334 Bacctum NM-64 B-58 Media: Germination medium Regeneration medium Transformation medium Selection medium

21. Seed Surface Sterilization Purpose to remove particles to prevent contamination Sterilization twice Seeds surface sterilization (modified): - Wash in de-ionized H2O & ivory soap ethanol bleach

22. Germination 1. Plated on MS Media 2. Placed in foil 3. Incubated for 7 days 4. 7 day old seedlings By: Forest Ross By: Charlene Carr

23. Tissue Culture

24. Summary of Regeneration

25. Transformation

26. Preparation of Cultures

27. Pictures by Charlene Carr Infiltration Studies

28. Transformation: Vacuum Infiltration

29. Stages of Transgenic Plantlets Collaborative effort from Chile Team CSG lab.

30. Gus Assay  Tissue - cotyledons, hypo-cotyledons, callus, and roots.  Positive Control – Tobacco  Negative Control – non transformed chili explant

31. Results

32. YearExperiment CultivarExperiment Percentages CotyledonsHypocotylsEmbryos 200719 **BacctumN/A 0% NM-64N/A 0% B-58N/A 12.77% 20 **B-58N/A 0 22 **NM-64N/A 45.16% SubichoN/A 9.21% CM -334N/A 0% 23 **SubichoN/A 0% NM-SN/A 19.35% 24 *NM-S17.86%0%N/A 25 **NM-64N/A 10% NM-SN/A 0% 200829 *NM-S100% N/A 30 ***NM-S11.61%N/A 31 *NM-S100% N/A 42 ***NM-S66.44%43.19%N/A * Regeneration values measured on medium: MS + ↓BA + ↓IAA +TIC + KAN ** Regeneration values measured on medium: MS + BA + IAA + TDZ + TIC + KAN *** Regeneration values measured on medium: MS +↓512 + TIC + KAN Percentage of Regenerated Transformed Plants for 2007 and 2008

33. StagesNM-S subjected to stage conditions Duration Germination Germination under dark conditions on MS medium7-14 days Tissue Culture Excised explants (cotyledons and hypo-cotyledons) and place on MS + acetosyringone 7-14 day old seedlings Vacuum Transformation Agrobacterium inoculation to introduce Gus reporter gene into chili cells by vacuum infiltration.2-3 days Explants were then placed on MS + acetosyringone medium to incubate. Washing Explants are washed with water plus Ticar to remove residual Agrobacterium.30 to 40 mins Explants are then placed on MS + 512 + Ticar medium to start the regeneration process. Protocol Standardized in 2008 by Charlene Carr Pepper Transformation and Regeneration

34. Pepper Transformation and Regeneration (continued) StagesNM-S subjected to stage conditions Duration Selection Explants transferred to selection medium containing antibiotics to select putative transformants.2-3 weeks Explants are placed on MS + 512 + Ticar + Kanyamycin. Embryo Formation Healthy explants are transferred to MS + low 512 + Tic + Kan for embryo formation.1-2 weeks Multi-shoot formation Healthy explants are transferred to MS + low BA + low IAA + Tic + Kan for plantlet formation.1-2 weeks and Elongation Rooting Healthy explants are transferred to MS + low IAA + Tic + Kan for root formation.1-2 weeks

35. Callus E1 G1 B2 Collaborative effort from Chile Team CSG lab.

36. Plantlets D1 B2 D1 B2 B3 D3 Collaborative effort from Chile Team CSG lab.

37. Putative Transformants:

38. Conclusion  Identified and established the NM chile lines with maximum regeneration capability in tissue culture (August 2007).  Standardized protocol for efficient gene delivery in chile plant cells using a reporter gene and have established an Agrobacterium strain and genotype combination (August 2007).  Established a whole plant transformation system in chile (January 2008).   We have generated several putative transgenic chile plants in tissue culture and they are being analyzed for the presence of the transgene (April 2008). Next: Initiate experiments to make gene constructs of interest for chile transformation.

39. Chile Biotechnology group  Melina Sedano, M.S., Research Associate  Charlene Carr (HHMI & MARC)  Carlos H  Brad Barrow (CREST)  Suman Bagga Ph.D. Dr. Champa S-Gopalan’s Lab

40. Project Supporters  Funding from HHMI 52005881 and MARC - NIH Grant GM61222  Funding from Chile Task Force, Chile growers association and ChIP (Chile Improvement Project) is acknowledged.  Dr Paul Bosland for his interest in this project and Dr Jit Baral for providing chile seeds.

41. Literature Cited  Arous S, Boussaid M, Marrakchi M (2001) Plant regeneration from zygotic embryo hypocotyls. In. Journal Applied Horticulture, pp 17-22  Gelvin SB (2005) Agricultural biotechnology: Gene Exchange by Design. In. Nature, pp 433, 583 - 584  Kyung Ko M, Soh H, Kim K-M, Kim Ys, Kyunghoan I (2007) Stable Production of Transgenic Pepper Plants Mediated by Agrobacterium tumefaciens. In. HortScience, pp 1425-1430  Ochoa-Alejo N, Ramirez-Malagon R (2001) In vitro chili pepper biotechnology. In Vitro Cellular and Developmental Biology Plant 37:701-729

42. Questions???