Development of an Efficient Transformation and Regeneration System for Chile (Capsicum annuum)

Charlene Carr Department of Plant and Environmental Science New Mexico State University Faculty Advisor Dr. Champa Sengupta-Gopalan

Road Map
I.

II. III. IV. V.

Plant Genetic Engineering Background a) Regeneration b) Transformation Previous Modified Plants vs Chili Research Objectives Materials and Methods Results

Regeneration

Plant Regeneration Technology

Collaborative effort from CSG lab.

Whole plants from single cells. Involves developing media and other growth conditions. Unique culturing conditions have to be developed for each plant.

Plant Tissues Used

(Ochoa-Alejo, et. al 2001)

Modified by Charlene Carr. By Suman Bagga

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.

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.

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.

Transformation

Transformation

Recombinant DNA delivery technologies (transformation)

The concept of using Agrobacterium tumefaciens

soil bacterium responsible for crown gall disease a vector to create transgenic plants

Plant Transformation

Agrobacterium tumefaciens

www.bio.davidson.edu. 2003

pCAMBIA Vector of Interest

-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

Tobacco

(www.nature.com .2006)

Arabidopsis thaliana

(www.zmbp.uni-tuebingen.de. 2007)

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. 2006)

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

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

Solanaceae - tobacco and tomato

Objectives
1.

Develop methodologies for in vitro regeneration of chile

Optimize conditions for Agrobacteriummediated transformation of chile.
a. Optimize DNA delivery to cells b. Standardize whole plant transformation

2.

Methods and Materials

1.

2.

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

Regeneration

Plant Materials

Chili Cultivars: NM-S

Media: Germination medium

Subicho CM-334

Regeneration medium Transformation medium Selection medium

Bacctum NM-64 B-58

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

Germination
1. 2. 3. 4.

Plated on MS Media Placed in foil Incubated for 7 days 7 day old seedlings

By: Forest Ross

By: Charlene Carr

Tissue Culture

Summary of Regeneration

Transformation

Preparation of Cultures

Infiltration Studies

Pictures by Charlene Carr

Transformation: Vacuum Infiltration

Stages of Transgenic Plantlets

Collaborative effort from Chile Team CSG lab.

Gus Assay

Tissue - cotyledons, hypo-cotyledons, callus, and roots. Positive Control Tobacco Negative Control non transformed chili explant

Results

Percentage of Regenerated Transformed Plants for 2007 and 2008

Year 2007 Experiment 19 ** Cultivar Bacctum NM-64 B-58 B-58 NM-64 Subicho CM -334 Subicho NM-S NM-S NM-64 NM-S NM-S NM-S NM-S NM-S Experiment Percentages Cotyledons Hypocotyls Embryos N/A N/A 0% N/A N/A 0% N/A N/A 12.77% N/A N/A 0 N/A N/A 45.16% N/A N/A 9.21% N/A N/A 0% N/A N/A 0% N/A N/A 19.35% 17.86% 0% N/A N/A N/A 10% N/A N/A 0% 100% 100% N/A 11.61% N/A N/A 100% 100% N/A 66.44% 43.19% N/A

20 ** 22 **

23 **
24 * 25 ** 2008 29 * 30 *** 31 * 42 ***

* 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

Pepper Transformation and Regeneration

Stages NM-S subjected to stage conditions Duration

Germination

Germination under dark conditions on MS medium

Excised explants (cotyledons and hypo-cotyledons) and place on MS + acetosyringone Agrobacterium inoculation to introduce Gus reporter gene into chili cells by vacuum infiltration. Explants were then placed on MS + acetosyringone medium to incubate. Explants are washed with water plus Ticar to remove residual Agrobacterium. Explants are then placed on MS + 512 + Ticar medium to start the regeneration process.

7-14 days 7-14 day old seedlings

Tissue Culture

Vacuum Transformation

2-3 days

Washing 30 to 40 mins

Protocol Standardized in 2008 by Charlene Carr

Pepper Transformation and Regeneration (continued)

Stages Selection NM-S subjected to stage conditions Explants transferred to selection medium containing antibiotics to select putative transformants. Explants are placed on MS + 512 + Ticar + Kanyamycin. Embryo Formation Healthy explants are transferred to MS + low 512 + Tic + Kan for embryo formation. Healthy explants are transferred to MS + low BA + low IAA + Tic + Kan for plantlet formation. 1-2 weeks 1-2 weeks Duration

2-3 weeks

Multi-shoot formation and Elongation Rooting

Healthy explants are transferred to MS + low IAA + Tic + Kan for root formation.

1-2 weeks

Callus
G1 B2

G1

E1

Collaborative effort from Chile Team CSG lab.

Plantlets

B3

B3

B3

D1

B2

D3

D1

B2

D3
Collaborative effort from Chile Team CSG lab.

Putative Transformants:

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.

Dr. Champa S-Gopalans Lab

Chile Biotechnology group Melina Sedano, M.S., Research Associate Charlene Carr (HHMI & MARC) Carlos H Brad Barrow (CREST) Suman Bagga Ph.D.

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.

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

Questions???