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)

Road Map

I. Plant Genetic Engineering Backgrounda) Regenerationb) Transformation

II. Previous Modified Plants vs Chili III. Research ObjectivesIV. Materials and MethodsV. Results

Regeneration

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.

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

Arabidopsis thaliana (www.zmbp.uni-tuebingen.de. 2007)Tobacco (www.nature.com .2006)

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

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

Objectives

1. Develop methodologies for in vitro regeneration of chile

2. Optimize conditions for Agrobacterium-mediated transformation of chile.

a. Optimize DNA delivery to cellsb. Standardize whole plant transformation

Methods and Materials

1. Regenerationa. Plant Materialsb. MS Mediac. Sterilization and Germinationd. Tissue Culture

2. Transformationa. Preparation of Culturesb. Infiltration Studies c. Vacuum Infiltrationd. GUS Assay

Regeneration

Plant Materials

• Chili Cultivars:• NM-S

• Subicho

• CM-334

• Bacctum

• NM-64

• B-58

• Media:• Germination

medium

• Regeneration medium

• Transformation medium

• Selection medium

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. Plated on MS Media

2. Placed in foil

3. Incubated for 7 days

4. 7 day old seedlings

By: Forest Ross

By: Charlene Carr

Tissue Culture

Summary of Regeneration

Transformation

Preparation of Cultures

Pictures by Charlene Carr

Infiltration Studies

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

Year Experiment Cultivar Experiment Percentages      Cotyledons Hypocotyls Embryos

2007 19 ** Bacctum N/A N/A 0%NM-64 N/A N/A 0%B-58 N/A N/A 12.77%

20 ** B-58 N/A N/A 022 ** NM-64 N/A N/A 45.16%

Subicho N/A N/A 9.21%CM -334 N/A N/A 0%

23 ** Subicho N/A N/A 0%NM-S N/A N/A 19.35%

24 * NM-S 17.86% 0% N/A25 ** NM-64 N/A N/A 10%

NM-S N/A N/A 0%2008 29 * NM-S 100% 100% N/A

30 *** NM-S 11.61% N/A N/A31 * NM-S 100% 100% N/A

  42 *** NM-S 66.44% 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

Stages NM-S subjected to stage conditions   Duration

GerminationGermination under dark conditions on MS medium 7-14 days

Tissue CultureExcised explants (cotyledons and hypo-cotyledons) and place on MS + acetosyringone

7-14 day old seedlings

Vacuum TransformationAgrobacterium inoculation to introduce Gus reporter gene into chili cells by vacuum infiltration. 2-3 daysExplants were then placed on MS + acetosyringone medium to incubate.

WashingExplants 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

Pepper Transformation and Regeneration (continued)

Stages NM-S subjected to stage conditions   DurationSelection

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 ElongationRooting

Healthy explants are transferred to MS + low IAA + Tic + Kan for root formation. 1-2 weeks

Callus

E1G1

G1 B2

Collaborative effort from Chile Team CSG lab.

Plantlets

D1 B2

D1 B2

B3 B3B3

D3

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.

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

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???