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???