urcas 2009

8/13/2019 URCAS 2009

1/42

Charlene Carr

Department of Plant and Environmental Science

New Mexico State University

Faculty Advisor Dr. Champa Sengupta-Gopalan

Development of an Efficient

Transformation and RegenerationSystem for Chile (Capsicum annuum)

8/13/2019 URCAS 2009

2/42

Road Map

I. Plant Genetic Engineering Background

a) Regeneration

b) TransformationII. Previous Modified Plants vs Chili

III. Research Objectives

IV. Materials and Methods

V.

Results

8/13/2019 URCAS 2009

3/42

Regeneration

8/13/2019 URCAS 2009

4/42

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.

8/13/2019 URCAS 2009

5/42

Plant Tissues Used (Ochoa-Alejo, et. al 2001)

Modified by Charlene Carr. By Suman Bagga

8/13/2019 URCAS 2009

6/42

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.

8/13/2019 URCAS 2009

7/42

Previous Regeneration Studies

Identification of plant growth Murashige and Skoogmedia (MS media) (1962)

Complimentary growth regulators (plant hormones)

Essential to the regeneration efficiency

Promotes callus, embryo, root, shoot and plantletformation

Callusing Multiple Embryo Root Development Whole Plant Formation


8/13/2019 URCAS 2009

8/42

Previous Growth Regulator Studies

Complimentary growth regulators (plant hormones)

BAP (benzylamino purine) at 5mg/L a syntheticcytokinin (shoot)

IAA (indole acetic acid) at 1mg/L is an auxin (celldivision)

GA (giberrillic acid) at 2mg/L - (Arous S et. al 2001)

Once the regeneration system is standardized, it can beintegrated with the transformation system.

8/13/2019 URCAS 2009

9/42

Transformation

8/13/2019 URCAS 2009

10/42

Transformation

Recombinant DNA delivery technologies(transformation)

The concept of usingAgrobacterium tumefaciens

soil bacterium responsible for crown gall

disease

a vector to create transgenic plants

8/13/2019 URCAS 2009

11/42

Plant

Transformation

8/13/2019 URCAS 2009

12/42

Agrobacterium tumefaciens

www.bio.davidson.edu. 2003
http://www.bio.davidson.edu/http://www.bio.davidson.edu/

8/13/2019 URCAS 2009

13/42

pCAMBIA Vector of Interest

8/13/2019 URCAS 2009

14/42

-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)
http://www.zmbp.uni-tuebingen.de/http://www.zmbp.uni-tuebingen.de/http://www.zmbp.uni-tuebingen.de/http://www.zmbp.uni-tuebingen.de/http://www.zmbp.uni-tuebingen.de/http://www.zmbp.uni-tuebingen.de/http://www.zmbp.uni-tuebingen.de/

8/13/2019 URCAS 2009

15/42

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)
http://www.ucsusa.org/http://www.ucsusa.org/

8/13/2019 URCAS 2009

16/42

Previous GMO vs. Chili

Previous GMOs have beenimproved with respect torotting, herbicide, insectresistance

Any plant tissue can be usedin tissue culture

Previous GMO crops havehigh regeneration capabilities

Solanaceae - tobacco andtomato

Many economically importantcrop species such as chili liesmany challenges

Low to produce wholeplants from cells intissue culture

Only colyledons andhypocotyledons

Protocols notrepeatable

Other reports are notcomplete

8/13/2019 URCAS 2009

17/42

8/13/2019 URCAS 2009

18/42

Methods and Materials

1. Regeneration

a. Plant Materials

b. MS Media

c. Sterilization and Germination

d. Tissue Culture

2. Transformation

a. Preparation of Culturesb. Infiltration Studies

c. Vacuum Infiltration

d. GUS Assay

8/13/2019 URCAS 2009

19/42

Regeneration

8/13/2019 URCAS 2009

20/42

Plant Materials

Chili Cultivars:

NM-S

Subicho

CM-334

Bacctum

NM-64

B-58

Media:

Germinationmedium

Regenerationmedium

Transformationmedium

Selection medium

8/13/2019 URCAS 2009

21/42

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

8/13/2019 URCAS 2009

22/42

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

8/13/2019 URCAS 2009

23/42

Tissue Culture

8/13/2019 URCAS 2009

24/42

Summary of Regeneration

8/13/2019 URCAS 2009

25/42

8/13/2019 URCAS 2009

26/42

Preparation of Cultures

8/13/2019 URCAS 2009

27/42

8/13/2019 URCAS 2009

28/42

8/13/2019 URCAS 2009

29/42

Stages of Transgenic Plantlets


8/13/2019 URCAS 2009

30/42

Gus Assay

Tissue - cotyledons, hypo-cotyledons, callus, and roots.

Positive ControlTobacco

Negative Controlnon transformed chili explant

8/13/2019 URCAS 2009

31/42

Results

P t f R t d T f d Pl t

8/13/2019 URCAS 2009

32/42

Year Experiment Cultivar Experiment Percentages

Cotyledons Hypocotyls Embryos2007 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 0

22 ** 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/A

25 ** NM-64 N/A N/A 10%

NM-S N/A N/A 0%

2008 29 * NM-S 100% 100% N/A30 *** NM-S 11.61% N/A N/A

31 * 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

8/13/2019 URCAS 2009

33/42

Stages NM-S subjected to stage conditions Duration

Germination

Germination under dark conditions on MS

medium 7-14 days

Tissue Culture

Excised explants (cotyledons and hypo-cotyledons) and place

on MS + acetosyringone

7-14 day old

seedlings

Vacuum Transformation

Agrobacteriuminoculation 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 residualAgrobacterium. 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

8/13/2019 URCAS 2009

34/42

Pepper Transformation and Regeneration

(continued)

Stages NM-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

8/13/2019 URCAS 2009

35/42

Callus

E1G1

G1 B2


8/13/2019 URCAS 2009

36/42

8/13/2019 URCAS 2009

37/42

Putative Transformants:

8/13/2019 URCAS 2009

38/42

Conclusion

Identified and established the NM chile lines with maximumregeneration capability in tissue culture (August 2007).

Standardized protocol for efficient gene delivery in chile plantcells using a reporter gene and have established anAgrobacteriumstrain and genotype combination (August 2007).

Established a whole plant transformation system in chile(January 2008).

We have generated several putative transgenic chile plants intissue culture and they are being analyzed for the presence of thetransgene(April 2008).

Next:Initiate experiments to make gene constructs of

interest for chile transformation.

8/13/2019 URCAS 2009

39/42

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-Gopalans Lab

8/13/2019 URCAS 2009

40/42

Project Supporters

Funding from HHMI 52005881 and MARC- NIH Grant GM61222

Funding from Chile Task Force, Chilegrowers association and ChIP (ChileImprovement Project) is acknowledged.

Dr Paul Bosland for his interest in this

project and Dr Jit Baral for providing chileseeds.

8/13/2019 URCAS 2009

41/42

Literature Cited

Arous S, Boussaid M, Marrakchi M (2001) Plantregeneration from zygotic embryo hypocotyls. In. JournalApplied Horticulture, pp 17-22

Gelvin SB (2005) Agricultural biotechnology: GeneExchange 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 PlantsMediated by Agrobacterium tumefaciens. In. HortScience,

pp 1425-1430

Ochoa-Alejo N, Ramirez-Malagon R (2001) In vitro chilipepper biotechnology. In Vitro Cellular and DevelopmentalBiology Plant 37:701-729

8/13/2019 URCAS 2009

42/42

Questions???

urcas 2009

Documents