Alfalfa Transformation Medicago

sativaBy: Andres Gonzalez

Graduate Student: Praveena Kanchupati Mentor: Yajun Wu

Alfalfa Today

Almost exclusively used for livestock feed.

Nutritious

Necessity for Project• Lower market price of alfalfa for

eventual cellulosic biofuel production

• Increase biomass yield to ensure profits to farmers

• Give alfalfa dual role as fuel and plant for cattle industry

Alfalfa

• Forage crop that can be grown worldwide

• Symbiotic nitrogen fixation with bacteria

• Perennial crop 5 year lifespan per plant

• Already mass produced in US

Objectives

• Delay flowering time• Give farmers choice of when to

harvest with purchasable cultivars• Cuf and Alfagraze

• Make alfalfa a viable dual use crop for sustainable global farming

Plant Transformation

• Agrobacterium mediated transformation

• Biologically mediated form of genetic alteration

Flowering Genes

• Flowering phase is delayed • Grow a taller and thicker plant• Targeted Genes:

• CONSTANS, FVE, and FCA

Suppress CONSTANS in

timed pathway

Suppress FVE and FCA

Combination of both pathways

will delay flowering and

promote growth

Arabidopsis flowering mutants

Jae-Woong, Yu et. al

Summary of methodsSeedling

• Excise cotyledon off of germinated seedlings and vortex in quartz.•Co-culture with agrobacteria.• Move to SGM for regeneration in antibiotics plant growth.

Leaf• Cut leaves off of plants in greenhouse.• Put leaves in B5h for infection.•Transfer to B5hKTc for callus formation. • Move embryos to MMSKTc.•Plantlets to MMSTc

Seedling Method1. Sterilization

2. Germinate seeds in MS Seed Germination

Medium

3. Excise cotyledon and vortex with

quartz and agrobacterium

4. Co-cultivation medium and bacterial infection

5. Seedling development medium with antibiotics for

plant growth

6. Transfer to soil

Seedling Method

* Fast and healthy plant generation in 2-3 weeks

* Regenerated plants were not transgenic

Leaf Method1. Cut leaves from plants and

surface sterilize

2. Wound leaves for exposure with agrobacterium in SHO

medium

3. Place leaves on B5h for infection

4. Transfer to B5hKTc for callus formation

5. Transfer to B5hOKTc for embryo formation and

regeneration6. Transfer embryos to

MMSKTc for formation of shoot

7. Move plantlets to MMSTc for root and shoot

development

8. Submerge bottom 2/3 of plant in water to “harden off”

the leaves. Then move to soil.

Leaf Method

* Leaves had problems surviving the sterilization process. Were weak

*Did not make it past the infection stage of method

Results

Troubleshooting

• Use younger cells more willing to transform genes.

• Increase surface wounding in stem for greater agrobacterium infection.

• Grow sterile plants that do not need to sterilize the leaf.

• Need a marker to indicate if genes transfer.

• GUS, a marker gene, was successfully transformed into leaf samples.• Blue color is made by transformed enzyme.

Current Research

• Seed method regenerates well but no transfer of genes yet.

• Leaf method now has gene transfer, need to get plant to regenerate from embryos.

Next Step

• Successful transformation methods to deliver CONSTANS, FVE, and FCA genes.

Discussion• Effects of altered pathways need

to be studied.• A transformed plant does not

guarantee increase in biomass yield nor a healthy plant.

• Will the transgenic plants be able to reproduce?

Acknowledgements

• Praveena Kanchupati• Dr. Wu• Eric Oines

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http://www.nepadbiosafety.net/abne/wp-cohttp://www.nepadbiosafety.net/abne/wp-content/uploads/2010/10/agrobacterium.jpgntent/uploads/2010/10/agrobacterium.jpg

http://ucanr.edu/repository/cao/landingpaghttp://ucanr.edu/repository/cao/landingpage.cfm?article=ca.v059n04p252&fulltext=ye.cfm?article=ca.v059n04p252&fulltext=yeses

http://www.yolofarmbureau.orghttp://www.yolofarmbureau.org

http://yeahiwasintheshit.tumblr.com/http://yeahiwasintheshit.tumblr.com/post/29694709966/alfalfa-was-murderedpost/29694709966/alfalfa-was-murdered