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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
SourcesSourceshttp://www.biomedcentral.com/1471-2229http://www.biomedcentral.com/1471-2229/12/81/12/81
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