Biotechnology Research

See also:

Samac, D. A., Tesfaye, M., Dornbusch M., Purev, S., and Temple , S. J. 2004. A comparison of constitutive promoters for expression of transgenes in alfalfa (Medicago sativa). Transgenic Res. 13:349-361. (PDF)

Somers, D. A., Samac, D. A. and Olhoft, P. M. 2003. Recent advances in legume transformation. Plant Physiol. 131:892-899. (PDF)

Alfalfa Transformation protocol (MS word file)

Agrobacterium -mediated transformation of alfalfa

Explants

Leaves of a highly regenerable genotype from Regen- SY are surface sterilized and cut into pieces approximately 1 cm square.

Co-cultivation

Leaf explants are co-cultivated with Agrobacterium tumefaciens strain LBA4404 containing for 7 days.

 
Tissue Culture

Callus induction

Bacteria are washed from explants which are then placed on callus induction medium containing kanamycin for selection of transformed cells.

Somatic embryo formation

Somatic embryos formed from callus cultures are removed and transferred to medium for further development.

 
Plantlet Conversion
 

Once plantlets have developed roots and shoots, they are transferred to soil in the greenhouse or growth chamber. Transformation and regeneration requires 10-12 weeks. Typically, each leaf piece produces one or more transgenic plants.

 
 
“Constitutive” Promoter Expression in Alfalfa
35S
CsVMV
ScBV
 

GUS expression in transgenic alfalfa. Highest expression of transgenes occurs with the CsVMV promoter. A, E, I: mature leaf. B, F, J: stem cross section. C, G, K: young roots. D, H, L: root nodules. 35S: cauliflower mosaic virus 35S promoter::GUS. CsVMV: cassava vein mosaic virus promoter::GUS. ScBV: Sugarcane badnavirus promoter::GUS.

 
 
Promoters for Vascular Cell Expression

P4::GUS

P4 is a -400 bp promoter deletion from PEP carboxylase. Pathirana et al.1997. Plant J. 12:293-304.

P-Atchit::GUS

P-Atchit is a class III chitinase promoter from Arabidopsis. Samac and Shah. 1991. Plant Cell 3:1063-1072.

 

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