Graduate Thesis Or Dissertation
 

Transformation of alfalfa using Agrobacterium tumefaciens

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  • Agrobacterium tumefaciens, the causative agent of crown gall disease, does not readily infect commercial varieties of alfalfa (Medicago sativa). Cloned virulence genes from the megaplasmid pTiBo542 have been shown to enhance Agrobacterium host range and infectivity on dicotyledonous plants. To obtain a gene transfer system for commercial alfalfa cultivars, an Agrobacterium strain containing a disarmed T-DNA, a cloned virG gene from pTiBo542, and an octapine Ti plasmid TiA6NC was constructed. This strain, A348 (pToK9, pGA472), was used to inoculate excised petioles of the alfalfa cultivar "Gladiator. Neomycin resistant callus tissue grew readily on 100 p.1/mIkanamycin and on appropriate media, leaf and embryo development was induced. DNA hybridization analysis of the transformed plant genomic DNA isolated from callus tissue confirmed the presence of the npt gene. VirG enhanced A. tumefaciens strains can thus be used in foreign gene transfer and expression in commercial cultivars of alfalfa.
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