Production of red clover transgenic for neomycin phosphotransferase II using Agrobacterium

Genetic variability for resistance to several of the more important diseases of red clover (Trifolium pratense L.) appears to be limited, Incorporation of resistant genes from other species via genetic transformation offers potential, This research was conducted to evaluate methodology for production of transgenic red clover plants via Agrobacterium tumefaciens mediated transformation. The effects of red clover genotypes and Agrobacterium strains on transformation success were evaluated in two experiments. Plants determined to be transgenic for the beta-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII genes were crossed with normal plants and the inheritance of GUS was determined, Putative transgenic callus and plants were produced on B5 medium containing 50 and 300 mg L(-1) kanamycin and carbenicillin, respectively. Both transformation experiments showed significant red clover genotype, Agrobacterium strain, and genotype x strain interactions (P < 0.05) for percentage of petiole pieces regenerating plants. Southern blots of DNA isolated from various GUS(+) plants showed evidence of single and multiple copies of the NPTII gene. Petiole pieces from various putative transgenic plants produced near normal amounts of callus when plated on B5 callus induction medium containing 50 mg L(-1) kanamycin. Progeny from crossing transgenic with normal plants segregated 1:1 as expected, The protocol outlined in this paper should be useful for incorporation of genes of agronomic importance into red clover.