GENETIC-TRANSFORMATION OF COMMERCIAL BREEDING LINES OF ALFALFA (MEDICAGO-SATIVA)

Bio-engineering technologies are now routinely used for the genetic im provement of many agricultural crops. However, breeding lines of Medic ago sativa are not easily amenable to genetic transformation and there fore cannot benefit from the molecular tools that have been developed for genetic manipulations. This paper describes a strategy that has be en developed to transfer DNA into commercially important breeding line s of winter-hardy alfalfa via Agrobacterium infection. Three highly re generative genotypes have been selected from ca 1000 genotypes within Ii breeding lines. They have been used as basic material for an extens ive genetic transformation trial. Combinations of genotypes (11.9, 8.8 , 1.5) expression vectors (pGA482, pGA643, pBibKan) and bacterial stra ins (C58, A281, LBA4404) were tested for their ability to produce stab le transgenic material. Putative transgenic plantlets were further scr eened by nptII-specific PCR amplification, Southern hybridization and recallusing assays. One genotype (1.5) gave only one transformant out of 432 individual trials. With the two other genotypes, efficiency of transformation (kanamycin-resistant calluses obtained/explant tested) ranged from 0 to 0.92 depending on the strain/vector combination used. Statistical interactions underline the possibility of obtaining good genotype-strain-vector combinations for alfalfa transformation. Predic ted transformation probability indicates that with strain LBA4404 cont aining the vector pGA482 and genotype 11.9, transformation efficiency is above 60% and 10% or more of the calluses retain embryogenic potent ial, PCR amplification and Southern hybridization of randomly chosen r egenerated plantlets demonstrated that all embryos developing on 50 mu g ml(-1) kanamycin had a stable genomic insertion of nptII. Sexual cr osses with untransformed genotypes showed that segregation of the tran sgenic trait followed Mendelian heredity.