PEG-MEDIATED TRANSFORMATION OF LEAF PROTOPLASTS OF SOLANUM-TUBEROSUM L CULTIVARS

As an alternative to Agrobacterium-mediated gene transfer, direct transformation of potato (Solanum tuberosum L.) cultivars was achieved by using the Mg2+/PEG protocol (Negrutiu et al. 1987) to stimulate DNA uptake into leaf protoplasts. The frequency of kanamycin-resistant clones varied between 1-12% from experiment to experiment independently of the genotype used. A deleterious effect of heat shock treatment (45-degrees-C for 5 min.) has been found on colony formation during optimization of the transformation procedure. The Mg2+ ion concentration (15-35 mM), the denaturation (100-degrees-C, 10 min. right before the treatment) and the form of the plasmid DNA (linear or circular) had no significant effect on the efficacy of the transformation. Application of denatured calf thymus DNA as carrier molecules (at concentration of 50-mu-g ml-1, however, resulted in a significant decrease in the number of the resistant cell colonies). Sixty to 80 percent of the kanamycin-selected callus tissues regenerated shoots. The presence and expression of the introduced neomycin phosphotransferase neo gene in regenerants with normal morphology and tetraploid chromosome number was proved by biological tests based on rooting and callus formation in the presence of the antibiotic and by NPT enzyme activity assay. Southern analysis revealed the integration of the introduced DNA molecules carrying the neo marker gene, into the genome of potato.