AGROBACTERIUM-MEDIATED TRANSFORMATION OF APPLE (MALUS X DOMESTICA BORKH) - AN ASSESSMENT OF FACTORS AFFECTING GENE-TRANSFER EFFICIENCY DURING EARLY TRANSFORMATION STEPS

The factors influencing transfer of an intron - containing beta-glucuronidase gene to apple leaf explants were studied during early steps of an Agrobacterium tumefaciens-mediated transformation procedure, The gene transfer process was evaluated by counting the number of beta-glucuronidase expressing leaf zones immediately after cocultivation, as well as by counting the number of beta-glucuronidase expressing calli developing on the explants after 6 weeks of postcultivation in the presence of 50 mg/l kanamycin. Of three different tested disarmed A. tumefaciens strains, EHA101(pEHA101) was the most effective for apple transformation. Cocultivation of leaf explants with A. tumefaciens on a medium with a high cytokinin level was more conducive to gene transfer than cocultivation on media with high auxin concentrations, Precultivation of leaf explants, prior to cocultivation, slightly increased the number of beta-glucuronidase expressing zones measured immediately after cocultivation, but it drastically decreased the number of transformed calli appearing on the explants 6 weeks after infection. Other factors examined were: Agrobacterium cell density during infection, bacterial growth phase, nature of the carbon source, explant age, and explant genotype.