AGROBACTERIUM-MEDIATED TRANSFORMATION OF APPLE (MALUS X DOMESTICA BORKH) - AN ASSESSMENT OF FACTORS AFFECTING REGENERATION OF TRANSGENIC PLANTS

We have previously developed a protocol for efficient gene transfer an d regeneration of transgenic calli following cocultivation of apple (c v. Jonagold) explants with Agrobacterium tumefaciens (De Bondt st al. 1994, Plant Cell Reports 13: 587-593). Now we report on the optimizati on of postcultivation conditions for efficient and reproducible regene ration of transgenic shoots from the apple cultivar Jonagold. Factors which were found to be essential for efficient shoot regeneration were the use of gelrite as a gelling agent and tilt: use of the cytokinin- mimicing thidiazuron in the selective postcultivation medium. Improved transformation efficiencies were obtained by combining the hormones t hidiazuron and zeatin and by using leaf explants from in vitro grown s hoots not older than 4 weeks after multiplication. Attempts to use pho sphinothricin acetyl transferase as a selectable marker were not succe ssful. Using selection on kanamycin under optimal postcultivation cond itions, about 2% of the leaf explants developed transgenic shoots or s hoot clusters. The presence and expression of the transferred genes wa s verified by beta-glucuronidase assays and Southern analysis. The tra nsformation procedure has also been succesfully applied to several oth er apple cultivars.