AGROBACTERIUM-MEDIATED TRANSFORMATION OF THE APPLE ROOTSTOCK MALUS MICROMALUS MAKINO WITH THE ROLC GENE

Malus micromalus Makino was genetically engineered with the rolC gene via Agrobacterium-mediated transformation. The antibiotics carbenicillin, hygromycin, and kanamycin (Km) inhibited shoot regeneration from in vitro leaf explants. The leaf segments were infected with Agrobacterium tumefaciens strain LBA4404 harboring one of the three different plasmids. Shoots were regenerated only from leaf segments infected with LBA4404 harboring the plasmid with the intron-containing neomycin phosphotransferase II gene as the selectable marker. Preculture of leaf segments on regeneration medium for 2 d before Agrobacterium treatment reduced formation of Km-resistant calluses. Transformation was confirmed by a histochemical β-glucuronidase (GUS) assay, amplification of the rolC gene by polymerase chain reaction (PCR), and by Southern blot analysis. The rolC-transformed M. micromalus shoots showed an increased rooting ability without auxin treatment, and reduced height, internode length and leaf areas. This research shows the potential application of using the rolC gene for developing dwarf apple rootstocks.