Transgenic plants from fragmented shoot tips of apple (Malus baccata (L.) Borkhausen) via agrobacterium-mediated transformation

Transgenic apple (Malus baccata (L.) Borkhausen) plants were obtained via Agrobaterium-mediated transformation of fragmented shoot tips. Our results showed that without wounding treatment or with wounding treatment (II, cutting shoot tips vertically into two parts), shoots generally regenerated from meristem tissues directly and adventitious shoot regeneration was rarely observed. Otherwise, when shoot tips were cut vertically into four parts, a high percentage of callus formation (89.2%) and of adventitious shoot regeneration (60.8%) was observed. Under 20 mg l−1 kanamycin selection pressure, over 51.7% fragmented shoot tips developed callus and seven transgenic plants with GFP (Green fluorescent protein) expression were obtained from about 120 explants (efficiency of 5.8%). No transgenic plant was obtained from agrobacteria mediate transformed leaves, even though 23.2% of which formed callus after co-cultivation and selection. Molecular analysis (PCR and RT-PCR) of the transformed lines with GFP expression confirmed integration and transcription of the transgene. Under fluorescence microscopy, areas with high density of transgenic cells were observed at the cutting edges of fragmented shoot tips, which indicated that shoot regeneration from transgenic cells should be a major factor inhibiting transformation efficiency. Our experiments also showed that with moderate or low selection pressure, transgenic shoots were obtained generally accompanied by a high numbers of chimeric shoots. While by using fluorescence microscopy observation of GFP expression, the transgenic and chimeric shoots could be detected and separated precisely for further transgenic plats regeneration or multiplication. This may be very useful for apple genetic breeding, as large numbers of transgenic plants could be obtained in a short time.

► Wounding treatment promotes adventitious shoot regeneration from fragmented shoot tips.
► Cells of fragmented shoot tips were more resistant to kanamycin selection compared to leaf cells.
► Transgenic apple plants were obtained from fragmented shoot tips.
► Shoot regeneration from transgenic cells was a major factor inhibiting transformation efficiency.