Studies On Transcription Factor CBF Gene Transformation Via Agrobacterium Tumefaciens In Apple (Malus Domestica Borkh)

The transcription factor CBF gene, which was considered to related to cold hardness induction, was transferred into apple stock M₂₆ and cultivar of Gala via Agrobacterium tumefaciens of strain EHA 105. The adventitious bud regeneration system and genetic transformation system have been modified by studying the effect of explant's types and status on the efficiency of regeneration and transformation, and the transgenic lines were obtained. The main results were as followed.1. The efficiency of adventitious bud regeneration was varied from the explants, with that of the albino stem segments was higher than that of the leaf disc. The optimal protocol for the adventitious bud regeneration from the albino stem segments was that: getting the albino stem segments by dark incubating the plantlets for 20 days, cutting the stems into pieces of 0.5 cm long containing 1² internodes, then laying them on the MS medium for dark incubating. The adventitious buds appeared 2³ weeks later. The rate of adventitious bud regeneration of M₂₆ and Gala was above 95%.2. The adding of BA 5.0 mg/L and TDZ 1.0 mg/L in the media could lead the rate of adventitious bud regeneration from albino stem segments to reach a high level, but the adding of TDZ could increased the occurring of vitrification in the adventitious buds. Though, the use of BA 5.0 mg/L is benefit to the adventitious bud regeneration from albino stem segments in M²⁶.3. The browning was more seriously in the albino stem segments than the leave. The supplying of Vc 100 mg/L or AgNO₃ 1 mg/L could limited the browning, and increased the efficiency of adventitious bud regeneration. But When the concentration of Vc was more than 400 mg/L or AgNC>3 was more than 3 mg/L, the supplying could decreased the efficiency, and that could lead the browning at the wounded area even more seriously.4. The efficiency of adventitious bud regeneration was varied greatly when the albino stem segments were sampled from the plantlets subcultured for different days. The highest efficiency was obtained when the segments were sampled from the plantlets that were subcultured for 20²5 days. If the subcultured time was less, the plantlets could be to short too be sampled, and if the time was more, the segments became brown easily and heavily.5. The optimal concentration of Kanamycin was 12 mg/L and 10 mg/L when the albino stem segments and the leave were used as the explants for the transformation mediated by Agrobacteria of strain EHA 105, respectively. The optimal concentration of Cef was 200 mg/L.6. Acetosyringone is able to induce the expression of vir gene, and accelerate the transformation of T-DNA. The supplying of Acetosyringone 30mg/L in the inoculation solution and cocultivation medium could increased the efficiency of gene transformation in apple.7. By comparing the factors on agrobacterium-mediated gene transformation, it suggests the optimal protocol for gene transformation in apple would be: at first, the explants should be pre-cultured for one day on the inducing medium;the bacterium concentration for inoculation would be OD6oo=0.6 around, the co-cultivation time is about 10 minutes, co-cultured for two days, and then, the explants were transferred onto the inducing medium that supplying with Km lOmg/L (orl2 mg/L) and Cef 200 mg/L.8. There were twelve transgenic lines passed the kanamycin-resistant test. The kanamycin-resistant test of transgenic lines was based on the three main aspects: ?The transgenic lines were able to be subcultured on media supplemented with 50mg/L kanamycin, continuously. ?The leaf pieces of the transgenic lines showed as same regeneration ability on media containing 30mg/L kanamycin as on media without kanamycin. ?The transgenic lines were able to root normally on media supplemented with 30mg/L kanamycin.9. The transgenic lines with the kanamycin-resistant ability were further tested by PCR analysis. The result was that one plant showed specific positive band as same as the plasmid control. The result indicated that the CBF gene has been integrated into the apple genome.10. The cold-resistance characteristics of the transgenic lines were tested by measuring the accumulation of proline and the changing of electric conductivity after treating the plants under 4°C condition for a certain time. The result showed that the proline content of transformed plants were higher than untransformed plants. It appeared that the transgenic lines showed the higher ability of cold-resistance.