| The effects of shoot proliferation and adventitious shoot regeneration on Malus spectabilis exposed to plant growth regulators with different combination were studied with leaves as explants in the present study. Meanwile, recombinant plant expressing vector pBI121-cry1Ab was constructed. Cry1Ab gene was introduced into M. spectabilis by Agrobacterium-mediated transformation. This study aimed to establish a high efficient leaves in vitro regeneration and genetic transformation system. The results as followed:(1) This experiment also studied the influence of different plant growth regulator to shoot proliferation and adventitious shoot regeneration of M. spectabilis. The shoots and leaves were used as explants by orthogonal experiment. The culture medium was MS supplemented with different quality concentration of 6-BA, TDZ and IBA combinedly. The results showed that MS adding with 6-BA 2.0 mgï¹'L-1 + IBA 0.1 mgï¹'L-1 is the optimum medium for shoot proliferation, the proliferation value reachs 8.11, and the average height of shoots is 2.14 cm; The highest percentage of regenerated shoots (100%) and the highest number of shoots per explant (6.73) occurred on MS medium containing 1.0 mgï¹'L-1 TDZ and 0.5 mgï¹'L-1 IBA; Rooting rate was above 65% on 1/2 MS medium with 0.3 mgï¹'L-1 TDZ; The leaf polarity test results showed that abaxial layer downside had the greatest regeneration efficiency.(2) The coding region of Cry1Ab was amplified from the Bt C3 strain by PCR, We amplified the coding region of Cry1Ab from the Bt C3 by PCR, the preliminary bioassay showed that expressed product of Cry1Ab had certain insecticidal activity. Then the recombinant plant expression vector pBI121-cry1Ab was constructed and could be applied in transgenic research.(3) The genetic transformation systerm of M. spectabilis was examined. The optimal critical concentrations of kanamycin (25 mgï¹'L-1) and Cefotaxime Na (200 mgï¹'L-1) inducing adventitious shoot regeneration of M. spectabilis were confirmed through the resistance testing of them.(4) On the basis of the establishment of efficient regeneration in M. spectabilis and combination of expression vector, we carried on the study of genetic transformation, mediated and induced by Agrobacteriunm tumefaciens. It was demonstrated that, 17.58% wounds was GUS-positive with GUS histochemical assay. Cry1Ab and GUS genes were transformed into the M. spectabilis and 2 positive plants were obtained by PCR preliminarily. |
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