| In this experiment, the plant regeneration systems of Phalaenopsis hybrid and Sedirea japonica were established, the antisense expression vector pBI-antiACO was constructed by inserting the fragment of ACO gene which obtained from total RNA of Phalaenopsis and DNA of Sedirea japonica by RT-PCR into pBI121, a high-efficiency and systematic transgenic procedure mediated by Agrobacterium with antisense ACO gene was developed from the protocorms, the transformants were verified by histochemical GUS staining,PCR and Southern blot. The main results were described as follows:1. The plant regeneration system of Phalaenopsis was established and optimized. The protocorm-like bodies (PLBs) and regenerating planets were obtained from the explants of embryos, cutting segments of flower-stem-knot, and young leaves by adjusting the factors such as different media, hormone combination and concentrations, natural additives etc. The results show that the suitable medium for germination of embryos was 1/3MS+ KT 0.5 mg/L + sucrose 20.0g/L + CW15% + AC 2.0 g/L + agar 8.0g/L. The most suitable media for induction of PLBs from cutting segments of flower-stem-knot and young leaves were Hyponex1 2.5g/L + Hyponex2 0.5g/L+TDZ 1.0mg/L +2,4-D 1mg/L + sucrose 30 g/L + CW 10% + AC 2.0 g/L. The optimizing medium for prolification of cluster sprouts was Hyponex1 2.5g/L + Hyponex2 0.5g/L + 6-BA 5.0mg/L + sucrose 20g/L + CW 10% + agar 8.0g/L. The rooting medium was Hyponex1 3.0 g/L + NAA 0.5mg/L + AC 2.0 g/L + sucrose 20.0 g/L + agar 8.0 g/L.2. The protocorms of Sedirea japonica were obtained from embryos by no-symbiosis germination. The concentration of major salts and the phytohormones in MS medium affect the germinative capacity of the embryos .The suitable medium for germination of embryos is 1/4 MS + KT 0.5 mg/L + CW 10% + sucrose 20g/L + agar 7.0g/L. It is all conductive to germination of embryos to add coconut water, peptone, potato and activated charcoal in MS medium. 1/3 MS + 6-BA 2 mg/L + NAA 0.01 mg/L + coconut water 10% + peptone 2.0 g/L + AC 2 .og/L + sucrose 20.0 g/L + agar 7.0g/L was the optimum medium for multiplication of PLBs. 1/2MS + NAA 0.5mg/L + IBA 0.5mg/L + sucrose 20.0g/L + AC 2.0g/L + agar 7.0 g/L was very suitable for differentiation and rooting.3. According to the reported sequences of ACO in Orchidaceae, The partial cDNA of the ACC oxidasegene was obtained from total RNA of Phalaenopsis hybrid and Sedirea japonica by RT-PCR. The antisense expression vector pBI-antiACO was constructed by inserting the fragment of ACO into pBI121 between the CaMV35S promoter and gus gene.4. The effect factors of Agrobacterium tumefaciens-mediated transformation of Phalaenopsis hybrid and Sedirea japonica were optimized. The best transient expression of GUS was obtained under the conditions as follows: pre-culture for 3 d,OD600≈0.4, infection for 15min, and co-culturing for 3d and appended 100 pmol/L AS.5. Transgenic plants were regenerated from the protocorms of two species orchids after being inoculated with Agrobacterium tumefaciens stains EHA105 harboring pBI-antiACO.6. The presence and expression of the transgenes in two orchids were assessed by histochemical GUS assay. The molecular assays, including PCR and Southern blot, were carried out to verify Phalaenopsis transformants.The aim of this work is to prolong display life of Phalaenopsis and Sedirea japonica flowers permanently. These results would be of some importance for the new method of the prolonging life of flowers and the technique of the transgenic research in Orchidaceae. |
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