| In this experiment, a high-efficiency and systematic transgenic procedure mediated by Agrobacterium tumefaciens with antisense ACS gene was developed from mature embryos in Phalaenopsis amabilis, and studies on cloning of ACS gene from the labellum of P. amabilis were also carried out. The main results were described as follows:1．The plant regeneration system of P. amabilis was optimized. The transgenic system of subtle yellow PLBs was established by adjusting the factors such as different hormone concentrations, natural additives, different sorbensations, inoculation disposation, light intensity and subcultural time, medium forms during the induction and growth of protocorm-like bodies of P. amabilis. The results showed that either the induced medium or the best multiplication medium of subtle PLBs was KC+6-BA6．0 mg/L + NAA0．2mg/L +AC0．5 mg/L+Pj100g/L in the dark for 50 days, PLBs cultured on KC+6-BA3．0 mg/L+ NAA0．5mg/L+AC1．0 mg/L+Pj 150g/L differentiated to shoot in the light, plantlets cultured on KC+6-BA2．0 mg/L+NAA 0．5mg/L+AC1．0 mg/L+Pj 150g/L rooted best. It was good for transgenic systems to use PLBs with incised growing point as the explants. The PLBs were not very sensitive to kanamycin, and PLBs of P. amabilis could be killed with kanamycin concerntration as high as 500 mg/L, while the concerntration of 150 mg/L for kanamycin which proved to be restricted rooting of transgenic plantlets was used for selecing transgenic plantlets.2．. The introduction of antisense ACS gene to P. amabilis was conducted, and the optimized parameters used in Agrobacterium-medialed transformation were obtained. The best transient expression of GUS was obtained under the conditions as follows: the PLBs which were not precultured but were pretreated with 0．2 mol/Lmannitol for 4 h; and the Agrobacterium suspension was with OD value of 0．3; the samples were infected for 20 mins, and then they were transferred onto the KC medium (pH 5．4) supplemented with 0．2 mg/L NAA and 6．0 mg/L6-BA for co-cultivation for 4 days at 25℃in the dark.3．A whole technical system of P. amabilis resistant PLBs, transgenic plant regeneration and transgenic assay was established. The indirect method of selecting resistant PLBs was that after co-culture, the protocorm-like bodies were cultured on KC+6-BA6．0mg/L+NAA 0．2mg/L+AC0．5 mg/L+Pj 100g/L+Cef200 mg/L for one month, then selected on KC+6-BA6．0 mg/L+NAA0．2 mg/L +AC0．5 mg/L+Pj 150g/L+ Km500 mg/L +Cef 200 mg/L for one month, transferred on KC + 6-BA3．0 mg/L+NAA0．5 mg/L+AC0．5mg/L+Pj150g/L+Cef200mg/L when PLBs were differenti-ated, rooted on KC+6-BA2．0mg/L+NAA0．5 mg/L+Cef 200 mg/L+Km150 mg/L. At last, 7 small resistant plantlets were maintained for further studies.4．The subsequent studies on the cloning of the ACS conservation region from the labellum of P. amabilis was carried out.. The modified Trizol for extraction and purification of total RNA from the labellum of P. amabilis was developed. The ACS conservation region in P. amabilis,which was specifically expressed in pollinational labellum, was obtained by reverse transcriptase-polymerase chain reaction(RT-PCR). Conservation region amplified by PCR was reclaimed, connected, transformed and sequence analysed.The results showed that the ACS cDNA of P. amabilis was 925bp and encoded a protein of 308 amino acids.The sequence had high identities with that of ACS gene which had been cloned.This experiment was of great importance for genetic improvement in P. amabilis, which would provide technical groudwork for the study on P. amabilis transformation. In addition, it also provided groundwork for the study on self-vector construction and express regulation.
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