Transformation Of D.phalaenopsis Banyan Pink And D.nobile With CHSA And AP1 Genes

As one of important ornamental plants, Orchids have an important commercial importance in world flower industry. Taxonomically, Orchidaceae is the largest family of flowering plants among monocotyledons, which occupies wide ranges of ecological habitats and exhibits highly specialized morphological, structural, and physiological characteristics. It is an ideal material to study flower pigmentation and ovule initiation and development. The inefficient transformation of orchids,however, hampered the investigation of orchid gene function and regulation in vivo. Orchid breeding by traditional sexual hybridization was time-consuming due to the long growth and reproductive cycle of these plants, mean while genetic engineering method offers an effective way to introduce novel characters into orchids. Until recently transgenic orchid plants have been reported for only nine genera of orchids.Dendrobium orchids are the firstly genera in Orchidaceae plants in terms of their amounts. Orchids are valuable ornamental plants, globally used as cut flowers and potted plants. We successfully transformed two Dendrobium orchids (Dendrobium phalaenopsis Banyan Pink and Dendrobium nobile ) with a relatively high efficiency via Agrobacterium-mediated transformation. The protocols provided here are simple, repeatable and effective. In order to alter the flower color and flowering time of Dendrobium orchids, we transformed the orchids of Dendrobium phalaenopsis and Den. nobile with the CHSA(chalcone synthase A) gene and the floral meristem identity gene API via Agrobacterium-mediated transformation of protocorm-like bodies (PLBs) and callus, respectively. The CHSA gene was cloned from Petunia hybrid, and the AP1 gene was cloned from Arabidopsis. A rather high transformation efficiency of 18.3% with AP1 and 16.7% with CHS was obtained. Integration of AP1 transgenes were confirmed in transgenic D. nobile orchid plants by Southern hybridization. The copy number of the integrated genes ranged from one to four, indicating a multiple insertion of the genes and independent transformation events and random integration. Southern blotting analysis showed that the CHSA inserted in thegenome DNA of transgenic lines with a single copy. Northern blotting analysis indicated the transcription of CHSA gene. We did not observe co-supression in transgenic lines. Our results suggested that long inoculation period is one of the major factors which reduced the tranformation efficiency significantly.