Orchids (Cymbidium spp., Oncidium, and Phalaenopsis).
Recent advances in genetic engineering have made the transformation and regeneration of plants into a powerful tool for orchid improvement. This chapter presents a simple and reproducible Agrobacterium tumefaciens-mediated transformation protocol and molecular screening technique of transgenics for two orchid species, Oncidium and Phalaenopsis. The target tissues for gene transfer were protocorm-like bodies (PLBs) derived from protocorms, into which constructed foreign genes were successfully introduced. To establish stable transformants, two stages of selection were applied on the PLBs co-cultivated with A. tumefaciens. About 10% transformation efficiency was achieved in Oncidium orchid, as 108 antibiotic resistant independent PLBs were proliferated from 1000 infected PLBs. In Phalaenopsis orchid about 11 to 12% of transformation efficiency was achieved by using the present protocol. Different molecular methods and GUS-staining used to screen putative transgenic plants to confirm the integration of foreign DNA into the orchid genome were also described in detail. The methods described would also be useful for transformation of desired genes into other orchid species.
Chan
Ming-Tsair
MT
Institute of BioAgricultural Sciences, ACADEMIA SINICA, 115 Academia Sinica Road, Taipei, Taiwan.
Chan
Yuan-Li
YL
Sanjaya
D016960:Agrobacterium tumefaciens / Q000166:cytology / Q000235:genetics*; D018929:Cell Culture Techniques; D003470:Culture Media; D018744:DNA, Plant / Q000737:chemistry; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D029595:Orchidaceae / Q000033:anatomy & histology / Q000235:genetics* / Q000502:physiology; D018749:RNA, Plant / Q000737:chemistry; D012038:Regeneration; D046509:Tissue Culture Techniques; D014170:Transformation, Genetic
D003470:Culture Media; D018744:DNA, Plant; D018749:RNA, Plant