Genetic Transformation and Detection of the Cymbidium hybridum Modified by Coat Protein Gene of ORSV

ObjectiveTo optimize the transformation system and identification methods for obtaining the germplasm resources of anti-virus Cymbidium hybridum by plant transgenic technology. MethodThe coat protein gene (CP) of ORSV was cloned from the cDNA of infected C. hybridum. After sequencing, the gene was constructed into pBI121 expression vector and transformed to the protocorm-like bodies (PLBs) of C. hybridum by Agrobacterium tumefaciens-mediated method. ResultThe genetic transformation system was optimized and the identification method of the transgenic plants was established by nest polymerase-chain-reaction (Nest-PCR). There were 32 transgenic plants of C. hybridum detected by Nest-PCR. ConclusionThe Agrobacterium tumefaciens-mediated transformation method of C.hybridum was optimized using PLBs as explants. The antibiotics(kanamycin)screening concentration was determined as 5% to 10% PLBs survival rate. Using the Nest-PCR to detected the transgenic plants was more sensitive and accurate than conventional PCR.