Transformation of Dendrobium orchid using Agrobacterium tumefaciens

Transformed orchids were recovered from the protocorm like bodies (PLBs) of Dendrobium when transformed with Agrobacterium tumefaciens strains LBA4404 and EHA105 harbouring the binary vectors pCAMBIA2301, pCAMBIA1301 and PBI121. The PLBs were generated in vitro by culturing the shoot apices on half strength MS medium. Then the PLBs were infected with Agrobacterium tumefaciens by gentle swirling of the PLBs with the bacterial cells obtained from the centrifugation of bacterial suspension, and also with the bacterial suspension diluted to 1/5 v/v with fresh medium. Then the explants were co-culfivafed with the bacteria. Initially recovered PLBs following co-cultivation were allowed for further selection with kanamycin. The transformed PLBs regenerated under kanamycin selection, while the untransformed cells could not survive under kanamycin. The transformed PLB's were allowed to grow further with subculturing at fortnightly intervals on selection medium containing 200 mgl⁻¹ kanamycin. The newly formed PLB's and leaves were subjected to histochemical GUS assay and PCR, to confirm the transformation. The PCR amplification of the genomic DNA of the transformed planlets revealed the presence of npt II gene and thus the genetic transformation with Agrobacterium tumefaciens is confirmed.