Exogenous Gene Transfer And Mutation Analysis In Phalaenopsis

In order to optimize the conditions for P. amabilis callus-inducement, several factors known to influence callus-inducement were examined. Exposure to sodium hypochlorite for 30min was found to be the most suitable disinfection duration; a 15 mg/L 6-BA was optimal for stem bud proliferation; face-up placement of explants in the medium resulted in a significant increase in the number of PLBs formed calli per leaf block (14 versus 7 in control); coconut milk, when supplied at 20%(v/v) and combined with TDZ could serve as a useful additive for elevating formation. An efficient genetic transformation system, using Agrobacterium tumefaciens-mediated T-DNA delivery, is described. Leaf-derived embryogenic calli were co-cultivated with A. tumefaciens strains harboring the binary vector pCAM2300. Transformation efficiency was affected by the size of calli, and the concentration of A. tumefaciens, and the duration of co-cultivation. The transformation frequency can achieve 12.16% when co-cultivation for 20min and 0.4 (OD600) of A. tumefaciens suspension with 1-2mm callus. Integration of T-DNA into the genome of putative transgenic plants was confirmed by PCR and subjected to cold-tolerance. In response to cold acclimation, five physiological indicators were measured with stepwise change of temperature, i.e., electrolyte leakage, activity of superoxide dismutase (SOD), content of malondialdehyde (MDA), proline and total soluble sugars. Conclusions were drawn that LTP gene is delivered to P. amabilis and weighs heavily for low temperature acclimation. In total, 89 transgenic plantlets containing LTP gene can be a firm proof for this approach of Agrobacterium-mediated P. amabilis transformation.In the chemical mutation experiment, three types of P. amabilis calli treated with different concentration of ethylmethanesulfonate (EMS) lead partial calli to be albino, brownish or even death. While an ideal portfolio lies in the material of 15 days'callus blocks by sub-cultivation and the treatment of 0.4% EMS for 2-6h, the survival percentage after mutation extends averagely 56% equivalent to the effect of semi-lethal dose. EMS mutation shares broad spectrum.