Evaluation of parameters for high efficiency gene transfer via particle bombardment in Indian mulberry.

ABSTRACT

Particle bombardment is a popular method of direct gene delivery into cell, tissue and organs since it requires minimum pre- and post-bombardment manipulation. In addition, this technique is much easier and fast to perform with intact tissue/organ and reduces the period of in vitro culture. Genetic transformation of mulberry, Morus indica cv. K2 was attempted by particle bombardment using hypocotyl, cotyledon, leaf and leaf callus explants. The effect of various physical and biological parameters during bombardment were studied by the histochemical localization of GUS reporter gene following two days of bombardment and by assessing the number of blue spots per explant. p35SGUSINT was used for optimization of different parameters. The percentage of GUS positive explants was very low with tungsten (20%) as compared to gold particles (36%) indicating tungsten toxicity to the tissue. Maximum GUS activity was observed at 1100 psi helium pressure and 9 cm target distance for hypocotyl, cotyledon and leaf. Double bombardment of explants with 10 microg of DNA loaded on macrocarriers clearly yielded a better (up to 56%) result as compared to a single bombardment (30%). Amongst the various plasmids tested, pBI221 gave the highest (100%) GUS positive explants in the leaf callus.