Study On Sugarcane By Microprojectile Bombardment-mediated Transformation With Bt Gene

Microprojectile bombardment is a transgenic method developed gradually at the end of 1980' The notable merit of this transformed method is that it is beyond the limit of receptors. So almost every material can be transformed by this method. The difficult course of producing and culturing protoplast is skipped. Simple tissue culture is enough. Microprojectile bombardment is a useful and convenient method of delivering foreign genes to plant cells, particularly members of monocotyledon which normally do not respond to agrobacterium-mediated vector delivery.E.coli plasmid pBIWIQ-5 containing Bt gene and kanamycin-resistant gene was as vector in this transformation. After harvesting from E.coli by alkaline lysis, plasmid DNA was proved having contained foreign gene by PCR. Then plasmid DNA was purified and adjusted to about lug/ul of concentration. Sugarcane embryogenic calli were bombarded with QJ-1000 particle gun using tungsten particles coated with plasmid DNA by CaCl2 and spermidine-3HCl. Transformed receptor materials were cultured on Ml medium for a week in order that bombarded materials recovered and expressed foreign gene sufficiently. Receptor materials were moved to M2 medium. New calli were cultured on M3 medium to induce sprouting. After 20 days, calli began differentiating shoots while non-transformed ones became brown and died or differentiated albino plants. Only partly calli, which had received plasmid DNA, formed embryoid and green plants. Tender plants were transferred to M4 medium when they were about 2 centimeters in height. Some plants, which rooted and formed obvious root system, were transplanted into soil.0.2-0.5 gram tender leaves were exacted total DNA by SDS when they were about 20 centimeters in length. Then PCR and southern analysis revealed most resistant plants had contained foreign gene. Anticipative signals of hybridization were appeared in resistant plants. The results showed that foreign gene Bt had been integrated into the genome of sugarcane successfully.Furthermore, factors affecting microprojectile bombardment-mediated transformation of sugarcane are discussed in this study. The future trends of transgenic technology applied in breeding of sugarcane are also discussed.