Studies On Marker-free Transgenic Insect-resistant Gene CpTI Maize

In the experiment, put the CpTI insect resistant gene and hyg selectable marker gene into two MAR sequences separately, used Agrobacterium super binal vector to transfer the embryo callus of maize inbred lines 18-599, which is used widely in yield. Co-cultivate three days and select three generations continuously in HygB culture medium. Obtained regenerated plants after differenttiation. Transformants were confirmed at the first time by PCR amplification, PCR-Dot blotting and PCR-Southem blotting hybridizations. In addition, co-cultivate temperature, co-cultivate pH and other factors which can effect the efficiency of transformation were discussed.The conclusions are as follows:1. The time that Agrobacterium reach to logarithmic growth in liquid medium is changeable. To observe and detect frequently is a stably and reliably approach to get the liquid medium which contain logarithmic growth Agrobacterium.2. The lower co-cultivate tempeture and co-cultivate pH can heighten the transformation efficiency to maize callus do not incarnate in the experiment, at least, the conclusion is incorrect to the maize genotype used in this experiment.3. The transformation efficiency is lower determined by the quantities of resistant callus.4. PCR amplification, PCR-Dot blotting and PCR-Southern blotting hybridizations can prove primarily that the target CpTI gene has integrated into the genome of regenerated maize plants, and gained T1 generation seeds.After the self-crossbreeding of transgenic maize plants, we can wipe off the plants contain selectable gene combined with molecular detection, and reserve the transgenic maize plants which have aimed gene merely , and get marker free transgenic plants(MFTPs) finally. It can enhance the security of food and environment of transgenic plants, and vail to more transgenic manipulation.