Studies On Insect Resistant Gene Transformation Of Populus Simonii×P.nigra Pollen Plantlets

In the base of optimal tissue culture system of Populus simoniiX P.nigra.: .to leaf-regeneration was MH+BA0.1mg/L; to root was MH+IBA0.4mg/L , genetic transformation experiment was processed.Agrobacterium mediated genetic transformation system was established: Firstly, the discs was cultured for 2 ~ 3 days; Secondly, dipped the leaves in the Agrobacteria(OD600 = 0.3 ~ 0.5), the infection temperature was 25 C ; Thirdly, co-culture without light for 2~4 days. Then, inoculate on the select medium which contain Kanamycin 40mg/L. In the same time, the select medium contained Cefazolin Sodium in order to inhibit the growth of Agrobacterium. At last, obtained Kanamycin resistant buds and inoculate them on the root medium (contained Kanamycin 40mg/L and cefazolin sodium 700mg/L).After established the genetic transformation system, the Spider toxin gene was used as foreign gene for this transformation experiment. Three transgenic plants were obtained and the transformation frequency was 0.62%, Gus detection showed that 75 percent were positive plants.The Molecule-Biology detection was also. 100 percent positive frequency was achieved by using PCR amplification; 35 percent positive frequency was obtained by southern blotting and the result of PCR southern blotting was 100%. The results showed that the foreign gene was transformed and integrated into the genome of Populus simonii X P. nigra .The insect feeding test indicated that the worm kill frequency was 62.5% and 92.5% when used Lymantria dispar 2 ages worm. The kill frequency of negative plants was 5% and 13.3%, lower than the positive plants. This result showed that the foreign gene could express in a high level. The growth of insects was restrained and the weight of control insects was 2.8 and 12.9 times to the treated ones.All the result showed that the foreign gene has integrated into the genome of Populus simonii XP.nigra and expressed normally.