Study On Salt Tolerance Gene (betA) Transformation For Populus Simonii×P.nigra Pollen Plants

In this study, salt tolerance gene- bet-A (choline dehydrogenase gene ) was transformed into Populus simonii X P. nigra plantlets medted by Agrobacterium tumefaciens ,which obtained from pollen. bet-A can compose betaine only in one step .In plant , betaine is an important osmotic regulator .Under salt stress , accumulation of betaine can protect the structure and function of protein in cell decreasing the flow of water ,and the plant can acquire high salt-tolerant ability .In this paper ,the factors that affect plant genetic transformation were discussed ,and the genetic transformation system of Populus simoniiX P. nigra plantlets was found also .The results of PCR amplification and Southern blotting hybridization show that the betA gene has been integrated into the genome of Populus simoniiX P. nigra .Under salt- stress condition , the transgenic plants have higher salt tolerance ability than those of control plants . The results it as follows:1. The select concentration of Kanamycin was 40mgL-1.2. The optimal concentration of antibiotics and its effects on leaves differentiationof Populus simonii X P. nigra were studied. The results showed that the antibiotics-cefazolin sodium which made in Shang Hai and Harbin were the best alternation and the optimal concentration was 700 mg-L-1.3. The transgenic system of Populus simoniiX P. nigra plantlets obtained from pollen was set up:Pre-culture: the leaves cultured in differentiation medium (MH+6-BA 0.5mg L-1+NAA 0.05 mgL-1+sucrose 25 g L-1+agar 6 g L-1) for 2days ;Dip: dipped the leaves on the Agrobacterium (OD600=0.1) ,the infection time was3~5 minutes ;Co-culture: Co-cultured in the differentiation medium for 2~3 days, Selective culture: the leaves in the select medium(MH+ 6-BA 0.5 mg L-1+ NAA 0.05 mg L-1+Km 40mg L-1+cefazolin sodium 700 mg L-1 + sucrose 25 gL-1+ agar 6 g L-1). We obtained 4 transgenic plants ,and analyzed by Southern blotting and PCR amplification .The results showed that the bet-A has been introduced into the genome of Populus simoniiX P. nigra ,the transformation rate was 1.23% . Rooting: On the select rooting medium (MH+0.2mg L-1 IBA+ Km 20mg L-1 +cefazolin sodium 300 mg L-1+ sucrose 20 g L-1 agar 6 g L-1), all the transgenic plants produce roots. The 485 transgenetic seeding survived in the green house.4. The salt tolerance test showed that all the transgenic plants have high in salt tolerance ability than those control plants .T1 andT2 can survive on the medium which contains 0.75% NaCl . T3 can survive in 0.65% ,T4 can survive in 0.70% NaCLThe sequence of salt tolerance ability from high to low was T2, T1, T4, T3.