Drought And Salt-alkali Resistance Gene P5CS Transformation Of Sunflower Inbred Lines

The drought and salt tolerance geneâ–³¹-pyrroline-5-carboxylate synthetase (P5CS) has been transfered into sunflower mediated by Agrobacterium tumefaciens. And transgenic plants were obtained and identified by PCR and PCR-Southern blot.This dissertation provides new experimental consult for sunflower salt-alkali stress breeding.1. The gene P5CS(1905bp) was amplified using the special primers by PCR from pBLP5CS-F 129A plasmid and cloned in plasmid pBS-T.The recombinant plasmid was digested by BamH I and Sal I ,the destination fragment reclaimed and inserted between the CaMV35S promotor and NOS terminater in expression vector-pCHF3.The plant expression vector pCHF3/P5CS was transfered into Agrobacterium tumefaciems strain LBA4404 by freeze-thaw method and the plant expression binary vector was constructed and identified by PCR of pCHF3/P5CS vector extracted from LBA4404.2. For genetic transformation of sunflower by LBA4404 with P5CS gene, maintainer line 75-33B and restorer line Ht80-97 were used. After transformation, transgenic plants were obtained and detected by PCR and PCR-Southern blot, PCR product of pCHF3/P5CS plasmid served as positive control and PCR product of DNA from non-transgenic sunflower as negative control.It was revealed that two transgenic plants from maintainer line 75-33B and four from restorer line Ht80-97 were positive. The frequency of genetic transformation are 3.17% and 4.65% respectively.3. In the experiment the concentration of Kan was 25 mg/L for screening of resistance shoots of sunflower, and concentration of agrobacterium was OD_600nm≈0.8, three infecting times: 10 min, 20 min and 30 min were used. The statistical analysis on the number of shoot regeneration indicated that the leaf discs of sunflower dipped in agrobacterium liquid for 30 min are better for buds differentiation and increasing transformation efficiencies.