Developing Anti-disease Material Of Potato(solanum Tuberosum L) By Bio-technology

Potato (solarium tuberosum L.) is one of the major food crops as well as vegetables which has been cultured world-wide for a long period. To promote the ability of disease-resistance is a primary target of potato breeding. In many tetraploid commercial cultivars, potato has a homophylic tetrasomic segregation patterns , which always result in male sterility and self-incompatibility. So it is very difficult to get a new variety with resistance to bacterial and fungal phytopathogens by traditional breeding method. Whereas plant genetic engineering, by which new efficient anti-phytopathogen genes can be introduced into potato, display a promising prospects in the improvement.In this study, an efficient transformation via Agrobacterium tumerfaciens system was developed. Through this system a synthetic gene GLU-CHI, which was resistant to most of fungal phytopathogens, and SPCEMA gene, which has a broad-spectrum resistance to bactrial and fungal phytopathogens, were introduced into potato cultivar "Taiwanhong".l.The efficient transformation systemsThe major elements controlling DNA delivery to potato wereinvestigated and optimal conditions for the cultivar " Taiwanhong" were founded. Stems and leaves, pre-cultured on MSI (MS+BA2.5mg/L+ 2,4-D0.5mg/L) for 4 days and 8 days, respectively. The OD60o of the Agrobacterium suspension was adjusted to about 0.7 and then stems and leaf discs were dropped into the suspension for 5 min. Then they were co-cultured on MS for 3 days under 23~25癈 in darkness. After co-culture all the explants were washed by sterile water on rotating bed with 120rpm for 30~60min. Subsequently, all of them were transferred to MSI (including Kan50mg/L and CeOOOmg/L) for inducing resistant calluses for 15~20d. Then theresistant calluses were cultured on MS2 (MS+ BA2.5mg/L+GA35.0mg/L+ZT1.0mg/L, including KanlOOmg/L and Cef300mg/L) for inducing resistant shoots for 20~40d. Finally, transformed plants were cultured on MS+ KanlOOmg/L to check if they were able to root, and then stems and leaves of which can root were used for another round of regeneration to check if they can induce Kan resistant shoots again.2. Confirmation of transgenic plantsIn the transformation of GLU-CHI gene, 135 leaf discs and 115 stems were used, from which 18 and 47 Kan-resistant shoots were obtained respectively, with the transformation frequency of 13.33% and 40.87%. As the SPCEMA gene, 120 stems were used and 12 Kan-resistant shoots were obtained. The transformation frequency is 10.00%.The transformants were confirmed by PCR .The results show that the target gene had been integrated into potato accompanying with NPTII gene.Crude enzyme liquid was extrated from 9 transformants of GLU-CHI gene and the activity of chitinase were qualitatively determined. Method of the multiple comparisons (LSD, least significant difference ) was carried on the level of a =0.01 to analysis these data. The result showed that, except for 2 of them, the activity chitinase of transgenic lines was badly obviously higher than that of control. It indicated that exogenous DNA had been expressed in transgenic plants.