Primary Study On Salt-tolerance Of Lisianthus Transformed By SeNHX1

High salinity is one of the major stresses effecting the crop plants growth, development and yield. At present, the population is expanding gradually; one-fifth of irrigated agriculture is adversely affected by soil salinity. Developing salt-tolerant crops and utilizing salty soil have been an urgent global question. The rapid advance in molecular biology promotes gene technology to become the focus in the field of salt-tolerant plants instead of traditional breeding techniques. Recent progress has been made in the identification and characterization of the mechanisms that allow plants to tolerate high salt concentrations. A lot of relevant salt-resistant genes have been cloned and expressed in other species successfully, and some transgenic plants with higher salt-tolerance have been reported.In this study, to develop a salt-tolerant Lisianthus, a vacuolar Na~+/H~+ antiporter gene SeNHX1 from Salicornia europaea L. is transferred into Lisianthus cultivar, using an Agrobacterium-mediated method. Firstly,the functional gene is cloned and inserted into the vector pBin438 with two 35S promoters and one enhancer, and a high efficient expression vector pBin438-SeNHX1 is obtained. The acceptor system Lisianthus is one of the top seven cut flowers in the world. Its regeneration system and related gene transformation have appeared in some literature. This research optimizes the leaf regeneration and Agrobacterium transformation system on the base of previous results. The optimal leaf differentiation medium is MS~+6-BA0.5mg/L~+ NAA0.1mg/L, with the regenerating rate of callus 70%; Shoot elongation medium is MS~+GA30.8mg/L~+NAA0.1mg/L~+IAA0.2mg/L; root regeneration medium is 1/2MS ~+6-BA0.5mg/L~+ NAA0.1mg/L, with regenerating rate of root 42%. The best Agrobacterium transformation condition is A.tumefaciens concentration: OD600=0.6, inoculated time: 20min, co-culture time: 3 days, concentration of Cef:300mg/L,concentration of Kan:40mg/L , concentration of AS:100μmol/L. 32 seedlings are gotten through the choice of 60mg/L Kan, and continuing choosing is made by 200mmol/L NaCl medium. According to PCR test, some Kan resistant plantlets are positive and preliminary results prove that gene had integrated into genomic DNA. The content of chlorophyll and proline in the leaf of Lisianthus dealt with 200mmol/L NaCl have been compared with control plants. Therefore, transgenic plantlets with SeNHX1 gene have enhanced their salt tolerance.