The Regeneration And Evaluation For Salt Tolerance Of Transgenic Sweetpotato Plants Expressing Cu/ZnSOD And APX

The embryogenic calluses derived from shoot apices of6sweetpotato (Ipomoea batatas (L.) Lam.)cultivars, including Xushu18, Xushu55-2, Xushu22, Xushu28, Xushu29and Xuzishu5, were used asmaterial. The induction efficiency of embryogenic callus, callus status, condition of suspension culturesand saving time were compared. The result showed that callus of Xushu55-2was excellent with highinduction efficiency and rapid proliferation. Xushu55-2was used to study the effect of ABA on somaticembryogenesis and plant regeneration. The result showed that ABA promoted the differentiation ofsomatic embryos and embryogenic callus treated by1.0mg/l ABA for7d then transferred to MSmedium could regenerate efficiently.The optimum antibacterial concentration of Cef was200mg/l. The selection pressure was Kan10,10,15, and5mg/l on solid medium selection, liquid medium selection, somatic embryo induction, andregeneration, respectively.Agrobacterium tumefaciens-mediated transformation system of sweetpotato was eatablished byusing embryogenic suspension culture of Xushu55-2. A. tumefaciens strain EHA105harboring a binaryvector pCAMBIA2300with Cu/Zn superoxide dismutase gene (Cu/ZnSOD), ascorbate peroxidase gene(APX). Neomycin phospho-transferase gene (NPTⅡ) was selectable marker gene. Embryogenicsuspension culture after subculture3d was cocultivated with the EHA105strain (OD600=0.5) for10min, then cocultivated for3d on MS medium contained with2.0mg/l2,4-D and20mg/lscetosyringone(AS). The cocultivated calluses were first antibacterial treated by600mg/l Cef for1hthen transferred to MS selection medium contained with2.0mg/l2,4-D,200mg/l Cef and10mg/l Kan.6~8w later,76Kanamycin-resistant embryogenic calluses were selected from1050embryogeniccalluses. Kanamycin-resistant embryogenic calluses were transfered to MS medium contained with1.0mg/l abscisic acid (ABA),15mg/l Kan then transferred to MS medium supplemented with5mg/l Kan7d later. Selected85transgenic plants randomly from598plantlets for PCR analysis. The result indicatedthat79regenerated plants were transgenic plants (92.9%). Southern blot analysis indicated the copynumber of foreign genes was1~3, which signified that the exogenous genes had been integrated into thegenome of sweetpotato.Transgenic plants of Xushu55-2were tested for salt tolerance identification. After incubated onnutritive soil with0.5%NaCl for2w, chlorophyll of transgenic plants declined slower, MDA andconductivity relatively lower compared with non-transgenic plants. The results showed that Cu/ZnSOD and APX improved stress tolerance of sweetpotato significantly. Three salt-tolerance strains ofXushu55-2were selected.