Establishment Of Eggplant Rootstock Grafted Solanum Torvum Genetic Transformation System Mediated By Agrobacterium

Solanum torvum Swartz,due to its resistance to diseases and insect pests,is a good rootstock grafted with eggplant.It is also a typical low accumulation of cadmium(Cd)plant.Thus,it's essential to investigate the physiological and molecular mechanism of S.torvum response to Cd,and will shed light on using biotechnology to breed low heavy metal accumulationcrops and vegetables.Furthermore,the underlying mechanisms of S.torvum StZIP5 and StZIP11 genes,the metal ZIP family transportersresponse to Cd accumulation,is still unclear.In this study,The following issues were investigated.Firstly,we established the explant regeneration system.Usingd hypocotyl of S.torvum as explant material,we studied the hormone ratio of callus induction,secondary culture of bud differentiation and rooting culture,and determined the screening pressure of hygromycin in callus-induced plant regeneration.Secondly,we set up the plant genetic transformation.Based on the optimized regeneration system of tissue culture,the effects of factors such as pre-culture time,concentration of bacteria solution and co-culture time on the genetic transformation efficiency of agrobacterium-mediated S.torvum were studied.Additionally,we revealed the preliminarily physiological and molecular mechanisms of StZIP5 and StZIP11 genes in tobacco.The effects of overexpression of S.torvum StZIP5 and StZIP11 genes in tobacco on the active chlorophyll content of antioxidant enzyme(SOD,POD,CAT and APX)in seedlings under the stress of Cd and the photosynthesis parameters were studied.The antioxidant enzyme(SOD,POD,CAT and APX),chlorophyll content,and photosynthesis parameters were studied in the overexpression of S.torvum StZIP5 and StZIP11 genes in tobacco under Cd stress.The main results were as followings:1.Based on the analysis of the ratio of 25 hormone combinations,the induction and bud differentiation of hypocotyl callus were conducted with 2 mg/L 6-BA + 0.1 mg/L IAA,with a proliferation coefficient of 6.62.In the secondary culture,the same hormone type and concentration ratio could maintain a high proliferation coefficient of 6.62.The best medium for rooting culture was 1/2 MS,and the rootingrate was up to 90% after 2 weeks of culture.The concentration of 8mg/L hygromycin was a suitable concentration for regeneration of Solanum torvum explants.After 1d of explant pre-culture,optical density of the bacterial solution was 0.4-0.6 and co-cultured for 2d,the explant grew normally with good conversion effect,and the conversion efficiency was up to 12.5%.2.We also transferred StZIP5 and StZIP11 genes of Solanum torvum into tobacco to obtain transgenic offsprings of tobacco,and carried out preliminary physiological,biochemical and phenotypic analysis on the offspring of StZIP11 transgenic tobacco.Physiological and biochemical analysis showed that chlorophyll content and light energy capture efficiency in the leaves of StZIP11(ZIP11OX)overexpressed transgenic tobacco seedlings were reduced under the stress of Cd.The antioxidant enzyme activity analysis showed that the relative activities of SOD and POD enzymes in the overexpressed ZIP11 OX tobacco leaves were significantly higher than those in the wild-type tobacco leaves.There was no significant differences in the relative activity of CAT enzyme.The relative activity of APX enzyme decreased significantly under the stress of 20?M Cd.On the other hand,the relative activity of SOD and POD enzyme in the overexpressed ZIP11 OX tobacco root decreased significantly compared with the wild-type.The relative activity of CAT enzyme was significantly increased in ZIP11 OX,but the difference between the two overexpressed lines was not significant.Under the stress of 20?M Cd,the relative activity of APX enzyme decreased significantly in ZIP11 OX strain.Our study successfully established an efficient callus induction and plant regeneration system of S.torvum tissue culture,which laid a foundation for agrobacterium-mediated genetic transformation of S.torvum.The molecular mechanism and physiological effects of S.torvum StZIP5 and StZIP11 genes overexpression in plants were preliminarily investigated,which provides a basis for further study the function of the two genes.