Construction Of Regeneration System In Vitro Using Cotyledonary Nodes As Explant And Explore On Genetic Transformation Of Salt-tolerance Gene StNHX1on Cucurbita Maxima Duch

Pumpkin (Cucurbita) is a traditional vegetable in China, which originate from Latin America andSouth America. There are abundant germplasm resources and more than500year cultivation history inChina. Its leaf, flower and fruit can be used as vegetable and its seed which has therapeutic diet andhealthy function can be fried. Besides, pumpkin is used as stock for other Cucurbites because of itsdeveloped root system, strong growth and strong resistance. There is remarkable difference of resistanceamong germplasm resources so that screening for high resistant stock is research hotspot currently. Invegetable production, the soil secondary salinization which is the main factor in lower yield and qualitybecomes more serious as the cultivation time goes longer in protected area. The existing stocks forCucurbites show weaker salt tolerance so the demand for grafting under soil salinization is not satisfied.There has not been obvious progress in traditional breeding for salt tolerance stock. Germplasminnovation and improvement using genetic engineering is thought to be an important route to amelioratesalt tolerance in pumpkin. The premise of genetic engineering is to establish regeneration system withhigh efficience and stabilization. Meanwhile, it is a base for gene function study and genetictransformation.This study has confirmed the factors of high efficient pumpkin regeneration system usingcotyledonary node as explant, explored the main factors of transformation (The gene StNHX1codingNa+/H+antiporter in vacuole from salt tolerance eggplant TorvumVigor) to C. maxima cv.‘Bei Guan’using Agrobacterium mediated transgenesis, built the regeneration system of ‘Bei Guan’ and themethod of gene StNHX1transformation. So this study is a foundation of pumpkin salt tolerance breeding. The main results and content are as follows:(1) Regeneration system of ‘Bei Guan’ using cotyledonary node: The best sterilization timeusing0.1%mercury bichloride is9minutes. The best medium of aseptic seedling is MS. The bestinoculation method is radicle insertion directly and pregermination previously. The best explants foraseptic seedling is green cotyledon when unfolding slightly. The best cutting method is half cotyledonarynode first and then cutting half lengthways. The best medium for indefinite bud differentiation is MS+1.0mg/L6-BA+4.0mg/L AgNO3and the best medium for root striking is MS. The indefinite bud with2-3roots can be transplanted for integrated plant.(2) The main factors of StNHX1gene transformation to C. maxima cv.‘Bei Guan’ usingAgrobacterium mediated transgenesis: The best annealing temperature for primers is54℃afterexploring annealing temperature using gradient PCR amplification. According to the primers,pCAMBIA3301-StNHX1is investigated through PCR, showing positive colony. After screen, the optimalcondition for transformation is as follows:3mg/L bialaphos,36h preincubation,10min inoculation timeusing OD600(0.3-0.5) Agrobacterium,250mg/L carbenicillin adding to sterile water3times and24hco-culture (Adding200μmol/L AS to medium).(3) Two positive plants were identified after GUS staining.