Optimization Of Regeneration System And Establishment Of Genetic Transformation System Of Pumpkin

Pumpkin is one of the most widely-planted crops in China,which has a good nutritive and ornamental value,also has a strong resistance capability and can be used as rootstock in cucurbit plants grafting.Diseases are very serious at present,which affect the yield and quality of pumpkin.Creating disease-resistant pumpkin variety by genetic engineering technologies plays an important role in solving such problems,and can also implement the variety improvement of pumpkin.But the past researches on pumpkin regeneration and genetic transformation is relatively limited.In this study,pumpkin was used as material,and 1/2 cotyledon near hypocotyl was used as explant to establish a high-efficiency regeneration system and explore the effects of different concentrations and proportions of phytohormone and AgNO₃ on pumpkin shoot inducing.Then investigating the agrobacterium-mediated genetic transformation of pumpkin based on the mentioned system.AtCYCD3.1 was used as target gene in this study to construct pC2300-35s-eGFP-AtCYCD3;1overexpression vector.We also make a study on the influence of preculture time,infection time and co-culture time on resistant shoots inducing in pumpkin genetic transformation process.The main results of this experiment are the followings:1.Different concentrations and proportions of phytohormone have dramatically effects on explants shoots inducing.Cotyledons were used as explants and cultured on MS supplemented with 35 different concentrations of phytohormone to induce shoots.Results showed that the frequency of regeneration plantlets was influenced by the concentrations and proportions of phytohormone in medium.Within a certain concentration,6-BA appeared to promote the efficiency of shoots regeneration.The addition of ABA in MS caused chlorosis problem of cotyledons,however,the combination of 6-BA and ABA could effectively promote the regeneration frequency of explants.The highest frequency of shoots regeneration?90.26%?was observed from explants cultured on MS containing 1.0 mg?L^-1 6-BA and0.5 mg?L^-1 ABA.The elongated shoots were cultured on MS for rooting,plantlets appeared healthy after transplanting.2.Preculture,infection and co-culture time have significant effects on pumpkin genetic transformation.In the process of agrobacterium-mediated pumpkin genetic transformation,exploring the effects of different preculture time,infection time and co-culture time on resistant shoots inducing on the basis of high-efficiency regeneration system,eventually determining the optimized experimental condition.The results indicate that when the preculture time of cotyledon was 24 h,the vacuum infection time of agrobacterium was 1.5 min twice,and the co-culture time between cotyledon and agrobacterium was 2 d,resistant shoots induced from explants were significantly higher than the others,and the frequency of resistant shoots regeneration was 14.64%.