Construction Of Melon Transgenic System Based On Somatic Embryogenesis

Transgenosis is one of the most effective techniques for functional analysis of plant genes,as well as a candidate approach for future crop molecular breeding.However,transgenosis could be hardly applied in most vegetable crops till now,due to factors such as immature regeneration systems.For example,the traditional regeneration system for melon,an important vegetable crop in China,is well known for its low efficiency and high chimerism probability.In this study,a high-efficiency melon regeneration system via somatic embryogenesis was established.The main results are as follows:1.A melon regeneration system based on somatic embryogenesis was extablished.Through the screening of hormone types and hormone concentrations,as well as to the different melon genotypes.,Cucumis melo L.var cantalupensis'Vedrantais' was found to exhihibt the highest efficiency of somatic embryogenesis under hormone combination 2,4-D and 6-BA.While,the other melon genotypes successed at callus formation,but hardly produce any somatic embryo.In addition,the optimal medium formula for somatic embryogenesis inducing(1L)is:MS + 30g sucrose + 2mg/L 2,4-D + 0.1mg/L 6-BA;rooting medium formula:MS +15g sucrose2.The super-agrobacterium based on pBBRacdS was constructed by dual plasmid method.To improve the efficiency of Agrobacterium-mediated melon transformation,the plasmid vector pBBRacdS was introduced to achieve ACC deaminase activity into Agrobacterium tumefaciens,which could reduce the ethylene content in explants or callus cells after Agrobacterium infection and finally improve the regeneration efficiency.Afterwards,the target gene CmWRKY27 overexpression vector was electrotransferred into Agrobacterium tumefaciens containing pBBRacdS by dual plasmid method to infect melon.3.Genetically modified melon was obtained via somatic embryogenesis pathway.Melon seeds were cut into pieces and pre-cultured for two days,and then co-cultured with Agrobacterium tumefaciens containing two vectors after infection.Liquid selection medium:El liquid medium +5 mg/L Hygromycin + 25 mg/L Timentin.Solid selective medium:MS+15g sucrose + 15mg/L Hygromycin + 100 mg/L Timentin.The transformation efficiency was about 0.18%.We found the system has relative higher screening efficiency than the traditional transgenic system in melon.In this study,a melon regeneration and transgenic system based on somatic embryogenesis was established to improve regeneration efficiency and reduce chimeric probability.It will contribute to the functional gene research of melon and provide an important technical for future molecular design breeding of Cucurbitaceae crops.