Studies On The Transformation Of Citrus With Chit42 And PhyB Genes

Citrus ranks the number one in the world fruit production, The conventionalbreeding program in citrus has long been hampered by such problems aspolyembryony, apomixis, long juvenility, high heterozygosity and arthenogenesis.Fortunately, genetic transformation technique is now providing us an "new, fastand direct" strategy to citrus genetic improvement. In this study, the transformation ofphyB gene into P. trifoliata epicotyl segments mediated by agrobacterium wasconducted. Genetic transformation of mature tissues can overcomes the long juvenileperiods. In this study, the gene chit42 was introduced into citrus. Mature citrus stemswere used as explants to research its regeneration and agrobacterium-mediatedtransformation system. The main results are as follows:1. The agrobacterium-mediated transformation of epicotyl segments wasopitimized. We confirmed that MS medium containing 1 mg/L 6-BA was the bestculture medium for bud bursting. Explants from 25-day-old seedlings showed highertransformation frequency than younger and older plant materials. The suitableconcentration of Kanamycin for selection was 75mg/L. A total of 27 grafting shootwere gained. PCR analysis indicated that the phyB and nptâ…¡genes were integratedinto P. trifoliata.2. Regeneration and transformation systems from mature plant material wasstudied. The result showed that the contamination rate of the pretreatment disinfectionmethod was the lowest and the intemode stem segments of citrus about 1cm long wasbest; The best culture medium for Newhall navel orange and Bingtang orange'sadventitious buds induction was MS+BA 3 mg/L; MS+BA 3 mg/L+0.5mg/L NAAis the best culture medium for Citrus reticulate Blanco. The suitable concentration ofKanamycin for the initial stage selection was 30mg/L.3. In vitro shoot-tip grafting of the regenerated mature shoots on seedlingrootstocks provided a rapid and efficient system for plant production. Bingtang sweetorange and ponkan obtained resistant shoot and Newhall obtained grafting shootwhich indicated that the nptâ…¡gene was integrated into by PCR analysis.