Studies On RT-PCR Detection System Of GLRaV-â…¢ And Shoot-tips Culture Technology Of Cherry Rootstock

The fruit trees viruses are threatening growth and development of fruit tree seriously.After the several factors that affected RT-PCR were examined, the RT-PCR detection systems for GLRaV-Ⅲwere established and optimized.The sequences of GLRaV-Ⅲwere analysised.The main research results were as following:1. The RT-PCR detection systems for GLRaV-Ⅲwere established. The highly efficient and lowly cost RT-PCR detection systems for GLRaV-Ⅲwere obtained by optimizing.The proper concentration of dNTPs and TaqE was 100μmol/L and 1U,respectively.The concentration of complementary primer and homologious primer should be 0.3umol/L-1.5umol/L. The concentration of MgCl2 was 0.9mmol/L. Reannealing time was 60 senconds and Reannealing temperature was between 52℃-58℃. Denaturation time and Denaturation temperature was 60 senconds and 94℃, respectively.Extention time was 2 minutes and the Cycles was 25 times.2. The fragments of the partial CPgene of GLRaV-Ⅲwere obtained by RT-PCR detection technique and were cloned to the pUCmT vectors successfully.Then,the inserted fragments were sequenced. The homologies of DNA sequence were up to 99.67%,compared with the published ones.So far the combination of shoot-tips culture and heat treatment is a effective way to eliminate the viruses of fruit trees.The establishment of the shoot-tips culture system is the basement of viruses elimination and rapid propagation of young virus-free plant. The major object of this study is to establish the technique systems of Colt shoot-tips culture. Faotors influencing propagation were optimized and discussed, A relatively optimal culture system for Colt shoot-tips is established. The optimized medium for shoot-tips initial culture was 1/2MS+ 1.0 mg/L BA + 0.1 mg/L IBA. In subculture multiplification, the optimized medium was 1/2MS+ 1.0 mg/L BA + 0.2 mg/L IBA+0.5 mg/L GA3 in which the propagation ratio is high and the vigor of trees is strong.The suitable value of pH is 5.2-6.0, Suitable density of sugar is 2-3%. The optimized medium of rooting was 1/2MS+ 0.5 mg/L IBA in which the ratio of rooting was high and roots grew quickly and strongly.