Studies On Elimination And Detection Of Viruses In Siberia

Lily is a perennial herb of Liliaceae and it's an important economic plant, widely cultivated in China. Many new varieties have been introduced into China in recent years , however viral diseases occur in some areas where lily is planted and the influence of viral diseases is becoming more and more obvious. Virus-infected trees show reduced growth, abnormal leaves and flowers. So it's important and urgent to eliminate severe viruses.CMV,LSV and LMoV are three viruses that damage lily seriously. Some research work has been done on Chinese lily and lily in Europe, in which biological or serological methods were adopted to detect the virus, but no virus elimination research has been carried out on Siberia. It has been proved that molecular biology methods are more sensitive than biological and serological methods which may lead to wrong conclusion because of low virus concentration in plant. Elimination of virus such as CMV,LSV and LmoV of Siberia and molecular biology detection methods have been researched systematically in this paper.1. The establishment of tissue culture system of Siberia. Results showed that MS was the most suitable culture medium for the lily tissue culture. The most suitable groups of various ingredients of the culture medium are listed as follows: The best induction culture medium of squama and stem section was MS+ 0.1～1.2 mg/L BA+0.2 mg/L NAA;MS+0.05～0.5 mg/L BA(or KT)+0.02 mg/L TAA or 0.05mg/L NAA is the best for multiplication induction; The best induction culture medium for roots is MS+ 0.05mg/L BA+0.5 mg/L NAA.2. The systemica study of four methods that can eliminate the viruses of CMV,LSV and LMoV .To be specific, they are stem point culture; stem point culture combined with heat treatment; stem point culture combined with ribavirin; stem point culture combined with ribavirin and heat treatment. On the comparison of the four methods and the consideration of the survival percentage and the rate of elimination, it can be concluded that the virus elimination rate of stem point culture combined with ribavirin and heat treatment method reaches the highest degree of 67.5%, with the stem point culture combined with heat treatment comes to the next, whereas the stem point culture combined with ribavirin is superior to stem point culture. 3. The application of electronic microscope and molecular biology method of RT-PCR technology to exam the effect of virus elimination. As the lily tissue contains rich polysaccharide, it's difficult to extract RNA of high quality , In this research paper, the author designed a new method of RNA extraction that has the following merits: it neither needs to dry the household utensils with the DEPC water treatment and high temperature, nor needs the fluid nitrogen to grind the material, which save a lot of preparatory work. The application of this method to extract RNA from the lily leaves without using RNasin in the whole process and its successive reaction will reduce the cost, save resources and time, and is more suitable for the extraction of the young tender lily leaves and test tube seedling RNA. What's more, the RNA extracted has a high quality, is suitable for the later RT-PCR examination,4. The optimization of the RT-PCR examination system of CMV, LSV, and LMoV. By using the one-stage process of RT-PCR, this method will save drugs and the operating process will not be contaminated easily. The optimized reacting system is M-MLV 5×Reaction Buffer 1μl,10×PCR buffer0.5μl,2. 5mM dNTPs (each) 0.8μl,10pmol/μl Down primer 0.2μl,10pmol/μl Up primer 0.2μl,M-MLVRT 200U/μl 0.1μl,2.5U/μl Taq polymerase 0.1μl; 0.4μl ;Infected materials such as extracted liquid RNA. Surplus part can be supplemented by aseptic double steam water to enables the reacting system to reach 10ul.