Study On Potatoes Virus Elimination Technology And Multiplex RT-PCR Method Of Virus Detection

As the potato being planted worldwidely, the planting areas have been increasingly expanded annually. The potato has a variety of characteristics of dietary fiber rich, potassium high, sodium low and nearly zero fat, therefore it makes more and more people calling potatoes as the "staple". Seed Potato after virus elimination by different methods will ensure the production amount stable in the main production areas, and will also ensures the potato quality, meanwhile to extend the utilization rate of the varieties. Preventing seed of potato’s degeneration caused by virus. Promoting reverse transcriptase virus gene amplification detection method can make the detection of potato field the workload and try reduce the dose of compound infection, multiple PT-PCR(m-RT-PCR) technology has will be put into actual production application of molecular biotechnology development potential.On the base of predecessors’ researches, the experiment is to delay the potato plants varieties of 4 plants virus infection from the Seeding Cultivation Room of Horticulture College of Jilin Agriculture University(JAU), and then making meristem-tip, heat-treatment, ribavirin, heat-treatment combined with meristem-tip and meristem-tip combined with ribavirin to eliminate virus and calculating the plant survival rate and seedling rate and a single virus rt-pcr detection procession of PVX, PVY, PVS and PLRV seedling rate, the following conclusions:1. The five virus-eliminated technologies are different in four kinds of virus elimination seedling. Meristem-tip to PVX elimination rate: 60%; PVY: more than 75%; PVS: 30%; PLRV: more than 60%; 38℃(4h)+25℃(20h) is most suited to remove PLRV In the heat-treatment. And the rate in the processing of PVS were less than 31% in the 16 groups, It shows that the heat-treatment is not fitable to remove PVS; Ribavirin to free virous was zero; Heat-treatment combined with meristem-tip to free PVS increase to more than 40%, PLRV increase to more than 80%; Using meristem-tip combined with ribavirin to eliminate PVX can over 60%, PVY is about 70%, and PVS is 50%, Howere PLRV canincrease to 90.28%. Generally,the combination of above two technologies is better than single technology.2. Various technologies are suitable for different viruses elimination. Meristem-tip combing with heat-treatment will get the higher efficiency rate of 69.86%; Heat-treatment combined with meristem-tip or meristem-tip combined with ribavirin to eliminate PVY were more than 75%; Meristem-tip combined with ribavirin to eliminate PVS canget 50.38%; PLRV’s best way ismeristem-tip combination with ribavirin, seedling rate is 90.82%.3. The designed specific primers as to four kinds of viruses has the most optimal Mgcl2(25mmol/L) concentration of 2 μL, and the dNTP concentration is 1.5 μL. The optimal reaction conditions of various virus PVS are as followings: 56.8℃; PVX: 55.6℃; PVY: 56.8℃; PLRV: 57.9℃, meanwhile the multiple RT-PCR primers concentration in the reaction system arePVX: PVY: PLRV : PVS = 0.5: 1:1:0.5.