Preliminary Study On Establishment And Optimization Of Rakkyo (Allium Chinense G. Don) Virus Elimination And Micropropagation System

Rakkyo (Allium chinense G. Don) is a traditional vegetable which is origated in China that can be used as edible bulb or medical herb. Recent years, because of the long-term asexual propagation by bulbs, rakkyo viral diseases became quite severer, therefore, it rendered germplasm devolution, quality decline and loss of yield. By now, it's a effective way to eliminate viruses and obtain virus-free rakkyo from infected bulbs by in vitro shoot-tip culture.This study focused on obtaining and proliferating of virus-free rakkyo plantlets and bulblets induction and swelling in vitro. The available approach to micropropagate rakkyo has been systematically investigeted. Using the powerful burgeon ability of bulb stem-disc and the adaptable ability of cuvette bulblet, we have established a high-multiplication and easily-manipulation technique system. The main results of our research are as follows:1. The high efficiently disinfecting process was established. The shoot apexes of rakkyoes were first dipped in 75% alcohol for 30 s, then sterilized by 2% Antiformin for lOmin with two drops of Tween-20, finally, washed 4-5 times with sterile water, it can reach the ideal result of lowering to 13.8% infecting ratio.2. In the rakkyo callus induction experiment, 0.5g/L PVP can effectively inhabitting the explant browning. By screening MS, LS and B₅ medium, we have obtained the best basic medium for rakkyo shoot-tip culture. Meantime, choosing the 0.5～1.0mm shoot-tip to culture can get the better virus-free result than others. Using secondary incision culture of rakkyo shoot apex can low down the diffculties of technique and maintain the virus-free ratio.3. In the virus detecting and checkup experiment, adopting sap transmission and molecular method to detect the ScaMV and virus-free effect judgement, CP gene fragments of virus were amplified by RT-PCR. The results showed that the RNA extracted from the Trizol regeat accorded with the demands of RT-PCR amplification, and the target amplification was the same as expected. It was concluded that the RT-PCR system worked smoothly. Through the extracting of infected rakkyo plants and the inoculated leaves, after reverse transtript, PCR and electrophoresis, then check the results.4. High efficient technique of shoot-tip culture was established based on the traditional shoot-tip virus-free experiments. The results showed, shoot-tip combining 32...