| Virus disease in fruit tree has adversely affected the growth and production of fruit trees in recent years. At present, there is no effective medicine to control fruit virus disease in apple production. Seedling quarantine and popularization of virus-free seedling are the keys to stop virus disease from the source. An effective and stable viruses-elimination system is the key to the production of virus-free seedlings. Heat treatment and shoot tip culture are commonly used in fruit tree. Low survival rate of shoot tip and high seedling mortality of heat treatment are the two major bottlenecks of viruses-elimination on tube seedlings. The experiment used different methods to eliminate viruses, screened the optimum culture conditions for different viruses-elimination methods, analyzed main factors affecting the efficiency of viruses-elimination, and detected viruses using PCR and ELISA methods to optimize and establish high efficient system of apple virus-free technology. The results were as followed:1. The 2/3 MS medium+6-BA 1.5mg/L+NAA 0.1 mg/L +Sucrose 30g/L + Agar 6g/L, and PH 5.8 is the most suitable for cultivation of ’gala’ stem tip, due to the differentiation rate and the multiplication coefficient were significantly higher than those of other media,reaching 56.67% and 6.36, respectively. The MS medium reded 1/3 macroelement was more suitable for shoot tip culture which browning rate was lower than the MS medium under the same conditions. In dark culture, the turning green rate of the shoot tip on 7d reached 70% was higher than 5d.2. In different conditions of heat treatment, the survival rate of treatment increasing temperature gradually(After 30℃ heat treatment 2d,the temperature increased of 1℃ daily until 36℃. After 36℃ heat treatment 2d, treatment at 37℃ for two days should be carried out. Finally, constant temperature treatment at 38℃ was supposed to conduct) declined the slowest and was the highest, up to 75.56%, which was significantly better than other treatment.3. Under the treatments of four different ribavirin concentrations, the survival rates of the materials were decreased with the increase of the ribavirin concentrations. The survival rates of 10mg/L and 20mg/L were 93.06% and 87.50% respectively, which were significantly higher than other treatments.4. By PCR technology, ’gala’ was detected with ASPV and ASGV two viruses, cultivar ’Nagafu 2’ with ASPV and rootstock ’B9’ with a virus ASGV. The virus-free rate was detected by PCR and ELISA two detection methods respectively, after using stem tip culture, heat treatment, heat treatment combined with shoot tip culture and ribavirin four methods eliminated viruses. The detection results showed that the virus-free rate of heat treatment combined with shoot tip culture was the highest that ASPV virus-free rate reached 100% and ASGV was 80.56%, significantly better than the other three single viruses-elimination methods. The viruses’ detection rate of PCR was higher than ELISA.5. ’gala’ tube seedlings in different hormone combinations medium for rooting treatment, experimental results showed that the auxin types and concentrations were significantly affected rooting rate of tube seedlings:1/2MS medium was significantly better than MS medium, and IAA was better than IBA and NAA.Exogenous SA can significantly improve the survival rate of transplanted tube seedlings, and there was a concentration effect. The suitable rooting medium was 1/2MS+ 0.2 IAA mg/L, and 0.8 mg/L SA treatment can significantly reduce the stomatal conductance,increased chlorophyll content and improve the survival rate of transplanting. |
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