Study On Apple Latentvirus Elimination And Detection Technique

The virus elimination in apple were achieved by the ways of shoot tip culture + heat treatment, shoot tip culture + heat treatment + Radix Isatidis and cryopreservation +shoot tip culture. Factors affecting elimination of virus were studied, the efficient elimination methods of apple virus were developed. New method of extracting total RNA from apple was posed; and the apple latent virus(ACLSV, ASGV, ASPV)were detection by reverse transcription polymerase chain reaction, and the RT-PCR detection system was optimized. Results are following:1. The ACLSV was detected by Dot-ELISA, The method of Dot-ELISA is simple and save time, The effect of passivation substance on ACLSV is milder than other method of serology.2. A rapid and simple procedure was posed for diffidently extracting total RNA from apple. The RNA can be used for RT-PCR. Acid phenol and 140mM NaCl were used to removal of DNA,insoluble PVP and 2-mercapto-ethanol were used to removal phenolic compounds, 15% ethanol and 1/5 volume of 3M KAc were used to removal polysaccharides. The procedure can be completed in less than four hours.3. The ACLSV, ASGV and ASPV were detected by reverse transcription polymerase chain reaction.. The RT-PCR method is sensitivity and save tune.4. The virus elimination in apple were achieved by the way of combining shoot tip culture with heat treatment. Before being heat treated, the shoots were cultured on new medium for 7-15 days, a higher survival rate was gained. The rate of survival is 11 % higher and the rate of elimination is 7.5% higher treamented by variable temperature than by invariable heat treament The regrowth rate changed when the shoot tips were cultured on mediums containing different concentration of hormone. Cultured on regeneration medium contain higher propotion by concentration of BA and NAA than stock medium, the regrowth tate of shoot tips is higher. The Nagafu-2 and Gold Spur Deli, would regenerate normally in medurm MS+BA1.0mg/L+NAA 0.1 mg/L, and New Jonagold, Orin would in medium MS+BA2.0mg/L+NAAO. 1 mg/L.5. Based on the method of shoot tip culture+heat treatment, the antivirus Radix Isatidis was applied into medium of tip culture, the results shows that the eliminationrate can attained 100%, but Radix Isatidis have strong inhibiting effect on apple tip growth.6. The apple virus can be successfully eliminated by cryopreservation of in vitro-grow shoot tips, the rate of elimination is 70%. Three compound cryopretectants were designed and the effects were investigated. The PVS2(35%glycerol+15% EG+35%sucrose MS liquid medium) and PVS3(40 % glycerol + 10% EG+ 45% sucrose +10 %DMSO) was more effective. Higher survival rate was gained by dehydration hi PVS3 for 90 min directly. The survival rate would higher by loading step by step in load solution (15%glycerol+15%sucrose)and diluted PVS3.