Apple Latent Virus Elimination And Detection By RT-PCR

Fruit tree virus are strong harmfulness,rapid spreading,diffusion and difficult to cure. The damage of apple virus is upswing in China apple production.The cultivation of non-virus plant is extremely urgent.Clump buds of tissue culture of apple SH40 are studied in this work.Heating treatment+darkness culture+shoop tip culture were used to eliminate apple latent virus.The main factor's effection on the growth increment,the proliferation coefficient,the death rate and the survival rate was searched.The optimized RT-PCR method to detect the ACLSV,ASGV and ASPV virus not only reduced the cost but also improved the efficiency.Five treatments were designed as follows:â‘ Apple shoot clusters were cultured 7 days in the culture room under lighting were put in the illumination culture box from 26/25℃to 38/32℃,by rising 1℃everyday,and then cultured for 30 days.â‘¡Apple shoot clusters cultured 7 days in the culture room under darkness were put in the box for darkness culture,conducted the above procedure.â‘¢Apple shoot clusters cultured 2 days in the culture room under darkness were directly put in the box for dark culture,the procedure is the ditto.â‘£Apple shoot clusters cultured 2 days in the culture room under darkness were directly cultured under 38/32℃in darkness for 30 days.⑤Apple shoot clusters cultured 7 days in the culture room under darkness were conducted same as treatment 4.Results were as followed:1.The survival rate,the height of the survival shoots,the proliferation coefficient of dark culture were prior to that of light culture:46%,2.1cm,2.1;65%,3.1cm,4.7;83%, 3.0cm,5.7;81%,2.7cm,7.2;70%,3.7cm,6.4.2.The effection of Pre-culture times and culture condition(light/dark)on treatments. It showed that treatment 4 was the best purpose:we put clump buds of tissue culture which were cultured 2-3 days in the room temperature in the box for darkness culture into 38/32℃high temperature,then cultivated them 30 days in darkness.Took 2 mm apple stem apex for subculture 30 days which could be used to RT-PCR detection.During heating treatment the darkness culture could increase the material's survival rate,and the height of the survival plant,but the proliferation coefficient was increased.It was 81%,2.7 cm and 7.2 respectivily.Heating treatment+darkness culture not only made the stem tip culture's drawing materials adding from 0.2 mm to 2 mm,decreased the Micromanipulation difficulty,but improved the elimination rate greatly.The elimimation rate of ACLSV, ASGV,and ASPV were 100%,93.3%and 100%.3.Extract total RNA of tissue culture by using RNAplant Extraction Kit.We removed DNA from RNA using DNaseâ… .Then use AMV reverse transcriptase to reverse transcript. The highly efficient RT-PCR detection systems of ACLSV,ASGV and ASPV were optimized.The best parameters of ACLSV were Mg²⁺:1.5mmol/L,2 U TaqE.Reannealing temperature was 58℃.The best parameters of ASGV were Mg²⁺:2mmol/L,1.5 U TaqE. Reannealing temperature was 55℃.The best parameters of ASPV were Mg²⁺:2mmol/L,1 U TaqE.Reannealing temperature was55℃.And the total RNA concentration was 1.0μl. Total volume of PCR was:ACLSV:20μl;ASGV and ASPV:25μl.The ACLSV,ASGV and ASPV were detected by this optimized transcription polymerase chain reaction.This system was ont only improved the amplification efficiency but reduced the cost greatly.