Studies On Elimination Of ACLSV And PNRSV By Combining Meristem Tip Culture With Thermotherapy In Cherry

The sweet cherry(Prw/ms.avmm L.)varieties 6-3, 6-7 and GiselaS (P. cerasus X P.canescens) were chosen as materials in this study. After several factors including medium, genotype and hermone were studied, the micropropagation system of cherry was obtained. Dormant buds and current shoot tips were used as explants. For meristem initiation culture, explants (l~2cm ) were washed with water (l~2h) and then placed in 70% ethanol for 15-30S, 0.1%HgCl for 5~10min, then, rinsed 5-6 times with sterilled water. Meristem domes (0.5~lmm, with lesf primodia) excised from buds of cherry 6-3, 6-7 and GiselaS were cultured on basic woody plant medium supplemented with BA0.5mg/l, IBA0.2mg/l and GA30.2mg/l. For shoot multiplication, the explants were subcultured on solid medium supplemented with BA1.0mg/l, IBA0.2mg/l, 2-3% sucrose, and pH5.2-6.0. In order to avoid the explants losing vigor, we used MS and FH medium at intervals. For root development, the cultures were cultured on FH rooting medium supplemented with NAA0.7mg/l and IBA1.0mg/l. After 18 days, high quality roots were obtained. Then, they were transferred to greenhouse. Up to 90% of plantlets were survied when the cultures were potted in vermiculite and placed in a high-humidity chamber for 3weeks.On the basis of the micropropagation system of cherry, elimination of ACLSV and PNRSV by combining thermotherapy with meristem tip culture were studied. Materials were treated under 38癈 for 3 weeks, 0.5~lmm length meristem tips were deprived from derilled buds and cultured on WPM medium. 3-4 months later, the plants were detected for ACLSV and PNRSV by RT-PCR and virus-free plants were obtained. The results were as follows: For 6-3, 6-7 and GiselaS, in method 1, 50 meristem tips of each variety was used, 24, 16 and 30 clones of them were established in tissue culture respectively. The numbers of ACLSV free clones were 7, 4 and 3, and PNRSV free clones were 11,7 and 9 respectively. For 6-3 and 6-7 in method 2, 20 and 25 meristem tips were used and 16, 24 clones were established in tissue culture respectively. The numbers of ACLSV free were 5 and 9, and PNRSV free were 10 and 14. Viruses can be detected in almost all clones in method 3. In a word, method 2 is best and method 1 is better than method 3.The method of RNA extraction and purification was developed. With this method, the reverse-transcritable RNA can be obtained in 3 hours. The costs accounting of RNA extraction has been reduced. The results showed that the method was suitable for RNA extraction from the tube clones and new leavies of cherry. The viruses materials were as check and primers were synthesized, the 358bp and 449bp fragments of the partial CP gene of ACLSV and PNRSV were obtained from the infected viruses materials, and no fragments be found in the virus free clones by RT-PCR.