Studies On Elimination Of Latent Viruses In Vitro-cultured Plants Of Pyrus Pyrifolia By Treatment With The Quercetin Combined With Shoot-tip Culture

Apple chlorotic leaf spot virus(ACLSV),Apple stem grooving virus(ASGV),and Apple stem pitting virus(ASPV) are three important viruses of apple and pear worldwide.In commercially cultivated apple and pear trees,these viruses do not usually cause obvious symptoms,but they decrease growth and productivity in infected trees.The application of certified healthy propagation material represents a useful measure for controlling these viruses.The chemotherapy of in vitro cultured plants using substances with antiviral activities could improve the efficiency of virus elimination.In this study,the efficiency of chemotherapy for virus elimination from in vitro cultured pear shoot tips was evaluated by both ELISA and dot-blot hybridization.The media for the root formation of in vitro pear plants were improved.Results are as following.1.In vitro pear plants of 'Huanghua' and 'Jinshui no.2' were re-checked for the persistence of ACLSV,ASGV and ASPV by RT-PCR.Plants with these three viruses were multiplied on MS medium for the following virus elimination treatments.2.The effect of pre-treatment by culturing in solutions containing 25μg/ml quercetin for 3 days and 4 days on titers of ACLSV,ASGV and ASPV were evaluated by dot-blot hybridization.Results showed that the hybridization signal for these three viruses was reduced from samples treated for three days,but there was not significant effect observed for plants treated for 3 days.3.In vitro pear plants pre-treated in 25μg/ml and 35μg/ml quercetin solutions were cultured on MS medium containing the same concentration of quercetin.After 45,50,55 and 60 days,shoot tips about 2mm,1mm and 0.5mm were cut from these plants treated for different periods and cultured on MS medium without quercetin.The regenerated plants were detected for the titers of ACLSV,ASGV and ASPV by PAS-ELISA.Results showed that the virus titers in plants regenerated from shoot tips treated for 45 days were greatly decreased.The efficiency of these treatments for virus elimination was evaluated by dot-blot hybridization.Results showed that the hybridization signal was greatly reduced from plants regenerated from shoot tips treated for more than 50 days. Pre-tratment for 4 days and culturing on MS containing 25μg/ml or 35μg/ml quercetin combined with 0.5mm shoot-tip culture could efficiently eliminate ACLSV and ASPV. Results also showed that higher concentration of quercetin(35μg/ml) could reduce virus hybridization signal,but did not reduce the periods for virus elimination.However, pre-treatment could shorten the treatment periods of virus elimination for about 5 days. The sizes of shoot tips for regeneration had a great effect on virus elimination and when 1 mm tips were used for plant regeneration virus hybridization signal was still detected from these plants.4.Six medium made by adjust the contents of phyto-hormones were used for the inducing of root from in vitro plants of Pyrus pyrifolia cv.5-6-45,Jinshui no.2 and Fengshui.More than 90%rooting rates were obtained for 5-6-45 and Jinshui no.2 on medium 1/2MS+2.0mg/L IBA+1.5mg/L NAA.On medium 1/2MS+0.5 IBA+0.5 NAA, Jinshui no.2 showed a lower rooting rate.All rooted plants were successful survived after transplanted into pots.