Combining Exogenous Application Of Melatonin And Cryotherapy For Eradication Of Apple Stem Grooving Virus (ASGV)

China is the largest apple-producing and consumptionin country of the whole world.Apple virus diseases has been threatening the sustainable development of the apple industry.Apple stem grooving virus(ASGV)is an important virus in apple and widely occurried in China.Use of virus-free plant material is an effective approache to combat virus and availability of efficient virus elimination technology is necessary for obtaining virus-free plants.This research used ASGV-infected in vitro ‘Gala' apple to investigate(1)combining exogenous of melatonin with cryotherapy for eradication ofASGV;(2)microtissue direct RT-PCR for ASGV detection.Results achieved in the present study are summarized as followings:1.Shoot segments(about 1.5 cm in length)were taken from the 4-week old in-vitro stock shootsand cultured on basic medium containing different concentrations of melatonin in the dark.Shoot tips(1.5-2 mm)containing 5-6 leaf primordia(LPs)were excised from melatonin-treated shoot segments after 1,2 and 3 months of treatments,and subject to cryotherapySurvival and regrowth ratescryotherapt-treated shoot tips were higher than those without melatonin treatment The highest survival and regrowth rates were achieved in shoot tips that had been treated with 10 and 15 ?M melatonin for 1 month,respectively.Histological immunological localization found that virus-free area of the shoot tips was enlarged in melatonin-treated shoot tips.2.RT-PCR was applied to detect virus sanitary in plantlets regenerated after 3 months of cryotherapy.Virus-free frequency was about 10% in plantlets that were produced by treatments of 15?M melatonin for 2 months and 20 ?M melatonin for 1 month.Cryotherapt alone failed to produced any virus-free plantlets.3.ASGV could be effectively detected by microtissue direct RT-PCR,compared with RT-PCR.The best results were obtained when 25 G of syringe needle was used for sampling.Reverse transcriptional Real-time fluorescen ce quantitative was used to detect the content of ASGV,melatonin of 15 ?M and 20 ?M can indeed reduce the concentration of the virus.Combining exogenous application of melatonin with cryotherapy opened a new approach to eliminating apple viruses,while microtissue direct RT-PCR provided a simple and efficient method for ASGV detection.