通过玻璃化超低温处理脱除草莓轻型黄边病毒(SMYEV)研究

以草莓品种明宝为材料,取茎尖做初代培养,继代扩繁5次后,取2mm左右的茎尖,利用玻璃化超低温技术对SMYEV进行脱除,并利用结合内标的多重RT-PCR技术对再生植株进行病毒检测。结果表明,在病毒脱除过程中,预培养蔗糖浓度为0.5mol/L,处理3d;装载处理为25℃,60min;玻璃化处理为0℃,120min;液氮处理60min后,进行40℃水浴2min,草莓茎尖的存活率为76%,而草莓轻型黄边病毒的脱除率为95%。只有通过液氮处理才可以脱除该病毒,液氮处理前的玻璃化处理脱毒率为0。利用超低温脱毒法可以简便有效地脱除SMYEV。

Preliminary study on the elimination of mild yellow-edge virus from in vitro shoot tips of Meihou strawberry cultivar by vitrification-cryop-reservation treatment

The surface-sterilized shoot tips of Fragaria ananassa cv.Meihou infected with strawberry mild yellow edge virus(SMYEV)were cultured five times in vitro and the shoot tips(about 2 mm in size) were cryopreserved.The SMYEV of regen-erated plants were detected by multiplex RT-PCR combined with internal control.The shoot tips were pre-cultured on basic medium with sucrose concentration of 0.5 mol/L for 3 days,then the pre-cultured shoot tips were loaded for 60 min at 25℃,vitrified with PVS2 for 120 min at 0 ℃,immersed directly in liquid nitrogen for 60 min and rewarmed in 40 ℃ for 2 min.The result showed that the percent of survival shoot tips was 76%,but the various steps taken before freezing in liquid nitrogen did not eliminate SMYEV.However,the freezing step could successfully eliminate SMYEV and the percentage of virus-free shoot tips reached 95%.