小滴玻璃化法脱除蝴蝶兰种苗建兰花叶病毒的研究

以感染建兰花叶病毒(Cymbidium mosaic virus,CymMV)的蝴蝶兰(Phalaenopsis aphrodite)品种‘满天红’为试材,通过筛选蔗糖预培养浓度、预培养时间、PVS2(Plant vitrification solution 2,PVS2)处理时间三个关键因素,建立蝴蝶兰茎尖小滴玻璃化超低温脱毒体系,将再生的茎尖诱导类原球茎,再分化成苗,经RT-PCR检测CymMV的脱除情况,阴性结果的再生植株进行增殖和诱导生根。结果显示:最佳预培养为:BM+0.6 mol·L-1蔗糖处理1~2 d,超低温茎尖的成活率为70%~76.7%,再生率为53.3%~56.7%;PVS2最佳处理时间为60~90 min,超低温茎尖的成活率为73.3%~76.7%,再生率为50.0%~56.7%。再生植株经RT-PCR检测,CymMV的脱除率为50%。该研究为兰科植物脱除CymMV提供了理论和技术基础。

Elimination of Cymbidium mosaic virus from in vitro Shoot Tips of Phalaenopsis aphrodite by Droplet Vitrification

In vitro shoots of Phalaenopsis Aphrodite‘Mantianhong’which were infected by Cymbidium mosaic virus(CymMV)were used as initial materials in the study.Droplet vitrification protocol of shoot tips was established by screening sucrose concentrations in preculture medium,preculture time and time of exposure to PVS2(Plant vitrification solution 2,PVS2).The regenerative shoot tips were induced to form protocorm-likebody(PLB)and differentiated to shoots.After detection by RT-PCR for CymMV,negative shoots were micropropagated and induced to form roots.The results showed that shoot tips being precultured on BM+0.6 mol·L^-1 sucrose for 1-2 days was the optimal,resulting 70%-76.7%survival rate and 53.3%-56.7%regeneration rate of cryopreserved shoot tips.The optimal time duration of PVS2 was 60-90 minutes,resulting 73.3%-76.7%survival rate and 50.0%-56.7%regeneration rate of cryopreserved shoot tips.Being tested by RT-PCR,half of the regenerated shoots were negative.The CymMV eradication frequency was 50%by droplet vitrification protocol,which laid a technical and theoretical foundation for CymMV eradication of orchids.