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摘要：超低温脱毒技术是目前培养无毒苗的一种新兴热门技术，弥补了传统茎尖培养中微小茎尖难取的局限。本试验选取五个不同品种的苹果材料，取约 2mm的茎尖进行玻璃化超低温处理，采用结合内标的 RT-PCR进行病毒检测。结果显示，最佳组培扩繁体系为：初代培养中所有外植体先用 75%酒精处理 45s，然后用0.1%升汞灭菌，水培促萌萌发的嫩芽处理 5min，实生苗幼苗茎段处理 10min，初代培养基为 1/2MS+1.0mg/L 6-BA+0.1mg/L NAA+1g/L PVP+30g/L蔗糖+7g/L琼脂粉，pH=5.8，继代增殖扩繁培养基为 MS+0.5mg/L 6-BA+0.1mg/L IBA+30g/L蔗糖+7g/L琼脂粉，pH=5.8。最佳玻璃化超低温脱毒体系为：预培养采用0.5mol/L蔗糖浓度的培养基处理2d，装载处理采用25℃处理60min，玻璃化处理采用 PVS-2 溶液0℃处理 90min，液氮冷冻 1h，40℃水浴2min解冻。苹果茎尖的存活率可达 78.33%，脱毒率可达 95.74%。 38025
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毕业论文键词：苹果病毒；超低温；玻璃化；病毒检测；脱毒
The Establishment of Cryopreservation Detoxification Technology of Apple
Abstract：Cryopreservation detoxification is an emerging technology for virus-free seedlings which makes up for the limitation of traditional shoot tip culture. In this study, we selected five different apple varieties, cut the shoot tips about 2mm long and made them vitrified and cryopreserved. The viruses in plants were detected by RT-PCR combined with internal control. The results showed that the optimum system of tissue culture propagation was as follows: in primary culture, all explants were sterilized by 75% alcohol for 45s, then the buds germinated by hydroponics were sterilized by 0.1% mercury dichloride for 5min and the stem segments of seedlings were sterilized by 0.1% mercury dichloride for 10min. The primary culture medium was 1/2MS+1.0mg/L 6-BA+0.1mg/L NAA+1g/L PVP+30g/L sucrose+7g/L agar powder, pH=5.8. The subculture medium was MS+0.5mg/L 6-BA+0.1mg/L IBA+30g/L sucrose+7g/L agar powder, pH=5.8. The optimum system of vitrified cryopreservation detoxification was as follows: the shoot tips were pre-cultured on the medium with sucrose concentration of 0.5mol/L for 2 days. Then the pre-cultured shoot tips were loaded for 60min at 25℃, vitrified with PVS-2 for 90min at 10℃, cryopreserved with liquid nitrogen for 1h and thawed by 40℃ water bath for 2min. The survival ratio of shoot tips was 78.33% and the virus-free ratio was 95.74%.
Keywords: Apple viruses; Cryopreservation; Vitrification; Virus detection; Detoxification
目录
摘要1
关键词1
Abstract1
Keywords1
引言1
1材料与方法3
1.1材料3
1.1.1植物材料3
1.1.2主要药品及试剂3
1.1.3主要仪器设备3
1.2方法4
1.2.1脱毒前病毒检测4
1.2.2初代、继代扩繁培养5
1.2.3茎尖玻璃化超低温处理6
1.2.4再生株系的病毒检测7
1.2.5生根培养与移栽炼苗7
2结果与分析7
2.1RNA的浓度和质量分析7
2.2RT-PCR检测7
2.3不同预培养蔗糖浓度对苹果茎尖存活率的影响9
2.4不同装载处理时间对苹果茎尖存活率的影响10
2.5不同PVS-2溶液处理时间对苹果茎尖存活率的影响10
2.6不同处理对苹果茎尖存活率及脱毒率的影响11
2.7超低温处理后茎尖形态变化12
2.8再生植株的RNA提取和病毒检测12
3讨论13
致谢13
参考文献14
图1不同苹果内源基因电泳结果3
图2‘烟富3号’水培促萌法示例图5
图3五种苹果材料初代示例图6
图4体视显微镜下切取的2mm茎尖6
图5体视显微镜下切取的2mm茎尖6
图6超低温处理后茎尖再生图8
图7‘烟富3号’结合内标的RT-PCR病毒检测电泳结果8
图8‘烟富10号’结合内标的RT-PCR病毒检测电泳结果8