Elimination of chrysanthemum stunt viroid and chrysanthemum chlorotic mottle viroid from infected chrysanthemum by cryopreservation
Elimination of chrysanthemum stunt viroid and chrysanthemum chlorotic mottle viroid from infected chrysanthemum by cryopreservation
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DOI：
10.1007/s00709-015-0874-6
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发表时间：
2016-07-01
期刊：
影响因子：
2.9
通讯作者：
Kim, Chang Kil
中科院分区：
文献类型：
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作者：
Jeon, Su Min;Naing, Aung Htay;Kim, Chang Kil
用逆转录聚合酶链反应（RT-PCR）证实，表现为菊花类矮病毒（CSVd）引起的矮化病症状的菊花‘Borami’和‘Secret Pink’和表现为菊花类绿斑驳病毒（CChMVd）引起的萎黄斑驳病症状的菊花‘Yellow Cap’分别被类病毒感染。实时荧光定量PCR结果显示，不同产地（漆谷、龟尾和庆山）的感染品种中类病毒浓度存在差异。我们采用冷冻保存方案，从龟尾采集的自然感染的‘Borami’中去除CSVd，通过改变几个因素，如植物玻璃化溶液（PVS2和PVS3）、暴露于液氮的时间、茎尖大小和低温处理，显示出最低的CSVd浓度。溶液（PVS2）和低温处理是决定类病毒清除效果的关键因素。我们将所有结果的最优因素组合在一起，对方案进行了优化，并在漆谷、庆山采集的‘Borami’和漆谷、龟尾、庆山采集的‘Secret Pink’中测试了方案的适用性，它们显示出不同的类病毒浓度。我们发现，根除率因品种和产地而异。当应用该方案从同一区域收集的“黄帽”中去除CChMVd时，观察到类似的结果。最后，我们发现巢式PCR检测类病毒比RT-PCR更可靠。总的来说，冷冻保存可用于消除感染菊花中的类病毒；然而，疗效取决于基因型和初始类病毒浓度。
Chrysanthemum morifolium 'Borami' and 'Secret Pink' showing symptoms of stunt disease caused by chrysanthemum stunt viroid (CSVd) and 'Yellow Cap' showing chlorotic mottle disease caused by chrysanthemum chlorotic mottle viroid (CChMVd) were confirmed to be infected by the respective viroids by using reverse transcription polymerase chain reaction (RT-PCR). Real-time PCR results showed that the viroid concentrations in the infected cultivars varied between the different regions of origin (Chilgok, Gumi, and Gyeongsan). We applied a cryopreservation protocol for elimination of CSVd from naturally infected 'Borami' collected from Gumi, showing the lowest concentration of CSVd, by varying several factors such as plant vitrification solutions (PVS2 and PVS3), duration of exposure to liquid nitrogen, shoot-tip size, and low-temperature treatment. The solution (PVS2) and low-temperature treatment were found to be critical factors determining the efficacy of viroid elimination. We optimized the protocol by combining of all resulted optimal factors and tested the applicability of the protocol in 'Borami' collected from Chilgok and Gyeongsan and in 'Secret Pink' from Chilgok, Gumi, and Gyeongsan, which displayed different viroid concentrations. We found that the elimination rates varied depending on the cultivar and region of origin. Similar results were observed when the protocol was applied to eliminate CChMVd from the 'Yellow Cap' collected from the same regions. Finally, we found that nested PCR is more reliable for viroid detection than RT-PCR. Overall, cryopreservation can be used to eliminate viroids from infected chrysanthemums; however, the efficacy depends on genotype and initial viroid concentration.