苹果茎尖超低温脱毒体系的建立
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摘要
病毒病是苹果栽培亟待解决的问题,不仅严重损坏苹果品质,还给树体带来毁灭性灾害。超低温脱毒技术的诞生和发展对果树脱毒苗的培育产生了重要影响。本试验研究出新型茎尖超低温脱毒技术体系并成功脱除苹果病毒ACLSV和ApMV。试验选取‘烟富3号’、‘烟富10号’、‘M9’、‘M9T337’四种苹果及砧木材料,提取总RNA进行RT-PCR病毒检测,发现‘烟富3号’携带ACLSV和ApMV,苹果砧木‘M9’和‘M9T337’携带ACLSV。对携带病毒材料进行初代、继代扩繁培养,然后取2 mm茎尖进行超低温处理,并用多重RT-PCR检测再生植株,获取脱毒苗后进行生根培养与炼苗移栽。结果表明,最佳玻璃化超低温处理为:预培养采用0.5 mol/L蔗糖浓度处理2 d,25℃下装载60 min,0℃下PVS2溶液处理90 min,液氮冷冻1 h,40℃水浴2 min解冻,苹果茎尖的存活率为78.33%,脱毒率可达95.74%。玻璃化超低温处理是产生脱毒效果的直接原因,运用超低温处理可以有效脱除苹果病毒ACLSV和ApMV。
Virus disease is one of the most urgent problems in apple cultivation. It not only seriously damages apple quality, but also br ings destructive disasters to apple trees. The birth and development of cryopreservation detoxification technology has brought good news for the cultivation of virus-free seedlings of fruit trees. In this experiment, a new technology system of cryopreservation detoxification technology of shoot tips was developed and apple virus ACLSV and APMV were successfully removed. Four different varieties of apples and rootstocks materials called 'Yanfu 3', 'Yanfu 10', 'M9', 'M9 T337' were selected to extract the total RNA for RT-PCR virus detection. We found that 'Yanfu 3' carried ACLSV and ApMV, and apple rootstocks 'M9' and 'M9 T337' carried ACLSV. We carried out the first generation and subculture propagation and culture of the viruses, and then took about 2 mm shoot-tips for cryopreservation treatment, then the regenerated plants were detected by multiplex RT-PCR. After the virus-free seedlings were obtained, rooting culture and seedling transplantation were carried out. Results showed that the best vitrification and cryopreservation treatment was as follows: shoot-tips were pre-cultured with sucrose concentration of 0.5 mol/L for 2 days, then loaded for 60 min at 25℃, treated with PVS2 for 90 min at 0℃, immersed in liquid nitrogen for 1 h, and thawed in water bath for 2 min at 40℃. The survival rate of apple shoot-tips was 78.33%, and the virus-free rate could reach 95.74%. Vitrification and cryopreservation treatment might be the immediate cause of detoxification, and it could effectively remove apple virus ACLSV and ApMV.
Virus disease is one of the most urgent problems in apple cultivation. It not only seriously damages apple quality, but also br ings destructive disasters to apple trees. The birth and development of cryopreservation detoxification technology has brought good news for the cultivation of virus-free seedlings of fruit trees. In this experiment, a new technology system of cryopreservation detoxification technology of shoot tips was developed and apple virus ACLSV and APMV were successfully removed. Four different varieties of apples and rootstocks materials called 'Yanfu 3', 'Yanfu 10', 'M9', 'M9 T337' were selected to extract the total RNA for RT-PCR virus detection. We found that 'Yanfu 3' carried ACLSV and ApMV, and apple rootstocks 'M9' and 'M9 T337' carried ACLSV. We carried out the first generation and subculture propagation and culture of the viruses, and then took about 2 mm shoot-tips for cryopreservation treatment, then the regenerated plants were detected by multiplex RT-PCR. After the virus-free seedlings were obtained, rooting culture and seedling transplantation were carried out. Results showed that the best vitrification and cryopreservation treatment was as follows: shoot-tips were pre-cultured with sucrose concentration of 0.5 mol/L for 2 days, then loaded for 60 min at 25℃, treated with PVS2 for 90 min at 0℃, immersed in liquid nitrogen for 1 h, and thawed in water bath for 2 min at 40℃. The survival rate of apple shoot-tips was 78.33%, and the virus-free rate could reach 95.74%. Vitrification and cryopreservation treatment might be the immediate cause of detoxification, and it could effectively remove apple virus ACLSV and ApMV.
引文
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