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首页> 外文期刊>Turkish Journal of Agriculture & Forestry >Clonal propagation and cryogenic storage of virus-free grapevine (Vitis vinifera L.) via meristem culture
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Clonal propagation and cryogenic storage of virus-free grapevine (Vitis vinifera L.) via meristem culture

机译：分生组织培养无病毒葡萄（Vitis vinifera L.）的克隆繁殖和低温保存

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A protocol for production of virus-free Vitis vinifera using meristem culture was developed. Meristems (0.1-0.2 mm) of V. vinifera infected with grapevine fanleaf virus and grape leafroll associated viruses were excised from 1-year-old growing vines. Shoot tips were cultured on half-strength Murashige and Skoog (MS) medium, supplemented with 0.02 mg L-1 of benzylaminopurine (BAP) and 0.01 mg L-1 of naphthalene acetic acid (NAA). BAP and kinetin resulted in differences in the number of new shoots per explant, shoot height, and number of new leaves per explant. BAP at 0.8 mg L-1 gave the highest in vitro multiplication rate, with 5.25 shoots per explant, whereas elongation was greatest in the presence of 0.2 mg L-1 of kinetin. Root initiation was tested on an MS medium supplemented with 0.0, 0.2, 0.4, 0.6, 0.8, or 1.0 mg L-1 of IBA (indole-3-butyric-acid), IAA (indole-3-acetic-acid), or NAA. Maximum root number was achieved using 0.6 mg L-1 of IBA. A survival rate of 95% was achieved when rooted explants were acclimatized ex vitro in a mixture of I soil: 1 perlite: 1 peat. Acclimatized plants grew in the greenhouse and were maintained as virus-free plants. Visual inspections as well as results of RT-PCR, using virus-specific oligonucleotide primers, showed that plants developed in vitro were free from grapevine fanleaf virus, grape leafroll associated virus-1, and grape leafroll associated virus-3 infections. Shoot tips from plantlets grown in vitro were cryopreserved by vitrification. Shoot tips were treated in a 2 mL cryotube filled with a solution of 5% (w/v) DMSO, 5% (w/v) glycerol, and 5% (w/v) sucrose at 25 degrees C for 20 min, then dehydrated with 1 mL of modified vitrification solution 2 (MPVS2) at 0 degrees C for 40 min. Maximum regrowth (55%) was obtained after cryogenic storage with cultures exposed to MPVS2 for 40 min at 0 degrees C. Cryopreserved shoot tips, after being warmed, resumed growth within 7 days and developed shoots directly without intermediate callus formation.
机译：开发了使用分生组织培养生产无病毒葡萄的方法。从1岁的正在生长的葡萄藤上切下感染了葡萄扇叶病毒和与葡萄卷相关的病毒的V. vinifera的分生组织（0.1-0.2 mm）。将茎尖在补充了0.02 mg L-1的苄氨基嘌呤（BAP）和0.01 mg L-1的萘乙酸（NAA）的半强度Murashige和Skoog（MS）培养基上培养。 BAP和激动素导致每个外植体的新芽数量，茎高和每个外植体的新叶片数量不同。 BAP浓度为0.8 mg L-1时，体外繁殖率最高，每个外植体有5.25枝，而当存在0.2 mg L-1激动素时，伸长率最大。在补充了0.0、0.2、0.4、0.6、0.8或1.0 mg L-1的IBA（吲哚-3-丁酸），IAA（吲哚-3-乙酸）或NAA。使用0.6 mg L-1 IBA可达到最大根数。当生根的外植体在I土壤：1珍珠岩：1泥炭的混合物中进行体外驯化时，存活率达到95％。适应环境的植物在温室中生长，并保持为无病毒植物。使用病毒特异性寡核苷酸引物的目视检查以及RT-PCR的结果表明，体外发育的植物没有受到葡萄扇叶病毒，与葡萄叶相关的病毒1和与葡萄叶相关的病毒3的感染。通过玻璃化冷冻保存体外生长的小植株的茎尖。在装有5％（w / v）DMSO，5％（w / v）甘油和5％（w / v）蔗糖的溶液的2 mL冷冻管中于25摄氏度下处理茎尖20分钟，然后在0摄氏度下用1 mL改性玻璃化溶液2（MPVS2）脱水40分钟。低温保存后，将培养物在0℃暴露于MPVS2 40分钟后，可获得最大的再生长（55％）。冷冻保存的芽尖变暖后，在7天内恢复生长，直接发育芽而没有中间愈伤组织形成。

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