Development of a seedling clone with high regeneration capacity and susceptibility to Agrobacterium in apple
Development of a seedling clone with high regeneration capacity and susceptibility to Agrobacterium in apple
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苹果具有高再生能力和对农杆菌敏感的幼苗克隆的开发
DOI：
10.1016/j.scienta.2013.09.033
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发表时间：
2013-12-17
影响因子：
4.3
通讯作者：
Zhang, Zhihong
中科院分区：
文献类型：
--
作者：
Dai, Hongyan;Li, Wenran;Zhang, Zhihong
农杆菌介导法已成为苹果遗传转化的首选系统。虽然目前已从多种基因类型中获得转基因株系，但转化效率不高。苹果在遗传上是高度杂合的，因此其幼苗群体分离严重。我们推测，可以从苹果幼苗群体中筛选出一个再生能力强、对农杆菌敏感的基因型。为了验证这一假设，本研究建立了苹果(Malus X Home Tica)的试管苗无性系，并比较了它们的再生能力和对根癌农杆菌的敏感性。种子来源选择了苹果遗传转化中广泛应用的皇家嘎拉品种。首先，从100个试管苗无性系中筛选出10个增殖能力强、叶形正常、叶面光滑的无性系，命名为GL-1~GL-10。对10个无性系的叶片切段不定芽再生能力进行了比较，结果表明GL-3具有极高的不定芽再生能力。对GL-3与其亲本的再生能力进行了比较，结果表明，GL-3的每外植体芽数是Royal Gala的2.6倍。通过监测GUS在叶片中的瞬时表达，比较了GL-3和Royal Gala对农杆菌的敏感性。GL-3的GUS阳性斑块率为46.7+/-1.1%，明显高于Royal Gala的25.1+/-7.2%。将携带AtmiR156a反义基因的根癌农杆菌EHA105菌株转化GL-3叶片后，获得了27.5%的抗卡那霉素外植体。综上所述，从苹果幼苗群体中筛选出一株再生能力强、对农杆菌敏感的品种，该策略可推广应用于其他多年生木本植物。(C)2013爱思唯尔B.V.保留所有权利。
Agrobacterium-mediated transformation has been the preferred system for transforming apples. Although transgenic lines have been obtained form a number of genotypes, the transformation efficiency was not high. Apple is highly heterozygous genetically, so its seedling populations segregate heavily. We speculate that a genotype with high regeneration capacity and sensitive to Agrobacterium could be screened from the seedling population Of apple. In order to verify this hypothesis, we developed in vitro seedling clones of apple (Malus x domestica) and compared their regeneration capacity and susceptibility to Agrobacterium tumefaciens in this study. Royal Gala, a genotype had been widely used in apple transformation, was selected as the source of seeds. First, 10 clones with higher multiplication capacity and normal leaf shape and smooth leaf surface, named GL-1 to GL-10, were screened out from 100 in vitro seedling clones. The capacity of adventitious bud regeneration from leaf segments were compared among the ten clones, and the result showed that GL-3 exhibited extremely higher adventitious regeneration capacity. GL-3 was also compared with its parent on the regeneration capacity, and the data showed that the number of buds per explant of GL-3 was 2.6 fold higher than that of Royal Gala. The susceptibility of GL-3 to Agrobacterium was compared with Royal Gala by monitoring the transient expression of GUS in leaf segments. For GL-3, the percentage of explants with large GUS-positive patches was 46.7 +/- 1.1%, which was obviously higher than that (25.1 +/- 7.2%) of Royal Gala. After the leaf segments of GL-3 were transformed with A. tumefaciens strain EHA105 carrying the AtmiR156a antisense gene, the percentage of explants with kanamycin-resistant buds reached 27.5%. In conclusion, a genotype with high regeneration capacity and sensitive to Agrobacterium was screened out from the seedling population of apple, and this strategy could be applied in other perennial woody plants. (C) 2013 Elsevier B.V. All rights reserved.