Establishment of a somatic embryo regeneration system and expression analysis of somatic embryogenesis-related genes in Chinese chestnut (Castanea mollissima Blume)
Establishment of a somatic embryo regeneration system and expression analysis of somatic embryogenesis-related genes in Chinese chestnut (Castanea mollissima Blume)
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板栗体细胞胚胎再生体系的建立及体细胞胚胎发生相关基因的表达分析
DOI：
10.1007/s11240-017-1250-3
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发表时间：
2017
影响因子：
3
通讯作者：
Qin Ling
中科院分区：
文献类型：
--
作者：
Lu Dan;Wei Wei;Zhou Wan;McGuigan Linda D.;Ji Fei-yang;Li Xiao;Xing Yu;Zhang Qing;Fang Ke-feng;Cao Qing-qin;Qin Ling
体细胞胚胎发生是一种可靠而重要的手段，控制体细胞胚胎发生的相关基因在体细胞胚胎发育的全过程中起着至关重要的作用。然而，板栗体细胞胚胎发生再生一直受到阻碍，其分子机制尚不清楚。因此，我们首先描述了体细胞胚启动、发育、成熟和萌发的方案。胚性愈伤组织在含1.8 μ M2，4-D和1.1 μ M6-BA的胚性起始培养基上获得，然后转移到不含任何激素的胚性发育培养基上至少4周，直至子叶胚出现。然后，将体细胞胚转移到含有0.5 μM NAA和0.5 μM 6-BA的Gamborg B-5基础盐混合物的胚成熟培养基中3周。最后，将成熟胚接种于WPM +0.5 μM NAA +0.5 μM 6-BA的胚萌发培养基中，获得了再生植株，转化率为2.1%。此外，还鉴定了8个胚胎发生相关基因，并通过实时定量RT-PCR（qRT-PCR）分析了这些基因在胚胎发生过程中的表达谱。CmSERK、CmLEC 1、CmWUS和CmAGL 15基因在胚胎发育初期表现出高表达，推测这些基因在胚胎发生的启动过程中发挥关键作用。对板栗体细胞胚胎发生相关基因的研究将为进一步阐明板栗体细胞胚胎发生的分子机制提供重要的理论依据。板栗体细胞胚再生体系的建立为板栗遗传转化体系的建立和遗传改良奠定了基础。
Somatic embryogenesis is a reliable and important tool, and the relevant genes controlling this process act as vital roles through the whole development of somatic embryos. However, regeneration via somatic embryogenesis in Chinese chestnut has been impeded and its molecular mechanism is not known. Therefore, firstly we described a protocol for somatic embryo initiation, development, maturation and germination. Embryogenic calli were obtained in embryo initiation medium containing 1.8 μM 2,4-D and 1.1 μM 6-BA, and then were transferred to embryo development medium without any hormones for at least 4 weeks, until cotyledonary embryos appeared. Next, the somatic embryos were transferred to embryo maturation medium containing Gamborg’s B-5 Basal Salt Mixture with 0.5 μM NAA and 0.5 μM 6-BA for 3 weeks. Finally, these mature embryos were germinated in embryo germination medium consisting of WPM with 0.5 μM NAA and 0.5 μM 6-BA, resulting in shoot regeneration with a 2.1% conversion rate. Additionally, eight embryogenesis-related genes were identified, and the expression profiles of these genes during embryogenesis were analyzed via quantitative real-time RT-PCR (qRT-PCR). The CmSERK, CmLEC1, CmWUS and CmAGL15 genes exhibited high expression in the initial embryo stages, which inferred that these genes played key roles during the initiation of embryogenesis. Studies on embryogenesis-related genes will provide an insight for further elucidating molecular mechanism during somatic embryogenesis of Chinese chestnut. Furthermore, the successful establishment of a somatic embryo regeneration system for Chinese chestnut will lay a significant foundation for a stable genetic transformation system and genetic improvement.