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体细胞胚胎发生(体胚发生)是规模化繁育林木良种的一项重要技术,相较其他无性繁殖方式,体胚发生具有彻底幼化、繁殖数量大及速度快等优点,至今已被广泛应用于云杉优良无性系繁育中。云杉(Picea asperata Mast.)是构成低纬度高海拔群落的主要针叶树种,在陆地生态系统中占有重要生态地位。此外,云杉具有单位面积蓄积量高,木质纤维长而柔软的特点,是高产优质的建筑及纸浆材原料。然而在实际生产中,云杉体胚的快繁技术仍面临萌发率和转换率低的问题。因此,解决云杉体胚萌发率低,实现高效的云杉规模化繁育体系对生态文明建设及木材可持续利用均具有重要意义。
植物体胚与合子胚均具有转变为完整植株的能力。通常合子胚,即种子发育的最后会经历成熟脱水阶段,获得脱水耐受性,提高萌发能力[1]。例如苜蓿(Medicago sativa L.)种子从果实中分离需处于成熟脱水阶段才能萌发[2],表明了脱水过程对于胚萌发的重要性。以合子胚获得脱水耐受性可提高萌发能力为参考,早期研究发现将云杉体胚置于干燥或高渗透压条件下,降低其含水量能够有效提高体胚萌发率,该方法被称为干化[3-5]。干化对体胚的整体外观和微观形态都有着显著影响,整体观察,干化能使云杉体胚颜色发生改变,根据色素沉积的多少可将干化体胚分为绿色子叶胚(胚根为红色,胚轴和子叶为绿色)和非绿色子叶胚(胚根、胚轴和子叶与干化前变化不大)两类。其中绿色子叶胚的萌发率要远远高于非绿色子叶胚,这种表型差异也被用以简单快捷地判断干化是否成功[6-7]。此外,可以观察到云杉体胚表层在干化后呈皱缩状[8],暗示了在干化过程中体胚细胞结构的变化。有研究表明,当裸子植物合子胚经历脱水时,细胞收缩产生的张力可能导致干燥敏感的细胞受到不可逆的损伤,但细胞壁折叠在整个脱水过程中能保持质膜和细胞壁的连接,从而确保细胞完整性[9-10]。这也是植物在生长发育过程中依赖改变细胞壁构象来应对各种环境刺激的机制。体胚和合子胚高度相似,如白云杉成熟体胚,其形态上除了胚的大小、子叶的取向和表面起皱等方面与合子胚存在一定差异外,其总体形态与发育阶段一致的合子胚非常相似[8]。然而,至今还没有可靠的科学证据表明细胞壁构型的动态变化是云杉体胚响应干化的一种模式。最近关于挪威云杉干化体胚解剖观察的研究显示,干化后体胚解剖结构变化显著,表现为细胞积累大量的脂质体和显著降低的淀粉粒[7],然而限于显微结构放大倍数的,该研究并未观察到干化细胞明显的细胞壁结构变化。
综合早期研究的证据,本课题组认为干化后云杉体胚特定的解剖结构,特别是细胞壁的可塑性可能是萌发率提高的原因之一。为了证实这一假设,本研究通过光学显微和电子显微观察精准地分析干化对体胚解剖结构的影响,建立了细胞形态变化与萌发的关系,同时分析了干化体胚子叶和胚根的差异表达转录本,旨在揭示干化影响粗枝云杉的关键表型和基因表达模式,最终为全面解析干化促进体胚萌发的机理奠定基础。
干化对粗枝云杉体胚解剖结构及细胞壁重塑基因表达的影响
Effects of Desiccation on Anatomical Structure of Somatic Embryo and Expression of Cell Wall Remodeling Gene of Picea asperata
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摘要:
目的 探究干化对云杉体胚解剖结构的影响,挖掘导致解剖结构改变的关键基因,初步明确干化促进体胚萌发的原因。 方法 以高胚性粗枝云杉细胞系为研究材料诱导体胚发生,干化处理不同时间后进行体胚表型观察及萌发率的测定。利用超景深显微镜、扫描电镜和透射电镜从不同层次观察干化体胚与未干化体胚的组织和细胞结构差异,并结合转录组测序分析干化前后体胚的基因表达变化。 结果 干化显著改变了粗枝云杉体胚形态,干化后体胚胚根呈红色,子叶呈绿色。显微观察发现干化后体胚的细胞呈现皱缩且分布不规则的状态,高倍视野下细胞壁不再具有刚性,表现出松弛扭曲状。转录组分析发现参与细胞壁重塑和影响细胞壁机械性能的多个XTHs、EXPAs和PMEs转录本在干化和未干化体胚中差异表达,且多上调表达,与显微观察发现干化体胚细胞壁形态变化的结果相符,这几类基因可能协同调控该过程,影响体胚萌发。 结论 干化过程中细胞壁重塑基因差异表达导致体胚细胞壁松弛,刚性结构消失,解除细胞分化和生长的限制,是干化促进体胚萌发的潜在原因。 Abstract:Objective To explore the effects of desiccation on the anatomical structure of somatic embryo of Picea asperata, identify the key genes that lead to the changes in anatomical structure, and preliminarily explore the reasons why desiccation promotes somatic embryo germination. Method The high embryonic cell line of Picea asperata was used to induce somatic embryogenesis. The phenotypic observation and germination rate of somatic embryos were determined after desiccation treatment for different time. Ultra depth of field microscope, scanning electron microscope and transmission electron microscope were used to observe the differences of tissue and cell structure and morphology between desiccated somatic embryos and non-desiccated somatic embryos, and transcriptome sequencing was combined to analyze the changes of gene expression between desiccated and non-desiccated somatic embryos. Results The somatic embryo morphology of spruce was significantly changed after desiccation. The radicle turned red and the cotyledon turned green after desiccation. Microscopic observation showed that the cells of the desiccated somatic embryo were shrunken and irregularly distributed. At high magnification, the cell wall was no longer rigid, showing a relaxed and twisted state. Transcriptome analysis found that several transcripts of XTHs, EXPAs and PMEs involved in cell wall remodeling and affecting the mechanical properties of cell walls were significantly differentially expressed between desiccated and non-desiccated somatic embryos, and their expressions were mostly up-regulated, which was consistent with the results of microscopic observation on the morphological changes of desiccated somatic embryos. These genes may synergically regulate the process of desiccation and affect the germination of somatic embryos. Conclusion The differential expression of cell wall remodeling genes during desiccation leads to the relaxation of somatic embryo cell wall, the disappearance of rigid structure, and the release of cell differentiation and growth restrictions, which is a potential reason for desiccation to promote somatic embryo germination. -
Key words:
- Picea asperata
- / somatic embryo
- / desiccation
- / anatomical structure
- / cell wall characteristics
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