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在体细胞胚胎发生过程中,油棕基因型的细胞反应涉及原形成层细胞的参与、DNA 去甲基化和生长素积累。

Cellular responses of oil palm genotypes during somatic embryogenesis involve participation of procambial cells, DNA demethylation, and auxin accumulation.

机构信息

CNPq/Embrapa Postoctoral Fellowship Program, Embrapa Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil.

Laboratório de Microscopia, Embrapa Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil.

出版信息

Plant Cell Rep. 2022 Sep;41(9):1875-1893. doi: 10.1007/s00299-022-02898-3. Epub 2022 Jul 1.

Abstract

Cell markers of somatic embryogenesis initiation from leaf tissues in oil palm involve the participation of procambial cells, DNA demethylation, and auxin accumulation. Low callogenesis and genotype-dependent response have been mentioned in the development of somatic embryogenesis protocols of Elaeis oleifera × E. guineensis elite hybrids, which requires more detailed investigations of the process. Thus, the initial cellular responses of immature leaves of adult genotypes of this hybrid were investigated for the first time, emphasizing histological, epigenetic, and endogenous auxin changes. Leaf segments from two genotypes, one responsive to somatic embryogenesis (B351733) and another non-responsive (B352933), were inoculated in Murashige and Skoog medium with 450 µM of 4-amino-3, 5, 6-trichloropicolinic acid. For anatomical analysis, samples of both genotypes were collected at 0, 20, 90, and 105 days of cultivation. Samples of both genotypes were also taken at different cultivation periods to analyze DNA methylation status (% 5-mC-5 methylcytosine) via ELISA test. Immunolocalization assays were performed with anti-indole-3-acetic acid and anti-5-methyl-deoxycytosine antibodies from samples of hybrid B351733. We distinguished two groups of cells reactive to the induction of embryogenic callogenesis, parenchymatous sheath cells, and procambial cells; however, only the latter are directly involved with the formation of calluses. The data obtained indicate that the formation of calluses in hybrid B351733 is related to DNA hypomethylation, while the non-responsiveness of leaf explants in hybrid B352932 is related to DNA hypermethylation. The in situ immunolocalization enabled the identification of initial markers of the callogenic process, such as IAA accumulation and hypomethylation. Identifying these events brings the possibility of establishing strategies for efficient manipulation of somatic embryogenesis protocols in palm trees.

摘要

油棕叶片体细胞胚胎发生起始的细胞标记物涉及原形成层细胞的参与、DNA 去甲基化和生长素积累。在油棕杂种(Elaeis oleifera×E. guineensis)优良杂交组合的体细胞胚胎发生培养体系的建立过程中,已提到愈伤组织发生能力低和基因型依赖性应答的问题,这需要对该过程进行更详细的研究。因此,首次对该杂种成年基因型未成熟叶片的初始细胞反应进行了研究,重点研究了组织学、表观遗传学和内源性生长素的变化。将两个基因型(一个对体细胞胚胎发生有反应(B351733),另一个无反应(B352933))的叶片片段接种到含有 450 μM 4-氨基-3,5,6-三氯吡啶酸的 Murashige 和 Skoog 培养基中。为了进行解剖分析,在培养的 0、20、90 和 105 天收集了两个基因型的样本。在不同的培养阶段还采集了两个基因型的样本,通过 ELISA 试验分析 DNA 甲基化状态(% 5-mC-5 甲基胞嘧啶)。从杂交种 B351733 的样本中进行了抗吲哚-3-乙酸和抗 5-甲基脱氧胞嘧啶抗体的免疫定位分析。我们区分了对胚胎发生愈伤组织诱导有反应的两组细胞,即薄壁鞘细胞和原形成层细胞;然而,只有后者直接参与愈伤组织的形成。获得的数据表明,杂交种 B351733 中愈伤组织的形成与 DNA 去甲基化有关,而杂交种 B352932 叶片外植体的无应答性与 DNA 高甲基化有关。原位免疫定位能够鉴定出愈伤组织发生过程的初始标记物,如 IAA 积累和去甲基化。鉴定这些事件为建立有效的掌状胚发生培养体系操作策略提供了可能性。

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