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以6份耐寒结球甘蓝杂交F1为试验材料,研究其胚胎发生过程及胚胎发生的诱导条件,为耐寒甘蓝材料的小孢子培养,以及其他芸薹属小孢子胚胎体系的建立及优化提供理论依据。采用4′,6-二脒基-2-苯基吲哚(DAPI)染色方法对小孢子体内和体外的发育及胚胎发生过程、特征进行观察;NLN-13培养基中添加阿拉伯半乳聚糖(Arabinogalactan,AG)和阿拉伯半乳糖蛋白(Arabinogalactan-protein,AGP)对小孢子培养胚胎发生率的影响进行统计分析;小孢子体内正常发育方向是单核小孢子经历2次不对称核分裂后发育成三核成熟花粉,小孢子单核晚期对应的花蕾长度主要集中在3.5~4.5mm;体外培养中,热激胁迫(32.5℃)诱导小孢子从配子体发育途径向胚胎发生途径转换,只有少数胚性细胞最终发育成为胚状体;供试材料中有5份材料获得胚状体,材料间出胚量差异较大,其中‘寒玉37’出胚量最高,为每蕾22.5个;在NLN-13培养基中添加10mg/L AG或10mg/L AGP,可以促进耐寒甘蓝胚状体发生。32.5℃热激胁迫1d可有效诱导耐寒甘蓝小孢子培养中的胚胎发生,10mg/L AG或10mg/L AGP促进胚胎诱导。
Six cold-resistant cabbage F1 hybrids were used as experimental materials to study the embryogenic process and embryogenic induction conditions, which provided theoretical basis for the establishment and optimization of microspore culture of Brassica napus embryos. . In vitro and in vivo development and embryogenesis of microspores were observed using 4 ’, 6-diamidino-2-phenylindole (DAPI) staining methods and their characteristics were observed. In NLN-13 medium, arabinogalactan The effect of Arabinogalactan (AG) and Arabinogalactan-protein (AGP) on the embryogenesis rate of microspore culture was analyzed statistically. The normal microspore developmental direction was that the microspore developed after two asymmetric mitoses In the in vitro culture, heat shock stress (32.5 ℃) induces the transformation of microspores from the path of gametophyte development to embryogenesis, and only a few embryos The cells eventually developed into embryoid bodies; 5 materials in the tested materials obtained embryoid bodies, and the amount of embryos produced among the materials differed greatly. Among them, ’Hanyu 37’ had the highest number of embryos at its birth, which was 22.5 per bud. In NLN- 13 medium supplemented with 10 mg / L AG or 10 mg / L AGP could promote the embryogenesis of cold-resistant cabbage. Heat shock at 32.5 ℃ for 1d effectively induced embryogenesis in microspore culture of resistant cabbage. 10mg / L AG or 10mg / L AGP promoted embryo induction.