【作者】 马月； 李宏宇； 孙红梅；

【Author】 MA Yue;LI Hongyu;SUN Hongmei;Key Laboratory of Protected Horticulture of Education Ministry, College of Horticulture, Shenyang Agricultural University;National and Local Joint Engineering Research Center of Northern Horticultural Facilities Design and Application Technology;

【机构】 沈阳农业大学园艺学院,设施园艺教育部重点实验室； 北方园艺设施设计与应用技术国家地方联合工程研究中心；

【摘要】 体细胞胚发生是植物表达细胞全能性的直接体现,因其具有再生植株结构完整、繁殖系数高和遗传稳定性强等诸多优点,被广泛应用于植物离体繁殖和种质创新。大多数物种中体胚发生仅限于特定的外植体状态和基因型。目前对不同基因型的体胚发生的分子机理尚不清楚。本研究以体胚高效发生的新疆百合(Lilium martagon var. pilosiusculum)以及难以形成体胚的朝鲜百合(L. amabile)为材料,对影响二者体细胞胚发生的差异进行了探索,以期挖掘影响百合体细胞胚性能力获得的关键因子,为顽拗型百合体胚的诱导提供理论和实践依据。组织切片观察细胞形态、细胞核形态和内含物染色情况,明确PIC诱导第6 d是新疆百合胚性细胞的发生的关键时间节点,也是朝鲜百合外植体细胞凋亡、褐化的起始时期。广靶代谢组分析表明,在胚性获得的关键节点,新疆百合比朝鲜百合积累了更多的氨基酸及其衍生物、糖类物质以及生物碱;而朝鲜百合积累了大量的酚酸类和黄酮类代谢物。KEGG富集分析表明,谷胱甘肽代谢途径富集了大量的差异表达物质。其中,还原型谷胱甘肽和氧化型谷胱甘肽的比值,可能是通过调节外植体细胞氧化还原稳态决定两种百合体细胞的脱分化和胚性获得能力差异的关键。外源添加还原型的谷胱甘肽GSH(0-0.6 mg/L)以及谷胱甘肽抑制剂BSO(0-0.3 mg/L),能够大幅度降低朝鲜百合鳞片外植体的褐化,并成功诱导外植体细胞脱分化产生愈伤组织。上述结果表明,不同基因型百合细胞调节氧化还原的能力对其胚性获得至关重要,为优化体胚发生困难物种的诱导条件提供了参考。更多还原

【Abstract】 Somatic embryogenesis is a direct process in which plant express cell totipotency.It has been widely used in in vitro propagation and germplasm innovation because of its complete structure, high reproduction coefficient and strong genetic stability. In most species, somatic embryogenesis occurs only in specific explant states and genotypes. At present, the molecular mechanism of embryo formation in different genotypes remains unclear. In order to explore the vital factors affecting the acquisition of embryogenicity in lily cells, Lilium martagon var. pilosiusculum with high somatic embryogenesis and L. amabile with difficult somatic embryogenesis were taken as materials in this study to explore the differences of somatic embryogenesis. Our results may provide theoretical and practical basis for the induction of recalcitrant lily somatic embryos. By observed cell morphology, nuclear morphology and inclusions staining in tissue sections, we confirmed that the 6th day after treated by PIC was the vital time point for acquisition of embryogenicity to L. martagon var. pilosiusculum. Meanwhile, L. amabile began to cell apoptosis and browning. Broad-target metabolite profiling showed L. martagon var. pilosiusculum accumulated more amino acids and derivatives, carbohydrates and alkaloids, while L. amabile accumulates a large number of phenolic acids and flavonoid metabolites in the acquisition of embryogenicity. KEGG enrichment analysis demonstrated that glutathione metabolism pathway enriched a large number of differentially expressed metabolites. The ratio of reduced and oxidized glutathione may be crucial to determine the lily cells dedifferentiation and acquisition of embryogenicity, because of which may regulate the oxidization and reduction of explants. Further experiments confirmed this result. Adding reduce glutathione GSH(0-0.6 mg/L) and inhibitor BSO(0-0.3 mg/L) in scale induction medium of L. amabile may significantly reduce the browning and successfully induced callus. In conclusion, we indicated that the ability of regulating reoxidation-reduction is crucial for inducing somatic embryogenesis for different lily genotypes. These results may provide a reference for optimizing the induction conditions of the species with difficulty in somatic embryogenesis.更多还原