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[目的]探索多年生黑麦草的愈伤组织诱导和植株再生,为多年生黑麦草的转基因研究奠定基础。[方法]以多年生黑麦草成熟种子为外植体,施以浓度不同的外源激素,探讨不同激素组合对愈伤组织诱导、继代培养以及植株分化的影响。愈伤组织诱导以MS培养基为基础培养基,添加300 mg/L水解酪蛋白,300 mg/L脯氨酸,300 mg/L谷氨酰胺,并附加不同浓度2,4-D(2、4、5、8、10 mg/L)和6-BA(0.025、0.100、0.200、0.300、0.400、0.500 mg/L),pH值调节至5.8。继代培养基为MS+2,4-D减半的诱导培养基。愈伤组织分化以MS培养基为基础培养基,添加300 mg/L水解酪蛋白,300 mg/L脯氨酸,300 mg/mL谷氨酰胺,并附加不同浓度6-BA(0.50、1.0、2.0、3.0 mg/L),pH值调节至5.8。[结果]黑麦草在附加了8 mg/L2,4-D和0.025 mg/L6-BA的MS培养基中诱导率最高,达56.42%。愈伤组织经过2～3次继代后分化,分化培养附加2.0 mg/L6-BA,分化率最高,达34.14%,且愈伤组织继代后的状态明显影响分化率,带有毛状根的愈伤组织分化能力明显低于表面光洁的愈伤组织。在大量元素减半的MS培养基中附加0.5 mg/L NAA进行生根培养,生根率达98%。[结论]建立了高频的遗传再生体系,移栽成活率可达98%。
[Objective] The research aimed to explore the callus induction and plant regeneration of perennial ryegrass and lay a foundation for the transgenic study of perennial ryegrass. [Method] With mature perennial ryegrass as explants, exogenous hormones with different concentrations were applied to investigate the effects of different combinations of hormones on callus induction, subculture and plant differentiation. Callus induced MS medium supplemented with 300 mg / L hydrolyzed casein, 300 mg / L proline and 300 mg / L glutamine supplemented with different concentrations of 2,4-D (2, 4,5,8,10 mg / L) and 6-BA (0.025, 0.100, 0.200, 0.300, 0.400, 0.500 mg / L) and pH adjusted to 5.8. Subculture medium is induction medium with MS + 2,4-D halved. Callus differentiation was based on MS medium supplemented with 300 mg / L hydrolyzed casein, 300 mg / L proline, and 300 mg / mL glutamine supplemented with different concentrations of 6-BA (0.50, 1.0, 2.0, 3.0 mg / L) and the pH was adjusted to 5.8. [Result] The highest induction rate of ryegrass in MS medium supplemented with 8 mg / L 2,4-D and 0.025 mg / L 6-BA was 56.42%. After 2 ~ 3 subcultures, the callus differentiated and differentiated and cultured with additional 2.0 mg / L 6-BA, the differentiation rate was the highest, reaching 34.14%. The callus subcultured significantly affected the differentiation rate and had hairy roots The callus differentiation ability was significantly lower than the callus surface smooth. 0.5 mg / L NAA was added to MS medium with a large number of elements reduced by half for rooting. The rooting rate was 98%. [Conclusion] The high frequency genetic regeneration system was established, the survival rate of transplanting up to 98%.