基于PGGB途径优化桫椤组织培养繁殖体系研究

Optimization of Tissue Culture and Propagation System of Alsophila spinulosa Based on PGGB Pathway

为优化桫椤(Alsophila spinulosa)快繁体系,提高其繁殖效率,以桫椤孢子为外植体,通过研究不同生长调节剂对孢子诱导配子体、原叶体绿色球状体(prothallus green globular body,PGGB)诱导、PGGB分化、孢子体生根4个过程的影响,进一步探讨不同基质对桫椤组培苗移栽的成活效果。结果表明,(1)孢子诱导配子体,1/8 MS+0.5 mg/L 6-BA+0.1 mg/L NAA+蔗糖20 g/L+琼脂10 g/L诱导效果最佳,诱导率64.62%,诱导天数23 d。(2)PGGB诱导,1/2 MS+0.3 mg/L 6-BA+0.4 mg/L NAA+蔗糖30 g/L+琼脂6 g/L诱导率达100%,PGGB鲜重1.13 g,增殖倍数11.04,直径2.01 cm。(3)PGGB分化,在1/2 MS+0.3 mg/L KT+0.4 mg/L IBA+蔗糖30 g/L+琼脂6 g/L的分化效果最佳,分化率92.12%,PGGB平均分化芽数41.67个。(4)孢子体生根,孢子体在1/2 MS+1.0 mg/L IBA+蔗糖30 g/L+琼脂6 g/L中的生根率100%。(5)炼苗,组培苗移栽在基质(红壤∶腐殖土∶泥炭土=2∶1∶1)中的成活率为96.3%。桫椤PGGB诱导及其再生体系的建立受植物生长调节剂影响,且基质对桫椤组培苗的成活率差异显著。研究成功构建了1个高效的桫椤繁殖再生体系,为进一步探究桫椤体外培养发育机制、新品种、规模生产及应用提供技术支持。

To optimize tissue culture techniques of Alsophila spinulosa to improve the success rate and productivity of tissue culture,we used A.spinulosa spores as explants to study the effects on the four processes:Spore-induced gametophytes by different growth regulators,prothallus green globular body(PGGB)induction,PGGB differentiation and sporophyte rooting.It was studied to further explore the survival effects of different substrates on the transplantation of A.spinulosa tissue culture seedlings.The results showed that,(1)spore-induced gametophytes,the best induction medium was 1/8 MS+0.5 mg/L 6-BA+0.1 mg/L NAA+sucrose 20 g/L+agar 10 g/L,with an induction rate of 64.62%and induction days of 23 d.(2)PGGB induction,the induction rate of 1/2 MS+0.3 mg/L 6-BA+0.4 mg/L NAA+sucrose 30 g/L+agar 6 g/L reached 100%,the fresh weight of PGGB was 1.13 g,the multiplication ratio was 11.04 and the diameter was 2.01 cm.(3)PGGB differentiation,the best differentiation result was obtained at 1/2 MS+0.3 mg/L KT+0.4 mg/L IBA+sucrose 30 g/L+agar 6 g/L,with a differentiation rate of 92.12%and a PGGB average of 41.67 differentiated buds.(4)Sporophyte rooting,the rooting rate of spores in 1/2 MS+1.0 mg/L IBA+sucrose 30 g/L+agar 6 g/L was 100%.(5)Acclimatization,the survival rate of tissue culture seedlings transplanted in the substrate(red soil∶Humus soil∶Peat soil=2∶1∶1)was 96.3%.The establishment of A.spinulosa PGGB induction and regeneration system was affected by plant growth regulators,and the survival rate of tissue culture seedlings was significantly different.This study successfully constructed an efficient reproduction and regeneration system of A.spinulosa,providing technical support for further exploring the development mechanism of A.spinulosa in vitro culture,new species,large-scale production,and application of A.spinulosa in soil matrix.