滇龙胆丛芽高效诱导与植株再生体系的建立

目的以滇龙胆Gentiana rigescens带叶茎尖、带芽茎段、叶片、根为外植体,建立滇龙胆高效、稳定的丛芽诱导体系,筛选出适宜的植株再生途径。方法以MS为基本培养基,在单因素实验的基础上,通过L16(45)正交及完全组合实验研究不同外植体和不同植物激素种类及其质量浓度对愈伤组织诱导、丛芽发生及植株再生的影响。结果带芽茎段为间接器官发生最佳材料,其在MS+6-BA 1.5 mg/L+NAA 1.5 mg/L+KT 0.05 mg/L中培养10 d、节上腋芽开始萌发后,基部可诱导出再分化能力极强的愈伤组织,得愈率为97.73%;15 d后愈伤组织开始分化出绿色丛芽,分化率100%;30 d后不定芽分化系数达18.65,增殖系数可达63.58。带叶茎尖为直接器官发生最佳材料,其在MS+6-BA 0.5 mg/L+NAA 1.0 mg/L+KT 0.5mg/L中培养30 d后,增殖系数亦可达44.36。2种材料培养获得的试管苗茎细瘦弱,不利于生根培养,试管苗在MS+6-BA1.5 mg/L+NAA 1.5 mg/L+KT 0.05 mg/L+PP333 10 mg/L中培养60 d后可获得健壮度大为改善的复壮苗。复壮苗生根则在1/2MS+6-BA 0.1 mg/L+NAA 2.0 mg/L中进行,45 d后可获得生长健壮的再生植株,生根率100%。再生苗移栽成活率达95%以上。结论建立的滇龙胆无性快速繁殖体系,为保护滇龙胆野生资源、优质种苗繁育提供了有效途径,也为其遗传转化研究奠定了实验基础。

Efficient induction system of adventitious shoot and plantlet regeneration in Gentiana rigescens

Objective An efficient and stable regeneration system was developed to select the optimal protocol for plant regeneration with various explants of Gentiana rigescens. Methods The study was designed as a L₉(3⁴) orthogonal experiment to explore the effects of different types and concentration of plant growth regulator combinations on adventitious shoot formation and plant regeneration, using different explants maintained on Murashige and Skoog (MS) medium after a single factor pre-experiment had been conducted. Results Stem with buds was the optimal explant for indirect organogenesis with a frequency of callus induction of up to 97.73% obtained on MS + 6-BA 1.5 mg/L + NAA 1.0 mg/L + KT 0.05 mg/L after 10 dwith the axillary shoots starting to germinate along with the emergence of callus with a strong capacity of differentiation from the base; The callus started to generate green multiple shoots with a 100% rate after 15 d and a coefficient of differentiation in the clump shoot of 18.65. The multiplication coefficient was up to 63.58. However, the stem tip was the best explant for direct organogenesis with a propagation coefficient of up to 44.36 cultured on MS + 6-BA 0.5 mg/L + NAA 1.0 mg/L + KT 0.5 mg/L 30 days later. The plantlet derived from two explants was too weak for rooting and the weak multiplication plantlet was maintained on MS medium supplemented with 6-BA 1.5 mg/L, NAA 1.0 mg/L, KT 0.05 mg/L, and PP₃₃₃ 10 mg/L to rejuvenation for 60 d. The regeneration plant with 100% rooting rate was obtained by culturing on 1/2 MS + 6-BA 0.1 mg/L + NAA 2.0 mg/L for 45 d and more than 95% of plantlets survived after transplanting. Conclusion The current study established a rapid propagation system which is helpful to protect the wild resources and high quality seedling propagation of G. rigescens, meanwhile providing scientific evidence for the research on genetic transformation.