川东獐牙菜体外高效再生体系的建立

为了构建川东獐牙菜(Swertia davidii Franch.)高效稳定的再生体系,以川东獐牙菜带叶茎尖、带芽茎段和叶片为材料,在单因素试验的基础上,通过完全组合及L₉(3⁴)正交试验,探究不同激素对其离体培养的影响。结果表明,川东獐牙菜3种外植体中,叶片适宜作为间接器官发生材料,在MS+2.0 mg·L^-1 6-BA+1.5 mg·L^-1 KT培养条件下,培养7 d便可见愈伤组织从切口处产生,培养30 d后即可分化出大量丛芽;不定芽在相同培养基中培养30 d后,增殖系数可达8.75。带芽茎段则适宜直接器官发生途径,其在MS +2.0 mg·L^-1 6-BA+1.0 mg·L^-1 NAA中培养7 d后,节上腋芽开始萌动,培养30 d后腋芽发生系数可达4.06;试管苗适宜的生根培养基为MS + NAA 0.05 mg·L^-1,培养30 d后即可获得再生植株,生根率为100%;生根苗经过炼苗,移栽30 d后成活率达90%以上。在川东獐牙菜的离体培养中,间接器官发生途径较直接器官发生途径效率更高。本研究通过叶愈伤组织-不定丛芽途径建立了川东獐牙菜高效再生体系,为保护其野生资源和种苗繁育提供了技术支撑,也为其遗传转化奠定了试验基础。

Establishment of a High-Effective Regeneration System for Swertia davidii Franch.

In order to establish a high-effective regeneration system for Swertia davidii, induction of callus and axillary buds were conducted using stem tips with leaves, stem with buds and leaves to explore the effects of different hormones on plant regeneration based on a single factor experiment, a complete combination test and a L₉(3⁴) orthogonal experiment. The results indicated that leaves were the most suitable materials for indirect organogenesis among three kinds of explants of S. davidii. The callus could be induced at the notch after culturing for 7 days and numerous adventitious shoots were differentiated after culturing for 30 days on the MS medium containing 6-BA 2.0 mg·L^-1 and KT 1.5 mg·L^-1. The multiplication coefficient of these shoots cultured on the same medium reached to 8.75. Stem with buds were suitable for direct organogenesis. Axillary buds began to grow after 7 days on the MS medium supplementing 6-BA 2.0 mg·L^-1 and NAA 1.0 mg·L^-1; the coefficient of axillary buds formation was 4.06. The optimal medium for rooting was found to be MS medium containing NAA 0.05 mg·L^-1. The rooting rate was 100% when cultured up to 30 days with whole plant regeneration. After acclimatization training, the survival rate of plantlets was more than 90% after transplanting into the field. Indirect organogenesis had a higher efficiency than that of direct organogenesis. Our present study provides a high-effective regeneration system for S. davidii from callus to adventitious shoot. The protocol constructed provides technical support for protecting the wild resource and seedling breeding, and also offers afoundation for the further genetic transformation.