半夏游离块茎诱导及其遗传稳定性SSR鉴定

Free Tuber Induction and Its Genetic Stability Identification of Pinellia ternata by SSR

为探究半夏疏松愈伤组织继代条件,建立半夏游离块茎悬浮培养体系,并验证其组织培养过程中是否发生体细胞无性系变异,以半夏叶柄为外植体,研究不同浓度的蔗糖与激素配比对其疏松愈伤组织增殖的影响,设计单因素试验和正交试验筛选诱导其游离块茎的适宜条件,通过SSR分子标记技术,检测游离块茎的遗传稳定性。结果表明,半夏疏松愈伤组织形成的适宜继代培养基为MS+2,4-D 2.0 mg·L^(-1)+6-BA 0.5 mg·L^(-1)+蔗糖30 g·L^(-1)+0.6%琼脂,以此继代可获得大量用于建立悬浮体系的半夏疏松愈伤组织;适于疏松愈伤组织分化形成游离块茎的培养基为MS+6-BA 0.5 mg·L^(-1)+IBA 0.5 mg·L^(-1)+蔗糖20 g·L^(-1),以此培养可获得大量用于人工种子制作或直接播种的半夏游离块茎;通过SSR标记,发现组织培养诱导形成的半夏游离块茎在DNA水平上未发生变异,仍保持其亲本的遗传稳定性。

To explore the subculture conditions of loose Pinellia ternate callus, establish suspension culture system of free P. ternate tuber, and verify whether somaclonal variation occurred during tissue culture, the P. ternate petioles were used as explants to study the effects of different concentrations of sucrose and hormone ratio on the proliferation of loose callus, and single factor test and orthogonal test were designed to screen the suitable conditions for inducing the free P. ternate tuber. Meanwhile, the genetic stability of free tuber was also detected by SSR markers. The results showed that MS+2.0 mg·L^(-1)2, 4-D+0.5 mg·L^(-1)6-BA+30 g·L^(-1)sucrose+0.6% agar was the optimal medium for the formation of loose P. ternate callus, which could obtain a large number of loose P. ternata callus by subculture with this medium. In addition, the medium suitable for the differentiation of loose callus into free tubers was MS+0.5 mg·L^(-1)6-BA+0.5 mg·L^(-1)IBA+20 g·L^(-1)sucrose, which could induce a large number of free P. ternate tubers for artificial seed production or direct sowing. Finally, it was found that the free P. ternate tuber induced by tissue culture did not change at DNA level, but still maintained the genetic stability of its parents based on SSR markers.