珍珠半夏(Pinellia ternate)高效再生体系的建立及其遗传稳定性检测

Efficient Plant Regeneration and Genetic Fidelity Assessment of in vitro-derived Pinellia ternate

本实验采用一年生珍珠半夏球茎为外植体,接种在MS+KT(1.0μM)+NAA(0.4μM)+蔗糖(3.0%)+琼脂(0.7%)培养基中,可在20~25 d内完成芽的萌发。再生芽在MS+NAA(0.4μM)+蔗糖(3.0%)+琼脂(0.7%)培养基中完成芽的伸长。伸长芽在MS+KT(1.0μmol/L)+NAA(0.4μmol/L)+蔗糖(3.0%)+琼脂(0.7%)培养基中,60 d内可以完成生根。将根放入1/2MS+NAA(2.0~5.0μmol/L)+IBA(2.0~5.0μmol/L)培养基中可完成生根。为建立稳定的高频离体快繁体系和适宜于遗传转化的再生体系,我们运用RAPD检测技术来确定离体培养过程中材料的体细胞遗传变异情况。本研究为山东省珍珠半夏的大规模生产和半夏的遗传改良提供技术平台。

Pinellia tripartita is of great interest because of its high value as a kind of elite herbage plant in Chinese traditional medicine. An efficient in vitro propogation system via protocorm-like body(PLB) induction has been developed using one year-old axillary node explants obtained from field-grown plants in the present research. The excised nodal buds began to sprout on Murashige and Skoog(MS) basal medium containing 1.0 μmol/L kinetin(KT)and 0.4 μmol/L naphthalene acetic acid(NAA) after 20~25 days in culture, and subsequently the well-developed vigorous shoots were obtained at the 60 th day after transfer. The regenerated shoots rooted well in 1/2MS basal medium when NAA(2.0~5.0 μmol/L) and 3-indole butyric acid(IBA, 2.0~5.0 μmol/L) were added. Axenic secondary explants were then transferred into new MS medium to which 0.4 μmol/L NAA was added for callus induction.The highest callus differentiation index(CDI) was scored from MS medium containing 0.5 μmol/L indolebutyric acid(IBA). Genetic fidelity of callus-derived shoots was assessed by performing PCR using 38 arbitrary primers. It was observed that all the primers displayed the same profiles between the stock plant and the randomly-selected shoots regenerated from the callus that had been subcultured within 15 cycles in MS medium containing 4.0 μmol/L NAA.