Shoot differentiation and gene-transfer in Periploca sepium Bunge

摘要以含橡胶元素的中国本土藤本植物黑枝栎（Periploca sepium Bunge）的子叶和下胚轴为茎的分化工具。采用ms培养基，BAP和NAA均作为芽分化激素。子叶和下胚轴均有分化，但下胚轴分化率较高。6μ mol/l IAA浓度为1/2MS，可识别根系分化。茎和叶用于芽分化。采用ms培养基，添加BAP和NAA作为芽分化激素。BAP 1μM ~ 3μM与NAA 0.1μ m组合和BAP 3μM ~ 10μM与NAA 0.1 ~ 1μM组合在茎部分化率较高。鉴定出高分化率，分化率在90% ~ 100%之间。介绍了农杆菌介导的基因转移法。以绿色荧光蛋白（GFP）基因为模式基因，以外植体为外植体。从茎中再生出一株在显微镜下具有绿色荧光的植株。采用PCR和Western分析法对DNA和GFP蛋白进行检测。从这两种分析结果来看，GPF基因在棘豆中的转移是成功的。介绍了农杆菌介导的铁蛋白基因转移方法。从茎部再生出两个植株，并通过PCR检测到两个植株的DNA片段。

Cotyledon and hypocotyl of Periploca sepium Bunge, which is a native liana tree species and contents rubber element in china, were used for shoot differentiation. MS-medium was used and both of BAP and NAA are used as hormones for shoot differentiation. Shoot differentiation was recognized in both cotyledon and hypocotyls, but differentiation rate was higher in hypocotyl. Root differentiation was recognized by using 1/2MS with 6μ mol/l IAA.Stem and leaf of Periploca were used for shoot differentiation. MS-medium was used and both of BAP and NAA were added as hormones for shoot differentiation. Differentiation rate in stem was higher in the combination of BAP 1μM〜3μM and NAA 0.1μM and the combination of BAP 3μM〜10μM and NAA 0.1〜1μM in leaf. High differentiation rate was recognized and that was in the range from 90% to 100%.Agrobacterium-mediated gene-transfer method was introduced for Periploca. GFP gene was used as a model-gene and stem from micropropagated shoot was used for explant. A plantlet that had green fluorescent under microscope was regenerated from a stem. PCR analysis and Western Analysis were used to investigate the DNA and GFP protein. From the result of both analysis, gene transfer of GPF gene had succeeded in Periploca.Agrobacterium-mediated gene -transfer method was introduced to Ferritin gene. Two plantlets had been regenerated from shoot stem, and DNA segment was detected in these two plantlets by PCR analysis.