Shoot differentiation and gene-transfer in Periploca sepium Bunge

以我国特有的藤本植物杠柳（PeriplocasepiumBunge）的子叶和下胚轴为外植体，进行芽分化研究。以MS为基本培养基，以BAP和NAA为激素诱导不定芽分化。子叶和下胚轴均能分化芽，但下胚轴分化率较高。根的分化用1/2 MS +6μ mol/lIAA进行鉴定，不定芽的分化用杠柳的茎和叶。以MS为基本培养基，附加BAP和NAA作为芽分化的激素。茎段分化以BAP 1μM × 3μM + NAA 0.1μM组合最高，叶片分化以BAP 3μM × 10μM + NAA 0.1 μM组合最高。采用农杆菌介导法对杠柳属植物进行了基因转移研究。以GFP基因为模型基因，以微繁殖的茎段为外植体。从茎再生出在显微镜下具有绿色荧光的植株。用PCR和Western分析检测DNA和GFP蛋白。从两种分析结果来看，GPF基因在杠柳属植物中的转基因已经成功。从茎段再生出2株再生植株，PCR检测到2株再生植株的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.