地锦与五叶地锦原生质体分离及培养研究

Isolation and Culture of Parthenocissus tricuspidata and P.quenquefolia Protoplasts

用纤维素酶、果胶酶、甘露醇的混合酶液提取地锦和五叶地锦无菌苗幼叶、五叶地锦胚乳愈伤组织及地锦幼胚愈伤组织的原生质体,对影响原生质体提取得率及活力因素进行分析,对不同材料原生质体得率进行比较,对提取的原生质体进行液体浅层、固液结合及固体等方式培养,对原生质体形成的愈伤组织以MS和改良B5培养基附加不同浓度NAA、6 BA、2,4 D、PEG、KT、ZT等进行分化培养。试验结果表明:纤维素酶对原生质体提取得率有极显著影响,适宜地锦幼叶原生质体提取的酶液组合为:纤维素酶0.5%、果胶酶0.3%、甘露醇0.6mol·L-1、酶解8h;不同材料原生质体提取得率有显著差异;仅五叶地锦胚乳愈伤组织来源的原生质体在固体培养基中形成新的愈伤组织,其它方式培养的不同材料原生质体均无分裂或仅形成数十个细胞的细胞团后便解体;用30余组配方进行了五叶地锦胚乳愈伤组织原生质体形成的新愈伤组织的分化培养,继代6～10次后仍无分化迹象。

Mixed enzyme liquid of cellulase R-10,Pectolyase Y_(23)and mannitol was used to extract the protoplasts from sterilized seedling leaves and endosperm callus of Parthenocissus tricuspidata and P.quenquefolia.The factors affectiong the yield and activities of portoplasts were analyzed and the yield of protoplast from different sources was compared.The protoplasts obtained were cultured by liquid,solid-liquid and solid culture methods.The differentiated cultures were conducted on the callus formed from protoplasts on MS and modified B5 media with different concentrations of NAA,6-BA,2,4-D,PEG,KT,and ZT.The results showed that cellulase R-10 had extremely singificant influences on the extract yield of protoplasts.The suitable enzyme liquid combination for extracting protoplast of P.tricuspidata was:cellulase R-10 0.3%,mannitol 0.6 mol·L^(-1),with enzymolysis time 8 hours.There existed significant differences among different sources in the yield of protoplast.Only the protoplast from P.quenquefolia endosperm callus could form new callus on soild media.While the protoplasts from other sources had no divistion or disintegrated after forming cell groups containing dozens of cells.The new callus tissues formed from endosperm callus protoplasts of P.quenquefolia were differentiated cultured with over thirty prescriptions,and no differentiation was found after 6-10 subculture.