播娘蒿与甘蓝型油菜的原生质体融合与植株再生

Production of asymmetric hybrids between Descurainia sophia and Brassica napus L utilizing an efficient protoplast-fusion system

以播娘蒿叶片和甘蓝型油菜子叶为材料提取原生质体。采用PEG—高pH、高钙附加DMSO融合方法,液体浅层静置培养,研究影响播娘蒿与甘蓝型油菜原生质体融合的因素,获得了再生植株。结果表明:4%纤维素酶+0.5%离析酶+5mmol/L MES酶解10h可获得高产率播娘蒿原生质体,1%纤维素酶+0.2%离析酶+3mmol/LMES酶解14h可获得高产率油菜原生质体;30%PEG+0.3mol/L葡萄糖+50mmol/L CaC l2.2H2O+15%DMSO可获得10%的融合率;改良B5培养基的原生质体培养效果最好;最佳分化培养基为MS+4.0mg/L 6-BA+0.3mg/LNAA+5.0mg/L AgNO3;最佳生根培养基为MS+0.1mg/L IBA+0.1mg/L NAA。

High yield of protoplast isolation was achieved from leaves of D. sophia and cotyledons of B. napus L. The isolated protoplasts were used for the establishment of an efficient protoplast-fusion system between the two cruciferous species. The best enzyme combination for isolating protoplast from D. sophia and B. napus was 4% Cellulase ＋ 0.5% Macerozyme ＋5mmoL/L MES and 1% Cellulase ＋ 0.2% Macerozyme ＋ 3mmoL/L MES, respectively. Using PEG-DMSO method, fusion percentage of 10.0% was obtained. Modified B5 media was the best for the culture of fused cells among the three liquid media B5, Km8p and MS. The best media for shoot regeneration from caUi was MS ＋4.0mg/L 6-BA ＋0.3mg/L NAA ＋ 5.0mg/L AgNO3 , and the best rooting media was MS ＋0.1 mg/L IBA ＋ 0.1 mg/L NAA.