枸杞、番茄体细胞原生质体培养体系研究

Study on the protoplast culture of somatic embryogenesis of Lycium barbarum. L. and Lycopersicon esculentum Mill.

利用组培技术,以宁杞1号枸杞和农大1号番茄种子的无菌苗叶片、下胚轴为外植体,脱分化培养分别获得其愈伤组织,经继代增殖和酶解处理获得质量较好的原生质体。结果表明,枸杞、番茄无菌苗的下胚轴分别在附加有1.0mg/L2,4-D＋1.5mg/LKT和1.0mg/L2,4-D＋1.0mg/LKT的MS固体培养基中诱导的愈伤组织（诱导率分别是68.45%、64.33%）无褐化或褐化率最低,产生的愈伤组织适合于原生质体培养且效果最好;将0.5%果胶酶、1%纤维素酶、0.2%离析酶R-10、0.2%半纤维素酶配合使用,在pH5.0~6.0、温度30℃下振荡酶解12~16h,所得枸杞、番茄原生质体均在Km8P液体培养基中纯化培养,原生质体及活力状态均可保持2d左右。

The sterilized seeds of ＂Ningqi 1＂ of Lycium barbarum L. and ＂Nongda 1＂ of Lycopersicon esculentum Mill were germinated on MS or 1/2MS medium to acquire aseptic seedlings, and then, using the seedlings to induce callus on MS medium supplemented. A stable cell suspension culture system was established with callus of L. barbarum L. and L. esculentum Mill after several rounds of subculture.The results showed that supplement of 1.0 mg/L 2, 4-D and 1.5 mg/L KT for L. barbarum L. and 1.0 mg/L 2, 4-D and 1.0 mg/L KT for L. esculentum Mill in MS solid culture medium are the best condition for inducing callus both of them, and achieved 68.45% and 64.33% induction rate respectively. Treating suspension cell of 0.5% enzyme（Pectinase）, 1% cellulase（Cellulase）, isolated and R-10 0.2%（Macerozyme R-10）, 0.2%, hemicellulase （Hemicellulase） at 30℃, pH 5.0-6.0 with a 12-16 h shaking resulted in the protoptast actvitity for 2 days.