穿心莲叶片原生质体制备方法的建立与优化

Optimization of Protoplasts Preparation of Leaves from Andrographis paniculata(Burm.f.)Nees

为优化穿心莲叶片原生质体制备方法,本研究运用酶解法考察不同酶组合、渗透压、MES浓度、液料比、植物生长情况等因素对穿心莲原生质体制备的影响。结果表明,穿心莲叶片原生质体制备的适宜材料为长约3 cm的新鲜穿心莲叶片;酶解液组成为1.5%纤维素酶R-10+0.75%离析酶R-10+0.6 mol/LD-甘露醇为渗透压调节剂+20 mmol/LMES+5 mmol/L CaCl_(2)+0.1%BSA作为质膜稳定剂,液料比为50∶1。本研究获得的穿心莲叶片原生质体制备优化方法,可为后续原生质体培养提供参考依据和为穿心莲种质创新提供新途径。

To order to improve the preparation method of Andrographis paniculata leaf protoplasts,the study,enzymolysis were used to investigate the effects of different enzyme combinations,osmotic pressure,MES concentration,liquid to material ratio,plant growth and other factors on the preparation of Andrographis paniculata.The results show that the suitable material for preparing protoplasts of Andrographis paniculata leaves is fresh leaves of Andrographis paniculata about 3 cm;the composition of the enzymolysis solution is:1.5%cellulase R-10+0.75%isolated enzyme R-10+at 0.6 mol/L D-mannitol is an osmotic pressure regulator+20 mmol/L MES+5 mmol/L CaCl_(2)+0.1%BSA as a plasma membrane stabilizer with a liquid-to-material ratio of 50∶1.Obtained an optimize the preparation of Andrographis paniculata leaf protoplasts,provide a reference for the subsequent protoplast culture,and provide a new way for Andrographis paniculata germplasm innovation.