黑果枸杞愈伤组织制备原生质体条件优化

Optimized Condition for Protoplast Isolation from Lycium Ruthenicum Murr

通过研究酶解时间、甘露醇浓度、酶液组成和离心速度对黑果枸杞愈伤组织制备原生质体分离效果的影响,并对制备原生质体用FAD染色检测活力.结果表明:黑果枸杞的原生质体分离最佳条件为酶解时间12h,甘露醇浓度0.4mol/L,酶液组成为纤维素酶1.8%+果胶酶0.3%+离析酶0.5%,离心速度800r/min,在此条件下原生质体产量为2.65×10~6个/gFW,FDA荧光检测活力在80%以上.

The factors effecting isolation of Lycium combinations of dissolved time, maunitol concentrations, ruthenicum Murr from, callus tissue, including different enzyme mixture,centrifugal speed and time are studied. The yield and viability of protoplast is measured through different combination of enzyme, different dissolved times, and when the separation being at low-speed of 800r/min. Then, the protoplast vitality are tested by fluorescein diac- etate staining method. The results indicates that the optimum enzyme soluteion for Lycium ruthenicum Murr leaves is to dissolve it 12 hours with a combination of Cellulase 1.8%＋Pectmase 0.3%＋macerozyme R-10 0.5%, and the mannitol concentrations is 0.6 mol/L. A plenty of vigorous protoptasts of Lycium ruthenicum Murr from leaves could be obtained at centrifugate 6 rain with 800 r/min. The protopl-ast yield is 2.65×106cells/gFW,and the vitality is more than 80% by using fluorescein diacetate staining method.