八仙花萼片有色原生质体游离及应用

为建立八仙花（Hydrangea macrophylla）有色原生质体游离体系，以八仙花萼片为试验材料，分别对材料的预处理方式、酶液组分、酶解时间、纯化离心力等影响酶解的关键因素进行研究。结果表明， 预处理方式选择分离花萼片上下表皮法，纤维素酶和离析酶水平分别为4%和0.4%（质量体积分数），甘露醇0.6 mol·L^-1，酶解液加热冷却后加入 0.1%（质量体积分数）BSA和10 mmol·L^-1 CaCl₂，酶解2 h，低温离心机4 ℃、1 426 r·min^-1离心5 min，为最优的八仙花萼片原生质体游离条件。利用所建的原生质体游离体系对4个不同品种的八仙花萼片进行有色原生质体游离，200× 镜下单视野内可获得13~35个有色的原生质体；对有色原生质体进行液泡的H⁺流测定，发现蓝色液泡较粉色液泡H⁺流的外排趋势明显，说明同一八仙花品种蓝色液泡的pH高于粉色液泡。

Isolation and Application of Sepal Colored Protoplast in Hydrangea macrophylla

In order to establish isolation system of sepals colored protoplast in Hydrangeamacrophylla， sepals of Hydrangeamacrophylla were used as experimental materials. The factors influencing enzymolysis were analyzed， such as treated methods of sepals， the components of enzymatic hydrolysates， enzymolysis time， centrifugal speeds etc. The results showed that the optimal procedure of enzymolysis was followed with the toring epidermis of sepals， 4% （w/v） cellulase， 0.4% （w/v） macerozyme， 0.6 mol·L^-1 mannitol and 0.1% （w/v） BSA， 10 mmol·L^-1 CaCl₂， enzymolysis for 2 h， and 1 426 r·min^-1 centrifuge at 4 ℃. This system was used to isolate sepal colored protoplast of 4 Hydrangea macrophylla cultivars， and 13~35 protoplasts per microscope vision at 200× were obtained. H⁺ flux measurement showed that the H⁺ effluxing tendency of blue vacuoles was more obvious than that of pink vacuoles in the same cultivar， it indicated that pH in blue vacuoles was higher than that in pink vacuoles of same cultivar.