小青杨叶肉原生质体分离条件的研究

Isolation of Mesophyll Protoplasts from Populus pseudo-simonii Kitag.

为获得大量、有活力的原生质体,建立起高效的小青杨(Populus pseudo-simonii Kitag.)原生体分离体系,本研究以小青杨无菌苗叶片为材料进行原生质体分离条件的研究。结果表明:酶的种类和浓度、渗透压稳定剂浓度、酶解时间、叶龄是影响原生质体分离的重要因素;将叶龄35天的无菌苗第2～3片叶片置于酶液组成为CPW+3.0%纤维素酶R-10+0.5%离析酶R-10+0.1%果胶酶Y-23+0.6mol/L甘露醇+0.6g/LMES+1g/LBSA中酶解8h,原生质体产量和活力分别为2.44×107个/g和78.7%。通过该分离体系稳定的获得了高产量、高活力的原生质体,可以满足进一步的原生质体培养等技术的要求。

To obtain high yield and high viability protoplasts,an efficiency protocol was presented here using leaves ofin vitro shoot culture ofPopulus pseudo-simonii Kitag..The yield of protoplasts depended on factors such as enzyme concentration,osmoticum concentration,incubation time and the age of leaves.The results showed that the optimal protocol for isolating protoplasts was treating 35 days aged leaves with CPW salts ＋ 3.0% Cellulase R-10 ＋ 0.5% Macerozyme R-10 ＋ 0.1% Pectolase Y-23 ＋ 0.6 mol/L mannitol ＋ 0.6 g/L MES＋ 1 g/L BSA for 8 h.Protoplast yields were up to 2.44×107 protoplasts per gram and the viability was 78.7%.This protocol that resulted in high yield and high viability of protoplasts could meet the demands of protoplast culture-based techniques.