牡丹叶片原生质体分离条件的研究

Study on the Conditions of Protoplast Isolation of Peony Leaves

以地方品种洛阳红牡丹叶片为材料,通过单因素和正交试验,研究了预处理时间、酶解时间、不同酶组合及酶液中甘露醇含量等因素对牡丹叶片原生质体分离的影响。结果表明:在蔗糖浓度为0.3mol/L的溶液中,预处理0.5h效果最好,(25±1)℃条件下,在0.5%纤维素酶+0.4%果胶酶+0.3%半纤维素酶+10%甘露醇、pH值5.8的混合酶液中,酶解6h所得的原生质体产量最高,有活力的细胞可达2.325×106个/g。

The leaves of the local variety Luoyangred were used as experiment materials.Uing single elemental and orthogonal experiment,the influences of pretreatment time,digestion time,different enzyme combinations and mannitol content in the mixed enzyme solution on protoplast isolation from leaves were studied.The results showed that the optimal pretreatment time was 0.5 h in the solution with 0.3 mol/L sugar and the leaves digested for 6 h in a mixture of cellulase 0.5%＋hemicellulase 0.5%＋pectinase 0.4%＋10% manitol at the temperature of 25 ℃,pH 5.8,producing active protoplast with yield of up to 2.325×106 cell per gram.