烟草NC_(89)叶肉细胞原生质体培养再生植株及影响因素的研究

Regeneration of Whole Plant From Tobacco NC 89 Mesophyll Protoplasts and Its Factor

生长１２０天的ＮＣ８９无菌苗叶片在ＯｎｏｚｕｋａＲ—１０和ＭａｃｅｒｏｚｙｍｅＲ１０的混合酶液中，酶解、收集、纯化，在修改的ＮＴ培养基上，应用液体—固体双层培养，原生质体经生长、分裂，形成愈伤组织，在多种分化培养基上，分化成再生植株。试验中对传统的酶解、收集方法作了改进，并对原生质体的分裂和愈伤组织的分化条件作了研究，对影响分化的多种因素作了深入的比较和探索，同时，本试验表明，叶片的生长状态和幼嫩程度是原生质体能否生长、分裂及再生的重要因素。

The protoplasts from NC 89 leaf(120 days) were collected and purified after enzyme digestion in the mixture of Onozuka R10 and Macerozyme R10 . 'Liquid-Solid' double layer method was used to culture the protoplasts in modified NT medium. The isolated protoplasts underwent development, division and formed callus, which regenerated plantlets after being transferred onto a differentiation medium. In the experiment, we improved the traditional methods of enzyme digestion and collection. Meanwhile we studied the conditions of protoplast division and callus differentiation, and also made a further comparison and explosion of the factors which affected the differentiation. The experiment showed that the developing status and delicate degree of the leaves are important factors which influence the protoplasts development and division.