马铃薯单细胞分离技术的研究

以马铃薯栽培品种“甘农薯1号”的无菌苗、子叶以及已建立的细胞悬浮培养系为材料,分别用酶解法和过滤细胞悬浮培养系的物理方法进行了马铃薯单细胞分离技术的研究。结果表明,酶解时,果胶酶浓度为0.20%～0.25%,解离4h获得的单细胞产量最高,且单细胞分裂频率可达16%以上;另外,酶解愈伤组织分离单细胞,比酶解子叶和叶片效果更好。而在用细胞悬浮培养物进行单细胞分离研究时,发现瓶壁法是一种最好的细胞悬浮液继代方法,可直接获得90%的单细胞频率,且细胞形状圆、胞质浓厚。采用过滤法分离单细胞,可得到98%的单细胞频率;3层细胞筛过滤法可有效提高所得单细胞的植板率。总体比较,过滤细胞悬浮培养物的物理操作方法获得的单细胞比酶解法获得的单细胞分裂能力更强。

TECHNIQUES ON ISOLATION OF SINGLE CELLS IN POTATO

The isolation of single cells from the sterile leaves,cotyledons and established cell suspen - sion of the potato cultivar “ Gannongshu1 ” was made by two different methods ,Enzyme-digestion method and physical method on suspension culture.The results of enzyme-digestion method showed that the suitable concen - tration of pectinase was0.20% ～ 0.25%,and the16%frequency of single cells division would be obtained after the suitable digestion time of4hours.Also ,enzyme digestion was more effective to isolate single cells from calli than that from cotyledons and leaves.As far as the physical method on cell suspension culture was concerned,the flask-wall method was found to be the optimal way to subculture the cell suspension system,and the single cell frequency of90%was directly reached by this method,meanwhile,these single cells had thick cytoplasm,round shape and great vigorous division ability.Furthermore,the filtering method was effective to isolate single cells.Moreover,the cells planting efficiency would be increased using three-layers cell sieves to purify the single cells.In summary,the physical method on suspension culture was more advantageous than enzyme-digestion to increase the division frequency of single cells.