板栗下胚轴原生质体的分离与纯化

Isolation and purification of protoplasts from chestnut hypocotyl

【目的】研究分离和纯化板栗下胚轴原生质体的影响因素。【方法】对分离板栗下胚轴原生质体的酶液组合、酶解时间、渗透压调节剂等因素进行优化,采用荧光增白剂(CFW)染色法和荧光素双醋酸酯(FDA)染色法分别对原生质体去壁和原生质体活力进行检测。【结果】分离板栗下胚轴原生质体最适宜的酶液组合为13%(w/v)甘露醇+2%(w/v)纤维素酶+0.5%(w/v)离析酶+1%(w/v)半纤维素酶,28℃温度下酶解6 h,原生质体产量达5.8×106个·g-1,且活力达94%。蔗糖浓度为23%(w/v)时,原生质体纯化效果最佳。【结论】建立了适合分离和纯化板栗下胚轴原生质体的方法。

[Objective]The method for isolation and purification of protoplasts from hypocotyl of chestnut was evaluated. [Methods]The hypocotyl of chestnut was used as materials to optimize the concentration and combination of cell-wall-digesting enzymes, hydrolysis time and osmotieum concentration, and the cell walls and viability of protoplasts were observed by using CFW staining and FDA staining methods. [Results]The suitable cell wall digesting enzymes for hypocotyl protoplasts were 13%（w/v） mannitol＋2% （w/v） cellulase＋0.5%（w/v） macerozyme＋l%（w/v） hemicellulase, the protoplast yield was 5.8-106.g- fresh weight and the viability of isolated protoplasts was about 94% when incubated at 28 ℃ for 6h. To obtain the best effect,the sucrose concentration for suspension purification of protoplast was set at 23%.[Conclu sion]The optimum method for purification and isolation of protoplasts from hypocotyl of chestnut was rec ommended.