Abstract: Hydrolysis parameters of protoplast with high yield and activity were determined using hypocotyl callus ofMelilotoides ruthenica(L.).The effects of enzyme combinations,mannitol concentration in the mixed enzyme solution,enzymolysis time,callus subculture and pretreatment time on protoplast isolations were studied.Results show that the optimum conditions for the highest yield and viability of protoplasts were with enzyme combinations containing 2% cellulase + 0.5% pectinase + 0.3% hemicellulase with 0.55 mol·L^-1 mannitol,after 24-hour dark pretreatment,followed by 12-hour enzymolysis and callus subculturing for 10 to12 days.

Key words: Melilotoides ruthenica(L) Sojak, Enzymolysis conditions, Protoplast

摘要： 以扁蓿豆(Melilotoides ruthenica(L.)Sojak)下胚轴愈伤组织为材料,研究酶液组合、酶解时间、酶液渗透压、继代培养时间及预处理措施对其原生质体分离效果的影响,以期确定能够酶解出高数量且高活力的原生质体的酶解条件。结果表明:获得有活力原生质体的最佳酶液组合为2%纤维素酶+0.5%果胶酶+0.3%半纤维素酶、酶解时间为12 h、酶液渗透压即甘露醇浓度为0.55 mol·L^-1,愈伤培养天数为10~12 d、预处理措施为黑暗24 h。

关键词: 扁蓿豆, 酶解条件, 原生质体