李智超++李琳玲++程华++程水源

摘要：为探究适宜豆腐柴（Premna microphylla Turcz）细胞悬浮培养条件，以豆腐柴叶片为材料，进行了愈伤组织的诱导、继代培养、愈伤组织分散、悬浮培养的研究，建立了摇瓶悬浮培养体系。结果表明，豆腐柴叶片诱导得到的愈伤组织形态各异，其中诱导最适合悬浮培养的松散型胚性愈伤组织的培养基为MS+0.4 mg/L 2，4-D+0.4 mg/L NAA+3%蔗糖+0.8%琼脂。通过纤维素酶和果胶酶分散愈伤组织块可以得到较好的分散细胞系，最适合悬浮细胞生长的培养基条件为MS+0.4 mg/L 2，4-D+0.8 mg/L KT+3%蔗糖，其悬浮培养细胞鲜重增加量平均每7 d可达6.46倍，暗培养相对更适合细胞的增殖。经最佳培养条件下培养21 d的豆腐柴悬浮细胞中果胶和蛋白质平均含量达6.57%和0.12%，远低于豆腐柴天然叶片中的含量。

关键词：豆腐柴（Premna microphylla Turcz）；愈伤组织；悬浮细胞

中图分类号：R282；Q813.1 文献标识码：A 文章编号：0439-8114（2017）01-0171-04

DOI：10.14088/j.cnki.issn0439-8114.2017.01.043

Embryoid Induction and Cell Suspension Culture of Premna microphylla Turca

LI Zhi-chao1，LI Lin-ling1，2，CHENG Hua1，2，CHENG Shui-yuan1，2

（1. College of Life Science， Huanggang Normal University， Huanggang 438000， Hubei，China；2.Economic Forest Germplasm Improvement and Comprehensive Utilization of Resources of Hubei Key Laboratory， Huanggang 438000， Hubei， China）

Abstract： In order to find the suspension culture conditions of Premna microphylla Turca，the leaf blades of the experimented Premna microphylla Turca，induced embryogenic callus and subculture，which established a good dispersion and a high dynamic suspension cell line. The results showed that the callus induction from leaves were different. Which induces the most suitable medium for suspension culture of loose embryogenic callus contained MS+0.4 mg/L 2，4-D+0.4 mg/L NAA+3% sucrose+0.8% agar. Cell lines can be dispersed better by cellulase and pectinase dispersed callus block. The most suitable medium for suspension cell growth contains MS+0.4 mg/L 2，4-D+0.8 mg/L KT+3% sucrose. And in this medium，the fresh weight of the suspension cultured cells could be increased by 6.46 times per 7 days. Dark culture was more suitable for cell proliferation. The average contents of pectin and protein were 6.57% and 0.12% in the suspended cells of Premna microphylla Turca，which had been cultured for 3 weeks under the optimum culture conditions.

Key words： Premna microphylla Turcz； callus； suspension culture

豆腐柴（Premna microphylla Turcz）又名臭黃荆、豆腐木、腐婢等，为马鞭草科豆腐柴属落叶直立灌木[1]。其叶片具有丰富的果胶，含量可达39.5%，酯化度73%～78%，可以作为优良的果胶提取原料[2]。此外，豆腐柴叶片中粗蛋白含量高达29.0%～34.1%，在目前已知植物中名列前茅；其根茎部含有木栓酮、木栓醇、十八碳酸、甾醇、胡萝卜甙、柚皮素、香草酸等多种药用成分[3，4]，被用于中国传统中医治疗皮肤伤口、感染、疟疾、痢疾、头痛和毒蛇咬伤等[4]。

目前，豆腐柴主要通过野外采集获得，由于野生条件下豆腐柴零星散布在海拔500～1 500 m向阳山坡疏林下、林缘、沟谷边、荒山、丘陵、灌木层中[3，4]，难以满足生产需要。此外，其生长缓慢，种子自然态萌发率低。……