滴水珠原生质体的分离纯化与植株再生  

Protoplasts isolation,purification and plant regeneration of Pinellia cordata

作　　者：杨仙[1] 马丹丹[1] 蒋福升[1] 陈铌铍[1] 丁滨[1] 金丽霞[1] 钱朝东[1] 丁志山[1] 

机构地区：[1]浙江中医药大学生命科学学院,浙江杭州310053

出　　处：《中国中药杂志》2014年第21期4211-4215,共5页China Journal of Chinese Materia Medica

基　　金：浙江省重点科技创新团队项目(2009R50042)

摘　　要：以滴水珠的幼嫩叶片为材料,对影响原生质体分离纯化、培养及植株再生的因素进行了研究。结果表明：适合滴水珠叶片的酶解体系为13%CPW（细胞原生质体洗液）＋1.0%纤维素酶＋0.1%果胶酶,p H 6.0,适宜酶解温度为25~28℃,酶解时间为4 h;滴水珠叶肉原生质体纯化以上浮法蔗糖梯度离心效果最佳,以25%的蔗糖做梯度材料,500 r·min^-1离心10 min;用于培养滴水珠原生质体的培养基为MS＋0.5 mg·L^-16-BA＋0.25 mg·L^-1NAA 13%甘露醇,培养3 d有细胞分裂启动迹象,30 d左右形成愈伤组织,转移至MS＋0.5 mg·L^-16-BA＋0.25 mg·L^-1NAA固体培养基上增殖培养,并继代数次,5~6个月后愈伤组织可分化出芽并再生植株。The main factors which affected the isolation, purification and cultivation of PineUia cordata protoplasts from leaves were studied. The results indicated that the optimum enzyme solution for P. cordata leaves was 13% CPW ＋ 1.0% Cellulose ＋0.1% Peetolase, at pH 6. O, temperature （25-28℃ ） for 4 h. The sucrose density gradient centrifugation was adopted to purifieate the proto- plasts collected, when 25% sucrose was used as mediator, centrifugating at 500 rpm for 10 min. When the protoplasts were shallow liquid and liquid-solid double layer cultured on the medium of MS ＋0.5 mg·L^-1 6-BA ＋0.25 mg· L^-1 NAA ＋ 13% mannitol at the density of 2.5 ×104 protoplasts/mL, or fed and nursed cultured at the density of 100-500 protoplasts/mL, cell division could be observed for 3 days; granular ealli appeared for 30 days. Calli was proliferated on the medium of MS ＋0.5 mg· L^-l 6-BA ＋0.25 mg· L^-l NAA solidified by 0.55% agar, and differentiated and regenerated after 5-6 months. Plant generation of P. cordata is successfully established.

关 键 词：滴水珠 原生质体分离 植株再生 

分 类 号：S567.239[农业科学—中草药栽培]