红姜花胚性细胞悬浮体系的建立和原生质体培养  

Establishment of Embryogenic Cell Suspension and Protoplast Culture of Hedychium coccineum

作　　者：肖望[1] 涂红艳[1] 邓崇会[1] 

机构地区：[1]广东第二师范学院生物系,广东广州510303

出　　处：《北方园艺》2015年第5期104-108,共5页Northern Horticulture

基　　金：广东省自然科学基金博士启动资助项目(10451030301004286);广东省高等院校学科与专业建设专项资金资助项目(2013KJCX0137);广州市科技计划资助项目(2014J4100151)

摘　　要：以红姜花(Hedychium coccineum)未成熟花丝和花药为试材,研究了红姜花胚性愈伤组织的诱导、细胞悬浮体系的建立以及原生质体的培养。结果表明:在MS+4mg/L 2,4-D+4mg/L NAA+1mg/L 6-BA+30g/L蔗糖+7g/L琼脂上经过120d培养诱导出了愈伤组织,愈伤组织在增殖培养基上经过继代筛选获得浅黄色、松散易碎的胚性愈伤组织。胚性愈伤组织通过3个月的悬浮培养,得到均质稳定的胚性细胞悬浮系。以胚性悬浮细胞(ECS)为起始材料分离原生质体,酶解试验表明,在原生质体分离过程中,用于原生质体分离的酶液需要保持一定的渗透压以保护原生质体不被破坏。当甘露醇的浓度为0.14mol/L时,原生质体产量最高,达到2.25×105个/mL PCV ECS(packed cell volume,PCV)。在看护培养系统中,原生质体经过7d的培养,细胞第一次分裂,经过14d培养,细胞分裂频率达到12.3%,28d时,细胞团形成频率达到4.2%。所形成的细胞团具有典型的胚性细胞特征。Embryogenci cell suspensions were established for the ornamental ginger Hedychium coccineum using filaments and anthers. Embryagenic indction, cell suspension culture system establishment and protolast culture o{ Hedychium coccineum were studied. The results showed that, after culture for 120 days, calli were induced on induced medium （Murashige and Skoog （MS） basal medium supplemented with 4 mg/L 2,4-D＋4 mg/L NAA＋1 mg/L 6-BA＋30 g/L sucrose＋7 g/L agar）. The calli were then transferred on proliferated medium （MS basal medium＋ 1 mg/L 2,4-D＋ 0. 25 mg/L NAA＋0. 25 mg/L 6-BA） and light yellow and friable embryogenic callus were obtained. These embryogenic callus were suspended in liquid medium, and after 3 months culture, a homogeneous and stable embryogenic ceil suspension （ECS）, composed of small cell aggregates, was established. Viable protoplasts were isolated from ECS in a enzyme mixture of 3.0% （w/v） cellulose R-10,2% （w/v） macerozyme R-10 and 0. 25% （w/v） pectinase Y-23 for 7 hours,enzyme solution with 0. 14 mol/L manntitol resulted in the highest yield of 2. 25 ×105 protoplasts per mL PCV ECS. Feeder layer culture systems were used for protoplast culture. Frequency of cell division at 14 days and colony formation at 28 days were 12.8 % and 4. 2 % respectively. However,all protoplast-derived cell colonies could not develop further.

关 键 词：红姜花 胚性细胞悬浮体系 原生质体 

分 类 号：S567.239[农业科学—中草药栽培]