仙茅愈伤组织诱导和细胞悬浮培养体系的建立  

Callus Induction and Establishment of Cell Suspension Culture System for Curculigo orchiodes

作　　者：邹璐 何钢[1] 刘贤桂[2] 张虹[1] 席飞飞 叶生宝 Zou Lu;He Gang;Liu Xiangui;Zhang Hong;Xi Feifei;Ye Shengbao(Life Science and Technology College,Central South University of Forestry and Technology,Changsha,410004;Forestry College,Central South University of Forestry and Technology,Changsha,410004)

机构地区：[1]中南林业科技大学生命科学与技术学院,长沙410004 [2]中南林业科技大学林学院,长沙410004

出　　处：《分子植物育种》2019年第16期5383-5389,共7页Molecular Plant Breeding

基　　金：国家林业公益性行业科技专项项目(201304807)资助

摘　　要：为初步建立仙茅(Curculigo orchiodes Gaertn)细胞悬浮培养体系。以仙茅幼叶为外植体,诱导产生愈伤组织后进行仙茅细胞悬浮培养研究。以MS为基本培养基,研究了植物生长调节剂对仙茅愈伤组织诱导和继代的影响,以及接种量,2,4-二氯苯氧乙酸(2,4-D),蔗糖,水解酪蛋白,2-氮吗啉乙烷磺酸(MES),L-脯氨酸(L-Pro)对仙茅细胞悬浮培养的影响。结果表明:仙茅愈伤组织诱导的最佳培养基为MS+6-BA 1.5 mg/L+NAA 0.5 mg/L,所得的愈伤组织颜色淡黄,质地疏松,诱导率达100%;最佳继代培养基为MS+6-BA 2.0 mg/L+NAA 0.5 mg/L,平均增殖倍数达10.8。仙茅细胞悬浮培养最佳培养基和培养条件为MS+2,4-D 0.5 mg/L+6-BA2.0 mg/L+NAA 0.5 mg/L+蔗糖20 g/L,接种量75 g/L,水解酪蛋白300 mg/L,MES 2 g/L,L-Pro 700 mg/L,摇床转速120 r/min,25℃光照培养,光照时间12 h/d,光照强度1 200 lx。该条件下悬浮细胞生长良好,最大生长量为(9.68±0.2)×10^5个/mL。初步选择出仙茅愈伤组织细胞的悬浮培养条件,也为仙茅细胞扩大培养及有效成分的提取提供物质基础。To preliminary establish the suspension culture system of C. orchiodes callus. Leaves were used as the explants to induce calli and study on the cell suspension of C. orchiodes. Using MS medium as the basic medium, effects of hormone on callus tissues induction and proliferation, as well as the inoculum amount Sucrose, Hydrolyzed casein, MES and L-Proline concentration on cell suspension culture of C. orchiodes were studied. The results showed that the optimal medium induction of C. orchiodes was MS+6-BA 1.5 mg/L+NAA 0.5 mg/L, the callus tissue color was pale yellow, the texture was loose and the induction rate was 100%;the best callus subculture medium was MS+6-BA 2.0 mg/L+NAA 0.5 mg/L, the average multiplication factor was 10.8. The best suspension culture medium for C. orchiodes was MS+2,4-D 0.5 mg/L+6-BA 2.0 mg/L+NAA 0.5 mg/L+20 g/L sucrose. The best culturing conditions were inoculum size: 75 g/L, hydrolyzed casein: 300 mg/L, MES: 2 g/L, L-Pro: 700 mg/L, gyratory shaker rotation speed: 120 r/min, temperature: 25℃ in lighting environment, Illumination time: 12 h/d, Illumination intensity: 1 200 lx. Under these conditions suspended cells grew well and the maximum amount of suspended cells was 9.68±0.2×10^5 cell/mL. The suspension culture conditions of callus cells of Curculigo orchiodes were selected, which also provided the material basis for the expanded culture and extraction of effective components of Curculigo orchiodes cells.

关 键 词：仙茅(Curculigo orchiodes Gaertn) 愈伤组织 诱导 悬浮培养 

分 类 号：S567.239[农业科学—中草药栽培]