《植物生理学报》 2016, 52(12): 1851-1860

华北绣线菊(Spiraea fritschiana Schneid)再生及遗传转化体系建立的研究

张娇, 刘计璇, 李也, 王子骐, 王可新, 谭继升, 刘慧民^*

东北农业大学园艺园林学院, 哈尔滨150030

通信作者：刘慧民；E-mail: liuhm0423@163.com

摘　要：

以花灌木华北绣线菊(Spiraea fritschiana)幼嫩健康的离体叶片为外植体, 进行愈伤组织诱导、不定芽诱导及生根培养; 再以华北绣线菊愈伤组织为受体材料, 筛选选择抗生素和抑菌抗生素浓度, 探讨菌液稀释倍数和侵染时间对根癌农杆菌遗传转化华北绣线菊的影响。结果表明, 愈伤组织最佳诱导培养基为: MS+1.0 mg•L^-1 6-苄基腺嘌呤(6-BA)+0.1 mg•L^-1萘乙酸(NAA)+1.0 mg•L^-1 2,4-二氯苯氧乙酸(2,4-D), 诱导率为94.16%; 愈伤组织最佳增殖培养基为: MS+1.0 mg•L^-1 6-BA+0.03 mg•L^-1 NAA; 不定芽最佳分化培养基为: MS+2 mg•L^-1 thidiazuron (TDZ)+0.1 mg•L^-1 NAA, 分化率为96.11%; 生根壮苗最佳培养基为: 1/2MS+0.4 mg•L^-1 NAA; 以60 mg•L^-1卡那霉素(kan)为选择压、150 mg•L^-1头孢霉素(cef)为抑菌浓度, 菌体用液体MS稀释2倍(OD₆₀₀=0.7), 侵染8 min, 为最优遗传转化体系, 诱导抗性芽8.7个, 遗传转化率为2%。本研究建立的华北绣线菊再生与遗传转化体系为其转基因研究及分子育种提供参考。

关键词：华北绣线菊; 再生体系; 诱导率; 分化率; 遗传转化体系; 转化率

收稿：2016-05-23   修定：2016-11-25

资助：中国博士后科学基金(20100480957)和黑龙江省教育厅科学技术研究项目(11551040)。 * 通讯作者(E-mail: liuhm0423@163.com)。

Establishment of regeneration and genetic transformation systems for Spiraea fritschiana Schneid

ZHANG Jiao, LIU Ji-Xuan, Li Ye, WANG Zi-Qi, WANG Ke-Xin, TAN Ji-Sheng, LIU Hui-Min^*

College of Horticulture and Landscape Architecture, Northeast Agricultural University, Harbin 150030, China

Corresponding author: LIU Hui-Min; E-mail: liuhm0423@163.com

Abstract:

Using young and healthy leaves as explants, the callus, adventitious buds and roots were induced of woody ornamental plant Spiraea fritschiana. Then the callus was used as receptor material to screen out the concentrations of selective antibiotic and bacteriostatic antibiotic, and the effects of genetic transformation on S. fritschiana by diluting the agrobacterium solution into different multiples and infecting them by different time were also discussed. The results show that the best medium for callus induction was MS+1.0 mg•L^-1 6-benzylaminopurine (6-BA)+0.1 mg•L^-1 naphthylacetic acid (NAA)+1.0 mg•L^-1 2,4-dichlorophenoxyacetic acid (2,4-D) with the inductivity of 94.16%. The medium for callus proliferation was MS+1.0 mg•L-1 6-BA+0.03 mg•L-1 NAA. The medium for adventitious buds induction was MS+2.0 mg•L^-1 thidiazuron (TDZ)+0.1 mg•L^-1 NAA, and using this medium the differentiation rate reached 96.11%. The medium for rooting and strengthening was 1/2MS+1.0 mg•L^-1 NAA. The selective antibiotic was kanamycin (kan) with the concentration of 60 mg•L^-1, and the bacteriostatic antibiotic was cefotaxime (cef) with the concentration of 150 mg•L^-1. When we diluted the Agrobacterium tumefaciens twice (OD₆₀₀=0.7) by liquid MS and infected the callus for 8 minutes, the transformation efficiency was the highest with 8.7 resistant buds and transformation rate of 2%. This study provides a basis for gene engineering and molecular breeding by building efficient regeneration system and genetic transformation system of S. fritschiana.

Key words: Spiraea fritschiana; regeneration system; inductive rate; differentiation rate; genetic transformation system; transformation rate