南方农业学报2024，Vol.55Issue(6)：1692-1699,8.DOI:10.3969/j.issn.2095-1191.2024.06.014
根癌农杆菌介导彩色马蹄莲遗传转化体系的建立及优化
Establishment and optimization of Agrobacterium tumefaciens mediated genetic transformation system for Zantedeschia hybrida Spr.
摘要
Abstract
[Objective]This study aimed to establish and optimize an Agrobacterium tumefaciens mediated genetic transformation system for Zantedeschia hybrida Spr.,providing theoretical support for improving the efficiency of genetic transformation and cultivating resistant varieties of Z.hybrida Spr.[Method]An orthogonal experiment was conducted using different explants(stem base,leaves,and adventitious buds)and different concentrations thidiazuron(TDZ)of 0.001,0.002 and 0.005 mg/L to determine the optimal explant and clustered bud proliferation medium.The pCAM-BIA2301 and pBI121 plasmids were introduced into A.tumefaciens LBA4404 competent cells using the liquid nitrogen freeze-thaw method.The Z.hybrida Spr.clustered buds were genetically transformed using β-glucuronidase(GUS)gene as a reporter gene for A.tumefaciens mediated transformation.Positive seedlings were verified by PCR amplification.The optimal transformation conditions were screened by orthogonal experiment with three factors:kanamycin(Kan)concen-trations(50,100 and 150 mg/L),infection time(25,30 and 40 min),and co-culture time(2 and 3 d).[Result]The in-duction rate of clustered buds using adventitious bud as an explant was extremely significantly higher than that from stem base and leaf(P＜0.01).When adventitious bud was used as explant and the TDZ concentration was 0.002 mg/L,the pro-liferation times of clustered buds(5.12 times)was significantly higher than that of 0.001 and 0.005 mg/L TDZ treatments(P＜0.05).The optimal medium for clustered bud proliferation was M6 medium+0.002 mg/L TDZ+30 g/L sucrose+6.25 g/L agar.The factors affecting the positive transformation frequency were ranked as:infection time＞Kan concentration＞co-culture time.The ideal infection conditions were:50 mg/L Kan,25 min infection time,and 3 d co-culture time,resulting in the highest GUS+clustered bud emergence frequency(54.0％)and PCR positive frequency(15.27％).[Conclusion]An efficientA.tumefaciens mediated genetic transformation system for Z.hybrida Spr.is successfully established and opti-mized.The optimal explant is adventitious bud,and the optimal transformation conditions are 50 mg/L Kan,25 min infec-tion time,and 3 d co-culture time.关键词
彩色马蹄莲/根癌农杆菌/遗传转化/优化Key words
Zantedeschia hybrida Spr./Agrobacterium tumefaciens/genetic transformation/optimization分类
园艺学与植物营养学引用本文复制引用
李菲,曹永琼,王纲,郭彦兵,李紫薇,吴红芝..根癌农杆菌介导彩色马蹄莲遗传转化体系的建立及优化[J].南方农业学报,2024,55(6):1692-1699,8.基金项目
National Natural Science Foundation of China(32160723,31960610) 国家自然科学基金项目(32160723,31960610) （32160723,31960610）
