摘要
【目的】探究农杆菌介导的建兰遗传转化体系。【方法】以建兰品种市长红(Cymbidium ensifolium‘Shizhanghong’)单龙根扩繁得到的根状茎作为试验材料,筛选影响其遗传转化的农杆菌菌株、乙酰丁香酮浓度和抗生素质量浓度,研究影响农杆菌介导建兰遗传转化效率和再生效率的主要因素。【结果】市长红遗传转化的最佳菌株为EHA105,乙酰丁香酮最适浓度为200μmol/L,潮霉素最佳质量浓度为40 mg/L。为保证高转化率和高再生率,市长红的最佳侵染体系为:预培养10 d后,在OD600值为0.4~0.6的农杆菌侵染液中振荡20 min,最后在黑暗条件下共培养3 d。通过筛选的最佳遗传转化条件,经绿色荧光蛋白鉴定、抗生素筛选、PCR和qRT-PCR检测,最终在180个市长红的根状茎中得到17株转基因阳性植株,转化率为9.44%。【结论】本研究建立了农杆菌介导的建兰品种市长红遗传转化体系,为其转基因育种奠定了基础。
[Purpose]To explore the genetic transformation system of Cymbidium ensifolium‘Shizhanghong’mediated by Agrobacterium.[Methods]The rhizomes of C.ensifolium‘Shizhang-hong’obtained from single pseudobulb propagation were used as experimental materials.Agrobac-terium strain,concentration of acetosyringone,and mass concentration of antibiotics were screened.The main factors affecting the genetic transformation efficiency and regeneration efficiency of C.en-sifolium‘Shizhanghong’mediated by Agrobacterium were studied.[Results]The optimal strain for genetic transformation of C.ensifolium‘Shizhanghong’was EHA105,the optimal concentration of acetosyringone was 200μmol/L,and the optimal mass concentration of hygromycin was 40 mg/L.To ensure a high transformation rate and high regeneration rate of C.ensifolium‘Shizhanghong’,the op-timal infection system was:after 10 days of pre-culture,the rhizomes were shaken in Agrobacterium infection solution(OD600=0.4-0.6)for 20 minutes,and then co-cultured in the dark for three days.Through the screening of the optimal genetic transformation conditions,the transgenic positive plants of C.ensifolium‘Shizhanghong’were identified by green fluorescent protein,antibiotic screening,PCR and qRT-PCR detection.Finally,17 transgenic positive plants of C.ensifolium‘Shizhanghong’were obtained from 180 rhizomes,with a transformation rate of 9.44%.[Conclusion]This study establishes a genetic transformation system mediated by Agrobacterium for C.ensifolium‘Shizhang-hong’,laying the foundation for transgenic breeding of this orchid cultivar.
作者
周彬
曹桦
陆琳
张钰莹
李涵
李东徽
ZHOU Bin;CAO Hua;LU Lin;ZHANG Yuying;LI Han;LI Donghui(College of Landscape and Horticulture,Yunnan Agricultural University,Kunming 650201,China;National Engineering Research Center for Ornamental Horticulture,Flower Research Institute,Yunnan Academy of Agricultural Sciences,Kunming 650205,China;Forestry(Flower)Industry Development Center,Zhouning County,Ningde City,Fujian Province,Zhouning 355400,China)
出处
《云南农业大学学报(自然科学版)》
北大核心
2025年第4期111-119,共9页
Journal of Yunnan Agricultural University:Natural Science
基金
云南省种子种业联合实验室项目(202205AR070001-05)。
关键词
建兰品种市长红
根状茎
农杆菌侵染
遗传转化
绿色荧光蛋白(GFP)
Cymbidium ensifolium‘Shizhanghong’
rhizome
Agrobacterium infection
genetic trans-formation
green fluorescent protein(GFP)
