东方百合高效转基因体系构建及EPSP基因的导入  

Establishment of Efficient Transgenic System and Introduction of EPSP Gene Into Lilium

作　　者：张洁[1] 郭文杰[1] 蔡宣梅[1] ZHANG Jie;GUO Wenjie;CAI Xuanmei(Biotechnology Institute,Fujian Academy of Agricultural Sciences,Fuzhou,Fujian 350003)

机构地区：[1]福建省农业科学院生物技术研究所,福建福州350003

出　　处：《北方园艺》2023年第4期46-53,共8页Northern Horticulture

基　　金：福建省属公益资助项目(2021R1027006);福建省农业科学院自由探索科技创新资助项目(ZYTS2021006);福建省农业科学院科技创新团队资助项目(CXTD2021009-3)。

摘　　要：以东方百合‘西伯利亚’无菌鳞茎超薄切片(0.5~1.0 mm)为试材,通过PIC和2,4-D不同浓度筛选愈伤诱导培养条件,采用超薄鳞片直接分化及愈伤分化2种转化方式,测试对Cef、Kan及Hgy的耐受性,分析不同干燥预处理以及MES、AS对转化效果的影响,并通过EPSP基因的导入,验证基因转化阳性率及转化效果,以期优化百合遗传转化的技术参数,建立高效遗传转化体系并进行EPSP基因的导入。结果表明:PIC对百合愈伤诱导有良好效果,MS+1 mg·L^(-1)PIC+0.3 mg·L^(-1)NAA为最佳愈伤诱导培养基;抗生素敏感性测试发现,培养基添加75 mg·L^(-1)Kan或20 mg·L^(-1)Hyg结合250 mg·L^(-1)Cef适宜抗性筛选;超薄鳞片及愈伤在60 min和30 min的干燥预培养下,阳性率均显著提高;MS+10 mmol·L^(-1)MES+200μmol·L^(-1)AS为重悬液处理,超薄鳞片获得48.3%的高效转化率,优于愈伤转化;同时,将EPSP基因转化百合,分子检测表示已获得转基因阳性株。该研究建立以超薄鳞片和愈伤为受体的2套高效遗传转化体系,并成功导入EPSP基因,获得800 mg·L^(-1)的草甘膦抗性,为转基因技术在后续的生产应用奠定技术基础。Ultrathin sections(0.5^(-1).0mm)of aseptic bulbs of Lilium orientalis‘Siberia’were used as test materials.Callus induction culture conditions were screened by PIC and 2,4-D at different concentrations.The tolerance to Cef,Kan and Hgy was tested by direct section differentiation and callus differentiation.The effects of different drying pretreatment,MES and AS on the transformation effect were analyzed,and the positive rate of gene transformation and transformation effect were verified by the introduction of EPSP gene,in order to optimize the technical parameters of Lilium genetic transformation,establish an efficient genetic transformation system and introduce EPSP.The results showed that PIC had a good effect on callus induction.MS+1mg·L^(-1)PIC+0.3mg·L^(-1)NAA was the best medium for callus induction.Antibiotic sensitivity test showed that 75mg·L^(-1)Kan or 20mg·L^(-1)Hyg combined with 250mg·L^(-1)Cef was suitable for resistance screening.The positive rates of ultrathin scales and callus were significantly increased after drying and preculture for60minutes and 30 minutes.MS+10mmol·L^(-1)MES+200μmol·L^(-1)AS treated with heavy suspension,the ultra-thin scales obtained an efficient conversion rate of 48.3%,which was better than callus transformation.At the same time,EPSP gene was transformed into Lilium,and molecular detection indicated that transgenic positive strains had been obtained.In this study,two sets of efficient genetic transformation systems of ultra-thin section and callus as the receptor were established,and EPSPgene was successfully introduced to obtain glyphosate resistance of 800mg·L^(-1),laying a technical foundation for the subsequent production and application of transgenic technology.

关 键 词：百合 EPSP基因 遗传转化 转基因 

分 类 号：S682.2[农业科学—观赏园艺]