超甜玉米转基因稳定高效再生体系的建立及转化植株PCR分析  

Establishment of stable and highly efficient plant regeneration system and PCR analysis of transformants in supersweet corn

作　　者：李余良[1] 胡建广[1] 苏菁[2] 刘建华[1] 

机构地区：[1]广东省农业科学院作物研究所,广东广州510640 [2]中山大学生命科学学院,广东广州510275

出　　处：《西北农林科技大学学报（自然科学版）》2007年第3期70-74,共5页Journal of Northwest A&F University(Natural Science Edition)

基　　金：广东省"十五"重大科技专项(A202004);广东省农业科学院院长基金项目(2004-02)

摘　　要：为了通过对影响超甜玉米再生因素的研究,建立超甜玉米转基因再生技术体系,获得抗虫资源。以超甜玉米幼胚为愈伤组织诱导外植体,利用基因枪法将苏云金杆菌杀虫蛋白基因转化胚性愈伤组织,通过优化再生和生根培养条件建立了稳定、高效的超甜玉米转基因再生成株体系。结果表明,随着愈伤组织继代时间的延长,再生成苗率降低,再生成苗越难;除草剂Basta对愈伤组织筛选的最适质量浓度为8 mg/L,愈伤组织预分化的最适培养基为MS+40 g/L蔗糖+20 g/L甘露醇+5.0 mg/L ABA,最适的再生培养基为MS+20 g/L蔗糖+2.0 mg/L6-BA+0.1 mg/L NAA,生根培养基为1/2 MS+0.1 mg/L NAA,对生根不好的小苗附加0.1 mg/L NAA+2.0mg/L IBA或0.2 mg/L NAA+3.0 mg/L IBA可以促进生根,生根率达93%以上。对部分再生转化植株进行PCR分析,部分植株能够扩增出426 bp片段,与阳性对照大小相同。试验结果初步证明Bt基因已经导入到玉米植株中。To establish regenration technique system for gene transformation in supersweet corn,and to obtain germplasm resources for insect resistance,the factors influencing plant regeneration were studied. The calli,derived from immature embryos,of the supersweet corn inbred line was transformed with the plasmid DNA containing Bt gene via the particle bombardment-mediated method. Through optimizing regeneration and rooting conditions in supersweet corn immature embryo culture,a stable, highly efficient plant post-transformation regenerating system has been established. The results showed that calli lost their regenerating ability after long-term subcultures. The optimal mass concentration of herbicide Basta was 8 mg/L to immature embryo calli selection ;the combination of MS＋ 40 g/L sucrose ＋ 20 g/L mannitol ＋ 5.0 mg/L ABA was the optimal medium for pre-differentiation of calli;the medium of MS＋20 g/L sucrose＋ 2.0 mg/L 6-BA ＋ 0. 1 mg/L NAA was the best for differentiation and regeneration ;the medium for rooting was 1/2 MS＋0.1 mg/L NAA,and the rooting rate was increased to above 93% when the medium of 1/2 MS＋0.1 mg/L NAA＋2.0 mg/L IBA or 1/2 MS＋0.2 mg/L NAA＋3.0 mg/L IBA was supplied. Genomic PCR analysis of the T0 individuals demonstrated that some seedlings could amplify 426 bp fragment and showed the same size in comparison with the postive check. The results confirmed that Bt gene has been integrated into the genomes of plants.

关 键 词：超甜玉米 转基因 幼胚 植株再生 再生体系PCR分析 

分 类 号：S513.035[农业科学—作物学]