Transformation of Medicago sativa L. Using a Ti Plasmid Derived Vector

A cultivar of Medicago sativa was transformed with Agrobacterium tumefaciens strain At81 binary-vector carrying the plasmid pKan3A, with the neomycin phosphotransferase gene under the control of the mannopine biosynthesis promoter. Stem segments were infected with the bacterium and kanamycin resistant calli were obtained; plant regeneration via somatic embryogenesis and polyembryogenesis was achieved only in media devoid of the antibiotic. Genetic transformation was confirmed by the presence of the structural gene through DNA-DNA hybridization and the enzymatic assay showed its functional expression. Mesophyll protoplasts, leaf calli and S1 progeny of transformed plants grew in the presence of kanamycin (100 μgml^-1). Results are discussed in relation to the use of kanamycin-resistant plants in somatic hybridization in the genus Medicago.     

几种抗生素对酸樱桃离体培养产生不定芽与再生根的影响

以酸樱桃zy-1无菌苗为试材,分别研究了在离体培养中羧苄青霉素(carbenicillin),头孢唑林钠(cefazolin)、卡那霉素(kanamycin)对zy-1生成不定芽、再生根的影响。结果表明:浓度为40mg/l的卡那霉素是酸樱桃ZY-1遗传转化选择压的最佳浓度,头孢唑林钠为适宜的抑菌性抗生素,且适宜的浓度范围为500mg/l~600mg/l。酸樱桃ZY-1的生根对卡那霉素极其敏感,头孢唑林钠次之,羧苄青霉素最轻。     

葡萄糖氧化酶基因转化棉花和抗性愈伤组织的获得

用葡萄糖氧化酶基因转化棉花 ,研究了影响转化和抗性愈伤组织获得的因素。结果表明 ,基因型和培养基构成是影响葡萄糖氧化酶基因转化棉花获得抗性愈伤组织的最关键因素 ,在本试验所采用的 8个基因型中 ,中棉所 1 3中 940 9(中棉所3 5)和中棉所 1 9较易诱导获得抗性愈伤组织 ,而珂字 2 0 1、中棉所 2 7等则较难 ;用于棉花遗传转化的感染时间以 7～ 1 0 min为宜 ,共培养时间以48h为宜 ;下胚轴比子叶易诱导获得抗性愈伤组织。在诱导愈伤组织的继代培养中 ,加入高浓度的卡那霉素是获得转化细胞的关键 ;用液体培养比用固体培养易筛选获得抗性转化愈伤组织     

Development of immunoassays for the detection of kanamycin in veterinary fields

Monoclonal antibody against kanamycin was prepared, and competitive direct ELISA and immunochromatographic assay were developed using the antibody to detect kanamycin in animal plasma and milk. The monoclonal antibody produced was identified to be IgG1, which has a kappa light chain. No cross-reactivity of the antibody was detected with other aminoglycosides, indicating that the monoclonal antibody was highly specific for kanamycin. Based on competitive direct ELISA, the detection limits of kanamycin were determined to be 1.1 ng/ml in PBS, 1.4 ng/ml in plasma, and 1.0 ng/ml in milk. The concentration of intramuscularly injected kanamycin was successfully monitored in rabbit plasma with competitive direct ELISA. Based on the colloidal gold-based immunochromatographic assay, the detection limits of kanamycin were estimated to be about 6-8 ng/ml in PBS, plasma, and milk. The immunochromatographic assay would be suitable for rapid and simple screening of kanamycin residues in veterinary medicine. Screened positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA. Therefore, the assays developed in this study could be used to complement each other as well as other laboratory findings. Moreover, instead of slaughtering the animals to obtain test samples, these methods could be applied to determine kanamycin concentration in the plasma of live animals.     

佛手组织培养和Km敏感性试验

以佛手的叶片、茎段为材料,建立了其组织培养系统.研究表明,B5比MS更适合作为诱导佛手愈伤组织的基础培养基,诱导愈伤组织效率较高的培养基配方为B5+2.0 mg·L-1 2,4-D+0.2 mg·L-1ZT和B5+1.5 mg·L-1 2,4-D+1.0 mg·L-1 NAA+0.4 mg·L-1 6-BA.在叶片对卡那霉素敏感性的试验中发现,叶片外植体预培养2 d后进行筛选的效果较好,卡那霉素的适宜筛选浓度为25 rng·L-1.     

防治紫花苜蓿真菌病害的生防菌筛选

本研究采用革兰氏染色、显微观察和分子生物学方法鉴定了2种具有拮抗潜力的微生物的分类地位,采用PDA培养基平板对峙法测定了其对苜蓿茎点霉(Phoma medicaginis)等9种常见的紫花苜蓿主要病原真菌的拮抗效果。研究结果表明这2种菌分别为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)和卡那霉素链霉菌(Streptomyces kanamyceticus)。前者对供试病原菌的抑菌率为24.02%~71.11%,其中对苜蓿尾孢菌(71.11%)、苜蓿匍柄霉(64.76%)和粉红镰孢(60.55%)抑菌效果最强;后者对供试病原菌抑菌率为49.95%~76.90%,其中对三叶草刺盘孢(76.90%)、苜蓿茎点霉(70.94%)、苜蓿匍柄霉(68.01%)和苜蓿小光壳(60.99%)抑制效果较强。两株菌对供试苜蓿9种病原真菌均表现出不同程度的拮抗作用,该研究结果为解淀粉芽孢杆菌和卡那霉素链霉菌作为紫花苜蓿多种病害的生物防治资源以及进一步开发利用提供了理论依据。     

利用卡那霉素间接鉴定法进行大规模的棉花转基因育种技术

:( 1 )将脱脂棉撕成小条沾取 0 .0 5%的卡那霉素溶液 ,粘附于棉花植株倒 2新生叶上。 5d后所有沾有卡那霉素溶液的感虫棉株的叶片均出现明显的黄色斑块 ,而转 Bt基因抗虫棉的叶片仍为正常绿色 ,无任何症状。 ( 2 )转 Bt基因抗虫棉种子接种于加有 1 0 0 0 mg· l- 1卡那霉素的 MS培养基中。四天后 ,抗虫棉子叶均表现为正常绿色 ,而常规品种子叶全为黄色 ,表明卡那霉素间接鉴定法是鉴定转 Bt基因抗虫棉的简便而又准确的方法     

Screening of Transgenic Soybean Transformed by Means of Pollen-tube Using Kanamycin

Kanamycin was used to screen T0 seeds of the variety Dongnong 46 transformed by means of pollen-tube method. The results showed that 400 mg·L-1 kanamycin could inhibit growth of non-transgenic plants, and 2 positive plants were gotten combined with Gus dyeing and PCR detection. It is proved that this method is e-conomic and effective in preliminary screening the transgenic plants.