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利用根外植体通过根癌农杆菌介导对茄子(茄属龙葵)进行转化

Agrobacterium tumefaciens-mediated transformation of eggplant (Solanum melongena L.) using root explants.

作者信息

Franklin G, Lakshmi Sita G

机构信息

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.

出版信息

Plant Cell Rep. 2003 Feb;21(6):549-54. doi: 10.1007/s00299-002-0546-9. Epub 2003 Jan 8.

Abstract

An efficient variety-independent method for producing transgenic eggplant (Solanum melongena L.) via Agrobacterium tumefaciens-mediated genetic transformation was developed. Root explants were transformed by co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBAL2 carrying the reporter gene beta-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Transgenic calli were induced in media containing 0.1 mg l(-1) thidiazuron (TDZ), 3.0 mg l(-1) N(6)-benzylaminopurine, 100 mg l(-1) kanamycin and 500 mg l(-1) cefotaxime. The putative transgenic shoot buds elongated on basal selection medium and rooted efficiently on Soilrite irrigated with water containing 100 mg l(-1) kanamycin sulphate. Transgenic plants were raised in pots and seeds subsequently collected from mature fruits. Histochemical GUS assay and polymerase chain reaction analysis of field-established transgenic plants and their offsprings confirmed the presence of the GUS and NPTII genes, respectively. Integration of T-DNA into the genome of putative transgenics was further confirmed by Southern blot analysis. Progeny analysis of these plants showed a pattern of classical Mendelian inheritance for both the NPTII and GUS genes.

摘要

开发了一种高效的、不依赖品种的通过根癌农杆菌介导的遗传转化生产转基因茄子(Solanum melongena L.)的方法。通过与携带二元载体pBAL2的根癌农杆菌菌株LBA4404共培养来转化根外植体,该二元载体pBAL2携带报告基因β-葡萄糖醛酸酶内含子(GUS-INT)和标记基因新霉素磷酸转移酶(NPTII)。在含有0.1 mg l(-1)噻二唑素(TDZ)、3.0 mg l(-1) N(6)-苄基腺嘌呤、100 mg l(-1)卡那霉素和500 mg l(-1)头孢噻肟的培养基中诱导产生转基因愈伤组织。推定的转基因芽在基础选择培养基上伸长,并在浇灌含100 mg l(-1)硫酸卡那霉素的水的营养土上高效生根。转基因植株在花盆中培育,随后从成熟果实中收集种子。对田间种植的转基因植株及其后代进行组织化学GUS检测和聚合酶链反应分析,分别证实了GUS和NPTII基因的存在。通过Southern杂交分析进一步证实了T-DNA整合到推定转基因植株的基因组中。这些植株的后代分析表明,NPTII和GUS基因均呈现经典的孟德尔遗传模式。

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