Krishnamurthy K V, Suhasini K, Sagare A P, Meixner M, de Kathen A, Pickardt T, Schieder O
Plant Tissue Culture Division, National Chemical Laboratory, Pune 411 008, India, , , , , , IN.
Institute for Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, 14195 Berlin, Germany e-mail:
Plant Cell Rep. 2000 Jan;19(3):235-240. doi: 10.1007/s002990050005.
Embryo axes of four accessions of chickpea (Cicer arietinum L.) were treated with Agrobacterium tumefaciens strains C58C1/GV2260 carrying the plasmid p35SGUSINT and EHA101 harbouring the plasmid pIBGUS. In both vectors the GUS gene is interrupted by an intron. After inoculation shoot formation was promoted on MS medium containing 0.5 mg/l BAP under a selection pressure of 100 mg/l kanamycin or 10 mg/l phosphinothricin, depending on the construct used for transformation. Expression of the chimeric GUS gene was confirmed by histochemical localization of GUS activity in regenerated shoots. Resistant shoots were grafted onto 5-day-old dark-grown seedlings, and mature plants could be recovered. T-DNA integration was confirmed by Southern analysis by random selection of putative transformants. The analysis of 4 plantlets of the T1 progeny revealed that none of them was GUS-positive, whereas the presence of the nptII gene could be detected by polymerase chain reaction.
用携带质粒p35SGUSINT的根癌农杆菌菌株C58C1/GV2260和含有质粒pIBGUS的EHA101处理四种鹰嘴豆（Cicer arietinum L.）品种的胚轴。在这两种载体中，GUS基因都被一个内含子打断。接种后，在含有0.5 mg/l 苄氨基嘌呤（BAP）的MS培养基上，在100 mg/l卡那霉素或10 mg/l草丁膦的选择压力下（取决于用于转化的构建体），促进了芽的形成。通过对再生芽中GUS活性进行组织化学定位，证实了嵌合GUS基因的表达。将抗性芽嫁接到5日龄黑暗培养的幼苗上，可以获得成熟植株。通过对推定转化体进行随机选择，用Southern分析证实了T-DNA的整合。对T1代后代的4个植株进行分析发现，它们均无GUS阳性，但通过聚合酶链反应可以检测到nptII基因的存在。
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