Abstract

Agrobacterium-mediated genetic transformation approach allows for introducing novel genes in cotton (Gossypium hirsutum L.). Development of efficient regeneration and transformation protocol is very important for recalcitrant plants like cotton. In the present study, five-day-old germinated mature embryo parts especially embryonic axis, hypocotyl and plumule of cotton ‘Lashata’ were wounded and inoculated with A. tumefaciens strain GV2260 harbouring plasmid p35S-GUS-INT. The binary plasmid p35S-GUS-INT contains neomycin phosphotransferase II (NPTII) gene driven by nopaline synthase (NOS) promoter and β-glucuronidase (GUS) gene controlled by cauliflower mosaic virus (CaMV) 35S promoter. After inoculation, explants were co-cultivated in liquid and agar-solidified MS medium for 48 h in dark condition. Agar-solidified co-cultivation medium increased transformation frequency as compared to liquid medium. The putative primary transformants were confirmed with histochemical GUS assay, PCR and RT-PCR. However, comparing the three culture explant, the embryonic axis explants had significant difference with embryonic hypocotyl and plumule explants in terms of number and percentage (%) of GUS, PCR and RT-PCR positive plant. The total transformation efficiency was recorded as 3% with varying GUS expression levels.

中文翻译：

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利用胚轴在棉花中高效农杆菌介导的遗传转化（陆地棉）

摘要

农杆菌介导的遗传转化方法允许在棉花中引入新的基因（棉（Gossypium hirsutum L.））。开发高效的再生和转化方案对于像棉花这样的顽re植物非常重要。在本研究中，将五天大的萌发的成熟胚部分，特别是棉花“ Lashata”的胚轴，下胚轴和胚芽受伤，并接种了带有质粒p35S-GUS-INT的根癌农杆菌GV2260。二元质粒p35S-GUS-INT包含由胭脂碱合酶（NOS）启动子和β-葡萄糖醛糖苷酸酶（GUS）驱动的新霉素磷酸转移酶II（NPTII）基因。）基因由花椰菜花叶病毒（CaMV）35S启动子控制。接种后，将外植体在液体和琼脂固化的MS培养基中于黑暗条件下共培养48小时。与液体培养基相比，琼脂固化的共培养培养基提高了转化频率。通过组织化学GUS测定，PCR和RT-PCR确认推定的一级转化体。然而，比较三种培养物外植体，在GUS，PCR和RT-PCR阳性植物的数量和百分数（％）方面，胚轴外植体与胚下胚轴和胚芽外植体具有显着差异。在变化的GUS表达水平下，总转化效率记录为3％。