棉花农杆菌基因转化体系的优化和抗黄萎病转基因植株的获得

为了优化根癌农杆菌介导的棉花下胚轴切段的基因转化体系，以苜蓿抗菌肽基因（alflafa antifungul peptide gene, alfAFP）为目标，利用GUS报告基因和潮霉素抗性基因的检测方法，分析和探讨了根癌农杆菌菌种、菌液浓度、浸染下胚轴的时间、共培养中乙酰丁香酮（AS）的浓度、共培养温度和时间等因子对基因转化的影响。优化分析表明，用农杆菌菌株LBA4404当OD600值为0.5-0.7的菌液浸染5-7日龄的棉花无菌苗下胚轴切段10-15分钟，在含200µmol/L AS的共培养基中21℃暗培养60h可更有效地提高转化率。经上述条件处理的下胚轴在含50mg/L潮霉素的愈伤诱导培养基上培养3周可产生转化率最高的愈伤。愈伤组织经分化培养获得15株再生棉花，经过PCR和southern-PCR分析，发现其中12株含有外源抗病基因alfAFP，可作为棉花抗病育种的种质。

Optimization of Agrobacterium-mediated cotton gene transformation and development of transgenic plants with antifungal gene

In order to develop a high effective gene transformation protocol of cotton hypocotyl pieces via Agrobacterium tumefaciens, an alflafa antifungul peptide gene (alfAFP) was used as a foreign gene, and effect factors of transformation, including Agrobacterium strains, bacterium density, infection time, acetosyringone (AS) concentration in co-culture medium, co-culture time and co-culture temperature, were devalued by the expression detection of GUS and hygromycin resistant gene in transgenic callus. Optimum conditions for transformation was that cotton hypocotyl pieces from 5 to 7 days-old sterile seedlings were dipped into Agrobacterium LBA4404 suspension (OD600=0.5-0.7) for 10-15 min, then were transferred on co-culture medium with 200µmol/L As and were co-cultured at 21℃ for 60h in the dark. The most amount of transgenic calli was gained by culturing hypocotyl pieces on callus introduction medium with 50mg/L hygromycin. 15 regenerative plants were developed from somatic embryos differentiated from transgenic calli on embryogenesis medium. PCR and PCR-southern detection proved that the antifungal gene was introduced in 12 regenerative plants. They might be used as germplasm resources for breeding resistant-fungal disease cotton in the near future.