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首页> 外文期刊>BMC Plant Biology >Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures
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Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures

机译：通过使用已建立的胚性愈伤组织培养物，农杆菌介导的阿拉伯咖啡（L.）的遗传转化大大增强

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Background Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica. Results We identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization. Conclusion Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes.
机译：背景技术在农作物的基因组测序之后，当今的主要挑战之一是确定所有预测基因的功能。当将基因验证方法用于木本物种时，主要障碍是从优良或商业品种中获得转基因植物的回收率低。胚性愈伤组织经常是转化的目标组织，但是生产或维持胚性组织的困难是许多木本植物，包括阿拉伯咖啡的遗传转化中遇到的主要问题之一。结果我们确定了在阿拉伯糖衣藻中成功长期增殖胚胎发生培养物所需的条件，并基于这些条件设计了高效，可靠的根癌农杆菌介导的转化方法。通过评估GFP检测不同参数对转化效率的影响，建立了带有pBin 35S GFP的LBA1119转化方法。使用胚性愈伤组织培养物，在20°C下与LBA1119 OD 600 = 0.6共同培养5天，即可实现可再现的转化。胚性愈伤组织培养物的维持条件，特别是高的生长素与细胞分裂素比率，培养物的年龄（最适增殖时间为7-10个月）和使用黄色的愈伤组织表型，是获得高效的最重要因素。转化（> 90％）。在组织学水平上，成功的转化与存在的前胚性肿块的数量有关。通过PCR和Southern印迹杂交证明所有选择的植物都被转化。结论通过优化用作转化目标组织的胚发生培养物的生产条件，已取得了提高咖啡转化效率的最大进展。这是第一次证明了文化时代对转化效率的强烈积极影响。我们的结果使农杆菌介导的胚发生培养物转化成为咖啡育种和农学上重要基因功能分析的可行和有用工具。

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