Tripathi Jaindra N, Oduor Richard O, Tripathi Leena
Bioscience Centre, International Institute of Tropical Agriculture Nairobi, Kenya ; Department of Biochemistry and Biotechnology, Kenyatta University Nairobi, Kenya.
Department of Biochemistry and Biotechnology, Kenyatta University Nairobi, Kenya.
Front Plant Sci. 2015 Nov 26;6:1025. doi: 10.3389/fpls.2015.01025. eCollection 2015.
Banana (Musa spp.) is an important staple food as well as cash crop in tropical and subtropical countries. Various bacterial, fungal, and viral diseases and pests such as nematodes are major constraints in its production and are currently destabilizing the banana production in sub-Saharan Africa. Genetic engineering is a complementary option used for incorporating useful traits in banana to bypass the long generation time, polyploidy, and sterility of most of the cultivated varieties. A robust transformation protocol for farmer preferred varieties is crucial for banana genomics and improvement. A robust and reproducible system for genetic transformation of banana using embryogenic cell suspensions (ECS) has been developed in this study. Two different types of explants (immature male flowers and multiple buds) were tested for their ability to develop ECS in several varieties of banana locally grown in Africa. ECS of banana varieties "Cavendish Williams" and "Gros Michel" were developed using multiple buds, whereas ECS of "Sukali Ndiizi" was developed using immature male flowers. Regeneration efficiency of ECS was about 20,000-50,000 plantlets per ml of settled cell volume (SCV) depending on variety. ECS of three different varieties were transformed through Agrobacterium-mediated transformation using gusA reporter gene and 20-70 independent transgenic events per ml SCV of ECS were regenerated on selective medium. The presence and integration of gusA gene in transgenic plants was confirmed by PCR, dot blot, and Southern blot analysis and expression by histochemical GUS assays. The robust transformation platform was successfully used to generate hundreds of transgenic lines with disease resistance. Such a platform will facilitate the transfer of technologies to national agricultural research systems (NARS) in Africa.
香蕉（芭蕉属）是热带和亚热带国家重要的主食作物和经济作物。各种细菌、真菌和病毒病害以及线虫等害虫是其生产的主要制约因素，目前正在破坏撒哈拉以南非洲的香蕉生产。基因工程是一种补充手段，用于将有用性状导入香蕉，以绕过大多数栽培品种生长周期长、多倍体和不育的问题。针对农民偏好品种建立稳健的转化方案对香蕉基因组学和改良至关重要。本研究开发了一种利用胚性细胞悬浮液（ECS）对香蕉进行遗传转化的稳健且可重复的系统。测试了两种不同类型的外植体（未成熟雄花和多个芽）在非洲当地种植的几个香蕉品种中形成ECS的能力。香蕉品种“卡文迪什·威廉姆斯”和“大麦克”的ECS通过多个芽形成，而“苏卡利·恩迪齐”的ECS通过未成熟雄花形成。ECS的再生效率约为每毫升沉降细胞体积（SCV）20000 - 50000株幼苗，具体取决于品种。利用gusA报告基因通过农杆菌介导的转化对三个不同品种的ECS进行转化，每毫升SCV的ECS在选择培养基上再生出20 - 70个独立的转基因事件。通过PCR、斑点杂交和Southern杂交分析确认了转基因植物中gusA基因的存在和整合，并通过组织化学GUS检测确认了其表达。该稳健转化平台成功用于产生数百个具有抗病性的转基因株系。这样一个平台将促进技术向非洲国家农业研究系统（NARS）的转移。
