A High-Throughput Regeneration and Transformation Platform for Production of Genetically Modified Banana

Abstract

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.

Efficient regeneration system applicable to five Musa cultivars

“…Mas (AA group). The most versatile method we found in the literature was reported by Tripathi et al [19] which was suitable for three banana cultivars, including Cavendish Williams, Gros Michel, and Sukali Ndiizi. In contrast, we report a simple method that uses only one kind of growth medium (MS with 8.9 mmol$L -1 BA and 9.1 mmol$L -1 TDZ) for regeneration of five kinds of immature male banana flowers that is reproducible and might overcome the problems associated with genotype dependency.…”

“…Another problem with banana regeneration is that it is time-consuming. Our method was able to produce welldeveloped plantlets from immature flowers in only 240-270 d; the shortest growth cycle using other methods was 344-478 d [19] . Hence, our method would serve to greatly improve regeneration efficiency.…”

“…First, our regeneration system is very simple, using only one medium (MS with 8.9 mmol$L -1 BA and 9.1 mmol$L -1 TDZ) for induction and differentiation of adventitious shoots. High-throughput regeneration and transformation using embryogenic cell suspension as reported by Tripathi et al [19] for example, required six different media to derive well-developed shoots from immature male flowers. Moreover, Hrahsel et al [20] reported in vitro propagation of M. acuminata (AAA) cv.…”

Application of genetic modification and genome editing for developing climate‐smart banana

“…Banana has been modified mainly using either Agrobacterium-mediated transformation or microprojectile bombardment. Agrobacterium-mediated transformation protocols have also been established for many cultivars of banana and plantains (Ganapathi et al, 2001;Khanna, Becker, Kleidon, & Dale, 2004;Tripathi, Mwaka, Tripathi, & Tushemereirwe, 2010;Tripathi, Muwonge, & Tripathi, 2012;Tripathi, Oduor, & Tripathi, 2015). Protocols for microprojectile bombardment technique are available for modifying banana and plantain (Becker, Dugdale, Smith, Harding, & Dale, 2000;Sagi et al, 1995).…”

Molecular analysis of somatic embryogenesis through proteomic approach and optimization of protocol in recalcitrant Musa spp.

“…The non‐availability of clean explant‐suckers (Singh et al ), in predominantly Fusarium wilt infested areas, make the high throughput system through the somatic embryogenic pathway by using male flower buds as explants a necessity. This is one of the tools capable of producing thousands of plantlets from 1 ml of embryogenic cell suspension (ECS) and a further advantage is the continued production with the establishment of viable cell suspension based on these plantlets (Schoofs , Strosse et al , Tripathi et al ).…”