Abstract
Genetic improvement of commercially accepted banana cultivars is strongly reliant on the ability to introduce genes that encode important agro-traits such as disease resistance. In most cases this can only be achieved using a transgenic approach. Public and regulatory acceptance of these events would greatly increase with “clean” single copy integration events free of the selectable marker gene and extraneous vector backbone. This would also allow for the successive addition of new genes and traits as they become available. In this study, we used the pMarker Free 1 (pMF1) vector containing the green fluorescent protein (gfp) reporter gene to assess the effectiveness of steroid-inducible recombination and positive/negative dual selection to regenerate transgenic Cavendish banana plants that were potentially free of the selectable marker gene. By examining the interaction of two different Agrobacterium strains with two different cultivars of Cavendish banana, namely Williams and Grand Naine, we describe a transformation and regeneration strategy that successfully produced marker-free, single transgene copy, gfp-expressing events. The system will provide a useful means of serially improving banana into the future.
Highlights
A major challenge facing global banana production is the development of disease resistant varieties
Bananas (Musa spp.) are threatened by a range of diseases, the most devastating being Black Sigatoka caused by Pseudocercospora fijiensis (Arango Isaza et al 2016), Panama disease or Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Ploetz 2015), banana Xanthomonas wilt caused by the bacterium Xanthomonas campestris pv. musacearum (Blomme et al 2013) and banana bunchy top disease caused by Banana bunchy top virus (BBTV) (Dale 1987)
Two Agrobacterium strains (LBA4404 and AGL1) were used to transform two cultivars of Cavendish banana (Williams and Grand Naine) with the pMarker Free 1 (pMF1)-GFP vector in order to assess whether virulence of the bacteria influences the number of T-DNA copies integrated into the banana genome, the prevalence of unwanted vector backbone and the effectiveness of DEX-mediated recombination and 5-FC-based negative selection
Summary
A major challenge facing global banana production is the development of disease resistant varieties. Molecular breeding of elite banana cultivars is, the most suitable strategy for the genetic improvement of current cultivars. Technologies that facilitate genetic modification and transgene expression in banana have advanced rapidly over the past twenty years. In this time, the development of efficient methods for plant transformation and regeneration (Khanna et al 2004), advances in pathogen resistance strategies (Shekhawat et al 2012; Tripathi et al 2014; Dale et al 2017) and sequencing of a diploid banana genome (D’Hont et al 2012) have collectively made engineered disease resistance in banana an achievable goal
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