Assessment of root-specific promoters in banana and tobacco and identification of a banana TIP2 promoter with strong root activity.

Genetic modification is one possible strategy to generate bananas (Musa spp.) with resistance to the soil-borne pathogen causing Fusarium wilt. The availability of banana root-specific promoters to target transgene expression to the sites of infection would be beneficial. We have assessed 18 promoter sequences derived from a range of plant species for their expression profiles in banana tissues to identify those with root-specific activity. Promoter sequences were isolated and fused to the ß-glucuronidase (GUS) gene to assess their expression levels and tissue specificity in both banana and the model plant tobacco. Two heterologous promoters conferring high root expression levels in banana were identified, including a ß-glucosidase 1 (GLU1) promoter from maize and the RB7-type tonoplast intrinsic protein (TIP)-2 promoter from strawberry. Further, a novel Musa TIP2-2 promoter sequence was isolated and characterized which, when fused to the GUS gene, conferred very high GUS expression levels in banana roots. These promoters will expand the options for the control of gene expression in genetically modified bananas, providing a tool to develop plants with resistance not only to soil-borne diseases such as Fusarium wilt, but also for the improvement of other traits, such as nematode resistance, nutrition or abiotic stress resistance.

Pro-vitamin A carotenoids in East African highland banana and other Musa cultivars grown in Uganda.

Bananas and plantains (Musa spp.) are an important staple and food security crop in sub-Saharan Africa. In Uganda, where the consumption of East African highland banana (EAHB) is the highest in the world, the population suffers from a high incidence of vitamin A deficiency (VAD). Since the consumption of pro-vitamin A carotenoids (pVAC) made available through the food staple can help alleviate these ailments, we set out to identify the most suitable banana variety to use in future biofortification strategies through genetic engineering. The study focussed on eight popular Musa cultivars grown in the heart of banana farming communities and across the three major agricultural zones of Uganda. The fruit pVAC concentration varied considerably within and across the cultivars tested. These variations could not be explained by the altitude nor the geographical location where these fruits were grown. More than 50% of the total carotenoids present in EAHB cultivars was found to comprise of α- and ß-carotene, while the retention of these compounds following traditional processing methods was at least 70%. Storage up to 14 days postharvest improved carotenoid accumulation up to 2.4-fold in the cultivar Nakitembe. The technical challenge for a successful biofortification approach in Uganda using genetically modified EAHB lies in guaranteeing that the fruit pVAC content will invariably provide at least 50% of the estimated average requirement for vitamin A regardless of the growing conditions.

Production of selectable marker gene-free Cavendish banana (Musa spp.) using a steroid-inducible recombinase platform.

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.

Banana21: From Gene Discovery to Deregulated Golden Bananas.

Uganda is a tropical country with a population in excess of 30 million, >80% of whom live in rural areas. Bananas (Musa spp.) are the staple food of Uganda with the East African Highland banana, a cooking banana, the primary starch source. Unfortunately, these bananas are low in pro-vitamin A (PVA) and iron and, as a result, banana-based diets are low in these micronutrients which results in very high levels of inadequate nutrition. This inadequate nutrition manifests as high levels of vitamin A deficiency, iron deficiency anemia, and stunting in children. A project known as Banana21 commenced in 2005 to alleviate micronutrient deficiencies in Uganda and surrounding countries through the generation of farmer- and consumer-acceptable edible bananas with significantly increased fruit levels of PVA and iron. A genetic modification approach was adopted since bananas are recalcitrant to conventional breeding. In this review, we focus on the PVA-biofortification component of the Banana21 project and describe the proof-of-concept studies conducted in Australia, the transfer of the technology to our Ugandan collaborators, and the successful implementation of the strategy into the field in Uganda. The many challenges encountered and the potential future obstacles to the practical exploitation of PVA-enhanced bananas in Uganda are discussed.

Transgenic Cavendish bananas with resistance to Fusarium wilt tropical race 4.

Banana (Musa spp.) is a staple food for more than 400 million people. Over 40% of world production and virtually all the export trade is based on Cavendish banana. However, Cavendish banana is under threat from a virulent fungus, Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) for which no acceptable resistant replacement has been identified. Here we report the identification of transgenic Cavendish with resistance to TR4. In our 3-year field trial, two lines of transgenic Cavendish, one transformed with RGA2, a gene isolated from a TR4-resistant diploid banana, and the other with a nematode-derived gene, Ced9, remain disease free. Transgene expression in the RGA2 lines is strongly correlated with resistance. Endogenous RGA2 homologs are also present in Cavendish but are expressed tenfold lower than that in our most resistant transgenic line. The expression of these homologs can potentially be elevated through gene editing, to provide non-transgenic resistance.

Golden bananas in the field: elevated fruit pro-vitamin A from the expression of a single banana transgene.

Vitamin A deficiency remains one of the world's major public health problems despite food fortification and supplements strategies. Biofortification of staple crops with enhanced levels of pro-vitamin A (PVA) offers a sustainable alternative strategy to both food fortification and supplementation. As a proof of concept, PVA-biofortified transgenic Cavendish bananas were generated and field trialed in Australia with the aim of achieving a target level of 20 µg/g of dry weight (dw) ß-carotene equivalent (ß-CE) in the fruit. Expression of a Fe'i banana-derived phytoene synthase 2a (MtPsy2a) gene resulted in the generation of lines with PVA levels exceeding the target level with one line reaching 55 µg/g dw ß-CE. Expression of the maize phytoene synthase 1 (ZmPsy1) gene, used to develop 'Golden Rice 2', also resulted in increased fruit PVA levels although many lines displayed undesirable phenotypes. Constitutive expression of either transgene with the maize polyubiquitin promoter increased PVA accumulation from the earliest stage of fruit development. In contrast, PVA accumulation was restricted to the late stages of fruit development when either the banana 1-aminocyclopropane-1-carboxylate oxidase or the expansin 1 promoters were used to drive the same transgenes. Wild-type plants with the longest fruit development time had also the highest fruit PVA concentrations. The results from this study suggest that early activation of the rate-limiting enzyme in the carotenoid biosynthetic pathway and extended fruit maturation time are essential factors to achieve optimal PVA concentrations in banana fruit.

Apoptosis-related genes confer resistance to Fusarium wilt in transgenic 'Lady Finger' bananas.

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana (Musa spp.). Apart from resistant cultivars, there are no effective control measures for the disease. We investigated whether the transgenic expression of apoptosis-inhibition-related genes in banana could be used to confer disease resistance. Embryogenic cell suspensions of the banana cultivar, 'Lady Finger', were stably transformed with animal genes that negatively regulate apoptosis, namely Bcl-xL, Ced-9 and Bcl-2 3' UTR, and independently transformed plant lines were regenerated for testing. Following a 12-week exposure to Foc race 1 in small-plant glasshouse bioassays, seven transgenic lines (2 × Bcl-xL, 3 × Ced-9 and 2 × Bcl-2 3' UTR) showed significantly less internal and external disease symptoms than the wild-type susceptible 'Lady Finger' banana plants used as positive controls. Of these, one Bcl-2 3' UTR line showed resistance that was equivalent to that of wild-type Cavendish bananas that were included as resistant negative controls. Further, the resistance of this line continued for 23-week postinoculation at which time the experiment was terminated. Using TUNEL assays, Foc race 1 was shown to induce apoptosis-like features in the roots of wild-type 'Lady Finger' plants consistent with a necrotrophic phase in the life cycle of this pathogen. This was further supported by the observed reduction in these effects in the roots of the resistant Bcl-2 3' UTR-transgenic line. This is the first report on the generation of transgenic banana plants with resistance to Fusarium wilt.

Inhibition of Agrobacterium-induced cell death by antiapoptotic gene expression leads to very high transformation efficiency of banana.

The death of plant cells in culture following exposure to Agrobacterium tumefaciens remains a major obstacle in developing Agrobacterium-mediated transformation into a highly efficient genotype-independent technology. Here, we present evidence that A. tumefaciens exposure induces cell death in banana cell suspensions. More than 90% of embryogenic banana cells died after exposure to A. tumefaciens and cell death was accompanied by a subset of features associated with apoptosis in mammalian cells, including DNA laddering, fragmentation, and formation of apoptotic-like bodies. Importantly, these cellular responses were inhibited in cells expressing the animal antiapoptosis genes Bcl-xL, Bcl-2 3' untranslated region, and CED-9. Inhibition of cell death resulted in up to 90% of cell clumps transformed with Bcl-xL, a 100-fold enhancement over vector controls, approaching the transformation and regeneration of every "transformable" cell. Similar results using sugarcane, a crop plant known for recalcitrance to Agrobacterium transformation, suggest that antiapoptosis genes may inhibit these phenomena and increase the transformation frequency of many recalcitrant plant species, including the major monocot cereal crop plants. Evidence of inhibition of plant cell death by cross-kingdom antiapoptotic genes also contributes to the growing evidence that genes for control of programmed cell death are conserved across wide evolutionary distances, even though these mechanisms are not well understood in plants.