East African highland cooking banana: towards an efficient selection of hybrids with user-preferred food quality traits.

BACKGROUND: Determinants of culinary qualities of East African highland cooking bananas (EAHCB) are not well known. This constrains the inclusion of user-preferred traits in breeding. The present study aimed to quantify key indicators of user-preferred characteristics to enable selection of acceptable hybrids. RESULTS: Qualitative characteristics that drive preference were big bunches (15-34 kg), long straight/slightly curved fingers (12-23 cm), yellowness and soft texture. Descriptive sensory analysis of the intensity of colour and texture the 23 genotypes revealed that landraces Kibuzi, Mbwazirume, Nakitembe and Mpologoma had higher intensity of yellowness and lower intensity of hardness (softer) and a low score (≤ 1.0) of astringency taste. A preference test showed that they had higher acceptability scores. Biochemical, instrumental and sensory data revealed correlations between sensory firmness and instrumental hardness (r = 0.5), sensory firmness and amylopectin (r = -0.54), suggesting that qualitative descriptions can be predicted by instrumental and biochemical indicators. Significant (P < 0.05) variations in amylose and total starch content were observed in different varieties. Moderate correlations between instrumental hardness and firmness in mouth (r = 0.55), cohesiveness and firmness in the mouth (r = 0.57), and adhesiveness and firmness in the mouth (r = 0.64) were observed. Surprisingly, carotenoids content was not correlated with yellowness in cooked matooke. However, positive correlations were observed between chroma (b*) parameters of raw matooke and sensorial assessed color on cooked samples. CONCLUSION: Qualitative characteristis; the bunch, pulp colour and texture; that drive users-preference in the EAHCB were quantified, paving way for breeders to use them to select genotypes with these attributes early in the breeding process. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Analysis of association of sensory and laboratory assessments for quality and consumer acceptability of steamed East Africa highland bananas.

BACKGROUND: The relevance of several characteristics for the acceptability of steamed East Africa bananas (matooke) was assessed using consumer-preferred characteristics, the overall liking scores, check-all-that-apply (CATA) and the Just About Right scale. The study was conducted in rural and urban locations in three banana growing regions of Uganda. Two landraces and two hybrids were processed into matooke. Twelve trained panellists evaluated color, taste and texture sensory characteristics. RESULTS: Consumers scored matooke from landraces as the most liked. The CATA test showed that the most important characteristics were: smooth mouthfeel, soft to the touch, not sticky, moldable, deep yellow color, attractive, good matooke taste and smell. Principal component analysis confirmed that most of the preferred sensory characteristics were associated with the local genotypes, whereas the less preferred characteristics were associated with hybrids. Correlation analysis revealed strong positive correlations between the consumer assessed characteristics, hardness by touch, softness to touch and yellowness, as well as quantitative laboratory characteristics (moldable, hardness by touch, softness and yellowness) of the steamed matooke. Color assessed by consumers was strongly correlated with the laboratory-assessed color indicators. CONCLUSION: The strong associations observed between laboratory-assessed and consumer-based characteristics (moldable by touch and yellowness) suggest the possibility of predicting consumer characteristics using quantitative laboratory sensory assessments. Matooke taste as assessed by consumer panel is strongly associated with smooth texture and deep yellow color, which were the characteristics associated with landraces in the laboratory sensory assessment. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Transgenic East African Highland Banana Plants Are Protected against Radopholus similis through Host-Delivered RNAi.

The burrowing nematode Radopholus similis is considered a major problem of intensive banana cultivation. It can cause extensive root damage resulting in the toppling disease of banana, which means that plants fall to the ground. Soaking R. similis in double-stranded (ds) RNA of the nematode genes Rps13, chitin synthase (Chs-2), Unc-87, Pat-10 or beta-1,4-endoglucanase (Eng1a) suppressed reproduction on carrot discs, from 2.8-fold (Chs-2) to 7-fold (Rps13). The East African Highland Banana cultivar Nakitembe was then transformed with constructs for expression of dsRNA against the same genes, and for each construct, 30 independent transformants were tested with nematode infection. Four months after transfer from in vitro culture to the greenhouse, the banana plants were transferred to a screenhouse and inoculated with 2000 nematodes per plant, and thirteen weeks later, they were analyzed for several parameters including plant growth, root necrosis and final nematode population. Plants with dsRNA constructs against the nematode genes were on average showing lower nematode multiplication and root damage than the nontransformed controls or the banana plants expressing dsRNA against the nonendogenous gene. In conclusion, RNAi seems to efficiently protect banana against damage caused by R. similis, opening perspectives to control this pest.

Use of timelapse photography to determine flower opening time and pattern in banana (Musa spp.) for efficient hand pollination.

Sterility and low seed set in bananas is the main challenge to their conventional genetic improvement. The first step to seed set in a banana breeding program depends on pollination at the right time to ensure effective fertilization. This study aimed at determining bract opening time (BOT) to enhance efficient pollination and seed set in bananas. A Nikon D810 digital camera was set-up to take pictures of growing banana inflorescences at five-minute intervals and time-lapse movies were developed at a speed of 30 frames per second to allow real-time monitoring of BOT. Genotypes studied included wild banana (1), Mchare (2), Matooke (4), Matooke hybrid (1), and plantain (1). Events of bract opening initiated by bract lift for female flowers (P < 0.01) started at 16:32 h and at 18:54 h for male flowers. Start of bract rolling was at 18:51 h among female flowers (P < 0.001) and 20:48 h for male flowers. Bracts ended rolling at 02:33 h and 01:16 h for female and flowers respectively (P < 0.05). Total time of bract opening (from lift to end of rolling) for female flowers was significantly longer than that of male flowers (P < 0.001). On average, the number of bracts subtending female flowers opening increased from one on the first day, to between one and four on the fourth day. The number regressed to one bract on day eight before start of opening of bracts subtending male flowers. There was a longer opening interval between bracts subtending female and male flowers constituting spatial and temporal separation. Bract rolling increased from partial to complete rolling from proximal to the distal end of the inflorescence among female flower. On the other hand, bracts subtending male flowers completely rolled. Differences in BOT of genotypes with the same reference time of assessment may be partly responsible for variable fertility. Hand pollination time between 07:00 and 10:00 h is slightly late thus an early feasible time should be tried.

First Evidence of Feeding-Induced RNAi in Banana Weevil via Exogenous Application of dsRNA.

Banana weevil (Cosmopolites sordidus) is the most devastating pest of banana and plantain worldwide, yet current control measures are neither effective, sustainable, nor environmentally sound, and no resistant farmer-preferred cultivars are known to date. In this paper, we examined the ability to induce RNA interference (RNAi) in the banana weevil via feeding. We first developed an agar- and banana corm (rhizome) flour-based artificial diet in a multi-well plate setup that allowed the banana weevils to complete their life cycle from egg through the larval instars to the pupal stage in an average period of 53 days. Adults emerged about 20 days later. The artificial diet allowed the tunneling and burrowing habits of the larvae and successful metamorphosis up to adult eclosion. Adding dsRNA for laccase2 to the artificial diet resulted in albino phenotypes, confirming gene-silencing. Finally, C. sordidus was fed with dsRNA against a selection of essential target genes: snf7, rps13, mad1, vha-a, vha-d, and lgl for a period of 45 days. 100% mortality within 9-16 days was realized with dssnf7, dsrps13, and dsmad1 at 200 ng/mL artificial diet, and this corresponded to a strong reduction in gene expression. Feeding the dsRNA targeting the two vha genes resulted in 100% mortality after about 3-4 weeks, while treatment with dslgl resulted in no mortality above the dsgfp-control and the water-control. Our results have implications for the development of RNAi approaches for managing important crop pests, in that banana weevils can be controlled based on the silencing of essential target genes as snf7, rps13, and mad1. They also highlight the need for research into the development of RNAi for banana protection, eventually the engineering of host-induced gene-silencing (HIGS) cultivars, given the high RNAi efficacy and its species-specific mode of action, adding the RNAi approach to the armory of integrated pest management (IPM).

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.

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.

Silencing of the Mitogen-Activated Protein Kinases (MAPK) Fus3 and Slt2 in Pseudocercospora fijiensis Reduces Growth and Virulence on Host Plants.

Pseudocercospora fijiensis, causal agent of the black Sigatoka disease (BSD) of Musa spp., has spread globally since its discovery in Fiji 1963 to all the banana and plantain growing areas across the globe. It is becoming the most damaging and economically important disease of this crop. The identification and characterization of genes that regulate infection processes and pathogenicity in P. fijiensis will provide important knowledge for the development of disease-resistant cultivars. In many fungal plant pathogens, the Fus3 and Slt2 are reported to be essential for pathogenicity. Fus3 regulates filamentous-invasion pathways including the formation of infection structures, sporulation, virulence, and invasive and filamentous growth, whereas Slt2 is involved in the cell-wall integrity pathway, virulence, invasive growth, and colonization in host tissues. Here, we used RNAi-mediated gene silencing to investigate the role of the Slt2 and Fus3 homologs in P. fijiensis in pathogen invasiveness, growth and pathogenicity. The PfSlt2 and PfFus3 silenced P. fijiensis transformants showed significantly lower gene expression and reduced virulence, invasive growth, and lower biomass in infected leaf tissues of East African Highland Banana (EAHB). This study suggests that Slt2 and Fus3 MAPK signaling pathways play important roles in plant infection and pathogenic growth of fungal pathogens. The silencing of these vital fungal genes through host-induced gene silencing (HIG) could be an alternative strategy for developing transgenic banana and plantain resistant to BSD.

Agrobacterium tumefaciens-Mediated Transformation of Pseudocercospora fijiensis to Determine the Role of PfHog1 in Osmotic Stress Regulation and Virulence Modulation.

Black Sigatoka disease, caused by Pseudocercospora fijiensis is a serious constraint to banana production worldwide. The disease continues to spread in new ecological niches and there is an urgent need to develop strategies for its control. The high osmolarity glycerol (HOG) pathway in Saccharomyces cerevisiae is well known to respond to changes in external osmolarity. HOG pathway activation leads to phosphorylation, activation and nuclear transduction of the HOG1 mitogen-activated protein kinases (MAPKs). The activated HOG1 triggers several responses to osmotic stress, including up or down regulation of different genes, regulation of protein translation, adjustments to cell cycle progression and synthesis of osmolyte glycerol. This study investigated the role of the MAPK-encoding PfHog1 gene on osmotic stress adaptation and virulence of P. fijiensis. RNA interference-mediated gene silencing of PfHog1 significantly suppressed growth of P. fijiensis on potato dextrose agar media supplemented with 1 M NaCl, indicating that PfHog1 regulates osmotic stress. In addition, virulence of the PfHog1-silenced mutants of P. fijiensis on banana was significantly reduced, as observed from the low rates of necrosis and disease development on the infected leaves. Staining with lacto phenol cotton blue further confirmed the impaired mycelial growth of the PfHog1 in the infected leaf tissues, which was further confirmed with quantification of the fungal biomass using absolute- quantitative PCR. Collectively, these findings demonstrate that PfHog1 plays a critical role in osmotic stress regulation and virulence of P. fijiensis on its host banana. Thus, PfHog1 could be an interesting target for the control of black Sigatoka disease in banana.

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.

Field resistance of transgenic plantain to nematodes has potential for future African food security.

Plant parasitic nematodes impose losses of up to 70% on plantains and cooking bananas in Africa. Application of nematicides is inappropriate and resistant cultivars are unavailable. Where grown, demand for plantain is more than for other staple crops. Confined field testing demonstrated that transgenic expression of a biosafe, anti-feedant cysteine proteinase inhibitor and an anti-root invasion, non-lethal synthetic peptide confers resistance to plantain against the key nematode pests Radopholus similis and Helicotylenchus multicinctus. The best peptide transgenic line showed improved agronomic performance relative to non-transgenic controls and provided about 99% nematode resistance at harvest of the mother crop. Its yield was about 186% in comparison with the nematode challenged control non-transgenic plants based on larger bunches and diminished plant toppling in storms, due to less root damage. This is strong evidence for utilizing this resistance to support the future food security of 70 million, mainly poor Africans that depend upon plantain as a staple food.

In vitro antifungal activity of synthetic dsRNA molecules against two pathogens of banana, Fusarium oxysporum f. sp. cubense and Mycosphaerella fijiensis.

BACKGROUND: A key challenge for designing RNAi-based crop protection strategies is the identification of effective target genes in the pathogenic organism. In this study, in vitro antifungal activities of a set of synthetic double-stranded RNA molecules on spore germination of two major pathogenic fungi of banana, Fusarium oxysporum Schlecht f. sp. cubense WC Snyder & HN Hans (Foc) and Mycosphaerella fijiensis Morelet (Mf) were evaluated. RESULTS: All the tested synthetic dsRNAs successfully triggered the silencing of target genes and displayed varying degrees of potential to inhibit spore germination of both tested banana pathogens. When Foc dsRNAs were applied to Foc spores, inhibition ranged from 79.8 to 93.0%, and from 19.9 to 57.8% when Foc dsRNAs were applied to Mf spores. However, when Mf dsRNAs were applied on Mf spores, inhibition ranged from 34.4 to 72.3%, and from 89.7 to 95.9% when Mf dsRNAs were applied to Foc spores. CONCLUSION: The dsRNAs for adenylate cyclase, DNA polymerase alpha subunit and DNA polymerase delta subunit showed high levels of spore germination inhibition during both self- and cross-species tests, making them the most promising targets for RNA-mediated resistance in banana against these fungal pathogens. © 2013 Society of Chemical Industry.

Cooking enhances but the degree of ripeness does not affect provitamin A carotenoid bioavailability from bananas in Mongolian gerbils.

Banana is a staple crop in many regions where vitamin A deficiency is prevalent, making it a target for provitamin A biofortification. However, matrix effects may limit provitamin A bioavailability from bananas. The retinol bioefficacies of unripe and ripe bananas (study 1A), unripe high-provitamin A bananas (study 1B), and raw and cooked bananas (study 2) were determined in retinol-depleted Mongolian gerbils (n = 97/study) using positive and negative controls. After feeding a retinol-deficient diet for 6 and 4 wk in studies 1 and 2, respectively, customized diets containing 60, 30, or 15% banana were fed for 17 and 13 d, respectively. In study 1A, the hepatic retinol of the 60% ripe Cavendish group (0.52 ± 0.13 µmol retinol/liver) differed from baseline (0.65 ± 0.15 µmol retinol/liver) and was higher than the negative control group (0.39 ± 0.16 µmol retinol/liver; P < 0.0065). In study 1B, no groups differed from baseline (0.65 ± 0.15 µmol retinol/liver; P = 0.20). In study 2, the 60% raw Butobe group (0.68 ± 0.17 µmol retinol/liver) differed from the 60% cooked Butobe group (0.87 ± 0.24 µmol retinol/liver); neither group differed from baseline (0.80 ± 0.27 µmol retinol/liver; P < 0.0001). Total liver retinol was higher in the groups fed cooked bananas than in those fed raw (P = 0.0027). Body weights did not differ even though gerbils ate more green, ripe, and raw bananas than cooked, suggesting a greater indigestible component. In conclusion, thermal processing, but not ripening, improves the retinol bioefficacy of bananas. Food matrix modification affects carotenoid bioavailability from provitamin A biofortification targets.

Expression of sweet pepper Hrap gene in banana enhances resistance to Xanthomonas campestris pv. musacearum.

Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum, is the most devastating disease of banana in the Great Lakes region of Africa. The pathogen's rapid spread has threatened the livelihood of millions of Africans who rely on banana fruit for food security and income. The disease is very destructive, infecting all banana varieties, including both East African Highland bananas and exotic types of banana. In the absence of natural host plant resistance among banana cultivars, the constitutive expression of the hypersensitivity response-assisting protein (Hrap) gene from sweet pepper (Capsicum annuum) was evaluated for its ability to confer resistance to BXW. Transgenic lines expressing the Hrap gene under the regulation of the constitutive CaMV35S promoter were generated using embryogenic cell suspensions of two banana cultivars: 'Sukali Ndiizi' and 'Mpologoma'. These lines were characterized by molecular analysis, and were challenged with Xanthomonas campestris pv. musacearum to analyse the efficacy of the Hrap gene against BXW. The majority of transgenic lines (six of eight) expressing Hrap did not show any symptoms of infection after artificial inoculation of potted plants in the screenhouse, whereas control nontransgenic plants showed severe symptoms resulting in complete wilting. This study demonstrates that the constitutive expression of the sweet pepper Hrap gene in banana results in enhanced resistance to BXW. We describe the development of transgenic banana varieties resistant to BXW, which will boost the arsenal available to fight this epidemic disease and save livelihoods in the Great Lakes region of East and Central Africa.