First report of banana bunchy top disease caused by Banana bunchy top virus in Uganda

Banana bunchy top virus (BBTV) that causes banana bunchy top disease (BBTD) is ranked among the top 100 invasive species in the world. Despite being omnipresent in the Democratic Republic of Congo (DR Congo) and South-Western Rwanda for over 2 decades, BBTV had not been reported in neighbouring Uganda. However, in 2020, banana plants with BBTD characteristic symptoms were observed in Arua City located in the North Western part of Uganda, bordering DR Congo. BBTV in these plants was confirmed using PCR and the comparison of the genomic DNA nucleotide sequence with nucleotide sequences in NCBI data base (https://www.ncbi.nlm.nih.gov/). The gDNA nucleotide sequence had 98-99% similarity with BBTV isolates form different countries in Africa, Asia, South Pacific and the USA. These results constitute the first confirmation of BBTD in Uganda. This infection is anticipated to have been introduced from DR Congo, mainly through infected planting materials. Once established, BBTD is very difficult to control on small-scale farms. It is therefore crucial to urgently understand the current spread of the virus, determine its risk to banana production in Uganda and devise proactive measures for its management

'NABIO808' (Syn. 'NAROBAN5'): A tasty cooking banana cultivar with resistance to pests and diseases

Abstract 'NABIO808' is a newly released, conventionally-bred triploid cooking banana cultivar in Uganda. It produces an average bunch yield of 54.5 t ha-1 yr-1 and is resistant to weevils, nematodes, and black Sigatoka. Additionally, its food is yellow, soft, and tasty, like that of most preferred landrace cultivars, making it more acceptable to end-users

Towards the biofortification of banana fruit for enhanced micronutrient content

In Uganda, vitamin A deficiency (VAD) and iron deficiency anaemia (IDA) are major public health problems with between 15-32% of children under 5 years of age showing VAD and 73% being anaemic. This is largely due to the fact that the staple food crop of the country, banana, is low in pro-vitamin A and iron, therefore leading to dietary deficiencies. Although worldwide progress has been made to control VAD and IDA through supplementation, food fortification and diet diversification, their long term sustainability and impact in developing countries such as Uganda is limited. The approach taken by researchers at Queensland University of Technology (QUT), Australia, in collaboration with the National Agricultural Research Organization (NARO), Uganda, to address this problem, is to generate consumer acceptable banana varieties with significantly increased levels of pro-vitamin A and iron in the fruit using genetic engineering techniques. Such an approach requires the use of suitable, well characterised genes and promoters for targeted transgene expression. Recently, a new banana phytoene synthase gene (APsy2a) involved in the synthesis of pro-vitamin A (pVA) carotenoids was isolated from a high â-carotene banana (F’ei cv Asupina). In addition, sequences of banana ferritin, an iron storage protein, have been isolated from Cavendish banana. The aim of the research described in this thesis was to evaluate the function of these genes to assess their suitability for the biofortification of banana fruit. In addition, a range of banana-derived promoters were characterised to determine their suitability for controlling the expression of transgenes in banana fruit. Due to the time constraints involved with generating transgenic banana fruit, rice was used as the model crop to investigate the functionality of the banana-derived APsy2a and ferritin genes. Using Agrobacterium-mediated transformation, rice callus was transformed with APsy2a +/- the bacterial-derived carotene desaturase gene (CrtI) each under the control of the constitutive maize poly-ubiquitin promoter (ZmUbi) or seed-specific rice glutelin1 (Gt1) promoter. The maize phytoene synthase (ZmPsy1) gene was included as a control. On selective media, with the exception of ZmUbi-CrtI-transgenic callus, all antibiotic resistant callus displayed a yellow-orange colour from which the presence of â-carotene was demonstrated using Raman spectroscopy. Although the regeneration of plants from yellow-orange callus was difficult, 16 transgenic plants were obtained and characterised from callus transformed with ZmUbi-APys2a alone. At least 50% of the T1 seeds developed a yellow-orange coloured callus which was found to contain levels of â-carotene ranging from 4.6-fold to 72-fold higher than that in non-transgenic rice callus. Using the seed-specific Gt1 promoter, 38 transgenic rice plants were generated from APsy2a-CrtI-transformed callus while 32 plants were regenerated from ZmPsy1-CrtI-transformed callus. However, when analysed for presence of transgene by PCR, all transgenic plants contained the APsy2a, ZmPsy1 or CrtI transgene, with none of the plants found to be co-transformed. Using Raman spectroscopy, no â-carotene was detected in-situ in representative T1 seeds. To investigate the potential of the banana-derived ferritin gene (BanFer1) to enhance iron content, rice callus was transformed with constitutively expressed BanFer1 using the soybean ferritin gene (SoyFer) as a control. A total of 12 and 11 callus lines independently transformed with BanFer1 and SoyFer, respectively, were multiplied and transgene expression was verified by RT-PCR. Pearl’s Prussian blue staining for in-situ detection of ferric iron showed a stronger blue colour in rice callus transformed with BanFer1 compared to SoyFer. Using flame atomic absorption spectrometry, the highest mean amount of iron quantified in callus transformed with BanFer1 was 30-fold while that obtained using the SoyFer was 14-fold higher than the controls. In addition, ~78% of BanFer1-transgenic callus lines and ~27% of SoyFer-transgenic callus lines had significantly higher iron content than the non-transformed controls. Since the genes used for enhancing micronutrient content need to be expressed in banana fruit, the activity of a range of banana-derived, potentially fruit-active promoters in banana was investigated. Using uidA (GUS) as a reporter gene, the function of the Expansin1 (MaExp1), Expansin1 containing the rice actin intron (MaExp1a), Expansin4 (MaExp4), Extensin (MaExt), ACS (MaACS), ACO (MaACO), Metallothionein (MaMT2a) and phytoene synthase (APsy2a) promoters were transiently analysed in intact banana fruit using two transformation methods, particle bombardment and Agrobacterium-mediated infiltration (agro-infiltration). Although a considerable amount of variation in promoter activity was observed both within and between experiments, similar trends were obtained using both transformation methods. The MaExp1 and MaExp1a directed high levels of GUS expression in banana fruit which were comparable to those observed from the ZmUbi and Banana bunchy top virus-derived BT4 promoters that were included as positive controls. Lower levels of promoter activity were obtained in both methods using the MaACO and MaExt promoters while the MaExp4, MaACS, and APsy2a promoters directed the lowest GUS activity in banana fruit. An attempt was subsequently made to use agro-infiltration to assess the expression of pVA biosynthesis genes in banana fruit by infiltrating fruit with constructs in which the ZmUbi promoter controlled the expression of APsy2a +/- CrtI, and with the maize phytoene synthase gene (ZmPsy1) included as a control. Unfortunately, the large amount of variation and inconsistency observed within and between experiments precluded any meaningful conclusions to be drawn. The final component of this research was to assess the level of promoter activity and specificity in non-target tissue. These analyses were done on leaves obtained from glasshouse-grown banana plants stably transformed with MaExp1, MaACO, APsy2a, BT4 and ZmUbi promoters driving the expression of the GUS gene in addition to leaves from a selection of the same transgenic plants which were growing in a field trial in North Queensland. The results from both histochemical and fluorometric GUS assays showed that the MaExp1 and MaACO promoters directed very low GUS activities in leaves of stably transformed banana plants compared to the constitutive ZmUbi and BT4 promoters. In summary, the results from this research provide evidence that the banana phytoene synthase gene (APsy2a) and the banana ferritin gene (BanFer1) are functional, since the constitutive over-expression of each of these transgenes led to increased levels of pVA carotenoids (for APsy2a) and iron content (for BanFer1) in transgenic rice callus. Further work is now required to determine the functionality of these genes in stably-transformed banana fruit. This research also demonstrated that the MaExp1 and MaACO promoters are fruit-active but have low activity in non-target tissue (leaves), characteristics that make them potentially useful for the biofortification of banana fruit. Ultimately, however, analysis of fruit from field-grown transgenic plants will be required to fully evaluate the suitability of pVA biosynthesis genes and the fruit-active promoters for fruit biofortification

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 β-carotene, while the retention of these compounds following traditional processing methods was at least 70%. Storage up to 14 days post-harvest 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

First evidence of feeding-induced RNAi in banana weevil via exogenous application of dsRNA

Simple Summary RNA interference (RNAi), a conserved mechanism in eukaryotic organisms, is initiated by the presence of double-stranded RNA (dsRNA) in the cells, regulating the expression of specific genes by degradation of their mRNA, and it is known as post-transcriptional gene-silencing (PTGS). RNAi can be employed to develop species-specific biopesticides where dsRNA is delivered to a pest insect via the oral route. 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. This study reports an artificial diet-based multi-well plate method and the first findings on the potential application of biotechnology in the control of the banana weevil using dietary RNAi, which would arguably provide the most sustainable and practical method for dsRNA delivery. A selection of target genes with effective RNAi leading to high and rapid mortality is presented. These results indicate the potential of RNAi-mediated management to suppress C. sordidus efficiently in the banana crop. 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)

Ploidy level of the banana (Musa spp.) accessions at the germplasm collection centre for the East and Central Africa

Banana Germplasm Collection serves as a source of useful genes for banana  breeding. However, insufficient and/or inaccurate information on the ploidy level of the germplasm renders its utilization in breeding difficult. The objective of this study was to determine and validate the ploidy level of 120 banana accessions in the ex situ germplasm collection centre for the East and Central Africa, located in Mbarara, Uganda. Flow cytometric analysis of the nuclear DNA content was used to determine the ploidy level of the accessions. Results indicate that accessions: Bura, Diana, Kambani-Rungwe, Paji and Pagatau, and Rungwe that were previously classified as diploids are actually triploids, whereas Selangor previously known to be a diploid is a tetraploid. Accessions such as Galeo, Mwitupemba and Ntindi 1 that were  previously classified as triploids were found diploids. GT, FHIA 25 and Muzungu Mwekundu that were considered as tetraploids, were found triploids. The information generated will guide correct placement of these accessions in the regional germplasm collection centre for the East and Central Africa and their utilization in banana breeding.Key words: Banana germplasm, breeding ex situ germplasm collection, flow  cytometry, ploidy

Techniques for measuring dynamic tire forces generated by heavy vehicles

Summary\ud \ud 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) β-carotene equivalent (β-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 β-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, as well as extended fruit maturation time, are essential factors to achieve optimal PVA concentrations in banana fruit

Isolation and functional characterisation of banana phytoene synthase genes as potential cisgenes

Carotenoids occur in all photosynthetic organisms where they protect photosystems from auto-oxidation, participate in photosynthetic energy-transfer and are secondary metabolites. Of the more than 600 known plant carotenoids, few can be converted into vitamin A by humans and so these pro-vitamin A carotenoids (pVAC) are important in human nutrition. Phytoene synthase (PSY) is a key enzyme in the biosynthetic pathway of pVACs and plays a central role in regulating pVAC accumulation in the edible portion of crop plants. Bananas are a major commercial crop and serve as a staple crop for more than 30 million people. There is natural variation in fruit pVAC content across different banana cultivars, but this is not well understood. Therefore, we isolated PSY genes from banana cultivars with relatively high (cv. Asupina) and low (cv. Cavendish) pVAC content. We provide evidence that PSY in banana is encoded by two paralogs (PSY1 and PSY2), each with a similar gene structure to homologous genes in other monocots. Further, we demonstrate that PSY2 is more highly expressed in fruit pulp compared to leaf. Functional analysis of PSY1 and PSY2 in rice callus and E. coli demonstrate that both genes encode functional enzymes, and that Asupina PSYs have approximately twice the enzymatic activity of the corresponding Cavendish PSYs. These results suggest that differences in PSY enzyme activity contribute significantly to the differences in Asupina and Cavendish fruit pVAC content. Importantly, Asupina PSY genes could potentially be used to generate new cisgenic or intragenic banana cultivars with enhanced pVAC content