Global banana disease management - getting serious with sustainability and food security

Much research in understanding plant diseases has been undertaken, but there has been insufficient attention given to dealing with coordinated approaches to preventing and managing diseases. A global management approach is essential to the long-term sustainability of banana production. This approach would involve coordinated surveys, capacity building in developing countries, development of disease outbreak contingency plans and coordinated quarantine awareness, including on-line training in impact risk assessment and web-based diagnostic software. Free movement of banana plants and products between some banana-producing countries is causing significant pressure on the ability to manage diseases in banana. The rapid spread of Fusarium oxysporum f. sp. cubense 'tropical race 4' in Asia, bacterial wilts in Africa and Asia and black leaf streak [Mycosphaerella fijiensis] in Brazil and elsewhere are cases in point. The impact of these diseases is devastating, severely cutting family incomes and jeopardising food security around the globe. Agreements urgently need to be reached between governments to halt the movement of banana plants and products between banana-producing countries before it is too late and global food security is irreparably harmed. Black leaf streak, arguably the most serious banana disease, has become extremely difficult to control in commercial plantations in various parts of the world. Sometimes in excess of 50 fungicide sprays have to be applied each year. Disease eradication and effective disease control is not possible because there is no control of disease inoculum in non-commercial plantings in these locations. Additionally, there have been enormous sums of money invested in international banana breeding programmes over many years only to see the value of hybrid products lost too soon. 'Goldfinger' (AAAB, syn. 'FHIA-01'), for example, has recently been observed severely affected by black leaf streak in Samoa. Resistant cultivars alone cannot be relied upon in the fight against this disease. Real progress in control may only come when the local communities are engaged and become actively involved in regional programmes. Global recommendations are long overdue and urgently needed to help ensure the long-term sustainable utilisation of the products of the breeding programmes.

Viabilidade econômica de manejos nutricionais na cultura de banana

O manejo adequado da adubação, visando à otimização da produtividade e qualidade dos frutos, é uma etapa representativa do processo produtivo para rentabilidade do empreendedor rural. Neste contexto, objetivou-se analisar a viabilidade econômica de cinco tipos de manejo nutricional de bananeiras de cultivares do subgrupo Cavendish, na região do Médio Paranapanema (SP), em 2009/2010. Foram utilizadas estruturas do custo operacional efetivo (COE) e custo operacional total (COT) e três indicadores de rentabilidade. Diferenças significativas foram observadas entre os manejos, sendo que o sistema de manejo que resultou em maior vantagem econômica para o produtor proporcionou índice médio de lucratividade 25,6% superior aos demais tratamentos, além de a receita líquida total ser, em média, 29,5% superior aos outros tipos de manejo. O custo por unidade produzida foi inferior no manejo nutricional mais lucrativo, apesar de a produtividade ter sido 9,5% inferior ao manejo que apresentou os maiores índices de produtividade.

Depósitos de calda obtidos com a aplicação aérea de defensivos na cultura da banana

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

O plantio associado entre banana (Musa acuminata Cultivar Cavendish "Grande Naine") e feijão-de-porco (Canavalia ensiformis (L.) D. C.): uma avaliação agronômica dos efeitos da competição por água, nitrogênio e energia luminosa

Nas regiões de clima tropical, o monocultivo da banana vem causando conseqüências ambientais desastrosas e, muitas vezes, impedindo uma exploração continuada de uma mesma área. A redução do rendimento é devido principalmente as limitações físico-químicas do solo e a rápida degradação do sistema radicular, agravada pela ação de parasitas do solo (nematóides, fungos, etc.). Em virtude destas limitações, várias iniciativas vem sendo buscadas para a minimização das perdas agronômicas e ambientais, destacando-se o melhoramento e a modificação genética, e a associação deste cultivo com espécies leguminosas. Porém uma das grandes dificuldades de avaliarmos os novos sistemas de cultivo alternativos concentra-se na falta de referenciais agronômicos relacionados principalmente com o funcionamento de sistemas de cultivos associados, especialmente relacionados aos fatores e condições que interferem diretamente na definição do rendimento da espécie principal. O presente estudo testou , em campo experimental, o uso de plantas de serviço associada a bananeira e seus efeitos na produção de biomassa durante seu o ciclo vegetativo. Isto porque é durante esta fase que a bananeira constrói sua capacidade de reservas de fotoassimilados e, consequentemente, define o potencial de produção e enchimento dos frutos. Além do monocultivo, definiu-se mais duas parcelas associadas com o feijão-de- porco: 1) o plantio simultâneo das duas espécies e; 2) o plantio de feijão-de-porco e, após 2 meses, a introdução da banana. Além de acompanhamento semanal das parcelas, realizou-se, bimensalmente, coletas destrutivas de dados sobre produção de matéria seca, superfície foliar e análise nutricional das plantas. Após a análise agronômica da fase vegetativa, aplicou-se a modelização dos sistemas de cultivo estudados e comparou-se os possíveis cenários sobre o rendimento final da bananeira, além de outros indicadores sobre os fatores de crescimento das plantas. Após o acompanhamento dos 7 primeiros meses do ciclo vegetativo, concluiu-se que a data de estabelecimento da associação foi determinante para o sucesso do cultivo associado. Podemos destacar que a associação entre a bananeira e o feijão-de-porco não causou limitações na produção de biomassa (4,2 ton/ha), quando comparada com o monocultivo (4,5 ton/ha). A redução do número de capinas também foi um indicador animador deste sistema de cultivo alternativo. Por outro lado, quando a bananeira foi plantada 60 dias após a leguminosa, a mesma representou uma séria limitação na produção de biomassa (2,7 ton/ha). Esta limitação deveu-se ao estado de forte competição devido a agressividade com que o feijão-de-porco recobria toda a parcela e alcançando uma altura (74 cm) superior que a muda de banana (29 cm). Em relação a primeira parte da metodologia aplicada - o diagnóstico agronômico -, a mesma foi eficiente para a avaliação do ciclo vegetativo da associação estudada, ficando a necessidade da continuidade do acompanhamento do ciclo reprodutivo, para a confirmação dos resultados em termos de formação e produção de frutos. Na fase de modelização, chegou-se a uma leitura dos resultados próxima dos resultados obtidos no campo. Em termos de rendimento em frutos, o monocultivo com adubação (400 kg/ha de nitrogênio) e irrigação (133 mm) teve um aumento na ordem de 50% no rendimento final (28 ton/ha) Quando comparada com a parcela nas condições reais do experimento (19,6 ton/ha). Já o rendimento em frutos da associação, apresentou o mesmo resultado com e sem adubação e irrigação (16 ton/ha). No tocante a contrução dos cenários, confirmou-se novamente algumas das vantagens da associação, principalmente na redução da adubação nitrogenada aplicada nos sistemas convencionais de cultivo. Finalmente, podemos imaginar a construção de várias formas de testar e otimizar o uso destes sistemas associados (cenários). Porém, confirma-se que a construção de novos referenciais agronômicos sobre sistemas de cultivo mais complexos (os cultivos associados) torna-se ainda muito necessário para a realização de avaliações mais precisas sobre estas alternativas. E, com estes novos referenciais técnicos, podemos imaginar, a médio e longo prazo, alguns dos benefícios das leguminosas sobre as propriedades físico-químicas do solo cultivado (cobertura viva, adubo verde, redução de adventícias, etc) e sobre a manutenção do rendimento dos cultivos (adubação verde).

Soil chemical attributes and leaf nutrients of Pacovan banana under two cover crops.

Banana is one of the most consumed fruits in the world, which is grown in most tropical countries. The objective of this work was to evaluate the main attributes of soil fertility in a banana crop under two cover crops and two root development locations. The work was conducted in Curaçá, BA, Brazil, between October 2011 and May 2013, using a randomized block design in split plot with five repetitions. Two cover crops were assessed in the plots, the cover 1 consisting of Pueraria phaseoloid es, and the cover 2 consisting of a crop mix with Sorghum bicolor, Ricinus commun is L., Canavalia ensiform is, Mucuna aterrima and Zea mays, and two soil sampling locations in the subplots, between plants in the banana rows (location 1) and between the banana rows (location 2). There were significant and independent effects for the cover crop and sampling location factors for the variables organic matter, Ca and P, and significant effects for the interaction between cover crops and sampling locations for the variables potassium, magnesium and total exchangeable bases. The cover crop mix and the between-row location presented the highest organic matter content. Potassium was the nutrient with the highest negative variation from the initial content and its leaf content was below the reference value, however not reducing the crop yield. The banana crop associated with crop cover using the crop mix provided greater availability of nutrients in the soil compared to the coverage with tropical kudzu.

The distribution of badnavirus-infected Musa germplasm from the International Transit Centre, Katholieke Universiteit Leuven, Belgium

Bioversity International is reviewing its moratorium on the distribution of virus-infected Musa germplasm from the International Transit Centre (ITC), and the ProMusa Crop Protection Working Group has been invited to comment on policy changes. This paper was written to form a basis of discussion among the working group members during the ISHS/ProMusa symposium. It argues that the distribution of Musa germplasm should be guided by the International Plant Protection Convention, which states that it is the responsibility of the importing country, not the exporter, to impose the phytosanitary measures. There may be special circumstances where the release of badnavirus-infected germplasm from the ITC could be justified.

Nematicidal activity of Solanum nigrum and S. sisymbriifolium extracts against the root-lesion nematode Pratylenchus goodeyi and its effects on infection and gene expression

The control of Pratylenchus goodeyi a common nematode parasite of banana crop in Madeira Island can benefit from searching for natural nematicides through plants extracts. With this aim we submitted Solanum nigrum and S. sisymbriifolium dried plants to a sequential extraction in the solvent sequence of dichloromethane, acetone, ethanol and water, and to na aqueous extraction of the fresh and dried plants. Analyses with the extracts at several concentrations were used to assess mobility and mortality on P. goodeyi. Results showed that the water extract and aqueous extracts from both plants at a concentration of 10 mg/mL affected nematode mobility and caused mortality but the acetone extract from S. nigrum was the most efficient, causing 100% mortality whereas dichloromethane had no effect on P. goodeyi. Determination of the lipophilic and phenolic compounds present in the two most effective Solanum extracts (acetone and water) and in dichloromethane extract revealed that some of these compounds had nematicidal activity. S. nigrum acetone extract (10 mg/mL) was used to find out the nematicidal potential following the effect at gene expression level and nematode behaviour. Genes coding for calreticulin and beta-1,4- endoglucanase related to parasitism and translocon-associated protein putatively connected to stress were obtained and its relative expression assessed in nematodes exposed to the extract. Results revealed that expression of Pg-CRT decreased showing to influence the infection, Pg-ENG remained steady and Pg-TRAPδ was induced over time exposure. Biological assays showed that P. goodeyi mobility and ability to infect the banana roots were affected as a decrease in the number of nematodes that reached the roots was obtained with the increased exposure time to the extract being implicated in the infection success. The information obtained from this thesis showed that S. nigrum has potential to be used for the development of a new control strategy against plant-parasitic nematodes.

Variação sazonal da concentração de nutrientes em folhas de bananeiras, no Vale do Ribeira-SP

The Vale do Ribeira, SP, main agricultural activity is the banana crop, which accounts for most of this fruit production in the State of São Paulo. The nutritional balance of the plant is one of the most important factors for the banana plant can complete the cycle and achieve high productivity. Aiming to evaluate the seasonal variation of leaf nutrient concentration in banana plants in Vale do Ribeira-SP, we used the results of 252 chemical analyses of plant tissue, collected from August 2009 to September 2010, in the 18 representative properties for the region, ten cultivated with subgroup Cavendish banana plant and eight of subgroup Prata banana plant. The largest variation between the macronutrient occurred for K and S, and among the micronutrients, especially for Fe and B. In some dates of evaluation, there was a higher leaf concentration of P, K, Ca and Zn, in subgroup Cavendish banana plants, while the subgroup Prata banana plants showed higher leaf concentration, especially of Mn, B and N. Climatic conditions, especially rain, influenced the leaf nutrient content, especially for K, N, S, B and Fe.

Viabilidade econômica da utilização dos resíduos da bananicultura na alimentação de cordeiros confinados

Aimed to analyze the economic feasibility of replacing hay Cynodon spp by hay banana crop residues in diet of lambs. We used 25 Santa Inês lambs, with an average age of five months and an average initial weight of 26.5kg, distributed in a completely randomized design with five treatments (40% hay Cynodon spp. + 60% concentrate, 20% hay banana leaf and 20% of Cynodon spp. + 60% concentrate, 40% of banana leaf hay + 60% concentrate, 20% hay pseudostem of banana and 20% of Cynodon spp. + 60% concentrate, 40% hay pseudostem of banana + 60% concentrate). After 69 days of experiment the animals were slaughtered. Possession of the cost of each diet and consumption of animals was calculated economic feasibility. Treatment with 40% of banana leaf hay + 60% concentrate showed the best economic indicators, based on the highest net income, the higher rate of return to higher profitability and marketing of live animals or slaughtered. The inclusion of banana crop residues in the diet of growing lambs increases the economic viability of the activity.

Variação sazonal da concentração de nutrientes em folhas de bananeiras, no Vale do Ribeira-SP

The Vale do Ribeira, SP, main agricultural activity is the banana crop, which accounts for most of this fruit production in the State of São Paulo. The nutritional balance of the plant is one of the most important factors for the banana plant can complete the cycle and achieve high productivity. Aiming to evaluate the seasonal variation of leaf nutrient concentration in banana plants in Vale do Ribeira-SP, we used the results of 252 chemical analyses of plant tissue, collected from August 2009 to September 2010, in the 18 representative properties for the region, ten cultivated with subgroup Cavendish banana plant and eight of subgroup Prata banana plant. The largest variation between the macronutrient occurred for K and S, and among the micronutrients, especially for Fe and B. In some dates of evaluation, there was a higher leaf concentration of P, K, Ca and Zn, in subgroup Cavendish banana plants, while the subgroup Prata banana plants showed higher leaf concentration, especially of Mn, B and N. Climatic conditions, especially rain, influenced the leaf nutrient content, especially for K, N, S, B and Fe.

The manipulation of apoptosis-related genes to generate resistance to Fusarium wilt and water stress in banana

Bananas are susceptible to a diverse range of biotic and abiotic stresses, many of which cause serious production constraints worldwide. One of the most destructive banana diseases is Fusarium wilt caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc). No effective control strategy currently exists for this disease which threatens global banana production. Although disease resistance exists in some wild bananas, attempts to introduce resistance into commercially acceptable bananas by conventional breeding have been hampered by low fertility, long generation times and association of poor agronomical traits with resistance genes. With the advent of reliable banana transformation protocols, molecular breeding is now regarded as a viable alternative strategy to generate disease-resistant banana plants. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi. Further, the transgenic plants showed increased resistance to a range of abiotic stresses. In this thesis, the use of anti-apoptosis genes to generate transgenic banana plants with resistance to Fusarium wilt was investigated. Since water stress is an important abiotic constraint to banana production, the resistance of the transgenic plants to water stress was also examined. Embryogenic cell suspensions (ECS) of two commercially important banana cultivars, Grand Naine (GN) and Lady Finger (LF), were transformed using Agrobacterium with the anti-apoptosis genes, Bcl-xL, Bcl-xL G138A, Ced-9 and Bcl- 2 3’ UTR. An interesting, and potentially important, outcome was that the use of anti-apoptosis genes resulted in up to a 50-fold increase in Agrobacterium-mediated transformation efficiency of both LF and GN cells over vector controls. Regenerated plants were subjected to a complete molecular characterisation in order to detect the presence of the transgene (PCR), transcript (RT-PCR) and gene product (Western blot) and to determine the gene copy number (Southern blot). A total of 36 independently-transformed GN lines (8 x Bcl-xL, 5 x Bcl-xL G138A, 15 x Ced-9 and 8 x Bcl-2 3’ UTR) and 41 independently-transformed LF lines (8 x Bcl-xL, 7 x BclxL G138A, 13 x Ced-9 and 13 x Bcl-2 3’ UTR) were identified. The 41 transgenic LF lines were multiplied and clones from each line were acclimatised and grown under glasshouse conditions for 8 weeks to allow monitoring for phenotypic abnormalities. Plants derived from 3 x Bcl-xL, 2 x Ced-9 and 5 x Bcl-2 3’ UTR lines displayed a variety of aberrant phenotypes. However, all but one of these abnormalities were off-types commonly observed in tissue-cultured, non-transgenic banana plants and were therefore unlikely to be transgene-related. Prior to determining the resistance of the transgenic plants to Foc race 1, the apoptotic effects of the fungus on both wild-type and Bcl-2 3’ UTR-transgenic LF banana cells were investigated using rapid in vitro root assays. The results from these assays showed that apoptotic-like cell death was elicited in wild-type banana root cells as early as 6 hours post-exposure to fungal spores. In contrast, these effects were attenuated in the root cells of Bcl-2 3’ UTR-transgenic lines that were exposed to fungal spores. Thirty eight of the 41 transgenic LF lines were subsequently assessed for resistance to Foc race 1 in small-plant glasshouse bioassays. To overcome inconsistencies in rating the internal (vascular discolouration) disease symptoms, a MatLab-based computer program was developed to accurately and reliably assess the level of vascular discolouration in banana corms. Of the transgenic LF banana lines challenged with Foc race 1, 2 x Bcl-xL, 3 x Ced-9, 2 x Bcl-2 3’ UTR and 1 x Bcl-xL G138A-transgenic line were found to show significantly less external and internal symptoms than wild-type LF banana plants used as susceptible controls at 12 weeks post-inoculation. Of these lines, Bcl-2 3’ UTR-transgenic line #6 appeared most resistant, displaying very mild symptoms similar to the wild-type Cavendish banana plants that were included as resistant controls. This line remained resistant for up to 23 weeks post-inoculation. Since anti-apoptosis genes have been shown to confer resistance to various abiotic stresses in other crops, the ability of these genes to confer resistance against water stress in banana was also investigated. Clonal plants derived from each of the 38 transgenic LF banana plants were subjected to water stress for a total of 32 days. Several different lines of transgenic plants transformed with either Bcl-xL, Bcl-xL G138A, Ced-9 or Bcl-2 3’ UTR showed a delay in visual water stress symptoms compared with the wild-type control plants. These plants all began producing new growth from the pseudostem following daily rewatering for one month. In an attempt to determine whether the protective effect of anti-apoptosis genes in transgenic banana plants was linked with reactive oxygen species (ROS)-associated programmed cell death (PCD), the effect of the chloroplast-targeting, ROS-inducing herbicide, Paraquat, on wild-type and transgenic LF was investigated. When leaf discs from wild-type LF banana plants were exposed to 10 ìM Paraquat, complete decolourisation occurred after 48 hours which was confirmed to be associated with cell death and ROS production by trypan blue and 3,3-diaminobenzidine (DAB) staining, respectively. When leaf discs from the transgenic lines were exposed to Paraquat, those derived from some lines showed a delay in decolourisation, suggesting only a weak protective effect from the transgenes. Finally, the protective effect of anti-apoptosis genes against juglone, a ROS-inducing phytotoxin produced by the causal agent of black Sigatoka, Mycosphaerella fijiensis, was investigated. When leaf discs from wild-type LF banana plants were exposed to 25 ppm juglone, complete decolourisation occurred after 48 hours which was again confirmed to be associated with cell death and ROS production by trypan blue and DAB staining, respectively. Further, TdT-mediated dUTP nick-end labelling (TUNEL) assays on these discs suggested that the cell death was apoptotic. When leaf discs from the transgenic lines were exposed to juglone, discs from some lines showed a clear delay in decolourisation, suggesting a protective effect. Whether these plants are resistant to black Sigatoka is unknown and will require future glasshouse and field trials. The work presented in this thesis provides the first report of the use of anti-apoptosis genes as a strategy to confer resistance to Fusarium wilt and water stress in a nongraminaceous monocot, banana. Such a strategy may be exploited to generate resistance to necrotrophic pathogens and abiotic stresses in other economically important crop plants.

Market access of Papua New Guinea bananas (Musa spp.) with particular respect to banana fly (Bactrocera musae (Tryon)) (Diptera: Tephritidae)

International market access for fresh commodities is regulated by international accepted phytosanitary guidelines, the objectives of which are to reduce the biosecurity risk of plant pest and disease movement. Papua New Guinea (PNG) has identified banana as a potential export crop and to help meet international market access requirements, this thesis provides information for the development of a pest risk analysis (PRA) for PNG banana fruit. The PRA is a three step process which first identifies the pests associated with a particular commodity or pathway, then assesses the risk associated with those pests, and finally identifies risk management options for those pests if required. As the first step of the PRA process, I collated a definitive list on the organisms associated with the banana plant in PNG using formal literature, structured interviews with local experts, grey literature and unpublished file material held in PNG field research stations. I identified 112 organisms (invertebrates, vertebrate, pathogens and weeds) associated with banana in PNG, but only 14 of these were reported as commonly requiring management. For these 14 I present detailed information summaries on their known biology and pest impact. A major finding of the review was that of the 14 identified key pests, some research information occurs for 13. The single exception for which information was found to be lacking was Bactrocera musae (Tryon), the banana fly. The lack of information for this widely reported ‘major pest on PNG bananas’ would hinder the development of a PNG banana fruit PRA. For this reason the remainder of the thesis focused on this organism, particularly with respect to generation of information required by the PRA process. Utilising an existing, but previously unanalysed fruit fly trapping database for PNG, I carried out a Geographic Information System analysis of the distribution and abundance of banana in four major regions of PNG. This information is required for a PRA to determine if banana fruit grown in different parts of the country are at different risks from the fly. Results showed that the fly was widespread in all cropping regions and that temperature and rainfall were not significantly correlated with banana fly abundance. Abundance of the fly was significantly correlated (albeit weakly) with host availability. The same analysis was done with four other PNG pest fruit flies and their responses to the environmental factors differed to banana fly and each other. This implies that subsequent PRA analyses for other PNG fresh commodities will need to investigate the risk of each of these flies independently. To quantify the damage to banana fruit caused by banana fly in PNG, local surveys and one national survey of banana fruit infestation were carried out. Contrary to expectations, infestation was found to be very low, particularly in the widely grown commercial cultivar, Cavendish. Infestation of Cavendish fingers was only 0.41% in a structured, national survey of over 2 700 banana fingers. Follow up laboratory studies showed that fingers of Cavendish, and another commercial variety Lady-finger, are very poor hosts for B. musae, with very low host selection rates by female flies and very poor immature survival. An analysis of a recent (within last decade) incursion of B. musae into the Gazelle Peninsula of East New Britain Province, PNG, provided the final set of B. musae data. Surveys of the fly on the peninsular showed that establishment and spread of the fly in the novel environment was very rapid and thus the fly should be regarded as being of high biosecurity concern, at least in tropical areas. Supporting the earlier impact studies, however, banana fly has not become a significant banana fruit problem on the Gazelle, despite bananas being the primary starch staple of the region. The results of the research chapters are combined in the final Discussion in the form of a B. musae focused PRA for PNG banana fruit. Putting the thesis in a broader context, the Discussion also deals with the apparent discrepancy between high local abundance of banana fly and very low infestation rates. This discussion focuses on host utilisation patterns of specialist herbivores and suggests that local pest abundance, as determined by trapping or monitoring, need not be good surrogate for crop damage, despite this linkage being implicit in a number of international phytosanitary protocols.

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.

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.