Abstract:

Over three billion people in developing countries face iron deficiency anaemia (IDA) (WHO,
2000). Further, nutritional deficiencies like iron (Fe24), zinc (Zn2+) and vitamin A account for
almost two-thirds of the child mortality worldwide (Bouis et al., 2011; Hossain and
Mohiuddin, 2013; Saltzman et al., 2013). These deficiencies are typically caused either by
diets with high intake of staple foods such as, banana that are low in micronutrients, or by
low intake of rich micronutrient sources like vegetables, fruits and fish products. In Uganda, IDA has been identified as a critical public health problem, where 73% of six to 59-month-old Ugandan children exhibit mild to severe anemia (MOH, 2002; UDHS, 2011). IDA causes a range of health problems in the human population, including pregnancy-related complications, brain damage in infants, and reduced productivity (Frossard et al., 2000). Traditional strategies to alleviate mineral deficiency in susceptible populations have relied on supplementation, food fortification and dietary diversification programs (Bouis, 1999; Bouis and Welch, 2010; Graham et al., 2001). These have seen limited success in alleviating IDA in developing countries like Uganda as they require reliable transport infrastructure and consistent policy support, which are currently unavailable. Biofortification has been reported to be a suitable option to complemento there approaches (Bouis, 2003; BouisetaL, 2011). Biofortification is the development of micronutrient-dense staple crops using traditional biotechnology or genetic modification (Nestel et al., 2006). Banana (Musa spp.) is one of the major staple foods in sub-Saharan Africa, and global production is approximately 97 million tons annually (FAOSTAT, 2010). Compared to other starchy staples, it is rich in many minerals but is deficient in nutrients like Fe, Zn and pro vitamin A. East African Highland bananas are an important group of genetically similar cultivars (Tushemereirwe et al., 2001) that have not changed over several generations as they are sterile and parthenocarpic triploids. These biological factors make their genetic improvement using classical breeding methods difficult. Therefore, the application of biotechnological tools has the potential to add desired traits without changing highly valued characteristics of the target crop. Such an approach requires well characterised genes and promoters. A number of genes involved in plant Fe assimilation have been isolated from Arabidopsisthaliana and other plants. Strategies to enhance Fe accumulation in edible plant parts include improving uptake (Connolly et al., 2002; Vasconcelos et al.,2006), Fe storage (Ravet et al., 2009b) and enhancing Fe transportation (Lee et al., 2009;
Masuda et al., 2009). However, there are no published studies on known banana cultivars with naturally elevated Fe accumulation in fruit pulp, and Fe metabolism in bananas is not well understood. Thus, in this study, cooking and dessert banana cultivars from different agro-ecological zones were investigated for the effect of genotype by environment interactions in addition to other environmental factors such as soil Fe, pH and texture on Fe and Zn accumulation in fruit pulp during development. The interaction of these factors was determined using principal components analysis and agglomerative hierarchical clustering. The effect of leaf position on Fe accumulation at vegetative, flowering and fruiting stages of development were also investigated. Biofortification of plants to enhance mineral accumulation in the edible plant parts has shown potential in plants like rice and cassava (lhemere et al., 2012; Narayanan, 2011; Masuda et al., 2012; Johnson et al., 2011). Recently transgenic bananas, cultivars 'Sukali Ndiizi', 'Nakinyika' and 'Cavendish', transformed with Fe enhancement genes were developed in Uganda and at Queensland University of Technology. Transgenic plants evaluated in the field harbor IRT1, FRO2, Sfer, FEA1, and OsNASl, whereas plants transformed with OsNAS2+OsYSL2 were only evaluated under glasshouse conditions. Detailed molecular and biochemical analyses were done on selected lines that produced fruits. The effect of gene expression on mineral accumulation was determined at different maturity stages. ICP-OES was used to determine mineral accumulation in the different plant tissues. The data shows that transgenic lines transformed with IRT1, FRO23nd FEA1 did not show significant amount of Fe in the fruit pulp while those transformed with ferritins, and OsNASl showed elevated amount ranging from 1.12 -1.8-fold as compared to wild type. All transgenic banana leaves accumulated more Fe compared to the wild type indicating that constitutive expression using 35S and Ubi promoters was more effective in the vegetative parts of banana plants compared to the fruit pulp. OsNASl enhanced Fe and Zn accumulation in both fruit pulp and leaf tissue although reduced Zn concentration was observed in the latter. In contrast, lines containing Sfer, FRO2, IRT1 and OsNAS2-OsYSL2 showed enhanced Fe content in both fruit and leaf tissue with reduced Zn content in these tissues. The results indicate that OsNAS genes are a desirable strategy particularly where both Fe and Zn are required in the diet. This research is part of a broader project currently being undertaken by researchers at Queensland University of Technology (QUT, Australia) and at the National Agricultural Research Organization (NARO, Uganda), to address Fe and pro-vitamin A micronutrient deficiency in consumer acceptable banana varieties via genetic engineering. Therefore, this study provides valuable information on how different genes affect Fe metabolism in fruit and leaf tissues and pinpoints specific insights for future development of Fe or Zn rich bananas.