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 anaemia (MOH, 2002; UDHS, 2011).
IDA causes a range of health problems in the human population, including pregnancyrelated
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 reportedtobe a suitable option tocomplementotherapproaches
(Bouis, 2003; BouisetaL, 2011). Biofortification isthe development of micronutrient-dense staple crops using traditional biotechnology orgenetic modification (Nestel et al., 2006).
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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
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 provitamin
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 theirgenetic
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).
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
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 componentsanalysis 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. Transgen ic
plants evaluated in the field harbour 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 transgeniclinestransformed 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 rangingfrom 1.12 -1.8 fold as compared to wild type. All
transgenicbanana 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.
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2006), Fe storage (Ravet et al., 2009b) and enhancing Fe transportation (Lee et al., 2009;
Masuda et al., 2009).
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 consumeracceptable banana varieties via geneticengineering. 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.