Defining the role of phytoene synthase in carotenoid accumulation of high provitamin A bananas

Vitamin A deficiency (VAD) is a serious problem in developing countries, affecting approximately 127 million children of preschool age and 7.2 million pregnant women each year. However, this deficiency is readily treated and prevented through adequate nutrition. This can potentially be achieved through genetically engineered biofortification of staple food crops to enhance provitamin A (pVA) carotenoid content. Bananas are the fourth most important food crop with an annual production of 100 million tonnes and are widely consumed in areas affected by VAD. However, the fruit pVA content of most widely consumed banana cultivars is low (~ 0.2 to 0.5 ìg/g dry weight). This includes cultivars such as the East African highland banana (EAHB), the staple crop in countries such as Uganda, where annual banana consumption is approximately 250 kg per person. This fact, in addition to the agronomic properties of staple banana cultivars such as vegetative reproduction and continuous cropping, make bananas an ideal target for pVA enhancement through genetic engineering. Interestingly, there are banana varieties known with high fruit pVA content (up to 27.8 ìg/g dry weight), although they are not widely consumed due to factors such as cultural preference and availability. The genes involved in carotenoid accumulation during banana fruit ripening have not been well studied and an understanding of the molecular basis for the differential capacity of bananas to accumulate carotenoids may impact on the effective production of genetically engineered high pVA bananas. The production of phytoene by the enzyme phytoene synthase (PSY) has been shown to be an important rate limiting determinant of pVA accumulation in crop systems such as maize and rice. Manipulation of this gene in rice has been used successfully to produce Golden Rice, which exhibits higher seed endosperm pVA levels than wild type plants. Therefore, it was hypothesised that differences between high and low pVA accumulating bananas could be due either to differences in PSY enzyme activity or factors regulating the expression of the psy gene. Therefore, the aim of this thesis was to investigate the role of PSY in accumulation of pVA in banana fruit of representative high (Asupina) and low (Cavendish) pVA banana cultivars by comparing the nucleic acid and encoded amino acid sequences of the banana psy genes, in vivo enzyme activity of PSY in rice callus and expression of PSY through analysis of promoter activity and mRNA levels. Initially, partial sequences of the psy coding region from five banana cultivars were obtained using reverse transcriptase (RT)-PCR with degenerate primers designed to conserved amino acids in the coding region of available psy sequences from other plants. Based on phylogenetic analysis and comparison to maize psy sequences, it was found that in banana, psy occurs as a gene family of at least three members (psy1, psy2a and psy2b). Subsequent analysis of the complete coding regions of these genes from Asupina and Cavendish suggested that they were all capable of producing functional proteins due to high conservation in the catalytic domain. However, inability to obtain the complete mRNA sequences of Cavendish psy2a, and isolation of two non-functional Cavendish psy2a coding region variants, suggested that psy2a expression may be impaired in Cavendish. Sequence analysis indicated that these Cavendish psy2a coding region variants may have resulted from alternate splicing. Evidence of alternate splicing was also observed in one Asupina psy1 coding region variant, which was predicted to produce a functional PSY1 isoform. The complete mRNA sequence of the psy2b coding regions could not be isolated from either cultivar. Interestingly, psy1 was cloned predominantly from leaf while psy2 was obtained preferentially from fruit, suggesting some level of tissue-specific expression. The Asupina and Cavendish psy1 and psy2a coding regions were subsequently expressed in rice callus and the activity of the enzymes compared in vivo through visual observation and quantitative measurement of carotenoid accumulation. The maize B73 psy1 coding region was included as a positive control. After several weeks on selection, regenerating calli showed a range of colours from white to dark orange representing various levels of carotenoid accumulation. These results confirmed that the banana psy coding regions were all capable of producing functional enzymes. No statistically significant differences in levels of activity were observed between banana PSYs, suggesting that differences in PSY activity were not responsible for differences in the fruit pVA content of Asupina and Cavendish. The psy1 and psy2a promoter sequences were isolated from Asupina and Cavendish gDNA using a PCR-based genome walking strategy. Interestingly, three Cavendish psy2a promoter clones of different sizes, representing possible allelic variants, were identified while only single promoter sequences were obtained for the other Asupina and Cavendish psy genes. Bioinformatic analysis of these sequences identified motifs that were previously characterised in the Arabidopsis psy promoter. Notably, an ATCTA motif associated with basal expression in Arabidopsis was identified in all promoters with the exception of two of the Cavendish psy2a promoter clones (Cpsy2apr2 and Cpsy2apr3). G1 and G2 motifs, linked to light-regulated responses in Arabidopsis, appeared to be differentially distributed between psy1 and psy2a promoters. In the untranscribed regulatory regions, the G1 motifs were found only in psy1 promoters, while the G2 motifs were found only in psy2a. Interestingly, both ATCTA and G2 motifs were identified in the 5’ UTRs of Asupina and Cavendish psy1. Consistent with other monocot promoters, introns were present in the Asupina and Cavendish psy1 5’ UTRs, while none were observed in the psy2a 5’ UTRs. Promoters were cloned into expression constructs, driving the â-glucuronidase (GUS) reporter gene. Transient expression of the Asupina and Cavendish psy1 and psy2a promoters in both Cavendish embryogenic cells and Cavendish fruit demonstrated that all promoters were active, except Cpsy2apr2 and Cpsy2apr3. The functional Cavendish psy2a promoter (Cpsy2apr1) appeared to have activity similar to the Asupina psy2a promoter. The activities of the Asupina and Cavendish psy1 promoters were similar to each other, and comparable to those of the functional psy2a promoters. Semi-quantitative PCR analysis of Asupina and Cavendish psy1 and psy2a transcripts showed that psy2a levels were high in green fruit and decreased during ripening, reinforcing the hypothesis that fruit pVA levels were largely dependent on levels of psy2a expression. Additionally, semi-quantitative PCR using intron-spanning primers indicated that high levels of unprocessed psy2a and psy2b mRNA were present in the ripe fruit of Cavendish but not in Asupina. This raised the possibility that differences in intron processing may influence pVA accumulation in Asupina and Cavendish. In this study the role of PSY in banana pVA accumulation was analysed at a number of different levels. Both mRNA accumulation and promoter activity of psy genes studied were very similar between Asupina and Cavendish. However, in several experiments there was evidence of cryptic or alternate splicing that differed in Cavendish compared to Asupina, although these differences were not conclusively linked to the differences in fruit pVA accumulation between Asupina and Cavendish. Therefore, other carotenoid biosynthetic genes or regulatory mechanisms may be involved in determining pVA levels in these cultivars. This study has contributed to an increased understanding of the role of PSY in the production of pVA carotenoids in banana fruit, corroborating the importance of this enzyme in regulating carotenoid production. Ultimately, this work may serve to inform future research into pVA accumulation in important crop varieties such as the EAHB and the discovery of avenues to improve such crops through genetic modification.

Molecular characterisation and carotenoid quantification of pro-vitamin A biofortified genetically modified bananas in Uganda

In Uganda, a significant proportion of the population depends on the micronutrient poor East African highland banana as a food staple. Consequently, micronutrient deficiencies such as vitamin A deficiency are an important health concern in the country. To reach most vulnerable rural poor populations, staple crops can be biofortified with essential micronutrients though conventional breeding or genetic engineering. This thesis provided proof of concept that genetically modified East African highland bananas with enhanced provitamin A levels can be generated and fully characterised in Uganda. In addition, provitamin A levels present in popular banana varieties was documented.

Generating transgenic banana (cv. Sukali Ndizi) resistant to Fusarium Wilt

Banana is one of the world’s most popular fruit crops and Sukali Ndizi is the most popular dessert banana in the East African region. Like other banana cultivars, Sukali Ndizi is threatened by several constraints, of which the Fusarium wilt disease is the most destructive. Fusarium wilt is caused by a soil-borne fungus, Fusarium oxysporum f.sp. cubense (Foc). No effective control strategy currently exists for this disease and although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by conventional breeding is difficult because of low fertility. Considering that conventional breeding generates hybrids with additional undesirable traits, transformation is the most suitable way of introducing resistance in the banana genome. The success of this strategy depends on the availability of genes for genetic transformation. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi, including Foc race 1 in banana cultivar Lady Finger. This thesis explores the potential of a plant-codon optimised nematode anti-apoptosis gene (Mced9) to provide resistance against Foc race 1 in dessert banana cultivar Sukali Ndizi. Agrobacterium-mediated transformation was used to transform embryogenic cell suspension of Sukali Ndizi with plant expression vector pYC11, harbouring maize ubiquitin promoter driven Mced9 gene and nptII as a plant selection marker. A total of 42 independently transformed lines were regenerated and characterized. The transgenic lines were multiplied, infected and evaluated for resistance to Foc race 1 in a small pot bioassay. The pathogenicity of the Ugandan Foc race 1 isolate used for infection was pre-determined and the spore concentration was standardised for consistent infection and symptom development. This process involved challenging tissue culture plants of Sukali Ndizi, a Foc race 1 susceptible cultivar and Nakinyika, an East African Highland cultivar known to be resistant to Foc race 1, with Fusarium inoculum and observing external and internal disease symptom development. Rhizome discolouration symptoms were the best indicators of Fusarium wilt with yellowing being an early sign of disease. Three transgenic lines were found to show significantly less disease severities compared to the wild-type control plants after 13 weeks of infection, indicating that Mced9 has the potential to provide tolerance to Fusarium wilt in Sukali Ndizi.

Health and prevention among East African women in the U.S.

African-born individuals in the U.S. face significant health challenges, including low utilization of preventive screening services. Using a community-based participatory research framework, we describe preliminary efforts at establishing a collaborative relationship with the East African communities of San Diego, identifying salient community health needs, and developing a framework for disseminating information and addressing identified health gaps. To this end, 40 East African-born women participated in focus groups with the purpose of eliciting community perspectives on U.S. health care services, beliefs about preventive screening, and to garner recommendations for future outreach. Qualitative analyses identified participants’ desire to engage in primary prevention techniques that incorporated best practices from their home countries and the U.S., and the need for health education programs to provide information on increasingly prevalent chronic diseases. The findings are discussed in connection with continued community-engaged efforts and the implications for health and resettlement policies to reduce inequities disfavoring resettled refugees.

Current kinematics and dynamics of Africa and the East African Rift System

Although the East African Rift System (EARS) is an archetype continental rift, the forces driving its evolution remain debated. Some contend buoyancy forces arising from gravitational potential energy (GPE) gradients within the lithosphere drive rifting. Others argue for a major role of the diverging mantle flow associated with the African Superplume. Here we quantify the forces driving present-day continental rifting in East Africa by (1) solving the depth averaged 3-D force balance equations for 3-D deviatoric stress associated with GPE, (2) inverting for a stress field boundary condition that we interpret as originating from large-scale mantle tractions, (3) calculating dynamic velocities due to lithospheric buoyancy forces, lateral viscosity variations, and velocity boundary conditions, and (4) calculating dynamic velocities that result from the stress response of horizontal mantle tractions acting on a viscous lithosphere in Africa and surroundings. We find deviatoric stress associated with lithospheric GPE gradients are similar to 8-20 MPa in EARS, and the minimum deviatoric stress resulting from basal shear is similar to 1.6 MPa along the EARS. Our dynamic velocity calculations confirm that a force contribution from GPE gradients alone is sufficient to drive Nubia-Somalia divergence and that additional forcing from horizontal mantle tractions overestimates surface kinematics. Stresses from GPE gradients appear sufficient to sustain present-day rifting in East Africa; however, they are lower than the vertically integrated strength of the lithosphere along most of the EARS. This indicates additional processes are required to initiate rupture of continental lithosphere, but once it is initiated, lithospheric buoyancy forces are enough to maintain rifting.

Microorganisms of the East African Great Lakes and their response to environmental changes

We have reviewed the phytoplankton composition and succession in the East African Great Lakes, their response to environmental changes, and the communities of microorganisms of the microbial food web. Recent studies in some great lakes, as well as progress in understanding phytoplankton succession and response to environmental factors, enable us to update knowledge of the phytoplankton ecology of these lakes. In particular, we present information indicating that phytoplankton composition in lakes Tanganyika and Kivu may reflect recent changes as a result of global warming or species introduction. We also stress the importance of microbes (at the base of the food web) in these systems and suggest that the microbial food web, which has been mostly overlooked until recently, may play a very large role in determining productivity and nutrient cycling in these large lakes.

A study on the trend of sports fishery of Mafia Island (Tanzania) along the East African coast from 1961-1972

Mafia waters in the western Indian Ocean on the east coast of Africa is a natural attractive area for fishing. It has extensive coral beds which harbour good fish life and attracts sport fishery in the area. About 12 commercially important fishes listed are caught by sports fishermen. The data indicates that this area can become an attractive centre for sports fishery almost throughout the year with peak season from November to February. Long-term planning of the fishery is necessary. The conservation measures should be evolved and gan fishing, dynamiting or any other kind of distructive fishing should be prohibited. This area has natural potential to become a sports fishing centre in the future and a great attraction for tourists and anglers.

East African Freshwater Fisheries Research Organization Annual Report 1967

Throughout the year the Fisheries Departments of Tanzania and Uganda continued to forward to EAFFRO data on the commercial fisheries of the lake. Regular records of commercial fishing activity In Kenya waters have not been received, appropriate information has been made available on request from the Chief Fisheries Officer. None of the research staff at EAFFRO have been assigned to a detail ed survey of the statistica1 data available, although severa1 research officers have taken the opportunity of analysing the data appropriate to their own research programmes. As recorded in the last Annual Report, an important feature of the UNDP Lake Victoria Fisheries Research Project will be to undertake the relevant statistical surveys essential to the proper.

East African Inland Fisheries Research Organization Annual Report for the year 1948

Introduction The laboratory at Jinja is being developed as a centre for hydro-biological research in East Africa, It has been built and is at present wholly financed from a grant from the Central Research allocation of the Colonial Development Fund. The building contains six laboratories in addition to a library, common room and general office. A maximum number of ten research workers could be given laboratory accommodation. However, shortage of living accommodation will limit the numbers who can work here for the next year or two.

East African Freshwater Fisheries Research Organization Director's report 1976

The Annual report covers Research work of the Organization carried out during the period 1975. Its explains the following research work: Haplochromis Studies, Electrophoresis,The Ecology of Haplochromis in the northern waters of Lake Victoria Limnology, Aquatic Pollution and Biochemical Studies The Biology of Synodontis in Lake Victoria Riverine Fisheries, Inshore Fishery of the Kenyan-Waters of Lake Victoria Biostatistics, the Inshore Fisheries of the Western part of Lake Victoria, The Offshore Fisheries of Kenyan Waters of Lake Victoria Lake Mobutu Sese Seko: Stock Assessment and Ecological Studies Fishery Economics, The Biology of Bagrus docmac in Lake Victoria Fishery Resource Surveys of Lake Wamala and Kijanebalola

A report on the fisheries of Uganda investigated by the Cambridge Expedition to the East African Lakes, 1930-31

There was very little previous information to use as a basis for work on Lakes Edward and George, but fortunately the region had been mapped in some detail by the Uganda-Congo Boundary Commission of 1906-08. This map served as a satisfactory foundation, but the western Congo shoreline of Lake Edward was inserted only by a dotted line, and a number of inaccuracies, particularly with regard to the islands and littoral of L. George, came to light during our survey.

Some remarks on the colour patterns of east African freshwater crabs

Very few accounts of African potamonids refer to natural colouration, and the author cannot recall any describing colour patterns. The generally undistinguished appearance of these crabs is well illustrated by Rathbun (1921) on the Congo Brachyura which includes collector's notes on a number of potamonid species. This article is based on remarks extracted from a communication sent to Dr. C. H. Fernando in connection with the article "Colour patterns in Ceylonese freshwater crabs (Patomonidae)", Bulletin of the Fisheries Research Station, Ceylon, v. 21.1(1970), pp. 1-4.

The history and research results of the East African Freshwater Fisheries Research Organization from 1946 - 1966.

Continuous research on the freshwater fisheries industry in East Africa, in common with most other African countries, did not commence until the end of the Second World War in 1945. Before the war some work on the fishes and hydrobiology of the lakes of East Africa had been undertaken by a series of scientific expeditions, which were sent out from Europe, between the years 1896 and 1939. These expeditions usually lasted for only a few months, and the collected data were published by young graduates and undergraduates, including such people as Mr. L. C. Beadle, (lately Professor Beadle of Makerere), Mr. V. E. Fuchs (now Sir Vivian Fuchs) and Mr. E. B. Worthington of Gonville and Caius College,Cambridge. Later. Dr. Worthington became Scientific Secretary to the Scientific Council for Africa, Scientific Secretary to the East African High Commission, and played a very large part in the furtherance of fisheries research and management in East Africa.

Annual Report of the East African Agricultural and Fisheries Research Council, 1961

In June, 1961, discussions were held in London to consider the future of the East Africa High Commission Services. It was agreed that the functions of the East Africa High Commission, with the exception of Defence, should be transfered to the East African Common Services Organization, which was subsequently established on 9th December, 1961, on the achievement of independence by Tanganyika. The Common Services Organization is controlled by an Authority, consisting of the principal elected Minister in each of the East African territories, which is responsible for the overall policy and direction of the Organization. The Authority is assisted by four Ministerial Committees, of which one is responsible for the Social and Research Services of the Organization