Genetic improvement of beta carotene in cassava (Manihot esculenta Crantz) landraces by Bright Boakye Peprah Submitted in accordance with the requirements for the degree Philosophiae Doctor in the Department of Plant Sciences (Plant Breeding) Faculty of Natural and Agricultural Sciences University of the Free State Bloemfontein, South Africa Promotor: Maryke T. Labuschagne (Prof) Co-promotors: Elizabeth Yaa Parkes (Dr) Angeline van Biljon (Dr) January 2020 DECLARATION I Bright Boakye Peprah declare that, the thesis hereby submitted by me, for the degree of Philosophiae Doctor in Plant Breeding at the University of the Free State, is my own independent work and has not previously, been submitted by me for a qualification at another institution of higher education. I furthermore, cede copyright of the thesis in favour of the University of the Free State. 04/01/2020 Bright Boakye Peprah Date i DEDICATION This study is dedicated to my adopted mother, Rev Dr Mrs Elizabeth Yaa Parkes, my wife, Mrs. Felicity Ababio (Adwoa kraa), my father Mr Yiadom Boakye Peprah (late), Lydia Annor (mother) and my wonderful children, Elizabeth Yaa Kyerewaah Peprah, Nana Kwame Boakye Peprah, Nana Afia Agyeiwaah Peprah and Nana Kofi Assim Berko and all my siblings who stood with me throughout the changing scenes of life and to see me arise and shine academically by the grace of God. ii ACKNOWLEDGEMENT This thesis could not have been produced without the help of Jehovah God Almighty, whom I serve, and many people and institutions that in diverse ways supported me prayerfully, technically, morally and financially. It may not be possible to list names of all individuals, institutions and organizations that have contributed or supported this study in diverse ways. I greatly appreciate and recognize every support or contribution given to this study. I wish to mention some of the contributors in the list below. WAAPP and BMGF for financial support. The Council for Scientific and Industrial Research (CSIR), the Crops Research Institute and the University of Ghana. The International Institutes, CIAT and International Institute of Tropical Agriculture (IITA) for supporting this work in diverse ways. I am so grateful to the University of the Free State (UFS) and in particular, the great people at Plant Breeding for creating an enabling environment for smooth academic work. Profound gratitude to Prof Maryke Labuschagne and family (I was blessed to have you as my supervisor). You have always been there for me and given excellent supervision with great patience and tolerance. Not forgotten are Dr Angeline van Biljon and Mrs. Sadie Geldenhuys for going every length to make things work out, you are awesome and my God will forever bless you. I also thank Rev Dr. Elizabeth Yaa Parkes and the family for taking me as a son, mum may Jehovah God bless you so much. Your encouragement and unique interest in keeping an eagle eye over me to see me through during my trying moments, was amazing. Drs. Peter Kulakow, Hernan Ceballos, Emmanuel Okogbenin, Robert Asiedu, Egesi CN, Robert Kawuki and William Esuma for your guidance and encouragements. I am grateful to Mr Lawrence Kent, Bill and Melinda Gates Foudation, and HarvestPlus for their financial support. Also, special thanks goes to all the great people including my course mates at the Plant Sciences Department at UFS who supported me in one way or the other with pieces of advice, technical and editorial assistance, e-mails and warm smiles to encourage me. iii I am also grateful to Prof Matilda Steiner Asiedu (UG), Dr Paul Ilona, Mr Peter Illeubey and Mr Afolabi (IITA - Ibadan). I thank Drs. John Asafu Agyei (blessed memory), Stella Ama Ennin, Hans Adu Dapaah (ex directors of the Crops Research Institute (CSIR-CRI), Dr. Mochiah (present director), Prof Emmanuel Otoo, Dr Ruth Prempeh, Mrs Benedicta Nsiah Frimpong, Mr Obed Harrison (UG), Elizabeth Afriyie Duah (UG), Dr Kwadwo Adofo, Mr Seth Frimpong (UG), Prof. Marian Quain, George Sefa Anane, Edem Lotsu (blessed memory), Ohene Gyan, Abigail Amoa- Owusu and the late Ampong Mensah for their immense support. The support and thanks to all staff at CSIR-CRI Fumesua (Root and Tuber division), Pokuase, Ejura and Ohawu station and MOFA staff at the various districts. I thank my wife and children, who supported me with their prayers and love; I also thank Pastor Collins for the prayers during my difficult times. Finally I thank the God of Israel, the Jehovah Nissi whom I served day and night ‘who promised to bring joy to my soul, Psalm 86:4’ for seeing me through to a successful end, may His name alone be forever praised. Amen. iv SUMMARY The aim of this study was to identify farmers’ adoption challenges, perceptions and preferences of yellow-flesh cassava. Combining ability and stability of these genotypes were also determined. Total carotenoid content (TCC), proximate values and hydrogen cyanide (HCN) of the yellow-flesh cassava were measured and the retention of carotenoids in boiled biofortified cassava was determined. This information will help breeders to identify genotypes with the best nutritional quality across the tested locations for planting and promotion in Ghana also could provide a basis for implementing a recurrent selection scheme for developing cassava varieties with high levels of carotenoids and dry matter. In all the locations visited, farmers’ knowledge on the improved cassava varieties (white flesh) and the yellow-flesh cassava were generally poor among the men and women interviewed, due to their inability to access planting materials, which could be improved by strengthening the cassava seed system for awareness, and increased availability of the varieties to farmers. Very few men and women cultivated improved varieties and yellow-flesh cassava. The young adults, who are the future of the agricultural sector, lacked access to improved varieties and they must be given extra attention to understand the activities of cassava breeding programmes, to empower them to make use of these materials. The general combining ability (GCA) was larger than specific combining ability (SCA) for cassava mosaic disease (CMD), harvest index (HI) and TCC, with predictability ratios (0.98, 0.88 and 0.92 respectively) close to one. Hence, there is a possibility for improvement of the characteristics by selection. Positive significant correlation between pulp colour and TCC (r=0.59) and pulp colour and cortex colour (r=0.58) were observed. Negative significant correlation were seen between CGM and HI (r=-0.50), CMD and RTN (r=-0.45), and HI and RTN (r=-0.51). It implies that these key traits could be effectively combined in a breeding program. In particular, breeders can rapidly screen for high TCC by visually assessing the pulp colour in addition selection for CMD symptoms (in a high disease pressure zone). The selected individuals for pulp colour at early stage screening can then be quantified for carotenoids at later stages, to save cost. Some of the yellow-fleshed genotypes (progenies) displayed comparable dry matter content (DMC) values as their white-flesh elite parents and were selected for multilocational trial testing towards commercial release in Ghana. Findings of this study demonstrated that it is possible to simultaneously select for yield and quality traits, such as DMC, at seedling stage. It was shown that the yellow flesh cassava varieties could be used in a hybridization scheme with local material to combine both TCC and DMC traits with high yield in a CMD resistance background. Carotenoid-rich varieties also v showed variation for important characteristics, which are key drivers of variety adoption in Ghana. In view of this, some cassava varieties, such as IBA090151 and IBA083774, are proposed for release in Ghana. The HCN content of the cultivars varied from location to location and the values observed were below 50 µg g-1 and hence can be classified as sweet cultivars (low HCN). The cultivars that were sweet were, however, above the range of the maximum acceptable HCN limit recommended by the World Health Organisation (WHO) and for that reason need to be processed before consumption (for example as fufu, konkonte, gari). Finally, it is recommended that cassava breeders review their breeding objectives to reflect the preferred traits of end-users, and pay attention to stakeholders’ perceptions of the yellow flesh cassava to develop demand driven varieties that will serve the need of end-users. Education to create awareness on the potential advantages and diverse uses of the improved biofortified cassava is also needed. Keywords: combining ability, cyanide, farmer-preferred traits, nutritional value, provitamin A, yellow flesh cassava vi TABLE OF CONTENTS Page DECLARATION i DEDICATION ii ACKNOWLEDGEMENTS iii SUMMARY v TABLE OF CONTENTS vii LIST OF TABLES xi LIST OF FIGURES xiii LIST OF ABBREVIATIONS xiv CHAPTER 1 GENERAL INTRODUCTION 1 References 5 CHAPTER 2 LITERATURE REVIEW 10 2.1 Importance of cassava 10 2.1.1 General importance 10 2.1.2 Importance of cassava in Ghana 12 2.1.3 Nutritional value of cassava 13 2.2 Yellow flesh cassava 14 2.3. Cassava biofortification 15 2.3.1 Importance of carotenoids and vitamin A 16 2.3.2 Dietary recommendations for vitamin A and carotenoids 18 2.3.3 Structure and genetics of beta carotene 18 2.3.4 Breeding for high beta carotene content 18 2.4 Growth and development of cassava 20 2.4.1 Dry matter partitioning and source–sink relationship 20 2.5 Variability in hydrocyanic acid content of cassava 22 2.5.1 Measures to control cyanide content in cassava 23 2.5.2 Effect of processing on the nutritional value of cassava 24 2.5.3 Impacts of processing on carotenoids 25 2.5.4 Bioavailability of carotenoids 26 2.6. Genotype by environment interaction 26 2.7 Heritability of characteristics in cassava 28 vii 2.8 Inheritance of nutritional traits 28 2.9 Mating designs and heterosis 29 2.9.1 2.9.2 Combining ability 30 2.9.2Diallel and North Carolina design II 2.9.3 Heterosis 31 2.10 Participatory plant breeding 32 References 33 CHAPTER 3 Awareness, perception and willingness to adopt yellow flesh cassava through participatory rural appraisal in coastal savannah and forest- 50 transition zones in Ghana Abstract 50 3.1 Introduction 50 3.2 Materials and methods 53 3.2.1 Brief description of area 53 3.2.2 Data collection procedures 54 3.2.3 Data analysis 55 3.3 Results 55 3.3.1 Characteristics of survey sample 55 3.3.2 Types of crops cultivated in study area 58 3.3.3 Importance of cassava 59 3.3.4 Access and control of productive resources 59 3.3.5 Knowledge of improved varieties and source of planting materials 60 3.3.6 Cassava varieties cultivated and preferred traits by participants by gender 61 3.3.7 Processors 62 3.3.8 Traits preferred by processors 62 3.3.9 Awareness, perceptions and willingness to adopt yellow fleshed 63 3.3.10 Factors affecting the adoption of new cassava varieties 65 3.4 Discussion 65 3.5 Conclusions and implications for cassava breeding 69 References 70 viii CHAPTER 4 Analysis of total carotenoid content, cassava mosaic disease, dry matter content, yield and its related components in F1 cassava families at two 76 locations in Ghana Abstract 76 4.1 Introduction 77 4.2 Materials and methods 77 4.2.1 Experimental site 77 4.2.2 Progeny development 78 4.2.3 Seedling nursery evaluation 79 4.2.4 Clonal evaluation trial 79 4.2.5 Statistical design and data analysis 80 4.3 Results 82 4.3.1 General combining ability 85 4.3.2 Specific combining abilities 86 4.3.3 Phenotypic correlation 87 4.3.4 Genetic parameters 87 4.4 Discussion 89 4.5 Conclusions 93 References 93 CHAPTER 5 Genetic variability, stability and heritability for quality and yield characteristics in provitamin A cassava varieties 98 Abstract 98 5.1 Introduction 98 5.2 Materials and methods 100 5.2.1 Varieties, experimental sites and design 100 5.3 Data collection 101 5.4 Data analysis 104 5.5 Results 104 5.5.1 Analysis of variance 104 5.5.2 Additive main effects and multiplicative interaction analysis 108 5.5.3 Correlations, genetic components and principle component analysis 108 5.5.4 GGE biplot for average dry matter content, fresh root weight, starch and stability of varieties 111 ix 5.5.5 The best performing genotype in each environment and mega- environments for dry matter content, fresh root weight and starch content 111 5.6 Discussion 114 5.7 Conclusions 115 References 116 CHAPTER 6 Proximate composition and cyanide content, and total carotenoid retention after boiling of yellow-fleshed cassava cultivars 119 Abstract 119 6.1 Introduction 119 6.2 Materials and methods 123 6.2.1 Varieties, field trials and sample preparation 123 6.2.2 Proximate analysis 124 6.3 Data analysis 128 6.4 Results 128 6.5 Discussion 135 6.6 Conclusions 138 References 139 CHAPTER 7 General conclusions and recommendations 145 Appendix 148 x LIST OF TABLES Page 2.1 World cassava production (million metric ton) between 2015 and 2018 10 2.2 Effect of different processing methods on the cyanide content of cassava 24 3.1 Characteristics of survey sample (qualitative statistics) 56 3.2 Summary statistics for producers (qualitative variables) 57 3.3 Summary statistics for processors (qualitative variables) 57 3.4a Gender groups’ perception on yellow-fleshed cassava 64 3.4b Gender groups’ expectations of the improved yellow-fleshed cassava 64 4.1 List of progenitors used in the study 78 4.2 Mean performance of progenitors and their F1 progenies evaluated across two locations in Ghana 84 4.3 Mean squares of crosses and sum of squares for combining ability effects of seven traits evaluated in 10 F1 families and seven parents across two 86 locations 4.4 General combining ability effects of cassava progenitors for seven traits at two locations in Ghana 87 4.5 Specific combining ability effects of parents for seven traits evaluated across two locations in Ghana 87 4.6 Phenotypic correlation of measured cassava characteristics evaluated across two locations 88 4.7 Genetic parameters for various traits studied across two locations in 88 Ghana 5.1 Provitamin A and white flesh cassava genotypes used for the study 101 5.2 Characteristics of the six trial environments 102 5.3 Analysis of variance and contribution of main effects to variation for measured characteristics across three environments in two growing 106 seasons 5.4 Means of five traits measured in two growing seasons (2015/2016 and 2016/2017) in 10 genotypes across six environments in Ghana 107 5.5 Mean values of nine traits measured in 10 genotypes across six environments in Ghana 108 5.6 Additive main effects and multiplicative interaction analysis of variance for measured characteristics 108 5.7 Phenotypic correlations coefficients for 10 traits measured on 10 cassava 109 genotypes across six environments in Ghana xi 5.8 Coefficients of variation, heritability and genetic advance for five traits of 10 cassava genotypes planted in six environments 109 5.9 Principal component analysis of 10 quantitative traits in 10 cassava genotypes showing eigenvectors, eigenvalues, individual and cumulative 110 percentage of variation explained by the first three principal component axis 6.1 Provitamin A and white cassava genotypes used for the study 124 6.2 Percentage moisture and carbohydrate content of fresh cassava varieties from three different locations 129 6.3 Protein and fat content of cassava varieties from three different locations 130 6.4 Crude fiber and ash content of fresh cassava varieties across three different locations 131 6.5 Comparison of hydrogen cyanide content of cassava genotypes from 134 different locations 6.6 Total carotenoid content of fresh and boiled cassava genotypes across 135 three different locations xii LIST OF FIGURES Page 5.1 Genotype by GE biplot showing (A) dry matter content and (B) fresh 112 root weight mean performance and stability of 10 cassava genotypes 5.2 Which wins where GGE biplot for best cultivars for (A) dry matter 113 content (B) fresh root weight in different environments 6.1 Hydrogen cyanide content of yellow flesh cassava from Cape-Coast 132 6.2 Hydrogen cyanide content of yellow flesh cassava from Ohawu 133 6.3 Hydrogen cyanide content of yellow flesh cassava from Fumesua 133 xiii LIST OF ABBREVIATIONS AGDP Agricultural gross domestic product AMMI Additive main effects and multiplicative interaction ANOVA Analysis of variance AOAC Association of official analytical chemists CBB Cassava bacterial blight CGM Cassava green mite CIAT International Center for Tropical Agriculture CMB Cassava mealy bug CMD Cassava mosaic disease CNG Cyanoglucoside CNP Cyanogenic potential CRI Crops Research Institute CSIR Council for Scientific and Industrial Research CSIR-CRI Council for Scientific and Industrial Research-Crops Research Institute CV Coefficient of variation DRC Democratic Republic of Congo DM Dry matter DMC Dry matter content EAR Estimated average requirement FAO Food and Agriculture Organization Fe Iron FRW Fresh root weight FSW Fresh shoot weight GCA General combining ability GCV Genotypic coefficient of variation GDHS Ghana Demographic Health Service GDP Gross domestic product GxE Genotype by environment GEI Genotype by environment interaction GGE Genotype and genotype by environment interaction GLSS Ghana Living Standards Survey GSS Ghana Statistical Service xiv IPAC Interaction principal component axis HCl Hydrogen chloride HI Harvest index HCN Hydrogen cyanide IFAD International Fund for Agricultural Development IITA International Institute of Tropical Agriculture LSD Least significant difference MAP Months after planting mg Milligram MOFA Ministry of Food and Agriculture NaOH Sodium hydroxide NCD II North Carolina design II NIRS Near infrared spectrophotometer PCA Principal component analysis PCV Phenotypic coefficient of variation PPB Participatory plant breeding PPD Post physiological deterioration PPMED Policy, Planning, Monitoring and Evaluation Division PRA Participatory rural appraisal pVA Provitamin A PVAC Provitamin A content RDA Recommended daily allowance RAE Retinol activity equivalents RCBD Randomized complete block design RTN Storage root number RTW Storage root weight SCA Specific combing ability TBC Total beta carotene TCC Total carotenoid content t ha-1 Ton per hectare TWT Total biomass USAID United State Agency for International Development UV Ultra violet VA Vitamin A xv VAD Vitamin A deficiency WAAPP West Africa Agricultural Productivity Programme WHO World Health Organization Zn Zinc xvi CHAPTER 1 GENERAL INTRODUCTION Cassava, the fifth most important staple crop in the world, is a widely grown and consumed root crop in Sub-Saharan Africa (Tan, 2015). It is also regarded as the most widely cultivated root crop in the tropical region and a crop that persistently contribute to food security mainly because of its ability to store its matured edible roots in the ground for about three years and unarguably the sixth most important crop (following crops like wheat, rice, maize, potato, and barley) in the world (Saranraj et al. 2019). Cassava is consumed in various forms such as boiled roots, fufu, and gari. Gari is very popular with urban dwellers as it is easy to prepare and can be stored for extended periods. In Nigeria, which is a major producer of cassava, it is also used for many industrial applications such as starch, glucose and ethanol production. In other countries of sub- Saharan Africa, including Ghana, the situation is similar, as 30 to 80% of the region’s inhabitants consume cassava (Otekunrin and Sawicka 2019). The world cassava production stands at 291 million tonnes in 2017 with leading countries like Nigeria (59 million), Congo DR (31 million), Thailand (30 million), Indonesia (19 million, Brazil (18.9 million), Ghana (18.4 million) ranked 1st, 2nd, 3rd, 4th, 5th and 6th respectively with production in the Africa (177 million in 2017) regarded as the world largest cassava growing region. (FAOSTAT 2019). The main nutritional component of cassava is carbohydrate, which derives from starch accumulated in the tuberous storage roots. On the other hand, the shoots and leaves of cassva are highly nutritious and are consumed as vegetables in many parts of Africa. It has high levels of protein (7 g per 100 g fresh material) and has high concentrations of lysine, minerals and vitamins (Hahn 1989; IITA 1990; Nweke et al. 1994; Fregene et al. 2000; Benesi 2005). The woody cassava stem cuttings are used commercially as planting materials (Ekanayake et al. 1997; Alves 2002). Cassava is adapted to a wide range of environments. It has good drought and acid soil tolerance, with good performance on degraded soils where other crops often fail (Jones 1959; Kawano et al. 1978; Jaramillo et al. 2005) and also resilience to climatic shocks (Jarvis et al. 2012). Cassava is an excellent alternative for maize for industrial processes in the tropics (Jaramillo et al. 2005). Cassava has a high yield potential and is better suited than cereals for production in areas where population pressure and crop failure are a challenge (Nweke 1996; Benesi 2005). A big advantage of cassava is the fact that it can be stored in the ground and harvested when needed, which contributes to food security (DeVries and Toenniessen 1 2001) and famine alleviation (Nweke et al. 2002). Cassava is a major staple crop, which contributes 22% to agricultural gross domestic product (GDP) (Policy, Planning, Monitoring and Evaluation Division (PPMED) 1991 Ministry of Food and Agriculture (MOFA) 2012) in comparison to the GCP contribution by other crops/products such as maize (5%), rice (2%), sorghum (14%) and millet (14%), cocoa (11%), forestry (7%), fisheries and livestock (5%) (Al-Hassan 1989; Dapaah 1996). It is grown in all 10 regions of Ghana (Okai 2001) and occupies over 90% of the country’s farming area (MOFA 2012). A study by Al-Hassan and Diao (2007) showed the potential of cassava to reduce poverty and promote economic growth in northern Ghana, which is among the poorest areas in the country. Cassava has been identified as an importnat commodity that can generate economic growth and fight poverty in a number of reports relating to Ghana’s economic growth and development (Dapaah 1991; 1996; Al-Hassan 1993; Nweke 2004). A limiting characteristic of cassava roots for human or animal consumption is their cyanogenic glucosides content (Kakes 1990). Traditional processing methods of grating, fermenting, boiling and/or drying removes most of the cyanide. Cassava roots are a good source of energy while the leaves provide protein, vitamins, and minerals. However, cassava roots and leaves are deficient in sulfur‐containing amino acids (methionine and cysteine) and some nutrients are not optimally distributed within the plant (Montagnac et al. 2009a). An additional constraint is the negligible amount of provitamin A content (PVAC) found in the white flesh varieties of cassava cultivated in Ghana. Beta carotene and other carotenoids are a dietary precursor of vitamin A, and are responsible for the yellow to orange flesh colour of storage roots (Degras 2003; Rodriguez-Amaya and Kimura 2004). Vitamin A (VA) is essential for good vision, and contributes to an effective immunity system, and is also involved in cellular differentiation, growth and reproduction. VA deficiency (VAD) is a widespread public health problem in 37 countries worldwide and affects a large percentage of people in areas where cassava is a staple crop, such as in sub-Saharan Africa, northeast Brazil, and Southeast Asia (Njoku et al. 2011). The 2014 Ghana Demographic and Health Survey (GDHS), which was carried out by the Ghana Statistical Service (GSS) and the Ghana Health Service, revealed that Ghana is characterised by rampant malnutrition and high incidence of nutrient deficiency-related diseases (GSS et al. 2015). They reported that more than three-quarters of children age 6- 59 months are anaemic. Anaemia rates were found to be higher in rural areas where cassava is the main source of livelihood compared to urban areas (72% vs. 58%). Anaemia was also higher in the northern, 2 Upper West and Ashanti regions. There is emerging evidence that improving the VA status of people has a synergistic effect on iron (Fe) and zinc (Zn) status. In Ghana, the group most at risk of VAD are pre-school children living in the northern part of the country and women in their childbearing years. In the remote areas of Ghana, where poverty is the most severe, VAD is an endemic problem. Nearly one million children in Ghana do not receive nutritional supplements. Beta carotene is the most abundant carotenoid in cassava and can be efficiently converted to VA. Beta carotene and vitamin E, ascorbic acid, enzymes and proteins make up the biological antioxidant network, which converts highly reactive radicals (•OH) and free fatty peroxy radicals to less active species. In this way they protect the body against oxidative cell damage (Packer 1992). In human nutritional studies, VA activity is expressed as retinol equivalent and 3.7 mg of cassava beta carotene has the biological (VA) activity of 1 mg retinol. According to Maziya-Dixon (2010) this refutes the previous estimate of about 12 mg of beta carotene in cassava being equivalent to 1 mg of retinol. The average daily requirements of beta carotene equivalent for children is 2.4 mg, for adults it is 3.5 mg while for lactating mothers it is 5.0 mg. (WHO 1995; Ukpabi and Ekeledo 2009). These dietary requirements are not adequately supplied by diets, especially in children, pregnant women, and the poor in several countries, including Ghana. Dietary diversification, food fortification and/or supplementation are the three strategies that have been used most frequently to prevent VAD. For a variety of reasons these strategies have not been effective to eradicate VAD (West 2003). Harvestplus, which involves a global alliance of research institutions, has initiated the development of micronutrient dense staple crops, also called biofortification, as a fourth strategy to eradicate VAD, with one of the initiatives being specifically the development of biofortified cassava clones with high PVAC in the roots (Dwivedi et al. 2012). Conventional breeding techniques can be applied for biofortification, by taking advantage of the genetic variability for micronutrients in different crops (Chavez et al. 2005), but genetic transformation is also an option (Welsch et al. 2010). The underlying factor to micronutrient problem is the consumption of deficient diet by people, and these techniques can be used to address this problem (Ceballos et al. 2013). Fortunately, the conversion of PVAC present in cassava roots into VA in humans has proven to be highly efficient (Thakkar et al. 2009). In a VA cassava biofortification breeding programme, the acceptability of its product by farmers and consumers, as well as the bioavailability of the beta carotene in the product should be considered (Njoku 2012). 3 In the southern part of Ghana, the adoption rates and adoption intensity of the improved cassava varieties in 2007 were 9 and 37% respectively (Dankyi and Adjekum 2007). This is because during the early stages of the breeding process, farmers and consumers were not included in the process (Nweke et al. 1994; Benesi 2005; Manu-Aduening et al. 2014). In 2017 1176, cassava farmers were interviewed and 80% were aware of the improved cassava varieties. Eighty seven percent (87%), 90%, 82% and 62% of farmers from the forest zone, Transition, coastal savannah and Guinea savannah respectively were aware of improved varieties. Forty one percent (41%) of cassava area was planted to improved cassava variety during the 2014/2015 major season (Acheampong et al. 2017) Plant breeding has shifted towards client-oriented participatory breeding. The principle is that farmers and scientists have equal inputs in the selection process, in a long-term collaborative process that leads to better products. Client-oriented participatory breeding improves breeding efficiency, accelerates adoption, leads to more acceptable varieties, promotes genetic diversity and saves cost through reduction of the breeding cycle (Morris and Bellon 2004; Witcombe et al. 2005; Mangione et al. 2006; Gyawali et al. 2007; Manu- Aduening et al. 2014). Therefore, to increase the acceptability and adoption rate for biofortified yellow-flesh cassava cultivars, farmers and consumers would of necessity have to be integrated at the early stages of the research and in the selection of varieties through participatory methods. The objectives of this study were to: 1. Identify farmers’ adoption challenges, perceptions and preferences for yellow flesh cassava through participatory rural appraisal. 2. Determine the combining ability for beta carotene, dry matter content (DMC), cassava mosaic disease (CMD), yield and its related components in some F1 cassava families. 3. Determine genetic variability, stability and heritability for quality and yield characteristics in provitamin A (pVA) cassava varieties. 4. Determine the total carotenoid content (TCC) and HCN in yellow flesh cassava cultivars and also to measure the retention of carotenoids during the processing of biofortified cassava into boiled cassava. 4 References Acheampong PP, Nimo- Wiredu A, Amengor NE, Nsiah- Frimpong B, Haleegoah J, Adu- Appiah A, Adogoba D (2017) Root and Tuber crops Technologies Adoption and Impact study in Ghana: The case of improved cassava Technologies. Report submitted to the West Africa Agriculture Productivity Programme (WAAPP- Ghana) Al-Hassan MR (1993) Cassava in the economy of Ghana in product development for root and tuber crops. Volume III – Africa and Latin America. Breeding projects work to stabilize productivity without increasing pressures on limited natural resources. BioSciences 43: 441-452 Al-Hassan R (1989) Cassava in the economy of Ghana. In: Nweke FI, Lynam J, Prudecio OY (eds.) Status of cassava research in Africa, COSCA working paper No.3. International Institute of Tropical Agriculture, Ibadan, Nigeria Al-Hassan RM, Diao X (2007) Regional disparities in Ghana: Policy options and public investment implications, IFPRI Discussion Papers 693, International Food Policy Research Institute (IFPRI) Alves AAA (2002) Cassava botany and physiology. In: Hillocks RJ, Thresh MJ, Bellotti AC (eds.) Cassava: Biology, production and utilisation. CABI International, Oxford. pp. 67-89 Benesi IRM (2005) Characterisation of Malawian cassava germplasm for diversity, starch extraction and its native and modified properties. PhD Thesis, Department of Plant Sciences (Plant Breeding), University of the Free State, Bloemfontein, South Africa Ceballos H, Morante N, Sanchez T, Ortiz D, Aragon I, Chavez AL, Pizarro M, Calle F, Dufour D (2013) Rapid cycling recurrent selection for increased carotenoids content in cassava roots. Crop Science 53: 2342-2351 Chávez AL, Sánchez T, Jaramillo G, Bedoya JMI, Echeverry J, Bolaños EA, Ceballos H, Iglesias CA (2005) Variation of quality traits in cassava roots evaluated in landraces and improved clones. Euphytica 143: 125-133 Cock JH (1985) Cassava: New potential for a neglected crop. Westview Press. Boulder, Colorado 5 Dapaah SK (1991) Contributions of root and tuber crops to socio-economic changes in the developing world: the case of Africa, with special emphasis on Ghana. In: Ofori F, Hahn SK (eds.) Tropical root crops in a developing economy. Proceedings of the ninth symposium of the international society for tropical root crops, 20-26 October, Accra, Ghana, pp. 21-24 Dapaah SK (1996) The way forward for accelerated agricultural growth and development. A paper presented to the government of Ghana on behalf of the Ministry of Food and Agriculture, Accra, Ghana Degras L (2003) Sweet potato. Macmillan, Oxford. England Denkyi AA, Adjekum AA (2007) determinats of the adoption of improved cassava varieties in Southern Ghana. Logistic regression analysis. Proceedings of the 13th International Society (ISTRC), Ghana West Africa 641-647 DeVries J, Toenniessen G (2001) Securing the harvest: biotechnology, breeding and seed systems for African crops. CABI publishing, Oxon, UK Dwivedi SL, Sahrawat KL, Rai KN, Blair MW, Andersson MS, Pfeiffer W (2012) Nutritionally enhanced staple food crops. Plant Breeding Reviews 36: 173-293 Ekanayake IJ, Osiru DS, Porto MCM (1997) Morphology of cassava. IITA Research Guide. Ibadan, Nigeria Food and Agriculture Oganization of the United Nation (FAO). FAOSTAT statistical database, statistical division Rome (2017) Fregene M, Bernal A, Duque M, Dixon AGO, Tohme J (2000) AFLP analysis of African cassava (Manihot esculenta Crantz) germplasm resistant to the cassava mosaic disease (CMD). Theoretical and Applied Genetics 100: 678-685 Ghana Statistical Service (GSS), Ghana Health Service (GHS), and ICF International. (2015) 2014 Ghana Demographic and Health Survey (DHS) Key Findings. Rockville, Maryland, USA: GSS, GHS, and ICF International. Gyawali S, Sunwar S, Subedi M, Tripathi M, Joshi KD, Witcombe JR (2007) Collaborative breeding with farmers can be effective. Field Crop Research 101: 88-95 Hahn SK (1989) An overview of African traditional cassava processing and utilization. Outlook in Agriculture 81: 110-118 IITA (International Institute of Tropical Agriculture) (1990) Cassava in Tropical Africa: A reference manual. Chayce Publications Services, Balding Mansell International, Wisbech, UK 6 Jaramillo G, Morante N, Pérez JC, Calle F, Ceballos H, Arias B, Bellotti AC (2005) Diallel analysis in cassava adapted to the midaltitude valleys environment. Crop Science 45: 1058-1063 Jarvis A, Ramirez-Villegas J, Campo BVH, Navarro-Racines C (2012) Is cassava the answer to Africa climate change adaptation. Tropical Plant Biology 5:9- 29 Jones WO (1959) Manioc in Africa. Stanford University Press, Stanford Kakes P (1990) Properties and functions of the cyanogenic system in higher plants. Euphytica 48: 25-43 Kawano K, Amanya A, Daza P, Rios M (1978) Factors affecting efficiency of selection in cassava. Crop Science 18: 373-376 Kawano K, Narintaraporn K, Narintaraporn P, Sarakarn S, Limsila A, Limsila J, Suparhan D, Sarawat V, Watananonta W (1998) Yield improvement in a multistage breeding program for cassava. Crop Science 38: 325-332 Mangione D, Senni S, Puccioni M, Grando S, Ceccarelli S (2006) The cost of participatory barley breeding. Euphytica 150: 289-306 Manu-Aduening JA, Peprah BB, Bolfrey-Arku G, Aubyn A (2014) Promoting farmer participation in client-oriented breeding: lessons from participatory breeding for farmer-preferred cassava varieties in Ghana. Advanced Journal of Agricultural Research 2: 8-17 Maziya-Dixon BB (2010) Beta carotene in cassava is easily converted to retinol. IITA Bulletin Ministry of Food and Agriculture (MOFA) (2012) Agriculture in Ghana. Facts and Figures, Statistics, Research and Information Directorate (SRID), Accra, Ghana Montagnac JA, Davis CR, Tanumihardjo SA (2009a) Nutritional value of cassava for use as a staple food and recent advances for improvement. Comprehensive Reviews in Food Science and Food Safety 8: 181-194 Morris ML, Bellon MR (2004) Participatory plant breeding research: opportunities and challenges for the international crop improvement system. Euphytica 136: 21-35 Njoku DN (2012) Improving beta-carotene content in farmers’ preferred cassava cultivars in Nigeria. Doctoral thesis, West Africa Centre for Crop Improvement, University of Ghana, Legon, Ghana Njoku DN, Vernon G, Egesi CN, Asante IK, Offei SK, Okogbenin E, Kulakow P, Eke- Okoro ON, Ceballos H (2011) Breeding for enhanced β-carotene content in cassava: constraints and accomplishments. Journal of Crop Improvement 25: 560- 571 7 Nweke FI (1996) Cassava: A cash crop in Africa. Collaborative study of cassava in Africa Working Paper No. 14, International Institute of Tropical Agriculture, Ibadan, Nigeria Nweke FI (2004) New challenges in the cassava transformation in Nigeria and Ghana. Discussion Paper No. 118, Environment and Production Technology Division, International Food Policy Research Institute, Washington, DC, USA. http://www.ifpri.org/divs/eptd/dp/papers/eptdp118.pdf (Accessed 19 January 2015) Nweke FI, Hahn SK, Ugwu BO (1994) Circumstances of rapid spread of cultivation of improved cassava varieties in Nigeria. Journal of Farming Systems Research and Extension 4: 93-120 Nweke FI, Spencer DSC, Lynam JK (2002) The cassava transformation: Africa’s best kept secret. Michigan State University Press, Lansing, Michigan, USA Okai E (2001) Genetic diversity in some local cassava cultvars in Ghana. MPhil Thesis. Crop Science Department, Faculty of Agriculture, University of Ghana, Legon, Ghana Otekunrin OA, Sawicka B (2019) Cassava, a 21st century staple crop: How can Nigeria harness its enorm/ous trade and potentials: ACTA Scientific Agriculture 3:195-202 Packer L (1992) Interactions among antioxidants in health and disease. Vitamin E and the redox cycle. Proceedings of the Society for Experimental Biology and Medicine 200: 271-276 PPMED (1991) Agriculture in Ghana, Facts and Figures. Ministry of Food and Agriculture, Accra, Ghana Rodriguez-Amaya DB, Kimura M (2004) HarvestPlus handbook for carotenoid analysis. HarvestPlus Technical Monograph Series 2. IFPRI, Washington, D.C. and CIAT, Cali Saranraj P, Behera SS, Ray RC (2019) Traditional foods from Tropical Root and Tuber crops: Innovations and challenges. In: Galanakis CM (ed.), Innovations in Traditional foods. Elsevier. pp. 159-191 Tan SL (2015) Cassava- silently, the tuber fills: the lowly cassava, regarded as a poor man’s crop, may help save the world from the curse of plastic pollution. UTAR Agriculture Science Journal, 1:12-24 Thakkar SK, Huo T, Maziya-Dixon B, Failla ML (2009) Impact of style of processing on retention and bioaccessibility of β-carotene in cassava (Manihot escultenta Crantz). Journal of Agricultural and Food Chemistry 57:1344-1348 8 Ukpabi UJ, Ekeledo EN (2009) Feasibility of using orange- f l e s h e d sweet potato as an alternative to carrot in Nigerian salad preparations. Agricultural Journal 4: 216- 220 Welsch R, Arango J, Bär C, Salazar B, Al-Babili S, Beltrán J, Chavarriga P, Ceballos H, Tohme J, Beyer P (2010) Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene. Plant Cell 22: 3348-3356 West KP Jr. (2003) Vitamin A deficiency disorders in children and women. Food and Nutrition Bulletin 24S: 78-90 WHO (1995) Global prevelence of Vitamin A defiency. Micronutrient deficiency information system. MDIS working paper No. 2 Witcombe JR, Joshi KD, Gyawali S, Musa AM, Johansen C, Virk DS, Sthapit BR (2005) Participatory plant breeding is better described as highly client-oriented plant breeding. 1. Four indicators of client-orientation in plant breeding. Experimental Agriculture 41: 299-319 9 CHAPTER 2 LITERATURE REVIEW 2.1 Importance of cassava 2.1.1 General importance Cassava (Manihot esculenta Crantz) is regarded as the most widely cultivated root crop in the tropical region (Saranraj et al. 2019) and it originated from several centres beginning from the southern edge of the Brazilian Amazon (FAO 2013). It serves as a food for over 900 million people in the tropics and sub-tropics (FAO 1996; Nassar 2005) and also as a source of calories in the human diet with 500 calories per day for more than 500 million people in sub-Saharan Africa, Asia and Latin America (Onwueme 2002). Among all staple crops in sub-Saharan Africa, cassava has been a major staple as it is grown mainly for its storage roots, being the economic part of the crop. As a 'crisis crop', it can be left in the ground for a period of time until shortages arise. Global cassava production in 2012 was 269.1 million ton and 149.4 million tons for Africa (Table 2.1). Table 2.1 World cassava production (million metric tons) between 2015 and 2018 2005 2016 2017 2018 World 293.0 288.5 279.3 277.8 Africa 172.7 172.8 168.3 169.7 West Africa 91.4 89.5 91.5 93.0 Nigeria 57.6 59.6 59.4 59.5 Ghana 17.7 17.8 19.0 20.8 FAOSTAT (2019) Cassava is ranked as the fifth most important staple crop in the world (Tan 2015) and one of the non-native crop in Africa that has achieved staple food status (Tewe 1992). Cassava roots are very rich in carbohydrate, which makes them an important source of dietry energy (FAO 2013). In 2017, the largest producing countries were Nigeria, Congo DR, Thailand, Indonesia, Brazil, Ghana, Vietnam, Cambodia, Angola, Mozambique, Cameroon, Malawi and China, with Africa producing more than half of the world’s total production (FAOSTAT 2017). Low yields have been due to production constraints and abiotic factors (Nweke 1996). Cassava is 10 tolerant to drought and acidic soils, with reasonable yield on degraded soils where other crops fail (Jaramillo et al. 2005) and is hence a very good crop for Africa (Nweke et al., 2002). Cassava can serve as an alternative to maize for industrial purposes in the tropics (Jaramillo et al. 2005) and is even more suitable than other grain staples in areas where population pressure and crop failure are a challenge (Al-Hassan 1993; Nweke 1996; Benesi 2005). Cassava roots can be stored in the soil and harvested when needed. This makes it a food security (DeVries and Toenniessen 2001) and famine reserve crop (Nweke et al. 2002), but also a good industrial crop (Dixon and Ssemakula 2008). Cassava production stretches through a wide belt from Madagascar in the southeast to Senegal and Cape Verde in the northwest (Raji et al. 2001; Benesi 2005). An increase in cassava production in Africa has been reported due to research and better use of agronomic practices, especially in Ghana and Nigeria, and rapid population growth forcing consumers to look for cheaper sources of calories (IFAD and FAO 2005). Cassava leaves and shoots are also used as vegetables in other parts of Africa because of its nutritional value for humans and animals (Ceballos et al. 2004) but have no market value in Ghana, since it is not consumed as a vegetable (Angelucci 2013). The seeds are used as medicine and in animal feed formulations (Fregene et al. 2000; Benesi 2005). The woody stems serve as cuttings for planting (Ekanayake et al. 1997; Alves 2002) and are sometimes sold to generate an income (Alves 2002; Popoola and Yangomodou 2006). Root crop production, especially cassava, can spur rural industrial development and raise incomes for producers, processors and traders. It will contribute to the food security status of its producing and consuming households (FAO and IFAD 2001).Cassava markets are being expanded in countries like Nigeria and Ghana for its products like; starch and its derivatives, ethanol, glucose syrup, composite flour and gari (Nweke et al. 2002; Nweke 2004). This has led to the growing demand for cassava, with cassava increasingly cultivated in large acreages of commercial farms and by farmers’ cooperatives (Nweke et al. 2002; Nweke 2004; Manu-Aduening et al. 2006). There are also excellent opportunities for product and market diversification in several other African countries (Benesi 2005; Al- Hassan and Diao 2007; Dixon and Ssemakula 2008). 11 2.1.2 Importance of cassava in Ghana Cassava adoption was very slow in Ghana in early 1980s after its introduction, due to the fact that most of the people in the forest belt preferred plantain, cocoyam whiles in the northern part people preferred sorghum, Maize and millet (Parkes 2011). Its cultivation and utilisation became important following the major crop failure in 1983, with cassava as the key exception to the catastrophe (Manu-Aduening et al. 2005). Currently, Ghana is the sixth largest producer of cassava in the world (FAOSTAT 2017), and cassava ranks first among the root and tuber crops in Ghana (IFAD and FAO 2005). This root crop is the main source of carbohydrates to meet the dietary requirements needed by people and is a regular source of income for most rural dwellers. Cassava is not only a food security crop but also an important industrial crop for the provision of cash and jobs for rural and urban communities (Nweke et al. 2002; Dixon and Ssemakula 2008). There is a growing importance of cassava in Ghana (Dapaah 1991; Al- Hassan et al. 1993; Manu-Aduening et al. 2006) and this has necessitated the development of cassava-based industries in the country. Cassava has tremendous potential in Ghana and Africa’s economy for food, feed and industrial uses, and provides cash and jobs for the rural communities (Nweke 2004; Dixon and Ssemakula 2008). Cassava roots can be consumed in a variety of forms (Amenorpe et al. 2006; Baafi and Sarfo-Kantanka 2008). The crop is used as starch and its derivatives, glucose syrup, flour and gari, for ethanol production and as animal feed (Nassar 2006). The cassava industry creates jobs for large numbers of people, mostly women, in sub-Saharan Africa (Haleegoah and Okai 1992; Thro et al. 1995). Fufu powder is produced from cassava, and in Ghana the true annual potential demand for this product is probably in the order of 1 000 to 17 100 metric ton. Even the lower limit would represent a substantial new opportunity for Ghanaian food manufacturers, albeit one that would not be easy to exploit. The estimated annual demand for fresh cassava roots translates to 2 000 to 34 200 metric ton, all of which could be supplied by Ghanaian farmers (Collinson et al. 2001). The agriculture-led economic growth has proven to reduce poverty more than non-agriculture-led growth (Al-Hassan and Diao 2007). 12 2.1.3 Nutritional value of cassava Cassava roots are mostly used as starchy product in the world and the fresh foliage is used in several countries as feed for animals and vegetables for human consumption (Cock 1985; Kawano et al. 1998). Among the starchy staples, cassava provides approximately 40% more of the carbohydrate consumed than rice and 25% more than maize (Nyerhovwo 2004). The root serves as a significant and cheap source of calories for both human and animal nutrition. Depending on the terrain, type, age of plant and climatic conditions, cassava has most of its nutrients in the roots and leaves, which are the edible parts of the plant. According to El- Sharkawy et al. (2012), cassava storage roots are predominantly used as a source of carbohydrate but less for protein, fat, minerals and vitamins. Consequently, cassava is of lower nutritional value than all cereals, legumes, and even some other root and tuber crops, such as yams. There are two types of cassava varieties; sweet varieties (having low HCN), which requires a low amount of processing, whilst bitter varieties require more processing because of its high total cyanide content or cyanogenic potential (CNP). The higher the CNP of a variety, the greater the need to process the root before consumption (Kakes 1990). Two types cyanogenic glycosides (linamarin and lotuaustralin) are synthesised in the leaves of the cassava plant. Cassava roots have a low level of protein, about 1-2% on fresh weight basis, and also a low level of essential amino acids (Mahungu 1987). The young leaves have a high crude protein content (170 to 400 g kg-1 on a dry matter (DM) basis), with almost 0.85% being true protein (Ravindran et al. 1983). Some wild relatives of cassava with high levels of protein have been discovered. Genes from these wild relatives have been introduced into Manihot esculenta, which has resulted in an increase in protein content of cassava storage roots (CIAT 2002; Ceballos et al. 2004; Olalekan A et al. 2011). Elsewhere in Africa (Nigeria and Uganda), introgression of genes for higher protein content into local farmers’ preferred varieties has started (Njoku 2012). Latif and Müller (2015) reported that cassava leaves are highly concentrated in vitamins B1, B2 and C, carotenoids, protein and minerals. Cassava leaves, depending on the variety, are also rich in Fe, Zn, manganese, magnesium and calcium and consumed by many as vegetable (Wobeto et al. 2006). While the leaves of cassava are nutrient rich, there are issues of bioavailability and crop acceptability in different parts of the world. The mineral content of the roots of cassava is reported to be two to five times lower than that of the plant leaves. A higher amount of VA in the form of pVA carotenoids are contained in the leaves of 13 cassava compared to its roots (Montagnac et al. 2009a). VA is an important micronutrient for the normal functioning of the visual and immune systems, growth and development, maintenance of epithelial cellular integrity and for reproduction (Huang et al. 2018). These carotenoids are also useful antioxidants. According to Gan et al. (2010), antioxidants are substances that fight free radicals, which cause oxidation of various biomolecules present in organisms. A diet highly concentrated in antioxidants strengthens the human body protection system (Blomhoff et al. 2006). Oxidative damage is a cell and tissue damage caused by free radicals (Gan et al. 2010). The most efficient way to get rid of free radicals is consumption of antioxidant nutrients such as vitamin C (ascorbic acid), vitamin E and beta carotene, which can be found in large quantities in yellow/orange coloured fruits and vegetables (Rahmat et al. 2003). Fiedor and Burda (2014) also suggested that carotenoids have some antioxidant properties. During the chain reaction mechanism of lipid oxidation, carotene reacts with active free radicals to form stable inactive products (Maziya-Dixon et al. 2000) thus preventing oxidative reaction from the production of off-flavours in foods and the potential damage of living cells in biological systems. This role played by carotene is independent of its VA activity (Ceballos et al. 2002). In the assessment of nutritional and anti-nutritional composition of cassava leaf protein concentrate from six cassava varieties for use in aqua feed using standard analytical techniques, Oresegun et al. (2016) reported the highest crude protein levels, beta carotene levels and lipid levels of 48.85%, 816.92 µg g-1 and 13.27%, respectively. The VA content of cassava leaves is comparable to that of carrots and is higher than that reported for legumes and leafy vegetables (Montagnac et al. 2009b). However, cassava leaves have some anti- nutritional and toxic substances. These substances interfere with digestibility and uptake of the nutrients, and they might present toxic effects, depending on the amounts consumed. 2.2 Yellow flesh cassava Yellow flesh cassava genotypes are planted on a small scale in Colombia, Philippines, Jamaica and other African countries like Nigeria, Uganda, Congo DR and Ghana (Oduro 1981). Research has shown that yellow flesh cassava varieties tend to have a low DMC (Akinwale et al. 2010), which is associated with poor cooking quality (Vimala et al. 2008). Most cassava breeding populations are white with only a few yellow flesh cassava populations found in Amazonia in Brazil (Njoku 2012). 14 Yellow-fleshed cassava genotypes have high levels of PVAC and their consumption have been suggested as a sustainable approach for addressing VA deficiencies. In cassava, intensity of yellow pigment in roots of some genotypes is strongly associated with beta carotene (Sánchez et al. 2006). Wide variation exists in root colour within the global yellow root germplasm, which has a range from pale yellow through orange to pink (Nassar 2007). This variation in root colour is corroborated by wide variation in carotenoid contents within the global cassava germplasm. Yellow flesh cassava has increased the different views on nutritional benefits associated with the crop; and beta carotene (pVArovitamin A) in yellow flesh cassava can sustainably address VAD through the dissemination of pVA cassava varieties in regions where the crop is a major staple (Makokha and Tunje 2005; Nassar and Ortiz 2010). Efforts in breeding yellow flesh cassava genotypes that are high in beta carotene content started in almost 20 years ago, with slow progress in its development and deployment to farmers (Welch and Graham 2005), which might be due to the negative correlation between beta carotene and DMC (Vimala et al. 2008; Akinwale et al. 2010). In Nigeria as well as Ghana, most of the cultivated landraces have white fleshed roots with a negligible amount of the pVA pigment. In 2012, the Crops Research Institute (CRI), Kumasi in Ghana, acquired some yellow flesh cassava genotypes with improved agronomic traits from the International Institute of Tropical Agriculture (IITA), in Nigeria. These genotypes are being used as a tool in fighting VAD in areas that lack VA rich food materials. VAD causes eye damage, mostly in children. About 60% of the dietary VA is produced from pVA or beta carotene and consumption of high beta carotene foods is the most effective way of fighting the deficiency. The amount of beta carotene the human body can absorb from VA cassava is more than twice the value or amount previously reported. (La Frano et al. 2012) and this was received with much hope to improve nutrition using food-based interventions. VA status in deficient populations could improve measurably if people switch to these new varieties with high levels of beta carotene (www.harvestplus.org). Since cassava is a major staple crop in Ghana, consumption of yellow flesh cassava varieties containing even moderate amounts of beta carotene can help reduce VAD in the country. 2.3. Cassava biofortification Biofortification is the process of incorporating micronutrient-dense traits in cassava varieties with good agronomic characters like fresh root yield/weight through conventional breeding or biotechnology. This method provides a more sustainable way of disseminating 15 micronutrients to rural/remote populations in developing countries. Both conventional and transgenic breeding methods are being employed to develop these varieties. Generally transgenic crops have tended to be a “political hot button” but crops that are a result of conventional breeding have found favour within communities on this account. Recently, there has been a shift in agriculture where the aim is not only to produce more calories to reduce hunger, but the use of more nutrient rich food in reducing hidden hunger (Saltzman et al. 2013). In many developing countries, cassava is regarded as a food security crop but with a number of liabilities. Montagnac et al. (2009a) reported that 500 g of cassava meal for an adult can provide an adequate amount of calories, but with an insufficient amount of pVA and protein. Some of the past efforts to improve the micronutrient content of cassava include programmes like Bio-cassava Plus (phase 1, 2005- 2010), dealing with traits like pVA, shelf life, cyanide content and diseases (Saltzman et al. 2013). Harvestplus, an international initiative involving a global alliance of research institutions in both developed and developing countries, seeks to improve nutritional status of vulnerable people in the society using plant breeding in developing staple crops rich in pVA, zinc and iron (Makokha and Tunje 2005). Under this initiative, targets are set such that vulnerable people will receive more than 50% of the estimated average requirement (EAR) using pVA cassava. Countries like Nigeria, Ghana, Uganda and Congo DR are benefitting from this scheme. Three cassava varieties with 25% of the EAR for women and pre-school children were released in Nigeria in 2011, with a possible release in Ghana by 2020. A few lines have been evaluated at the on-farm stage and scientists are currently waiting for their assessment by the national variety release committee. EMBRAPA, a research institution in Brazil, also released three cassava varieties with about 9 ppm pVA, and planting materials have been distributed to farmers in the country (Saltzman et al. 2013). 2.3.1 Importance of carotenoids and vitamin A Carotenoids are described as richly coloured molecules, which are the sources of the yellow, orange and red colours of many plants (Rodriguez-Amaya and Kimura 2004). In the human diet, carotenoids are obtained from fruits and vegetables. According to Clagett- Dame and Knutson (2011), carotenoids are also found in some fungi, bacteria and algae. Green leafy and yellow-orange vegetables and fruits provide significant amounts of beta carotene (pVA carotenoids) (Veda et al. 2007). The essential role of carotenoids in humans is pVA 16 carotenoids serving as precursors of VA (Chávez et al. 2005) and is important for optimal growth and cell and tissue differentiation. In human plasma, the most common carotenoids include beta carotene, alpha carotene, beta cryptoxanthin, lutein and lycopene. They have health-promoting effects together with zeaxanthin. Among the carotenoids , beta carotene has the highest pVA potential and is also the most wide spread (Rodriguez-Amaya and Kimura 2004). Thus, alpha carotene and beta cryptoxanthin exhibit about 50% of the VA activity of beta carotene. Carotenoids have several beneficial effects on human health, including the enhancement of immune response, reduction in the risk of diseases such as cancer, cardiovascular diseases, cataracts, and mascular degeneration. It is also essential for optimal growth and lung development of the newborn during pregnancy. About 40% increase in VA intake for pregnant women, was recommended and a 90% intake for breast feeding women (Njoku 2012). The president of Ghana in 2005 launched a special initiative aimed at promoting cassava for starch production as well as a potential source of feeds in the livestock industry. The potential of cassava as food and feed can even be increased with enhanced VA, Zn, Fe and protein content (Njoku 2012). This can be a unique opportunity to increase production, and also prove highly nutritious animal feed, flour, food and chips for both the local, and export markets. In Latin American countries, especially Brazil, water extracted from high carotene cassava during starch production is highly nutritious and serves as additional feed for animals (Njoku et al. 2011). VA is a fat-soluble vitamin that exists in three forms; retinol, retinal and retinoid in animal source foods and as pVA carotenoids, (mostly beta carotene), in plant source foods (Wardlaw et al. 2004). The retinol is the storage form and is found in the liver until needed by the body. VA is important in the functioning of the immune system and for good vision (FAO/WHO 2002). Latif and Müller (2015) that, VA status, when improved in deficient children, can help improve their resistance to diseases and hence reduce their mortality and illness from infections significantly also reported it. Furthermore, improving VA status in deficient children aged from 6 months to 6 years increases their chances of staying alive longer (UNICEF 2014). It is further reported that possible mortality from measles is reduced by approximately 50%, 40% from diarrheoa and 25-35% overall. VA sources include breast milk, animal milk, liver, eggs, fish, butter, palm oil, mangoes, pawpaw, carrots, orange flesh potatoes and dark green leafy vegetables (Pan American Health Organization, 2005). 17 The Institute of Medicine Food and Nutrition board (2000) reported that VA activity of beta carotene in foods is one-twelfth of retinol preformed VA. Another advantage of pVA is that it is only converted to VA when the body needs it, to avoid potential toxicity from an overdose of VA (Clagett-Dame and Knutson 2011). Carotenoids have also been shown to be related to the improvement of immune system and lowered risk of degenerative diseases such as cancer, cardiovascular diseases, muscular degeneration and cataracts (Njoku et al. 2011). 2.3.2 Dietary recommendations for vitamin A and carotenoids The recommended dietary allowance (RDA) of VA depends on the amount required to maintain adequate accumulation to support normal functions of the body. The RDA for VA for infants and children is 400-600 µg retinol activity equivalents (RAE; 1333 - 2000 IU). For adolescents (14-18 years) and adults 19 years and above it is 700 µg RAE (2 333 IU). The RDA for females; 900µg RAE (3000 IU) for pregnant women; 750-770 µg RAE (2333 - 2 567 IU) with the upper limit for pregnant women being 3000 µg RAE or 10000 IU for lactating mothers and for women 1200-1300 µg RAE (4000 - 4333 IU) (Institute of Medicine Food and Nutrition board 2000; Institute of Medicine Food 2001). The retinol activity equivalent is used as a measure of VA equivalence in foods. A mixed diet of 12 µg of all trans beta carotene or 24 µg of other pVA carotenoids (alpha carotene, cis-beta- carotene, beta-cryptoxanthin) is equivalent to 1 µg of retinol (Dary and Mora 2002). 2.3.3 Structure and genetics of beta carotene Structurally, half of VA (retinol) is essentially beta carotene molecules. Alpha carotene and beta cryptoxanthin comprise the remaining half of VA activity. Beta carotene exists as a mixture of trans and cis forms with highly significant levels of the cis isomers compared to the trans form, but with lower VA activity (Rodriguez-Amaya and Kimura 2004). The quantitative variability of root colour observed in cassava clones suggests that carotenoid transport and accumulation are governed by a number of genes each with a small effect (Ferreira et al. 2008). Akinwale et al. (2010) reported that there are no maternal or cytoplasmic effects in the inheritance of carotene. A segregation ratio of 9:3:3:1 was observed when white root cassava was crossed with yellow flesh cassava, which indicates that beta carotene is controlled by two or more genes. Njenga et al. (2014) on the other hand, reported the presence of maternal and cytoplasmic influence on beta carotene inheritance in cassava 18 Akinwale et al. (2010) also reported a negative correlation between DMC and carotenoid while studying African cassava germplasm, but the two traits had a weak positive correlation in Latin American cassava germpalsm (Ortiz et al. 2011). The negative correlation reported in Africa germplasm may be due to linkage of the carotenoid genes with that for low DMC in cassava roots (Njoku 2012). It is believed that with time, the linkage will be broken through recombination and selection (Ceballos et al. 2013). 2.3.4 Breeding for high beta carotene content Research was carried out to increase the concentration of bioavailable PVAC in the edible portion of staple crops such as rice, wheat, maize and cassava (Graham and Welch 1996). A broad distribution of concentration less than 0.1 to 2.4 mg carotene/100 g fresh roots has been reported when more than 632 (Iglesias et al. 1997) and 2457 (Chávez et al. 2005) cassava clones were evaluated. Molecular marker-assisted selection was employed in the development of quick, inexpensive ways for screening micronutrients in staple crops (Wong et al. 2004). This could enhance the introgression of genes into locally adapted cassava varieties. The importance of such a breeding activity will be driven by factors related to bioavailability of type of micronutrient and also willingness on the part of farmers to adopt such varieties. Carotenoid content can be improved simultaneously with yield and its related characteristics. Beta carotene makes the cassava pulp to have yellow to orange colour. It is generally same with other major staple crops (e.g. sweet potato). Yellow flesh cassava roots are thus a good source of carotenoids. Jos et al. (1990) reported the potential of increasing carotene content in cassava storage roots through recurrent selection. They could increase the carotenoid concentration of fresh storage roots of cassava in a base population from 4.2 mg kg-1 to 14 mg kg-1 after two cycles of selection and recombination. A database with more than 3000 samples of cassava genotypes was used to evaluate the potential of near infrared spectrophometry (NIRS) and spectrophotometer devices to predict root quality traits. Maximum TCC and total beta carotene (TBC) were 25.5 µg g-1 and 16.6 µg g-1 respectively on fresh weight basis (Sánchez et al. 2014). A number of screening factors/parameters are required for better nutritional quality selection and it includes selection of a phenotype with agronomic micronutrient efficiency (Graham and Welch 1996), food processing concerns (Ceballos et al. 2012), bioavailability of the nutrients in improved cassava. 19 Breeding for higher carotene content in cassava could also reduce or delay post physiological deterioration (PPD). Reduced PPD in roots of carotene-rich cassava varieties was attributed to antioxidant property of carotenoids, particularly those of beta carotene, which is the predominant carotenoid in cassava roots (Safo-Kantanka et al. 1984). Sánchez et al. (2006) and Morante et al. (2010) have also reported reduced PPD. 2.4 Growth and development of cassava 2.4.1 Dry matter partitioning and source–sink relationship During cassava growth, carbohydrates are needed to ensure good development of the leaves (source) to be able to produce DM in the storage roots, stem and growing leaves (sink) (Alves 2002). The amount of DM in cassava roots depends on the genotype as well as environmental factors and DM can vary from 15-45% (Graham et al. 1999). On average, about 90% of storage root DM is carbohydrate, and the other components are 4% crude fiber, 3% ash, 2% crude protein and 1% fat (Kawano et al. 1978). High root DMC is important, especially when roots are used as food, feed and industrial raw materials (Tan and Mak 1995). High DMC thus improves the extraction efficiency and economic value of products of home-based and industrial processing. Price differentials for roots are usually paid on the basis of DMC or starch content; hence, improvement of these traits would greatly increase farm income (Kawano et al. 1978). Cassava DM is mainly translocated from leaves into the stems and storage roots of the cassava plant. However, it decrease in amounts with time in the leaves during crop growth in the leaves. Between 60 and 75 days after planting, cassava DMC are higher in the leaves compared with stems and storage roots. After that period, the DMC in storage roots increase rapidly, reaching 50 to 60% of the total DM around 120 days after planting (Tavora et al. 1995). At harvest (12 months after planting or MAP) DM is highest in the roots, followed by stems and leaves (Alves 2002). The dry matter content in the storage roots are mostly lower during the vegetative and higher during rest period (Edvaldo et al. 2006). Excess moisture stress increased dry matter accumulation in rootsock, fibrous and storage roots, but decreased partitioning to stems and leaves (Lahai and Ekanayake 2009). Mtunda (2009) reported that root DMC at 7 MAP was higher than 11 and 14 MAP. In addition, Kawano et al. (1987) observed that root DMC tended to be higher at 8 than 12 MAP, and that higher contents were seen at the beginning of the dry season than at the beginning of the wet season. During this period, starch is hydrolysed as a source of energy for the growing leaves. 20 Maximum levels of DM accumulation depends on genotypes and environmental conditions (Oelslige 1975; Howeler and Cadavid 1983). The importance of growing conditions in determining the maximum levels for DM accumulation suggests that germplasm should be evaluated under different environments to estimate the possible effects of genotype by environment interaction (GEI) during selection. DM accumulation also depends on photo- assimilate availability (source activity) and sink capacity of storage parts. Sink capacity is determined by the number of storage roots and their mean weight. Photosynthetic rate positively correlates with root yield, total biomass and leaf area index, interception of radiation and biomass production. This indicates that photosynthesis increases when photo- assimilates demand is high (Ramanujam 1990). Cassava has diverse uses and most of the criteria for selecting quality are also diverse, but high starch content and quality (physico-chemical properties) are always required (Benesi 2005). The amount of starch in cassava is usually estimated from DMC, and both are highly correlated (r = 0.810; IITA 1974; CIAT 1975), but the quicker method is to determine the root’s specific gravity, which is related to both DMC and starch content (Ellis et al. 1982). Specific gravity is obtained by weighing unpeeled cassava roots in air and water using a suitable balance. Estimation of both traits is based on the principle of a linear relationship between specific gravity with DM and starch content (Kawano et al. 1987). DMC is known to be relatively highly heritable, although it is influenced by temperature and rainfall patterns. A number of genes with predominantly additive gene effects apparently controls DMC. Simple breeding techniques such as phenotypic mass selection can be used to exploit the additive variations in DMC (Mtunda 2009), and selection for DMC can be highly effective in cassava breeding (Hershey 1987). Cassava varieties with 30% and more DMC are said to have high DMC (Braima et al. 2000). High DMC is associated with post-harvest deterioration (van Oirschot et al. 2000; Chavez et al. 2005), although the reason for this is unknown. This could have serious consequences for commercial outlets, but not in subsistence agriculture where roots are immediately utilised. Mahungu (1998) reported that there is a shift in the paradigm factor and root yield alone is not sufficient to justify the production of a particular cassava variety. Root DMC is a critical factor, among others. 21 2.5 Variability in hydrocyanic acid content of cassava Cassava contains a cyanogenic glucoside, linamarin (2-β-D-glucopyranosyl-oxy- isobutronitrile) in its leaves and tuberous roots which, when acted upon by linamarase (EC3.2.1.21: linamarin β-D-glucohyrolase) in the plant, is hydrolysed into cyanohydrins, which are further hydrolyzed to give HCN (Fukuba et al. 1983). Potentially toxic compounds, cyanogenic glucosides can cause acute poisoning and death in humans and animals when consumed in high quantities. Cassava cultivars with less than 50 ppm HCN are harmless (Endris 2007). Cyanogenic potential of some known cassava varieties ranges from less than 10 mg kg-1 to more than 500 mg kg-1 as HCN on fresh weight basis (O’Brien et al. 1994) and it is therefore important to evaluate cassava cultivars for cyanogenic potential. Various reports have suggested strategies to reduce the cyanide content of processed cassava; improved processing methods during the production of cassava products, such as flour (Cardoso et al. 2005) and heap fermentation, can remove twice as much linamarin as does sun drying. In order to produce cassava flour with 10 mg HCN equivalents per kg of flour (ppm) which is the World Health Organization (WHO) safe level, one should use sweet cassava roots having 32 ppm linamarin or less (Bradbury 2004). The amount of HCN in cassava varies strongly according to genotype, environmental conditions and various parts of the same plant (Food Safety Network 2005; Endris 2007). HCN content of cassava roots have been reported to be lower with potassium fertilisation (El-Sharkawy and Cadavid 2000, Susan et al. 2005; Endris 2007). Contrary to that, Attalla et al. (2001) reported high HCN level in cassava tuber tissues with increasing rates of potassium fertilizer (K2SO4). Production of cyanide in various cultivars is affected by soil, weather and other geographical conditions (Bokanga et al. 1994), same cultivars may produce high cyanide in one location and significantly lower value in another (Charles et al. 2005). It is therefore necessary to assay cyanide content of cassava root by characterizing for location and genotype before use for human consumption. The recommended WHO maximum acceptable level of cyanide in foods meant for human consumption is below 10 mg kg-1 (Bradbury and Egan 1992). The cyanide level of all cassava tubers were found to be above this recommended level (White et al. 1998). Cyanide is usually removed from tubers by different processing methods such as fermentation, boiling, steaming, drying and roasting (Cardoso et al. 2005). 22 2.5.1 Measures to control cyanide content in cassava Regardless of the usefulness of cassava, it has a major drawback as food such as its perishability, its low protein content and its potential toxicity, which are a crucial limitation on its sustainability as a food source in the tropics. Upon crushing the plant tissues, its inherent cyanogenic glucosides are catalytically hydrolyzed to release HCN (Oloya et al. 2017). Due to the potential cyanogen toxicity, research efforts have been channeled to mitigate this effect. Three major strategies have been adopted and these are (i) breeding of acyanogenic cassava varieties; (ii) controlling its metabolism and (iii) removal of cyanogens through processing (Nambisan 2011). Development of acyanogenic varieties through breeding is probably the ultimate of the three listed approaches; however, it is a long-term solution as research efforts are still underway on this. Both genetic improvement and effective processing methods can be implemented to control cyanogenesis in cassava. There is also the option to manipulate the linamarin metabolism in roots, but this is also a long-term process. Processing remains the most efficient way of controlling cassava cyanogens in the short term (Nambisan 2011). For populations which rely on cassava as a staple, cultivars with low cyanogen (linamarin) content should be grown as far as possible, or alternatively, if high cyanogen or bitter cultivars are used, adequate processing should be done to reduce the cyanogen content to safer levels. The processing methods used in cassava consuming communities are very diverse. These may include peeling and slicing fresh tubers then boiling, baking, steaming, drying, deep frying, fermentation, grating/pounding followed by drying or roasting. Most of these processing methods are effective in reducing cyanoglucoside (CNG) content to some extent (Bradbury 2006; Nambisan 2011). Depending on the processes used, they usually either lead to hydrolysis of CNG to release acetocyanohydrin and cyanide, which are volatilized and subsequently lost, or the highly soluble CNG and its hydrolytic products are leached out in the water (Nambisan 2011). When cassava is cut into chunks and boiled, about 80% of the CNG content is removed (Table 2.2). It is therefore important to decant the water after boing, as the CNG in the roots leaches into it. The volume of water should be enough for optimum dissolution of CNG. When bitter tubers are cooked, it is customary to decant the water a few times until the bitterness is reduced to the maximum extent. Sun drying of cassava chips of 10 mm 23 thickness also removes 80% of CNG. Processes such as baking, steaming and frying result in only around 20% loss of CNG, due to inactivation of linamarase and stability of linamarin at high temperature. As seen in Table 2.2, the process of grating/pounding followed by sun drying is the very effective since it facilitates the enzyme reaction and results in 95–99% removal of CNG (Nambisan 2011). The most efficient processing method by far is fermentation, which removes about 80 to 98% of the cyanogens. Table 2.2 Effect of different processing methods on the cyanogen content of cassava Processing method Cyanogen retained (%) Mechanism of removal Boiling 20-50 Leaching Blanching and drying 50 Leaching Baking, frying, steaming 80 Thermal degradation Sun drying 20-50 Enzyme action Oven drying 30-50 Enzyme action Crushing and sun drying < 5 Disintegration and enzyme action Grating/fermentation, < 2 Disintegration and enzyme action dewatering, drying Nambisan (2011) In Africa, fermentation is frequently adopted in making products like gari (roasted grated cassava), fufu (boiled and pounded), lufan, casaba and farina. These foods are prepared by a combination of grating/soaking, fermentation, dewatering and drying/roasting. The level of cyanogens in the final processed product is influenced by both the original CNG content and the processing methods applied. This suggests that different methods must be used for processing high and low cyanide varieties. 2.5.2 Effect of processing on the nutritional value of cassava To increase the shelf stability of products, to facilitate conveyance and sales, reduce cyanogenic content and improve palatability and nutrient bio-availability, cassava must be processed into different products (Nyirenda et al. 2011). As with cyanide, simple processing of cassava such as drying cause a reduction in moisture and volume roots (Obilie et al. 2004) is drying and it causes a reduction in moisture, volume and cyanide concentration of roots. This prolongs product shelf life (Westby 2002). One of the traditional methods of processing cassava is fermentation (Cardoso et al. 2005) and is reported to enhance the nutrient content 24 of foods through biosynthesis of vitamins, fibre digestibility as well as enhancing micronutrient bioavailability. It also aids in degrading anti-nutritional factors. Bioavailability of micronutrients like vitamin B6, thiamin and carotenoids can also be improved through thermal processing like boiling, as it discharges them from cell walls in the plant matrix (Montagnac et al 2009b). It has been reported that processing cassava affects the nutritional composition of cassava roots through alteration and losses in essential nutrients (Montagnac et al. 2009a; 2009b). Analysis of nutrient retention has shown that raw and boiled cassava roots keep the majority of high-value nutrients, except riboflavin and Fe (Onyenwoke and Simonyan 2014). The extent of soaking of cassava significantly affects nutrient retention and content in the processed samples. The longer the soaking, the lower the nutrient retention, especially the minerals. Lafun and fufu processing methods seemed to retain more of the minerals than other processing methods. The gari processing method, which involves grating, predisposes the minerals to easy leaching through draining water. Significant mineral losses in the final products were reported after repeated washings with rehydration and draining in abacha. Fermentation and cooking enhanced nutrient content in prepared eba, fufu and amala. To improve the nutrient contribution of gari and eba for consumers, two days of fermentation of raw cassava was suggested (Adepoju et al. 2010). With regards to cassava leaves, proximate components showed no difference between the ash, lipid, protein, starch and fiber contents of non-processed, pounded or ground cassava leaves. However, the free sugar component was reduced compaired to non-processed leaves. Processing has been reported to have no effect on the calcium, magnesium, potassium, sodium, phosphorus, copper, manganese and Zn contents of cassava leaves except for Fe (Achidi et al. 2008). 2.5.3 Impact of processing on carotenoids The stability of food nutrients according to Montagnac et al. (2009b), is affected by processing and preservation. Processes such as roasting and fermentation have been reported to have severe impacts on carotene (Halvorsen et al. 2006). Other processes such as dehydration, blanching and canning may also have effects on the antioxidant property of the carotenoids of some edible plants (van Het Hof et al. 2000). However, there is an increased 25 antioxidant activity upon boiling and steaming of some vegetables such as carrots, lettuce, peppers, potatoes, tomatoes and cabbages (Halvorsen et al. 2006). It has also been reported by Negi and Roy (2000) that beta carotene is sensitive to heat and oxidation during blanching and drying. High temperatures can cause losses of pVA and therefore excessive boiling, roasting and frying at high temperatures affects pVA (Thakkar et al. 2009) but low boiling for a few minutes, soaking and chopping can improve bioavailability with little carotenoid loss (La Frano et al. 2013). 2.5.4 Bioavailability of carotenoids The percentage of consumed nutrient that is assimilated and reaches the point of being absorbed during blood circulation is termed as bioavailability (Maiani et al. 2009) whilst the proportion of a carotenoid that is transferred from the food matrix to micelles during digestion and made accessible for intestinal absorption, is termed as bioaccessibility (Stahl et al. 2000). The fraction of bioavailable pVA that can be changed into retinol is known as bioconversion (Ceballos et al. 2013). The efficiency of the process that ingested dietary pVA carotenoids are absorbed and converted to retinol is termed bioefficacy (van Lieshout 2001). In an animal source, vitamin A is readily taken as preformed retinol but from plant sourced foods, it is taken as pVA and therefore needs to be converted into retinol in the human body (Institute of Medicine 2001). Bioavailability and bioconversion of carotenoids are affected by a number of factors, which include the quantity of fat available in the food (Thurnham et al. 2003), carotene types, molecular bonds, amount of carotene contained in a meal, nutritional status of the organism, genetic conditions and organism related conditions (Parada and Aguilera 2007). 2.6. Genotype by environment interaction GEI occurs when cultivars perform significantly different with marked changes in pattern under different environmental conditions (Dixon et al. 1994). It can also be defined as the failure of genotypes to achieve the same relative performance in different environments (Fernandez 1991). Large GEI variation impairs the accuracy of yield estimation and reduces the relationship between genotypic and phenotypic values, thereby reducing progress from selection. It also prevents the extrapolation of results of agronomic evaluations from one location to another, thus requiring expensive trials at multiple locations (Shaffi et al. 1992). Hence, it is essential to examine different lines or crosses in several environments to determine their genetic potential and also for releasing genotypes with adequate adaptation 26 (Baiyeri et al. 2008). The relative performance of genotypes across environments defines the importance of GEI for the traits of interest. Cassava as a crop generally is widely adapted to different ecologies but an individual cultivar can vary in adaptation because cassava cultivars are very sensitive to GEI (Ssemakula et al. 2007). Also, the response of an individual genotype to different environments may follow a diverse pattern due to the influence of the climate and soil variations. It is on this pattern that selection for high root yield, pest and disease resistance, and stable root yield is based (Dixon et al. 1994). Quantitative traits in cassava associated with root qualities, for example, carotene content, DMC, starch and HCN content may show sizeable interaction with the environment. Therefore, the testing of new lines in different locations (environments) to establish their genetic potential is crucial in cultivar development. In addition to high mean yields, stability of a genotype’s performance in different environments is necessary to assist breeders in selecting superior cultivars to meet varying growing conditions. One approach is to reduce the number of replications used in a single field trial, assuming that performance can still be evaluated accurately. The rate of adoption of biofortified cassava genotypes will largely depend on their agronomic performance, including fresh and dry storage root yield, resistance to major pests and diseases, DMC, HCN, and the stability of these traits over time and space. Although cassava as a crop is widely adapted to a variety of environmental conditions, most of the white fleshed varieties show narrow adaptation with large GEI effects (Dixon et al. 1994; Dixon and Nukenine 1997). Ssemakula et al. (2007) and Maroya et al. (2012) reported a significant GEI for total carotene content and they reported higher impact of genotypic effect than both environment and GEI, indicating fewer environments are necessary to distinguish genotypes with high and stable performance. In contrast, Maroya et al. (2010) found a non- significant GEI on beta carotene when evaluating nine yellow flesh cassava genotypes in Ghana. Kawano et al. (1978), Ssemakula and Dixon (2007), Aina et al. (2009), Maroya et al. (2012) and Thompson (2013) reported significant GEI for DMC. Maroya et al. (2012) found higher environmental effects on DMC in contrast to the higher genotypic effects reported by others. Tan and Mak (1995) found that GEI effects were significant for cyanide content when studying the relative influence of genotype, environment and GEI effects on this trait in Malaysia. 27 In recent years, research has progressively placed attention on increased storage root production. However, agriculture must now not only produce more calories to reduce hunger but also more nutrient-rich food to reduce hidden hunger (Kennedy et al. 2003). 2.7 Heritability of characteristics in cassava Heritability estimates how much variation in phenotypic traits in a population is due to genetic variation among individuals in that population (Klug and Cummings 2005). The two commonly used measures of heritability are broad-sense and narrow-sense. Narrow- sense heritability is more useful than broad-sense since its estimation does not involve dominance and interaction variances. There are many approaches for estimating heritability. Some researchers use the parent-offspring regression approach, or by comparing full-sibs. An analysis of variance (ANOVA) approach can also be used in estimating heritability, correlation and regression. Heritability estimates are crucial in cultivar development and GEI testing strategies. Studies have shown that PVAC is controlled by a few (~2) major genes and is highly heritable (Menkir and Maziya-Dixon 2004; Grüneberg et al. 2005). However, heritability can be overestimated if studies contrast non-pVA and high pVA genotypes (Ewool and Akromah 2017). Both minerals and pVA are mainly driven by additive genes implying good general combining ability for these traits. Important characters such as harvest index (HI) and resistance to diseases such as cassava bacterial blight (Xanthomonas manihotis) and Cercospora leaf spot (C. henningsii) are highly heritable traits (Kawano et al.1978) indicating that they will be easily transmitted relatively to progenies. 2.8 Inheritance of nutritional traits The colour of root parenchyma seems to be simply inherited. A report by Hershey and Ocampo (1989) established that a partially dominant gene determined root colour (white/cream/yellow). Homozygous dominant individuals have yellow roots while homozygous recessive individuals have white roots while the heterozygous individuals have cream roots (Hershey and Ocampo 1989). The inheritance of beta carotene concentration in storage root thus appear to be determined primarily by two genes, one controlling transport of shoot precursors to roots and one responsible for the biochemical processes affecting the accumulation of beta carotene in the root (Akinwale et al. 2010). Although that was true for parents they used, recent studies have shown that the inheritance of root colour, and by implication carotene content may be more complex (Akinwale et al. 28 2010). Although major genes are responsible for the transport and accumulation of carotene in the storage roots, the quantitative variability observed within root colour classes suggested that a number of genes with smaller effects are involved in the accumulation process. The biochemical pathway leading to the synthesis of carotenes has also been long established (Spurgeon and Porter 1980) to support the genetics of this trait. Single breaks in the pathway could culminate in lack or reduced levels of carotene formation. According to Iglesias et al. (1997), there is variation in beta carotene concentration in cassava storage roots in germplasm collection (630 genotypes) and also in the global cassava germplasm collection (about 5 500 genotypes). The authors further reported that sufficient genetic variability exists within the available cassava germplasm, which could help crop breeders to develop cassava varieties that contain enough beta carotene to meet the daily requirements of adults (i.e. 6 mg d-1 beta carotene) depending on bioavailability of beta carotene in cassava storage roots. Sánchez et al. (2006) reported a range in beta carotene concentrations in fresh roots from 0.1 to 2.4 mg 100 g-1. The relationship between root colour and heritability, as well as the stability of root beta carotene content, to different root- processing techniques, has been studied. Visual screening by using intensity of orange colour to estimate beta carotene content seems feasible. However, it is possible that other forms of carotenoids could also be responsible for the deep yellow colour observed in accessions that have intermediate beta carotene concentrations. 2.9 Mating designs and heterosis Various mating designs are used by crop breeders and geneticists to develop improved lines through plant breeding. Successful plant breeding schemes involve the correct choice of design and suitable progenitors (Khan et al. 2009). Mating design is used to provide genetic information of a trait under investigation and to generate breeding populations to be used as a basis for variety development. It also provides information for evaluating parents, estimation of genetic parameters and gain (Acquaah 2012). Mating design refers to the methods employed by plant breeders to produce progenies in plant breeding. However, the choice of a mating design for estimating genetic variances should be dictated by the objectives of the study, time, cost, space and other biological limitations (Nduwumuremyi et al. 2013). Some commonly used mating designs in cassava include polycross, North Carolina (I, II, III), diallel (methods I, II, III, IV) 29 2.9.1 Combining ability Combining ability is defined as the performance of hybrid combinations (Kambal and Webster 1965). It helps in the selection of parents for hybrid development and genetic studies (Duvick 2001). Combining ability can be estimated by using the genetic parameters of the parents and the hybrid components (GCA and SCA, respectively) of diallel analysis (Griffing 1956). Sprague and Tatum (1942) first explained the concepts of GCA and specific combining ability (SCA) that underlies the genetic and breeding attributes of a genotype and therefore important in designing an efficient breeding strategy. GCA refers to the average performance of a line in a hybrid combination and is mostly due to additive gene effects. SCA is due to non-additive gene effects and is the case where combinations do relatively better or worse than would be expected based on the average performance of the lines involved (Falconer and Mackay 1996). The relative amount of improvement to be obtained from GCA and SCA is proportional to their variances and it shows the type of gene action controlling a particular trait (Quick 1978). Thus the relative sizes of the predictability ratio [(2GCA/(2GCA:SCA)] have been used to assess the relative importance of GCA and SCA. The closer the ratio is to one, the more important the additive gene effects (Baker 1978). GCA was more important in controlling CMD resistance (Lokko et al. 2005; Parkes 2011) and dominance plays an important role in fresh root weight (FRW) but was of little importance in some traits such as DMC (Cach et al. 2006). Njenga et al. (2014) reported significant GCA for root pulp colour. Selection of crop varieties based on combining ability estimates is useful to identify the most valuable progenitors and families for breeding and cultivar improvement. Both diallel and NCD II mating designs have been used to interpret genetic information on important traits in cassava (Jaramillo et al. 2005; Cach et al. 2006; Parkes 2011; Njoku 2012). These studies used crosses between heterozygous cassava clones as parents given that inbred lines don’t exist in cassava. Rajendran (1989) reported additive gene action and non-additive gene action for storage root yield and yield components (such as HI, storage root number (RTN) and storage root weight) respectively. Root quality traits, namely starch, DMC and HCN content are predominately non-additive (Amma et al. 1995). Parkes et al. (2013) reported additive gene action for root number and plant height as well as for CMD and cassava bacterial blight in the forest zone of Ghana. Peninah et al. (2014) reported additive gene action for root pulp colour in yellow flesh cassava varieties from IITA and root pulp colour correlates directly with beta carotene. They also reported that improving local white cassava varieties for beta carotene does not affect yield. They further suggested that GCA and SCA of selected parents should also be considered due to the additive and non-additive gene effects involved in the inheritance of the trait. Combining 30 ability of parents and their performance are very important when formulating specific breeding programmes (Rajendran 1989). Diallel crosses are also used to investigate GCA of parents and to identify superior parents for use in cultivar development (Ortiz et al. 2001; Yan and Hunt 2002). The usefulness of both additive and non-additive gene action in the inheritance of yield and yield components has been reported in different studies (Maris 1989; Ruiz de Galarreta et al. 2006). However, such information is scant for the yellow flesh cassava breeding population in Ghana. Selecting individuals and families for high DMC and TCC in a population improved for CMD resistance could be an important strategy in breeding for yield stability towards under food and nutrition security. 2.9.2 Diallel and North Carolina design II The diallel and North Carolina design II (NCD II) mating designs provide genetic information such as combining ability of parents and the inheritance of quantitative traits (Kang 1994). The NCD II mating scheme is a cross-classification design whereby different sets of parents are used as males and females. The design can accommodate more parents than the diallel and provides the same type of genetic information (Hallauer and Miranda 1988). The main effects of males and females correspond to GCA and the female x male interaction to SCA (Jaramillo et al. 2005; Cach et al. 2006). Both diallel and NCD II mating designs have been used to interpret genetic information on important traits in cassava (Jaramillo et al. 2005; Cach et al. 2006; Parkes 2011; Njoku 2012). 2.9.3 Heterosis Crossbreeding or hybridization of two parents often result in positive fitness-related effects in F1 progeny (Akah 1992; Sprague 1983) and this has been exploited in plant breeding for years. The performance of F1 progeny that exceed the average parental performance is mostly referred to as hybrid vigour or heterosis (Shull 1908; Parkes 2011) or superiority in performance of hybrids over their parents. It is the interpretation of increased vigour, size, speed of development, resistance to disease and pest, manifested by crossbred lines as compared with their parents. There are two predominant theories that are used to define heterosis; dominance and over- dominance hypothesis (Crow 1952). Dominance hypothesis is produced when deleterious recessive alleles from one individual parent are masked by dominant or partially dominant alleles from in the second individual (second parent) in the hybrid (F1), while over-dominance hypothesis is due to the heterozygous superiority of the 31 F1 (hybrid) over homozygous individuals. Therefore, increased vigour is proportional to the amount of heterozygosity (Lamkey and Edwards 1999). Heterosis is due to the combined action and interaction of allelic and non-allelic genes and is usually closely and positively correlated with heterozygosity (Burton 1968). There are different types of heterosis, such as mid-parent, better-parent and standard heterosis. Mid- parent heterosis is defined as the difference between the hybrid and the mean of the two parents (Falconer and Mackay 1996). 2.10 Participatory plant breeding Farmers as well as professional plant breeders have important knowledge and skills that could complement one another. Participatory Plant Breeding (PPB) is based on this concept, and is defined as a range of approaches that involve a mix of participants (including scientists, breeders, farmers and other stakeholders) in different plant breeding stages. Depending on whether researchers or farmers are controlling the breeding process, and the scale on which the work is undertaken (community-centred or research to extrapolate results), two categories can be identified, which may be either farmer-led or formal-led PPB. These concepts are not well understood and are difficult to pinpoint using conventional market research methods. Farmers are often uneducated and unschooled in plant breeding, but they have, in several years, dominated crop production and selected superior cultivars of different crop species (Harlan 1992). They were able to recover and successfully propagate genetic recombinants that exhibited desirable traits (Jauhar 2006) through the collection of volunteer eedlings, which they selected and added to their cultivated genotypes (Manu-Aduening et al. 2005; Kizito et al. 2007; Peroni et al. 2007; Pujol et al. 2007). Under formal plant breeding programmes has been low and scientists are yet to fully utilize their knowledge and this has resulted into low adoption of modern varieties bred (Witcombe et al. 1999). Farmers have different perceptions and priorities to those of plant breeders (Manu- Aduening et al. 2005). Farmers often use specific stable morphological traits like height at first apical branching, petiole colour and culinary attributes such as taste, to differentiate and name varieties (Kizito et al. 2007) as well as to indicate preferences. Farmers were reported to consistently base their selections on distinctive traits which they can observe, such as canopy shape, stem colour and root skin colour (Manu-Aduening et al. 2014). High CMD incidence and good extension services usually had to increase farmer adoption of improved varieties. Cassava breeding efficiency tend to improve when farmers participate in the early stages of breeding (Dixon et al. 2007). Farmer participation in breeding also enhanced farmer ownership of newly developed cultivars, leading to their acknowledgement that they are the ultimate beneficiaries, which often resulted in the rapid adoption of new 32 improved cultivars (Manu-Aduening et al. 2014) References Achidi AU, Ajayi OA., Maziya-Dixon B, Bokanga M (2008) The effect of processing on the nutrient content of cassava (Manihot esculenta Crantz) leaves. Journal of Food Processing and Preservation 32: 486-502 Acquaah G (2012) Principles of plant genetics and breeding. 2nd ed. Wiley-Blackwell, Oxford Adepoju OT, Adekola YG, Mustapha SO, Ogunola SI (2010) Effect of processing methods on nutrient retention and contribution of cassava (Manihot spp) to nutrient intake of Nigerian consumers. African Journal of Food, Agriculture, Nutrition and Development 10: 2099-2110 Aina OO, Dixon AGO, Ilona P, Akinrinde, E (2009) G x E interaction effects on yield and yield components in the Savanna region of Nigeria. African Journal of Biotechnology 8: 4933-4945 Akah SMK (1992) Crossbreeding, additive and heterotic effects on production traits in Jersey crossbred cattle at agricultural research station, Legon. Thesis submitted to the department of Animal Science, Faculty of Agriculture-University of Ghana in partial requirement for an MPhil Akinwale MG, Aladesanwa RD, Akinyele BO, Dixon AGO,Odiyi AC (2010) Inheritance of ß-carotene in cassava (Manihot esculenta Crantz). International Journal of Genetics and Molecular Biology 2: 198-201 Al-Hassan MR (1993) Cassava in the economy of Ghana in product development for root and tuber crops. Volume III – Africa and Latin America. Breeding projects work to stabilize productivity without increasing pressures on limited natural resources. BioSciences 43: 441-452 Al-Hassan RM (1989) Cassava in the Economy of Ghana. In: Nweke FI, Lynam J, Prudencio CY (eds.) Status of cassava research in Africa. COSCA working paper No. 3. International Institute of Tropical Agriculture, Ibadan, Nigeria Al-Hassan RM, Diao X (2007) Regional disparities in Ghana: Policy options and public investment implications, IFPRI Discussion Papers 693, International Food Policy Research Institute (IFPRI) Alves AAA (2002) Cassava botany and physiology. In: Hillocks RJ, Thresh MJ, Bellotti AC (eds.) Cassava: Biology, production and utilisation. CABI International, Oxford. pp. 67-89 33 Amenorpe G, Carson G, Tetteh JP (2006) Ethnobotanical characterization of cassava (Manihot esculenta Crantz) in Western Region of Ghana. Journal of Agricultural Science 39: 123-130 Amma CSE, Sheela MN, Pillai PKT (1995) Combining ability, heterosis and gene action for three major quality traits in cassava. Journal of Root Crops 21: 24-29 Angelucci F (2013) Analysis of incentives and disincentives for cassava in Ghana. Technical notes series, MAFAP, FAO, Rome Attalla AR, Greish MHM, Kamel AS (2001) Effect of potassium fertilizer rates and row spacing on some cassava varieties (Manihot esculenta, Cranz.), under new reclaimed soil. Journal of Agricultural Science 26: 4731-4707 Baafi E, Sarfo-Kantanka K (2008) Agronomic and processing attributes of some cassava (Manihot esculenta Crantz) genotypes affected by location and age at harvest in Ghana. International Journal of Agricultural Research 3: 211-218 Baiyeri KP, Edibo GO, Obi IU, Ogbe FO, Egesi CN, Eke-Okoro ON, Okogbenin E, Dixon AGO (2008) Growth, yield and disease responses of 12 cassava genotypes evaluated for two cropping seasons in a derived savannah zone of south-eastern Nigeria. Journal of Tropical Agriculture, Food, Environment and Extension 7: 162- 169 Baker RJ (1978) Issues in diallel analysis. Crop Science 18: 533-536 Benesi IRM (2005) Characterisation of Malawian cassava germplasm for diversity, starch extraction and its native and modified properties. PhD Thesis, Department of Plant Sciences (Plant Breeding), University of the Free State, South Africa Blomhoff R, Carlsen MH, Andersen LF, Jacobs DR (2006) Health benefits of nuts: potential role of antioxidants. British Journal of Nutrition 96: S52-S60. Bokanga M, Ekanayake IJ, Dixon AG, Porto MCM (1994) Genotype-environment interactions for cyanogenic potential in cassava. Acta Horticulturae 375: 131-139 Bradbury HJ (2004) Processing of cassava to reduce cyanide content. Cassava cyanide diseases Network Newsletter 3 Bradbury JH (2006) Simple wetting method to reduce Cyanogens content of cassava flour, Journal of Food Composition and Analysis 19: 388-393 Bradbury JH, Egan SV (1992) Rapid screening assay of cyanide content of cassava. Phytochemical Analysis 3: 91-94 Braima J, Neuenschwamder H, Yaninek F, Cudjoe JP, Exhendu N, Toko M (2000) Pest control in cassava farms: IPM field guide for extension agents. Wordsmiths Printers, Lagos, Nigeria 34 Burton GW (1968) Heterosis and heterozygosis in pearl millet forage production. Crop Science 8: 229-230 Cach NT, Lenis JI, Pérez JC, Morante N, Calle F, Ceballos H (2006) Inheritance of useful traits in cassava grown in sub-humid conditions. Plant Breeding 125: 177-182. Cardoso AR, Mirione E, Ernesto M, Massaza F, Cliff J, Haque MR, Bradbury HJ (2005) Processing of cassava roots to remove cyanogens. Journal of Food Composition and Analysis 18: 451-460 Ceballos H, Chávez AL, Sánchez T, Bedoya JM, Echeverri J, Tohme J (2002) Genetic potential to improve carotene content of cassava and strategies for its deployment. The Journal of Nutrition 132: 2988S Ceballos H, Iglesias CA, Pérez JC, Dixon AGO (2004) Cassava breeding: opportunities and challenges. Plant Molecular Biology 56: 503-516 Ceballos H, Luna J, Escobar AF, Pérez JC, Ortiz D, Sánchez T, Pachon H, Dufour D (2012) Spatial distribution of dry matter in yellow-fleshed cassava roots and its influence on carotenoids retention upon boiling. Food Research International 45: 52-59 Ceballos H, Morante M, Sánchez T, Ortiz D, Aragón I, Chávez AL, Pizarro M, Calle F, Dufour D (2013) Rapid cycling recurrent selection for increased carotenoids content in cassava roots. Crop Science 53: 2342-2351 Charles A, Chang Y, Ko W, Sriroth K, Huang T (2005) Influence of amylopectin structure and amylose content on the gelling properties of five cultivars of cassava starches. Journal of Agriculture and Food Chemistry 53: 2717-2725 Chávez AL, Sánchez T, Jaramillo G, Bedoya JM, Echeverry J, Bolaños EA, Ceballos H, Iglesias A (2005) Variation of quality traits in cassava roots evaluated in landraces and improved clones. Euphytica 143: 125-133 CIAT (1975) Annual Report of the Centro International de Agricultural Tropical, Cali, Colombia CIAT (2002) CONDOR 2, beta 1 version. CIAT-CAF-CI. PE-4 Project annual report Clagett-Dame M, Knutson D (2011) Vitamin A in reproduction and development. Nutrients 3: 385-428 Cock JH (1985) Cassava: New potential for a neglected crop. West View Press, Boulder, CO, USA Collinson C, van Dyk G, Gallat S, Westby A (2001) Urban marker opportunities for high quality cassava products in Ghana. Paper based on poster presentation at the International Society of Tropical Root and Tuber Crops - African Branch (ISTRCAB) 12-16 Nov 2001 35 Crow FJ (1952) Dominance and over dominance in heterosis. In: Gowen JW (ed.) Iowa State College Press, USA. pp. 282-297 Dapaah SK (1991) Contributions of root and tuber crops to socio-economic changes in the developing world: the case of Africa, with special emphasis on Ghana. In: Ofori F, Hahn SK (eds.) Tropical root crops in a developing economy. Proceedings of the ninth symposium of the International Society for Tropical Root Crops, 20-26 October, Accra, Ghana. pp. 21-24 Dapaah SK (1996) The way forward for accelerated agricultural growth and development. A paper presented to the Government of Ghana on behalf of the Ministry of Food and Agriculture Dary O, Mora JO (2002) Food fortification to reduce vitamin A deficiency: International Vitamin A Consultative Group recommendations. Journal of Nutrition 132: 2927S- 33S DeVries J, Toenniessen G (2001) Securing the harvest: biotechnology, breeding and seed systems for African crops. Chapter 13: Cassava. CABI Publishing Oxon, UK and New York. pp. 147-156 Dixon AGO, Akoroda MO, Okechukwu RU, Ogbe F, Ilona P, Sanni LO, Ezedinma C, Lemchi J, Ssemakula G, Yomeni MO, Okoro E, Tarawali G (2007) Fast-track participatory approach to release of elite cassava genotypes for various uses in Nigeria’s cassava economy. Euphytica 160: 1-13 Dixon AGO, Asiedu R, Hahn SK (1994). Genotypic stability and adaptability: analytical methods and implications for cassava breeding for low-input agriculture. Acta Horticulturae 380: 130-137 Dixon AGO, Nukenine EN (1997) Statistical analysis of cassava yields with the Additive Main Effects and Multiplicative Interaction (AMMI) model. African Journal of Root and Tuber Crops 3: 46-50 Dixon AGO, Ssemakula G (2008) Prospects for cassava breeding in Sub-Saharan Africa in the next decade. Journal of Food Agriculture and Environment 6: 256-262 Duvick DN (2001) Biotechnology in the 1930s: the development of hybrid maize. Nature Reviews, Genetics 2: 69-74 Edvaldo S, Filho PSV, Pequeno GM, Vidigal MCG, Scapim AC, Kvitschal MV, Maia RR, Rimoldi F (2006) Effect of harvest period on foliage production and dry matter distribution in five cassava cultivars during the second plant cycle. Brazilian Archives of Biology and Technology, 49:6 36 Ekanayake IJ, Osiru DSO, Porto MCM (1997) Agronomy of cassava. IITA Research Guide 60. Training Programme, IITA. Ibadan, Nigeria. First Edition Ellis RH, Hong TD, Roberts EH (1982) An investigation of the influence of constant and alternating temperature on the germination of cassava seed using a two- dimensional temperature gradient plate. Annals of Botany 49: 241-246 El-Sharkawy MA, Cadavid LF (2000) Genetic variations within cassava germplasm in response to potassium. Experimental Agriculture 36: 323-334 El-Sharkawy MA, De Tafur SM, Lopez Y (2012) Eco-physiological research for breeding improved cassava cultivars in favourable and stressful environments in tropical/sub- tropical bio-systems. Environmental Research Journal 6: 143-210 Endris S (2007) Cyanogenic potential of cassava cultivars grown under varying levels of potassium nutrition in South Western Ethiopia. In: Ortiz R, Nassar N (eds.) Proceedings: first international meeting on cassava breeding, biotechnology and ecology. pp. 163-170 Ewool MB, Akromah R (2017) Genetic variability, Coefficient of Variance, Heritability and Genetic Advance of Pro-Vitamin A maize hybrids. International Journal of Agriculture Innovation and Research, 6:84-90 Falconer DS, Mackay TFC (1996) Introduction to quantitative genetics. Fourth edition. Longman Scientific and technical Co., Essex, England FAO (1998) Storage and Processing of Roots and Tubers in the Tropics http://www.fao.org/3/x5415e/x5415e00.htm#Contents FAOSTAT (2014) Food and Agriculture Organization, Agricultural data. Crops and products domain FAOSTAT (2017) Food and Agriculture Organization, Agriculture data. Crops and products domain FAO/WHO - Food and Agricultural Organization of the United Nations/World Health (2002) Vitamin A. In: Human Vitamin and Mineral Requirements. Report of a Joint FAO/WHO Expert Consultation. FAO, Rome. pp. 87-107. Fernandez GCJ (1991) Analysis of clone x environment interaction by stability estimates. Horticultural Science 26: 947-950 Ferreira CF, Alves E, Pestana KN, Junghans DJ, Kobayashi AK, SantosVJ, Silva RP, Soares E, Fukuda W (2008) Molecular characterization of cassava (Manihot esculenta Crantz) with yellow-orange roots for beta-carotene improvement. Crop Breeding and Applied Biotechnology 8: 23-29 37 Fiedor J, Burda K (2014) Potential role of carotenoids as antioxidants in human health and disease. Nutrients 6: 466-488 Food Safety Network (2005) Safe food from farm to fork. http://www.food safetynetwork.ca (accessed on January 2015) Food and Agriculture Organization of the United Nations (FAO). Save and Grow: Cassava, A guide to sustainable production and intensification, Roome (2013) Food and Agriculture Organization of the United Nations (FAO) FAOSTAT 2019 FAO and IFAD (2001) The Global cassava development stratergy and implementation plan, volume 1. Proceedings of the validation forum on the Global cassava development stratergy, Rome Fregene M, Bernal A, Duque M, Dixon AGO, Tohme J (2000) AFLP analysis of African cassava (Manihot esculenta Crantz) germplasm resistant to the cassava mosaic disease (CMD). Theoretical and Applied Genetics 100: 678-685 Fukuba H, Igarashi O, Briones CM, Mendoza EMTT (1983) Determination and detoxification of cyanide in cassava and cassava products. Phillipine Journal of Crop Science 7: 170-175 Gan RY, Xu XR, Song FL, Kuang L, Li HB (2010) Antioxidant activity and total phenolic content of medicinal plants associated with prevention and treatment of cardiovascular and cerebrovascular diseases. Journal of Medicinal Plants Research 4: 2438-2444 Graham RD, Senadhira D, Beebe S, Iglesias C, Monasterio I (1999) Breeding for micronutrient density in edible portions of staple food crops: Conventional approaches. Field Crops Research 60: 57-80 Graham RD, Welch RM (1996) Breeding for staple-food crops with high micronutrient density: Agricultural strategies for micronutrients.Working Paper No. 3.1996. IFPRI, Washington, D.C Griffing B (1956) Concept of general and specific combining ability in relation to diallel crossing systems. Austrian Journal of Biological Science 9: 463-493 Grüneberg WJ, Manrique K, Zhang D, Hermann M (2005) Genotype x environment interactions for a diverse set of sweetpotato clones evaluated across varrying ecogeographic conditions in Peru. Crop Science 45: 2165-2171 Haleegoah J, Okai E (1992) The role of women in root crop production for food security in Africa. In: Akorada MO (ed.) Root Crops for Food Security in Africa. Fifth triennial Symposium of the International Society for Tropical Root Crops. Kampala, Uganda. November 22-28 38 Hallauer AR, Miranda JB (1988) Quantitative genetics in maize breeding. 2nd ed. Ames: Iowa State University Press Halvorsen BL, Carlsen MH, Phillips KM, Bohn SK, Holte K, Jacobs DR Jr, Blomhoff R (2006) Content of redox-active compounds (i.e., antioxidants) in foods consumed in the United States. The American Journal of Clinical Nutrition 84: 95-135 Harlan JR (1992) Crops and man, 2nd ed. American Society of Agronomy, Crop Science Society of America, Madison Hershey CH (1987) Cassava germplasm resources. In: Hershey CH (ed.) Cassava Breeding: A multi-disciplinary review. Proceedings of the Cassava Workshop held in Philippines, 4-7 March, 1985. CIAT, Cali, Colombia. pp. 96-151 Hershey CH, Ocampo C (1989) New marker genes found in cassava. Cassava Newsletter 13: 1-5 Howeler RH, Cadavid LF (1983) Accumulation and distribution of dry matter and nutrients during a 12 months growth cycle of cassava. Field Crops Research 7: 123-139 Huang Z, Liu Y, Guangying Q, Brand D, Zheng SG (2018) Role of vitamin A in the immune system. Journal of Clinical Medicine 7: 258 IFAD and FAO (2005) A review of cassava in Africa with country case studies on Nigeria, Ghana, Tanzania, Uganda and Benin. Proceedings of the validation forum on the global cassava development strategy, Rome, Volume 2 Iglesias CA, Mayer J, Chavez L, Calle F (1997) Genetic potential and stability of carotene in cassava roots. Euphytica 94: 367-373 IITA (International Institute of Tropical Agriculture) (1974) Annual Report of the International Institute of Tropical Agriculture IITA, Ibadan, Nigeria Institute of Medicine, Food and Nutrition Board (2000) Β-carotene and other carotenoids. Dietary reference intakes for vitamin C, vitamin E, selenium, and carotenoids. Washington, D.C.: National Academy Press. pp. 325-400 Institute of Medicine (2001) Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc National Academy Press, Washington, D.C. Jaramillo G, Morante N, Pérez JC, Calle F, Ceballos H, Aria B, Bellotti AC (2005) Diallel analysis in cassava adapted to the midaltitude valleys environment. Crop Science 45: 1058-1063 Jauhar PP (2006) Modern biotechnology as an integral supplement to conventional plant breeding: the prospects and challenges Crop Science 46: 1841-1859 39 Jos JS, Nair SG, Moorthy SN, Nair RB (1990) Carotene enhancement in cassava. Journal of Root Crops 16: 5-11 Kakes P (1990) Properties and functions of the cyanogenic system in higher plants. Euphytica 48: 25-43 Kambal AE, Webster OJ (1965) Estimates of general and specific combining ability in grain sorghum, Sorghum vulgare Pers. Crop Science 5: 521-523 Kang MS (1994) Applied quantitative genetics. Baton Rouge, LA, USA Kawano K, Daza P, Amaya A, Rios M, Goncalvez MF (1978) Evaluation of cassava germplasm for productivity. Crop Science 18: 377-380 Kawano K, Fukuda WMG, Cenpukdee U (1987) Genetic and environmental effects on dry matter content of cassava root. Crop Science 27: 69-74 Kawano K, Narintaraporn K, Narintaraporn P, Sarakarn S, Limsila A, Limsila J, Suparhan D, Sarawat V, Watananonta W (1998) Yield improvement in a multistage breeding program for cassava. Crop Science 38: 325-332 Kennedy G, Nantel G, Shetty P (2003) The scourge of '' hidden hunger'': global dimensions of micronutrient defiencies. Food Nutrition and Agriculture 32: 8-16 Khan SA, Ahmad H, Khan A, Saeed M, Khan SM, Ahmad B (2009) Using line x tester analysis for earliness and plant height traits in sunflower (Helianthus annuus L.). Recent Research in Science and Technology 1: 202-206 Kizito EB, Chiwona-Karltun L, Egwang T, Fregene M, Westerbergh A (2007) Genetic diversity and variety composition of small-scale farms in Uganda: an interdisciplinary study using genetic markers and farmer interviews. Genetica 130: 301-318 Klug WS, Cummings MR (2005) Essentials of genetics. 5th ed. Upper Saddle River, New Jersey. Pearson Education, Inc La Frano MR, Woodhouse LR, Burnett DJ (2013) Bio-fortified cassava increases β- carotene and vitamin A concentrations in the TAG-rich plasma layer of American women. British Journal of Nutrition 110: 310-320 La Frano MR, Woodhouse LR, Burnett DJ, Burri BJ (2012) Biofortified cassava increases β-carotene and vitamin A concentrations in the TAG-rich plasma layer of American Women. British Journal of Nutrition 21: 1-11 Lahai MT, Ekanayake IJ (2009) Accumulation and distribution of dry matter in relation to root yield of cassava under a fluctuating water table in inland valley ecology. African Journal of Biotehnology 8:4895-4905 40 Lamkey KR, Edwards JW (1999) The quantitative genetics of heterosis. In: Coors JG, Pandey S (eds.) Proceedings of the International symposium on genetics and exploitation of heterosis in crops, CIMMYT Mexico 1997 ASA, CSSA and SSSA. pp. 31-48 Latif S, Müller J (2015) Potential of cassava leaves in human nutrition: A review. Trends in Food Science and Technology 44: 147-158 Lokko Y, Danquah EY, Offei SK, Dixon AGO, Gedil MA (2005) Molecular markers associated with a new source of resistance to the cassava mosiac disease. African Journal of Biotechnology 4: 873-881 Mahungu NM (1987) Selection for improved root quality in cassava. In: Hershey CH (ed.) Cassava breeding: A multidisplinary review. Proceedings of a workshop held in the Philippines, 4-7, March, 1985. Centro International de Agricultura Tropical (CIAT), Cali, CO. pp. 89-103 Mahungu, NM (1998) Cassava germplasm enhancement in Southern Africa. In: Akoroda, MO and Teri, JM (eds.) Food security and crop diversification in SADC countries. The role of cassava and sweet potato. Proceedings of the Scientific Workshop for the Southern Africa Root Crops Research Network held at Pamodzi hotel, Lusaka, Zambia. pp. 100-109 Maiani G, Castón MJ, Catasta G, Toti E, Cambrodón IG, Bysted A, Granado-Lorencio F, Olmedilla-Alonso B, Knuthsen P, Valoti M, Böhm V, Mayer-Miebach E, Behsnilian D Schlemmer U (2009) Carotenoids: actual knowledge on food sources, intakes, stability and bioavailability and their protective role in humans. Molecular Nutrition & Food Research 53 Suppl 2: S194-218 Makokha AO, Tunje TK (2005) Potential for alleviating vitamin A deficiency in East Africa through cassava and sweet potato tubers. African Crop Science Conference Proceedings 7: 643-646 Mann C (1997) Reseeding the green revolution. Science 277: 209-220 Manu-Aduening JA, Peprah BB, Bolfrey-Arku G, Aubyn A (2014) Promoting farmer participation in client- oriented breeding: lessons from participatory breeding for farmer- preferred cassava varieties in Ghana. Advanced Journal of Agricultural Research 2: 8-17 Manu-Aduening J, Lamboll R, Dankyi A, Gibson R (2005) Cassava diversity in Ghanaian farming systems. Euphytica 144: 331-340 41 Manu-Aduening J, Lamboll RI, Ampong Mensah G, Lamptey JN, Moses E, Dankyi AA, Gibson RW (2006) Development of superior cassava cultivars in Ghana by farmers and scientists: The process adopted outcomes and contributions and changed roles of different stakeholders. Euphytica 150: 47-61 Maris M (1989) Analysis of an incomplete diallel cross among three ssp. tuberosum varieties and seven longday adapted ssp. andigena clones of the potato (Solanum tuberosum L.). Euphytica 41: 163-182 Maroya NG, Asante IK, Dixon AGO (2010) Genotype by environment interaction effect in beta carotene of yellow root cassava (Manihot esculenta Crantz) genotypes in Ghana. Proceedings, 11th ISTRC- AB Symp. Kinshasa, DR Congo Maroya NG, Kulakow P, Dixon AGO, Maziya-Dixon BB (2012) Genotype x environment interaction of mosaic disease, root yields and total carotene concentration of yellow- fleshed cassava in Nigeria. International Journal of Agronomy 2012: 1-8 Maziya-Dixon B, Kling JB, Menkir A, Dixon A (2000) Genetic variation in total carotene, iron, and zinc contents of maize and cassava genotypes. Food and Nutrition Bulletin 21: 410-413 Menkir A, Maziya-Dixon B (2004) Influence of genotype and environment on β-carotene content of tropical yellow-endosperm maize genotypes. Maydica 49: 313-318 MOFA (Ministry of Food and Agriculture) (2012) Agriculture in Ghana. Facts and Figures, Statistics, Research and Information Directorate (SRID), Accra, Ghana Montagnac JA, Davis CR, Tanumihardjo SA (2009a) Nutritional value of cassava for use as a staple food and recent advances for improvement. Comprehensive Reviews in Food Science and Food Safety 8: 181-194 Montagnac JA, Davis CR, Tanumihardjo SA (2009b) Processing techniques to reduce toxicity and antinutrients of cassava for use as a staple food. Comprehensive Reviews in Food Science and Food Safety 8: 17-27 Moorthy SN (1994) Tuber crops starches. Central Tuber Crops Research Institute. Technical Bulletin Series: 18. St. Josephs Press, Cotton Hill, Thiruwanathapuram, Kerala, India Morante N, Sánchez T, Ceballos H, Calle F, Pérez JC., Egesi C, Cuambe CE, Escobar AF, Ortiz D, Chávez AL, Fregene M (2010) Tolerance to postharvest physiological deterioration in cassava roots. Crop Science 50: 1333-1338 Mtunda KJ (2009) Breeding, evaluation and selection of cassava for high starch content and yield in Tanzania. Doctoral thesis. African Centre for Crop Improvement, University of KwaZulu-Natal, South Africa 42 Nambisan B (2011) Strategies for elimination of cyanogens from cassava for reducing toxicity and improving food safety. Food and Chemical Toxicology 49: 690-693 Nassar NMA (2005) Cassava: Some ecological and physiological aspects related to plant breeding. Gene Conserve 3: 229-245 Nassar NMA (2006) Cassava genetic resources: extinct everywhere in Brazil. Genetic Resources and Crop Evolution 53: 975-983 Nassar NMA (2007) Cassava genetic resources and their utilization for breeding of the crop. Genetics and Molecular Research 6: 1151-1168 Nassar, NMA, Ortiz R (2010) Breeding cassava to feed the poor. Scientific American Journal 302: 78-82 Nduwumuremyi A, Tongoona P, Habimana S (2013) Mating designs: helpful tool for quantitative breeding analysis. Journal of Plant Breeding and Genetics 01: 117- 129 Negi PS, Roy SK (2000) Effect of blanching and drying methods on β-carotene, ascorbic acid and chlorophyll retention of leafy vegetables. LWT-Food science and Technology 33: 295-298 Njenga P, Edema R, Kamau J (2014) Combining ability for beta-carotene and important quantitative traits in a cassava F1 population. Journal of Plant Breeding and Crop Science 6: 24-30 Njoku, D, Gracen V, Egesi CN, Asante I, Offei SK, Okogbenin E, Kulakow P, Eke-Okoro ON, Ceballos H (2011) Breeding for enhanced b-carotene content in cassava: constraints and accomplishments. Journal of Crop Improvement 25: 560-571 Njoku DW (2012) Improving Beta-carotene content in farmers’ preferred cassava cultivars in Nigeria. Doctoral thesis, West Africa centre for crop improvement, University of Ghana, Legon, Ghana. NRCRI Annual Report. 2008. National Root Crops Research Institute, Umudike Nweke FI (1996) Cassava: A cash crop in Africa. Collaborative study of cassava in Africa Working Paper No. 14, International Institute of Tropical Agriculture, Ibadan, Nigeria Nweke FI (2004) New challenges in the cassava transformation in Nigeria and Ghana. Environment and production technology division. Discussion paper NO 118. IFPRI, USA Nweke FI, Spencer DSC, Lynam JK (2002) The cassava transformation: Africa’s best kept secret. Michigan State University Press, Lansing, Michigan, USA Nyerhovwo JT (2004) Cassava and the future of starch. Electronic Journal of Biotechnology 7: 5-8 43 Nyirenda DB, Chiwona-Karltun L, Chitundu M , Haggblade S, Brimer L (2011) Chemical safety of cassava products in regions adopting cassava production and processing – Experience from Southern Africa. Food and Chemical Toxicology 49: 607-612 O’Brien GM, Wheatley CC, Iglesias C, Poulter NH (1994) Evaluation, modification, and comparison of two rapid assays for cyanogens in cassava. Journal of the Science of Food and Agriculture 65: 391-399 Obilie EM, Tano-Debrah K, Amoa-Awua WK (2004) Souring and breakdown of cyanogenic glycosides during the processing of cassava into akyeke. International Journal of Food Microbiology 93: 115-121 Oduro KA (1981) Some characteristics of yellow pigmented cassava. In: Terry ER, Oduro KA, Caveness F (eds.) Proceedings of first tropical symposium of the international Society for Tropical Root Crops - Africa Branch (ISTRC-AB). pp. 42-44 Oelsligle DD (1975) Accumulation of dry matter, nitrogen, phosphorus and potassium in cassava (Manihot esculanta Crantz). Turrialba 25: 85-87 Okai E (2001) Genetic diversity in some local cassava cultivars in Ghana. MPhil. Thesis, Crop Science Department, Faculty of Agriculture, University of Ghana, Legon, Ghana Olalekan A, Labuschagne M, Fregene M (2011) Increased storage protein from interspecific F1 hybrids between cassava (Manihot esculenta Crantz) and its wild progenitor (M. esculenta ssp. Flabellifolia). Euphytica 183:303-311 Oloya B, Adaku C, Ntambi E, Andama M (2017) Detoxification of Nyar-Udota Cassava Variety in Zombo District by Fermentation. International Journal of Nutrition and Food Sciences 6: 118-121 Onwueme IC (2002) Cassava in Asia and the Pacific. In: Hillocks RJ, Thresh JM, Bellotti AC (eds.) Cassava: Biology, Production and Utilization. CABI Publishing Oxon, UK and New York, USA. pp. 55-65 Onyenwoke CA, Simonyan KJ (2014) Cassava- post harvest processing and storage in Nigeria: A Rewiew. African Journal of Agricultural Science 9: 3853-3863 Oresegun A, Fagbenro OA, Ilona P, Bernard E (2016) Nutritional and anti-nutritional composition of cassava leaf protein concentrate from six cassava varieties for use in aqua feed. Food Science and Technology 2: 1147323 Ortiz D, Sánchez T, Morante N, Ceballos H, Pachon H, Duque MC, Chavez AL, Escobar AF (2011) Sampling strategies for proper quantification of carotenoid content in cassava breeding. Journal of Plant Breeding and Crop Science 3: 14-23 44 Ortiz R, Madsen S, Wagoire WW, Hill J, Chandra S, Stolen O (2001) Additive main effect and multiplicative interaction model for diallel-cross analysis. Theoretical and Applied Genetics 102: 1103-1106 Pan American Health Organization (2005) Providing vitamin A supplements through immunization and other health contacts for children 6–59 months and women up to 6 weeks postpartum: A guide for health workers, 2nd edition Parada J, Aguilera JM (2007) Food microstructure affects the bioavailability of several nutrients. Journal of Food Science 72: R21-R32 Parkes EY (2011) Assessment of genetic diversity, combining ability, stability and farmer preference of cassava germplasm in Ghana. PhD thesis, Department of Plant Sciences (division of Plant Breeding), University of the Free State, South Africa Parkes EY, Fregene M, Dixon AGO, Peprah BB and Labuschagne MT (2013) Combining ability of cassava genotypes for cassava mosaic disease and cassava bacterial blight, yield and its related components in two ecological zones in Ghana. Euphytica 194: 13-24 Peninah N, Edema R, Kamau J (2014) Combining ability for beta-carotene and important quantitative traits in a cassava f1 population. Journal of Plant Breeding and Crop Science 6: 24-30 Peroni N, Kageyama PY, Begossi A (2007) molecular differentiation, diversity and folkclassification of “sweet” and “bitter” cassava (Manihot esculenta) in Caicara and Caboclo management system (Brazil). Genetic Resources and Crop Evolution 54: 1333-1349 Popoola TOS, Yangomodou OD (2006) Extraction, properties and utilization potential of cassava seed oil. Biotechnology 5: 38-41 Pujol B, Renoux F, Elias M, Rival L, Mckey D (2007) The unappreciated ecology of landraces population: Conservation consequences of soil seed banks in cassava. Biological Conservation 136: 541-551 Quick JS (1978) Combining ability, interrelationship among an international array of durum wheat. In: Ramanujam S (ed.) Proceedings of the fifth international wheat genetic symposium. New Delhi. pp. 634-647 Rahmat A, Kumar V, Fong LM, Endrini S, NSani HA (2003) Determination of total antioxidant activity in three types of local vegetables shoots and the cytotoxic effect of their ethanolic extracts against different cancer cell lines. Asia Pacific Journal of Clinical Nutrition 12: 308-311 45 Rajendran PG (1989) Combining ability in cassava. Journal of Root Crops 15: 15-18 Raji AA, Dixon AGO, Fawole I, Gedil M (2001) Diversity analysis of African landraces of cassava as assessed with agro botanical traits and molecular markers. In: Fauquet CM, Taylor NJ (eds.) Cassava: An ancient crop for modern times. Proceedings of the fifth International Meeting of the CBN. CD3, CBN-V Video Archive-S6-25 Ramanujam T (1990) Effect of moisture stress on photosynthesis and productivity of cassava. Photosynthetic 24: 217-224 Ravindran V, Kornegay ET, Cherry JA (1983) Feeding values of cassava tuber and leaf meals. Nutrition Report Intentional 28: 189-196 Rodriguez-Amaya DB, Kimura M (2004) HarvestPlus handbook for carotenoid analysis. HarvestPlus Tech. Monograph series 2. International Food Policy Research Institute (IFPRI) and International Center for Tropical Agriculture (CIAT). Washington, DC and Cali, Colombia Ruiz de Galarreta JI, Ezpeleta B, Pascualena J, Ritter E (2006) Combining ability and correlations for yield components in early generations of potato breeding. Plant Breeding 125: 183-186 Safo-Kantanka O, Aboagye P, Amartey SA, Oldham JH (1984) Studies on the content of yellow-pigment scassava. In: Terry ER, Doku EV, Arene OB, Mahungu NM (eds.) Tropical Roots crops production and uses in Africa. IDRC, Ottawa, Canada. pp. 103- 104. Saltzman A, Birol E, Bouis H, Boy E, De Moura F, Islam Y, Pfeiffer W (2013) Bio fortification: progress toward a more nourishing future. Global Food Security 2: 9- 17 Sánchez T, Ceballos H, Dufour D, Ortiz D, Morante N, Calle F, Zum-Felde T, Dominguez M, Davrieux F (2014) Prediction of carotenoids, cyanide and dry matter contents in fresh cassava root using NIRS and Hunter color techniques. Food Chemistry 151: 444-451 Sánchez T, Chávez AL, Ceballo H, Rodriguez-Amaya DB, Nestel P, Ishitani M (2006) Reduction or delay of post-harvest physiological deterioration in cassava roots with higher carotenoids content. Journal of the Science of Food and Agriculture, 86: 634- 639 Shaffi B, Mahler KA, Price WJ, Auld DL (1992) Clone x Environment interaction effects on winter rape seed yield and oil content. Crop Science 32: 922-927 Shull GH (1908) The composition of a field of maize. American Breeders Association 4: 296-301 46 Sprague GF (1983) Heterosis in maize: theory and practice. In: Frankel R (ed.) Heterosis. Monographs on Theoretical and Applied Genetics 6: 47-70 Sprague GF, Tatum LA (1942) General versus specific combing ability in single crosses of corn. Journal of the American Society of Agronomy 34: 923-932 Spurgeon SL, Porter JW (1980) Carotenoids. In: Stumpf PK, Conn EC (eds.) The Biochemistry of Plants. Academic Press volume 4. pp. 420-483 Ssemakula G, Dixon AGO (2007) Genotype x environment interaction, stability and agronomic performance of carotenoid- rich cassava clones. Scientific Research and Essay 2: 390-399 Ssemakula G, Dixon AGO, Maziya-Dixon B (2007) Stability of total carotenoid concentration and fresh yield of selected yellow- fleshed cassava (Manihot esculenta Crantz). Journal of Tropical Agriculture 45: 14-20 Stahl W, Heinrich U, Jungmann H, Sies H, Tronnier H (2000) Carotenoids and carotenoids plus vitamin E protect against ultraviolet light induced erythema in humans. The American Journal of Clinical Nutrition 71: 795-798 Susan JK, Ravindran CS, George J (2005) Long-term fertilizer experiments: three decades of experience in cassava. Central Tuber Crops Research Institute. Sreekariyam, Thiruvanathapuram, Kerala, India Tan SL, Mak C (1995) Clone x environment influence on cassava performance. Field Crops Research 42: 111-123 Tavora FJAF, Melo FIO, Pinho JLN, Queiroz de GM (1995) Yield, crop growth rate and assimilatory characteristics of cassava at the coastal area of Ceara. Revista Brasileira de Fisiologia Vegetal 7: 81-88 Tewe OO (1992) Detoxification of cassava products and effects of residual toxins on consuming animals. Roots, tubers, plantains and bananas in animal feeding. David Machin, Solveig Nyvold, Series, FAO Animal Production and Health Papers 95: 221 Thakkar SK, Huo T, Maziya-Dixon B, Failla ML (2009) Impact of style of processing on retention and bio-accessibility of β-carotene in cassava (Manihot esculanta, Crantz). The American Journal of Clinical Nutrition 57: 1344-1348 Thompson RNA (2013) Genetic analysis of postharvest physiological deterioration in cassava (Manihot esculenta Crantz) storage roots. PhD thesis, WACCI, University of Ghana, Legon 47 Thro AM, Msabaha M, Kulembeka H, Shengerow, Kapande A, Mlingi M, Hemed L, Digges P, Cropley J (1995) Proceedings of the 2nd International scientific meeting of the Cassava Biotech Network. In: Thro AM, Roca WM (eds.) Bogor Indonesia working document 150 CIAT Cali Colombia. pp. 28-35 Thurnham DI, McCabe GP, Northrop-Clewes CA, Nestel P (2003) Effect of subclinical infection on plasma retinol concentrations and assessment of prevalence of vitamin A deficiency: meta-analysis. Lancet 362: 2052-2058 UNICEF (2014) United Nations children’s Fund. UNICEF East Asia and Regional Office (EAPRO) van Lieshout M (2001) Bioefficacy of β-carotene dissolved in oil studied in children in Indonesia. American Society for Nutrition 73: 949-958 van Oirschot OEA, O’ Brien GM, Duffour DO, El- Sharkawy MA, Musa E (2000) The effect of pre-harvest pruning of cassava upon most deterioration and quality characteristics. Journal of the Science of Food and Agriculture 80: 1866-1873 Veda S, Platel K, Srinivasan K (2007) Varietal differences in the bioaccessibility of β- carotene from mango (Mangiferaindica) and papaya (Carica papaya) fruits. Journal of Agricultural and Food Chemistry 19: 7931-7935 Vimala, B, Nambisan B, Theshara R, Munnikrishnam (2008) Variability of carotenoids in yellow-flesh casava (Manihot esculenta Crantz). Gene Conserve 31: 676-685 Wardlaw GM, Hampl JS, DiSilvestro RA (2004) Perspective in Nutrition. Sixth edition McGraw-Hill College Welch RM, Graham RD (2005) Agriculture: The real nexus for enhancing bioavailable micronutrients in food crops. Journal of Trace Elements in Medicine and Biology 18: 299-307 Westby A (2002) Cassava utilization, storage and small-scale processing. In: Hillocks RJ, Thresh JM, Bellotti AC (eds.) Cassava biology, production and utilization. CABI Publishing, Wallingford, UK. pp. 281-300 White WLB, Arias-Garzon DI, McMahon JM, Sayre RT (1998) Cyanogenesis in cassava, the role of hydroxynitrile lyase in root cyanide production. Plant Physiology 116: 1219-1225 Witcombe JR, Petre R, Jones S, Joshi A (1999) Farmer participatory crop improvement. The spread and impact of rice varieties identified by varietal selection. Experimental Agriculture 35: 471-487 48 Wobeto C, Correa AD, de Abreu CMP, dos Santos CD, de Abreu JR (2006) Nutrients in the cassava (Manihot esculenta, Crantz) leaf meal at three ages of the plant. Cienc Technol Aliment 26: 865-869 Wong JC, Lambert RJ, Wurtzel ET, Roncherford TJ (2004) QTL and candidate genes phytoene synthase and f-carotene desaturase associated with the accumulation of carotenoids in maize. Theoretical and Applied Genetics 108: 349-359 Yan W, Hunt A (2002) Biplot analysis of diallel data. Crop Science 42: 21-30 49 Chapter 3 Awareness, perception and willingness to adopt yellow flesh cassava through participatory rural appraisal in coastal savannah, forest and forest-transition zones in Ghana Abstract Deficiency of micronutrients, such as vitamin A, constitutes a major public health problem in Ghana, which affects mainly children and women. Qualitative and participatory approaches were employed to investigate preferences of producers and processors, their perceptions of, willingness to adopt, and inhibitory factors to adoption of newly developed biofortified cassava cultivars. A total sample of 113 producers and processors participated in 12 focus groups in three cassava growing districts within three agro-ecological zones (forest, transition and coastal) using a semi-structured interview schedule. The results showed that knowledge and use of improved varieties and the new yellow flesh cassava was generally low among men and women farmers in all the three locations visited. Participants preferred other traits such as climate smart varieties, long storability in the soil and mult- purpose varieties in addition to the high yielding trait. Factors that contributed to low technology adoption were lack of awareness, access and availability of new varieties, gender stereotyping, access to extension services and perception of the new varieties. It is recommended that cassava breeders focus more on education of producers and consumers in terms of potential benefits of the biofortified varieties. They should also incorporate the identified trait preferences in their breeding objectives and adopt a multi- stakeholder approach in improving the cassava seed systems in Ghana. 3.1 Introduction Cassava is grown primarily for its starchy storage roots and is an important staple for more than 800 million people, mostly in sub-Saharan Africa and other parts of the world (Anna et al. 2010). In terms of calories consumed, it is the second most important food staple (Tarawali et al. 2012) and mostly called Africa's food insurance crop because it gives appreciable yield even in the face of drought and poor soil management (Dixon et al. 2003). However, current cassava varieties mainly cultivated in Ghana produce roots which are high in DM but low in VA, protein, fat, minerals and other micronutrients (Ceballos et al. 2007; Thakkar et al. 2009). VAD constitutes a public health problem and affects mainly children and women. The HarvestPlus, a development programme involving a global alliance of 50 research institutions, initiated the development of micronutrient-dense staple crops (Bouis et al. 2011; Dwivedi et al. 2012). Among these initiatives is the development of biofortified cassava clones with high content in the roots (yellow colouration). VA is involved in the normal functioning of the human visual and immune systems and is an essential vitamin. It also affects growth and development, and is involved in the reproduction process and the maintenance of epithelial cellular integrity (ACC/SCN 2000; Combs 1998). Mortality in children can be reduced by 23 to 30% when their VA status is improved (Beaton et al. 1993). It is estimated that 75 to 251 million children have subclinical symptoms of VAD (WHO 2009). Four strategies have been developed and applied to improve the status of VA, especially among the vulnerable groups in Africa. These are dietary diversification, food fortification, supplementation and biofortification. The first three approaches are relatively cost-effective, but have failed to eradicate the problem of VAD, for a variety of reasons (West 2003). Biofortification is an approach that relies on conventional plant breeding and modern biotechnologies to increase the micronutrient density of staple crops, including cassava (Bouis 2003; Nestel et al. 2006). It holds promise for improving the nutritional and health status of poor populations in both rural and urban areas of developing countries like Ghana and Nigeria. Cassava is one of the crops targeted for biofortification, as is consumed daily by large numbers of people in sub-Saharan Africa. Significant progress has been made in pVA cassava research in Ghana in the past few years and cultivars are at an advanced stage of release to Ghanaian farmers. The challenge is stimulating wider uptake of the varieties and incorporation into the food systems. A number of plant breeding programmes in developing countries such as Ghana haven’t been impactive due to lack of consideration of farmers’ needs and concerns, leading to low adoption rates of released varieties (Kamau et al. 2011). Witcombe (2009) reported that the active participation of target clients such as farmers in breeding can help breeders select important traits needed in hybridization, but this aspect is mostly ignored, a phenomenon, which, in part, explains why many farmers at times continue to grow landraces, which have farmer-preferred attributes as opposed to the new officially, released varieties (Witcombe 2009). PPB has been used by various researchers to bring all stakeholders together with the aim of addressing this challenge and narrowing the communication gap between scientists and farmers. These gaps have important implications for productivity and food security. Many scientists have successfully implemented PPB in selecting superior cassava cultivars in various countries. Manu-Aduening et al. (2006) implemented a successful PPB 51 programme for developing superior cassava cultivars in Ghana by involving farmers early in the process (the seedling nursery trial stage). Schofield et al. (2009) similarly involved farmers in participatory varietal selection of cassava varieties in the Great Lakes area. Kamau et al. (2011) also implemented PPB in Kenya resulting in the successful selection of 30 genotypes (which combined early-bulking and high root quality traits) after a preference tests by farmers in KARI-Kiboko farm (Eastern Kenya),. Several authors, including Morris and Bellon (2004), Mangione et al. (2006), Gyawali et al. (2007) and Manu-Aduening et al. (2014) have confirmed that participatory breeding improves breeding efficiency, accelerate adoption, lead to more acceptable varieties, promotes genetic diversity and saves cost through reduction in breeding cycle. PPB utilizes many approaches such as participatory varietal selection, surveys, key informant interviews and focus group discussions. In social science, this is termed Participatory Rural Appraisal (PRA) approaches, which is heavily reliant on community participation. This method is designed in a way to involve farmers as partners. Farmers are therefore responsible for gathering and analyzing information, meaning they are not just only serving as sources of information but as analyst. These two aspects of the process provide vital information required by farmers (Kamau et al. 2011; Were et al. 2012) to guide the uptake of new varieties. The involvement of farmers at some breeding stages could change their conservative behavior and promote the adoption of new genotypes by incorporating their preferred traits (Nduwumuremyi et al. 2016). Understanding factors that affect famers' adoption of new cassava varieties, especially yellow flesh cassava, could be useful to plant breeders in designing their research programmes. Thus, breeding objectives targeting the end-user preference could therefore enhance the adoption of new varieties. 3.1.1 Status of yellow flesh cassava in Ghana Cassava is a perennial crop grown mainly for its storage roots and belongs to the economically important family Euphorbiaceae (Boampong and Sarfo-Kantanka 2014; Amenu 2015). Different varieties (both white and yellow flesh cassava) have been cultivated for many decades across the various suitable cassava growing regions in Africa and within Ghana. However, yellow flesh cassava is known to be cultivated on a limited scale in Colombia, Philippines, Jamaica and some African countries, and Ghana is no exception (Okoro 2015). In the past, local germplasm was collected in parts of Ashanti, Eastern, Volta, Greater Accra, Western and Central regions under the National Agricultural Research Project Root and Tuber Crops Research Programme. These collections were characterised 52 at CSIR-Crops Research Institute (CRI), CSIR-Plant Genetic Resources Research Institute and the Department of Crop Science, University of Cape Coast (Annor-Frimpong 1991). This germplasm may have contained the common local yellow variety called “Bankye borodee” literally translated as “cassava-plantain” due to its pulp colour. In 1998, Boampong and Sarfo-Kantanka (2014) made a total collection of 212 landraces, from which they selected 36 accessions to generate the ethno-botanical information. Among these accessions, was the common local yellow variety “Bankye borodee” which was found in farmers’ fields. This shows that up to the 20th century, this local yellow variety had been common, though the germplasm kept eroding. With constant genetic improvement and the development of new varieties, farmers adopted new varieties that meet their needs and offer them more satisfaction and livelihood security. As they adopted new varieties, landraces and old varieties were dropped. New developments in the cassava sector led to the loss of most of the landraces, old improved varieties and the popular local yellow flesh variety. Currently, in Ghana, very few farmers, especially the adults, have few stands of the yellow flesh cassava on their farms with very limited planting materials, as identified in this study. The objectives of this study were i) to assess and determine the importance of cassava select Ghanaian communities in threeecologies; ii) to identify cassava preferred traits by farmers; iii) to investigate farmer/stakeholders' awareness, perception and preference for yellow flesh cassava cultivars and iv) to examine factors that could possibly affect adoption of new cassava varieties in the select communities of Ghana. 3.2 Materials and methods 3.2.1 Brief description of area The study was conducted in Techiman municipal, Agona East and Ketu North districts in the forest-transition, forest and coastal savannah zones of Ghana, respectively. Geographically, Techiman Municipal lies between longitudes 1°49′ east and 2°30′ west and latitude 8°00′ north and 7°35′ south, Agona East at latitudes 5o 30″ and 5o 50″ north and longitudes 0o35″ and 0o55″ west and Ketu North districts at latitude 6o03’N and 6o20’N and longitude 0o49’E and 1o05’E. (GSS, 2015). These three districts were selected based on the high volumes of cassava produced and the high processing activities that occurs in these locations. 53 3.2.2 Data collection procedures Prior to the main field work, there were several engagements with the extension agents and the research team at the various districts, to assist in the mobilization of farmers and processors drawn for the study. All preliminary desk reviews on selecting participants for focus group and key informant interviews and guidelines for conducting focus group to ensure effective field work as described by Elias (2013), were carried out. A mixed method approach to data collection was adopted to collect both qualitative and part of the quantitative data, which focused on demographic and farm/processing level characteristics. The PRA approach was, however, the main method used in the data collection. This approach was adopted to create awareness and enhance the uptake of the yellow flesh cassava varieties in future, when released (Turyahikayo 2014). The qualitative tool used was the focus group discussions, key informant interviews and personal observations. The study instruments were peer-reviewed and enumerators trained on them prior to data collection. Within each district, four cassava growing villages and 10 participants from each village were sampled for the group discussions. Participants were then conveyed to a central location for the meeting. Four focus group discussions were held in each district, involving producers and processors. A total of 12 focus groups, involving 8-10 persons per group, were held across the three districts. The study combined all three types of questions (engagement, exploration and exit) during the group discussions and the key informant interviews. Conscious efforts were made to have a representation of adult males, adult females and young adults for the discussion. A total of 113 producers and processors participated, representing a 94.2% rate of response. From the social perspective, adults and young adults were defined based on the community’s accepted definition; that is that a young adult was someone between the ages of 18-45 years and adult; above 45 years. Qualitatively, an adult was described as a married person or someone who has given birth and the young adult as the unmarried or an active male or female living under the parents’ roof. In Techiman Municipal and Agona East districts, the adults were more than the young adults, so groups were disaggregated based on sex (male and female), however, at Ketu North district, the project team sticked to the planned design (disaggregation based on gender; adults and young adults) and this was the first district visited. 54 The disaggregation was done to allow full participation of all the targeted direct participants and to elicit gender-based responses. Ten participants made up of five producers and five processors were sampled from 12 communities, with gender considerations in the sampling design. Data collected covered land tenure arrangements, crops grown in the communities, cassava varieties cultivated and processed by men and women, cassava traits preferred by men and women, importance of cassava to study groups, access to productive resources, adoption and dis-adoption of varieties, awareness, preferences and perception of yellow flesh cassava and demographic variables of respondents. 3.2.3 Data analysis The data collected was analyzed using a content analysis approach as described by Elo and Kyngas (2008), which was also adopted by Esuma et al. (2019) in their study on “Men and women’s perception of yellow flesh cassava among rural farmers in eastern Uganda” and descriptive statistics. Primarily, the field notes and audio recordings were first transcribed for each of the groups and key informants. The responses were coded to identify emerging themes, categorized and analyzed separately for adult producers, young adults, producers, adult processors and young adults processors. Lastly, comparative analysis across the districts were done to examine consistency in results and draw inferences from the study. The descriptive statistics were used to describe the basic features of the demographic information collected. These included frequencies and means distributions. 3.3 Results 3.3.1 Characteristics of survey sample Some similarities existed between the two groups; producers and processors, and results are presented in Tables 3.1, 3.2 and 3.3. The study recorded a high percentage of young adults’ participation (based on communities’ definition); 53.8% of producers and 62.5% of processors, respectively. 55 Table 3.1 Characteristics of survey sample (qualitative variables) Characteristics Producers Processors Frequency Percentage Frequency Percentage (N=65) % (N=48) % Sex Male 55 84.6 15 31.2 Female 10 15.4 33 68.8 Social groups (as defined by community) Adults (>45 years) 30 46.2 18 37.5 Young adults (18-45 yrs) 35 53.8 30 62.5 Head of household Yes 53 81.5 20 41.7 No 12 18.5 28 58.3 Marital status Married 53 81.5 41 85.4 Single 11 16.9 1 2.1 Divorced - - 3 6.2 Widowed 1 1.5 3 6.2 Highest level of education No formal education 10 15.4 12 25.0 Basic 46 70.8 34 70.8 Secondary 8 12.3 1 2.1 Tertiary 1 1.5 1 2.1 Residential status Native 45 69.2 19 39.6 Permanent settlers 20 30.8 29 60.4 Tenancy arrangements Family land 12 18.5 Ownership 16 24.6 Purchased 1 1.5 Rent 25 38.5 Sharecropping 11 16.9 Main occupation Farmer 65 100 - - Farmer-processor - - 10 20.8 Processor - - 38 79.2 Source of labour Hired 33 50.8 11 22.9 Family 8 12.3 12 25.0 Both (hired and family) 24 36.9 25 52.1 56 Table 3.2 Summary statistics for producers (quantitative variables) Variables Minimum Maximum Mean Standard deviation Age (years) 21 70 43.28 12.01 Farming experience (years) 1 45 17.26 11.31 Cassava farm size (hectares) 0.40 4.80 1.22 0.76 Total cropped land (hectares) 0.40 8.40 2.36 1.54 Years in school 0 15 8.23 4.00 Household size 1 20 7.57 4.04 Net income from cassava 150 7000 1524.62 1485.75 (GH¢) ($ 291.32) Net income from all crops 150 7500 2598.85 2132.67 grown (GH¢) ($ 418.17) Exchange rate: $1=GH¢5.1 (As at 25th APRIL, 2019) Table 3.3 Summary statistics for processors (quantitative variables) Variables Minimum Maximum Mean Standard deviation Age (years) 30 75 43.83 9.05 Processing experience (years) 2 30 10.44 7.72 Years in school 0 15 5.77 4.09 Household size 1 21 6.69 3.39 Net income from processing 480 4500 2105.0 1042.45 ($ 204.40) Exchange rate: $1=GH¢5.1 (As at 25th APRIL, 2019) Farming (100%) and processing as already envisaged by the study, provided livelihoods for farmers (100%), and most of processors (79.2%) in the communities. About 21% of processors cultivated cassava to feed their factories. Most producers were defined as small holders with land holdings of less than 2.5 hectares for all cultivated crops. Cassava production alone covered about 52% of the total land area under crop production, which showed the importance of cassava to these communities. Any significant change in the sector could impact a lot of lives. A small percentage (26.1%) of participants had right of 57 ownership of land compared to 73.9% who either farmed on family lands, who rented or practiced sharecropping. A number of differences were found between producers and processors. Most of the producers were males (84.6%) while the majority of the processors were females (68.8%). With regards to education, the majority of the producers (70.8%) and processors (70.8%) had attained basic level education, and 12.3% and 2.1% respectively had reached secondary level. Transition from the basic level to the secondary level showed a 58.5% and 68.7% drop for both the producers and processors in the community. Similar results were reported in Uganda, by Esuma et al. (2019). Considering the years spent in school, producers had spent more years (8.23) in school than processors (5.77). The majority of the producers (81.5%) were heads of their households compared to 41.7% of the processors. The majority (69.2%) of the producers are indigenes of the study communities compared to 60.4% of processors, who were migrant permanent settlers. 3.3.2 Types of crops cultivated in study area Farmers in the study area cultivated a variety of crops, both food and cash crops. The crops varied slightly between men and women. Several crops were cultivated in the community by men, women and young adults, which included cassava, plantain, cocoyam, sweetpotato, rice, pepper, maize, yam, tomatoes, cowpea, garden eggs, onion, okro, water melon, oil palm, cocoa, orange, cashew, banana, pawpaw and mango. Cassava ranked first, followed by maize, as food crops in all districts by all gender groups, since cassava was seen to perform well on poor soils and under harsh weather conditions. From the discussions and personal observations, it was realised that it is currently difficult to classify a crop as mainly a “food crop” since all the known food crops are targeted for cash. The tree crops could, however, be classified exclusively as cash crops, which were cultivated mostly by men. A male key participant at Techiman Municipal admitted that the women in the community were very hard working and cultivated all crops, and men also have “muscled” to cultivate everything. Both men and women groups narrated this: 58 “What makes us grow the cassava and cashew is that, I cannot cultivate the whole of my farm land, so I start with cassava which is 2 years. By the time I harvest, the cashew I added would have started sprouting. I do this because of insufficient capital”. Rebecca: cassava farmer in Techiman North. “It can be used for brewing, binding books and used for the textile industry in particular” Silvanus, male young adult in Ketu North. “When it comes to farming here, both men and women mostly grow crops equally. Women grow what they want, for instance pepper was previously for women but the men have taken over.” Male farmer “The men have taken over all the crops. They grow every crop here; cassava, vegetable (pepper, okro), cocoyam, plantain” Male participant. 3.3.3 Importance of cassava The pair-wise ranking was employed to examine the importance of cassava as a food or cash crop. Cassava was confirmed as an important food security and income-generating crop, adapted to different farming systems like intercropping and suitable for multiple food and industrial uses. Cassava was intercropped for several reasons, such as reduced labour cost for weeding and preparing new fields, as mentioned by both men and women, or to provide shade for the tree crops, as mentioned by men. This is also done as a sign of security and to generate extra income, especially among late maturing tree crops. 3.3.4 Access and control of productive resources The main productive resource considered in this study was land access related to the land acquisition process and size of operation. Investigating the access of men, women and young adults to land and tenure arrangements is critical for any variety uptake. Generally, access to land for farming in the study communities ranged from rights acquired through renting, to the right of permanent use of a piece of land through inheritance, which is termed as “family lands” or by total purchase. There were also some participants who were engaged in sharecropping and this was found common for the majority of the young adults. Participants in the focus group and the other key informants 59 “We have the mentality that men are stronger than women, so in allocating land, men are definitely given larger areas than women.” Male adult farmer. interview s m entioned th is. A fter a length y disc ussi on, it was rea lized that consid ering lan d as a nom inal ent ity, bot h me n and wom en h ad right of ac cess, how ever, the level of access differed by community. While in some communities, women could only access land through their husband or a relative, in other places there were no barriers, as reflected in the supporting quotes below: Women, however, farmed small sizes of land due to perceived lack of strength and capital resources, as reflected in the quotes in the box below - an issue of stereotyping by the communities. “If women want land they will get it, as long as they are not lazy they can get land. Women have access and control over inherited lands even in their married homes. Women do not need a man to lead them when renting land.” Male adult farmers in Techiman North. These statements were confirmed by the women and men groups at Techiman Municipal. “I know that if the land is very big the man will get it. Because they perceive women are not strong enough to cultivate big lands.” Female farmer. 3.3.5 K nowled ge o f im pr oved v ar ieties and sou rce of plant ing m ate ria ls In all the loca tion s vi sited , most men and w omen h ad little knowledge of the improved varieties, since they could not access the planting material. In addition, respondents had the perception that the improved varieties normally referred to as “Agric” were not 60 poundable and therefore did not serve their fundamental purpose of using it for “fufu”. Very few adult males and females cultivated the improved varieties, whose planting materials were received from extension officers or agricultural volunteers in the community. Thereafter, planting materials were mainly sourced from their own farms or from fellow farmers. Very few farmers had contact with agricultural extension officers in the localities. 3.3.6 Cassava varieties cultivated and the preferred traits by participants on gender basis The varieties cultivated by both producers and processors and their preferred traits, were examined in the districts and it was realised that there was a high level of agreement between men, women and young adults on several varieties, with slight differences among locations. In Techiman, several local and improved varieties, were cultivated. The local varieties included Bensere, Wenchi bankye, Afosa, Nkuruwa, Dakware, Esiabayaa, Bankye kokoo, Nkomti, Bankye borodee (with yellow root), Agoro and Bankye ahoofe. The improved varieties were Afisiafi, Nkabom and Bankyehemaa. The women, especially adults, cultivated more improved varieties than the men. Very few farmers (less than 5%) had stands of yellow flesh improved cassava varieties, and these were mainly cultivated by adults. Only very few young adults were aware of the local yellow flesh cassava, which was known in local language as “Bankye borodee”, as majority of the young adults had never seen it. The characteristics preferred by women include early maturing (6-12 months), tolerance to abiotic stresses (very harsh weather conditions), quick canopy closure (thus labour saving on weeding), and suitability for multiple food uses (such as fufu, gari, ampesi and kokonte). Men tend to prefer varieties that yielded higher (with higher income), easy-to-harvest, high DM and having good cooking qualities throughout the season. In Ketu North District, varieties cultivated by all gender groups includes Kpenyevi, Hushivi, Dogbovi and Bosomensia. Adult males and male young adults grew improved varieties such as Abasafitaa, Ampong and Tech bankye. The females had difficulty accessing these improved planting materials but the men received it through contact with extension agents in this district. This is due to the fact that, women need a man or her husband in some cases to lead them All gender groups preferred varieties that are early maturing (6 months), poundable and high yielding (big roots). Adult male and young male adults preferred varieties that could save labour by closing its canopy early, since males normally did the weeding in that district. Adult females and young female adults needed varieties that could easily be processed domestically into starch while the adult male and young male adults 61 preferred varieties suitable for industrial uses (for brewing, starch and biscuits) to earn higher income. In general male participants requested for more research on their preferred varieties and landraces to improve the poundability and the storability in preferred materials in the soil. In Agona East District, the common varieties grown by all gender groups include Madumaku, Bosomensia, Esiabaayaa, Duafra, Nkonmono, Agage and Wodziawonye. Adult males and females cultivated improved varieties such as Sika bankye and Bankyehemaa. The study however showed that young adults did not have access to improved planting materials. In this district, the preferred traits relative to adults did not differ significantly by gender. The prime traits of interest were poundability and multiple uses such as gari, starch, kokonte, ampesi and other forms of products for industrial uses. Men generally prefer traits like good storability in the soil, ease of harvesting, early canopy formation (for weed suppression), and suitability for industrial uses. Women on the other hand tend to prefer traits such as ease of peeling, high starch content and early canopy formation. 3.3.7 Processors There were no significant differences in the varieties preferred by producers and processors, as most processors also had farms that fed the processing unit. Men and women processors handled similar varieties. The five important varieties mostly processed were Bensere, Wenchi bankye, Agric (improved), Bankyehemaa and Agyiribaa. The yellow flesh cassava was not common among the processors in the Agona East district. 3.3.8 Traits preferred by processors Cassava processing was dominated by women. The men who were involved, mostly young adults, depended on hired labour, mostly women, to perform the different stages of processing which they supervised. Some men also processed jointly with their spouses. The main trait of interest for processing by men and women processors was high DM or less moisture content. Not all the varieties had this trait, given that cassava storage roots are sometimes used for “fufu” or “konkonte”. Women processors also preferred varieties that could be used for starch and biscuits while the men tend to prefer varieties that have high level industrial value use for starch and ethanol production as well as brewing. 62 3.3.9 Awareness, perceptions and willingness to adopt yellow-flesh cassava 3.3.9.1 Awareness The majority (90%) of the adult men and women farmers and processors were aware of yellow flesh cassava, compared to the young adults (2%). Within the young adults, awareness was skewed towards the young male adults who were mostly cultivating family farms. On average, the local yellow variety (Bankye borodee) had been known in the community for over 20 years through ancestral lineage. However, at the time of the study, the local yellow flesh cassava variety was described as almost extinct, since only a few men and women were still cultivating it (bankye borodee) on their farms. None of the young adult farmers had the material in their field. The absence of the local yellow flesh cassava variety in the communities was mainly attributed to the lack of planting material of the variety. The absence of yellow flesh cassava planting material constrained processors to normally add palm oil to white fleshed root for customers requesting for yellow “gari” product. 3.3.9.2 Perceptions During the discussions, two categories of people were identified based on when it came to their perceptions of yellow flesh cassava. There were some, who had seen and used the varieties before, and there were others whom had never used it. In order to avoid lack of response to study questions (i.e. questionnaire) and inadequacy of information during the elicitation, the team took note of the assumptions and expectations of those who had never had experience with the local yellow flesh cassava variety. This was done because a similar study by Gonzalez et al. (2011) recorded high lack of response, which indicated that respondents did not have strong perceptions of the new varieties that were evaluated by the research team and farmers through a participatory approach. Overall, farmers asserted that, if the planting materials were made available, yellow flesh cassava cultivation could increase their income due to the high likelihood and propensity by food vendors/restaurants and processors to use it in their food processing, with higher prospect for increased market share. Some men also said the yellow flesh cassava roots do not store well in the soil. The processors admitted that the processed cassava product “yellow gari” command higher premium price than the white or cream type, but having low market demand, and therefore small market share. They have therefore suggested that action be taken to increase the market demand for easy uptake. This was found laudable and could be included in future promotional campaigns to enhance adoption. The other perception information of the yellow flesh cassava are presented in Table 63 3.4. These perceptions were given based on participants’ experience with the local yellow variety or what they have heard from yellow flesh cassava farmers or processors. Table 3.4a Gender groups’ perception on yellow flesh cassava Perception Gender group Substitute for plantain, oil palm and colour Adult male and adult female additives added Mealy Adult male and adult female Low yielding Adult male and adult female in Techiman Municipal and Agona East districts Thinner cassava sticks which may affect Adult female and female young adults yield Difficulty in accessing planting material All gender groups Less starch Adult female and female young adults Small market share All gender groups Has more nutrients than the white type Adult male The stakeholders who had never cultivated the cassava type could only relate to it by providing their expectations and assumptions Table 3.4b Gender groups’ expectations of the improved yellow flesh cassava Expectation/ assumptions Gender group Market share of the crop type increased through consumer All gender groups sensitization Consumers may perceive colour change of cassava product Adult female processors as addition of colour additives or oil palm May taste bitter just like some other improved varieties Female young adult processors The beta carotene level must be high (i.e. “must be as Male and female young yellow as plantain or yellow corn”) adults Have qualities of a good cassava (“if possible, improve on All genders local varieties”) Not serve multiple purposes “yellow cassava can only be All genders used for fufu and gari not products like starch/brewing” 64 3.3.9.3 Willingness to adopt yellow flesh cassava When the willingness of men and women producers and processors to accept the yellow flesh cassava was assessed, the majority of the participants were found to be willing to accept it (92.5%). This is predicated on a number of factors among which are the ready market (for processors and food vendors), for gari processing and reduced cost associated with the non-use of palm oil to enhance yellow color in cassava. There wasn’t much information or evidence about adoption based on the nutritional and health benefits, so participants were educated briefly to facilitate adoption in future, when introduced. Few had reservations about the local yellow variety because of previous experience, which included its low yield, root colour and slender planting materials. Some of the men were willing to accept the new yellow flesh cassava only if the planting materials could be distributed free of charge, since they hardly pay for cassava stakes which appears not to be commercialized in the districts. All gender groups wanted planting materials to be made easily accessible and available in large quantities and a lot of advocacy to be done to further increase market share. 3.3.10 Factors affecting the adoption of new cassava varieties Several factors stemming from social, cultural, economic, psychological and institutional considerations have been reported to affect technology uptake (Gonzalez et al. 2011). The study found similar factors such as cultural beliefs and norms, land tenure arrangements and allocation, extension contacts, awareness, perception, participants preferred traits and production/processing objective as factors impeding the dissemination and adoption of the yellow flesh cassava. These variables are thus the key drivers of adoption. 3.4 Discussion The importance of cassava in Ghana cannot be over emphasized, as it is the most important root crop, followed by yams and cocoyams, in terms of quantity produced, but ranks second to maize in terms of area planted. Being a staple for over 800 million people in the world and described as Africa’s insurance crop, as it ensures improved welfare and food security for small-holder farmers who cultivate it (Anna et al. 2010). The crop has other special attributes such as its ability to adapt to different farming systems, reduce cost of farm operations when intercropped, and its capacity to perform well on marginal soil (Dixon et al. 2003). Notwithstanding its importance as a food, the crop is low in essential micronutrients, indicating challenges related to nutritional security for the many people who depend on the crop. A high risk could therefore be perceived, as the majority of resource- 65 poor who typically are people, usually based in rural areas, rarely consume balanced diets, which are necessary to provide them with the required nutrients for good health (Esuma et al. 2019). This is then a developmental issue, which, if not curtailed, could affect agricultural and labour productivity as well as the human capital development (McGovern et al. 2017). The biofortification of Ghanaian staple crops is therefore a matter of urgency, as the other nutrition enhancement mechanisms are not readily sustainable (Bouis 2003; West 2003; Nestel et al. 2006) So breeding programmes/teams must be readily and rapidly facilitated to expedite action on biofortification of staple crops for the benefit of small holder farmers and consumers. Beside cassava, farmers intensified crop diversification through the cultivation of other roots crops, cereals, vegetables and tree crops to ensure household security. Diversification is adopted by farmers as a coping strategy to reduce food security risk and stabilize food stocks and welfare. Crop diversification to be more advantageous than sole cropping (Mango et al. 2018; Makate et al. 2016). Crop diversification is thus explored as a means of developing agricultural resilience, in addition to being the most ecologically feasible, cost- effective and rational ways of reducing uncertainties in agriculture including providing farm households with diet diversity, income and some nutrient levels”. Cassava cultivation is intertwined with several factors such as ethnicity, access to resources (including labour, cash and land), gender and wealth (Adjei-Nsiah and Sakyi- Dawson 2012). The results showed gender differences in access to, and control of land resources. The differences were as a result of economic and cultural factors. The study was conducted in Southern Ghana, but the findings showed that ethnicity and location played a role in the access to and control of resources. While among the Akans, both adult males and females had access to and control of land, and hence could take decisions regarding resource investment and allocation, the “Ewe” woman needed a man/her husband in some cases to lead her to the chief before accessing the land. Previous research has demonstrated that there is an association between the decision-making powers women enjoy and the quantity (and quality) of land rights they hold in every society (Chigbu 2019). The study found that women farmers are heterogeneous, have different needs, and are affected differently, especially by their culture, location and economic status in society as realised by this study. Size of land as an economic resource has been found an important determinant of adoption (Lavison 2013). However, in both tribes (Akan and Ewe), women farm sizes were relatively smaller than their male counterparts’ farms. This dimension is partly rooted in cultural beliefs and practices though the Intestate Succession (PNDC) Law 111, 1985 and 1998 Children’s Act 66 560 have been enacted. The study areas belonged to the two lineage systems in Ghana; Matrilineal and Patrilineal. In the Akan family system, inheritance is matrilineal; which bonds a child to the mother than the father. Here, women may have right to their lineage lands but in varied proportions compared to that of men (FAO, n.d.; Kutsoati and Morck, 2012). This is because the lineage authority; mainly men allocate more land to males who are heads of households. On the other hand, in the patrilineal system; which the Coastal Savannah belonged, women only access land through marriage and hold it only when the marriage is in force. Whilst in marriage women loose acces to their lineage lands (FAO, n.d.). Land is passed on to the young male adult which gives him the right of access and use.The unmarried young female adult however becomes constrained in accessing land. In Africa and for that matter Ghana, customary laws discriminate against women’s rights to land (Fonjong et al. 2012). In Ghana, in both lineage systems, the culture styreotype against women; looking upon them as inferior hence the limited access to large proportions of land. The results indicated adult farmers had little knowledge of the improved varieties previously released by researchers and they complained of the difficulty in accessing the planting materials. Most of the young adults, on the other hand, had never heard of the improved varieties. Majority of men and women farmers expressed reservations about the attributes of most improved varieties, as the varieties did not often meet their preferences. These gaps may be a result of poor awareness creation for farmers on the attributes and uses of the released varieties by the appropriate institutions responsible for the development of the varieties and extension services. It is incumbent on research and extension institutions to involve all gender groups in their field trials and demonstrations, and also develop sustainable mechanisms to widely disseminate new varieties widely to end users in order to create more awareness about the good values of new varieties. A new variety or technology can only make a positive contribution to economic growth if it is widely diffused and used by the target group (Uaiene et al. 2009). The government of Ghana must make funds available to research institutions and the Ministry of Food and Agriculture to multiply breeder and foundation planting materials for distribution of improved varieties (including yellow cassava) to private seed companies, for further multiplication to generate copious quantities to improve availability to farmers. Producers and processors, depending on their respective objectives of production, have distinct varietal trait preferences. Farmers are generally assumed to be identical in their trait preferences but differences existed between men, women and young adults (Kamau et al. 2011; Traoré et al. 2016), since farmers could have multiple objectives. Young adults were 67 much more interested in cassava with industrial qualities, while women preferred cassava that could be used for domestic processing/multiple food purposes. The men preferred high yielding cassava varieties, which could return more cash and are easy to harvest to reduce drudgery and labour costs. This confirms the heterogeneity of farmers and differentiation in trait preferences (Smale et al. 2001; Christinck et al. 2017; Teeken et al. 2018). Breeders must be concerned about additional preferred traits and include them as additional breeding objectives in order to broaden the genetic base, while, being mindful of the trade-offs that may be expected, too. Despite the great potential of agricultural varieties and technologies, its adoption by the resource poor smallholder farmer has been slow. Awareness and perception of technology are seen as important determinants that facilitate its uptake (Meijer et al. 2015), but unfortunately the knowledge and cultivation of the improved varieties and yellow flesh cassava (pVA cassava) were low, especially among the young adults, who are the future of agriculture. The adults who were aware of yellow flesh cassava did not cultivate it and complained of lack of planting materials in addition to their negative perception of the yellow flesh cassava. Okoro (2015) found that farmers preferred local varieties more to improved varieties, and a similar pattern was observed in this study. Having an understanding of these factors are essential for economists (studying the determinants of growth) and for the developers and disseminators of such technologies (Uaiene et al. 2009). Most studies (Uaiene et al. 2009; Akudugu et al. 2012; Loevinsohn et al. 2012; Mwangi and Kariuki 2015; Ponguane and Mucavele 2018) tend to emphasize extrinsic factors such as demographics and farm level characteristics of the adopter as other factors that influence the adoption of innovations. In addition, intrinsic factors such as awareness, perception and attitudes have been found as important drivers of adoption (Meijer et al. 2015; Mawusi 2004). A combination of both the intrinsic and extrinsic factors must be studied and considered by economists, breeders and policy makers in making informed decisions. With the virtual extinction of the landraces with yellow flesh colour, which used to be widely planted about three decades ago (Boampong and Safo- Kantanka 2014), almost all landraces and improved varieties cultivated and processed are white fleshed, with the exception of “Lamesese” (yellow flesh) which was released in 2015, unfortunately Lamesese though not extensively cultivated, and the carotenoid levels have not been quantified. There is therefore a niche for improved yellow flesh cassava varieties (having high beta carotene) in Ghana. Such varieties must be released and disseminated nationally to improve nutrition security 68 and health for both farmers and consumers. A high percentage of the farmers and processors willing to accept the yellow flesh cassava, which is heart-warming. However, their expectations and doubts about the yellow cassava varieties need to be addressed through extensive education for wide diffusion. Poor functioning markets hinder resource poor farmers from accessing modern technologies; hence an increased access to credit markets could enable farmers and private sector acquire improved varieties, other inputs and access information on modern technologies. This would thus improve the market performance of Domestic cassava commodity markets. It can also be improved through market surveys so as to help identify specific segments for developed product.The excellence in Breeding and Gender in Breeding are developing gender plus tools that will capture customer profiling and market segmentation to give a clearer picture on traits and prefences by consumers for new cassava varieties developed (Orr et al. 2018). The product launching of released varieties will also enhace adoption and utilization of biofortified cassava. 3.5 Conclusions and implications for cassava breeding White fleshed varieties, cassava, most of which are landraces are cultivated across the different agro-ecologies of Ghana. There is limited farmer knowledge on improved varieties including yellow flesh cassava varieties and there is need to improve this by strengthening the farmers in Ghana to enhance accessibility and availability of these varieties by farmers cassava seed systems. Very few men and women are cultivating improved varieties, and yellow flesh cassava. The young adults, who are the future of the agricultural sector, are severely lacking in access to improved varieties. These cadres of farmers require extra attention to enhance their knowledge and access to new varieties including in addition to improving their capacity to use these materials. Numerous similarities were seen in the preferred traits of men and women; though slight differences were observed between districts. Men preferred varieties that could be stored longer in the soil, are easy to harvest all year round, could compete favorably with weeds and are suitable for industrial processing. On the other hand, women preferred climate smart varieties, and varieties that are labour saving and could easily be processed domestically into starch and “gari”. Participants have expressed willingness to cultivate and process the yellow flesh cassava if there is improvement in the availability of planting materials of these varieties, increased awareness and market demand. This study successfully demonstrated the importance of qualitative and participatory approaches to research. It is recommended that cassava breeders review their breeding objectives to reflect the preferred traits of farmers and end users. There is need to pay attention to perception issues of the yellow flesh cassava in order 69 to develop demand driven varieties that will best meet the need of end users. Education to create awareness on the potential advantages and diverse uses of the improved biofortified cassava is also required. Further studies are proposed to investigate and assign measures/ratings to men and women for the qualitative traits evaluated in this work. References ACC/SCN (2000) Fourth report on the world nutrition situation. Administrative Committee on Coordination, Subcommittee on Nutrition. Food Policy Research Institute, United Nations, Geneva, Switzerland Adjei-Nsiah S, Sakyi-Dawson O (2012) Promoting cassava as an industrial crop in Ghana: Effects on soil fertility and farming system sustainability. Applied and Environmental Soil Science 2012: 940954 Akudugu M, Guo E, Dadzie S (2012) Adoption of modern agricultural production technologies by farm households in Ghana: what factors influence their decisions? Journal of Biology Agriculture and Healthcare 2: 1-13 Amenu E (2015) Effect of post-harvest management on cassava production, processing and quality of starch produced during gari-making. Master of Philosophy. Department of Horticulture, Faculty of Agriculture, KNUST Anna B, Gleadow R, Cliff J, Zacarias A, Cavagnaro T (2010) Cassava: the drought, war and famine crop in a changing world. Sustainability 2: 3572-3607 Annor-Frimpong C (1991) A survey of cassava cultivation practices in Ghana. Tropical root and tuber crops in a developing economy. In: Ofori F, Hahn SK (eds.) Proceedings of the ninth symposium of the International Society for Tropical Root Crops. 20- 26th October, 1991 Beaton GH, Martorell R, Aronson KJ, Edmonston, McCabe BG, Ross AC (1993) Effectiveness of vitamin A supplementation in the control of young child morbidity and mortality in developing countries. ACC/SCN State of the Arts Series, Nutrition Policy Paper 13. World Health Organization, Geneva, Switzerland Boampong EY, Safo-Kantanka O (2014) Ethno botany of some cassava germplasm in Ghana. International Knowledge Sharing Platform (Globus). V3 Bouis HE (2003) Micronutrient fortification of plants through plant breeding: Can it improve nutrition in man at low cost? Proceedings of the Nutrition Society 62: 403- 411 70 Bouis HE, Hotz C, McClafferty B, Meenakshi JV, Pfeiffer WH (2011) Biofortification: A new tool to reduce micronutrient malnutrition. Food and Nutrition Bulletin 32: S31- S40 Ceballos H, Fregene M, Pérez JC, Morante N, Calle F (2007) Cassava genetic improvement. In: Kang MS, Priyadarshan PM (eds.) Breeding major food staples. Blackwell Publishing, Ames, IA. pp. 365-391 Chigbu EU (2019) Masculinity, men and patriarchial issues aside: How do women's actions impede women's access to land? Matters arisng from a peri-rural community in Nigeria. Land Use Policy 81: 39-48 Christinck A, Weltzien E, Rattunde F, Ashby J (2017) Gender differentiation of farmer preferences for varietal traits in crop improvement: evidence and issues. Working Paper No. 2. CGIAR Gender and Agriculture Research Network; CGIAR System Management Office and International Center for Tropical Agriculture (CIAT). Cali, Colombia Combs GF (1998) The vitamins. Fundamental aspects in nutrition and health. Academic Press, London, UK Dixon AGO, Bandyopadhyay R, Coyne D, Ferguson M, Ferris RSB, Hanna R (2003) Cassava: from poor farmers’ crop to pacesetter of African rural development. Chronicle of Horticultural Journal 43: 8-15 Dwivedi SL, Sahrawat KL, Rai KN, Blair MW, Andersson MS, Pfeiffer W (2012) Nutritionally enhanced staple food crops. Plant Breeding Reviews 36: 173-293 Elias M (2013) Practical tips for conducting gender-responsive data collection. Bioversity International, Rome Elo S, Kyngas H (2008) The qualitative content analysis process. Journal of Advanced Nursing 62: 107-115 Esuma W, Nanyonjo AR, Miiro R, Angudubo S, Kawuki RS (2019) Men and women’s perception of yellow‑root cassava among rural farmers in eastern Uganda. Agriculture and Food Security 8: 10 FAO, n.d. Gender and Land Rights Database. Ghana: Customary norms, religious beliefs and social practices that influence gender-differentiated land rights. http://www.fao.org/gender-landrights-database/country-profiles/countries- list/customary-law/en/?country_iso3=GHA. Accessed, 15th April, 2020. Fonjong L, Sama-Lang IF, Fon Fombe L (2012) Implications of Customary Practices on Gender Discrimination in Land Ownership in Cameroon, Ethics and Social Welfare, 6:3, 260-274, DOI: 10.1080/17496535.2012.704637 71 Gonzalez C, Perez S, Cardoso CE, Andrade R, Johnson N (2011) Analysis of diffusion strategies in northeast Brazil for new cassava varieties with improved nutritional quality. Experimental Agriculture 47: 539-552 Gyawali S, Sunwar S, Subedi M, Tripathi M, Joshi KD, Witcombe JR (2007) Collaborative breeding with farmers can be effective. Field Crops Research 101: 88-95 Ghana Statistical Service (GSS), Ghana Health Service (GHS), and ICF International. (2015) 2014 Ghana Demographic and Health Survey (DHS) Key Findings. Rockville, Maryland, USA: GSS, GHS, and ICF International. Kamau J, Melis R, Laing M, Derera J, Shanahan P, Eliud C, Ngugi K (2011) Farmers’ participatory selection for early bulking cassava genotypes in semi-arid Eastern Kenya. Journal of Plant Breeding and Crop Science 3: 44-52 Kutsoati E, Morck R (2012) Family Ties, Inheritance Rights, and Successful Poverty Alleviation: Evidence from Ghana. SSRN Electronic Journal. DOI: 10.2139/ssrn.2020542 Lavison R (2013) Factors Influencing the adoption of organic fertilizers in vegetable production in Accra. MSc Thesis, Accra, Ghana Loevinsohn M, Sumberg J, Diagne A (2012) under what circumstances and conditions does adoption of technology result in increased agricultural productivity. Protocol. London: EPPI Centre, Social Science Research Unit, Institute of Education, University of London Makate C, Wang R, Makate M, Mango N (2016) Crop diversification and livelihoods of smallholder farmers in Zimbabwe: adaptive management for environmental change. Springerplus 5: 1135 Mangione D, Senni S, Puccioni M, Grando S, Ceccarelli S (2006) The cost of participatory barley breeding. Euphytica 150: 289-306 Mango N, Makate C, Mapemba L, Sopo M (2018) The role of crop diversification in improving household food security in central Malawi. Agriculture and Food Security 7: 7 Manu-Aduening JA, Lamboll RI, Ampong Mensah G, Lamptey JN, Moses E, Dankyi AA, Gibson RW (2006) Devel opment of superior cassava cultivars in Ghana by farmers and scientists: The process adopted, outcomes and contributions and changing roles of different stakeholders. Euphytica 150: 47-61 72 Manu-Aduening JA, Peprah B, Bolfrey-Arku G, Aubyn A (2014) Promoting farmer participation in client-oriented Breeding: Lessons from participatory Breeding for farmer-preferred cassava varieties in Ghana. Advanced Journal of Agricultural Research 2: 8-17 Mawusi SE (2004) Farmers’ knowledge and perception towards a sustainable adoption of sugar beet in Kenya. A thesis submitted in partial fulfillment of a Master of Science degree in environmental science at Lund University, Sweden McGovern ME, Krishna A, Aguayo VM, Subramanian SV (2017) A review of the evidence linking child stunting to economic outcomes. International Journal of Epidemiology 46: 1171-1191 Meijer SS, Catacutan D, Ajayi OC, Sileshi GW, Nieuwenhuis M (2015) The role of knowledge, attributes and perceptions in the uptake of agricultural and agroforestry innovations among smallholder farmers in Sub-Saharan Africa. International Journal of Agricultural Sustainability 13: 40-54 Morris ML, Bellon MR (2004) Participatory plant breeding research: Opportunities and challenges for the international crop improvement system. Euphytica 136: 21-35 Mwangi M, Kariuki S (2015) Facrors determining Adoption of new Agricultural technology by smallholder farmers in developing countries. Journal of Economics and Sustainable Development 6: 258-216 Nduwumuremyi A, Melis R, Shanahan P and Asiimwe T (2016) Participatory appraisal of preferred traits, production constraints and postharvest challenges for cassava farmers in Rwanda. Food Security 8: 375-388 Nestel P, Bouis HE, Meenakshi JV, Pfeiffer W (2006) Biofortification of staple food crops. Journal of Nutrition 136: 1064-1067 Okoro P (2015) Developing superior cassava [Manihot esculenta (Crantz)] varieties using partial inbreds. Doctor of Philosophy Thesis, WACCI, University of Ghana, Legon Orr A, Cox CM, Ru Y, Ashby J (2018) Gender and social targeting in plant breeding. Lima (Peru). CGIAR Gender and Breeding Initiative. GBI Working Paper. No. 1. Available from: www.rtb.cgiar.org/gender-breeding-initiative (accessed 10.10.2019). Ponguane S, Mucavele, N (2018) Determinants of agricultural technology adoption in Chókwè District, Mozambique. MPRA Paper No. 86284. Online at https://mpra.ub.uni-muenchen.de/86284/ 73 Schofield J, Ntawuruhunga P, Kanju E, Mkamilo G, Ndyetabula I, Jeremiah S, Obiero H, Ogecha J, Tatahangy W, Bigirimana S, Gashaka G (2009) Proceedings of the programme for Africa’s seed systems. The march toward a green revolution in Africa: Improving the lives of farmers through stronger seed systems. 5-8 October, 2009, Bamako, Mali Smale M, Bellon M, Gomez J (2001) Maize diversity, variety attributes and farmers’ choices in South-Eastern Guanajuato, Mexico. Economic Development and Cultural Change 50: 201-225 Tarawali G, Iyangbe C, Udensi UE, Ilona P, Osun T, Okater C, Asumugha GN (2012) Commercial-scale adoption of improved cassava varieties: A baseline study to highlight constraints of large-scale cassava based agro-processing industries in Southern Nigeria. Journal of Food, Agriculture and Environment 10: 689-694 Teeken B, Olaosebikan O, Haleegoah J, Oladejo E, Madu T, Bello A, Parkes E, Egesi C, Kulakow P, Kirscht H, Tufan HA (2018) Cassava trait preferences of men and women farmers in Nigeria: implications for breeding. Economic Botany 72: 263- 277 Thakkar SK, Huo T, Maziya-Dixon B, and Failla ML (2009) Impact of style of processing on retention and bioaccessibility of b-carotene in cassava (Manihot esculenta Crantz). Journal of Agricultural and Food Chemistry 57: 1344-1348 Traoré SA, Markemann A, Reiber C, Piepho HP, Valle Zárate A (2016) Production objectives, trait and breed preferences of farmers keeping N’Dama, Fulani Zebu and crossbred cattle and implications for breeding programs. Animal, page 1 of 9 © The Animal Consortium 2016 Turyahikayo E (2014) Resolving the qualitative-quantitative debate in healthcare research. Medical Review 5: 6-15 Uaiene R, Arndt C, Masters W (2009) Determinants of agricultural technology adoption in Mozambique. Discussion papers No. 67E Were W, Shanahan P, Melis R, Omari O (2012) Gene action controlling farmer preferred traits in cassava varieties adapted to mid-altitude tropical climatic conditions of western Kenya. Field Crops Research 133: 113-118 West KP Jr (2003) Vitamin A deficiency disorders in children and women. Food and Nutrition Bulletin 24: S78-S90 WHO (2009) Global prevalence of vitamin A deficiency in populations at risk 1995–2005. World Health Organization, Geneva, Switzerland 74 Witcombe JR (2009) Methodologies for generating variability. In: Ceccarelli S, Guimaraes EP, Weltzien E (eds.) Part 3: The development of base populations and their improvement by recurrent. Plant breeding and farmer participation, FAO, Rome, Italy. pp. 139-157 75 Chapter 4 Analysis of total carotenoid content, cassava mosaic disease, dry matter content, yield and its related components in F1 cassava families at two locations in Ghana Abstract Cassava is currently ranked as the number one food staple and is the most widely cultivated crop in Ghana. Cassava production in Ghana is around 18.5 million tons with more than 70% of the country’s farmers engaged in its production. Since 1993, 26 improved varieties have been released in Ghana and distributed to farmers. In most cases, these varieties were white-fleshed with low or negligible amounts of carotenoids, but were bred and selected for processing into intermediary products (gari, konkonte, starch). Malnutrition is endemic in cassava producing regions of Africa, partly due to the low micronutrient content of this storage root crop, given that cassava is a major component of most household diets. It is for this reason that the development of nutrient dense cassava varieties require much attention to reduce the effect of malnutrition among the poor in a less expensive and more sustainable way. The current study was designed to generate genetic information and also to develop cassava clones that combine good total carotenoid content (TCC) and high dry matter content (DMC) using a 2 x 5 North Carolina II breeding scheme. Ten F1 families were generated and evaluated across two different locations in Ghana. Results indicate general combining ability (GCA) mean squares were larger than their respective specific combining ability (SCA) mean squares for harvest index, cassava mosaic disease (CMD) and TCC, with over 70% of the total variation explained. These traits are therefore highly driven by additive genetic effects. The positive significant correlations that were observed between pulp colour and TCC, TCC and CMD, pulp colour and CMD, and pulp colour and cortex colour makes screening of large numbers of progenies possible and relatively easier in cassava breeding programme. The female parental used are improved cassava lines selected in IITA with CMD reistance background. This could allow breeders to combine both TCC and CMD at the early stages through visual assessment of pulp colour and CMD symptoms. Through visual screening, large number of genotypes can be screened down to a small number and the few selected can then be quantified for TCC at the later stages of breeding scheme to save costs. One of the parental materials used in this study, P6, showed positive GCA effects for TCC, DMC, CMD and storage root weight (RTW), hence could be crossed into a high DMC background to increase chances of generating clones that combine both TCC and DMC. 76 4.1 Introduction The populations of underdeveloped and developing countries often suffer undernourishment and so-called “hidden hunger” as a result of micronutrient deficiencies. Areas in Africa, including Ghana, where cassava is widely consumed, are characterized by rampant malnutrition because its storage roots are low in nutrients such as VA (Ssemakula and Dixon 2007). It is for this reason that the development of nutrient dense cassava varieties needs and deserve much more attention to eliminate the ramifications of malnutrition among the poor in an inexpensive and sustainable way. VAD constitutes a major public health problem and affects mainly children and women. Several programmes in nutrition security have been initiated, for example HarvestPlus, which is a global alliance involving several research institutions, tasked with initiating initiated the development of micronutrient-dense staple crops (Bouis et al. 2011; Dwivedi et al. 2012) under a joint partnership of the International Center for Tropical Agriculture (CIAT) and IITA. Among its several initiatives is the development of biofortified cassava clones with high PVAC in the roots. On this score, the national cassava breeding programme at CSIR-CRI initiated a breeding programme with the aim of developing high yielding cultivars with high levels of TCC, DMC and disease resistance. Deployment of such cassava varieties could sustainably improve nutrition and reduce prevalence of VAD in communities that heavily depend on cassava, especially the rural populations (Nassar and Ortiz 2010; Esuma et al. 2016). Adoption of biofortified cassava genotypes in Ghana will largely depend on their agronomic performance, including storage fresh roots, DMC, resistance to major pests and diseases and the stability of these traits over time and space (Njoku 2012). DMC influences texture after boiling and is also a key parameter in the production of gari (a popular cassava product consumed in Ghana). The strong negative correlation that has been reported between DMC and TCC in African cassava germplasm (Akinwale et al. 2010; Njoku et al. 2015; Esuma et al. 2016) could present a potential challenge for the breeding programmes that are aiming to improve both traits. Chávez et al. (2005) and Sánchez et al. (2014) found that correlations between TCC and DMC were not statistically significant when analyzing Latin American cassava germplasm. 4.2 Materials and methods 4.2.1 Experimental site The crossing block for this study was first planted in May, 2015 at the CSIR-CRI in the semi-deciduous forest zone of Ghana located at 1°30’0’’W and 6°42’0’’N, 186 m above sea level, with a bimodal rainfall distribution, with two rainy and one dry seasons. A second 77 crossing block was established at the same location in 2016 due to a limited number of botanical seeds collected during the first hybridization. The soil of the experimental area (Fumesua) is Asuansi series, a ferric Acrisol with sandy loam top soil over sandy clay. 4.2.2 Progeny development Seven genetically diverse clones (five yellow flesh cassava at advanced selection stages by IITA and two white-fleshed selected from farmers' fields in Ghana) were used as progenitors (Table 4.1). These progenitors were planted in the crossing block at Fumesua under rain fed conditions. Planting was done using disease-free stakes planted in three row plots of five plants/row with a plot size of 15 m2. Blocking was used to allow all matings involving a single group of males (two white-fleshed) to a single group of females (five yellow flesh cassava) to be kept intact as a unit (Acquaah 2012). Spacing between and within rows was 1.5 m to ease movement during the pollination process. Weeding was done as deemed necessary. Controlled pollinations were carried out by hand as described by IITA (1990). The seven parents were crossed in a NCD II to produce 10 F1 families without reciprocals. The seed viability was tested by the floatation method (CIAT 2003). The dry seeds were mixed with water and floated. Non-viable seeds were discarded. At least 110 seeds from each cross were germinated in seed trays in the screen house at the Fumesua station in 2017. Table 4.1. List of progenitors used in the study Genotype Code Source RFC Salient traits Debor P1 Farmer White High DMC Wenchi Alata P2 Farmer White High DMC IBA061635 P3 IITA Light yellow CMD resistance, pVAC IBA070539 P4 IITA Yellow CMD resistance, pVAC IBA070593 P5 IITA Yellow CMD resistance, pVAC IBA090090 P6 IITA Yellow CMD resistance, pVAC IBA070536 P7 IITA Light yellow CMD resistance, pVAC IITA = International Institute of Tropical Agriculture, RFC = root flesh colour, DMC = dry matter content, CMD = cassava mosaic disease, pVAC = provitamin A carotenoids 78 4.2.3 Seedling nursery evaluation Seedlings from the 10 F1 families were transplanted to the seedling nursery at the Fumesua station in the minor rainy season of August 2017 for the purpose of generating planting materials for clonal evaluation. The seedlings were established in single rows at 50 cm by 1 m spacing between and within rows, respectively. Data were collected from each individual stand per family for severity ratings of CMD taken at 1, 3 and 6 MAP and cassava green mite at 6 MAP using a scale of 1 to 5 (1 = no symptoms; 5 = severe symptoms) according to IITA (1990). The seedlings (progenies) were evaluated alongside the progenitors. At harvest (10 MAP), root cortex and pulp colour for individual stands were scored based on a standard colour chart developed by IITA (1990). Individuals were selected based on the ability to produce enough (≥ 12) standard-size cuttings, about 25 cm (4-6 nodes). Clonal evaluation trial Selected individuals were planted in a randomized complete block design (RCBD) with two replications at two sites: Fumesua and Ejisu. The soils for the trial sites were: Asuasi series, a ferric acrisol with sandy loam topsoil over sandy clay at Fumesua and Amantin series, chronic lixisol with sandy loam topsoil at Ejura. Annual rainfall for the sites during the trial period was 1205 mm at Fumesua and 1311 mm at Ejura. Each individual was represented by a single row plot of three plants per rep. with 2 m alleys between blocks. Planting was done at a spacing of 1 m x 1 m between and within rows. The clonal trials were planted in July 2018 and harvested in June 2019 for root measurements. Progenies were evaluated alongside the progenitors in the clonal trials. Weeding was done as deemed necessary and trials were rain fed. 4.2.3.1 Agronomic and morphological characteristics measured During the growth period, data were collected on severity of CMD, cassava green mite (CGM) and cassava bacterial blight (CBB). The incidence and severity of CMD, CGM and CBB were scored using a scale of 1-5, where 1 represented no symptoms and 5 severe damage (IITA 1990). The scoring was done at 1, 3 and 6 MAP for CMD; 6 and 9 MAP for CGM; 1 and 3 MAP for CBB and an average score for analysis was determined. Plant vigour was also measured at 3 MAP. At harvest (12 MAP), all plants in a row were uprooted and the biomass bulked to estimate yield components by separately weighing the 79 fresh roots (kg plot-1) and foliage (kg plant-1) using a Salter Brecknell suspended weighing scale calibrated in kilograms. HI was measured as a ratio of fresh root weight (FRW) to total biomass as: FRW HI = (FRW + FSW) Fresh root yield was measured as root weight (RTW) assessed on a minimum of two plants per genotype. DMC was determined by measuring the weight of the storage roots in water and air and the values calculated according to Kawano et al. (1998). The iCheck analytical kit developed by BioAnalyt laboratory was used for measuring the TCC at the laboratory. The extraction was done following the procedure by Esuma et al. (2016). Five gram of cassava root sample was taken from each clone, pounded and ground into a smooth and fine paste using a mortar and pestle at the laboratory. Twenty ml distilled water was added to the sample to ease grinding. The resulting solution was transferred into 50 ml calibrated Falcon tubes and shaken thoroughly and 0.4 ml of the solution injected into the iExTM Carotene vial using the syringe and needle provided with the kit. The vials were placed on a smooth surface and left to stand for 5 min, it was then shaken again and allowed to stand until two solution phases were seen. The absorbance of the upper phase in the vial was measured using the iCheck kit (reading). TCC (µg g-1) calculated as: (Vs/ Ws) x R Where Vs= volume of the solution transferred to the falcon tube Ws= weight of a sample measured R= final reading by the kit at 450nm wavelength 4.2.4 Statistical design and data analysis ANOVA was done to determine the significance of genetic differences for the traits/variables measured in the 2 x 5 balanced NCD II experiment; within and across locations. The RCBD model of NCD II 80 was used for the genetic analysis and it considered the effect of location, both male and female parents, their interaction and each interaction with the location and all of their interactions together. GCA and SCA effects were also estimated based on the parental effects, and their interaction. All the analyses were done using AGD-R v5.0 (Analysis of Genetic Design with R for Windows) software (Rodriguez et al. 2015). The NCD II Multi-locational RCBD statistical model is: yijkd = µ + Ld + repk(Ld)+gij+Ld*gij+ eijkd = µ + Ld + repk(Ld)+ mi+ fj+ mi* fj+Ld*mi+Ld*fj+Ld*mi*fj+eijkd yijkd is the observed value µ is the mean Ld locational effect repk(Ld) is the effect of replicate k nested in location d (k=1, 2) mi is the male effect (i= 1, 2) fj is the female effcet (j= 1, 2,...5) eijkd residual eijkd residualVC Henderson (balanced) σ2m (MSm-MSmf)/srm σ2f (MSf-MSmf)/srf σ2mf (MSmf- MSe)/sr σ2m (m-1)MSm+(f-1)MSf-(m+f-2)MSmf Sr(2mf-m-f) σ2A 4σ2g σ2 2D 4σ mf σ2 σ2 2E gxe + σ e s sr h2b σ2A+ σ2D σ2A+ σ2 2D+σ E h2 2n σ A σ2 2 2A+ σ D+σ E 81 Variation due to males, females, and males x females were denoted as GCAm, GCAf, and SCA variation, respectively. For CMD, GCA and SCA effects were negative, as the preference is for low values. For all other traits, positive estimates of GCA and SCA effects were used to identify genotypes with high yield and yield components. The relative importance of additive (GCA) and non-additive (SCA) genetic effects in explaining the performance of the progeny for each of the traits was determined by individually expressing the GCAf mean square, GCAm mean square, and the SCA mean square as a percentage of the treatment (crosses) mean square as shown in the formula below (Baker 1978; Hirut et al. 2017). GCA/(GCA + SCA)*100 = 2MS GCApooled/(2MS GCApooled +MS SCA) x 100 MS GCApooled= (f-1)MS GCAf + (m-1)MS GCAm/(m+f-2) Where; MS GCApooled = mean squares for GCA; MS SCA = mean squares for SCA; f = number of female parents; m = number of male parents; MS GCAf = mean square of GCAf; MS GCAm = mean square of GCAm, respectively. Average degree of dominance = (Dominance variance/Additive variance)1/2 4.3 Results For all genotypes of the 10 F1 families, TCC ranged from 1.20 to 9.10 ug/g, with the highest mean (6.14 ug/g) recorded for family P1 x P6 and the lowest (2.98 ug/g) for family P1 x P7 (Table 4.2). Individual DMC values for the evaluated genotypes ranged from 19.70% to 42.4%. DMC with means ranging from 26.86% for family P2 x P5 to 36.28% for family P1 x P3. Family P1 x P6 recorded the highest mean RTW (18.35 kg plot-1) while family P2 xP5 recorded the lowest mean RTW (14.34 kg plot-1). At the parental level, genotypes P1 and P2 recorded the highest value for DMC and CMD (35.4%; 2.05 and 34.20%; 1.99 respectively) but they had the lowest levels of TCC and RTW compared to the yellow parental genotypes (P4-P7). The GCA mean squares for female progenitors were highly significant for all traits measured except for RTN (Table 4.3). The SCA mean squares for DMC, RWT and TCC were also highly significant. The GCA mean squares for the CMD and total biomass (TWT) for male progenitors were highly significant as well, while the SCA mean squares were 82 not. The GCA effects for both male and female and SCA effects were significant for RWT and HI, indicating both additive and non-additive gene effects for this trait. The male and female GCA mean squares, as well as SCA mean squares, showed various levels of significance for CMD, HI, RTN, RTW and TWT, indicating the importance of both additive and non- additive gene effects. Chikoti et al. (2016) reported significant GCA and SCA effects for CMD as found in this study for CMD and other traits. The GCA sum of squares for female progenitors accounted for more of the total variation than the GCA sum of squares for male progenitors for DMC. The SCA sum of squares accounted for 63.03% of DMC cross sum of squares. The GCA: SCA ratio for all the traits were higher than 1 except for DMC (1.00) where there were higher SCA than GCA effects (Table 4.3). The coefficient of variation (CV) ranged from 4% for HI to 39% for TCC (Table 4.2). Low to high CVs for different traits have been reported in several studies. Pasajee et al. (2016) reported high values for TCC (44%). Vasconcelos et al. (2017), Tumuhimbise et al. (2014) and Njoku (2012) also reported low values of 4%, 5% and 10.8% respectively for DMC. 83 Table 4.2. Mean performance of progenitors and their F1 progenies evaluated across two locations in Ghana Parent/family No CMD DMC HI RTN RTW TCC TWT % kg µg g-1 kg P1 (Debor) 2.05 35.40 0.45 28.60 15.77 0.80 20.20 P2 (W.Alata) 1.99 34.20 0.42 27.13 15.36 0.75 22.06 P3 (IBA061635) 1.57 31.66 0.46 26.75 16.77 3.53 19.22 P4 (IBA070539) 1.56 31.84 0.42 29.76 16.42 5.41 22.60 P5 (IBA070593) 1.99 27.40 0.43 26.78 16.09 5.70 20.23 P6 (IBA090090) 2.05 32.27 0.45 26.69 17.15 5.33 21.84 P7 (IBA070536) 1.29 27.69 0.44 29.34 17.45 3.28 22.99 P1 x P3 20 1.39 36.28 0.46 26.39 15.25 3.34 18.47 P1 x P4 17 1.53 27.64 0.43 30.06 15.77 5.68 21.32 P1 x P5 16 1.91 27.94 0.45 29.25 16.38 5.61 20.26 P1 x P6 18 1.90 32.70 0.46 27.18 18.35 6.14 21.93 P1 x P7 16 1.19 27.80 0.46 30.13 18.08 2.98 21.51 P2 x P3 15 1.74 27.04 0.45 27.12 16.29 3.72 19.98 P2 x P4 19 1.59 36.03 0.42 29.46 17.07 5.13 23.88 P2 x P5 14 2.08 26.86 0.40 24.30 14.34 5.79 20.20 P2 x P6 14 2.21 32.24 0.43 26.20 15.94 4.50 21.75 P2 x P7 11 1.39 27.58 0.41 28.59 16.81 3.57 24.49 Grand mean 1.69 30.74 0.44 27.87 16.43 4.19 21.38 S.e.m 0.07 0.83 0.005 0.40 0.25 0.40 0.39 CV% 17.59 11.16 4.39 5.97 6.24 39.57 7.46 CMD = Cassava mosaic disease; DMC = Dry matter content; HI = Harvest index; RTN = Storage root number; RTW= storage root weight; TCC = Total carotenoid content; TWT = Total biomass; CV = coefficient of variation 84 Table 4.3. Mean squares of crosses and sum of squares for combining ability effects of seven traits evaluated in 10 F1 families and seven parents across two locations Source df CMD DMC HI RTW TCC TWT RTN Locations 1 0.779*** 9.555ns 0.0103ns 162.409*** 272.745*** 45.689ns 62.500ns Genotype 9 6.630*** 963.891*** 0.033*** 97.620*** 87.770*** 208.877*** 233.368** Male 1 7.829*** 10.216ns 0.151*** 55.311* 2.259ns 322.891*** 248.039** Female 4 12.478*** 798.463*** 0.024*** 106.284*** 173.947*** 325.607*** 323.104ns SCA 4 0.494ns 1367.74*** 0.013** 99.534*** 25.221*** 63.642ns 139.965** Error 618 0.279 8.276 0.005 10.892 1.050 28.986 37.298 GCAm 13.12 0.12 50.50 6.30 0.28 17.17 15.11 GCAf 83.57 36.85 31.89 48.38 87.09 69.28 78.24 SCA 3.31 63.03 17.61 45.32 12.63 13.54 6.15 GCA:SCA 4.96 1.00 3.87 1.14 1.02 2.27 3.46 Baker ratio 0.98 0.48 0.88 0.66 0.92 0.91 0.81 *P≤0.05; ** P≤0.01: *** P≤0.001; CMD = Cassava mosaic disease; DMC = Dry matter content; HI = Harvest index; RTN = Storage root number; RTW= storage root weight; TCC = Total carotenoid content; TWT = Total biomass 4.3.1 General combining ability effects P2 (W.Alata), a white-fleshed progenitor with negligible TCC, had a negative TCC GCA effect of -0.13 (Table 4.4). This indicates the unsuitability of this parent as a good combiner when targeting high carotenoid in the progeny. In the female parents, P5 showed a significant positive effect for TCC but high negative effect for DMC, although not significant (Table 4.4). In contrast, parents P3 (IBA061635) and P7 (IBA070536) showed negative GCA effects for TCC. Results also showed yellow flesh cassava female parents as having positive positive GCA effects for DMC. However, P7 (IBA070536) was found to have a negative GCA effect for both DMC (but not significant). High positive GCA effects for RTW were identified with genotypes P6 (IBA090090) and P7 (IBA070536). 85 Table 4.4 General combining ability effects of cassava progenitors for seven traits at two locations in Ghana Progenitor CMD DMC HI RTN RTW TCC TWT P1 -0.10 -0.17 0.01 0.69 0.31 0.08 -0.58 P2 1.68 -0.61 -0.02 -0.78 -0.36** -0.13* 0.78 P3 -0.11 1.09 0.02* -1.16 -0.68 -1.14 -2.06 P4 -0.12 1.27 -0.02 1.85 -0.03 0.73 1.32* P5 0.31* -3.16 -0.01 -1.14 -1.10 1.02* -1.05 P6 0.37** 1.71 0.006 -1.22 0.69 0.64** 0.56 P7 -0.39 -2.87 -0.002 1.45 1.00 -1.40 1.72 *P≤0.05; ** P≤0.01: *** P≤0.001; CMD = Cassava mosaic disease; DMC = Dry matter content; HI = Harvest index; RTN = Storage root number; RTW= storage root weight; TCC = Total carotenoid content; TWT = Total biomass 4.3.2 Specific combining ability Cross P1 x P3 had positive non-significant SCA for DMC but a negative effect for TCC and RWT (Table 4.5). Three crosses, P1 x P6, P2 x P7 and P1 x P3, had significant SCA effects for TCC; the first two (P1 x P6 and P2 x P7) were positive while the last cross (P1 x P3) had a negative significant SCA effect for the trait. Four families (P1 x P4, P1 x P7, P2 x P3 and P2 x P5) showed positive SCA effects for TCC (although not significant). Results showed that Family P1 x P3 and P1 x P6 had significant negative effects for CMD. Baker (1978) explained that when SCA means are not significant, the hypothesis is that performance of single cross progeny can be adequately predicted on the basis of the GCA. When the SCA mean squares are significant, the relative importance of GCA and SCA should be determined by estimating the components of variance to predict the progeny performance (Fasahat et al. 2016). The closer the ratio of 2GCAMS/(2GCAMS+SCAMS) is to 1, the more important the additive gene effects. The predictability (or Baker ratio) in this study varied from 0.48 for DMC to 0.98 for CMD (Table 4.3). All the studied traits had a ratio closer to one for the combined data except for DMC, indicating the importance of GCA and additive gene effects for most of the traits. 86 Table 4.5 Specific combining ability effects of parents for seven traits evaluated across two locations in Ghana Family CMD DMC HI RTN RTW TCC TWT P1 x P3 -0.08*** 4.79 -0.009 -1.05 -0.83 -0.26*** -0.17 P1 x P4 0.07 -4.03** -0.009 -0.39 -0.96 0.20 -0.69 P1 x P5 0.01 0.71 0.008** 1.79 0.70** -0.17 0.61** P1 x P6 -0.06*** 0.20 0.007 -0.20 0.89** 0.74*** 0.67 P1 x P7 0.0004 0.28 0.01 0.08* 0.32 0.37 -0.91 P2 x P3 0.06 -4.01* 0.01 1.14* 0.88 0.32 0.02 P2 x P4 -0.09 4.81 0.01 0.48 1.01* -0.14 0.71 P2 x P5 -0.03 0.07 -0.004* -1.69 -0.66 0.22 0.50* P2 x P6 0.04** 0.58* -0.003 0.29 0.85 -0.69 -0.81 P2 x P7 0.02*** 0.50 -0.009 0.01 -0.27 0.43*** -0.87 *P≤0.05; ** P≤0.01: *** P≤0.001; CMD = Cassava mosaic disease; DMC = Dry matter content; HI = Harvest index; RTN = Storage root number; RTW= storage root weight; TCC = Total carotenoid content; TWT = Total biomass 4.3.3 Phenotypic correlation Some of the traits were significantly correlated (Table 4.6). However, there was no significant correlation between TCC and HI, RTW, RTN, TWT (yield components) but TCC showed positive significant (P<0.01) correlation with pulp colour (colour intensity) and CMD. Positive significant (P<0.001) correlation was recorded for pulp colour and CMD. Negative significant correlation were seen between CGM and HI, CGM and RTN, CMD and RTN, and HI and RTN. Positive and significant (P<0.01) correlation was seen between CMD and DMC, pulp colour and cortex colour, TWT and RTW, RTN and RTW and DMC correlated positively with TCC, though not significantly. 4.3.4 Genetic parameters Broad sense heritability ranged from 0.68 (DMC) to 0.99 (TCC) and narrow heritability sense across the locations ranged from 0.61 (HI) to 0.91 (CMD, TCC) (Table 4.7). Heritability estimates were classified according to Bhateria et al. (2006) as high (>0.50), medium (0.30 - 0.50) and low (< 0.30). All the traits studied recorded high heritability, which indicates that selection could be done using direct recurrent selection to improve the traits. 87 All the traits had high narrow and broad sense heritability. High broad sense heritability indicated that the traits had high genetic variance, both additive and non-additive. Narrow sense heritability is important for breeding programmes as it estimates the relative importance of the additive portion of the genetic variance that can be transmitted to the next generation. Table 4.6 Phenotypic correlation of measured cassava characteristics evaluated across two locations CGM CMD COR PULP HI RTN RTW TCC TWT CMD 0.17ns COR 0.12ns 0.40ns PULP 0.23ns 0.77*** 0.58** HI -0.50* 0.18ns 0.14ns 0.28ns RTN -0.04ns -0.45* -0.28ns -0.26ns -0.51* RTW -0.51* -0.23ns -0.20ns -0.03ns 0.38ns 0.49ns TCC -0.02ns 0.55** 0.34ns 0.59** 0.01ns -0.05ns 0.23ns TWT -0.23ns -0.26ns -0.33ns -0.32ns 0.09ns 0.43ns 0.71*** 0.29ns DMC -0.06ns 0.60** 0.10ns 0.34ns 0.00ns -0.13 0.00ns 0.20ns 0.18ns *P≤0.05; ** P≤0.01: *** P≤0.001; CGM= Cassava green mite; CMD= Cassava mosaic disease; COR= Colour of the cortex; PULP= Pulp colour; HI-= Harvest index; RTN= Storage root number; RTW= storage root weight; TCC= Total carotenoid content; TWT= Total biomass; DMC= Dry matter content; Table 4.7 Genetic parameters for various traits studied across two locations in Ghana Variances/ traits CMD DMC HI RTN RWT TCC TWT GCAmale 0.13 0.40 0.005 89.83 14.86 7.19 18.44 GCAfemale 0.46 20.26 0 2.27 0.90 27.25 1.32 SCA 0.005 0.33 0.0001 4.26 1.27 1.33 1.25 Genotype 0.21 8.57 0.0006 64.73 11.49 13.89 12.37 Additive 0.83 34.29 0.002 258.92 45.96 55.56 49.49 Dominance 0.02 1.32 0.005 17.02 5.06 5.31 4.99 Environmental 0.07 16.91 0.001 20.28 5.37 0.22 8.21 Broad 0.93 0.68 0.75 0.93 0.90 0.99 0.87 Narrow 0.91 0.65 0.61 0.87 0.81 0.91 0.79 Degree of 0.16 0.20 0.5 0.26 0.33 0.31 0.32 dominance CMD = Cassava mosaic disease; DMC = Dry matter content; HI = Harvest index; RTN = Storage root number; RTW= storage root weight; TCC = Total carotenoid content; TWT = Total biomass 88 4.4 Discussion Mean TCC values in this study varied from 2.98 to 6.14 µg g-1, with a grand mean of 4.19 µg g-1, which is comparable to values reported by Esuma et al. (2016) in Uganda, and Maroya et al. (2012), Njoku (2012) as well as Ssemakula and Dixon (2007) in Nigeria. The mean is, however, lower than those reported by Ortiz et al. (2011) and Ceballos et al. (2013), both in Colombia. The differences observed could be as a result of many years of breeding for TCC at CIAT (Colombia), the age of the plant as reported by Ortiz et al. (2011), and the nature of the parental lines used in generating the breeding populations. In most of the studies reported in Africa, parental lines used were selected solely based on either TCC and DMC (white-fleshed) and used in crosses as either male or female parent for each trait, in a design to combine both DMC and TCC to meet stakeholder demands. In other words, a DMC parent crossed to TCC parent. Mean DMC ranged from 26.86 to 36.28% with a grand mean of 30.19%, comparable to values reported by Esuma et al. (2016) and Tumuhimbiase et al. (2014), but lower than values reported by Kamau et al. (2010). Although the DMC grand mean is lower in this study compared to those reported for landraces grown in Ghana by farmers, some individual genotypes evaluated had higher DMC than that of the commonly grown varieties. These evaluated individuals could be selected and further tested towards release, since the trait is one of the key drivers of cassava variety adoption in Ghana. Earlier PRA work done in this study suggested that farmers would be willing to adopt yellow flesh cassava. The mean RTW ranged from 14.34 to 18.35 kg plot-1. These mean values were higher than that of farmer-grown varieties, hence could be adopted by farmers in Ghana, but need to be tested in famers’ fields on larger plot sizes. Cassava varieties with high TCC and DMC will be rejected by the National Release Committee if they are susceptible to CMD in Ghana. This confirms the importance of selecting clones that are resistant or tolerant to CMD. In the current study, all families recorded CMD values less than 2 (resistant) as shown in Table 4.2. Some phenotypic correlations in this study are of special importance for selecting high TCC cassava clones in breeding programmes. Firstly, the positive significant correlation between pulp colour and TCC (Chávez et al. 2005; Esuma et al. 2016), are good for screening large numbers of progenies of elite breeding lines in the cassava breeding programme. This is because pulp colour is directly impacted by carotenoids thus an indicator for Vitamin A. TCC and CMD, pulp colour and CMD, pulp colour and cortex colour. The correlation between CMD and pulp color (TCC) would need more studies to explain the basis underlying this observation. Edoh et al. (2015) also reported positive correlation between 89 TCC and CMD when evaluating high beta carotene cassava genotypes at advanced trial in Nigeria. Most elite parent materials in breeding programmes in Africa have been pre- selected for CMD resistance and explains why the parent materials and progenies showed good CMD resistance. It shows both TCC and CMD could be combined and selected for at the early stages by visually assessing the pulp colour and CMD symptoms. The reduced number of selected individuals allow for a comfortable size that could be subjected to more demanding quantitative screening analysis for TCC in the later stages of the programme to save cost. Mbusa et al.(2018) reported that beta carotene (TCC for our case) can be measured almost quantitatively through a colour chart (visual assessment) estimates since its field estimates (based on the chart TCC values) significantly correlated with those from the laboratory analysis (quantification) in sweet potatoes. The positive but not significant correlation (0.20) between TCC and DMC is useful for developing cassava varieties that could combine both traits. This could be due to the fact that the female parents used in these studies have been selected over years at IITA-Ibadan. Esuma et al. (2016) and Mbusa et al. (2018) reported negative correlations between TCC and DMC in both cassava and sweet potatoes. Ceballos et al. (2013) reported an initial negative regression between DMC and TCC in CIAT cassava germplasm, which turned into a positive regression after years of recombination and selection. Hence, several years of recombination and selection could help cassava breeders combine both traits in African breeding programmes. Negative correlation was observed between CMD, CGM and the yield components (RTN, TWT and RTW). The negative correlation is due to the fact that CMD severity (and other pests and diseases) are scored low index for high resistance and vice versa (i.e. low resistance receives high severity index). So good disease response (low scores) go with high yield response (including yield components). It implies therefore that yield and yiled components respond to selection for resistance to pest and diseases. This is the result of the negative impact of diseases and pests on cassava root yield (Hahn et al. 1980; Fokunang et al. 2000; Ssemakula and Dixon 2007; Parkes et al. 2013). The positive significant correlation between RTN and RTW, and TWT and RWT, has also been reported by several other authors (Akinwale et al. 2010; Ntawuruhunga and Dixon 2010; Parkes et al. 2013; Chikoti et al. 2016). This indicates that the higher the root number, the higher the root weight, and eventually the higher the root yield. The non-significant correlation between RWT and DMC suggested that there was no pleiotropic effect between them, and that they 90 can be selected for independently. However, there is need for breeding to combine both traits to enhance dry root yield which is critical to the commercialization of the crop. Recombination and selection will therefore need to be fixed through crosses and then selected for at early stages of a breeding programme. Combined yield and DMC selection should therefore be possible at the seedling stage (Tumuhumbiase et al. 2014). Lastly, the negative correlation between pulp colour and RTN and RTW has also been reported by Ojulong (2006) who stated that colour is highly correlated with beta carotene and negatively with RWT, which is a key driver for adoption. This means that improving the colour may compromise the root yield. Again, breeding to improve simultaneous selection for both traits into a single genotype should be a breeding objective and should be done for both root color and yield for value addition to the crop. Genetic information was generated in the current study in order to estimate GCA and SCA values for traits of interest. Proportion sum of squares (SS) of both the GCA and SCA as percentage of total sum of squares were calculated to help determine the relative importance of additive and non-additive effects (Falconer and Mackay 1996). This information is critical in the selection of appropriate progenitors and breeding methods for efficient cassava breeding for CMD, DMC and TCC. GCA SS were higher than their respective SCA SS for CMD, HI and TCC, explaining more than 70% of the total variation. This suggests that additive gene effects are more important for the three traits. Esuma et al. (2016) reported that GCA accounted for significantly larger SS than SCA SS for HI and TCC. Baafi et al. (2016) reported larger GCA than SCA SS in sweet potatoes for beta carotene. Tumuhimbise et al. (2014) also reported larger GCA SS than SCA SS for CMD severity and HI. The relative importance of additive gene action for TCC, CMD and HI was confirmed by the higher Baker's ratios (more than 0.5) for these traits. The results suggest that both TCC and CMD could be enhanced through recurrent mass selection due to the additive nature and high heritability for these traits (Ceballos et al. 2013). Improved varieties released in the 1980s and 1990s by IITA explored additive genetic effects for CMD resistance. The discovery of a dominant CMD2 gene (Akano et 2002) has since facilitated the rapid development of CMD resistance in cassava and breeding programs in Africa have explored this gene in the development of most varieties releases in 2000s and subsequently. The use of both additive genetic variance and dominance gene could enhance the development of more durable and stable CMD resistance genetic background for the introgression of other traits such as TCC and DMC. Further studies need to be done on the 91 parental lines used in this study to ascertain if any of the parental lines have a pedigree for the CMD2 gene. However it might be difficult to combine with both DMC and TCC due to the larger SCA SS for the former, an indication of non-additive effects. Kamau et al. (2010) and Tumuhumbiase et al. (2014) reported that DMC is under the influence of non-genetic effects. Ngailo (2015) also reported larger SCA SS for DMC when breeding sweetpotato for improved yield and related traits in Eastern Tanzania. However, the presence of some yellow-fleshed genotypes having DMC values in the same range as the white-fleshed progenitors is an indication that it is possible to breed for both TCC and DMC in a breeding programme. However, Chikoti et al. (2016) reported larger SCA SS (67.9%), indicating the influence of non-additive gene action. GCA and SCA mean squares were significant for RTN and RTW, which implies that these traits showed significant additive and dominance genetic variances. Chiona (2009) and Balcha (2015) also reported significant GCA and SCA effects for yield and yield parameters in Malawi and Ethiopia respectively, but this contrasted a report by Mbusa et al. (2018). GCA SS was larger than their respective SCA SS for RTN and RWT, suggesting the predominance of additive genetic effects, as also reflected in their mean squares. Progenitor P6 (IBA090090) showed positive GCA effects for TCC, DMC, RTW and CMD, hence could be crossed to a genetic background of high DMC to increase chances of generating clones that combine both TCC and DMC. Narrow sense heritability for the traits were high in general. Lestari et al. (2010) reported high broad sense heritability of 87% for number of storage roots. Chiona (2009) also reported high broad sense heritability of 96.9% for the same trait. Heritability in both for narrow and broad sense were high for DMC in this study. Shumbusha et al. (2014), Parkes et al. (2013) and Chiona (2009), all reported high broad-sense heritability for this trait. Broad sense heritability was generally higher than their respective narrow sense heritability for all traits, indicating the presence of non-additive gene effects for their expression. A high broad sense heritability as found for most traits implies that these traits have a highly heritable portion of variation due to both additive and non-additive gene effects with relatively lower influence from the environment. High heritability of a characteristic can be exploited by plant breeders through selection (Akinwale et al. 2010). Narrow sense heritability is more important as it measures the relative importance of the additive portion of the genetic variance that can be transmitted to the next generation of the offspring (Fehr 92 1991). Hence, the high narrow sense heritability observed for all the traits were good for the breeding programme. 4.5 Conclusions Data generated from this study can be applied for planning an efficient cassava breeding strategy for breeding of yellow flesh cassava in Ghana. The analysis of variance and the GCA:SCA ratio indicated that the GCA was larger than SCA for CMD, HI and TCC and also with predictability ratios close to 1, indicating the presence of additive gene effects and a possibility for improvement of the characters by selection. Some yellow-fleshed genotypes having DMC values in the same range as the white-fleshed progenitors is an indication that it is possible to breed for both TCC and DMC in a breeding programme in Africa, and this was confirmed by the positive (though not significant) correlation between the two traits. Progenitor P6 (IBA090090) showed positive GCA effects for TCC, DMC, RTW and CMD, hence could be crossed to a genetic background of high DMC to increase chances of generating clones that combine both TCC and DMC. Findings of this study showed that yield and quality characteristics can be selected simultaneously, such as DMC early stages of the breeding cycle. References Acquaah G (2012) Principles of plant genetics and breeding. 2nd ed. Wiley-Blackwell, Oxford Akano A, Dixon A, Mba C, Barrera E, Fregene M (2002) Genetic mapping of a dominant gene conferring resistance to cassava mosaic. Theoretical and Applied Genetics, 105: 521- 525 Akinwale MG, Aladesanwa RD, Akinyele BO, Dixon AGO, Odiyi AC (2010) Inheritance of β- carotene in cassava (Manihot esculenta Crantz). International Journal of Genetics Molecular Biology 2: 198-201 Baafi E, Ofori K, Carey EE, Gracen EV, Blay ET, Manu-Aduening J (2016) Genetic control of beta-carotene, iron and zinc content in sweetpotato. Journal of Plant Studies 6: 1- 10 Baker RJ (1978) Issues in diallel analysis. Crop Science 18: 533-536 Balcha FG (2015) Breeding of sweet potato for improvement of root dry matter and β- carotene contents in Ethiopia. PhD Thesis, University of KwaZulu-Natal, South Africa 93 Bhateria S, Sood SP, Panthania A (2006) Genetic analysis of quantitative traits across environments in linseed (Linum usitatissimum L.). Euphytica 150: 185-194 Bouis HE, Hotz C, McClafferty B, Meenakshi JV, Pfeiffer WH (2011) Biofortification: A new tool to reduce micronutrient malnutrition. Food Nutrition Bulletin 32: S31- S40 Ceballos H, Morant N, Sanchez T, Ortiz D, Aragon I, Chavez AL, Pizarro M, Calle F, Dufour D (2013) Rapid cycling recurrent selection for increased carotenoids content in cassava roots. Crop Science 53: 1-10 Chávez AL, Sánchez T, Jaramillo G, Bedoya JMI, Echeverry J, Bolaños EA, Ceballos H, Iglesias CA (2005) Variation of quality traits in cassava roots evaluated in landraces and improved clones. Euphytica 143: 125-133 Chikoti CP, Shanahan P, Melis R (2016) Combining ability analysis of cassava (Manihot esculenta Crantz) genotypes for cassava mosaic disease in cassava. Australian Journal of Crop Science 10: 956-963 Chiona M (2009) Towards enhancement of β-carotene content of high dry mass sweetpotato genotypes in Zambia. PhD thesis, University of Kwazulu-Natal, South Africa Chipeta MM, Bakosi JM, Saka VW, Benesi IRM (2013) Combining ability and mode of gene action in cassava for resistance to cassava green mite and cassava mealy bug in Malawi. Journal of Plant Breeding and Crop Science 5: 195-202 CIAT (2003) Project IP3 Improved cassava for the developing world. Annual report. Cali, Colombia DaSilva AMZ (2008) Breeding potential of cassava (Manihot esculenta Crantz) in Mozambique. PhD Thesis. University of the Free State, South Africa Dwivedi SL, Sahrawat KL, Rai KN, Blair MW, Andersson MS, Pfeiffer W (2012) Nutritionally enhanced staple food crops. Plant Breeding Reviews 36: 173-293 Edoh NL, Adiele J, Ndukwe I, Ogbokiri H, Njoku DN, Egesi CN (2015) Evaluation of high beta carotene cassava genotypes at advanced trial in Nigeria. The Open Conference Proceedings Journal. 7:144-148 Esuma W, Kawuki RS, Herselman L, Labuschagne MT (2016) Diallel analysis of provitamin A carotenoid and dry matter content in cassava (Manihot esculenta Crantz). Breeding Science 66: 627-635 Falconer DS, Mackay TFC (1996) Introduction to quantitative genetics. Fourth edition. Longman Scientific and technical Co., Essex, England. Fasahat P, Rajabi A, Rad JM, Derera J (2016) Principles and Utilization of combining ability in plant breeding. Biometrics and Biostatistics International Journal 4:1-22 94 Fehr WR (1991) Principles of cultivar development (vol. 1): Theory and Technique. Iowa, USA: Macmillian Publishing Company Fokunang C, Ikotun T, Dixon A, Akem C (2000) Field reaction of cassava genotypes to anthracnose, bacterial blight, cassava mosaic disease and their effects on yield. African Crop Science Journal 8: 179-186 Hahn SK, Terry ER, Leuschner K (1980) Breeding cassava for resistance to cassava mosaic disease. Euphytica 29: 673-683 Hirut B, Shimelis H, Fentahun M, Bonierbale M, Gastelo M, Asfaw A (2017) Combining ability of highland tropic adapted potato for tuber yield and yield components under drought. PLoS ONE 12(7): e0181541 IITA (International Institute of Tropical Agriculture) (1990) Cassava in Tropical Africa: A reference manual. Chayce Publications Services, Balding Mansell International, Wisbech, UK Kamau J, Melis R, Laing M, Derera J, Shanahan P, Ngugi E (2010) Combining the yield ability and secondary traits of selected cassava genotypes in the semi-arid areas of Eastern Kenya. Journal of Plant Breeding and Crop Science 2: 181-191 Kawano K, Narintaraporn K, Narintaraporn P, Sarakarn S, Limsila A, Limsila J, Suparhan D, Sarawat V, Watananonta W (1998) Yield improvement in a multistage breeding program for cassava. Crop Science 38: 325-332 Lestari SM, Hapsari I, Sutoyo S (2012) Improving storage root protein content in sweetpotato through open-mating pollination. Agrivita Journal of Agricultural Science 34: 225-232 Maroya NG, Kulakow P, Dixon AGO, Maziya-Dixon BB (2012) Genotype x Environment Interaction of mosaic disease, root yields and total carotene concentration of yellow- fleshed cassava in Nigeria. International Journal of Agronomy 2012: 1-8 Mbusa HK, Ngugi K, Florence MO, Kivuva BM, Muthoni JW, Nzuve FM (2018) The inheritance of yield components and beta carotene content in sweet potato. Journal of Agricultural Science 10: 71-81 Nassar NMA, Ortiz R (2010) Breeding cassava to feed the poor. Scientific American Journal 302: 78-82 Ngailo ES (2015) Breeding sweetpotato for improved yield and related traits and resistance to sweetpotato virus disease in Eastern Tanzania. PhD Thesis, University of Kwazulu- Natal, South Africa 95 Njoku DN, Gracen VE, Offei SK, Asante IK, Egesi CN, Kulakow P, Ceballos H (2015) Parent offspring regression analysis for total carotenoids and some agronomic traits in cassava. Euphytica 206: 657-666 Njoku DW (2012) Improving Beta-carotene content in farmers’ preferred cassava cultivars in Nigeria. Doctoral thesis, WACCI, University of Ghana, Legon, Ghana Ntawuruhunga P, Dixon AGO (2010) Quantitative variation and interrelatiohip between factors influencing cassava yield. Journal of Applied Biosciences 26:1594-1602 Ojulong HF (2006) Quantitative and molecular analysis of agronomic traits in cassava (Manihot esculenta Crantz). PhD Thesis in Plant Breeding, University of the Free State, South Africa Ortiz D, Sánchez T, Morante N, Ceballos H, Pachon H, Duque MC, Chavez AL, Escobar AF (2011) Sampling strategies for proper quantification of carotenoid content in cassava breeding. Journal of Plant Breeding and Crop Science 3: 14-23 Parkes EY, Fregene M, Dixon AGO, Peprah BB, Labuschagne MT (2013) Combining ability of cassava genotypes for cassava mosaic disease and cassava bacterial blight, yield and its related components in two ecological zones in Ghana. Euphytica 194: 13-24 Pasajee K, Kittipadakul P, Phumichai C, Lertsuchatavanich U, Petchpoung K (2016) Path analysis of agronomic traits of Thai cassava for high root yield and low cyanogenic glycoside. Pertanika Tropicak Agricultural Science 39: 197-218 Rodriguez F, Gregorio A, Pacheco A, Crossa J, Burgueno J (2015) Analysis of genetic designs with R (AGD- R), version 5.0. CIMMYT Research Software Sánchez T, Ceballos H, Dufour D, Ortiz D, Morante N, Calle F, Zum Felde T, Dominguez M, Davrieux F (2014) Prediction of carotenoids, cyanide and dry matter contents in fresh cassava root using NIRS and hunter color techniques. Food Chemistry 151: 444-451 Shumbusha D, Tusiime G, Edema R, Gibson P, Adipala E, Mwanga ROM (2014) Inheritance of root dry matter content in sweetpotato. African Crop Science Journal 22: 69-78 Ssemakula G, Dixon AGO (2007) Genotype x environment interaction, stability and agronomic performance of carotenoid- rich cassava clones. Scientific Research and Essay 2: 390-399 Tumuhimbise R, Shanahan P, Melis R, Kawuki RS (2014) Combining ability analysis of storage root yield and related traits in cassava at the seedling evaluation stage of breeding. Journal of Crop Improvement 28: 530-540 96 Vasconcelos LM, Brito AC, Carmo CD, Oliveira PHGA, Oliveira EJ (2017) Phenotypic diversity of starch granules in cassava germplasm. Genetics and Molecular Research 16 (2): gmr16029276 97 Chapter 5 Genetic variability, stability and heritability for quality and yield characteristics in provitamin A cassava varieties Published as: Peprah, B.B., Parkes, E., Manu-Aduening, J., Kulakow, P., van Biljon, A., Labuschagne, M.T., 2020. Genetic variability, stability and heritability for quality and yield characteristics in provitamin A cassava varieties. Euphytica 216:31. https://doi.org/10.1007/s10681-020-2562-7. Abstract Cassava is widely consumed in many areas of Africa, including Ghana, and is a major part of most household diets. These areas are characterised by rampant malnutrition, because the tuberous roots are low in nutritional value. Provitamin A (pVA) biofortified cassava varieties have been developed by the International Institute for Tropical Agriculture, but adoption of these varieties in Ghana will largely depend on their agronomic performance, including fresh root weight, dry mater content (DMC), resistance to major pests and diseases, mealiness, starch content and the stability of these traits. Eight pVA varieties with two white checks were planted in three environments for two seasons to determine stability and variability among the varieties for important traits. There were significant variations in performance between varieties and between environments for cassava mosaic disease, storage root number, fresh root weight and starch content. High broad-sense heritability and genetic advance were observed for all traits, except DMC, and could be exploited through improvement programmes. This study identified the best performing enhanced pVA varieties for traits, which are key drivers of variety adoption in Ghana. In view of this, some varieties can be recommended for varietal release after on-farm testing. The study also showed the possibility of tapping heterosis after careful selection of parents. 5.1 Introduction The populations of underdeveloped and developing countries often suffer undernourishment and “hidden hunger” as a result of micronutrient deficiencies (Maroya et al. 2010). Areas in Africa, including Ghana, where cassava is widely consumed, are characterised by rampant malnutrition because the tuberous roots are low in nutrients such as VA (Ssemakula and Dixon 2007). It is for this reason that the development of nutrient 98 dense cassava cultivars needs more attention to eliminate the ramifications of malnutrition among the poor in an inexpensive and sustainable way. VAD constitutes a public health problem and affects mainly children and women. Recently, different programmes such as HarvestPlus, involving a global alliance of research institutions, initiated the development of micronutrient-dense staple crops (Bouis et al. 2011; Dwivedi et al. 2012). Among these initiatives is the development of biofortified cassava clones with high PVAC in the roots. Adoption of biofortified cassava varieties in Ghana will largely depend on their agronomic performance, including FRW, DMC, resistance to major pests and diseases, starch content and the stability of these traits over time and space. DMC influences texture after boiling, and is also a key parameter in the production of gari (a popular cassava food in Ghana). According to Ceballos et al. (2017), there is no negative relationship between carotenoids and DMC, thus, making it possible to identify varieties with high PVAC and acceptable levels of DMC. GEI is the result of inconsistent performance of varieties across environments. The expression of genes that control key agronomic traits in cassava is influenced by both abiotic and biotic stresses, which can lead to GEI (Kang 2002). Breeders face the GEI challenge by evaluating genotypes in several environments to ensure that they have good and stable performance (Acquaah 2012). Several statistical models have been developed to interpret GEI data to understand stability. Scientists have highlighted weaknesses and strengths of these models, which includes commonly used ones like additive main effects and multiplicative interaction (AMMI) as well as genotype and genotype by environment interaction (GGE) biplots. Several studies on cassava have used AMMI for assessment of GEI effects for traits and storage root yield (Kvitschal et al. 2006; Aina et al. 2007), carotenoid and DMC (Maroya et al. 2010; Esuma et al. 2016) and early bulking of storage roots (Agyeman et al. 2015). The AMMI model was reported to capture a large portion of the GGE sum of squares and uniquely separates main and interaction effects as required for most agricultural research purposes (Gauch 2006). Yet, the AMMI biplot does not have the most important feature of a true biplot, namely the inner-product property and this biplot does not display the discriminating ability and representativeness view of a biplot, which is effective in evaluating test environments. Hence, the GGE biplot has been proposed to effectively identify the best-performing genotypes across environments, identify the best genotypes for mega-environment delineation, whereby specific genotypes 99 can be recommended for specific mega-environments and evaluate the yield and stability of genotypes (Yan and Kang, 2003; Yan and Tinker, 2006). This study was designed to evaluate yellow flesh cassava clones across locations for DMC, CMD, CGM, starch content, yield and its related characteristics; to determine the magnitude of genotype, environment, and GEI effects on these traits, and to identify stable and high performing clones for DMC and FRW using GGE biplots. 5.2 Materials and methods 5.2.1 Varieties, experimental sites and design Ten varieties were evaluated, of which eight were selected from sets of yellow flesh cassava clones previously acquired from IITA and the other two varieties were white- fleshed landraces obtained from farmer fields in Ghana (Table 5.1). Trials were conducted over two seasons, May 2015 - May 2016 and June 2016 - June 2017 at three locations situated in different agroecological zones. Fumesua (forest), Ejura (forest transition) and Kokroko (transition). Each planting season was considered an environment, giving a total of six environments. Temperature and rainfall data were recorded during the experimentation period as well as soil nutrient profile of the fields prior to planting the trials (Table 5.2). Trials were laid out in a RCBD with three replications, each consisting of four rows of seven plants, giving a plot size of 28 plants. Planting was done at a spacing of 1 × 1 m. To increase chances of sprouting and uniform plant establishment, all stakes used for planting were generated from the middle portions of mature stems. Replications were separated by 2 m alleys. Weeding was done when necessary and experiments were entirely rain fed. 100 Table 5.1 Provitamin A and white flesh cassava genotypes used for the study Genotype Code Status Source Pulp colour IBA090090 G1 Improved IITA Yellow IBA090151 G2 Improved IITA Yellow IBA070557 G3 Improved IITA Yellow IBA085392 G4 Improved IITA Yellow Debor G5 Landrace Farmer White IBA083774 G6 Improved IITA Yellow IBA070593 G7 Improved IITA Yellow IBA070539 G8 Improved IITA Yellow UCC G9 Released CSIR-CRI White IBA083724 G10 Improved IITA Yellow 5.3 Data collection The varieties were evaluated at monthly intervals, starting at 1 MAP to 9 MAP, for their reaction to CMD and CGM. Damage symptoms were scored on a scale of 1 - 5, where 1 = no symptoms and 5 = very severe symptoms (IITA, 1990). Only the score of the most severely affected plants were recorded in a plot. For each trial, TCC, DMC, FRW and HI were measured at 12 MAP. The inner two rows of each experimental plot constituted a net plot of 10 plants for measurement of the traits. Biomass from harvested plants was bulked to estimate yield components by separately weighing the FRW and foliage (FSW). HI was computed from the measure of FRW and FSW as: HI = FRW/(FRW + FSW). Root samples from each plot (5 kg) were weighed in air (Wa) using a balance after cleaning the soil and other debris from the roots. The root samples were again weighed in water (Ww). The same container was used to weigh the sample in both air and water. Specific gravity was calculated as: X = Wa/(Wa – Ww) DMC and starch content were calculated using the following formulas: DMC = 158.3 x specific gravity - 142 (Kawano et al., 1987) Starch content = (210.8 x specific gravity) - 213.4 (Howeler, 2014). 101 The mealiness was measured by taking a small portion of the boiled sample and pressing it between the thumb and the index finger. When it is soft and can form a sticky paste, it is considered mealy and suitable for ‘ampesi’ (that is boiled and eaten) or for ‘fufu’. On the other hand, the hard and difficult to press root will not form a sticky paste and is considered non-mealy. However, non-mealy genotypes can be used for cassava dough ‘agbelima’, or dried for ‘konkonte’, cassava chips or processed into gari. Mealiness is measured on a scale of 1 - 4 (1 = non-mealy 2 = mealy, 3 = very mealy and 4 = excellent) (Parkes, 2011). The plant vigour was measured 3 MAP in terms of how the plants germinated. The scale for measurement was 1 - 4, (1 = very poor, 2 = poor, 3 = good, 4 = very good). Table 5.2 Characteristics of the six trial environments Season 1 (May 2015 – May 2016) Season 2 (June 2016 – June 2017) Parameter Edubiase Kokroko Pokuase Edubiase Kokroko Pokuase (Env1) (Env2) (Env3) (Env4) (Env5) (Env6) pH 5.3 6.2 7.1 5.6 5.7 6.9 OM (%) 2.0 0.9 2.2 2.4 0.4 2.0 N (%) 0.3 0.03 0.1 0.1 0.1 0.2 P (ppm) 3.4 3.1 5.8 3.9 4.6 4.7 Ca (ppm) 3.0 1.8 5.1 3.1 2.3 5.8 Mg (ppm) 2.3 1.2 1.7 1.1 2.1 1.4 K (ppm) 0.8 0.6 0.1 0.1 3.2 0.1 Zn (ppm) 13.9 1.5 34.1 13.1 1.8 44.0 B (ppm) 0.4 0.6 0.2 0.3 0.8 0.2 Cu (ppm) 25.9 0.9 44.0 24.0 0.7 54.0 Fe (ppm) 17365.6 3775.6 5939.38 18365.6 3752.6 6139.28 Mn (ppm) 1618.63 470.98 1367.72 1508.63 533.72 1478.71 Rainfall (mm) 2100.0 892.9 1072.4 2350 1160.9 1420.0 Min T (oC) 21.7 32.0 30.2 21.3 32 31.0 Max T (oC) 29.1 35.0 35.8 31.1 34 34.8 Latitude 4°40’0’’N 7°39’1.57’’N 5°42’0’’N Longitude 1°38’0’’W 1°56’56.48’’W 0°16’36’’W Altitude 136.1 482.1 45.7 OM = Organic matter content; Min T = Minimum temperature; Max T = Maximum temperature 102 TCC was measured following the method of Rodriquez-Amaya and Kimura (2004). Fresh cassava roots of three different sizes; small, medium and large, were washed with tap water to remove dirt and debris, allowed to dry and then peeled. The peeled roots were washed with deionized water to avoid contamination and dried with tissue under a subdued light to protect carotenes. Root samples and extracts were protected from the light as much as possible. Roots were cut longitudinally in half and then the two halves were cut again longitudinally into quarters. Each quarter would therefor include tissue from the periphery, mid-parenchyma and core of the root, as well as proximal, central and distal sections (Chávez et al., 2008). The two quarters of each root were then ground and mixed for a uniform sample. The sample was then packaged into aluminum foil, placed into a whirl pack and labeled. Ten gram of the test sample was transferred into a clean dried mortar, and about 3 g of Celite was added to the test sample to ease maceration of the cassava tissues as well as filtration. Cold acetone (50 ml) was first added in the mortar. The mixture was crushed with a pestle until fine and then filtered. Extraction was repeated three times with cold acetone to ensure complete extraction. The extract was filtered using a Buchner funnel with 90 mm filter paper and rinsed with cold acetone.The combined extract was transferred into a separation funnel with 5 ml distilled water and 20 ml petroleum ether. Deionized water (500 ml) was dispensed through the walls of the separation funnel to wash the acetone down. Brine solution was added to break any emulsion formed in the ether extract. The petroleum ether extract containing the carotenoids was partitioned in the upper layer in the separating funnel, and the aqueous layer was gradually discarded. The extract was then transferred gradually into a 25 ml volumetric flask using a small funnel with sodium sulfate on top of cotton wool to dry any excess water. Petroleum ether was added to the extract in the volumetric flask and transferred into a 30 ml glass bottle. Aliquots of the extracts were transferred into a cuvette and was read using an UV-Vis spectrophotometer at wavelength of 450 nm from which absorbance readings was obtained and TCC (µg g-1) calculated as: TC = [A x volume (ml) x 104]/[A1% 1xcm s ample weight (g)] where A = absorbance; volume = total volume of extract 25 ml, A1%1cm = absorption coefficient of beta carotene in PE (2592). 103 All procedures for carotenoid extraction and measurement were performed in subdued light and samples were analyzed within 24 hours of harvesting. TCC was measured only in one year without replication to confirm status of genotypes as pVA enriched. 5.4 Data analysis Data were subjected to ANOVA and AMMI analysis for FRW, DMC, RTN and starch of 10 cassava varieties obtained per plot across environments, using Genstat software Release 17.0 (2011). Genetic effects were considered fixed, and location and season effects random. The GGE biplot method outlined by Yan (2002) was used to display the G and GEI patterns in the data in a biplot. The which-won-where pattern, which is an intrinsic property of the GGE biplot rendered by the inner-product property of the cassava genotype environment data set, was also visually presented. In addition, the GGE biplot was used to identify high yielding and adapted cassava varieties as well as suitable test environments. Stable varieties for each environment were selected from AMMI analysis and principal component (PCA) axes were extracted and statistically tested by Gollob’s (1968) F-test procedure (Vargas and Crossa 2000). Phenotypic correlation coefficients and PCA and its biplots were analysed using Genstat software Release 17.0. Trait components and magnitude of variation responsiveness to selection were calculated according to Okwuagwu et al. (2008). Expected genetic advance of the mean for each trait was calculated according to Allard (1960). Genotypic and phenotypic variances were calculated according to Obilana and Fakorede (1981). 5.5 Results 5.5.1 Analysis of variance In the combined ANOVA (Table 5.3) the main effects (genotype, location and year) were highly significant (P < 0.001) for all traits evaluated except CMD and significant (P < 0.05) for location and year. Values for separate seasons were only presented for the five main yield components, as the focus was on the data of the seasons combined. Values of DMC, FRW and HI were significantly higher in the second season than the first (Table 5.4) due to more favourable growing conditions in the second season. Combined over two seasons FRW ranged from 18.99 to 32.67 t ha-1 with a mean of 23.43 t ha-1 (Table 5.5). Genotype IBA083774 had the highest yield of 32.67 t ha-1, while the lowest 104 value of 18.99 t ha-1 was recorded by IBA085392. DMC ranged from 23.19% to 30.26% with a mean of 27.38%. The local cultivar recorded the highest (30.25%) DMC, followed by IBA083774 (29.39%) and IBA085392 recorded the lowest value (23.19%). CMD scores ranged from 1.0 to 2.17 with a mean of 1.15. All the yellow flesh cassava varieties had a severity score of 1.0 with the exception of IBA070593 (1.17). The local cultivar recorded the highest severity score (2.17) to CMD. All the elite cassava genotypes from IITA recorded higher TCC values than the local check used. Genotype IBA083774 with the highest FRW recorded the lowest TCC values among the IITA materials and the local check with the highest DMC recorded the lowest TCC value. 105 Table 5.3 Analysis of variance and contribution of main effects to variation for measured characteristics across three environments in two growing seasons Source Df SS MS % of total SS CMD Genotype 9 21.45 2.38*** 65.10 Location 2 0.23 0.12ns 0.70 Year 1 0.27 0.27* 0.82 Gen.loc 18 1.43 0.08ns 4.34 Gen.year 9 1.67 0.19*** 5.07 Loc.year 2 0.21 0.11ns 0.64 Gen.loc.year 18 1.68 0.09* 5.10 Error 118 6.00 0.05 DMC Genotype 9 883.66 98.19*** 39.96 Location 2 245.34 122.67*** 11.10 Year 1 189.11 189.11*** 8.55 Gen.loc 18 191.27 85.75*** 8.65 Gen.year 9 57.47 6.39* 2.60 Loc.year 2 171.50 85.75*** 7.76 Gen.loc.year 18 86.49 4.80ns 3.91 Error 118 380.27 3.22 Starch Genotype 9 443.13 49.24*** 39.96 Location 2 123.03 61.52*** 11.10 Year 1 94.83 94.83*** 8.55 Gen.loc 18 95.91 5.33*** 8.65 Gen.year 9 28.82 3.20* 2.60 Loc.year 2 86.01 43.00** 7.76 Gen.loc.year 18 43.38 2.41ns 3.91 Error 118 190.70 1.62 FRW Genotype 9 2758.90 306.40*** 12.02 Location 2 4413.48 2206.74*** 19.22 Year 1 3029.08 3029.08*** 13.19 Gen.loc 18 1499.40 83.30ns 6.53 Gen.year 9 671.50 74.61ns 2.92 Loc.year 2 2189.76 1094.88*** 9.54 Gen.loc.year 18 1648.67 91.59* 7.18 Error 118 6685.96 56.66 RTN Genotype 9 9060.10 1006.70*** 16.39 Location 2 3390.30 1695.20*** 6.13 Year 1 1566.50 1566.50*** 2.83 Gen.loc 18 10419.90 578.90*** 18.85 Gen.year 9 3031.20 336.80ns 5.48 Loc.year 2 1073.10 536.50ns 1.94 Gen.loc.year 18 5854.50 325.20* 0.11 Error 118 20725.90 175.60 *P≤0.05, ** P≤0.01 ***P≤0.001; CMD = cassava mosaic disease; DMC = dry matter content; FRW = fresh root weight; RTN = storage root number 106 Table 5.4 Means of five traits measured in two growing seasons (2015/2016 and 2016/2017) in 10 genotypes across six environments in Ghana Traits DMC FRW CMD HI RTN Genotype Year % t ha-1 IBA90090 1 26.00a 18.71a 1.0a 0.36a 39.22a 2 27.91b 20.67a 1.0a 0.36a 43.11a IBA090151 1 26.14a 21.00a 1.00a 0.38a 55.24a 2 29.10b 29.06b 1.00a 0.41a 56.89a IBA070557 1 28.93a 19.11a 1.00a 0.34a 44.78a 2 28.60a 26.06b 1.00a 0.43b 42.89a IBA085392 1 22.61a 12.78a 1.00a 0.28a 29.11a 2 23.77b 25.06b 1.00a 0.33b 56.00b IBA083724 1 27.95a 21.63a 1.00a 0.28a 53.56a 2 30.37b 27.61a 1.00a 0.43b 60.22b IBA083774 1 28.82a 28.28a 1.00a 0.45a 53.56a 2 29.96a 37.06b 1.00a 0.47a 59.44a IBA070593 1 24.59a 16.71a 1.11a 0.37a 34.89a 2 28.15b 21.67a 1.22a 0.34a 36.00a IBA070539 1 23.03a 15.79a 1.00a 0.42a 34.22a 2 24.75b 29.17b 1.00a 0.52b 48.00b UCC 1 26.97a 23.89a 1.33a 0.55a 44.22a 2 29.48b 29.06b 1.00b 0.46b 43.89a Local check 1 28.54a 15.43a 2.44a 0.42a 45.22a 2 31.98b 29.94b 1.89b 0.46b 46.11a Mean 1 26.36a 19.33a 1.19a 0.38a 43.36a 2 28.41b 27.53b 1.11a 0.42b 49.26a DMC = dry matter content (%); FRW = fresh root weight (t ha-1); CMD = cassava mosaic disease; HI = harvest index; RTN = storage root number (t ha-1). Means followed by the same letter are not statistically different 107 Table 5.5 Mean values of nine traits measured in 10 genotypes across six environments in Ghana Genotype FRW RTN TWT DMC Starch HI CMD CGM Mealy TCC IBA090090 19.69 41.17 35.79 26.95 13.24 0.45 1.00 1.27 1.94 10.37 IBA090151 25.03 56.06 39.04 27.62 13.72 0.34 1.00 1.05 1.67 12.73 IBA070557 22.58 43.83 34.42 28.76 14.52 0.39 1.00 1.11 1.89 7.78 IBA085392 18.99 42.56 40.62 23.19 10.59 0.31 1.00 1.16 1.50 11.74 IBA083724 24.62 56.67 30.23 29.16 14.81 0.36 1.00 1.12 1.89 6.58 IBA083774 32.67 56.50 39.97 29.39 14.97 0.46 1.00 1.50 1.06 3.12 IBA070593 19.19 35.44 33.72 26.37 12.83 0.35 1.17 1.61 1.94 16.00 IBA070539 22.48 41.11 23.27 23.89 11.08 0.47 1.00 1.38 1.66 13.79 UCC 26.47 44.06 24.84 28.23 14.15 0.51 1.17 1.33 2.50 3.13 Local 22.69 45.67 38.64 30.26 15.68 0.44 2.17 1.67 3.33 0.78 Grand mean 23.43 46.31 34.04 27.38 13.54 0.41 1.15 1.33 1.93 8.60 S.e.d 6.15 10.82 7.89 1.47 2.06 0.05 0.18 0.47 0.76 1.56 CV % 32.10 28.60 28.40 6.60 9.40 15.50 19.60 43.20 48.20 59.70 FRW = fresh root weight (t ha-1); RTN = storage root number (t ha-1); TWT = total biomass (t ha-1); DMC = dry matter content (%); HI = harvest index; CMD = cassava mosaic disease; CGM = cassava green mite; TCC = total carotenoid content (µg g-1) 107 5.5.2 Additive main effects and multiplicative interaction analysis Combined AMMI ANOVA (Table 5.6) showed that genotype, environment and GEI effects were highly significant (P<0.001) for CMD, DMC, FRW, RTN and starch. IPCA1 mean squares were highly significant (P<0.001) for all traits except FRW, which was significant at P<0.01. IPCA1 and IPCA2 accounted for more than 70% of the total variation observed in GEI, which was confirmed by the significant (P<0.001) GEI effects for all traits Table 5.6 AMMI analysis of variance for measured characteristics Source df CMD DMC RTN FRW Starch Genotype 9 2.38*** 98.18*** 1006.7*** 306.5*** 49.24*** Environment 5 0.14*** 121.19*** 1206.0*** 926.5*** 60.77*** GEI 45 0.11*** 7.45*** 429.0*** 84.9*** 3.74*** IPCA1 13 0.28*** 15.83*** 770.3*** 127.5** 7.94*** IPCA2 11 0.07ns 3.72ns 396.8*** 89.4* 1.87ns Residual 21 0.02 4.210 234.60 56.10 2.11 % GEI due to IPCA1 76.29 61.40 51.87 43.40 61.39 % GEI due to IPCA2 15.89 12.23 22.61 25.76 12.20 *P≤0.05, ** P≤0.01 ***P≤0.001, CMD = cassava mosaic disease; DMC = dry matter content; RTN = storage root number; FRW = fresh root weight; GEI = genotype by environment interaction; IPCA = interaction principal component axis 5.5.3 Correlations, genetic components and principle component analysis RTN and TWT, FRW and TWT, TWT and vigour, HI and RTN, HI and FRW, CMD and mealiness, RTN and FRW and CGM and HI were highly significantly positively correlated. Significant negative correlations were observed between TWT and HI as well as between vigour and HI showed (Table 5.7). The magnitude of the phenotypic coefficient of variation (PCV) was higher than their corresponding genotypic coefficient of variation (GCV) for all the traits studied. The PCV ranged between 8.55% and 26.09%, with CMD showing the highest value, followed by TWT and with DMC recording the lowest value. Heritability was generally high for all characters and varied from 41.34% for RTN to 88.89% for CMD (Table 5.8). 108 Table 5.7 Phenotypic correlations coefficients for 10 traits measured on 10 cassava genotypes across six environments in Ghana Traits CGM CMD DMCc HI Mealy RTN FRW TWT CMD 0.13 DMC 0.03 0.18* HI 0.33 0.05 0.08 Mealy -0.01 0.29*** -0.001 -0.01 RTN 0.20** 0.03 0.16* 0.45*** -0.07 FRW 0.19* -0.05 0.07 0.62*** -0.18* 0.69*** TWT -0.18* -0.13* -0.02 -0.43*** -0.13 0.30*** 0.36*** Vigour -0.12 -0.04* 0.14 -0.22** -0.03 0.07 0.05 0.33** *P≤0.05, ** P≤0.01 ***P≤0.001, CGM = cassava green mite; CMD = cassava mosaic disease; DMC = dry matter content; HI = harvest index; RTN = storage root number; FRW = fresh root weight; TWT = total biomass Table 5.8 Coefficients of variation, heritability and genetic advance for five traits of 10 cassava genotypes planted in six environments Traits Genetic parameters Mean GCV PCV (%) H2b Gas FRW 25.33 15.58 17.63 78.39 26.27 RTN 46.31 10.39 16.15 41.34 13.76 TWT 34.04 14.04 18.10 60.10 22.40 Starch 13.55 11.44 13.14 75.95 20.55 DMC 27.38 8.00 8.55 87.55 15.41 CMD 1.15 24.35 26.09 88.89 47.77 GCV = genotypic coefficient of variation (%); PCV = phenotypic coefficient of variation (%); H2b = broad-sense heritability (%), Gas = expected genetic advance of the mean; FRW = fresh root weight; RTN = storage root number; TWT = total biomass; DMC = dry matter content, CMD = cassava mosaic disease From the PCA (Table 5.9) the first three principal components (PC) had eigenvalues higher than one and accounted for 83.93% of the total variation. PC1 accounted for 40.50% variation and RTN, DMC and starch were the principal contributors. PC2 accounted for 27.51% of the variation with TWT, vigour and CGM contributing most to 109 the variation. PC3 accounted for 15.92% of variation with FRW, mealiness, CMD and HI being the main contributing factors. Table 5.9 Principal component analysis of 10 quantitative traits in 10 cassava genotypes showing eigenvectors, eigenvalues, individual and cumulative percentage of variation explained by the first three principal components axis Characters Eigenvectors PC1 PC2 PC3 RTN 0.34 0.27 0.06 FRW 0.35 0.23 0.47 TWT -0.09 0.52 -0.32 Mealy 0.28 -0.33 -0.41 DMC 0.43 0.16 -0.14 Starch 0.43 0.16 -0.14 Vigour 0.12 0.49 -0.13 CMD 0.34 -0.27 -0.37 CGM 0.24 -0.31 -0.06 HI 0.32 -0.19 -0.56 Eigenvalue 4.05 2.75 1.50 Individual 40.50 27.51 15.92 Cumulative 40.50 68.01 83.93 RTN = storage root number; FRW = fresh root weight; TWT = total biomass; DMC = dry matter content; CMD = cassava mosaic disease; CGM = cassava green mite; HI = harvest index 110 5.5.4 GGE biplot for average dry matter content, fresh root weight, starch and stability of varieties The biplot (Figure 5.1) showed that PCA1 and PCA2 explained 91% of variation for DMC. DMC was highest in genotype IBA083724 (G10), followed by IBA083774 (G6) and local (G5). IBA085392 (G4) had the lowest DMC value. Varieties IBA090151 (G2) and IBA070539 (G8) were more stable with genotype IBA070593 (G7) being the most unstable. Genotype IBA083774 (G6) had the highest mean for FRW, followed by UCC (G9), IBA090151 (G2), while IBA070593 (G7) ranked the lowest (Figure 5.1). In terms of stability, varieties IBA090090 (G1) and UCC (G9) were most stable. 5.5.5 The best performing genotype in each environment and mega-environments for dry matter content, fresh root weight and starch content PC1 explained 77% and PC2 14% of variation, both reflecting 91% of the DMC variation (Figure 5.2). PC1 explained 53% and PC2 17% of variation in FRW, reflecting a total 70% of variation. A convex-hull drawn on the varieties from the origin of the biplot gave five sections with IBA070593 (G7), IBA085392 (G4), IBA090090 (G1), IBA083774 (G6) and IBA083724 (G10) as vertex varieties. G10 was the best variety in three environments (Edubiase, Env 4; Pokuase, Env 3; and Kokroko, Env 2) and G6 was best in three other environments (Edubiase, Env 1, Kokroko, Env 5 and Pokuase, Env 6) for DMC (Figure 5.2A). The biplot grouped all the environments into two groups, suggesting two mega-environments. The first mega-environment had environments Env 4, Env 3, Env 2 and Env 6 with varieties IBA083724 (G10), UCC (G9), Local (G5), IBA090151 (G2) and IBA083774 (G6) as the best performers and the second mega-environment had environments Env 5 and Env 1, with genotype IBA070557 (G3) performing best. For FRW, IBA083774 (G6), IBA070539 (G8), IBA070593 (G7), IBA085392 (G4) and IBA083724 (G10) were the vertex varieties for the five sections of the biplot (Figure 5.2B). The biplot grouped all the environments into two groups, suggesting two mega- environments. The first mega-environment had environments Env 1, Env 2, Env 3, Env 5 and Env 6 with varieties UCC (G9), IBA090151 (G2) and IBA083774 (G6) as the best performers and the second mega-environment had environment Env 4, with genotype Local (G5) performing the best. 111 B A Figure 5.1 GGE biplot showing (A) dry matter content and (B) fresh root weight mean performance and stability of 10 cassava genotypes 112 B A Figure 5.2 Which wins where GGE biplot for best cultivars for (A) dry matter content (B) fresh root weight in different environments 113 5.6 Discussion DMC, FRW, starch, CMD, mealiness and RTN are key drivers for cassava variety adoption (Abdoulaye et al. 2014; Esuma et al. 2016) in Ghana. All the yellow flesh cassava varieties in this study had higher TCC values than the local and improved check. Three of the yellow flesh cassava varieties (IBA090I51, IBA083774 and IBA083724) recorded higher FRW than the checks. In terms of DMC, the local variety was not statistically different from varieties IBA083774 and IBA083724, which recorded the highest FRW and CMD score. There were significant variations in mean performance of varieties for CMD, RTN, FRW and starch, which are some of the most important traits for consumer acceptance (Owusu and Donkor 2012), in different environments. TCC-rich cassava cultivars could be selected using on-station trials in one location and selected cultivars can be subjected to multi- location evaluation where the focus is on other important traits of cassava for variety adoption (Esuma et al. 2016) The significant genotype effects observed for the traits studied indicated that varieties were significantly different; hence, genetic improvement could be achieved through hybridization. The significant GEI (from AMMI analysis) for CMD, DMC, RTN, FRW and starch, indicated variation in genotypic responses to different environments and this underlined the importance of the multi-environment testing of newly developed varieties. The combined ANOVA for CMD, DMC and starch indicated that genotype main effect accounted for 65.10%, 39.96% and 39.96% of variation, respectively. This was confirmed by the small difference between their PCV and GCV values. Selection for such traits could be fairly easy due to the close association between the genotype and the phenotype Cassava breeding aimed at selecting desired genotypes is linked with GCV, heritability estimates, genetic advance as percentage of the population mean and other genetic parameters for important traits (Idahosa et al. 2010). The magnitude of the heritability of the selected traits studied were generally high. Pradeepkumar et al. (2001) reported that heritability estimates together with genetic advance contribute to improved selection response. The low PCV values for DMC, in this study have also been reported by other authors (Kundy et al. 2015; Ewa et al. 2017). The generally higher values of PCV than 114 their corresponding GCV values for traits indicated the considerable role of the environment in the expression of these traits; hence, the variation in the varieties are due to both genotype and the environment. The high heritability values for the measured traits indicate the presence of a larger portion of heritable variation, which would aid selection. RTN with quite high heritability and low genetic advance could pose a challenge if selection is based only on this trait. Esuma et al. (2016) confirmed a strong negative correlation between DMC and TCC. Ceballos et al. (2013) reported simultaneous gains for both TCC and DMC through rapid selection. There is need in Ghana to combine these two important traits in the breeding programme. The best yellow flesh cassava varieties identified in this study could be the starting material for this improvement. Correlations among traits play an important role in plant breeding as it can improve selection efficiency. The positive significant correlation between FRW and RTN, TWT, HI, DMC and starch, suggests that an increase in mean value of any one of these character pairs would significantly increase the mean of the other (Akinwale et al. 2010). The negative significant correlation of HI and TWT is very important in cassava breeding, where the ultimate focus is on yield/weight (storage roots) which correlates positively with HI. However, varieties must also produce prolific stems from planting material which is also related to TWT. The negative correlations between CMD and FRW, TWT and vigour confirms the potential storage root yield losses that can be caused by the disease, which was confirmed by Parkes et al. (2013). There was also a significant positive correlation between CMD and mealiness. Landraces are more susceptible to CMD and most landraces in Ghana are mealy. 5.7 Conclusions This study showed the best performing TCC-rich varieties also have variation for important traits of cassava, which are key drivers of variety adoption in Ghana. In view of this, varieties IBA090151, IBA083774 and IBA083724 can be considered for varietal release after on-farm testing. The study also revealed that the yellow flesh cassava varieties could be used in a hybridization scheme with the local material to combine both TCC and DMC traits with high yield in a CMD resistance background. 115 References Abdoulaye T, Abass A, Maziya-Dixon B, Tarawali G, Okechukwu R, Rusike J, Alene A, Manyong V, Ayedun B (2014) Awareness and adoption of improved cassava varieties and processing technologies in Nigeria. Journal of Development and Agriculture Economics 6: 67-75 Acquaah G (2012) Principles of Plant Genetics and Breeding. 2nd ed. John Willey and Sons Ltd, UK Agyeman A, Parkes EY, Peprah BB (2015) AMMI and GGE biplot analyses of root yield performance of cassava genotypes in forest and coastal ecologies. International Journal of Agriculture Policy Research 3: 122-132 Aina OO, Dixon AGO, Akinrinde EA (2007) Additive Main Effects and Multiplicative Interaction (AMMI) Analysis for yield of cassava in Nigeria. Journal of Biological Sciences 7: 796-800 Akinwale MG, Aladesanwa RD, Akinyele BO, Dixon AGO, Odiyi AC (2010) Inheritance of β- carotene in cassava (Manihot esculenta Crantz). International Journal of Genetics and Molecular Biology 2: 198-201 Allard RW (1960) Principles of plant breeding. John Wiley and Sons Inc. New York Bouis HE, Hotz C, McClafferty B, Meenakshi JV, Pfeiffer WH (2011) Biofortification: A new tool to reduce micronutrient malnutrition. Food and Nutrition Bulletin 32: S31- S40 Ceballos H, Davrieux F, Talsma EF, Belalcazar J, Chavarriaga P, Andersson MS (2017) Carotenoids in cassava roots. In: Cvetkovic D (ed.) Carotenoids. InTech, https://www.intechopen.com/books/carotenoids/carotenoids-in-cassava-roots Ceballos H, Morante N, Sanchez T, Ortiz D, Aragon I, Chavez AL, Pizarro M, Calle F, Dufour D (2013) Rapid cycling recurrent selection for increased carotenoids content in cassava roots. Crop Science 53: 2342-2351 Chávez AL, Ceballos H, Rodriguez-Amaya DB, Pérez JC, Sánchez T, Calle F, Morante N (2008) Sampling variation for carotenoids and dry matter contents in cassava roots. Journal of Root Crops 34: 43-49. Dwivedi SL, Sahrawat KL, Rai KN, Blair MW, Andersson MS, Pfeiffer W (2012) Nutritionally enhanced staple food crops. Plant Breeding Reviews 36: 173-293 Esuma W, Kawuki RS, Herselman L, Labuschagne MT (2016) Stability and genotypes by environment interaction of provitamin A carotenoid and dry matter content in cassava in Uganda. Breeding Science 66: 434-443 116 Ewa F, Nwofia E, Egesi C, Olasanmi B, Okogbenin E (2017) Genetic variability, heritability and variance components of some yield and yield related traits in second backcross population (BC2) of cassava. African Journal of Plant Science 11: 185-189 Gauch HG (2006) Statistical analysis of yield trials by AMMI and GGE. Crop Science 46: 1488-1500 GenStat for Windows (2011) 14th Edition. VSN International Ltd., Hemel Hempstead, UK Gollob HF (1968) A statistical model which combines features of factor analytic and analysis of variance techniques. Psychometrika 33: 73-115 Howeler R (2014) Sustainable soil and crop management of cassava in Asia: a reference manual. CIAT Publication no 389, CIAT, Colombia Idahosa DO, Alika JE, Omoregie AU (2010) Genetic variability, heritability and expected genetic advance as indices for yield and yield components selection in cowpea (Vigna unguiculata (L.) Walp. Academia Arena 2: 22-26 IITA (International Institute of Tropical Agriculture) (1990) Cassava in Tropical Africa: A reference manual. Chayce Publications Services, Balding Mansell International, Wisbech, UK Kang MS (2002) Quantitative genetics, genomics, and plant breeding. CABI Publishing, New York, USA Kawano K, Fukuda WMG, Cenpuckdee U (1987) Genetic and environmental effects on dry matter content of cassava root. Crop Science 27: 69-74 Kundy C, Mkamilo GS, RN Misangu (2015) Genetic variability among six traits in twelve cassava (Manihot esculenta Crantz) genotypes in Southern Tanzania. Journal of Natural Sciences Research 5: 33-38 Kvitschal MV, Vidigal Filho PS, Scapin CA, Goncalves-Vidigal MC, Pequeno MG, Sagrilo E, Rimoldi V (2006) Evaluation of phenotypic stability of cassava clones by AMMI analysis in North-western Parana State. CBAB 6: 236-241 Maroya NG, Asante IK, Dixon AGO (2010) Genotype by environment interaction effect on beta-carotene of yellow root cassava (Manihot esculenta Crantz) genotypes in Ghana. Proc 11th ISTRC-AB Symp, Kinshasa, DR Congo, 4-8 October 2010 Obilana A, Fakorede MAB (1981) Heritability: A treatise. Samaru Journal of Agricultural Research 1: 72-82 117 Okwuagwu CO, Okoye MN, Okolo EC, Ataga CD, Uguru MI (2008) Genetic variability of fresh fruit bunch yield in Deli/duru x tenera breeding populations of oil palm (Elaeis guineensis Jacq.) in Nigeria. Journal of Tropical Agriculture 46: 52-57 Owusu V, Donkor E (2012) Adoption of improved cassava varieties in Ghana. Agricultural Journal 7: 146-151 Parkes EY (2011) Assessment of genetic diversity, combining ability, stability and farmer preference of cassava germplasm in Ghana. PhD thesis, Department of Plant Sciences (division of Plant Breeding), University of the Free State, South Africa Parkes EY, Fregene M, Dixon AGO, Peprah BB, Labuschagne MT (2013) Combining ability of cassava genotypes for cassava mosaic disease and cassava bacterial blight, yield and its related components in two ecological zones in Ghana. Euphytica 194: 13-24 Pradeepkumar T, Bastian D, Joy M, Radharkrishnam NV, Aipe KC (2001) Genetic variation in tomato for yield and resistance bacterial wilt. Journal of Tropical Agriculture 39: 157-158 Rodriquez-Amaya DB, Kimura M (2004) HarvestPlus handbook for carotenoid analysis. International Food Policy Research Institute (IFPRI) and International Center for Tropical Agriculture (CIAT), Washington, DC and Cali Ssemakula G, Dixon AGO (2007) Genotype × environment interaction, stability and agronomic performance of carotenoid-rich cassava clones. Scientific Research and Essay 2: 390-399 Vargas M, Crossa J (2000) The AMMI analysis and graphing the biplot. Biometrics and Statistics Unit, CIMMYT Yan W (2002) Singular-value partitioning in biplot analysis of multi-environment trial data. Agronomy Journal 94: 990-996 Yan W, Kang MS (2003) GGE Biplot Analysis: A Graphical Tool for Breeders, Geneticists, and Agronomists, CRC Press, Boca Raton, Fla, USA. Yan W, Tinker Y (2006) Biplot analysis of multi-environment trial data: principles and applications. Canadian Journal of Plant Science 86: 623-645 118 Chapter 6 Proximate composition and cyanide content, and total carotenoid retention after boiling of yellow-flesh cassava cultivars Abstract Biofortified yellow-flesh cassava is important in countries with high cassava consumption, to improve the vitamin A status of its population. In this study, 10 cultivars were evaluated over three locations for proximate composition and cyanide content as well as retention of carotenoids after boiling. There were significant differences (p<0.001) in the crude fiber, fat, protein and ash content of all the cassava genotypes investigated. All yellow flesh cassava genotypes recorded higher protein values than the local cultivars across all locations, except for cultivar IBA085392. The cyanide content of the cultivars varied between locations, but was within the range of sweet cassava cultivars, but in excess of the maximum acceptable limit recommended by the WHO. However, the cyanide can be reduced in cassava cultivars with several processing methods such as roasting, frying, boiling, and fermentation. Micronutrient retention is an important aspect of research on biofortified crops, because a loss of micronutrients during processing and cooking reduces the nutritional value of biofortified foods. TCC of fresh and boiled cassava was measured by spectrophotometer. TCC of fresh peeled cassava was 1.18 - 18.81 µg g-1 on a fresh weight basis, whereas in boiled cassava TCC was 1.01 - 13.36 µg g-1. All the yellow flesh cassava genotypes recorded higher TCC values in both the fresh and boiled state than the white- fleshed varieties used as checks. 6.1 Introduction Cassava roots are a staple food that provides carbohydrates and energy for more than 2 billion people in the world, while representing the main source of carbohydrate and energy for the approximately 700 million people living in the tropical and sub-tropical areas (Ferraro et al. 2015). VAD is a widespread nutritional disorder in low-income countries and is still a public health concern globally. The insufficient intake of VA over a long period of time causes xerophthalmia that may lead to irreversible blindness. Subclinical deficiency of VAD can aggravate diseases such as diarrhea and other infectious diseases (FAOSTAT 2003). 119 Yellow flesh cassava cultivars rich in pVA are part of the outputs of an international biofortification effort by HarvestPlus, the IITA, CIAT and other National Agricultural Research institutions, to reduce VA and other micronutrient deficiencies through the development of staple food crops with enhanced micronutrient content. Replacing the white- fleshed cassava varieties grown by most farmers with new high pVA (yellow) cassava varieties to address micronutrient and health needs of people, could benefit an estimated 20 million children under 6 years of age, who are currently at risk from diseases associated with VAD. Biofortified staple crops with higher micronutrient density, including yellow flesh cassava varieties biofortified with pVA carotenoids, have been developed to improving food and nutrition security reducing micronutrient deficiencies across the world (Saltzman et al. 2016). These biofortifies crops directly contributes to the attainment of Sustainable Development Goal 2 in eradicating all forms of hunger, including hidden hunger. Biofortified cassava varieties developed by conventional breeding techniques have been released in the main cassava- producing countries of Africa, such as the DRC, Ghana and Nigeria. In 2013, 15 yellow-fleshed cassava cultivars with TCC levels between 4 - 18 μg g-1 were obtained from IITA-Nigeria. These cultivars have been tested across the various agro- ecologies in Ghana for their agronomic performance by the CRI towards possible commercial release in 2020. Good cooking quality of cassava storage roots is an important parameter in selecting cassava for human consumption. Other factors important for selection are hydrocyanic acid content, starch, fiber, cooking time, flavour, consistency and cooked pulp texture (Wheatley 1987). Cassava roots are the main source of calories in communities where it grown and consumed and their nutritional composition is therefore important. The roots consist mostly of starchy flesh (about 80% to 90% of the total weight of the root) with water making up a large proportion of this flesh (Wheatley and Chuzel 1993; Harris and Koomson 2011). The water content for cassava is in the range of 60.3% to 87.1% (Padonou et al. 2005). In cassava flour the moisture is much lower and was reported to vary from 9.2% to 12.3% (Charles et al. 2005) and 11% to 16.5% (Shittu et al. 2007). Moisture content is very important in shelf life of cassava flour since levels higher than 12% allows for microbial growth, which significantly reduce its shelf life (Padonou et al. 2010; Harris and Koomson 2011). Cassava contains very low levels of protein of about or 1 - 3% on a DM basis (Buitrago 1990; Charles et al. 2005) and between 0.4 and 1.5 g 100-1 g fresh weight (Bradbury and Holloway 1988). 120 So cassava is therefore much more starchy than cereals such as maize and sorghum that have about 10 g protein 100 g-1 fresh weight (Montagnac et al. 2009). Cassava plants are very valuable, as they produce more weight of carbohydrate per unit area than other staple food crops under comparable agro-climatic conditions. Unfortunately, the roots have very low nutritional value due to low protein content and high starch content. About 50% of the crude protein in the roots consists of whole protein and the other 50% of free amino acids (Zvinavashe et al. 2011). Raw cassava root has more carbohydrate than potatoes and less carbohydrate than wheat, rice, yellow maize, and sorghum on a 100 g basis (Montagnac et al. 2009). The aim of the HarvestPlus program is the improvement of micronutrient content of crops to such an extent that it will impact on human nutritional and health status in a way that can be measured. Equally important is not to negatively affect agronomic characteristics of the crop, such as yield and disease resistance. The process of developing biofortified crops include factors such as nutrient retention after harvesting, how much of the crop is consumed, and whether the biofortified crop is acceptable to the consumer. The bioconversion to provitamin A to retinol in the case of pVA rich foods (called bioavailability), is also an important factor. The mechnisms must also be in place for large- scale dissemination of the biofortified crop, which may differ in specific target countries (Boy and Miloff 2009). Carotenoids are very sensitive to light, heat and physical handling, which leads to losses during the processing of yellow flesh cassava roots into commonly consumed products (Maziya-Dixon et al. 2015). Total carotenoid retention is therefore largely dependent on specific cultivars and processing methods used to prepare products (Jaramillo et al. 2018). The pVACs target level for cassava, set to reach 50% of the EAR for children and pregnant women in the DRC and Nigeria, assumes that up to 50% of pVACs in peeled roots is lost during processing, storage, and cooking (Saltzman et al. 2013; Anderson et al. 2017). Carotenoid retention higher than 50% in boiled cassava has been reported in different studies (Chavez et al. 2007; De Moura et al. 2015). A study in Kenya demonstrated that feeding 2 – 4 year old children with boiled yellow fleshed cassava improved their VA status (Talsma et al. 2016). Cassava is mainly traded in Ghana either as dry pieces of fermented cassava roots, konkonte, that are milled into cassava flour to prepare banku, or as fermented cassava paste, bankye mole, used to prepare koko. Cassava is also boiled and pounded with plantain to prepare fufu. 121 Generally, fufu in Ghana is prepared by cooking peeled cassava in boiling water, whereas chikwangue is prepared by precooking and steaming fermented cassava paste (Avouampo et al. 1995; Humpal et al. 2012). In Nigeria, a study found that apparent carotenoid retention in fufu prepared with fermented cassava flour was 17 – 32%, but no information on true retention was presented (Omodamiro et al. 2012). The same study also found that apparent retention of carotenoids was 86 – 90% when fufu was prepared with a wet paste without a drying step. Another study in Nigeria reported true carotenoid retention between 12 and 36% when processing biofortified cassava roots into fufu, using fermented cassava paste without a drying step (Maziya-Dixon et al. 2015). Although there is some level of information on carotenoid retention in cassava in other parts of the world, it is limited for a country like Ghana, making it difficult to estimate its potential impact on VA status of children and women in the country. Despite its nutritional and commercial benefits, cassava contains toxic substances that limit its utility, the most important being cyanogen, which is responsible for the bitter taste of some cassava cultivars (FAO 2000). Cassava cultivars are therefore classified into two major types: bitter and sweet (Ubwa et al. 2015) on the basis of the cyanogenic content. “Sweet” cassava variety roots contain less than 50 mg kg-1 HCN on fresh weight basis, whereas those classified as “bitter” varieties may contain up to 400 mg kg-1 HCN (FAO 2008). However, the level of cyanide in the cassava roots can be effectively reduced with different processing and fermentation methods (Emurotu et al. 2012). Cyanide is the result of the enzymatic hydrolysis of molecules such as linamarin, lotaustralin, and acetone cyanohydrin (Marcus and Adesina 2001; Asegbeloyin and Onyimonyi 2007). Cyanide is stored in vacuoles of cassava cells, and is known to be more concentrated in leaves and the root cortex compared to root parenchyma (Cardoso et al. 2005). Linamarin and linamarase react when cassava cells are mechanically damaged during harvesting. They then release acetone cyanohydrin, and this then decomposes to release cyanide (Omotioma and Mbah 2013), either by hydroxyl nitrile lyase or spontaneously when the pH is higher than 5 (Orjiekwe et al. 2013). Several neurological diseases, including ataxic neuropathy, cretinism, and xerophthalmia are seen in areas where cassava is the staple food, and this has been attributed to cyanide poisoning (Emmanuel et al. 2012; Abraham et al. 2016). Cyanide can also cause thyroid disorders, goiter and stunting in children (Mburu et al. 2013). Cassava toxicity levels vary depending on altitude, geographic location, period of harvesting, crop variety and seasonal conditions (Ndam et al. 2019). Several cases of 122 cassava poisoning has been recorded in Nigeria, all resulting from improper fermentation and processing of cassava. Cyanide exposure of more than 50 mg kg-1 caused symptoms such as headache, weakness, changes in taste and smell, irritation of the throat, vomiting, lacrimation, abdominal colic, pericardial pain and nervous instability (Ifeabunike et al. 2017). Cyanide content of cassava is higher during drought periods due to water stress in the plant (Ojo et al. 2013). In Mozambique, more than 55% of fresh sweet roots became extremely toxic during drought periods, a trend which was also observed in the Democratic Republic of Congo (Gitebo et al. 2009) and other countries in Africa (Cardoso et al. 2005). Cassava must therefore be processed to make it safe for consumption. Numerous processing techniques are used in cassava eating countries. These techniques often improve palatability, extend shelf life but also decrease cyanogenic potential of cassava (Bradbury 2006). The aim of the present study was to determine the TCC, proximate values and HCN in yellow flesh cassava cultivars and to measure the retention of carotenoids during the processing of biofortified cassava into boiled cassava. This will help breeders to identify genotypes with the best nutritional quality across the tested locations for planting and promotion. 6.2 Materials and methods 6.2.1 Varieties, field trials and sample preparation The same varieties used in Chapter 5 were used for this study, with the only difference being that Cape Vars, a commercially released white fleshed variety, was used as one of the controls, together with a white fleshed local landrace (Husivi). Trials were conducted from May 2015 - May 2016 at three locations situated in different agroecological zones. Fumesua (forest), Cape Coast (Rain forest) and Ohawu (Coastal savannah). Trials were laid out in a RCBD with three replications, each consisting of four rows of five plants, giving a plot size of 20 plants. Planting was done at a spacing of 1 × 1 m. Replications were separated by 2 m alleys. Weeding was done when necessary and experiments were entirely rain fed. The soils for the trial sites at Fumesua is Asuasi series, a ferric acrisol with sandy loam top soil over sandy clay. At Cape Coast, it is Benya series, Acrisol with deep yellowish red to yellowish brown, well to moderately well drained alluvial clays and Ohawu have Toje- Alajo series, a loamy top soil over sandy loam soil. Annual rainfall for the sites during the trial period was Fumesua (1205 mm), Cape Coast (1295 mm) and Ohawu (1024 mm). 123 The fresh roots were sampled randomly for each variety before they were washed and peeled. The samples were transported immediately to the laboratory from the fields. Samples from the apical, middle and distal portions of the roots were cut into small cubes, packed and heat-sealed in laminated bags of 1 kg each, and stored in a cool place until used. A total of 60 samples from each location were analyzed for various characteristics (30 fresh samples and 30 boiled samples obtained from ten genotypes with three replications) (Table 6.1). Table 6.1 Provitamin A and white cassava genotypes used for the study Genotype Code Status Source Pulp colour IBA090090 G1 Improved IITA Yellow IBA090151 G2 Improved IITA Yellow IBA070557 G3 Improved IITA Yellow IBA085392 G4 Improved IITA Yellow Husivi G5 Landrace Farmer White IBA083774 G6 Improved IITA Yellow IBA070593 G7 Improved IITA Yellow IBA070539 G8 Improved IITA Yellow Cape Vars G9 Released CSIR-CRI White IBA083724 G10 Improved IITA Yellow 6.2.2 Proximate analysis Determination of moisture and dry matter content The moisture and DM of the fresh cassava genotypes were determined using the AOAC (1990) method. Two gram of each sample was weighed (W1) into a dry evaporating dish of known weight and the sample spread evenly within the dish with a spatula. The dish (partially open) was placed in the air oven and dried for three hours at 105oC. After drying, the dish was closed and transferred to the desiccator, and then reweighed (W2) after cooling to determine the weight of the sample. Measurements were taken in duplicate and were calculated as follows: Moisture (%) = (W1- W2)/W1 Where W1= Weight (g) of sample before drying W2= Weight (g) of sample after drying 124 DMC was obtained by subtracting the percentage moisture from the total percentage: DMC = 100% - Percentage moisture. Determination of crude protein The solution turned green and clear. The sample solution was then transferred into a 100 ml volumetric flask and made up to the mark with distilled water. Twenty-five ml of 2% boric acid was pipetted into a 250 ml conical flask and two drops of a mixed indicator (20 ml of bromocresol green and 4 ml of methyl orange) was added; and placed into the decomposition chamber of the distillation apparatus, 15 ml of a 40% NaOH solution was added. Ten ml of the digested sample solution was then introduced into a Kjeldahl flask. The condenser tip of the distillation flask was then dipped into the boric acid in the conical flask. The ammonia in the sample solution was then distilled into the boric acid until it changed completely too bluish green. The distillate was then titrated with 0.1 N HCl solution until it became colourless. The percentage total nitrogen and the crude protein were determined. Measurements were done in triplicate. Percent total nitrogen (% N) = (Titre value - Blank value) x 0.01 x 14 x 100 x 100% 1000 x 5 x weight of sample Percentage protein (% Protein) = Percent total nitrogen (% N) x 6.25 Where: atomic weight of nitrogen = 14, volume of titrant = 5 ml, blank value = 0.45, Molarity of acid (HCl) = 0.01, Volume of digest = 100 ml Determination of crude fat Crude fat was determined based on the Sohxlet extraction method of the AOAC (1990). Two g of the sample was weighed into a muslin thimble and inserted into the extraction column with the condenser connected. Petroleum ether as extracting solvent (200 ml) was poured into a 250 ml round bottom flask of known weight and fitted into the extraction unit. The flask was then heated at 60oC for 2 hours on a hot plate. Losses of the solvent due to heating were prevented by the condenser, which cooled and refluxed the evaporating solvent. After extraction, the thimble was removed, and the solvent salvaged by distillation. The round bottom flask containing the fat and the residual solvent was placed on a water bath to evaporate the solvent, followed by further drying in an oven at 103oC for 30 min to completely evaporate the solvent. The flask was then cooled in a desiccator and weighed. Measurements were done in triplicate. 125 Percentage fat = (W1-W2) / W1 Where W1 = weight of the sample before heating W2 = weight of sample after heating Determination of crude fiber Crude fibre was determined by the AOAC (1990) method. The defatted sample (from crude fat determination) was transferred into a 750 ml Erlenmeyer flask and 0.5 g of asbestos was added. Boiling 1.25% H2SO4 (200 ml) was added and the flask was immediately set on a hot plate and a condenser connected to it. Boiling occurred within 1 min and the sample was digested for 30 min. The content of the flask was passed through a filter paper into a funnel and subsequently washed with boiling water until the washings were no longer acidic. The sample was washed back into the flask with 200 ml boiling 1.25% NaOH solution. The condenser was again connected to the flask and the content of the flask was boiled for 30 min. It was then filtered through the filter paper and thoroughly washed with boiling water until washing was no longer alkaline. The residue was transferred to a clean crucible with a spatula and the remaining particles washed off with 15 ml ethanol into the crucible. The crucible with its content was then dried in an oven overnight and cooled in a desiccator and weighed. The crucible with its content was then ignited in a furnace at 600oC for 30 min, cooled and reweighed. Measurements were done in triplicate. Percentage fibre = (W1-W2) / W1 Where W1 = weight of the sample before heating W2 = weight of sample after heating Determination of ash content Ash content was determined by using the AOAC (1990) method. Each sample of 2g was weighed into a weighed porcelain crucible. The crucible with its content was placed in a furnace and preheated to 600oC for 2 hours. The sample was then allowed to cool in the furnace to 250oC. The crucible and the ash were then transferred into an oven at 100oC for 30 min cooling. The crucible with its contents were then cooled in a desiccator. The weight of the crucible and its content was recorded. Measurements were done in triplicate. Percentage ash = (W1-W2) / W1 126 Where W1 = weight of the sample before heating W2 = weight of sample after heating Determination of carbohydrate content Total percentage carbohydrate was determined by adding the total values of crude protein, crude fat, crude fibre, moisture and ash constituents of the sample and subtracting it from 100% (Onyeike and Oguike 2003). Percentage carbohydrate =100 – (% moisture + % ash + % crude protein + % crude fat + % crude fibre) Determination of TCC using the spectrophotometer One gram of each fresh sample of yellow flesh cassava cubes was weighed and ground using a mortar and pestle. Pyrogalol was added to facilitate the grinding and to prevent oxidation. Methanol (25 ml) was then added and the mixture was transferred into a conical flask corked with filter paper by vacuum filtration. Acetone (25 ml) was added to the residue to ensure maximum extraction of carotenoids. The volume of the filtrate was recorded as the volume after first extract. The filtrate was then poured into a separating funnel (where the tap was closed) fixed to a retort stand. Petroleum ether (20 ml) was placed into the separating funnel before the extract was added. Distilled water was finally added. A separation of yellowish coloured organic solvent and colourless inorganic solvent was observed. The tap of the separating funnel was opened for the inorganic solvent to run out into a beaker placed under the funnel, and finally discarded. Distilled water was again added to the sample for washing. The inorganic solvent was collected and discarded. Washing was repeated several times until the carotenoid solution was clear. All excess distilled water was discarded. A funnel was then placed in a conical flask under the separating funnel and the organic solvent containing the carotenoids was collected. The volume of the organic solvent was recorded as the volume of the second extract. A glass cuvette was filled with the organic solvent extract and the absorbance was read at 450 nm. The extraction was read in triplicate using a T80 UV/VIS spectrophotometer. TCC was then calculated using the formula: TCC (µg g-1) = A ×V (ml) × 104 A1%1cm×Ƥ (g) Where A = is the absorbance 127 V = Total extract volume after second extraction Ƥ = Sample weight A1%1cm = 2592 (beta carotene extinction coefficient in petroleum ether) The TCC of the boiled root samples of the yellow flesh cassava was also determined using the spectrophotometry method. First the cubes were placed in laminated polythene bags and boiled in water at 100oC for 20 min. One gram of each boiled sample was ground using a mortar and pestle. Pyrogalol was added to facilitate the grinding and to prevent oxidation. Methanol (25 ml) was then added and the mixture transferred into a conical flask corked with filter paper/vacuum filtration. Acetone (25 ml) was also added to the residue to ensure maximum extraction of carotenoids. The volume of the filtrate was recorded as the volume after first extract. The filtrate was then poured into a separating funnel and 20 ml petroleum ether was already added to the separating funnel. Distilled water was finally added. The same procedure was then followed as for the raw samples 6.3 Data analysis Data was subjected to ANOVA using SPSS, version 21. Results were presented as means ± standard deviations. Differences between means were considered significant at p<0.05 using the Duncan multiple range test (Eleazu and Eleazu 2012). 6.4 Results Moisture content The moisture content (Table 6.2) ranged from 50.48% to 83.84% at Cape Coast, 62.20% to 80.07% at Fumesua and 56.31% to 90.43% at Ohawu. Genotype Husivi (local) recorded the overall highest moisture content per fresh weight at the Ohawu locations. At Cape- Coast, genotype IBA085392 had the highest moisture content and Cape Vars the lowest content. At Fumesua and Ohawu, genotypes IBA070539 and Husivi (local) had the highest moisture content, respectively. Carbohydrate content Carbohydrate content of samples from Cape-Coast, Fumesua and Ohawu ranged from 12.85% to 45.79%, 14.90% to 40.41% and 6.85% to 38.82% respectively (Table 6.2). The highest value was recorded for genotype Cape Vars (white-fleshed) across the three locations. The local (white fleshed) and improved variety (Cape Vars) recorded higher carbohydrate content than most of the yellow flesh cassava genotypes. 128 Table 6.2 Percentage moisture and carbohydrate content of fresh cassava varieties from three different locations Moisture content (%) Carbohydrate content (%) Variety Cape-Coast Fumesua Ohawu Cape Coast Fumesua Ohawu I090090 70.4±13.1abcd 76.6±0.2c 70.3±1.0bc 26.9±13.1abc 20.1±0.1ab 25.8±0.8de I090151 79.5±10.3cd 66.9±0.3ab 64.8±2.0b 17.9±9.8ab 29.0±0.1cd 6.9±1.9ef I070557 66.9±0.78abcd 62.2±3.7a 66.9±0.2b 30.7±0.4bcd 34.2±0.2d 27.3±1.7de I085392 83.8±4.45d 64.7±7.0a 69.7±0.9bc 12.9±4.4a 30.2±9.5cd 27.2±1.0de I083724 58.2±11.7ab 67.3±0.3ab 76.6±6.3cd 38.9±11.8cd 28.5±0.6bcd 19.9±6.2bcd I083774 66.2±0.26ab 62.2±3.7a 67.5±5.0b 34.4±0.1bcd 34.9±1.3d 24.5±10.9cde I070593 66.8±3.1abcd 73.3±5.1bc 82.8±3.5de 29.0±5.7abcd 24.5±5.1bc 15.0±3.4abc I070539 50.7±2.0a 80.1±0.5c 83.7±4.3cde 45.6±2.1d 14.9±1.9a 13.0±4.4ab Cape Vars 50.4±11.8a 54.8±1.1ab 56.3±5.7ab 45.8±11.6d 40.4±0.8bcd 38.8±5.4f Husivi 66.2±2.6abc 63.1±0.2a 90.4±2.0a 30.7±2.6bcd 34.3±0.2d 33.0±2.6a p-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Values are presented in means and ± standard deviations. Means along the same column with different superscripts are statistically different (P<0.05) Protein and fat contents Protein content ranged from 0.01 to 1.45% with genotype IBA070557 recording the highest value among the samples from Cape Coast, followed by genotype I090151 which recorded 1.32% and 1.26% at Fumesua and Ohawu respectively. (Table 6.3). The protein content of the white fleshed variety (husivi) was generally lower than most of the yellow flesh cassava genotypes across all locations. Fat content ranged from 0.05% to 1.24% with genotype I070539 recording the highest value at Cape Coast and Fumesua followed by genotype I070557 at Ohawu (1.16%). Generally, samples from Ohawu recorded the highest protein content followed by those from Fumesua and Cape Coast. For Fat content, genotypes from Cape Coast had the highest, followed by Fumesua and Ohawa. Crude fiber and ash content Crude fiber is the part of food made up of cellulose and lignin. Genotype IBA083724 (2.62 %) had the highest fiber followed by genotypes IBA085392 and I083774 (2.57%) all at the same location, Fumesua (Table 6.4).Genotypes from Fumesua recorded the highest crude fibre, followed by those from Ohawu and Cape Coast. Ash content is indicative of inorganic constituents (such as minerals) and for cassava, it generally ranges from 1% to 2%. Genotype IBA083724 (Ohawu) had the lowest ash content 129 of 0.02% and is therefore likely to contribute less minerals in the diet when consumed. Almost all the yellow flesh cassava genotypes had higher ash content relative to the white fleshed variety (local). Generally, genotypes from Cape Coast had the highest ash content, followed by those from Fumesua and Ohawa Table 6.3 Protein and fat content of cassava varieties from three different locations Protein content (%) Fat content (%) Variety Cape-Coast Fumesua Ohawu Cape Coast Fumesua Ohawu I090090 0.24±0.01d 0.28±0.01d 0.45±0.01e 0.92±0.1cd 0.07±0.04a 0.74±0.1cd I090151 0.32±0.01f 1.32±0.01a 1.26±0.01c 0.72±0.1ab 0.87±0.3cd 0.05±0.001a I070557 1.45±0.01a 0.58±0.01f 0.85±0.01g 0.94±0.2cd 1.16±0.04d 1.14±0.5d I085392 0.01±0.01a 0.37±0.01a 1.12±0.01h 0.96±0.1cd 0.27±0.3ab 0.12±0.03ab I083724 0.31±0.01f 0.25±0.01c 0.67±0.01f 1.05±0.04de 0.12±0.1a 0.47±0.24bc I083774 0.19±0.01c 0.17±0.01a 0.84±0.01g 0.96±0.03cd 0.74±0.1bcd 0.84±0.1cd I070593 0.08±0.01b 0.18±0.01b 0.27±0.01c 0.67±0.03ab 0.40±0.4abc 0.07±0.03a I070539 0.27±0.01f 0.26±0.01c 0.37±0.01d 1.24±0.1e 1.22±0.02d 0.30±0.10ab Cape Vars 0.02±0.01a 0.10±0.01a 0.23±0.01b 0.54±0.001a 0.45±0.3c 0.27±0.10ab Husivi 0.03±0.01a 0.18±0.01b 0.13±0.01a 0.82±0.03bc 0.17±0.2a 0.05±0.0001a p-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Values are presented in means and standard deviations. Means along the same column with different superscripts are statistically different (P<0.05) 130 Table 6.4 Crude fiber and ash content of fresh cassava varieties across three different locations Crude fibre Ash content (%) Variety Cape-Coast Fumesua Ohawu Cape Coast Fumesua Ohawu I090090 0.75±0.1ab 2.53±0.2c 2.07±0.03d 0.72±0.1ab 0.42±0.1a 0.62±0.3ab I090151 1.04±0.2abc 2.48±0.1c 1.33±0.2ab 1.02±0.3ab 0.60±0.4a 0.65±0.4ab I070557 0.47±0.3a 1.24±0.1ab 1.72±0.04c 1.020.7ab 0.59±0.08a 2.11±2.0ab I085392 0.94±0.21abc 2.57±0.2c 1.49±0.01bc 1.39±0.1ab 1.94±2.0a 0.37±0.04ab I083724 1.04±0.5abc 2.62±0.04c 2.23±0.1de 0.82±0.3ab 1.17±0.3a 0.02±0.003a I083774 1.02±0.1abc 2.57±0.2c 1.47±0.04bc 1.22±0.3ab 0.47±0.04a 2.34±2.30b I070593 1.34±0.6bc 1.16±0.4a 1.03±0.04a 2.09±1.9b 0.42±0.1a 0.77±0.04ab I070539 1.42±0.03c 2.53±0.2c 1.72±0.04c 0.72±0.1ab 2.07±2.4a 0.90±0.001ab Cape Vars 1.02±004abc 1.74±0.3b 2.54±0.4a 0.72±0.04ab 0.69±0.3a 0.79±0.003ab Husivi 1.04±0.5abc 1.65±0.3b 2.1±0.01d 0.52±0.4ab 0.57±0.04a 0.45±0.10ab p-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Values are presented in means and standard deviations. Means along the same column with different superscripts are statistically different (P<0.05) Hydrogen cyanide content The highest and lowest HCN content of the fresh cassava from Cape Coast were 47.76 µg g-1 and 23.88 µg g-1 in Cape Vars and IBA083774, respectively (Figure 6.1). The HCN of samples significantly differed (p>0.05). For Fumesua, the highest HCN content (43.1 µg g- 1) was recorded in genotype IBA070557 and IBA070593 with genotype IBA085392 having the lowest value (9.9 µg g-1) (Figure 6.2). The highest HCN content was found genotype 131 Figure 6.1 Hydrogen cyanide content of yellow flesh cassava from Cape-Coast P<0.05 Key: 101 to 110 represents varieties I090090, 1090151, I070557, I085392, I083724, I083774, I070593, I070539, Cape Vars and Husivi IBA085392 (47.8 µg g-1) and the lowest in genotype IBA090090 (23.9 µg g-1) in Ohawu. Across the three sites, genotypes Cape Vars from Cape Coast, IBA070593 and IBA070557 both from Fumesua, and IBA085392 from Ohawu had the highest HCN levels of 47.8 µg g-1, 43.1 µg g-1 and 47.8 µg g-1 respectively. Whilst samples IBA083774, IBA085392 and IBA090090 from Cape Coast, Fumesua and Ohawu respectively had the lowest HCN values of 23.9 µg g-1, 9.9 µg g-1 and 23.9 µg g-1. Ohawu recorded the highest mean value for HCN (36.40 µg g-1), followed by Cape Coast (30.69 µg g-1) with the lowest from Fumesua (29.73 µg g-1). There was no statistically significant differences in HCN levels across the three locations (30.00±7.04, 37.78±7.02, 29.73±10.02 at P=0.10). Genotype Cape vars recorded the highest mean value (39.63 µg g-1) for HCN, followed by genotypes IBA070557 (36.00 µg g- 1) and IBA070593 (36.00 µg g-1) across all the three locations. Genotype IBA083724 recorded the lowest HCN mean (25.77 µg g-1). 132 Figure 6.2 Hydrogen cyanide content of yellow flesh cassava from Ohawu p<0.05 Key: 101 to 110 represents varieties I090090, 1090151, I070557, I085392, I083724, I083774, I070593, I070539, Cape Vars and Husivi Figure 6.3 Hydrogen cyanide content of yellow flesh cassava from Fumesua P<0.05 Key: 101 to 110 represents varieties I090090, 1090151, I070557, I085392, I083724, I083774, I070593, I070539, Cape Vars and Husivi 133 Table 6.5 Comparison of hydrogen cyanide content of cassava genotypes from different locations Variety Cape-Coast Ohawu Fumesua Mean±SD P-value I090090 36.90 23.90 30.10 30.30±6.50 0 .75 I090151 32.20 26.00 36.30 31.50±5.19 I070557 28.00 36.90 43.10 36.00±7.59 I085392 26.00 47.80 9.90 27.90±19.02 I083724 30.10 28.00 19.20 25.77±5.78 I083774 23.90 37.40 26.00 29.10±7.26 I070593 26.00 41.00 4 3.10 36.70±9.33 I070539 28.00 41.00 34.50±9.19 Cape Vars 47.80 41.00 30.10 39.63±8.93 Local 28.00 41.00 30.10 33.03±6.98 Mean±SD 30.69±7.04 36.40±7.82 29.77±10.72 0.20 Total carotenoid content The colour of the cut crosss section of fresh roots show colour ranges that depicts the level of TCC, this is visually assessed by colour chat. Actual levels are determined in the labouratory. For this study, the concentration of TCC in the fresh roots ranged from 1.18 µg g-1 (Cape Vars) for samples from Cape Coast to 18.81 µg g-1 (I070539) for samples from Ohawu. For the boiled analysis, TCC ranged from 1.01 µg g-1 (Cape Vars) for samples from Cape Coast to 13.86 µg g-1 (I083724) for samples from Fumesua. Boiling was found to decrease the total carotenoid content of the different cultivars across all three locations. For both boiled and fresh samples, there were differences in TCC content across the three locations. Fresh samples in Fumesu recorded the highest, followed by Ohawu and then Cape Coast (10.71±4.27 µg g-1, 10.61±4.27, 5.87±3.16; p= 0.02 respectively). The same trend was observed after boiling. 134 Table 6.6 Total carotenoid content (µg g-1) of fresh and boiled cassava genotypes across three different locations Fresh Boiled Variety Cape- Fumesua Ohawu P- Cape Fumesua Ohawu P- Coast value Coast value I090090 6.11±004 14.56±0.04 10.00±0.04 5.09±0.04 11.29±0.04 8.43±0.04 I090151 5.98±0.04 11.44±0.04 8.52±0.04 5.98±0.04 10.64±0.04 7.08±0.04 I070557 11.99±0.2 11.78±0.07 9.87±0.05 5.22±0.09 11.49±0.06 6.44±0.04 I085392 4.80±0.04 14.05±0.04 11.51±0.04 4.64±0.04 10.51±0.04 8.70±0.04 I083724 4.63±0.08 14.52±0.04 11.70±0.07 3.42±0.04 13.86±0.07 11.50±0.04 I083774 6.81±0.04 13.84±0.04 9.89±0.04 5.22±0.04 11.81±0.04 7.63±0.04 I070593 8.15±0.04 10.11±0.04 18.81±0.08 7.20±0.04 9.21±0.04 16.91±0.06 I070539 7.81±0.04 14.06±0.04 15.74±0.04 4.71±0.2 12.38±0.04 12.08±0.04 Cape 1.18±0.04 1.34±0.04 5.14±0.08 1.01±0.04 1.00±0.04 4.74±0.04 Vars Local 1.49±0.04 1.36±0.04 4.90±0.04 1.04±0.04 1.02±0.04 4.56±0.08 M±SD 5.87±3.16 10.71+5.15 10.61±4.27 0.02 4.27±2.0 9.10±8.85 8.85±3.78 0.02 p-value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 6.5 Discussion The results of the proximate analysis of the different cassava genotypes samples from three different locations revealed good variation for all traits with ranges of 50.48 - 90.4% for moisture content, 6.85 - 45.79% for carbohydrate, 0.01 - 1.26% for protein, 0.07 - 1.24% for fat, 0.47 - 2.62% for fibre and 0.37 - 2.34% for ash. Significant differences (p<0.001) were found amongst the genotypes for each of the proximate analysis parameters. In general, the observed ranges were below values reported by Otache et al. (2017) and Rajapaksha et al. (2017). The maximum limit for the crude fibre and fat content observed agreed with values reported by Eleazu and Eleazu (2012). However, the values observed for fat content was higher than the values reported by Adegbola et al. (1992) and Ifeabunike et al. (2017). The carbohydrate values obtained in this study were lower than values reported by other studies such as Okpako et al. (2008) which had a range of 62.0-72.4%, Christopher et al. (2016) (87-89%) and Otache et al. (2017) with 85-89%. The results in this study generally indicated that yellow flesh cassava tend to have less carbohydrate than white-fleshed 135 varieties. Crude ash content is usually indicative of inorganic constituents (minerals such as K, Zn and Ca) and for cassava, it generally ranges from 1% to 2%. Ash contents represents the total mineral content in food after it has been burned at a very high temperature. The ash and protein contents were lower than values reported by other studies (Idugboe et al. 2015; Otache et al. 2017), but were similar to those reported by Emmanuel et al. (2012) and Eleazu and Eleazu (2012) from six yellow and white cassava varieties cultivated in Umudike, Nigeria. Cyanide concentrations vary in different cassava genotypes according to the altitude, geographical location and seasonal and production conditions (Oluwole et al. 2007). Reports have shown that age, variety and environmental conditions influence the occurence and concentration of cyanide in various parts of the cassava plant and at different stages of development (Charles et al. 2005), hence the genotypes need to be tested at different ages of maturity for further inferences. Cassava is classified as sweet or bitter based on a total cyanide content of less or more than 50 mg kg-1, respectively. In drought conditions, there is an increased total cyanide content due to water stress (Cardoso et al. 2005). Thus, a variety is considered to be “sweet” under one set of conditions may be “bitter” in a different geographical location or climatic conditions (Hadayat et al. 2016). Values from 15 - 400 mg kg-1 fresh weight of total cyanide in cassava roots have been reported in different studies (FSANZ 2008), and there were reports of even higher levels, depending on where the crop was grown (Oluwole et al. 2007; Cardoso et al. 2005). However, the rates can be reduced in cassava with different processing and fermentation methods. The observed levels of cyanide obtained in the present study showed that all the genotypes sampled could be classified as sweet varieties. The values were lower than those reported by Ubwa et al. (2015) but it is not advisable to eat it raw since the range is above the acceptable limit (10 mg kg-1). A loss of micronutrients during processing and cooking is undesirable, as it reduces the nutritional value of biofortified foods. Hence, micronutrient loss must be considered, and breeding targets set appropriately so that biofortified foods will add sufficient micronutrients to the diet to have a positive impact. It is therefore very important that biofortified crops should be able to retain sufficient levels of micronutrients after typical processing, storage 136 and cooking practices for biofortification to be successful. All eight tested genotypes had TCC higher than the farmer preferred varieties (Husivi) and the improved check (Cape Vars) in both fresh and boiled states. TCC of fresh peeled cassava of the evaluated genotypes measured by spectrophotometer was 1.18 µg g-1 to 18.81 µg g-1 on fresh weight basis whereas in boiled cassava it was lower between 1.01 µg g-1 and 13.36 µg g-1 across the three locations. In general, there was a decrease in TCC level during boiling. This is in contrast to findings by van Jaarsveld et al. (2006) reported that carotenoid retention was better when sweet potatoes were boiled for the shortest possible time compared to methods like drying, frying and roasting that caused reduced retention. Boy and Miloff (2009) also reported that boiling has higher TCC retention compared to other processing techniques in sweet potatoes. Different factors separately or combined, such as heat, light, oxygen and enzymes, can lead to major or minor losses of carotenoids in yellow cassava during processing into consumable products (Chavez et al. 2007; Eyinla et al. 2019). The losses observed in the study for boiled roots could also be due to carotenoid isomerization and oxidation, which is the breakdown of trans-carotenoid to their cis-isomers due to increased content with moisture and heat treatment during boiling (Bendich 1993). Gari is also one of the most popular products of cassava processing in Ghana and sub-Saharan Africa and it has been reported that extended roasting during its processing results in higher carotene content (De Moura et al. 2015). Gari may therefore be a useful way of bioefficiently utilizing biofortified cassava in VA deficient population. Further studies on more varieties commonly used for cassava dough, fufu, konkonte and gari may be needed to ascertain how yellow flesh cassava varieties may may respond for TCC during processing into Ghanaian food forms. Findings by other authors like Eyinla et al. (2019), Bechoff et al. (2018) and Taleon et al. (2019) confirm TCC loss patterns in cassava products consumed in sub-Saharan Africa. The result further suggests that the current yellow flesh cassava genotypes being evaluated could provide more VA in diets and contribute to reduction of health challenges associated with VAD, which is widespread in Ghana and sub-Saharan Africa. Following the agricultural transformation agenda in Ghana (Modernization of Agriculture in Ghana), which has resulted in the availability of improved varieties (including biofortified cassava) there is a great need to scale up micronutrients in staple foods produced in the country (Edoh et al. 2016). 137 Even though the impact of consuming yellow flesh cassava products on VA serum concentration is not yet fully established in VAD populations in Ghana, the results give an indication that yellow flesh cassava varieties are better than white-flesh in terms of carotenoid and protein contents and have the chance of reducing VAD in Ghanaian populations where it is still endemic. Beta carotene in cassava storage roots does not play a role in photosynthesis, as it is located in the cell chloroplasts (Czygan 1980) where it is found in lipid droplets or bound to a protein that is released during cooking, thereby enhancing its bioavailability. 6.6 Conclusions In view of the importance of cassava to the economy of Ghana, and given its role as a major crop in alleviating hunger in Africa, genetic improvement of the crop to address food and nutritional needs of Ghana and the continent has been recognizied. The crop has to be improved for productivity, proximate composition and safe cyanide content. The yellow flesh cassava varieties evaluated have better TCC levels than varieties grown by Ghanaian farmers. With increasing awareness on the toxicity of cyanide in cassava and given that cassava is mainly consumed as processed food, yellow-fleshed cassava are effectively safe for consumption as food to enhance vitamin A. Biofortification of cassava cultivars presents a viable and promising intervention for tackling VAD in disease-burdened populations of sub-Saharan Africa. The WHO is advocating food and nutrition security and yellow flesh cassava could be a key driver for this purpose. Considering that cassava is consumed principally as processed foods, further studies are needed to ascertain the TCC losses in processing of the various food forms the crop is consumed. There is also the need to develop new farmer-preferred cassava varieties that combine high TCC with high DMC to increase their rate of adoption, since most of these varieties/landraces already possess some important traits like high DMC and stability, which are key drivers of cassava adoption. Further breeding of cassava varieties with higher TCC is ongoing in Ghana and in many other countries. It is therefore expected that more varieties will be available in the nearest future with increased adoption rates and increased pVA intake. 138 References Abraham K, Buhrke T, Lampen A (2016) Bioavailability of cyanide after consumption of a single meal of foods containing high levels of cyanogenic glycosides: a cross over study in humans. Archives of Toxicology 90: 559-574 Adegbola OB, Smith JB, Okeudo MJ (1992) Responses of West African Dwarf Sheep fed cassava peel and poultry manure baked diets. Department of Animal Science, Obafemi Awolowo Anderson ME, Saltzman A, Virk PS, Pfeiffer WH (2017) Progress update: crop development of biofortified staple food crops under Harvest-Plus. African Journal of Food, Agriculture Nutrition and Development 17: 11905-11935 AOAC (1990) Official Methods of Analysis of AOAC International, Gaithersburg. 15th Edition Asegbeloyin JN, Onyimonyi AE (2007) Effect of different processing methods on the residual cyanide of ‘Gari’. Pakistan Journal of Nutrition 6: 163-166 Avouampo E, Gallon G, Treche S (1995) Influence de la variété et de l’ordre de réalization de l’épluchage et du rouissage sur l’aptitude à la transformation des raciness de manioc. In: Egbe TA, Brauman A, Griffon D, Treche S (eds.) Transformation Alimentaire du Manioc. ORSTOM Editions, Paris. pp. 429-447 Bechoff A, Tomlins, KI, Chijioke U, Ilona P, Westby A, Boy E (2018) Physical losses could partially explain modest carotenoid retention in dried food products from biofortified cassava. PLoS ONE 13: e0194402 Bendich A (1993) Biological functions of dietary carotenoids. Annals of New York Academy of Sciences 691: 61-67 Boy E, Miloff A (2009) Pro-vitamin A carotenoid retention in orange sweet potato. A review of the literature. Magazine issue 3/2009 Bradbury JH (2006) Simple wetting method to reduce cyanogens content of cassava flour. Journal of Food Composition and Analysis 19: 388-393 Bradbury JH, Holloway WD (1988) Cassava, M. esculenta. Chemistry of tropical root crops: significance for nutrition and agriculture in the Pacific. Australian Centre for International Agricultural Research, monograph nr 6, Canberra, Australia. pp. 76- 104 Buitrago AJA (1990) La yucca en la alimentacion animal. Centro Internacional de Agricultura Tropical, Cali, Colombia 139 Cardoso AR, Mirione E, Ernesto M, Massaza F, Cliff J, Haque MR, Bradbury HJ (2005) Processing of cassava roots to remove cyanogens. Journal of Food Composition and Analysis 18: 451-460 Charles, A.L. Sriroth K, Huang, TC (2005) Proximate composition, minerial contents, hydrogen cyanide and phytic acid of 5 cassava genotypes. Food Chemistry 92: 615- 620 Chavez AL, Sanchez T, Ceballos H, Rodriguez-Amaya DD, Nestel P, Tohme J, Ishitani M (2007) Retention of carotenes in cassava roots submitted to different processing methods. Journal of the Science of Food and Agriculture 87: 388-393 Christopher IN, Enyinnaya CO, Okolie JI, Nkwoada A (2016) The proximate analysis and biochemical composition of the waste peels of three cassava cultivars. Internationl Journal of Scientific Engineering and Applied Science 2: 64-71 Czygan FZ (1980) Pigments in Plants. Gustav Fischer Verlag, Stuttgart De Moura F, Milo A, Boy E (2015) Retention of provitamin A carotenoids in staple crops targeted for biofortification in Africa: Cassava, maize and sweet potato. Critical Reviews in Food Science and Nutrition 55: 1246-1269 Edoh NL, Adiele J, Ndukwe I, Ogbokiri A, Njoku DN, Egesi CN (2016) Evaluation of high beta carotene cassava genotypes at advanced trial in Nigeria. The Open Conference Proceedings Journal 7: 144-148 Eleazu CO, Eleazu KC (2012) Determination of the proximate composition, total carotenoid, reducing sugars and residual cyanide levels of flours of 6 new yellow and white cassava (Manihot esculenta Crantz) varieties. American Journal of Food Technology 7: 642-649 Emmanuel OA, Clement A, Agnes SB, Chiwona-karltun L, Drinah BN (2012) Chemical composition and cyanogenic potential of traditional and high yielding CMD resistant cassava (Manihot esculenta Crantz) varieties. International Food Journal 19: 175- 181 Emurotu JE, Balehdeen UM, Ayeni OM (2012) Assessment of heavy metals level in cassava flour sold in Ayigba market Kogi state, Nigeria. Advances in Applied Science Research 3: 2544-2548. Eyinla ET, Maziya-Dixon B, Alamu EO, Sanusi AR (2019) Retention of pro- vitamin A content in products from new biofortified cassava varieties. Foods (MDPI) 8: 177 FAO (2000) Food and Agriculture Organization. Défendre la cause du manioc. http://www.fao.org/nouvelle/2000/000405-f.htm 140 FAO (2008) Cyanide poisoning and cassava food: Safety focus. Committee on World Food Security 34th Session number 19. http://www.fao.org/unfao/bodies/cfs/ cfs34/index_en. htm FAOSTAT (2003) Statistical Database FAOSTAT. http://faostat.fao.org/ Ferraro V, Piccirillo C, Tomlins K, Pintado ME (2015) Cassava (Manihot esculenta Crantz) and Yam (Dioscorera Spp.) crops and their derived foodstuffs: Safety, Security and Nutritional value. Critical Review in Food Science and Nutrition, 56:2714-2727 FSANZ (2008) Proposal P1002 hydrocyanic acid in ready-to-eat cassava chips. http://www.foodstandards.gov.au/foodstandards/proposals/proposalp1002hydrocy3 848.cfm Gitebo DN, Banea-Mayabu JP, Matadi RN, Tylleskar T, Gebre-Medhin MRosling MH (2009) Geographical and seasonal association between linamarin and cyanide exposure from cassava and the upper motor neuron disease Konzo in DRC. CCDN News 14: 215 Hidayat A, Zuraida N, Hanarida I (2016) The cyanogenic potential of roots and leaves of ninety-nine cassava cultivars. Indonesian Journal of Agricultural Science, 3: 25-32. Harris MA, Koomson CK (2011) Moisture-pressure combination treatments for cyanide reduction in grated cassava. Journal of Food Science 76: T20-T24 Humpal D, Musangu B, Tunieka M (2012) Cassava value chain assessment: Bas-Congo, Kinshasa, and Bandundu provinces. US Agency for International Development, Washington, DC. Available http://pdf .usaid.gov/pdf_docs/pnady673.pdf Idugboe OD, Nwokoro SO, Imasuen JA (2015) Chemical composition of cassava peels collected from four locations (Koko, Warri, Okada and Benin City), Brewers' spent yeast and three grades os caspeyeast. International Journal of Science and Research 6: 1439- 1442 Ifeabunike OB, Nwaedozie JM, Aghanwa CI (2017) Proximate analysis, hydrogen cyanide and some essential mineral content of sweet cassava variety (Manihot utilisima) and bitter cassava variety (Manihot palmata) cultivated in Kachia Local Government Area of Kaduna State, Nigeria. International Journal of Biochemistry Research and Review 19: 1-12 Jaramillo AM, Londono FL, Orozco CJ, Patino G, Belalcazar J, Davrieux F, Talsma EF (2018) A comparison study of five different methods to measure carotenoids in biofortified yellow cassava (Manihot esculenta). PLoS ONE 13: e0209702 141 Marcus AA, Adesina BS (2001) The effect of cooking time on root mealiness and taste of cassava tuber. Nigeria Journal of Food Science and Technology 1: 113 Maziya-Dixon B, Awoyale W, Dixon A (2015) Effect of processing on the retention of total carotenoid, iron and zinc contents of yellow-fleshed cassava roots. Journal of Food and Nutrition Research 3: 483-488 Mburu FW, Swaleh S, Njue W (2013) Potential toxic levels of cyanide in cassava (Manihot esculenta Crantz) grown in Kenya. African Journal of Food Science 6: 416-420 Montagnac JA, Davis CR, Tanumihardjo SA (2009) Nutritional value of cassava for use as a staple food and recent advances for improvement. Comprehensive Review in Food Science and Food Safety 8: 181-188 Ndam YN, Mounjouenpou P, Kansci G, Kenfack MJ, Meguia MPF, Natacha NS, Eyenga N, Akhobakoh MM, Nyegue A (2019) Influence of cultivars and processing methods on the cyanide contents of cassava (Manihot esculenta Crantz) and its traditional food products. Scientific Africa 5: e00119 Ojo RT, NObi NP, Akintayo CO, Adebayo-Gege GI (2013) Evaluation of cyanogen contents of cassava and cassava-based food products in Karu, Nasarawa state, North- Central Nigeria, IOSR Journal of Environmental Science, Toxicology and Food Technology 6: 47-50 Okpako CE, Ntui VO, Osuagwu AN, Obasi FI (2008) Proximate composition and cyanide content of cassava peels fermented with Aspergillus niger and Lactobacillus rhamnosus. Food, Agriculture and Environment 6: 251-255 Oluwole OSA, Onabolu AO, Mtunda K, Mlingi N (2007) Characterization of cassava (Manihot esculenta Crantz) varieties in Nigeria and Tanzania and farmers perception of toxicity of cassava. Journal of Food Composition and Analysis 20: 559-567 Omodamiro RM, Oti E, Etudaiye HA, Egesi C, Olasanmi B, Ukpabi UJ (2012) Production of fufu from yellow cassava roots using the odourless flour technique and the traditional method: evaluation of carotenoids retention in the fufu. Advances in Applied Science Research 3: 2566-2572 Omotioma M, Mbah GO (2013) Kinetics of natural detoxification of hydrogen cyanide contained in retted cassava roots. International Journal of Express Research 1: 9. Onyeike EN, Oguike JU (2003) Influence of heat processing methods on the nutrient composition and lipid characterization of groundnut (Arachis hypogaea) seed pastes. Biokemistri 15: 43-43 142 Orjiekwe CL, Solola A, Iyen E, Imade S (2013) Determination of cyanogenic glycosides in cassava products sold in Okada, Edo State, Nigeria. African Journal of Food Science 7: 468-472 Otache MA, Ubwa ST, Godwin AK (2017) Proximate anlysis and mineral composition of peels of three sweet cassava cultivars. Asian Journal of Physical and Chemical Science 3: 1-10 Padonou SW, Nielsen DS, Akissoe NH, Hounhouigan JD, Nago MC, Jakobsen M (2010) Development of starter culture for improved processing of Lafun, an African fermented cassava food product. Journal of Applied Microbiology 109: 1402-1410 Padonou W, Mestres C Nago MC (2005) The quality of boiled cassava roots: instrumental characterization and relationship with physicochemical properties and sensorial properties. Food Chemistry 89: 261-270. Rajapaksha KDSCN, Somendrika MAD, Wickramasinghe I (2017) Nutritional and toxicological composition analysis of selected cassava processed products. Slovak Journal of Food Sciences 11: 35-42 Saltzman A, Andersson MS, Asare-Marfo D, Lividini K, De Moura FF, Moursi M, Oparinde A, Taleon V (2016) Biofortification techniques to improve food security. Reference Module in Food Science 1: 1-9 Saltzman A, Birol E, Bouis H, Boy E, De Moura F, Islam Y, Pfeiffer W (2013) Biofortification: progress toward a more nourishing future. Global Food Security 2: 9-17 Shittu TA, Sanni LO, Awonorin SO, Maziya-Dixon B, Dixon A (2007) Use of multivariate techniques in studying the flour making properties of some CMD resistant cassava clones. Food Chemistry 101: 1606-1615 Taleon V, Sumbu D, Muzhingi T, Bidiaka S (2019) Carotenoids retention in biofortified yellow cassava processed with traditional African methods. Journal of the Science of Food and Agriculture 99: 1434-1441 Talsma EF, Brouwe ID, Verhoef H, Mbera GNK, Mwangi AM, Demir AY, Maziya- Dixon B, Boy E, Zimmermann MB, Melse-Boonstra A (2016) Biofortified yellow cassava and vitamin A status of Kenyan children: a randomized controlled trail. American Journal of Clinical Nutrition 103: 258-267 Ubwa ST, Otache MA, Igbum GO, Shambe T (2015) Determination of cyanide content in three sweet cassava cultivars in three local government areas of Benue State, Nigeria, Journal of Food and Nutrition Sciences 6: 1078-1085 143 van Jaarsveld PJ, Marais DW, Harmse E, Nestel P, Rodriguez-Amaya DB (2006) Retention of β-carotene in boiled, mashed orange-fleshed sweet potato. Journal of Food Composition and Analysis 19: 321-329 Wheatley CC (1987) Preservation of cassava roots in polythylene bags. Cali: Centro Internacional de Agricultura Tropical, 33p (Serie 045c-07-06) Wheatley CC, Chuzel G (1993) Cassava: the nature of the tuber and use as a raw material. In: Macrae, R., Robinson, R.K. and Sadler, M.J. (eds.) Encyclopedia of Food Science, Food Technology and Nutrition. Academic Press, San Diego, California. pp. 734-743 Zvinavashe E, Elbersen HW, Slingerland M, Kolijn S, Sanders JPM (2011) Cassava for food and energy: exploring potential benefits of processing of cassava into cassava flour and bioenergy at farmstead and community levels in rural Mozambique. Biofuels, Bioproducts and Biorefining 5: 151-164 144 Chapter 7 General conclusions and recommendations Cassava is currently ranked as the number one food staple and the most widely cultivated crop in Ghana. Cassava production in Ghana is around 18.4 million ton per year and more than 70% of farmers are engaged in its production, contributing about 22% to agriculture GDP. Several improved varieties (26 in total) have been released and disseminated to farmers in Ghana since 1993. In most cases, the released varieties were white-fleshed with low or negligible amounts of carotenoids but were bred and selected for processing into intermediary products (gari, flour, konkonte). Malnutrition is endemic in cassava producing regions of Africa, partly due to the low micronutrient content in this root crop, which is a major component of most household diets. It is for this reason that the development of nutrient dense cassava cultivars should be prioritized and given more attention to eliminate or minimize malnutrition among the poor, in a sustainable inexpensive way. Several white fleshed varieties, most of which are traditional landraces, are still cultivated across the different agro-ecologies of Ghana. Knowledge of these improved varieties especially for the yellow flesh cassava are highly limited. This could be easily addressed by strengthening and improving the cassava seed systems. Very few men and women farmers cultivate improved varieties and yellow-flesh cassava. Information indicates that young adult farmers, who typical represent the future of Ghanaian agriculture, are lacking access to improved varieties and must be given extra attention in all cassava programmes to enhance their knowledge and capacity to efficiently optimize cultivation of improved varieties. In general, several similarities were found among men and women for preferred traits that makes it easy to breed for the same traits for both sexes although slight differences were found for preferred traits across locations. Men typically tend more to prefer varieties that could be stored longer in the soil, easily harvested all year round, favorably competitive with weeds and highly suitable for industrial processing. On the other hand, women tend to more prefer varieties which are climate smart (well adapted), labour saving (canopy dense for weed suppression) and easily suited for processing into starch and “gari”. With increased availability of planting material and improved awareness and market demand for the new improved, farmers have high interest and willingness to adopt, cultivate and process yellow flesh cassava. 145 The mean TCC values in this study varied from 2.98 to 6.14 µg g-1, with a grand mean of 4.19 µg g-1, which is comparable to values reported in Uganda and Nigeria. The mean is however, lower than those reported in Colombia. Although the DMC grand mean is lower in this study as compared to those reported for landraces grown in Ghana by farmers, some individual genotypes evaluated in this study had higher DMC than that of the commonly grown varieties. These genotypes could be selected and further tested towards release, since the trait is one of the key drivers of cassava variety adoption in Ghana. Earlier PRA work done in this study suggested that farmers would be willing to adopt yellow flesh cassava varieties if FRW and DMC are high. The study also generated relevant information to understand the genetics of key traits of interest to farmers to aid the implementation of efficient cassava breeding programme for Ghana to meet the needs of the cassava community. The ANOVA and the GCA:SCA ratio indicated that the GCA was larger than SCA for CMD, HI and TCC with predictability ratios close to 1, indicating the presence of additive gene effects and a possibility for improvement of the characteristics by selection. The phenotypic correlations among the studied traits revealed a positive significant correlation between pulp colour and TCC, TCC and CMD, pulp colour and CMD, and pulp colour and cortex colour. This study identified materials that were good for TCC and CMD resistance implying both traits could be jointly selected for and improved at the early breeding stages including the ability to visually assess the pulp colour (for TCC) and CMD symptoms. Visual assessment of pulp color could rapidly reduce the number of materials at the seedling nursery or clonal evaluation trials rapidly to just few numbers which then be quantitatively assessed to reduce cost at the later stages of breeding. Some yellow flesh cassava progenies were found to showed favorable good response for increased DMC values. This was evident in the positive correlation found between TCC and DMC (though not significant). This is an important finding, as it confirms the report by Ceballos et al. (2013) that DMC, which is a key driver for cassava adoption, can be improved alongside TCC. One of the parents utilized in this study, P6 (I090090) showed positive GCA effects for TCC, DMC, RTW and CMD, and would be very suitable for use in crosses (with another parent with good complementary genetic background) to develop improved cassava genotypes combining both TCC and DMC efficiently. Findings of this study also demonstrated that it is possible to simultaneously select for yield and quality traits, such as DMC at early stages. The study also showed that some of the TCC- rich varieties evaluated showed very good performance and meets other key traits of 146 farmer interest critical for their adoption in Ghana. In view of this, varieties IBA090151, IBA083774 and IBA083724 can be considered for varietal release after on-farm testing. The study further revealed that these yellow flesh cassava varieties can be used as parents in crosses with the local material to improve landraces grown by farmers for TCC, DMC, high yield and CMD resistance. The WHO is advocating for improvement of food and nutrition security worldwide. Yellow flesh cassava could be a key driver for this purpose. This reiterates the need for research to reduce micronutrient losses related to processing of biofortified crop products and to further increase the level of TCC concentration in farmer preferred landraces/varieties to increase rate of adoption, given that a number of varieties or landraces grown by farmers already possess other key important traits like DMC and stability. Further breeding initiatives to develop higher TCC in cassava varieties is ongoing in Ghana as with other parts of the world. It is expected that these efforts will provide superior varieties highly elevated TCC. The results indicate that with increased consumption of boiled biofortified cassava there could be improvement in pVA intake in areas where VAD exists. People would also have to be educated on best processing methods that retain more TCC so that they can maximize benefits from the consumption of biofortified foods. The HCN level in the yellow flesh cassava tested in this study were well within the permissible levels recommended by WHO for human consumption for the various food products in Ghana. This study demonstrated the importance of exploring participatory approaches to research for impactive results. It is recommended that cassava breeders review their breeding objectives to reflect the preferred traits of end users, and pay attention to stakeholders’ perceptions of yellow flesh cassava to develop demand driven varieties that will serve the need of end users. Education to create awareness on the potential advantages and diverse uses of the improved biofortified cassava is also needed. Further studies are proposed to investigate and assign measures and ratings to men and women for the qualitative traits recorded. It is also recommended that, in developing high TCC and DMC varieties, breeders should carefully select good parental lines with high breeding values for both traits and other farmer prime traits of interest. 147 Appendices Appendix 1: Colour chart for visual assessment of carotenoid content based onpigmentation of root parenchyma Source: Dr Elizabeth Yaa Parkes, harvestPlus cassava breeder, IITA- Ibadan, Nigeria 148 Appendix 2: Baseline study on trait preferences and perception of yellow flesh cassava by men and women chain actors A checklist through participatory approach Date of interview: Time of interview: Name of interviewer: Name of recorder: Location of interview: Tool 1: Farmers’ checklist A. Socio-demographics characteristics of cassava farmers Name Sex Age Marital status Level of education Farming experience Farm size for cassava Land tenure system Main occupation Labour source Residential status (Native or settler) Head of household Note: For the land tenure system, probe: who are entitled to ownership of land in the community (men and women, settlers and native) 149 B. Farm level characteristics Farm production 1. What was the average total food crops farmed last year? 2. List the main food and cash crops grown by men and women (Use pair-wise ranking) 3. Importance of cassava in the community 4. Percentage of the farm grown to cassava 5. Do men and women have equal access to and control over cassava farms? If no, what actions can be taken to increase women access and ownership of land 6. Challenges in expanding the cassava field Varietal information in the community 1. Please list the cassava varieties grown as given by each participant 2. Kindly give the meanings of the variety names given 3. Rank varieties per participant and as a group 4. Is cassava cultivated as a sole or intercrop and why? 5. Types of cassava roots colour grown and why? 6. Which type of cassava roots are mostly liked by customers (white or yellow) and why? 7. For each of the variety grown, what do you like about it? 8. For each of the variety grown, which do you want improved. 9. What traits do you prefer in cassava varieties (eg. what do you like to see in new varieties)? 10. Tell us about some traits that are distinct for men and women (probe for the reasons). 11. Tell us about the kinds of activities men and women engage in during the cassava cropping calendar (Start from land search to marketing). 12. Which aspects of cassava cultivation are believed to be difficult for men and women Information on planting material and credit access 1. Source of planting material 2. In the last five years, has anyone received any new varieties? (Please tell us how you got these varieties) 150 3. In the last five years, has anyone bought any new varieties? (Please which varieties and why) 4. Source of information on improved varieties? (probe for opportunities and challenges in accessing those varieties) 5. Do you produce your own cassava stems, buy some, or get it from somewhere, why? 6. If you buy or get it from somewhere, why 7. What are the challenges in accessing new varieties 8. Are there opportunities of credit for cassava production? If yes, what are the sources? 9. How easy is the process of accessing credit by men and women? (probe for who gets credit easily and why) Adoption and dis-adoption 1. How long do you use a particular variety before discarding (local and improved), provide examples. 2. In this group how many have ever used improved cassava varieties? 3. If you have never used it, why not? 4. How many are still using it? 5. For those who have stopped, why? 6. Which varieties have you abandoned? Preferences, perception and willingness to accept yellow flesh cassava 1. How many of you are aware of yellow flesh cassava? 2. Since when did you become aware (take per participant) of it? 3. For those who are aware, how many have ever cultivated yellow flesh cassava? 4. How many are still cultivating it? 5. What did you like / still like about the yellow flesh cassava? 6. What do you want improved? 7. Where do you get planting materials? 8. What are the challenges in accessing planting materials? 9. How do you perceive the yellow flesh cassava compared to white in terms of: • Taste 151 • Texture (when cooked) • Appearance: Colour • Nutrients • Marketability (which one sells faster) • Price (which one has higher price) • Uses 10. How many are willing to cultivate yellow flesh cassava and why? 11. If not willing to cultivate, why? Appendix 3 Tool 2: Processors’ checklist C. Socio-demographics characteristics of cassava processors Question/Aspect Responses Name (optional) Sex Age Marital status Level of education Processing experience Amount of roots/ bags processed every week Main occupation Labour source Residential status (Native or settler) Head of household Yes [1] No [0] Nett income (estimate): (After expenses) Note: For the land tenure system, probe: who are entitled to ownership of land in the community (men and women, settlers and native) D. Background 1. Which kind of jobs/activities do women and men do in the processing business (Activity profiling) 152 2. Describe the process for accessing cassava roots (probe for the differential opportunities or challenges for accessing the sources of these roots) 3. Who are the key stakeholders in the business of cassava roots (probe for the women and men in the trade and why) 4. Identify gender based constraints in the cassava processing 5. What challenges are in expanding the cassava processing business Varietal information in the community 1. Please list the cassava varieties processed as given by each participant 2. Kindly give the meanings of the variety names given 3. Rank varieties per participant and as a group 4. Do you process each cassava variety separately or together and why 5. Types of cassava roots colour processed and why 6. Which type of cassava roots are mostly liked by customers (white or yellow) and why 7. For each of the variety grown, what do you like about it 8. For each of the variety grown, which do you want improved 9. What traits do you prefer in cassava varieties (Eg. what do you like to see in new varieties) 10. Tell us about some traits that are distinct for men and women (probe for the reasons) 11. Which aspects of cassava processing are believed to be difficult for men and women Information on planting material and credit access 1. Do you have your own farm 2. If yes, source of planting material 3. In the last five years, has anyone received any new varieties? (Please tell us how you got these varieties) 4. Source of information on improved varieties? (probe for opportunities and challenges in accessing those varieties) 5. Do you produce your own cassava stems or buy some or get it from some why? 6. If you buy or get it from somewhere, why 7. What are the challenges in accessing new varieties 153 8. Are there opportunities of credit for cassava processing? If yes, what are the sources? 9. How easy is the process of accessing credit by men and women? (probe for who gets credit easily and why) Adoption and dis-adoption 1. How long have you process a particular variety before discarding (local and improved), provide examples 2. In this group how many have ever processed improved cassava varieties? 3. If you have never processed, why not? 4. How many are still processing? 5. For those who have stopped, why? 6. Which varieties have you abandoned Preferences, perception and willingness to accept yellow flesh cassava 1. How many of you are aware of yellow root cassava? 2. Since when did become aware (take per participant) 3. For those who are aware, how many have ever processed yellow root cassava? 4. How many are still processing? 5. What did you like / still like about the yellow flesh cassava 6. What do you want improved 7. Where do you get yellow flesh roots to process? 8. If from your own farm, where did you get planting materials? 9. What are the challenges in accessing yellow flesh roots/ its planting material?s 10. How do you perceive the yellow flesh cassava compared to white in terms of: • Taste • Texture (when cooked) • Appearance: Colour • Nutrients • Marketability (which one sells faster) • Price (which one has higher price) • Uses 11. How many are willing to process yellow flesh cassava and why? 154 12. If not willing to process, why? Appendix 4: Scoring the pulp colour using colour chat Appendix 5A and 5B: Eliciting information from stakeholders through a PRA 155 Appendix 6: Pearson phenotypic correlation for some cassava traits Pearson Correlation Coefficients, N = 20 Prob > |r| under H0: Rho=0 CGM CMD CORT_COL PULP_COL HI RTN RTW TCC TWT DMC CGM 1 0.16688 0.12345 0.2271 -0.49521 -0.03972 -0.51294 -0.02271 -0.23443 -0.057 CGM 0.4819 0.6041 0.3356 0.0264 0.868 0.0207 0.9243 0.3198 0.8113 CMD 0.16688 1 0.3957 0.77168 0.17597 -0.44956 -0.23005 0.54842 -0.25133 0.59704 CMD 0.4819 0.0842 <.0001 0.458 0.0467 0.3292 0.0123 0.2851 0.0054 CORT_COL 0.12345 0.3957 1 0.57959 0.14467 -0.28015 -0.19977 0.33654 -0.33167 0.09944 CORT_COL 0.6041 0.0842 0.0074 0.5428 0.2316 0.3984 0.1468 0.1531 0.6766 PULP_COL 0.2271 0.77168 0.57959 1 0.27598 -0.25987 -0.03062 0.58975 -0.32291 0.33529 PULP_COL 0.3356 <.0001 0.0074 0.2389 0.2685 0.898 0.0062 0.1649 0.1484 HI -0.49521 0.17597 0.14467 0.27598 1 -0.50741 0.3841 0.01111 -0.09499 0.00351 HI 0.0264 0.458 0.5428 0.2389 0.0224 0.0945 0.9629 0.6904 0.9883 RTN -0.03972 -0.44956 -0.28015 -0.25987 -0.50741 1 0.49008 -0.05152 0.42546 -0.12906 RTN 0.868 0.0467 0.2316 0.2685 0.0224 0.0283 0.8292 0.0615 0.5876 RTW -0.51294 -0.23005 -0.19977 -0.03062 0.3841 0.49008 1 0.22903 0.70884 0.00344 RTW 0.0207 0.3292 0.3984 0.898 0.0945 0.0283 0.3314 0.0005 0.9885 TCC -0.02271 0.54842 0.33654 0.58975 0.01111 -0.05152 0.22903 1 0.28791 0.20481 TCC 0.9243 0.0123 0.1468 0.0062 0.9629 0.8292 0.3314 0.2184 0.3864 TWT -0.23443 -0.25133 -0.33167 -0.32291 -0.09499 0.42546 0.70884 0.28791 1 0.16712 TWT 0.3198 0.2851 0.1531 0.1649 0.6904 0.0615 0.0005 0.2184 0.4813 DMC -0.057 0.59704 0.09944 0.33529 0.00351 -0.12906 0.00344 0.20481 0.16712 1 DMC 0.8113 0.0054 0.6766 0.1484 0.9883 0.5876 0.9885 0.3864 0.4813 156 Appendix 7: Scoring for mealiness 157