ABSTRACT
Understanding seed biology and germination ecology of plants is critical for domestication of neglected and underutilized species (NUS). However, this information is not readily available for Tylosema fassoglense [Family: Fabaceae]; despite its potential as a future crop. This research is primarily intended to benefit small-holder farmers of Kenya, by improving nutrition and enhancing sustainable agriculture, while contributing to good health and well-being. The main objective of this study was to evaluate the seed germination requirements of T. fassoglense by determining dormancy type, the effects of light, temperature and water potential on germination of scarified seeds as well as assessing the correlations among seed functional traits. Seed lots of T. fassoglense were collected from Busia, Migori and Siaya counties in Kenya. Seed dormancy was determined by water imbibition rates and germination tests on scarified and non-scarified. Scarified seeds were germinated under 12/12 plus 0/24 hour photoperiod as well as over a range of temperatures from 10 to 45 °C with intervals of 5 °C and at varied water potentials; 0, -0.25, -0.5, -1.0, -1.5 Megapascal (MPa). Water imbibition rates (g), germination percentages as well seed mass, oil and morphology were measured. Percentage data were normalized by arcsine transformation, subjected to analysis of variance (ANOVA) and the means separated with Tukey’s HSD (p<0.05) using SAS Version 2002-2003 statistical software. Scarification significantly (p<0.05) improved water uptake by 4 to 22 times as well as germination percentage from 30% to 90% (Migori seed lot) and from 70% to ≥90% (both Busia and Siaya seed lots). Seeds germinated well under light/dark or dark photoperiods while relative light germination index (RLG) ranged from 0.46 to 0.57. Seed germination was significantly (p<0.05) reduced at 10 °C and 40 °C whilst germination was zero at 45 °C in all the seed lots. The calculated base, optimum and ceiling temperatures (Tb, To and Tc; 50th percentile) ranges were 4.05-8.0 °C, 33.61-35.75 °C and 46.54-47.24 °C respectively while thermal time (θT(50)) suboptimal ranged from 76.19 to 89.02 degree Celsius days (°C d). Low water potential of -0.5 MPa significantly (p<0.05) reduced final germination to less than 50% in all seed lots while germination was zero at both -1.0 and -1.5 MPa. The base water potential (Ψb) and hydro time (θH(20)) ranges was -0.92 to -0.97 MPa and 3.95 - 4.11 Megapascal days (MPa d) respectively. Siaya seed lot were significantly (p<0.05) heavier than the other lots. Negatively significant (p<0.05) correlations were observed between Tb and θT; Ψb and θT while non-significant correlations were observed between germination and physical traits. Scarification improved water imbibition as well as final germination percentage and seeds of T. fassoglense are probably neutral photoblastic. The optimum temperature range for germination was 30-35 °C while seed germination was tolerant to low water potential up to -0.5 MPa. There were interrelationships among seed functional traits of T. fassoglense. The findings of this study will be useful in future research contributing towards the domestication of T. fassoglense as a future crop to enhance a sustainable agriculture and nutrition in Kenya and potentially worldwide.
Keywords: dormancy; germination; light; potential crop; physical traits; temperature; Tylosema fassoglense; water potential
Table of Contents
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
List of Figures viii
List of Tables x
List of Acronyms xi
GENERAL ABSTRACT xii
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background 1
1.2 Problem Statement 3
1.3 Justification 5
1.5 Main Objective 6
1.5.1 Specific objectives 6
1.5.2 Hypotheses 7
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1.1 Taxonomy and botany of Tylosema fassoglense 8
2.1.2 Ecological distribution 9
2.1.3 Traditional uses 10
2.1.4 Nutritional composition 10
2.1.5 Tylosema fassoglense seed 10
2.2 Seed maturity, collecting and seed characteristics 11
2.3 Seed germination and dormancy 12
2.4 Environmental factors that influence seed germination 14
2.4.1 Role of light on seed germination 14
2.4.2 Effect of temperature on germination 15
2.4.3 Effect of water potential on germination 16
2.5 Role of maternal environmental on seed functional traits 17
CHAPTER THREE
3.0 Dormancy and the role of light on germination of Sprawling bauhinia (Tylosema fassoglense)
3.1 Abstract 19
3.2 Introduction 19
3.3 Material and methods 21
3.3.1 Seed source information 21
3.3.2 Seed lot characteristics 24
3.3.3 Imbibition of water 25
3.3.4 Effect of manual scarification on germination 25
3.3.5 Seed germination under light and darkness 25
3.4 Germination evaluation 26
3.5 Data analyses 26
3.6 Results 27
3.6.1 Seed lot characteristics 27
3.6.2 Imbibition of water 27
3.6.3 Effect of manual scarification on germination 29
3.6.4 Role of light on germination 29
3.7. Discussion 30
3.7.1 Seed dormancy and dormancy-breaking 30
3.7.2 Sensitivity to light presence 32
3.8 Conclusion 33
CHAPTER FOUR
4.0 Germination response to temperature and water potential of Sprawling bauhinia (Tylosema fassoglense)
4.1 Abstract 34
4.2 Introduction 35
4.3. Materials and Methods 37
4.3.1 Seed lot information 37
4.3.2 Experiments on seed germination 37
4.3.3 Germination evaluation 38
4.3.4 Calculations 38
4.4 Data analysis 40
4.5. Results 40
4.5.1 Seed germination response to temperature 40
4.5.2 Seed germination response to water potential 44
4.6. Discussion 47
4.6.1 Thermal threshold 47
4.6.2 Hydro threshold 50
4.7 Conclusions 51
CHAPTER FIVE
5.0 Interrelationships among seed functional traits of sprawling bauhinia (Tylosema fassoglense)
5.1 Abstract 52
5.2 Introduction 52
5.3 Materials and method 54
5.3.1 Seed material information 54
5.3.2 Seed physical traits 55
5.3.2.1 Seed (Length, width, thickness) and shape 55
5.3.2.2 Seed oil content 55
5.3.2.3 Seed mass 55
5.3.3 Correlation among seed traits 56
5.4 Results 56
5.4.1 Seed physical traits 56
5.4.3 Interrelationships between seed traits 57
5.5 Discussion 57
5.5.1 Seed physical traits 57
5.5.2 Interrelationships between seed traits 58
5.6 Conclusion 60
CHAPTER SIX
6.0 GENERAL DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS
6.1 GENERAL DISCUSSION AND CONCLUSIONS 62
6.2 RECOMMENDATIONS 65
REFERENCES 67
LIST OFAPPENDICES
Appendix: 1: Mean seed mass increment (g) by water imbibition of scarified and non-scarified seed of Tylosema fassoglense from three seed lots 80
Appendix 2: Estimated regression line parameters: y = ax + b for germination rates (1/T50) plotted against temperature for four replicates of the different seed lots of Tylosema fassoglense 81
Appendix 3: Estimated regression parameters: y = ax + b for germination rates (1/T20) plotted against water potential for four replicates of the different seed lots of Tylosema fassoglense 81
List of Figures
Figure 1:1: Map of agroecological zones, AEZ of Kenya (Source: FAO System: Infonet Biovision Home) 3
Figure 2:1: Tylosema fassoglense root tuber partially excavated (Photo: V. Otieno) 9
Figure 2:2: Tylosema fassoglense flowers (Left) and seeds (Right) (Photo: V. Otieno) 9
Figure 2:3: A sectioned seed of Tylosema fassoglense [Left] and T. esculentum [Right] showing the cotyledon, radicle and seed coat (Photo: Pablo-Barreiro Gomez) 11
Figure 3:1: Map of counties where seeds of Tylosema fassoglense were collected in Kenya (Map: Abiud Sayah) 22
Figure 3:2: Seed x-ray radiograph of Tylosema fassoglense (Radiograph: V. Otieno) . 28
Figure 3:3: Water uptake by imbibition at 25°C plotted against time for scarified (continuous line) and non-scarified (dotted line) seeds of Tylosema fassoglense from three seed lots. Blue= Busia, Red=Migori and Black=Siaya seed lots. Points with asterisks (*) differ (p<0.05, n=10) statistically between scarified and non-scarified seeds, vertical bars are standard error (±se) 28
Figure 3:4: Germination percentages of scarified (clear bars) and non-scarified (grey bars) seeds of Tylosema fassoglense incubated under 12/12 hr photoperiod and 25 °C. Bar graphs with asterisks (*) differ statistically (p<0.05, n=4) and vertical error bars are the standard error (±se) 29
Figure 3:5: Germination percentages of scarified seeds of Tylosema fassoglense seed lots under 12/12 hour (clear bars) and 0/24 hour (grey bars) photoperiod. No significant (p>0.05, n=4) difference between the two photoperiods and vertical error bars are standard error (±se) 30
Figure 4:1: Cumulative germination time course of Tylosema fassoglense seeds germinated at different temperatures under 12/12 hour photoperiod for Busia seed lot 40
Figure 4:2: Cumulative germination time course of Tylosema fassoglense seeds germinated at different temperatures under 12/12 hour photoperiod for Migori seed lot 41
Figure 4:3: Cumulative germination time course of Tylosema fassoglense seeds germinated at different temperatures under 12/12 hour photoperiod for Siaya seed lot 41
Figure 4:4: Final germination percentage ( ) and germination rate ( 1/t50) of Tylosema fassoglense seed germinated at varied temperature for Busia seed lot. Points with * and ƚ are significantly (p<0.05) different and vertical bars are standard error (±se) 42
Figure 4:5: Final germination percentage ( ) and germination rate ( 1/t50) of Tylosema fassoglense seed germinated at varied temperature for Migori seed lot. Points with * and ƚ are significantly (p<0.05) different and vertical bars are standard error (±se). 43
Figure 4:6: Final germination percentage ( ) and germination rate ( 1/t50) of Tylosema fassoglense seed germinated at varied temperature for Siaya seed lot. Points with * and ƚ are significantly (p<0.05) different and vertical bars are standard error (±se). 43
Figure 4:7: Cumulative germination time course of Tylosema fassoglense seeds germinated at different water potentials, 25 °C and 12/12 hour photoperiod for Busia seed lot 45
Figure 4:8: Cumulative germination time course of Tylosema fassoglense seeds germinated at different water potentials, 25 °C and 12/12hour photoperiod for Migori seed lot 45
Figure 4:9: Cumulative germination time course of Tylosema fassoglense seeds germinated at different water potentials, 25 °C and 12/12hour photoperiod for Siaya seed lot 46
Figure 4:10: Effect of varied water potential on final germination percentage of Tylosema fassoglense from three seed lots. Points with differ (p<0.05) statistically at seed lot level, error bars are standard error (±se) 47
List of Tables
Table 3:1:Information of Tylosema fassoglense seed lots showing seed source, seed collection months (grey cells) and the average monthly precipitation (mm), maximum, mean and minimum temperatures for the collection sites (Source: Kenya Met. Dept. Satellite 23
Table 3:2: Seed characteristics of the three seed lots of Tylosema fassoglense: moisture content, equilibrium relative humidity eRH, Variance and Viability(Cut test) 27
Table 4:1: Seed germination thresholds for three seed lots of Tylosema fassoglense; Cardinal temperatures (base Tb, optimal To and ceiling Tc) and thermal time (θT) in degree Celsius days (°Cd) at suboptimal and supraoptimal range for four replicates at 50th percentile (1/t50) 44
Table 4:2: Mean seed germination rate at 20th percentile for four replicates of Tylosema
fassoglense seed from three seed lots germinated at different water potentials 46
Table 4:3: Hydro time model parameters (base water potential and hydro time) for the three seed lots of Tylosema fassoglense calculated at the 1/t20 germination percentile 47
Table 5:1: Comparison of seed physical traits for Tylosema fassoglense seeds collected from three counties in Kenya 57
Table 5:2: Correlations matrix between Tylosema fassoglense seed functional traits 57
List of Acronyms
AEZ Agro-ecological Zones
ANOVA Analysis of Variance
ASALs Semi-Arid and Arid Lands
DNA Deoxyribonucleic Acid
eRH Equilibrium Relative Humidity
FAO Food and Agriculture Organisation of the United Nations
IPCC Inter-governmental Panel on Climate Change
IPGRI International Plant Genetic Research Institute
ISTA International Seed Testing Association
KEFRI Kenya Forestry Research Institute
MC Moisture Content
MSB-P Millennium Seed Bank Partnership
NUS Neglected and Underutilised Species
RH Relative Humidity
RBG Kew Royal Botanic Gardens, Kew
SDGs Sustainable Development Goals
SSA Sub Saharan Africa
UPP Useful Plants Project
UN-DESA United Nations Department of Economic and Social Affairs
UN United Nations
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background
The current food security is threatened by rising population, coupled with climate change impacts such as droughts, heat waves and floods (Lal, 2013; Wheeler and Von Braun, 2013; Rosenzweig et al., 2014; Valin et al., 2014; UN DESA, 2015). Nowhere is this challenge more formidable than in the sub-Saharan Africa (SSA) countries where population growth rates are among the highest in the world while basic infrastructure is inadequate and fertilisers are extremely costly (Cordell et al., 2009). To address food security and mitigate climate change effects, there is need to adopt climate-resilient, low-input crops for SSA countries.
Adapting mainstream crops to climate change phenomena involves conventional crossing and selection approaches, sometimes aided by biotechnological as well as genomic means, or through crossing with old landraces and crop wild relatives (CWRs) (Lopes et al., 2015; Castañeda-Álvarez et al., 2016; Kissoudis et al., 2016). However, mainstream crops are adapted to relatively favourable moisture and nutrient conditions, which can be improved incrementally by breeding but not to the extent required to be productive in harsh, infertile environments in the marginal production areas (Figure 1.1). Therefore, adoption of neglected and underutilised legume crops such as Tylosema fassoglense, Vigna subterranea, V. unguculata, is a more promising approach to sustainable agriculture, since they are nutritionally rich, highly adapted to severe climates and capable of growing in infertile soils (Padulosi et al., 2013).
The family Fabaceae to which cultivated legumes belong, exhibits great diversity in terms of distribution, life form (liana, shrub, tree) and morphological variation in seed size, shape as well as dormancy (Baskin and Baskin, 2014). Despite cultivated grain legumes being second to cereals as major plant-protein sources on a world scale, their production does not meet the ever increasing demand (FAO Statistics, 2016). Tylosema fassoglense is a nutritionally rich neglected and underutilized grain legume for Africa. Its drought-tolerance (Brink, 2006) and excellent nutrient content comparable to commercially cultivated crops (soybean, peanut, common bean), has the potential to provide sustainable food and feed in the future (Otieno et al., 2015). The seeds of T. fassoglense are nutritionally rich in protein (30-40%), lipids (24- 40%), minerals and starch (Dubois et al., 1995; Okumu, 2011; Otieno et al., 2015), therefore can contribute towards the country’s protein demand partially or completely substitute other animal proteins in the household’s diet. Most legume plants also fix atmospheric nitrogen allowing them to colonise diverse habitats of poor soils and when intercropped with food crops provides reprieve to farmers on nitrogen fertilizer application (Graham and Vance, 2003). Wild plants also display diverse adaptive traits to their habitat that permit seed germination and seedling establishment under unpredictable conditions unlike crops that are adapted to favourable environments (Dürr et al., 2015; Castillo-Lorenzo et al., 2019b).
Despite, a number of plants including legumes having been domesticated several decades ago, a large proportion with great socioeconomic importance still remain in the wild with unrealized potential (Ulian et al., 2017). The lack of sufficient knowledge on how to propagate these wild species also hampers their utilisation in livelihood and conservation programmes (Rodríguez- Arévalo et al., 2017). Seed collecting and tuber extraction of T. fassoglense from the wild is not only unsustainable but also hampers natural regeneration and negatively impacts on natural populations. The understanding of seed functional traits of this neglected and underutilised grain legume is important for its domestication, conservation and sustainable utilization through propagation for cultivation and reintroduction to their natural habitat. Furthermore, the wide distribution of T. fassoglense provides an opportunity for diversity due to genetics, environment and interactions between these two factors resulting in variation of seed germination, genetic and seed composition traits which are essential for selecting suitable germplasm for future breeding and promotion. However, despite the immense potential for adoption and cultivation in marginal production areas, T. fassoglense is still in the wild. Therefore, there is a need for intensive agronomic research to bring it into cultivation and to utilize its potential as a cash crop.
1.2 Problem Statement
Research on T. fassoglense has focussed on its economic importance and utilisation with little emphasis on its propagation. The protein content in T. fassoglense seed is comparable to soybean while oil content is more than that in peanut (Dubois et al., 1995; Okumu, 2011; Otieno et al., 2015). The species is adapted to drylands of sub-Saharan Africa, SSA (Castro et al., 2005; Sinou et al., 2009). However, information on seed germination and seedling establishment of T. fassoglense that is important in its regeneration and propagation has not been generated. Therefore, understanding seed biology and germination ecology of T. fassoglense is crucial in its production for consumption and commercialisation. Furthermore, “true seed” is the most preferred planting material under crop production over vegetative material due to ease of storage and as a commodity for trade.
Physical dormancy, PY which is simply the presence of water impermeable seed/fruit coat is an adaptive trait found in 18 angiosperm plant families including Fabaceae (Baskin and Baskin, 2014). Breeding (selection) has eliminated dormancy in cultivated grain legumes, however it still remains a challenge in propagating wild species (Bewley and Black, 1994; Cheng and Bradford 1999). While PY has been reported in T. esculentum (Fabaceae) (Travlos et al., 2007) seed dormancy and germination of the other four species in this genus including T. fassoglense seeds has not been documented. It has therefore been difficult to promote this nutritious grain legume species for adoption by small-holder farmers in Kenya.
Seed physical traits (mass and shape) have been used in predicting light requirements for germination, soil seed bank formation and dispersal (Baskin and Baskin, 2014). Seed sowing depth is often related to crop emergence, however light filtering through canopy and burial under leaf litter or cracks in the soil affects species regeneration ability in their natural habitat (Fenner, 1991). Baskin and Baskin, (2014) proposes three groups of plants depending on their light requirement for germination: positive photoblastic (those that require light), negative photoblastic (those that don’t require light) and neutral photoblastic (those insensitive to light). Under cultivation crop seeds are buried in the soil, however germination ecology of T. fassoglense in relation to light requirement remains unknown despite its potential as a future crop.
Tylosema fassoglense a NUS growing in the wild have adaptive mechanisms displayed by varied seed functional traits that interact with the environmental factors to ensure regeneration and survival within a habitat (Dürr et al., 2015). Therefore, knowledge of germination traits (hydro and thermal thresholds) is important for predicting its environmental limits (Ghaderi- Far et al., 2010) and formulating adoption strategies (Hardegree et al., 2016). Seed vigour, as a descriptor of regeneration potential (ISTA, 2017) can also be inferred from the germination traits; that is germination rate, thermal as well as hydro times quantified by characterisation of the germination-physiological processes. This important information to guide the domestication and conservation of T. fassoglense has not been generated.
Seed maternal environment has been shown to influence seed physical traits (i.e. mass, size, composition) which are often correlated to germination parameters (i.e. hydro and thermal thresholds) (Norden et al., 2009; Dürr et al., 2015; Gardarin et al., 2016; Castillo-Lorenzo et al., 2019a, b). The understanding of correlations between seed physical and germination traits and interrelationships among germination traits is not only important for germplasm sourcing and future breeding but also the environmental range for adoption (Ghaderi-Far et al., 2010; Dürr et al., 2015). However, literature on interrelationships amongst seed functional traits for T. fassoglense has not been generated. The current research aimed at determining the type of seed dormancy and effects of light, temperature and water potential on germination as well as assessing the interrelationships among seed traits of T. fassoglense.
1.3 Justification
The major challenge to the exponential human population growth is malnutrition which has created two spectra of populations, calorie deficient (hungry) and calorie excess (obese). For example the undernourished population in Kenya is estimated at 10 million (IFAD FAO and UNICEF, 2017). The need to address food insecurity through conversion of forest land to increase conventional crop production has resulted in biodiversity loss. Furthermore, about two thirds of Kenya’s landmass is unarable and support wild life or livestock production. Therefore, there is need to produce nutritious foods sustainably while protecting the environment.
During plant domestication (7,000-12,000 years ago) several species were cultivated, however less than 200 have been extensively cultivated leading to narrow genetic diversity (Padulosi et al., 2013). Currently, rice, wheat and maize provide about 40% of the world’s calorie intake (Ulian et al., 2020). Intensification of agriculture during the “Green Revolution of 1960s to 1980s” resulted in unforeseen challenges such as land degradation, environmental pollution, increased pests and diseases as well as decreased dietary diversity and neglect of traditional food crops (Webb and Eiselen, 2009 in Ulian et al., 2020). However, there is currently increased recognition of neglected and underutilized species of plants to improve agrobiodiversity, livelihoods as well as sustainable agriculture and food security (Ulian et al., 2020).
Tylosema fassoglense is a neglected drought-tolerant grain legume native to ASALs of sub- Saharan Africa (Castro et al., 2005; Brink, 2006). It produces seeds rich in protein (30-45%) and oil (24-40%) as well as carbohydrate-rich tubers with potential as a source of food and feed (Dubois et al., 1995; Okumu, 2011; Otieno et al., 2015). Its’ root tubers are highly valued for medicinal properties (Adongo et al., 2012; Maundu et al., 1999), which threatens its natural population. Therefore adopting T. fassoglense into mainstream agriculture has the potential to contribute towards agrobiodiversity, food security, ensure sustainable agriculture and reduce exposure to climate change impacts which supports the Government of Kenya, Big 4 Agenda and United Nations Sustainable Development Goals, (UN SDGs) of 2015. In addition, its cultivation will increase seed production, provide an opportunity to understand harvest maturity of its starch-rich tuber which will also support conservation and sustainable utilization of germplasm resources according to the Convention on Biological Diversity (CBD).
There is need to create awareness on the potential of T. fassoglense to public institutions, farmers, non-governmental organisations (NGOs) as well as other organisations engaged in crop development and germplasm conservation, however there is no literature on seed biology and germination ecology to guide its propagation. To contribute to promotion and adoption of
T. fassoglense as a climate-resilient, low input grain legume by resource-poor farmers in Kenya, the study envisaged to generate information on the species domestication and conservation through determination of seed dormancy type, light requirement for germination; to characterise seed germination response to temperature and water potential as well as to examine the interrelationships among seed functional traits of T. fassoglense with a view to determine conditions for optimal seed germination. The knowledge generated in this study will help Government institutions, NGOs, farmers and genebank managers in domestication, conservation and germplasm testing of T. fassoglense.
1.5 Main Objective
To determine optimal germination conditions of Tylosema fassoglense for enhanced adoption as a climate-resilient, low input grain legume by small-holder farmers in Kenya.
1.5.1 Specific objectives
This study was designed with the following specific objectives:
1. To evaluate seed dormancy condition and role of light on germination of scarified seeds of T. fassoglense
2. To determine the effects of temperature and water potential on germination of scarified T. fassoglense seed
3. To assess interrelationships among seed functional (physical and germination) traits of T. fassoglense
1.5.2 Hypotheses
1. Tylosema fassoglense seeds are dormant while germination is not influenced by light
2. The optimum temperature for germination of T. fassoglense seeds is between 25°C and 30°C while germination process have a high tolerance to low water potentials
3. There are correlations amongst seed functional traits of T. fassoglense
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