ABSTRACT
Boswellia sacra is a frankincense-producing tree found in the Arabian Peninsula that includes Oman and southern Somaliland (Northern Somalia). In Somaliland, it is mainly distributed in the Sanaag region including Cel Afweyn where many families depend on its value chain for a living. There is a high global demand for frankincense gums and resin products due to its medicinal, cultural and cosmetic importance leading to overexploitation of existing stands, leading to failure of trees to regenerate attributed to excessive tapping on tree barks causing damage that increases pest and disease infestation. The establishment of the species is hindered by the poor seed germination rate as well as grazing of young seedlings by both wildlife and livestock. This study sought to address the decrease in B. sacra numbers through regeneration via tissue culture and avail information on genetic diversity of two B. sacra populations in Af yare Dawl-dawl and Exdad in Somaliland for conservation and germplasm enhancement. Seed viability was determined by germination rate and tetrazolium tests, which revealed 4.15 % germination rate and 3.3% viability. These values were low but comparable to previously reported percentages of less than 10%, due to lack of viable embryos attributed to self-incompatibility within the species and high tapping frequency. Clonal propagation using tissue culture applied leaf and axillary bud explants for direct and indirect in-vitro regeneration using plant growth regulators, such as TDZ, BAP, NAA, in efforts to develop a micropropagation protocol for mass propagation. Direct in- vitro regeneration did not produce any regenerants while indirect regeneration produced callus on MS media containing 5 µm TDZ. Somatic embryogenesis was initiated in MS media containing 1 µm BAP+ 0.25 µm IAA, where shoot regenerants were produced. Genetic diversity determined using morphological and molecular markers to establish diversity within and between germplasm to enhance breeding programs. Morphological characterization of B. sacra genotypes from Exdad and Af Yare Dawl-dawl had no significant (p>0.05) differences in height, height to branching, number of stems and tree bottom swelling, however, stem type was strongly correlated to height. Hierarchical cluster analysis of principle components extracted from morphological data revealed two main clusters each with two sub-clusters with genotypes from both regions distributed within these clusters, thereby showing high similarity. Molecular characterization was done on Af Yare Dawl-dawl genotypes using SRAP markers and they revealed very low heterozygosity, He=0.053, indicative of very low genetic diversity. PCoA analysis of pairwise genetic distance matrix led to formation of three major clusters with 35.14%, 11.42% and 9.08% variation, indicating existence of three major groups of genotypes with different parentage. This variation indicates that genotypes from the different clusters can be used as progenitors for hybridization and domestication purposes. Low genetic diversity observed calls for urgent conservation measures of the tree species as well as introductions from other areas to enhance the germplasm found in Af Yare Dawl-dawl. Breeding programs for enriching genetic diversity in the study area should be initiated since the current population is faced with the threat of extinction should a sudden environmental change occur. Sustainable frankincense harvesting practices should be enforced to ensure sustainable exploitation of this important resource.
TABLE OF CONTENTS
DECLARATION i
ABSTRACT iii
ACKNOWLEDGEMENTS v
LIST OF FIGURES xi
LIST OF TABLES xii
LIST OF IMAGES xiii
LIST OF ABBREVIATIONS xiv
CHAPTER ONE: INTRODUCTION
1.1 Background information 1
1.2 Statement of the problem 3
1.3 Justification 3
1.4 Objectives 5
1.4.1 General Objective 5
1.4.2 Specific objectives 5
1.5 Hypothesis 5
CHAPTER TWO: LITERATURE REVIEW
2.1 Distribution of Boswellia sacra 6
2.2 Biology of Boswellia sacra 6
2.3 Importance and utilization of Boswellia sacra 7
2.3.1 Utilization of frankincense in the ancient world 7
2.3.2 Present Utilization of Frankincense 8
2.4 Propagation of Boswellia sacra 9
2.5 Tissue culture and its applications 9
2.5.1 Choice of explant 11
2.5.2 Growth regulators 11
2.6 Importance of genetic diversity studies 12
2.6.1 Assessing genetic variation in plants 13
2.6.1.1 Restriction Fragment Length Polymorphism (RFLP) 14
2.6.1.2 Random Amplified Polymorphic DNA (RAPD) 15
2.6.1.3 Amplified Fragment Length Polymorphism (AFLP) 15
2.6.1.4 Simple Sequence Repeats (SSR) 16
2.6.1.5 Inter Simple Sequence Repeats (ISSR) 16
2.7 Biotechnology in tree breeding 17
CHAPTER THREE
Abstract 19
3.1 Introduction 20
3.2 Materials and methods 21
3.2.1. Seed collection sites 21
3.2.2 Seed extraction 22
3.2.3 Boswellia sacra seed viability tests 22
3.2.4 Micro-propagation of B. sacra 24
3.2.4.1 Source of explants 24
3.2.4.2 Media preparation 24
3.2.4.3 Explant preparation and sterilization 25
3.2.4.4 Inoculation of B. sacra explants in media 25
3.2.4.5 Growth culture conditions 25
3.3 Results 26
3.3.1 Boswellia sacra seed tests 26
3.3.1.1 Determination of B. sacra 1000 seed weight 26
3.3.1.2 Tetrazolium test on B. sacra seeds 29
3.3.1.3 Germination test on B. sacra seeds 31
3.3.2 Effect of different hormones, combinations and explants on shoot production via direct organogenesis 31
3.3.2.1 Effect of BAP, meta-topolin riboside and thidiazuron on leaf explants 31
a) Effect of 6-Benzylaminopurine on B. sacra leaf explants 32
b) Effect of Meta-topolin riboside on B. sacra leaf explants 33
c) Effect of Thidiazuron on leaf explants 34
3.3.2.2 Effect of PVP on media non-browning 36
3.3.2.3 Effect of BAP and TDZ on survival of axillary bud explants 37
a) Effect of BAP on axillary bud explants 37
b) Effect of TDZ on axillary bud 38
3.3.2.4 Effect of different hormones on axillary bud breaking 39
a) Effect of BAP on axillary bud breaking 39
b) Effect TDZ on axillary bud breaking 40
3.3.3 Effect of different hormones and combinations on calli production for indirect organogenesis 41
3.3.3.1 Effect of TDZ on callus induction from leaf explants 42
3.3.3.2 Effect of Meta-methoxytopolin on callus induction from leaf explants 43
3.3.3.3 Effect of MemTR and TDZ combinations on callus induction from leaf explants 45
3.3.3.4 Effect of BAP on callus induction from leaf explants 47
3.3.3.5 BAP and IAA combinations on callus induction from leaf explants 48
3.3.4 Effect of BAP and NAA on somatic embryogenesis from calli. 50
3.3.4.1 Effect of BAP on somatic embryogenesis from calli 50
3.3.4.2 Effect of BAP and NAA on somatic embryogenesis from calli 51
3.3.5 Effect of different hormones and combinations on shoot production via somatic embryogenesis 52
3.3.5.1 Effect of BAP and IAA on shoot development from somatic embryos 52
3.4 Discussion 54
3.4.1 Germination and viability of B. sacra seeds are affected by lack of embryos 54
3.4.2 Low regeneration of B. sacra via organogenesis 56
3.4.3 Regeneration via somatic embryogenesis 57
3.5 Conclusion and recommendations 60
CHAPTER FOUR
Abstract 62
4.1 Introduction 63
4.2 Materials and Methods 65
4.2.1 Morphological characterization of B. sacra from Exdad and Af Yare Dawl-dawl sites 65
4.2.1.1 Study areas, sampling and data collection 65
4.2.2 Molecular characterization of B. sacra genotypes from Exdad and Af Yare Dawl- dawl 68
4.2.2.1 Sample collection 68
4.2.2.2 DNA extraction and quantification 68
4.2.2.3 ISSR primer optimization 69
4.2.2.3 SRAP marker optimization 70
4.2.3 Data analysis 71
4.2.3.1 Data collection and analysis for morphological data 71
4.2.3.2 Molecular data analysis 71
4.2.3.3 Combined morphological and molecular data analysis 72
4.3 Results 73
4.3.1 Morphological characterization 73
4.3.1.1 Stem type 73
4.3.1.2 Number of stems 73
4.3.1.3 Tree height 74
4.3.1.4 Height to branching 75
4.3.1.5 Swelling at the base of the stem 75
4.3.1.6 Growth surfaces of B. sacra trees in Exdad and Af Yare Dawl-dawl 76
4.3.1.7 Correlation between traits of B. sacra trees in Af Yare Dawl-dawl site 77
4.3.1.8 Correlation between traits of B. sacra trees in Exdad 77
4.3.1.9 Correlation between traits of B. sacra trees in Exdad and Af Yare Dawl-dawl. 78
4.3.1.10 Hierarchical cluster analysis for morphological data 79
4.3.2 Molecular characterization 82
4.3.2.1 DNA quantification 82
a) Agarose gel quantification 82
b) Nanodrop DNA quantification 84
4.3.2.1 ISSR primer optimization 85
4.3.2.2 SRAP marker optimization 88
4.3.2.3 SRAP marker analysis 88
a) SRAP marker analysis for Af Yare Dawl-dawl subpopulations 90
4.3.3 Combined molecular and morphological characterization 92
4.4 Discussion 94
4.4.1 Morphological diversity 94
4.4.2 DNA quality was affected by polyphenolic compounds and moisture build-up in silica gel 95
4.4.3 SRAP markers generated polymorphism for Exdad DNA samples 96
4.4.4 Low heterozygosity of the B. sacra genotypes indicated low genetic diversity 97
4.5 Conclusion and recommendations 98
CHAPTER FIVE
5.0 General discussion, conclusion and recommendations 100
References 105
Appendices 114
LIST OF FIGURES
Figure 3.1: Mean percentage number of B. sacra capsules with seed collected from Af Yare Dawl-dawl and Exdad. 28
Figure 3.2:1000 seed weight of seeds collected from Af Yare Dawl-dawl and Exdad3.3.1.2 Tetrazolium test on B. sacra seeds 28
Figure 3.3: Effect of BAP on leaf explant survival for direct shoot organogenesis 32
Figure 3.4: Effect of BAP and mTR on explant survival for direct organogenesis 34
Figure 3.5: Effect of TDZ on leaf explant survival for direct organogenesis 35
Figure 3.6: Effect of PVP on reduction of phenolic compounds on media of B. sacra explants.37
Figure 3.7: Effect of BAP on axillary bud explant survival for direct shoot organogenesis 38
Figure 3.8: Effect of TDZ on axillary bud explant survival for shoot organogenesis 39
Figure 3.9: Effect of BAP on axillary bud breaking 40
Figure 3.10: Effect of TDZ on axillary bud breaking 41
Figure 4.1: Distribution of single and multiple stems in B. sacra trees in Exdad and Af Yare Dawl- dawl. 73
Figure 4.2: Mean number of stems on B. sacra trees sampled in Exdad and Af Yare Dawl-dawl. 74
Figure 4.3: Performance in height for the sampled trees from Exdad and Af Yare Dawl-dawl. 74
Figure 4.4: Mean height to branching on B. sacra trees in Exdad and Af Yare Dawl-dawl. 75
Figure 4.5: Percentage number of trees with and without base swelling in Exdad and Af Yare Dawl-dawl. 76
Figure 4.6: Growth surface characteristic of B. sacra trees in Exdad and Af Yare Dawl-dawl . 76
Figure 4.7: Dendrograms showing hierarchical cluster analysis of accessions from i) Af Yare 80
Figure 4.8: Dendrogram showing hierarchical cluster analysis of similarity matrix obtained from six morphological traits in B. sacra ecotypes from Af Yare Dawl-dawl (BD) and Exdad (AD) showing two major clusters (1 and 2) each with two (A, B, C, D) subclusters. All clusters consisted of genotypes from both Af Yare Dawl-dawl and Exdad. 81
Figure 4.9: PCoA analysis from genetic data matrix of 31 B. sacra genotypes from Af Yare Dawl- dawl with three main clusters. 89
Figure 4.10: Unweighted neighbor-joining dendrogram for 31 B. sacra accessions from Af Yare Dawl-dawl. 90
Figure 4.11: Principal coordinates analysis from genetic distance matrix of B. sacra genotypes. 92
Figure 4.12: Dendrogram showing hierarchical cluster analysis of a combined data set consisting morphology and molecular attributes of B. sacra genotypes from Af Yare Dawl-dawl. Two main clusters (1 and 2) each having two subclusters (A, B, C and D) are shown. Most genotypes fell on cluster 1. 93
LIST OF TABLES
Table 3.1: Fruit capsules of B. sacra collected from trees in Exdad and Af Yare Dawl-dawl regions, Somaliland 23
Table 3.2: Callus induction frequency on leaf explants of B. sacra under varying TDZ levels of TDZ over time 43
Table 3.3: Performance of leaf explants on different concentrations of MemTR over time 45
Table 3.4: Performance of leaf explants on different combinations of MemTR and TDZ over time. 46
Table 3.5: Performance of calli under different concentration levels of BAP over 5 weeks of culture 48
Table 3.6: Effect of BAP and IAA on callus induction on leaf explants. 49
Table 3.7: Development of somatic embryos on calli cultured in various levels of BAP and NAA over time 52
Table 3.8: Shoot regeneration under different combinations of BAP and NAA 53
Table 4.1: UBC primers screened and their sequence 69
Table 4.2: Primer sequences for primers used to assess of genetic diversity in B. sacra genotypes70
Table 4.3: Correlation matrix between stem type, number of stems, tree height, bottom swelling and growth of B. sacra trees in Af Yare Dawl-dawl. 77
Table 4.4: Correlation matrix between stem type, number of stems, tree height, bottom swelling and growth of B. sacra trees in Exdad 78
Table 4.5: Correlation matrix between stem type, number of stems, tree height, bottom swelling and growth of B. sacra trees in Exdad and Af Yare Dawl-dawl. 79
Table 4.6: Quantity and quality of DNA extracted from leaf samples collected in Af Yare Dawl- dawl (BD) and Exdad (AD) 84
Table 4.7: ISSR primer optimization results 85
Table 4.8: SRAP primer pairs selected for assessment of genetic diversity for B. sacra genotypes88 Table 4.9: Mean and SE over loci for B. sacra genotypes from Af Yare Dawl-dawl. 89
Table 4.10: Mean and SE over loci for multiple-stemmed and single-stemmed genotypes from Af Yare Dawl-dawl 91
Table 4. 11: Analysis of variance for single-stemmed and multi-stemmed B. sacra genotypes from Af Yare Dawl-dawl. 91
LIST OF IMAGES
Image 3.1: Boswellia sacra fruit capsules. 2. One B. sacra seed (b) extracted from a single capsule with pericarps dehisced (a). B. sacra seeds after extraction from fruit capsules 27
Image 3.2: Dissected B. sacra seeds showing: 1,2) color change to pink, 3,4) no color change after tetrazolium test. 30
Image 3.3: a) B. sacra seed germinating after 12 days b) B. sacra seedling, 24 days after sowing 31
Image 3.4: Browning of media and death of leaf explants after 5 weeks of culture on media containing BAP. 33
Image 3.5: Browning of media and death of leaf explants after five weeks of culture on media containing TDZ 35
Image 3.6: Axillary buds performance on media (1) explants cultured in MS media with no PGR + 1 g/L PVP showing no signs of bud breaking after 3 weeks. (2) explants cultured on MS media with 44.39 µm BAP having two leaves after 2 weeks (a) vigorous growth (b) signs of necrosis 41
Image 3.7: Dead explants (a) on media with no PGRs and explants forming callus (b) on media with 5µm MemTR after four weeks of culture 43
Image 3.8: Calli on 1. petiole 2. midribs 3. After 4 weeks of culture 4. After 8 weeks of culture 50
Image 3.9: Various stages of somatic embryogenesis (a) globular stage, (b) heart stage and (c) cotyledonary stage 51
Image 3.10: Developing shoot (a) after two weeks of culture and (b) after four weeks in MS media with no PGRs. Shoot (c) after two weeks and (d) after five weeks of culture in MS media with 1 µm BAP + 0.25 µm IAA. Note the hyperhydricity in (b) and (d) 53
Image 4.1: Morphological and growth surface differences between B. sacra trees. a, c and d have multiple stems originating from base in contrast to b. a and d are growing on faces of a cliff and rock respectively while c is growing on a flat rock surface. Also note the bottom swelling in a and c 67
Image 4.2: Gel images showing the quantity of DNA from 55 samples from Af Yare Dawl-dawl (BD) and Exdad (AD). Note the smearing, an indication of DNA shearing due to degradation. 83
Image 4.3: Gel images showing polymorphism and amplification of UBC ISSR primers 808,822,824, 825, 802, 806, 808, 811, 813, 817,818, 820, 829 and 849. Although
there were several primer amplifications, note the inconsistencies in band intensity 87
Image 4.4: Gel image of 12 SRAP marker pairs over two samples with primers 1, 2, 4, 7,8,10,11 and 12 showing polymorphism 88
Image 4.5: PCoA analysis from genetic data matrix of 31 B. sacra genotypes from Af Yare Dawl- dawl with three main clusters. Error! Bookmark not defined.
LIST OF ABBREVIATIONS
2,4-D - 2,4 - Dichlorophenoxyacetic acid
AFLP - Amplified Fragment Length Polymorphism BAP - 6-Benzylaminopurine
BSA -Bovine Serum Albumin
CRD - Completely Randomized Design DNA - Deoxyribonucleic Acid
dNTPs – Deoxyribonucleic triphosphates IAA – Indole Acetic Acid
IBA – Indole-3-butyric Acid IB – Isolation buffer
ITS – Internal transcribed spacer
ISSRs - Inter-Simple Sequence Repeats
IUCN – International Union for Conservation o Nature MAS – Marker assisted selection
MS - Murashige and Skoog NAA- 1-Naphthaleneacetic acid
RAPD- Random Amplified Polymorphic DNA SSRs- Simple Sequence Repeats
TDZ- Thidiazuron
SRAP - Sequence-related Amplified Polymorphism mTR- Meta-Topolin Riboside
MemTR - 6-(3-methoxybenzylamino) purine-9-riboside (meta-methoxytopolin) PCR - Polymerase chain reaction
PCoA- Principal Co-ordinate Analysis PCA- Principal Component Analysis PEG – Polyethylene glycerol
PGR – Plant growth regulator TAE – Tris-Acetate-EDTA Buffer TBE -Tris-Borate-EDTA Buffer TDZ- Thidiazuron
WPM – Woody plant media
CHAPTER ONE
INTRODUCTION
1.1 Background information
Increasing demand for plants and plant products such as food, fuel, medicines, fibre, construction materials and industrial raw materials is undermining the available supplies all over the world (Giri et al., 2004; Ayensu, 1983). The changing climatic patterns and globalization of trade have made things worse for the natural plant products with high demand (Thuiller et al., 2005). Traditional plant propagation and germplasm conservation methods are playing a key role in trying to maintain a balance between demand and supply but have not been entirely successful (Engelmann and Engels, 2002). It is clear that radical measures are required if the increasing demand for plants products is to be met sustainably (Rao, 2004). One of these plants whose utilization has led to rapid decrease in its population is Boswellia sacra (Groenendijk et al., 2012).
Boswellia sacra is one of the olibanum producing trees in Somaliland (Farah, 1994). Olibanum or frankincense refers to dried resin exudates obtained from Boswellia species (Ben-Yehoshua et al., 2012). These species include B. sacra, B. frerreana Birdwood, B. serrata Roxb., B. papyrifera Hoscht, B. thurifera Roxb., B. rivae Engl. and B. neglecta S. Moore (Tisserand and Young, 2014). The frankincense is obtained through making incisions on the barks of these trees, a practice commonly known as ‘tapping’ (Farah, 1994). In Somaliland, two species, Boswellia sacra Flueck. and Boswellia frerreana Birdwood are used for frankincense production. Frankincense from Boswellia sacra is known locally as beeyo while that from B. frerreana Birdwood is known as meydi (Farah, 1994). Frankincense obtained from B. sacra is of high quality due to its high concentration of boswellic and earns a premium price (Farah, 1994). The olibanum is used in its unprocessed form for making perfumes, performing sacred religious rituals and making traditional medicines (Coppen, 1995). Processed frankincense, however, has found numerous uses throughout the world which include manufacture of cosmetics and perfumes (Crow, 2006). Recent advances in medical research have shown that boswellic acid, a component of frankincense oil has anti- inflammatory and anti-carcinogenic properties (Suhail et al., 2011).
The numerous uses of frankincense in pharmaceutical, perfumery and cosmetic industries have created a rise in demand for frankincense (Crow, 2006). This, in turn, has translated frankincense trade in Somaliland becoming a major economic activity earning the second position after livestock as the region’s leading exports (Muhumed, 2012.). Various activities from frankincense value chain, harvesting to processing, have created employment opportunities to a large number of communities in Somalia ( Farah, 1994). B. sacra trees grow wildly in vast regions of Northern Somalia, which are owned by clans and controlled by clan elders in terms of frankincense production and management (Farah, 1994). Harvesting is normally done by men while sorting out of the gum into the various grades for marketing based on size, purity and colour is done by women (Crow, 2006).
Frankincense production in Somaliland has faced several challenges since the fall of the government in 1991, where the control over plant exploitation and governing policies have been weakening. This has led to overexploitation of the trees, thereby increasing their mortality rate (UNEP, 2005). The frankincense trade is currently unregulated, leaving harvesters at the mercy of exporters and brokers (Crow, 2006). Lack of local processing facilities has locked out local communities from the benefits of value addition (ICPALD, 2011). The increasing demand amid low productivity levels calls for interventions that will seek to support protection of the existing trees while seeking to develop appropriate propagation mechanisms if sustainability of the resource is to be assured. This forms the basis for the present study, to seek to employ propagation technologies and characterisation of the available species to inform conservation efforts.
1.2 Statement of the problem
High demand for frankincense globally has led to over-exploitation of frankincense trees through over-tapping of already declining tree populations. The present practice has been creating too many incisions in one plant which makes the trees fail to regenerate and eventually die. Too many wounds have also exposed the trees to infections and pests, which affect their normal physiology. These factors have led to the decline in population of frankincense trees (Groenendijk et al., 2012). As a result, Boswellia sacra is now considered a threatened species in Somalia (IUCN, 2021).
Another key factor undermining the population of Boswellia forests is the low seed germination rate. This coupled with the harsh environmental conditions where the trees grow and feeding of young seedlings by animals reduces the likelihood of success in propagation from seed under both natural and modified environments (Raffaelli et al., 2003, Swartout and Solowey, 2018).
Information on genetic variability within B. sacra populations in Somaliland is also missing, which could provide information in support to the existing population conservation and propagation. Therefore, the morphological differences within these populations cannot be attributed accurately to environmental or genetic factors (Thulin and Warfa, 1987). Further, this information gap also greatly affects the choice of genotypes to be used as progenitors in conservation and domestication efforts as well as establishment of breeding programs for the species.
1.3 Justification
Most of the communities in Somaliland, over 70%, earn their livelihood along the frankincense value chains (Farah 1994), making the trees very crucial to the economy with an annual export value estimated at $7.3 million. Boswellia sacra is also considered sacred by the Somali people and is an important part of their culture, However, its overexploitation has threatened the tree population and therefore much effort is needed to address the propagation challenges if sustainable production is to be achieved. Efforts to propagate the tree through seed have led to frustrations from failures due to the poor germination rates of less than 10% (Swartout and Solowey, 2018). Mass propagation through cuttings has also been attempted but poor rooting and high mortality rates have been reported (Raffaelli et al., 2008). Therefore, there is a need to investigate the possibility of mass propagation of B. sacra through tissue culture technology.
It is also very crucial that the genetic variability within Boswellia sacra populations from different regions in Somaliland should be determined to support ongoing conservation and propagation efforts. Frankincense from B. sacra populations from different regions shows visual differences in colour and size (Svoboda et al. 2001). This variation has been attributed to environment, tree age, post-harvest storage or annual rainfall (Svoboda et al. 2001; Crow, 2006). Many morphological differences between B. sacra trees have been described (Thulin and Warfa, 1987) but the effect of genetic variation between the populations has not been investigated, hence the importance of this study. Previous studies in Oman used ISSR and ITS markers on B. sacra (Coppi et al., 2010), but this has not been done in Somaliland. There is no information available regarding use of SRAP markers on B. sacra population studies, in which this study seeks to explore.
Development of an efficient micropropagation protocol and the determination of the genetic diversity between and within these populations will greatly aid conservation efforts. This in turn safeguard the income of communities dependent on this important resource as well as earning the country revenue through foreign exchange.
1.4 Objectives
1.4.1 General Objective
To determine seed viability, genetic diversity and micropropagation of B. sacra populations to support conservation and mass propagation efforts in Somaliland
1.4.2 Specific objectives
1. To determine seed viability and explant micro-propagation using different plant growth regulators (PGRs) to enhance propagation in genetic improvement of B. sacra.
2. To assess genetic diversity using morphological and molecular markers of select B. sacra genotypes from Af Yare Dawl-dawl and Exdad regions of Somaliland to enhance breeding programs.
1.5 Hypothesis
1. There is no effect of plant growth regulators on direct organogenesis or somatic embryogenesis on B. sacra leaf and axillary bud explants.
2. There is no genetic diversity between and within populations of B. sacra from Exdad and Af Yare Dawl-dawl regions of Somaliland that can be revealed by morphological and molecular markers
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