ADDIS ABABA UNIVERSITY SCHOOL OF GRADUATE STUDIES DEPARTEMENT OF BIOLOGY CHROMOSOME STUDY ON SEVEN ENDEMIC ALOE SPECIES (ALOACEAE) OF ETHIOPIA A Thesis Submitted to the School of Graduate Studies of Addis Ababa University in Partial Fulfillment of the Requirements for the Degree of Master of Science in Biology (Applied Genetics) BY ALEMAYEHU MEKURIA November 2007 Addis Ababa Declaration I, the undersigned, declare that this thesis is my original work. It has never been presented for a degree in any other institution and that all sources of material used in it have been duly acknowledged. Name: ______________________________ Signature: ____________________________ Date: _______________________________ This thesis has been submitted for examination with our approval as university advisors. _________________ Kifle Dagne (Dr) ___________________ Sebsebe Demissew (prof.) Table of Contents Page Acknowledgement………………………………………………………………………...i List of Tables……………………………………………………………………………..v List of Figures…………………………………………………………………………....vi List of Appendices……………………………………………………………………. viii Abstract………………………………………………………………………………….ix 1. Introduction……………………………………………………………………………1 1.1. Taxonomic position of Aloe…………………………………………………..1 1.2. Origin and Geographic Distribution of Aloe………………………………….1 1.2.1. Geographical Distribution and Classification of Endemic Aloe Species in Ethiopia ..........................................................................4 1.2.2.1. Aloe kefaenisis M.G.Gilbert & Sebsebe Demissew ……...5 1.2.2.2. Aloe harlana Reynolds…………………………………...5 1.2.2.3. Aloe trigonantha Leach…………………………………...5 1.2.2.4. Aloe yavellana Reynolds………………………………….6 1.2.2.5. Aloe debrana Christian…………………………………...6 1.2.2.6. Aloe schelpei Reynolds…………………………………...7 1.2.2.7. Aloe species from Bale……………………………………7 1.3. Botanical Description of the Genus Aloe …………………………………….7 1.3.1. Reproduction Mechanism…………………………………………..8 ii 1.3.2. Ecology of Aloe species…………………………………………….9 1.4. Economic and Medicinal Importance of Aloe Species……………………...10 1.4.1. Pharmacology, therapeutic applications and other uses of Aloe species……………………………………………………………10 1.4.2. Traditional Medicine………………………………………………12 1.5. Conservation………………………………………………………………...13 1.6. Molecular study of Aloe species in Ethiopia………………………………...13 1.7. Cytogenetic study……………………………………………………………14 1.7.1. Chromosome number ……………………………………………..15 1.7.1.1. Polyploidy……………………………………………….15 1.7.1.2. Genome size……………………………………………..16 1.7.2. Chromosome morphology………………………………………...16 1.7.2.1.Centromere ………………………………………………17 1.7.2.2. Secondary constriction and satellites……………………18 1.7.3. Karyotype …………………………………………………………18 1.7.4. Chromosome Cytology of the Genus Aloe………………………..20 2. Objectives of the study……………………………………………………………….24 2.1. General objectives………………………………………………………….24 2.1.1. Specific objectives………………………………………………...24 3. Materials and Methods………………………………………………………………25 3.1. Plant Materials………………………………………………………………25 3.2. Somatic chromosome analysis………………………………………………27 3.2.1. Pretreatment ………………………………………………………27 iii 3.2.2. Slide preparation…………………………………………………..28 3.2.2.1. Air dry Technique……………………………………….28 3.2.2.2. Squash Technique ………………………………………28 3.2.3. Karyotype Analysis ……………………………………………….29 4. Results ………………………………………………………………………………..31 4.1. Aloe species from Bale………………………………………………………32 4.2. A. yavellana…………………………………………………………………33 4.3. A. trigonantha……………………………………………………………….34 4.4. A. schelpei…………………………………………………………………...35 4.5. A. harlana…………………………………………………………………...36 4.6. A. debrana…………………………………………………………………..37 4.7. A. kefaenisis…………………………………………………………………38 5. Discussion…………………………………………………………………………….41 6. Conclusions and recommendations…………………………………………………45 6.1. Conclusions…………………………………………………………………45 6.2. Recommendations…………………………………………………………..45 7. References……………………………………………………………….47 iv List of Tables Page Table 1. Total number and number of endemic species of Aloe and % of endemism by country in Africa…………………………………………………..3 Table 2. Endemic or near endemic Aloe species in Ethiopia and their geographic distribution……………………………………………………………4 Table 3. List of Aloe species studied, sites of collection and their collection number…..27 Table 4. Summary of chromosome number counts of the studied species ……………..32 Table 5. Chromosome size of the studied species……………………………………….40 v List of Figures Page Fig.1.Pictures of Aloe species used in the present study: A. Aloe yavellana; B. A. kefaenisis; C. A. trigonantha; D. A. harlana; E. A. schelpei; F. A. debrana; G. Aloe species from Bale……………………………………...26 Figure 2- Chromosomes of an Aloe species from Bale. A. somatic metaphase chromosomes (2n = 14); B. The karyogram prepared from chromosomes shown in A. Bar = 10µm………………………………………………………..33 Figure 3- Chromosomes of Aloe yavellana. A. metaphase plate with 14 chromosomes; B. partial complement with one chromosome showing a satellite at the tip of its long arm (arrow); C. the karyogram constructed from A. Bar = 10µm…………………………………………………………………..34 Figure 4 - Chromosomes of A. trigonantha. A metaphase plate with 14 chromosomes; B. the karyogram constructed from A. Bar = 10µm……………………………35 Figure 5- Somatic chromosomes of A. schelpei; A. metaphase plate (2n = 14+1 additional chromosome arrow); B. the karyogram constructed from A. Bar = 10µm……………………………………………………………..36 Fig 6- Somatic chromosomes of A. harlana, A. Metaphase plate with 14 chromosomes; B. Prophase cell showing 4 satellites (arrows); C. Karyogram prepared from chromosomes shown in A. Bar = 10µm………...37 Fig- 7. Somatic chromosomes of A. debrana; A. Metaphase plate (2n=14) B. Part of prophase chromosomes with 3 satellites (arrows); C. Karyogram prepared from chromosomes shown in A. Bar = 10µm………...38 vi Figure 8- Somatic chromosome of A. kefaenisis; A. Metaphase plate (2n = 14) B. 2 satellites arrowed, C. Karyogram prepared from chromosomes Shown in A. Bar = 10µm ……………………....................................................39 vii List of Appendices Page Appendix 1. Chromosome measurements, arm ratios and position of centromere in Aloe species from Bale……………………………………………….51 Appendix 2. Chromosome measurments, arm ratios and position of Centromere in Aloe yavellana……………………………………………………….52 Appendix 3. Chromosome measurements, arm ratios and position of Centromere in Aloe trigonantha…………………………………………………….53 Appendix 4. Chromosome measurements, arm ratios and position of Centromere in A. schelpei…………………………………………………………..54 Appendix 5. Chromosome measurements, arm ratios and position of centromere in A. harlana…………………………………………………………….55 Appendix 6. Chromosome measurements, arm ratios and position of Centromere in A. debrana…………………………………………………………...56 Appendix 7. Chromosome measurements, arm ratios and position of Centromer in A. kefaenisis…………………………………………………………..57 viii Abstract Seven endemic Aloe species, five of which were collected from their respective geographical locations in Ethiopia and two species obtained from the Aloe Garden at the Sciense Faculty, Addis Ababa University were studied. They include Aloe kefaenisis, Aloe harlana, Aloe trigonantha, Aloe yavellana, Aloe debrana, Aloe schelpei, an Aloe (not yet described) species from Bale. Chromosome preparations were made from root tip cells after pretreatment with colchicines or 8- hydroxyquinoline or cold treatment in ice water. Maceration was done in 4 % cellulase + 4 % pectinase solution at 37 0C for about 1 hr which was followed by air dry slide preparation. Chromosomes were stained with Giemsa in Sorensen’s phosphate buffer (PH= 6.8). Root tips were hydrolyzed with 1N HCl for about 10 minutes at 60 0C in a water bath, the chromosomes were stained either in toluidine blue or aceto–orcein which was followed by squash preparation. The result showed that all species have 2n=14, and one plant of Aloe schelpei have 2n = 14 + 1, the additional chromosome being a small metacentric element. The number of satellites observed in different species was varied from one to four. All species showed bimodal karyotype which consists of four pairs of large chromosome (18.26 – 13.82 µm long) and three pairs of small chromosomes (4.11 – 6.00 µm long). The classification of the chromosomes according to centromere position, showed subterminal (st) for the large chromosomes; submedian (sm) and median (m) for the small chromosomes. In the present study no significant chromosomal differentiation in size and morphology between the Aloe species investigated were observed. As a result it is not possible to infer species relationships on the basis of chromosome cytology. It was recommended that future cytological studies in Aloe should include meiotic analysis in interspecific hybrids and molecular cytogenetic techniques such as FISH and GISH. Keywords: Aloe, Endemic species, Chromosome number, Karyotype. ix 1. Introduction 1.1.Taxonomic Position of Aloe The genus Aloe and related genera were placed in Class Hexandria, Order Monogynia, due to the 6 stamens and a single pistil (Linnaeus, 1753). Subsequently, they have been treated as one of the 28 tribes, namely Aloeae in the family Liliaceae. Later they were included in the alooid genera, along with Kniphofia Moench, in the family Aloaceae. Then, the alooid genera were classified into the sub-family Alooidaeae in the family Asphodelaceae. The currently widely accepted position of the alooid plants is in a separate family the Aloaceae, with seven genera: Aloe L., Gasteria Duval, Haworthia Duval, Lomatophyllum Willd., Chortolirion A. Berger, Poellnitzia Uitew., and Astroloba Uitew