Morphological, Histological and Molecular Identification of Some Cucurbits in Khartoum State, Sudan. By: Ismail Ahmed Mohammed Ahmed B.Sc. (Honours) in Agriculture (Agricultural Biotechnology) August 2002 A Thesis Submitted to the University of Khartoum in fulfillment for the degree of Master of Science (Agric). Supervisor: Dr. Abdel Gabbar Nasir Gumaa Department of Botany and Agricultural Biotechnology Faculty of Agriculture University of Khartoum June 2007 ﻗﺎل ﺗﻌﺎﻟﻰ: (ﻭﻫﻮ ﺍﻟﱠﺬِﻱ ﺃﹶﻧﺰﻝﹶ ﻣِﻦ ﺍﻟﺴﻤﺎﺀِ ﻣﺎﺀً ﻓﹶﺄﹶﺧﺮﺟﻨﺎ ﺑِـﻪِ ﻧﺒـﺎﺕ ﻛﹸﻞﱢ ﺷﻲﺀٍ ﻓﹶﺄﹶﺧﺮﺟﻨﺎ ﻣِ ﻨ ﻪ ﺧﻀِﺮﺍﹰ ﻧﺨﺮِ ﺝ ﻣِ ﻨ ﻪ ﺣﺒﺎﹰ ﻣﺘﺮﺍﻛِﺒـﺎﹰ ﻭﻣِﻦ ﺍﻟﻨﺨﻞِ ﻣِﻦ ﻃﹶﻠﹾﻌِﻬﺎ ﻗِﻨﻮﺍﻥﹲ ﺩﺍﻧِﻴﺔﹲ ﻭﺟﻨﺎﺕٍ ﻣ ﻦ ﺃﹶﻋﻨﺎﺏٍ ﻭﺍﻟﺰ ﻳ ﺘﻮﻥﹶ ﻭﺍﻟﺮﻣﺎﻥﹶ ﻣﺸﺘﺒِﻬﺎﹰ ﻭﻏﹶﻴﺮ ﻣﺘﺸﺎﺑِﻪٍ ﺍ ﻧ ﻈﹸ ﺮ ﻭ ﺍﹾ ﺇِﻟِـﻰ ﺛﹶﻤﺮِﻩِ ﺇِﺫﹶﺍ ﺃﹶﺛﹾﻤﺮ ﻭﻳﻨﻌِﻪِ ﺇِﻥﱠ ﻓِـﻲ ﺫﹶﻟِ ﻜﹸ ـ ﻢ ﻵﻳـﺎﺕٍ ﻟﱢﻘﹶـﻮﻡٍ ﻳﺆﻣِﻨﻮﻥ) (اﻷﻧﻌﺎم: 99) i TABLE OF CONTENTS Item Page No Dedication ................................................................................. i List of Tables .......................................................................... ii List of Figures ......................................................................... iii Acknowledgement .................................................................. iv Abstract (English) .....................................................................v Abstract (Arabic) .................................................................... vi CHAPTER ONE: INTRODUCTION ...................................... 1 CHAPTER TWO: STUDY AREA ............................................4 1. Location .................................................................................................. 4 2. Climate ................................................................................................... 4 2.1 Temperature ........................................................................................ 4 2.2 Rainfall .................................................................................................. 4 2.3 Winds ..................................................................................................... 7 2.4 Humidity .............................................................................................. 7 3. Geology .................................................................................................. 7 4. Geomorphology .................................................................................... 8 5. Soil .......................................................................................................... 8 6. People .................................................................................................. 10 7. Water resources .................................................................................. 11 CHAPTER THREE: MATERIALS AND METHODS..........12 1. Morphological methods ....................................................................... 12 2. Histological methods ............................................................................ 12 2.1 Preparation of plant material ........................................................... 12 2.2 Fixation ................................................................................................ 13 2.3 Washing ............................................................................................... 13 ii 2.4 Dehydration ........................................................................................ 13 2.5 Clearing ............................................................................................... 13 2.6 Embedding in paraffin ....................................................................... 14 2.7 Blocking ............................................................................................... 14 2.8 Trimming ............................................................................................ 15 2.9 Blocking ............................................................................................... 15 2.10 Sectioning ......................................................................................... 15 2.11 Staining ............................................................................................. 15 2.12 Mounting .......................................................................................... 16 3. Molecular methods ............................................................................. 16 3.1 Preparation of solutions ................................................................... 16 3.1.1 CTAB (3x) ...................................................................................... 16 3.1.2 Washing buffer .............................................................................. 16 3.1.3 TE buffer ........................................................................................ 16 3.1.4 Chloroform isoamyl alcohol .......................................................... 16 3.1.5 TAE buffer ..................................................................................... 16 3.1.6 Liquid nitrogen .............................................................................. 17 3.1.7 Loading buffer ................................................................................ 17 3.1.8 Preparation of agarose gel............................................................. 17 3.2 Preparation of plant tissue .............................................................. 17 3.3 DNA extraction by CTAB (3x) method ............................................ 17 3.4 Quantification of DNA ...................................................................... 18 3.5 RAPD analysis .................................................................................... 18 3.6 Data analysis ...................................................................................... 19 CHAPTER FOUR: LITERATURE REVIEW ...................... 20 1. Morphological study ............................................................................ 20 2. Histological study .................................................................................. 24 3. Molecular study .................................................................................... 28 3.1 DNA markers ...................................................................................... 28 3.2 Uses of DNA markers ........................................................................ 29 iii 3.3 Random Amplified Polymorphic DNA (RAPD) marker ............... 29 3.4 Polymerase chain reaction (PCR) ..................................................... 30 3.5 Molecular analysis of Cucurbits ...................................................... 30 CHAPTER FIVE: RESULTS AND DISCUSSION .............. 34 1. Genera and species descriptions ........................................................ 34 2. Histological analysis ............................................................................. 66 3. Molecular analysis ............................................................................... 72 CONCLUSIONS AND RECOMMENDATIONS .................... 90 REFERENCES ......................................................................... 92 iv Dedication This work is dedicated to the soul of my father, may Allah be pleased with him, my mother, Second father Mohammed, all Brothers and Sisters with love. v LIST OF TABLES Table No. Page No. 1. Composition of clearing solution (I & II) .......................................... 14 2. DNA concentration and purity of plant accessions ........................... 80 3. Number of total and polymorphic fragments using RAPD marker ................................................................................................... 81 vi LIST OF FIGURES Figure No. Page No. 1. Location of the study area...................................................................... 5 2. Vegetation of the study area .................................................................. 6 3. Adenopus breviflorus Benth. ................................................................ 37 4. Citrullus lanatus (Thunb.) Mansf. ...................................................... 38 5. Coccinia grandis L. ............................................................................... 40 6. Colocynthis vulgaris Schrad. ............................................................... 42 7. Ctenolepis cerasiformis (Stcks.) Hook. ............................................... 44 8. Cucumis dipsaceus Spach. .................................................................... 46 9. Cucumis melo var flexuosus Naud. ..................................................... 47 10. Cucumis melo var reticullatus L. ....................................................... 49 11. Cucumis metuliferus E. ...................................................................... 50 12. Cucumis prophetarum L. .................................................................... 51 13. Cucumis sativus L. .............................................................................. 53 14. Cucurbita maxima Duch. .................................................................... 55 15. Cucurbita moschata Poir. ................................................................... 57 16. Cucurbita pepo L. ................................................................................ 58 17. Lagenaria siceraria Molina. ............................................................... 60 18. Luffa aegyptiaca Mill. ......................................................................... 62 vii 19. Momordica balsamina L. ...................................................................