STUDIES ON DATE PALM IN TISSUE CULTURE

STUDIES ON DATE PALM IN TISSUE CULTURE By GEHAN MOHAMED YOUSRY SALAMA B.Sc. Agric. Sci. (Horticulture), Fac. Agric., Ain Shams Univ., 1990 M.Sc. Agric. Sci. (Ornamental Horticulture), Fac. Agric., Cairo Univ., 2004 THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY In Agricultural Sciences (Ornamental Horticulture) Department of Ornamental Horticulture Faculty of Agriculture Cairo University EGYPT 2012

APPROVAL SHEET STUDIES ON DATE PALM IN TISSUE CULTURE Ph.D. Thesis In Agric. Sci. (Ornamental Horticulture) By GEHAN MOHAMED YOUSRY SALAMA B.Sc. Agric. Sci. (Horticulture), Fac. Agric., Ain Shams Univ., 1990 M.Sc. Agric. Sci. (Ornamental Horticulture), Fac. Agric., Cairo Univ., 2004 APPROVAL COMMITTEE Dr. HUSSEIN ABD EL HAQ BOSILA... Professor of Floriculture, Medicinal and Aromatic Plants, Fac. Agric., Al- Azhar University, Cairo. Dr. MOHAMED ABDEL-KHALIK AL KHATEEB... Professor of Ornamental Horticulture, Fac. Agric., Cairo University Dr. AFAF MAHMOUD HABIB... Associate Professor of Ornamental Horticulture, Fac. Agric., Cairo University Dr. EL- SAADY MOHAMED BADAWY... Professor of Ornamental Horticulture, Fac. Agric., Cairo University Date: 20 / 11 / 2012

SUPERVISION SHEET STUDIES ON DATE PALM IN TISSUE CULTURE Ph.D. Thesis In Agric. Sci. (Ornamental Horticulture) By GEHAN MOHAMED YOUSRY SALAMA B.Sc. Agric. Sci. (Horticulture), Fac. Agric., Ain Shams Univ., 1990 M.Sc. Agric. Sci. (Ornamental Horticulture), Fac. Agric., Cairo Univ., 2004 SUPERVISION COMMITTEE Dr. EL- SAADY MOHAMED BADAWY Professor of Ornamental Horticulture, Fac. Agric., Cairo University Dr. AFAF MAHMOUD HABIB Associate Professor of Ornamental Horticulture, Fac. Agric., Cairo University Dr. ABDEL-MONEM EL-BANA Professor of Date palm, Director of Agric. Research Center, Giza

Name of Candidate: Gehan Mohamed Yousry Salama Degree: Ph.D. Title of Thesis: Studies on Date Palm in Tissue Culture Supervisors: Dr. EL- Saady Mohamed Badawy Dr. Afaf Mahmoud Habib Dr. Abdel- Monem El- Bana Department: Ornamental Horticulture Approval: 20 / 11 / 2012 ABSTRACT This study had been carried out at the Ornamental Horticulture, Department, Faculty of Agriculture, Cairo University, during the period from 2007 to 2012. The experiments had been conducted in the Centre Laboratory for Date Palm Research, Plant Biotechnology Department, Agriculture Research Center, Ministry of Agriculture, Giza, Egypt. The aim of this study was to investigate a suitable protocol to improve the production and development of somatic embryos of date palm cv. Sakkoty, through suspension culture technique and to indentify appropriate conditions for protoplast isolation and fusion between Sakkoty and Khalas cv. Studying the factors affecting growth and development of friable calli induced from shoot tip explants including mesh filter size, 2,4-D, vitamins, amino acids and thidiazurone (TDZ) at different concentrations, in separate experiments. The obtained results revealed that globularization degree and somatic embryo number were enhanced using filter mesh size at 500 ϻ M especially in combination with liquid medium enriched with 0.1 mg/l 2,4-D. Thiamin HCl at 2.5 mg/l was superior in enhancing somatic embryo formation than other tested vitamins when added to MS liquid medium + 0.1 mg/l 2,4-D + 0.1 mg/l NAA. Glutamine had stimulatory effects on cell suspension culture of date palm compared with asparagine and argenine. The addition of TDZ to suspension culture medium at 1.0 mg/l promoted somatic embryo production. Biochemical components varied according to different developmental stages. Globular stage had a highest significant content of total soluble sugars, while cotyledonary stage contained high phenols content. No significant differences in free amino acids and indoles were observed through different stages. Enzyme concentrations and incubation time affected positively on protoplasts yield. The best yield was obtained from addition of cellulose 1.5 %, hemicellulase 0.5 % and Macerozyme at 0.5 % and incubation time for 12 hours. The effects of ABA, GA 3, sucrose different concentrations, temperature degree during maturation and germination stages and histological studies during callus growth and development were investigated. Key words: Date palm, somatic embryogenesis, callus, 2,4-D, biotin, TDZ, Temperatures, GA 3, ABA, cell suspension and protoplast.

ACKNOWLEDGEMENT Thanks to ALLAH I would like to express my sincere gratitude and admiration to my teacher Prof. Dr. EL- SAADY MOHAMED BADAWY Professor of Ornamental Horticulture, Faculty of Agriculture, Cairo University for suggesting the problem; his supervision teaching, support and patience. I am particularly grateful to him for his valuable instructions during carrying out of the experiments and correction of the manuscript. I have been greatly assisted by Dr. AFAF MAHMOUD HABIB Associate Professor of Ornamental Horticulture, Faculty of Agriculture, Cairo University, is thanks for her supervision, support and advice during my studies. Prof. Dr. Abdel-Monem El-Bana Head of Research of Date palm Lab., ARC, Giza is acknowledged for his supervision and providing excellent library facilities at the date palm Research Lab. Sincere thanks are extended to Dr. Ahmed Gabr (National Research Center-Cairo, Egypt) for his advice and careful help in protoplast fusion work, I would like to express my deepest thanks and sincere appreciation to Prof. Dr. Mona Hassan,Dr. Amal Fathy, Dr. Rehab Sidky and Dr. Eman Mostafa Researcher in the Date Palm Tissue Culture Lab for their advice helping in the laboratory work. Also great thanks to Dr. Mohamed Abd El-Rasool for his helping in the histological work. Thanks too, to all staff members of Ornamental Horticulture, Department, Faculty of Agriculture, Cairo University. Thanks go out to all members of the Date Palm Lab. for their friendly advice, support and unlimited help during these years. Sincere thanks and gratitude to my parent s soul, husband and sister for encouragement and providing all the facilities throughout this work.

CONTENTS Page INTRODUCTION... 1 REVIEW OF LITERATURE... 4 1. Establishment of callus suspension cultures... 7 a. Plant materials... 7 b. Sterilization... 8 c. Callus induction... 9 2. Auxin and cytokinin... 9 3. Somatic embryos maturation... 12 4.High frequency and synchronous somatic embryogenesis... 13 a. Cell suspensions... 13 b. Effect of vitamins... 16 c. Effect of amino acids... 19 d. Effect of thidiazurone... 21 e. Effect of abscissic acid... 24 f. Effect of gibberellic acid... 29 g. Effect of sucrose... 31 h. Effect of temperature degrees... 34 5. Biochemical and structural analyses... 36 6. Protoplast induction, culture and fusion... 40 MATERIALS AND METHODS... 44 1. Establishment of calli cultures... 44 2. Maturation stage... 45 a. Effect of different mesh filter diameters (callus sizes) and 2,4-D concentrations... 46 b. Effect of vitamin concentrations and culture media... c. Effect of different concentrations of amino acid... 47 d. Effect of different concentrations of 2,4-D and TDZ on date palm suspensions cultures... 47 e. Effect of incubation under different temperature degrees... 3. Germination stage... 48 I 46 48

4. Rooting and Pre-acclimatization... 48 5.Extraction and determination of carbohydrate components and free amino acid... 49 a. Extraction... 49 b. Determination of total soluble sugars... 49 c. Determination of reducing sugars... 50 d. Determination of free amino acids... 50 6. Extraction and determination of total phenols and indoles... 51 a. Determination of total phenols... 51 b. Determination of total indoles... 51 7. Histological studies... 52 8. protoplast isolation... 52 a. Plant materials... 53 b. Protoplast isolation... 53 c. Purification of protoplast... 53 d. Protoplast culture... 54 e. Protoplast fusion and culture... 54 RESULTS AND DISCUSSION... 58 2. Maturation stage... 58 a. Effect of 2,4-D concentrations and mesh filter diameter of callus suspensions cultures on 1.Globularization degree... 58 2. Number of somatic embryos... 59 b. Effect of vitamins and culture media on 1.Globulrization degree... 63 2.Number of somatic embryos formed from embryogenic callus cultured in three media... 65 c. Effect of different concentrations of amino acids on growth and development of date palm callus suspension culture... 69 Experiment 4 2.6. Effect of 2,4-D and TDZ concentrations on growth and development of date palm callus suspension culture... 75 a. growth value... 75 b. Globularization degree... 75 II

c. Number of somatic embryos... 76 d. Vitrification... 77 Experiment 5 2.7.Effect of ABA and temperatures on growth and development of date palm during maturation and germination stage... 81 2.7.1. Maturation stage... 81 a. Growth value... 81 b. Globularization degree... 82 c. Number of somatic embryos... 84 d. Vitrification... 84 2.7.2. Germination stage... 86 a. Number of secondary somatic embryos... 87 b. Number of germinated embryos... 88 c. Number of roots/embryo... 90 d. Vitrification degree... 91 2.8. Effect of GA 3 and temperatures on growth and development of date palm during maturation and germination stage... 96 2.8.1. Maturation stage... 96 a. Growth value... 96 b. Globularization degree... 97 c. Number of somatic embryos... 99 d. Vitrification... 99 2.8.2. Germination stage... 100 a. Number of secondary somatic embryos... 100 b. Germinated embryos... 101 c. Number of roots/ embryo... 102 d. Vitrification... 103 2.9. Effect of sucrose and temperatures on growth and development of date palm during maturation and germination stage... 107 2.9.1. Maturation stage... 107 a. Growth value... 107 b. Globularization degree... 108 III

c. Number of somatic embryos... 108 d. Vitrification... 110 2.9.2. Germination stage... 112 a. Number of secondary somatic embryos... 112 b. Germinated embryos... 114 c. Number of roots/embryo... 114 d. Vitrification... 115 2.10. Rooting and Pre-acclimatization... 117 3. Biochemical components... 120 3.1. carbohydrate components... 120 3.2. Free amino acids, indoles and phenols components... 122 a. Free amino acids... 123 b. Indoles... 124 c. Phenols... 125 4. Histological studies... 126 5. Protoplasts isolation, culture and fusion of two date palm cultivars cv. Sakkoty and Khalas... 130 5.1. Protoplasts isolation... 130 5.2. Protoplasts culture... 134 5.3. Protoplasts fusion... 135 SUMMARY... 137 REFERENCES... 149 ARABIC SUMMARY... IV

No 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. LIST OF TABLES Title Mixture of enzyme solutions used in protoplast isolation from embryogenic calli of date palm cultivars Sakkoty and Khalas... Composition of incubation mixture (CPW)... Modified Kao and Michayluk medium (1975)... Effect of 2,4-D concentrations and mesh filter diameter of callus suspensions on globularization degree of somatic Effect of 2,4-D concentrations and mesh filter diameter of callus suspension on number of somatic embryos Effect of vitamins and culture media on globularization degree of callus... Effect of vitamins and culture media on number of somatic embryos formed from embryogenic callus cultured in three media... Effect of different concentrations of amino acid on growth and development of date palm callus suspension... Effect of 2,4-D and TDZ concentrations on growth and development of date palm callus suspension... Effect of abscisic acid and temperatures on growth and development of date palm callus suspension... Effect of abscisic acid and temperature on number of secondary somatic embryos, germinated embryos, number of roots and vitrification... Effect of gibberellic acid and temperature on growth and development of date palm callus suspension... Effect of gibberellic acid and temperature on number of secondary somatic embryos, germinated embryos, number of roots and vitrification... Page 56 56 57 59 61 64 67 73 80 83 89 98 105 V