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Transcript of STUDIES ON DATE PALM IN TISSUE CULTURE · STUDIES ON DATE PALM IN TISSUE CULTURE By GEHAN MOHAMED...
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, GA3, 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, GA3, 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.
I
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 .............. 46
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 ...... 48
3. Germination stage ............................................................... 48
II
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
III
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 GA3 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
IV
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 .......................................................
V
LIST OF TABLES
Page Title No
56
Mixture of enzyme solutions used in protoplast isolation
from embryogenic calli of date palm cultivars Sakkoty and
Khalas ....................................................................................
1.
56 Composition of incubation mixture (CPW) ........................... 2.
57 Modified Kao and Michayluk medium (1975) ...................... 3.
59
Effect of 2,4-D concentrations and mesh filter diameter of
callus suspensions on globularization degree of somatic
4.
61
Effect of 2,4-D concentrations and mesh filter diameter
of callus suspension on number of somatic embryos
5.
64
Effect of vitamins and culture media on globularization
degree of callus ......................................................................
6.
67
Effect of vitamins and culture media on number of somatic
embryos formed from embryogenic callus cultured in three
media ......................................................................................
7.
73
Effect of different concentrations of amino acid on growth
and development of date palm callus suspension ..................
8.
80
Effect of 2,4-D and TDZ concentrations on growth and
development of date palm callus suspension .........................
9.
83
Effect of abscisic acid and temperatures on growth and
development of date palm callus suspension .........................
10.
89
Effect of abscisic acid and temperature on number of
secondary somatic embryos, germinated embryos, number
of roots and vitrification ........................................................
11.
98
Effect of gibberellic acid and temperature on growth and
development of date palm callus suspension .........................
12.
105
Effect of gibberellic acid and temperature on number of
secondary somatic embryos, germinated embryos, number
of roots and vitrification ........................................................
13.