A STUDY ON THE PRODUCTION AND ACCUMULATION OF BETALAINS IN CULTURED CELLS OF Beta vulgaris By Samia Jamal Kalkatawi A thesis presented in fulfilment of the requirements for the degree of Doctor of Philosophy University of Edinburgh 1997 DECLARATION I hereby declare that this thesis was composed by my self and the work described herein to be my own, except where indicated otherwise 11 ACKNOWLEDGEMENTS Praise be to Allah. Lord of the worlds, by his will the accomplishment of this study is made possible; and may his blessing and peace be upon His Prophet Mohammed. Throughout the period of this research work, many people rendered invaluable assistance to me in one form or another. I therefore most gratefully acknowledge their help and kindness. First and foremost I wish to express my deep and sincere appreciation to my former supervisor Professor M. M. Yeoman for his advice, guidance encouragement and invaluable criticism throughout the experimental work and the preparation of this thesis. I would also like to thank my current supervisors Dr. A. Hudson and Dr. S. Smith, and all members of the Botany Department, in particular those in Lab 205 for their help and friendship which has enabled me to enjoy the time I have spent in this department. Furthermore, I would also like to thank Dr. Marysia Miedzybrodzka for her help in parts of the experimental work. I wish to express my gratitude to King Abdul Aziz University, Jeddah, Saudi Arabia, for the financial support which made this study possible. Finally, a particular debt to my parents to whom this thesis is dedicated, my sister, my husband Dr. Khairy Abideen, my son Ammar and my daughter Dania for their patience and continuing encouragement. 1111 ABBREVIATIONS A absorbance BA benzyladenine B5 Gamborg'sB5 °C degree centigrade ca circa (approximately) cv. cultivar cm centimetre cont. control d. days 2,4-D 2,4-dichiorophenoxyacetic acid d.wt. dry weight DOPA dihydroxyphenylalanine eds. editors e.g. for example et al. etalia f.wt. fresh weight g gram h. hour(s) HC1 hydrochloric acid HPLC high performance liquid chromatography i.e. that is i-N inorganic nitrogen i-P inorganic phosphorus Kin kinetin 1 litre wavelength M molar m metre mg milligram max. maximum mm. minimum ml millilitre mm millimolar nun millimetre mol. mole NAA naphthalene acetic acid NaOH sodium hydroxide nm nanometre no. number O.D. optical density P probability PCV packed cell volume PPC percentage of pigmented cells iv r correlation coefficient R A 2 coefficient of determination RF distance moved by a compound relative to the solvent front rpm revolutions per minute Rt retention time S. second se. standard error of the mean suc. sucrose TLC thin layer chromatography Tyr. tyrosine micrometre p1 microlitre pmo1 micromole uv ultraviolet v/v volume per volume vis visible w/v weight per volume xg. gravitational force V CONTENTS Title i Declaration 11 Aknowledgment iii Abbreviations iv Contents vi Abstract X CHAPTER ONE: INTRODUCTION 1 CHAPTER TWO: MATERIALS AND METHODS 28 2.1 Cell and Tissue Culture 29 2. 1.1 Culture Material 29 2.1.2 Preparation of Culture Media 29 2.1.2.1 Standard growth medium 29 2.1.2.2 Media with different concentrations of nitrogen or phosphorus 30 2.1.2.3 Growth regulators stock solutions 30 2.1.3 Sterilisation and Aseptic Techniques 32 2.1.4 Maintenance of Cell Cultures 32 2.1.5 Single Cell Cloning and Selection of Cell Lines 32 2.2 Characterisation of Culture Growth and Cell Viability 35 2.2.1 Determination of Fresh Weight 35 2.2.2 Determination of Dry Weight 35 2.2.3 Determination of Packed Cell Volume (PVC) 37 2.2.4 Determination of Cell Population Density 37 2.2.5 Determination of Cell Viability 38 Vi 2.2.6 Determination of the Proportion of Pigmented Cells 38 2.2.7 Calculation of Relative Growth Rate 39 2.3 Extraction of Betalains from Cells of Suspension Cultures 39 2.4 Analysis of Betalains by Spectrophotometry 39 2.5 Analysis of Betalains by Thin-Layer Chromatography (TLC) 40 2.6 Analysis of Betalains and Related Compounds by High Performance Liquid Chromatography (HPLC) 41 2.6.1 High Performance Liquid Chromatography System 41 2.6.2 Preparation of the Mobile Phases 42 2.6.3 Preparation of Samples for HPLC Analysis 42 2.6.4 Operating Conditions for HPLC Analysis 43 2.6.5 Identification of Peaks 44 2.7 Preparation of Standard Solutions 44 2.7.1 Betacyanin Standards 44 2.7.2 Miraxanthin V Standard 44 2.7.3 Betalamic Acid Standard 49 2.7.4 Tyrosine and Dopamine Standards 51 2.8 Quantification of Compounds 51 2.9 Estimation of Tyrosine Hydroxylase Activity 53 2.9.1 Preparation of Crude Enzyme Extract 53 2.9.2 Determination of Soluble Protein Content in the Enzyme Extract 53 2.9.3 Assay of Tyrosine Hydroxylase Activity 55 2.10 Statistical Analysis 57 Vii CHAPTER THREE: RESULTS 3.1 Characterisation of the Growth and Betalain Accumulation in Suspension Cultures of Beta vulgaris 62 3.2 Effect of Culture Conditions on Growth and Betalain Accumulation in Suspension Cultures of Beta vulgaris 67 3.2.1 Effect of Manipulation of Major Nutrients on Growth and Betalain Production 70 3.2.1.1 Effect of the concentration of inorganic nitrogen in the medium on growth and betalain accumulation 70 3.2.1.2 Effect of the concentration of inorganic phosphorus in the medium on growth and betalain accumulation 76 3.2.1.3 Effect of sucrose concentration on growth and betalain accumulation 83 3.2.2 Effect of Growth Regulators Supplementation on Growth and Betalain Production 91 3.2.2.1 Effect of different concentrations of 2,4-D on growth and betalain production 91 3.2.2.1.1 Effect of 2,4-D at concentrations ranging from 0 to 0.2 mgI' 92 3.2.2.1.2 Effect of 2,4-D at concentrations ranging from 0 to 0.002 mg.F' on growth and betalain production over 3 passages 96 3.2.2.1.3 Effect of 2,4-D at concentrations ranging from 0.002 to 0.01 mg.r' 103 3.2.2.2 Effect of various concentrations of kinetin on growth and betalain production 110 3.2.3 Effect of Precursors Feeding on Growth and Betalain Production 114 3.2.3.1 Effect of supplementation of tyrosine on growth and betalain production 114 3.2.3.2 Effect of supplementation of DOPA on growth and betalain production 118 3.3 Investigation into Betalain Production in Relation to Cellular Heterogeneity 126 3.3.1 Single Cell Cloning and Characterisation of the Derived Cell Lines 127 VIII 3.3. 1.1 Establishment and characterisation of cell lines 127 3.3.1.2 Investigation of the stability of the selected cell lines 143 3.3.2 Effect of Culture Conditions on Betalain Production in the Violet Cell Line 150 3.3.2.1 Effect of 2,4-D on growth and betalain production in cell cultures of the violet cell line 151 3.3.2.2 Effect of sucrose on growth and betalain production in cell cultures of the violet cell line 155 3.3.2.3 Effect of light on growth and betalain production in cell cultures of the selected violet cell line 157 3.3.2.3.1 Effect of light and dark treatments on growth and betalain production 158 3.3.2.3.2 Effect of irradiation level on the growth and betalain content in cell cultures of the violet cell line 163 3.3.3 Effect of Culture Conditions on Betalain Production in the Yellow Cell Line 166 3.3.3.1 Effect of 2,4-D on growth and betalain production in cultures of the yellow cell line 166 3.3.3.2 Effect of added betalamic acid on growth and betalain production in cultures of the yellow cell line 171 3.4 Investigation into the Possible Role of Tyrosine Hydroxylase Activity on Betalain Production 179 3.4.1 The Activity of Tyrosine Hydroxylase in Cell Cultures of the White and Violet Cell Lines 180 3.4.2 The Activity of Tyrosine Hydroxylase in Cell Cultures of the Violet Cell Line Grown at Two Different Concentrations of Sucrose 182 3.4.3 The Activity of Tyrosine Hydroxylase in Cell Cultures of the Violet Cell Line Grown either in the Light or in the Dark 184 CHAPTER FOUR: DISCUSSION 187 CHAPTER FIVE: REFERENCES 218 ix ABSTRACT The aim of this project was to study the accumulation of betalains in suspension cultures of Beta vulgaris. Betalains were found to accumulate in suspension cultures of Beta vulgaris in association with growth. The onset of pigment synthesis occurred prior to, or immediately after, the start of cell division. The production of betalains could be influenced by the conditions of culture differently from growth. Varying the concentration of inorganic nitrogen (i-N) or inorganic phosphorus (i-P); above or below that in B5 medium, did not increase either growth or pigment production. 3% sucrose was shown to be optimum for betalain accumulation while the fresh weight of culture was not affected, comparing to the control (2% sucrose). Lowering the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) favoured betalain production. The highest pigment content per culture was achieved at 0.006 mg.i' 2,4-D at which concentration the culture growth was not significantly affected, as compared to the control 0.02 mg.1-1 2,4-D. Altering the concentration of kinetin in the medium in the range of 0.05-1 mg.r' did not increase the production of betalains.