A STUDY on the PRODUCTION and ACCUMULATION of BETALAINS in CULTURED CELLS of Beta Vulgaris

A STUDY ON THE PRODUCTION AND ACCUMULATION OF BETALAINS IN CULTURED CELLS OF Beta vulgaris

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. Administration of either tyrosine or dihydroxyphenylalanine (DOPA) did not significantly affect either growth or pigment production.

A single cell plating technique was used successfully to isolate a number of cell lines (violet, orange, yellow, and white) with different qualitative and quantitative characteristics with regard to betacyanin and betaxanthin content. HPLC analysis of the betalains produced by the selected cell lines and the mother culture, revealed that betanin and miraxanthin V were the main betalains produced by the mother culture and the derived pigmented cell lines. These cell lines underwent changes in pigment production during a period of non-selective subculturing (15 months). The production of betalains in these cell lines was investigated in relation to cellular heterogeneity and the study showed that the cellular population of the cell lines contained a limited proportion of cells that accumulated the pigment. The proportion of pigmented cells had little effect on determining the yield of pigment in cultures while the capacity of these cells to accumulate the pigment played the most important role in governing the productivity of the culture. There was a maximum limit for the proportion of the "productive" cells which was a property of the cell line and was heritable. Below this limit the proportion of the productive cells could be modified by the manipulation of culture conditions.

The relationship between the activity of tyrosine hydroxylase and the final yield of betalain in cultures was explored and the study suggested that the enzyme could play a regulatory role in betalain biosythesis.

x CHAPTER ONE

INTRODUCTION 1.1 The Betalains

Betalains are a group of naturally occurring water soluble nitrogenous pigments which are a products of secondary metabolism. They are often classified as alkaloids but they are not really alkaloids because of the presence of several carboxyl groups which render the molecules acidic in nature. Betalains include two groups of pigments, the red- violet betacyanins and the yellow-orange betaxanthins. Both pigments are characterised by one moiety which is derived from betalamic acid (see Fig. 1.1) and differ from each other by the part bound to the betalamic acid residue. The attachment of cyclo-DOPA, to the moiety and its o-glycosidation or acylation results in the formation of a large variety of betacyanins, while condensation of betalamic acid with an amine or amino acid

(protein or non-protein amino acid) gives rise to different betaxanthins. The isolation of a number of betacyanins and betaxanthins from plants and elucidation of their chemical structure was reviewed by Steglich and Strack in 1990.

Figure 1.1 The general structure of betalains (a) betalamic acid moiety (b) R'-N-R 2 , residue of cyclo-DOPA in betacyanins (c) R 1-N-R2, residue of amino acid or amine in betaxanthins.

R1 ® R2 b or c 1.1.1 Occurrence in Nature

The occurrence of betalains is restricted to the plant order Caryophyllales

(Centrospermae). They do not occur in animals or micro-organisms, except in one mushroom, the Fly Agaric (Amanita muscaria) (see Dopp et al., 1982 and Mabry

1980). Betalains accumulate in the vacuoles of cells and can be found in almost every

structure in the plant, including the subterranean organs. Both betacyanins and betaxanthins are normally found together in various proportions. However, betacyanins are usually dominant (see Mabry 1980, Piattelli 1981, Böhm and Rink

1988).

1.1.2 Functions of the Betalains

Generally, the presence of bright coloured pigments may function as a signal

or warning to other organisms (Luckner 1990). However, betalains in flowers and

fruits may act as an attractant for vectors that serve in pollination and seed dispersal.

Betalains may also play a role in a self-defence mechanism, as formation of the

pigment was found to be induced in injured tissues (Obrenovic 1990), and betalains

have been shown to inhibit viral reproduction (Sosnova 1970). In addition, betanin-

containing beet seedlings were found to have a higher resistance to "damping off' and

when the fungus responsible for this disease was grown in liquid culture containing 50

mgf' of betanin, growth was reduced by 50% (see Mabry 1980). Betalains have also

been shown to inhibit IAA oxidase and may counteract the inhibitory effects of IAA

3 on the elongation of wheat roots, however, there was no indication that betalains play a role in regulating the activity of IAA (Stenhid 1976).

1.1.3 The Biosynthesis of Betalains

A scheme for the biosynthetic pathway leading to the betalains (Fig. 1.2), has been suggested by Schutte and Liebisch (1985) based on results from tracer experiments with intact plants and plant parts. Judging by the structure of betacyanin and betaxanthin and the incorporation of labelled tyrosine and DOPA

(dihyroxyphenylalanine) into both types of betalains (see Impehlizzeri and Piattelli

1972, Zryd et al., 1982, and Endress et al., 1984), it seems that both betalains share the same pathway.

It has been shown from feeding experiments with radio-active tyrosine, that radioactivity also appears in DOPA and cyclodopa in addition to the betalains (Zrd et al., 1982). Moreover, in colourless cells, no incorporation of labelled tyrosine into L-

DOPA was observed (Constabel and Haala 1968, and Zryd et al., 1982). These results provide some evidence that L-DOPA is an intermediate in betalain synthesis. It is also believed that the entry point to the biosynthetic pathway leading to the betalains, is the hydroxylation of L-tyrosine into L-DOPA which is catalysed by tyrosine hydroxylase

(see Endress 1977, 1979, Wichers etal., 1983,1984, and MUller et al., 1993, 1994).

Figure 1.2 The proposed biosynthetic pathway for betalains (after SchUtte and Liebisch,

1985)

COOH H2N'

HO - 2 COO H O 0 COOH Tyrosine Stizolobic acid 'Jr HO / Recycli- \ 201 ionS NH2 HO2NcooH o DOPA o

Amino acid—+ + oromine

HO-- N'" COOH HOOC COOH HO0C''COOH H H cycto-DOPA BetaIam ic acid Betoxon thin e.g.Vulgaxan thin l

Betanidin, RH Betanin, RGIucose

5 Betalamic acid has also been suggested as an intermediate to all the betalains, and it is the chromophore of the pigment (see Piattelli 1976). The occurrence of this compound as a natural constituent of a number of the Centrospermae (Kimler et al.,

1971) would support this suggestion. Further support was provided by Chang et al.

(1974) who demonstrated the incorporation of labelled L-DOPA into betalamic acid.

The formation of betalamic acid is postulated to occur through an extradiol cleavage of the aromatic ring in the DOPA molecule, at the position between 4 and 5, and subsequent closure of the resultant intermediate to a heterocyclic system (Fischer and

Dreiding 1972, Impellizzeri and Piattelli 1972, Chang et al., 1974, Girod and Zryd

1991, Terradas and Wyler 1991, and Hinz et al., 1995). The reaction sequence between L-DOPA and betalamic acid has also been studied in the mushroom Amanita muscaria. Terradas and Wyler (1991) reported that 4,5 secodopa was an intermediate in the above step, and suggested that an enzyme similar to the metapyrochatechase found in bacteria and animals was responsible for this reaction. Recently, the enzyme,

DOPA 4,5 dioxygenase, which catalyses the formation of 4,5 secodopa has been isolated from Amanita muscaria (Fivaz et al., 1994, and Hinz 1995) and Portulaca grandlora (Fiva.z et al., 1994). On the other hand, Girod and Zryd (1991) have reported that cchydroxyl--alanine muconic-c-semialdehyde is formed after the extradiol fission of the ring of the DOPA molecule, before hydrogenation and recyclisation occur, and they (the authors) isolated the enzyme 3,4-DOPA 4,5 dioxygenase which catalyses such conversion. These different results show the present state of uncertainty of how the betalains are synthesised.

6 1.2 Colours in the Food Industry

1.2.1 The Use of Food Colourings

It is generally believed that the use of added colours has a direct bearing on the acceptance of many foods. Indeed, colour in food is an early signal of freshness and readiness for consumption (see Goldenberg, 1977, and Klaui and Bauernfeind, 1981).

In the UK, for example, about 75% of all foods are processed in some way before they reach the consumer (Timberlake and Henry, 1986) and colours are added to food for a number of reasons (see Goldenberg, 1977, and Timberlake and Henry 1986):

To make food more attractive and hence stimulate the appetite and enhance

enjoyment.

To replace natural colour lost during food processing, e.g. by high

temperatures.

To provide colour to otherwise colourless or dull looking foods.

To ensure uniformity of colour of different batches of a product.

In general, the addition of colouring agents does not change the nutritional quality of the processed food and the majority of our food would still have the same quality and flavour without added colours (Goldenberg, 1977), however, it does influence the customers' preference.

7 1.2.2 Problems with Food Colours

In the past synthetic dyes have been largely used in the food industry because of their stability and ease of use. However, many of these colorants present a health hazard and have been banned in some countries, for example, FD and C red No. 2 and

No. 4 in the USA (Pasch and Von Elbe, 1978). Among the currently used colorants, the red dye Amaranth (B 123), and yellow dyes Sunset yellow (El 10) and Tartrazine

(E102) have also been associated with allergies. In particular, tartrazine is known to cause asthma and is implicated in hyperactivity especially in children (see Timberlake and Henry, 1986). The fear aroused by such adverse effects of synthetic dyes has shifted the attention to natural colours. For example, in the period 1969-1984, 356 papers were issued on natural colourants and only 71 on synthetic dyes (Francis,

1987), a natural colour can be defined as a colour extracted from a plant or animal in such way that keeps the pigments chemically unchanged (Timberlake and Henry,

1986).

1.2.3 Plants as a Source of Food Colours

Plant pigments are ideal for use as food colourings since they are essentially the same as those contained in the original plant material and have a long history of consumption over thousands of years. However, despite this, the toxicology of such compounds must be evaluated before permission for use in food can be given. This is because some secondary products might be toxic even at low concentrations and therefore the risk involved must be weighed against the benefits of using them in food

8 (Busch, 1991). The major plant pigments currently in use as natural food colourings

include carotenoids, chlorophylls, anthocyanin, betalains, saffron, gardenia, curcumin,

and carthamins. These natural food colours are discussed in detail by Bauernfeind

(1981), Klaui and Bauernleind (1981), Timberlake and Henry (1986), Francis (1987), and Knewstubb and Henry (1988).

1.2.4 Beetroot Red

Beetroot red (E162) is used as a safe food additive (Conforti-Froes et al.,

1990) in different food products (Von Elbe and Maing, 1973). The commercial preparation of the pigment is obtained from the storage root of Beta vulgaris by extraction under acidic conditions and evaporating the extract to dryness. The resulting powder contains betacyanin and a lesser amount of betaxanthin (Timberlake and Henry, 1986). Beetroot red is water-soluble and stable in the pH range 3-7 (see

Nilsoon, 1970). However, it tends to be degraded by heat (Steglich and Srack, 1990), light and oxygen and therefore it is used mostly in short-life dairy products such as sausages, yoghurt, ice cream, sherbet, gelatin dessert powder and maraschino cherries

(see Von Elbe etal., 1974, Pasch etal., 1975, Pasch and Von Elbe 1978).

9 1.3 Plant Cell Cultures as a Source of Desirable Secondary Products

1.3.1 The Potential of Plant Cell Culture

Higher plants are capable of synthesising a large range of compounds useful to

man. Many of these compounds are a product of secondary metabolism and have

properties which are of social and commercial importance. For example, plant

secondary metabolites are used in the pharmaceutical, fragrance and food industry

"flavours and dyes" (see Collin 1987, Fowler 1987, Phillipson 1990, and Wink 1990).

Such substances can be extracted directly from plants. However, there are difficulties

in obtaining these plants as the majority of them grow in tropical regions (Collin,

1987). Although some compounds previously obtained from plants are now

chemically synthesised (Fowler, 1987) many other products cannot be chemically

synthesised for several reasons such as cost or complexity of the product mixture

(Wink, 1990). Therefore, biological production of the compound still presents the

more reliable and economic way. Indeed, about 25% of the active ingredients in prescriptions in the USA and Western Europe are derived from higher plants (Curtin

1983, Phillipson 1990, and Fowler et al., 1990).

Tissue culture systems provide a predictable alternative source of secondary products. There are several advantages in producing desirable compounds by cultured cells (see Yeoman, 1986):

1. Freedom from constraints imposed by various environmental factors,

including climate, pests, and diseases.

10 Elimination of geographical and seasonal constraints so that the product can

be produced wherever and whenever required.

Rapid growth of cultured cells permits production of the compound in a

shorter time than that needed for raising the whole plant.

Growing cells under controlled conditions enables controlled production of

the product according to demand.

Production of novel compounds not normally detected in the plant.

Biotransformation of specific compounds, e.g. the conversion of the cardiac

glycoside n-methyl digitoxin to 3-methyl digoxin by cell cultures of

Digitalis lanata (Alfermarm et al., 1983).

However, despite the advances made in the field of plant tissue and cell culture, the production of secondary metabolites by cultured cells at the industrial level has been limited and only two compounds, shikonin and berberine, are being produced commercially from cultures of Lithospermum erythrorhizon and Coptis japonica respectively (Fujita 1988). The problem with plant tissue culture is that in most instances the yields of secondary products are much lower than those in the plants from which cultures were derived. Despite this, there are some reports of cultures accumulating secondary metabolites at levels which equal those produced by the plant (Kinoshita et al., 1990 and Villarreal et al., 1993) or even higher (Kunakh,

1994) (see also Fowler et al., 1990). In general, low yield of product is the major barrier to the commercial production of compounds by plant tissue cultures.

11 1.3.2 The Relationship Between Growth and Development and Product

Accumulation

The growth rate of cultures and biomass yield together with product yield are important determinants for system productivity (Fowler, 1987). In most cases accumulation of secondary metabolites in plant cell cultures occurs when growth has ceased or slowed down. For example, Lindsey and Yeoman (1983) showed that alkaloid accumulation in a number of Solanaceous species occurred at the end of the growth phase and was associated with tissue organisation. Also, the production of anthocyanin in Vitis sp. takes place at the stationary phase of growth (Hirose et al.,

1990, Böhm et al., 1991, and Sakuta et al., 1994). This inverse relationship between growth and product accumulation has been explained on the basis of a switch from primary to secondary metabolism (Phillips and Henshaw 1977, Yeoman et al., 1980).

At a time when primary and secondary metabolism compete for common precursors, the slowing down of growth results in the diversion of precursors into secondary metabolic pathways (Phillips and Henshaw 1977, Lindsey and Yeoman 1984, Lindsey

1985 & 1986, Hall and Yeoman 1991). Alterations in enzyme/substrate compartmentalisation (Lindsey and Yeoman 1985) and/or changes in enzyme activities (Yeoman et al., 1980) must be involved in this regulation. Thus, organised cultures, which usually grow more slowly, might be expected to have a higher rate of secondary metabolism and therefore accumulate higher yields of secondary compounds.

12 However, there are also some instances in which growth and secondary

metabolism are positively correlated, for example, betacyanin accumulation was

found to take place during the active growth phase in Phytolacca americana (Sakuta

et al., 1986, 1994, Hirano and Komamine 1994), Chenopodium rubrum (Hirose et al.,

1990) and Beta vulgaris (Jaafar 1992). Maximum production of secondary products

during the logarithmic phase of growth was also reported for volatile oils in Ruta

graveolens (Corduan and Reinhard, 1972), podophyllotoxin in Podophyllum

(Kadkade 1982), sapogenin in Agara wightii (Sharma and Khanna 1980) and

berberine in Thalictrum rugosum. Also, Reinhard et al. (1989) have shown that the

conversion of -methyldigitoxin to -methy1digoxin was highest at the logarithmic

growth phase and lower at the lag and stationary phases.

In a number of culture systems, the formation of secondary products occurs

during the lag phase, a period after subculture when growth and cell division have not

yet started and while the cells are adapting to a new environment. For instance, the

production in cell cultures of geomichrysone in Cassia (Noguchi and Sankawa 1982), phenolics in tobacco (Cvikrova et al., 1988) ferruginol in Salvia (Miyasaka et al.,

1985) and carotenoids in Bixa orellana (Boyd 1991). However, this might be a

continuation of metabolite production from the previous stationary phase (Noguchi

and Sankawa 1982).

13 1.3.3 Cellular Heterogeneity and Secondary Metabolite Production

Cellular heterogeneity, with regard to product formation, within plant cell

cultures has been observed both directly and indirectly. Direct demonstration of the heterogeneity of a cellular population in culture is made by visual observation, using a microscope, when the cells accumulate a coloured metabolite or a metabolite that can be visualised e.g. intracellular staining and/or UV microscopy. Indirect demonstration can be made through the isolation of cell lines with a different productivity for the metabolite of interest. For example, Colijn et al. (1981) and Yamakawa et al. (1982) demonstrated, using light microscopy, that only a limited number of cells in Petunia and Vitis cultures respectively accumulated anthocyanin pigment to visible levels. In

Catharanthus roseus, Knobloch et al. (1982) also observed that anthocyanin accumulation was distinctly heterogeneous and only ca 5% of the cells contained the pigment. In another study on the same species Hall and Yeoman (1986b) reported that approximately 10% of the cells regularly accumulated detectable levels of anthocyanin and there was a considerable variation within this "productive" population. According to these authors, the variation in intracellular anthocyanin content and concentration, as estimated by using microdensitometry, in cell cultures was much greater than that observed within tissues of mature plants. Sato and Yamada

(1984) have observed, using fluoromicroscopy, that cell cultures of Coptis japonica are heterogeneous in nature and they (the authors) could derive various cell lines, from the original culture, with different productivities of berberine. Dougall et al.

(1980) also isolated cell lines from wild carrot cultures with varying capacities for anthocyanin accumulation. According to Weller and Lasure (1981), different cell lines

14 could be obtained from the same stock culture of Beta vulgaris. After a series of

selections, the red callus contained 40% red cells, 6% yellow cells, 4% brown cells and 50% non-pigmented cells, whereas, the yellow callus contained only 1-2% yellow cells and the rest were non-pigmented except for a few brown cells. In addition, pigment intensity varied remarkably from cell to cell. Such cellular heterogeneity in red beet cultures was also reported by Girod and Zryd (1987).

The mechanisms controlling cellular heterogeneity is not filly understood therefore the control of this phenomenon may either be genetical or environmental or both. However, the productivity of a culture may be stabilised by repeated selection and maintenance under defined conditions (see Dougall et al., 1980, Sato and Yamada

1984, Yamamoto et al., 1989, and Sakamoto et al., 1994).

1.4 Increasing the Yield of Secondary Products in Plant Cell Cultures

As already mentioned, low product yield is the main barrier for commercial exploitation of cell cultures. There are, however, several ways in which the yield of product from cell cultures may be increased:

Selection of high yielding cell lines.

Manipulation of culture conditions (physical or chemical).

Manipulation of the genome.

15 1.4.1 Selection of High Yielding Cell Lines

The fact that cultures are derived from tissues that may include cells of a different genetic 'make up' and give rise to cultures displaying genotypic variation

(Yeoman and Forche, 1980) provides an opportunity for the selection of cells with certain characteristics such as high production of a particular secondary product.

Selection of cells with desirable properties can also be made after a treatment to induce mutation, for example, by treating cells with physical or chemical agents such as ultraviolet light or coichicine (see Collin and Dix, 1990). Cell lines selected from callus, cell suspensions and protoplasts have been successfully exploited for the improvement of product yield of cultures and crop plants (see Tabata et al., 1978,

Fujita 1988, and Collin and Dix 1990). Stable cell lines with high productivity for anthocyanin have been isolated from cell cultures of Catharanthus roseus (Hall and

Yeoman 1987), Euphorbia millii (Kinoshita et al., 1990) and Arabica carodata

(Sakamoto et al., 1994). Sato and Yamada (1984) and Fujita (1988) also obtained high yielding cell lines for berberine production from Coptis japonica cell cultures and for shikonin production from Lithospermum erythrorhizon cultures respectively.

However, selected high yielding cell lines may be unstable and undergo a reduction in product yield with time (Deus-Neumann and Zenk, 1984). In such cases, repeated selection is necessary to sustain the high productivity of the culture. Indeed, repeated selection has been used effectively to obtain stable cell lines (see Watanabe et al., 1982, Sato and Yamada 1984, and Sakamoto et al., 1994). Moreover,

16 maintenance of the cell line in a proper medium can also improve and stabilise the productivity of the culture (see Yamamoto et al., 1989).

1.4.2 Manipulation of Culture Conditions

Using this approach the culture conditions are manipulated to provide an optimum environment for the operation of the biosynthetic pathway leading to the compound of interest. In fact, manipulation of culture conditions has been used successfully to increase culture yield (El-Bahr etal., 1989, Panda etal., 1992, Tamura et al., 1994, and Hilton and Rhodes 1994). Such manipulations include alterations to the source and concentration of nutrients (macro and micro nutrients), changing the type, concentration and balance of growth regulators, varying the pH of the medium, supplementation of a precursor (s) to the product, addition of biotic and abiotic elicitors, and manipulation of the physical conditions such as light and temperature

(for review see Yeoman and Yeoman 1996).

The most important nutrients and those that have received most attention are nitrogen, phosphorus and carbohydrate (sugar). In general, the effect of these nutrients on secondary metabolite production in cultures is related to growth e.g. altered concentrations which contribute to a reduction in growth can result in an increase in the amount of metabolite accumulated where cultures exhibit an inverse relationship between growth and product accumulation (see Lindsey, 1985). However, there are cases in which these nutrients influence metabolite production independently from growth. For example limitation of nitrogen can result in a reduction in growth and

17 enhance the production of a secondary metabolite (Yamakawa et al., 1983, Lindsey

1985, and Ishikura et al., 1989). Whereas, in Lithospermum eythorhizon cell cultures, the growth and formation of shikonin derivatives increased with a rise in the total nitrogen in the medium within the range of 67-104 mM, (Mizukami et al., 1977). In

Beta vulgaris, nitrogen (ammonium and nitrate) at levels higher than that of the original B5 medium changed the growth rate but not the betacyanin content

(Constabel and Nassif-Makki 1971) and also in Chenopodium rubrum (Berlin et al.,

1986). The nitrogen source in some cases, had a critical effect on the production of secondary metabolites (see Fujita et al., 1981, Wichers et al., 1985, and Demeyer and

Dejaegere 1992). For example, pigment production in immobilised cultures of

Lavandula vera was completely suppressed when ammonium chloride was the sole nitrogen source, independent of its' concentration, whereas, when potassium nitrate was used as a source of nitrogen cells produced greater amounts of pigment

(Nakajima et al., 1989). BOhm and Rink (1988) also showed that replacement of ammonium with nitrate substantially increased the accumulation of betacyanin in

Portulaca grandlora. On the other hand, the ratio of ammonium to nitrate has been shown to be important in determining growth and secondary metabolite production.

Ikeda et al. (1976) showed that a high ammonium to nitrate ratio increased product accumulation and reduced growth. On the contrary, Jung et al. (1994) reported that growth and catharanthin production in hairy root cultures of Catharanthus roseus was increased by increasing the concentration of nitrate. In Vitis cell cultures, anthocyanin production was maximal when the molar ratio of NH4 :NO3 was 1:1 (Yamakawa et al., 1983). The ratio of carbon to nitrogen C/N in the medium has also been shown to

18 have an effect on product accumulation in some instances (Yamakawa et al., 1983, Do and Cormier 1991a, Deliu et al., 1992, and Ballica et al., 1993). According to Ballica et al. (1993), the yield of tropane alkaloid in Datura stramonium was substantially increased (ca 100%) at a C/N ratio of 100 whereas it decreased at a C/N ratio of 70.

The effects of phosphorus have also been studied in a number of species (see

Knobloch and Berlin 1983, Obata-Sasamoto and Komamine 1983, Lindsey 1985,

Berlin et al., 1986, Sakuta et al., 1986, Hirose et al., 1990, Deliu et al., 1992, Jung et al., 1994, Smolov and Oleinikova 1994, and Monroy et al., 1994). Deliu et al. (1992) reported that there is a direct link between the consumption of phosphate and the induction of alkaloid production in Berberis parvfolia. Similarly, Monroy et al.

(1994). observed that the absence of phosphate from the medium correlated with an increase in betalain accumulation in Beta vulgaris cell cultures. Hirose et al. (1990) have shown that phosphate starvation totally suppressed cell division and betacyanin accumulation in Phytolacca americana but when phosphate was added to the medium cell division and betacyanin synthesis took place. In contrast, lack of phosphate has been reported to increase the production of alkaloids and phenolics in Catharanthus roseus cell cultures (Knobloch and Berlin 1983) and DOPA in Stizolobium hassjo callus (Obata-Sasamoto and Komamine 1983).

Sucrose is the most common source of carbon in the medium of cell cultures and has been shown to have a considerable effect on growth and secondary product accumulation. High concentrations of sucrose have been reported to enhance the

19 production of secondary metabolites (see Knobloch and Berlin 1980, Cormier et al.,

1990, and Panda et al., 1992). In Vitis vinfera, for instance, anthocyanin production was significantly enhanced at high concentrations of sucrose (Cormier et al., 1990).

Such an effect was related to the high osmotic stress caused by high concentration of sugar (Do and Cormier 1990, 1991b). However, a stimulation in product formation as well as growth by high concentrations of sucrose (7-10%) was reported by Kitamura et al. (1991) for tropane alkaloid production in Duboisia myoporoides and Duboisia leichhardtii root cultures. Dupraz et al. (1994) also showed that 5% sucrose was most appropriate for growth and alkaloid production in transformed roots of Datura quercfolia.

Among the micronutrients studied, Choi et al. (1994) reported that calcium and magnesium affected the production of saponin in callus of Panax ginseng. In contrast, these ions did not influence alkaloid and polyphenol accumulation in

Catharanthus roseus (Knobloch and Berlin 1980). Copper has been shown to inhibit betacyanin accumulation in Portulaca grandflora callus. However, this component was considered to be a co-factor rather than a nutrient (Endress 1976).

Plant growth regulators, particularly the auxins and cytokinins are important components of culture media. Although, there are a number of cultures which can grow without added phytohormones (see for example Zryd et al., 1982 and Berlin et al., 1986), in general, the type and concentration as well as the balance of auxin and cytokinin is often critical in determining growth and secondary metabolite production

20 (see Dougall 1980, Mantel! and Smith 1983, Ishikura et al., 1989, El-Bahr et al.,

1989, Panda et al., 1992, and Choi et al., 1994). For instance, El-Bahr et al. (1989) found that in Datura stramonium cultures growth was better with NAA and BA than with NAA and Kin, whereas, alkaloid production (hyoscine and hyoscyamine) was highest with kinetin alone. Ishikura et al. (1989) reported that maximum growth and phenol production in Prunus yedoensis, was obtained at 0.3 mg.F' of both 2,4-D and kinetin. Constabel and Nassif-Makki (1971) showed that the exclusion of 2,4-D from the medium was necessary for betacyanin formation in callus of Beta vulgaris and

Amaranthus caudatus. Further, they have shown that replacement of 2,4-D with NAA induced pigment formation in both cultures. Kinetin has been shown to enhance the production of betacyanin in the dark (see Leathers et al., 1992) and in the light (Rudat and Goring 1995). Girod and Zryd (1991) have shown that by changing the ratio of auxin to cytokinin, the phenotypic character (colour) of Beta vulgaris cells could be altered. They suggested that plant growth regulators induce changes in the secondary metabolic pathways in cultured cells

Precursor (s) feeding (see Fowler 1983, Lindsey and Yeoman 1984, Ballica et al., 1993, and Sugimoto et al., 1994) have also been shown to induce and direct the biosynthetic pathways leading to the formation of several compounds. Application of precursors of betalain (such as tyrosine and DOPA) have been used to study the biosynthetic pathway leading to the pigment (e.g. Zryd et al., 1982) but specific studies on the use of precursors for increasing betalain production have not been reported.

21 Application of biotic or abiotic elicitors has also been used as a means to increase the yield of secondary products (see Eilert et al., 1987, Holden et al., 1988a, b, Eilert 1989 and Vazquez-Flota et al., 1994). It is known from extensive studies on the microbial invasion of plants that modification of hosts' metabolism results in the accumulation of secondary metabolites as a defence response against the micro- organism (see Keen et al., 1972, Wolters and Eilert 1983). It has been also shown that compounds from the pathogens can cause the same response in plants. These compounds have been described as elicitors (Keen et al., 1972) and together with those constitutive elicitors from the host plant (Dixon et al., 1989) comprise the group of biotic elicitors. Physical and chemical stresses such as UV radiation, exposure to cold or heat, ethylene, fungicides, antibiotics, salts of heavy metals or high salt concentrations, have been defined as abiotic elicitors (Eilert 1989). In fact, elicitation has been shown to increase the yield of secondary metabolites in many cell cultures; for example, increasing the yield of berberine in Thalictrum rugosum cultures (Funk et al., 1987) and tropane alkaloid in Datura stramonium (Ballica 1993).

Amongst the physical conditions light, temperature, pH, and aeration have also been manipulated to increase product yield. Light has been shown to influence the production of several secondary metabolites (see review by Mantell and Smith 1983,

Towers and Yamamoto 1985). Contrasting effects of light on the production of secondary metabolites have been reported, for example, the inhibition of nicotine accumulation in tobacco cell cultures (Ohta and Yatazawa 1978, Hobbs and Yeoman

1991) and the stimulation of anthocyanin production by Haplopappus gracilis

22 cultures (Stickland and Sunderland 1972) and berberine production in cell culture of

Thalictrum rugosum (Kim et al., 1988). For betalain in particular, although light was shown to be essential for pigment production in some cases, e.g. in Portulaca grandflora cell cultures (Liebisch and Böhm 1981) and callus (Kishima et al., 1991) and in cell cultures of Beta vulgaris (Girod and Zryd 1985, 1987). In other cases, synthesis of betalain in the dark was observed, however, the pigment accumulation was increased by light, e.g. cell cultures of Chenopodium rubrum (Berlin et al., 1986) and Beta vulgaris (Jaafar 1992). On other hand, betacyanin production was reported to be higher in the dark than in the light in callus cultures of Portulaca grandfiora

(Endress et al., 1984) and Chenopodium rubrum (Berlin et al., 1986). The effect of light on betalain was reviewed by Leathers et al. (1992). Few studies were made on the effect of light quality and quantity on betalain production in tissue cultures. One of these, made by Berlin et al. (1986), showed that betacyanin production was increased by 30% in cell cultures of Chenopodium rubrum grown under blue light (16 h. photoperiod), compared to the control (white light grown cultures). Rudat and Goring

(1995) showed that illumination with UV-light or UV-containing light (blue and white) was necessary for betacyanin formation in the cell culture of Chenopodium rubrum. Kishima et al. (1995) also reported that blue and blue/UV light induced betalain pigmentation in callus culture of Portulaca.

The effect of temperature on product accumulation has received limited attention. For example, Fowler (198 8) reported a dramatic effect of temperature on the growth and product accumulation in Catharanthus roseus cell cultures. Morris (1986)

23 observed that optimum growth in cell cultures of Catharanthus roseus was at 35°C while optimum serpentine production was obtained between 20°C and 25°C, and optimum accumulation of ajamalicine was at 25°C. Hilton and Rhodes (1994) also studied the effect of temperature on the growth and hyoscyamine accumulation in

Datura stramonium. However, they found that altering the temperature, in the range of 20°C -30°C had only a small effect on the product accumulation.

pH of the medium can affect the uptake of nutrients and precursors, the permeability of cell membranes (Butenko 1968), and release of products from the vacuole to the culture medium (Brodelius 1990, Matile 1990) and so it may influence the production of secondary metabolites. Döller et al. (1976) found that an initial pH of 5.5 was optimum for the production of serpentine in Catharanthus roseus. One particular cell line of the same species accumulated more alkaloids when grown in production medium with pH 7.0 than with pH 5.5 (Kutney et al., 1981). Jaafar (1992) showed that initial pH in the range of 5.4-6 has no effect on betalain production in cell cultures of Beta vulgaris.

Last but not least, among the components of the physical environment, is the aeration and mixing of cells. This is vital especially in large-scale production systems in fermentors or bioreactors (see reviews by Mantel! and Smith 1983, Fowler 1988,

Westphal 1990, Payne et al., 1991, and Schlatmann et al., 1993).

24 1.4.3 Genetic Manipulation

The recent advances in molecular biology and recombinant DNA technology provide a powerful tool for cloning genes and introducing them into plant cells.

Although their use in the field of secondary metabolite production has not been widely studied, the potential for applying such techniques seems to be significant (for review see Yeoman and Yeoman 1996). If a particular enzyme in a pathway is shown to be regulatory it may be possible to clone the gene for the enzyme and amplify or modify its activity. A culture of cells containing the modified gene could then express enhanced activity in the pathway, leading to an increased amount of the particular secondary metabolite. The over-expression of enzymes of a pathway has been studied by a number of researchers. For example, Songstad et al. (1990, 1991) found that the proto alkaloids tryptamine and tyramine were accumulated at greater levels in tobacco when a DNA clone encoding tryptophan decarboxylase (TDC) from Catharanthus roseus, driven by the CaMV35S promoter, was introduced. In contrast, Napoli et al.

(1990) and van der Krol et al. (1990) found that introduction of extra copies of the genes encoding chalcone synthase (CHS) and dihydroflavonol-4-reductase (DFR) into

Petunia, under the transcriptional control of CaMV35S, resulted in a reduced, rather than increased pigmentation, owning to a reduced level of transcription of both foreign and native "chs" and "dfr" genes. In a similar type of a study, Elkind et al.

(1990) attempted to overexpress a bean (PAL) gene in transgenic tobacco, in the expectation that this would lead to an increase in flux through the phenylpropanoid pathway and an increase in the lignin content of transgenic plants. However, lignin and soluble phenolics content was reduced and this was believed to be the result of the disruption of the regulation of the native tobacco PAL enzyme activity by the effect of the foreign gene on native control mechanisms. These examples demonstrate the potential of using recombinant DNA technology to produce high yielding systems for a particular secondary metabolite by interfering with or enhancing regulatory pathways. However, it is clear that much more research on metabolic regulation is necessary before this approach can be used successfully.

Other approaches involving genetic manipulation could also prove useful in increasing the yields of secondary metabolites. One possibility is the use of antisense

RNA to block the formation of an enzyme which could degrade the desired product or to suppress the activity of enzymes that compete for common precursors, thus eliminating side chain reactions (see van der Krol et al., 1988 and Mol et al., 1990).

Another possibility is to modify the promoter sequence of developmentally regulated genes so that they would always be active and the product would be synthesised continuously (see Yeoman et al., 1990).

26 1.5 Aims and Objectives

The aim of this investigation was to study the production of betalain pigments in suspension cultures of Beta vulgaris as a means to increase product yield. The following objectives were attempted:

To determine the relationship between growth and betalain accumulation.

To improve the product yield by modifying the culture conditions.

To select cell lines with high productivity of either betacyanin or betaxanthin.

To identify the betacyanins and betaxanthins produced by the cell lines.

To investigate the production of betalains in relation to cellular heterogeneity.

To explore the role of tyrosine hydroxylase in determining the yield of pigment.

27 CHAPTER TWO

MATERIALS AND METHODS

28 2.1 Cell and Tissue Culture

2.1.1 Culture Material

Suspension cultures of Beta vulgaris used in this study were kindly given by

Dr. C. S. Hunter, Plant Sciences Group, University of the West of England.

2.1.2 Preparation of Culture Media

2.1.2.1 Standard Growth Medium

Gamborg's 135 medium (Gamborg et al., 1968), supplemented with sucrose and growth regulators (Table 2.1.2.1) was used for the routine culture of cell and tissue cultures of Beta vulgaris. The medium was prepared by dissolving 3.71 g.1 -1 of

Gamborg's B5 medium (Imperial Laboratories, Twyford, England), 20 g1 1 sucrose

(BDH Ltd., UK), 0.1 mg.1' Kinetin and 0.02 mg.r' 2,4-Dichlorophenoxyacetic acid

(2,4-D) (both obtained from Sigma Chemicals Limited, UK) in distilled water. The kinetin and 2,4-D were added from stock solutions prepared as described in 2.1.2.3.

The medium was adjusted to pH 5.8 using M KOH or M HC1 (BDH Ltd., Poole,

Dorset, England) and then dispensed into 250 ml Erlenmeyer flasks (Pyrex, England),

50 ml per flask. The flasks were covered loosely with a double layer of aluminium foil and autoclaved.

To solidify, 0.6 % w/v of Bacto agar (Difco Laboratories, Detroit, USA) was added to the liquid medium after the pH had been adjusted. After autoclaving the

29 medium was cooled to ca 60°C and then poured into 5 cm disposable Petri dishes

(Bibby Sterilin Ltd., Staffs, UK), (ca 8 ml/dish) and allowed to solidify before use.

2.1.2.2 Media with different concentrations of nitrogen or phosphorus

B5 media with different levels of nitrogen and phosphorus were used in

experiments (3.2.1.1) and (3.2.1.2) respectively. Media free of KNO 3 , (NH4 )2 504,

NaH2PO4, sucrose, and growth regulators, were prepared at 100 times the normal concentration (see list in Table 2.1.1). The media were stored at -40°C as 10 ml portions. When required, the concentrated stock medium was allowed to thaw and the

desired amounts of KNO3 , (NH4)2 SO4, and NaH2PO4 were added to attain the desired level of nitrogen and phosphorus. Sucrose and growth regulators were added at the concentration used in the standard medium. The final volume was then made up to 1 litre and the pH of the media was adjusted to 5.8.

2.1.2.3 Growth regulator stock solutions

Stock solutions of 100 mg.1 1 of kinetin and 2,4-D were prepared by dissolving

10 mg of each separately in 5 ml of 0.1 M KOH and the volume of each solution was made up to 100 ml with distilled water. The stock solutions were stored at 4°C in the refrigerator and renewed every three months.

30 Table 2.1.1 Constituents of Supplemented Gamborg's B5 medium used throughout the investigation

Constituent mg.1'

CaC12 .2H20 150.0

KNO3 3000

MgSO4.7H20 250.0

NaH2 PO4.21120 169.6

(NH4 ) 2 SO4 134.0

FeNa EDTA 40.0

CoC12 .6H20 0.025

CuSO4.51420 0.025

H3B03 3.00

K! 0.75

MnSO4.41-120 13.20

Na2Mo04.2H20 0.25

ZnSO4.7H20 2.00

i-inositol 100.0

Nicotinic acid 1.00

Pyridoxin HC1 1.00

Thiamine HC1 10.0

2,4-Dichiorophenoxyacetic acid 0.02

Kinetin 0.1

Sucrose 20,000

31 2.1.3 Sterilisation and Aseptic work

Sterilisation of culture media, glassware and instruments was carried out by autoclaving at 121°C for 20 mm. at a steam pressure of 15 psi. The aseptic manipulation of cultures, was carried out in a laminar air flow cabinet. Before use the surfaces of the cabinet were sprayed with absolute ethanol and then wiped off with a paper towel. Instruments were kept in absolute ethanol and flamed immediately before use.

2.1.4 Maintenance of Suspension Cultures

Suspension cultures of Beta vulgaris were maintained in Gamborg's B5 medium prepared as described in (2.1.2.1). Cultures were incubated at 25 ± 1°C on an orbital shaker rotating at 86 rpm under continuous illumination of 15 ± 3 .tmol.m 2.s, supplied by Thorn natural light fluorescent tubes (Thorn EMI, UK). They were subcultured every 13 or 14 days by transferring 1 g. wet weight of cells, using a perforated spoon, into a 250 ml Erlenmeyer flask containing 50 ml of liquid medium.

2.1.5 Single Cell Cloning and Selection of Cell Lines

The technique of plating out cell suspensions onto agar plates was adapted from the method described by Street (1977). The principle of the method is to plate out at low density and select only colonies originating from single cells, or small aggregates (2-6 cells) which are likely to have been formed from a single cell.

32 Using this approach, pooled cells of 7 day old suspension cultures were first filtered aseptically through a 500 p.m nylon mesh, to remove the large aggregates, and then through a 200 p.m nylon mesh. The filtrate which consists predominantly of single cells and small aggregates (2-6 cells) , was allowed to settle for 30 min and the supernatant discarded. After the cellular density of this cell suspension had been determined, series of dilutions (four) were made, using cell free conditioned medium obtained from a 7 day old cell culture, in order to attain four different cellular densities. Each cell suspension was incubated at 35°C for 5 min and then mixed with an equal volume of standard medium (2.1.2.1) containing 1.2% w/v agarose (Sigma,

UK). 12 ml of each preparation was then poured into a 9 cm Petri dish. The final cellular densities of these preparations were 1 2x 1 0, WO 3x 1 and 1. 5x 1 ce1l.m1. After gelling the plates were sealed with a double layer of Paraflim, and then viewed under a binocular microscope from the underside of the plate. Single cells of four colours violet, orange, yellow and white were marked with a fine needle. The marks were confirmed using coloured pens and the plates incubated in the dark at 25

±1 °C. After 28 days, growth of colonies was observed on all plates at each cell

density (Fig. 2.1.1). However, the plates with an initial density of 1 .5x10 3 cell.mr1 were the best, since the space between colonies was wide enough to select individual colonies with ease. Colonies of ca 0.5 mm in diameter which had arisen from marked cells were picked up with a sterile needle and transferred onto the surface of 20 ml of the same medium used for plating contained in a 9 cm Petri dish, up to 36 colonies per plate were subcultured. The plates were then sealed with Paraflim and incubated at 25

± 1°C under 7 p.mol.s 1 .m 2 of illumination. When the colonies reached a size of about

33 Figure 2.1.1 The appearance of plated cells grown for 28 days in the dark on 50% conditioned medium. The initial cellular density of A, B, C and D were 12x10 3,

6xl03, 3x103, and 1.5x103 cell.mF' respectively.

34 4 mm in diameter, they were transferred separately onto the surface of 8 ml of

medium in 5 cm plates (Bibby Sterilin Ltd, Staffs, UK) (2.1.2.1). Four types of cell

lines, according to their colours, were isolated, violet, orange, yellow and white. Five

plates of each type were subcultured and the plates incubated at 25 ± 1°C in the light

(15 ± 1 imol.s .m 2). After four weeks, one plate of each isolated cell line was chosen

to represent the selected cell line (Fig. 2.2.1). Suspension cultures of each selected cell

line were initiated by transferring ca 1 g. f.wt. of callus into 250 ml Erlenmeyer flasks

containing 50 ml of standard liquid medium (2.1.2.1). Cultures were maintained under

the conditions described in (2.1.4).

2.2 Characterisation of Culture Growth and Cell Viability

2.2.1 Determination of Fresh Weight

Either 30 ml or the entire culture (50 ml) was vacuum filtered through

Whatman filter paper using a Buchner funnel. Cells were washed twice with distilled water, filtered and then weighed.

2.2.2 Determination of Dry Weight

After determination of fresh weight, the same samples were dried in an oven at

75°C overnight (16 h). They were then cooled in a desiccator and weighed. In some experiments, samples were freeze dried for 2-3 days and then weighed.

35 Figure 2.2.1 The appearance of isolated cell lines 4 weeks after the first transfer to the

standard solid medium. Cultures were grown under 15 ± 1 imol.s'.m 2 illumination at

25 ± 1°C. A, B, C and D are the violet, orange, yellow and white cell lines

respectively.

C [!]

Mgt

36 2.2.3 Determination of Packed Cell Volume (PCV)

10 ml samples taken from well mixed suspension cultures were poured into 15 ml graduated conical centrifuge tubes and centrifuged for 5 min at 1000 xg. The PCV per culture was then calculated.

2.2.4 Determination of Cell Population Density

Cell population density was determined using a Hawskley Crystallite haemocytometer (grid volume 1.8 il). Maceration of cells was carried out according to the method of Reinert and Yeoman (1982). 100 mg of freshly harvested cells were suspended in 2 ml of a solution containing 12% w/v chromium trioxide and 10% v/v hydrochloric acid. Cells were gently dispersed with a glass rod to break up large aggregates and then left for 4 h at room temperature. Finally the cell suspension was incubated in a water bath at 60°C for 20 min and agitated on a vortex mixer for 15 seconds. Appropriate dilutions were made in order to attain ca 200 cells per grid.

Cells were counted under a microscope, four grids for each sample. Cell population density is presented as cell number per ml after calculation using the following formula:

Cell no. = Vol. of macerate x cell count Vol. above grid

37 2.2.5 Determination of Cell Viability

The method of Gaff and Okong'o-Ogola (197 1) was used to determine the

viability of cultured cells. The method is based upon the loss of semi-permeability of

dead cells and the penetration of the pigment "Evan's blue" into cells. A drop of cell

suspension was placed on a microscope slide and mixed with a drop of Evan's blue

pigment (0.25% w/v), then covered with a glass cover slip and left for 5 minutes

before counting. The number of stained, dead, cells was counted in random samples of

ca 600 cells and the percentage of viable cells was calculated.

2.2.6 Determination of the Proportion of Pigmented Cells

2 ml of suspension culture was transferred to a 20 ml test tube and the large

aggregates in the sample were broken down very gently using a glass rod. The cell

suspension was then mixed thoroughly and ca 0.1 ml was removed using Pasteur pipette and placed on a microscope slide with a grid (S-7 England Finder, Graticules

Ltd, Kent, UK). A cover slip was then placed on top of the preparation and the cells

counted using a microscope. The pigmented cells in a population of 500-1000 cells were counted. The percentage of pigmented cell (PPC) was calculated using the formula:

PPC = No. of pigmented cells x 100 Total of cells counted

38 2.2.7 Calculation of Growth Rate

The growth rate was determined by the following equation:

GR=Log W2 - Log Wi T2-Tl

Where GR = Growth rate.

WI = Initial fresh weight of cells.

W2 = Final fresh weight of cells.

T2 - Ti = Time that the cells were in culture.

2.3 Extraction of Betalains from Cells of Suspension Cultures

Freshly harvested cells were homogenised in ca 2 ml of 80% v/v methanol

with a pestle and mortar. The homogenate was then poured into a 10 ml centrifuge

tube together with the washings from the pestle and mortar, the volume made up to 4

ml and the tube centrifuged at 2000 xg for 10 mm. The pellet was then re-extracted

twice more with 80% v/v methanol and centrifuged as above. The final volume was

made up to 10 ml and the pigment measurements were performed immediately after

extraction.

2.4 Analysis of Betalains by Spectrophotometry

The absorption spectrum of the cell extracts and the optical density of the

betalain pigments were determined using a Beckman D U-64 spectrophotometer.

Betacyanin and betaxanthin were measured at wavelengths of 535 and 472 nm

39 respectively. As the betacyanin absorbs light at 472 nm with ca 52% of that absorbed

at 535 nm, the optical density of betaxanthin at 472 nm was calculated according to

the following formula:

Optical density of betaxanthin at 472 nm = A472 - (A535 x 0.52)

Where: A472 = Absorbance at 472 nm.

A535 = Absorbance at 535 nm.

2.5 Analysis of Betalains by Thin-Layer Chromatography (TLC)

Betalain pigments were separated by thin layer chromotography using a multiple development technique described by Bilyk (1981). Cell extracts, prepared as described in (2.3) were spotted onto cellulose-coated glass plates (Merck, Darmstadt,

Germany). The spotted plates were dried under a nitrogen stream and then run twice in two different solvents in the same direction (see below).

Constituents Amount in ml Solvent I Solvent II

Isopropanol 30 55

Ethanol 35 20

Distilled water 30 20

Acetic acid 5 5

40 The plates were run up to 10 cm in solvent I, then dried and rerun in solvent II up to

15 cm, during development the TLC tanks were covered with aluminium foil to shield the plates from light to avoid any possible degradation of pigments. After drying the plates, betacyanin and betaxanthin were readily visible under white light as a purple and a yellow spot/band respectively.

2.6 Analysis of Betalains and Related Compounds by High Performance Liquid

Chromatography (HPLC)

High performance liquid chromatography (HPLC) (see Girod and Zryd 1991) was employed to ensure accurate qualitative and quantitative analysis of cell extracts.

2.6.1 High Performance Liquid Chromatography System

HPLC analysis was performed using a Gilson 302 liquid chromatography system fitted with a hyperchrom column (250 x 4.6 mm ID) containing Hypersil ODS

(C18) (Capital HPLC Limited, Scotland, UK) and a Gilson holochrome uv/vis detector. 20 p1 of each sample was injected into the system using a Gilson auto sampling injector 231-401 controlled by an IBM PS2 model 50 microcomputer using the Gilson HPLC system controller 714 vi .2. All data handling and processing were performed using the same software.

41 2.6.2 Preparation of the Mobile Phases

The two solvents of the mobile phase were prepared using HPLC grade' constituents or equivalents. Solvent A consisted of 50 mM sodium dihydrogen orthophosphate (Fisons, England) and 2.5 mM triethylamine (BDH., Chemicals Ltd,

Poole, England). The pH of the solution was adjusted to 4.2 with phosphoric acid

(BDH, Chemicals Ltd, Poole, England). Solvent B consisted of 40% v/v acetonitrile

(Sigma Aldrich, England, UK). Both solvents were filtered through 0.45 tm nylon-66 membrane filters (Whatman, England, UK) under reduced pressure. Prior to use, filtered solvents were degassed for ca 15 min by passing a stream of helium through them via a sparger. This was to prevent air bubbles from entering the HPLC system as a result of large pressure differences being applied to the mobile phase.

2.6.3 Preparation of Samples for HPLC Analysis

Fresh cells were harvested from cultures and frozen in liquid nitrogen, then lyophilised in a freeze dryer for 2-3 days under vacuum at -5 7°C. The freeze dried cells were kept in a refrigerator until required. Immediately before analysis, 200 mg of dried cells was grounded to a powder, on ice, using a pestle and mortar. The powder was then extracted for 20 min in 10 ml of 80% methanol v/v acidified with 0.2% HCl v/v. The extract was then centrifuged at 5000 xg for 30 min at 2°C and the pellet reextracted three more times under the same conditions. The Pooled supernatants were then evaporated to dryness using a rotary evaporator over a warm water bath at 28°C.

The residue was dissolved in water (HPLC grade, BDH, England, UK) and

42 centrifuged at 20,000 xg for 5 mm. The aqueous extract was then filtered through a

0.45 tm nylon membrane filter (Whatman) held in a Millipore filter unit (Millipore,

UK Ltd, Watford) attached to a syringe . Samples of 1 ml of cell extract were collected in 2 ml vials with a crimp top (Phase separation, Clwyd, UK). Samples were stored at -20 °C and analysed within three days.

2.6.4 Operating Conditions for HPLC Analysis

The methods described by Girod and Zr'yd (1991) were used for HPLC analysis of betalains and related compounds. The two solvents of the mobile phases, prepared as described in (2.6.2), were pumped at a flow rate of 1 ml.min' using the following analytical gradient program:

Time in mm. % Solvent A % Solvent B

0 100 0

15 70 30

20 40 60

25 20 80

30 20 80

The total time for each run was 45 min which also enabled the system to equilibrate between injection of samples. The separated components were detected by uv/vis detector at a wavelength of 535 nm for betacyanins, 472 nm for betaxanthins, 424 nm for betalamic acid and 280 nm for tyrosine, DOPA, and dopamine.

43 2.6.5 Identification of Peaks

Peaks were identified by the use of external standards as well as internal standards, which were used for confirmation of the identification. Some of the standards were purchased commercially while others were prepared in the laboratory.

2.7 Preparation of Standard Solutions

2.7.1 Betacyanin Standards

Betanin and isobetanin were purchased from APIN Chemical Ltd, Tokyo.

Betanidin and isobetanidin were produced by enzymatic hydrolysis of betanin and isobetanin respectively. Enzymatic hydrolysis was performed using 10 mg.mr' of 3-

Glucosidase (from Almond, Sigma, UK) in citrate buffer (0.1 M, pH 4.5). 0.2 ml of this enzyme solution was added to 0.8 ml of betaninlisobetanin solution(100 mg.mr') and the mixture incubated at 37°C for 5, 10, and 30 mm. The HPLC analysis of the products in shown in Fig. 2.7.1. The average retention times of betanin, isobetanin, betanidin and isobetanidin are 10.4, 12.7, 12.0, and 14.6 min respectively. Fig. 2.7.2 shows the chemical structure of the compounds (see Nilsson, 1970).

2.7.2 Miraxanthin V Standard

Miraxanthin V (see Fig. 2.7.3) standard was prepared according to the method described by Trezzini and Zryd (1991). Miraxanthin V was synthesised by a spontaneous conjugation between betalamic acid (Pigment chromophor) and the added amino acid dopamine. Betalamic acid was prepared by the alkaline hydrolysis

44 Figure 2.7.1 HPLC chromatograms of a betanin and isobetanin mixture (A) and the

products of enzymatic hydrolysis after 5 mm (B), 10 mm (C), and 30 mm (D). Peaks

of betanin, isobetanin, betanidin and isobetanidin are marked as a, b, c, d respectively.

100 a, 100

90 90

80 80

70 70

60 60

50 50

40 40

30 io

20 20

10 10 tn 0 0 S.00 10.00 15.00 20.00 Zbt 100 100 U 90 90

0 80 rj 80

70 70

Be 60

so 50

40 40

30 30

20 20

10 10

0 0

0.00 5.00 10.00 IS.00 20.00 0.00 5.00 10.00 15.00 20.00

Retention Time (mm)

Figure 2.7.2 The chemical structure of Betanin (A) and Betanidin (B). The isomer of betanin and betanidin is the C-1 5 epimer of the corresponding compound (see

Nilsson, 1970).

(A)

6' CH,OH

COOH 19 1 20 H

(B)

46 of betanin 50 mg of betanin powder (Apin, Chemical Ltd, Tokyo) was dissolved in 10 ml of distilled water and after filtration, the solution was hydrolysed for 30 min at room temperature with 20 ml of 1.5 M ammonia (Fisons, England). After the mixture had turned completely yellow, the visible spectrum of the solution was measured to confirm that betanin had been completely hydrolysed to betalamic acid (maximum absorption at 424 run). Then 5 mg of dopamine were added to 5 ml of the prepared betalamic acid. The preparation was stirred for 15 min and the pH adjusted to 6 with

HC1, the solution reduced to dryness in a rotary evaporator at 28°C and then the residue redissolved in water (HPLC grade, BDH, England, UK). The absorption spectrum and HPLC analysis (see 2.6) of the preparation are shown in Fig. 2.7.4 (A) and (B) respectively. The maximum absorption of miraxanthin V was at 472 nm and retention time of miraxanthin V was recorded at 16.94 mm, with little variation between different runs.

Figure 2.7.3 The chemical structure of miraxanthin V as elucidated by Steglich and

Strack (1990).

OH Lç OH

CH2 CH2 HN

eO: . 1 CoH

47 Figure 2.7.4 The visible spectrum of a miraxanthin V standard (A) prepared

according to the method described in 2.7.2, and the HPLC chromatogram, monitored

at 472 nm, (B) of the preparation according to the method described in 2.6.

(A)

0.35 472 0.3 0.25 C) 0.2 0.15 U, 0.1 0.05 0 400 450 500 550 600 650 Wave length (nm)

100

30 I 10

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

Retention Time (mm)

48 2.7.3 Betalamic Acid Standard

Betalamic acid (see Fig. 2.7.5) standard was prepared by the alkaline hydrolysis of betanin as described in 2.7.2. After hydrolysis was completed the solution was reduced to dryness in a rotary evaporator at 28°C and then dissolved in water (HPLC grade, BDH, England, UK) and analysed by HPLC using the methods described in 2.6. The visible spectrum and HPLC chromatogram of the prepared betalamic acid are shown in Fig. (2.7.6) (A) and (B) respectively. The compound has a maximum absorption at 424 nm, and a retention time of 6.97 min with little variation between different runs.

Figure 2.7.5 The chemical structure of betalamic acid (see Böhm and Rink, 1988).

HOOC COOH

49 Figure 2.7.6 The visible spectrum of a betalamic acid standard (A) prepared as

described in 2.7.2, and the HPLC chromatogram monitored at 424 nm (B) of the

preparation as analysed according to the method described in 2.6.

(A)

0.7 424 0.6 0.5 0 U C 0 0.4 .0 0 0.3 (6) .0 .( 0.2 0.1

350 400 450 500 550 600 Wave length (nm)

(B) 100

40 LZ rA 30

0.00 b.O1i 10.00 lb.00 L?LôIO 30.0fl0 35.00 40.00

Retention Time (mm)

50 2.7.4 Tyrosine and Dopamine Standards

Tyrosine and dopamine (see Fig. 2.7.7) standards were purchased from Sigma

(Chemical ltd, UK). The HPLC chromatograms of these compounds are shown in Fig.

2.7.8. The average retention times of tyrosine and dopaniine are 7.04 min and 7.86 min respectively.

Figure 2.7.7 The chemical structureoftvrosine donamine

(A) (B)

HO__/__ CH2 CH2 NH2

",I HO H2N COOH HO

2.8 Quantification of Compounds

The amounts of betacyanin, miraxanthin V, and betalamic acid in samples were determined according to their molar extinction coefficients based on the laws of

Lambert and Beer (see Dawes 1957) using the following formula:

C=OD C where; C = Molar concentration of pigment

OD = Optical density

C Molar extinction coefficient 56,000 for betacyanins, 48,000 for betaxanthins and 24,000 for betalamic acid (Girod and Zryd 1991)

CfL S5 I Figure 2.7.8 HPLC chromatogram monitored at 280 nm of tyrosine (A), and dopamine (B) standards analysed under the conditions described in 2.6.

100

00 r-1

0 rID 100 10 Es Be

U • vu a. U.9 IU.VJ I n .IOJ W.WIO .IOO iO.O 3.00 40.00

Retention Time (mm)

52 The amounts of tyrosine and dopamine in samples were determined using the calibration curves presented in Fig. 2.8.1.

2.9 Determination of Tyrosine Hydroxylase Activity

2.9.1 Preparation of Crude Enzyme Extract

All steps in the preparation of tyrosine hydroxylase were carried out at 2-4°C.

Cells from 14 day old suspension cultures were harvested, weighed and then stored at

-20°C. At the time of analysis, frozen cells (6 g f.wt.) were allowed to thaw and then homogenised in a pestle and a mortar, on ice, with 8 ml of 0.1 M phosphate buffer

(pH6) containing 0.5 M sodium chloride and 25 mM sodium ascorbate. The homogenate was then centrifuged at 1600 xg for 30 mm. The pellet was then washed twice in 5 ml of the same buffer and centrifuged for 30 mm. The pooled supernatants were then collected and the final volume was adjusted to 20 ml.

2.9.2 Determination of Soluble Protein Content in the Enzyme Extract

Protein content of the enzyme extract was determined using the method of

Bradford (1976). The method is based on the fact that Coomassie Brilliant Blue G-

250 exists in two forms of different colours, red and blue. The red form is converted to the blue form as a result of binding to protein. The protein dye complex has a high extinction coefficient at 595 nm which facilitates the sensitive measurement of protein.

53 Figure 2.8.1 Calibration curves for tyrosine (A) and dopamine (B) standards used for the estimation of these compounds.

y = - 4.1654e-3 + 0.19839x R"2 = 1.000

3.0

2.5

a, 2.0 C U, 0 >.

E 1.0

0.5

0.0 0 2 4 6 8 10 iw; Peak area (x unit) A. 280 nm

y = 2.2338e-2 + 9.0012e-2x RA2 = 1.000

3.0

2.5

C E ca 1.5 0

0.5

0.0 0 5 10 15 20 25 30 -4 Peak area (xlO )A.280nm

54 Dye solution was prepared by dissolving 100 mg of Coomassie Brilliant blue

G-250 (United States Biochemical Corporation, Cleveland, Ohio) in 50 ml of 95% ethanol followed by addition of 100 ml of 85% w/v phosphoric acid (BDH, England,

UK). The final volume of the solution was made up to 1 litre and the dye solution was stored in the dark at room temperature.

Protein content of the enzyme extract was estimated by addition of 5 ml of dye solution to 0.1 ml of enzyme extract. The mixture was vortexed for 20 seconds, allowed to stand for 5 min and the absorption was measured at 595 tun. The amount of protein in the sample was estimated from a calibration curve made with a solution of Bovine Serum Albumin (10-100 rig) (Sigma, UK) (Fig. 2.9.1).

2.9.3 Assay of Tyrosine Hydroxylase Activity

A modification of the method of Arnow (193 7) was used for the estimation of tyrosine hydroxylase activity. The principle of the method is that in acid solution, 3,4 dihydroxyphenylalanine (DOPA), the product of the enzyme, reacts with nitrous acid to give a yellow colour, this colour changes to orange-red, with a maximum absorption at 510 nm, in the presence of excess sodium hydroxide.

The assay was carried out by the addition of 0.2 ml of enzyme extract to 0.8 ml of 2 mM tyrosine in 0.1 M phosphate buffer at pH 6. The mixture was incubated at

27°C for 15 mm, after which the enzyme reaction was stopped by the addition of 0.5 ml of 0.5 M HC1. Then 1 ml of nitrite molybdate, freshly prepared by dissolving 1 g

55 Figure 2.9.1 Calibration curve for the estimation of protein using Bovin Serum

Albumin as a standard (Bradford 1976).

y= -10.472+108.29x RA2=0.990

120

iL'Iø •i C E n 80 ca C > 0 60 S C

I- 0 0) ce

ft 0.2 0.4 0.6 0.8 1.0

ODat595nm

56 of sodium nitrite and 1 g of sodium molybdate in 10 ml distilled water, was added. The mixture was vortexed for 30 seconds, left to stand for 5 min and then 0.5 ml of 2 M

NaOH was added. Immediately after the addition of NaOH the absorption at 510 nm was measured. The same assay mixture with inactivated enzyme (inactivated by the addition of 0.5 ml of 0.5 M HC1 to 0.2 ml of enzyme extract), at time zero was used as a control. The amount of DOPA produced by the enzymatic reaction was estimated from a calibration curve made with standard solutions of DOPA (Sigma, UK) (Fig.

2.9.2). Enzyme activity is expressed as units (U). One unit is the amount of enzyme that catalyses the formation of 1 tmol of DOPA per minute. The specific activity was calculated by dividing the activity of enzyme by the amount of protein in mg.

2.10 Statistical Analysis

Statistical analysis (see Gomez and Gomez 1984) was carried out by a computer using a statistical package program, Minitab "Release 10" (see Minitab Manual 1994).

Unless otherwise stated, three replicates, at least were employed in experiments and the mean was then calculated. Standard error of the mean (se.) was calculated in order to estimate the variation within the group of replicates in each case.

Data expressed as percentages were subjected to arcsine transformation, whenever the percentage value in a set of data falls out of the range (30-70), and statistical manipulations were carried out on the transformed values (see Sokal and Rohlf 1981).

This was done because percentage values do not conform to the normal distribution and

57 Figure 2.9.2 Calibration curve made with standard solutions of DOPA for the estimation of DOPA produced by tyrosine hydroxylase in the enzyme assay (Arnow

1939).

y=62.142+ 1671.8x RA2=0.983

1200

1000

800 . c'j Q. 0 1, 600 . 0 E 400

200

0 0.0 0.1 0.2 0.3 0.4 0.5 0.6

ODat5lOnm

58 so statistical methods which assume that sample data were normally distributed about a mean cannot be applied to their analysis. Tables found in Sokal and Rolf (1969) were used for transformations.

Analysis of variance and multiple comparison of the means (FISHER multiple comparison) were applied in order to assess the significancy in the differences between means. T-test was employed for comparison between two treatments. The correlation between two variables was determined by the correlation coefficient (R).

59 CHAPTER THREE

RESULTS

60 This chapter is divided into four main sections (3.1, 3.2, 3.3, and 3.4). In

section 3.1 the growth and betalain accumulation in suspension cultures of Beta

vulgaris was characterised. In the following section (3.2) the effect of culture

conditions on growth and betalain production was investigated. Subsection 3.2.1 dealt

with the effect of manipulation of major nutrients (nitrogen, phosphate, and sucrose),

subsection 3.2.2 examined the effect of growth regulators supplementation (2,4-D and

kinetin), and in subsection 3.2.3 the application of the precursor tyrosine and DOPA

was tested. In section 3.3 the production of betalain in cell cultures of Beta vulgaris

was investigated in relation to cellular heterogeneity. This section includes three

subsections 3.3.1, 3.3.2, and 3.3.3. In subsection 3.3.1 various cell lines (violet,

orange, yellow, and white) were derived from the original culture of Beta vulgaris, by

cloning single cells of different colours. The cell lines were characterised with regard

to growth, betalain content, betalain related compounds content, and the proportion of pigmented cells. The stability of the cell lines with regard to betalain production and the proportion of pigmented cells was also examined over a period of 15 months. In

subsection 3.3.2 the production of betalain in the violet cell line under modified culture conditions was investigated focusing on the relationship between the production of betalain and cellular heterogeneity. Subsection 3.3.3 examined the effect of culture conditions on the production of betalain and cellular heterogeneity in the yellow cell line. Finally, in section 3.4 the relationship between the activity of tyrosine hydroxylase and the final yield of betalain in Beta vulgaris cultures was explored.

61 3.1 CHARACTERISATION OF GROWTH AND BETALAIN

ACCUMULATION IN SUSPENSION CULTURES OF Beta vulgaris

The accumulation of the majority of secondary products in plant cell cultures occurs at the end of the growth cycle (stationary phase) when cell division has ceased or slowed down (e.g. Lindsey and Yeoman 1983, Hirose et al., 1990, Böhm et al.,

1991, and Sakuta et al., 1994). This inverse relationship between the growth and secondary metabolite accumulation has been related to the competition between primary and secondary metabolism for common precursors (Phillips and Henshaw

1977, and Yeoman et al., 1980). In such cases limitation of growth, i.e. by manipulation of culture conditions, may result in an enhanced production of secondary metabolites (see Lindsey and Yeoman 1984, Lindsey 1985, 1986, Hall and

Yeoman 1991). However, there are some instances in which production of secondary metabolites takes place during the active growth phase (see for example Corduan and

Reinhard 1972, Sharma and Khanna 1980, Kadkade 1982, Hirose et al., 1990, and

Sakuta 1994). Betacyanin in particular has been reported to accumulate and reach the maximum levels during the logarithmic phase of growth in Phytolacca amricana cell cultures (Sakuta et al., 1986, 1994 and Hirose et al., 1990). In Beta vulgaris cell cultures Jaafar (1992) also showed a positive correlation between growth and betalain production and the maximum betalain was achieved when cell proliferation was maximal. In contrast, Monroy et al. (1994) reported that betalain production in Beta vulgaris cell cultures was not growth related. So, different secondary metabolites may have different patterns of accumulation in cultured cells. In addition, the accumulation

62 of the same product may vary in different cultures. In this experiment, therefore, growth and betalain production in suspension cultures of Beta vulgaris, was characterised in order to determine the relationship between growth and pigment accumulation throughout the growth cycle of the culture.

Cultures were initiated by inoculating 1 g (wet weight) of cells, obtained from a 13 day old stock cell culture (see 2.1.1), into 50 ml of standard growth medium (see

2.1.2.1) contained in a 250 ml Erlenmeyer flask. The cultures were then placed on an orbital shaker and incubated under the conditions specified in 2.1.4. The growth of cultures and betalain production as well as the changes in the proportion of pigmented cells were followed by taking samples on days 0, 2, 5, 9, 13, 15, 17 and 21. At each sampling time three flasks were harvested and measured. The growth of the culture was assessed by determining the fresh weight, dry weight, packed cell volume, cell number and cell viability of cultures according to the methods described in 2.2. The betalain content was extracted as indicated in 2.3 and analysed according to the method described in 2.4 and 2.5. The proportion of the pigmented cells was determined as described in 2.2.6.

Results presented in Fig. 3.1.1 show the changes in fresh weight (A), dry weight (B), packed cell volume (C), cell number (D) and cell viability (E) of the cultured cells over a period of 21 days. As shown by the figure, the fresh weight

(f.wt.) and packed cell volume (PCV) remained almost unchanged during the first two days. After this brief lag phase the f.wt. and PCV increased steadily until day 15 after which no significant increase was observed. After day 17 the f.wt. and PCV decreased

63 Figure 3.1.1 Characterisation of the growth of a Beta vulgaris suspension culture; (A)

culture fresh weight, (B) culture dry weight, (C) packed cell volume, (D) cell population

density per ml of the culture, (E) proportion of viable cells. Each value is s mean of 3

replicates ± Se. (A) (B) 10 0.5

8 0.4

0.3

LD 4 02

0 2 0.1

01. ,.i . i . ..,.i . ,...i . .. I 0.0 "I"_1 I 0 2 4 6 8 10 12 14 18 18 20 22 0 2 4 6 8 10 12 14 16 18 20 22 Time from subculture (d) Time from subculture (d)

24 12

10.

> 16 8 0 0 LD 12 2 6

8 4.

4. 2

TU.U.I.,U I,', o ' i.i...Ii...T. 0 2 4 6 8 10121416182022 0 2 4 6 810121416182022 1 Time from subculture (d) Time from subculture (d)

(E)

100 70

90 65 0

80 60

70 55 -0-- Percent. value $ Armin value 60 50 0 2 4 6 8 10 12 14 16 18 20 22 Time from subculture (d)

64 until the end of experiment. The patterns of dry weight (d.wt.) and cell number (cell

no.) change were also similar to that of f.wt. and PCV, nevertheless the dry weight

increased slightly on day 2 but the cell number did not. In addition there was no

significant increase between day 9 and 13. However, that was followed by a sharp

increase between day 13 and 15 after which it remained almost constant. The

proportion of viable cells increased during the first five days, this probably resulted

from the death of some cells during subculture. However, after day 5 the viability of

cells remained almost constant (Ca 82%) until day 17. After that it decreased up to the

end of culture period.

The betalains produced by the cultures were separated by TLC (see 2.5) of a

cell extract, in 80% methanol, obtained from a sample taken at time 0. Figs. 3.1.2 (A) and (B) show the separated pigment bands on a TLC plate and their corresponding visible spectra. Two distinct pigment bands were separated, one violet (a) and one yellow (b). The violet pigment band (RF = 0.3) was identified as betacyanin and has an absorption maximum at 535 nm. The yellow pigment (RF = 0.7) was identified as betaxanthin and has an absorption maximum at 472 nm.

Fig. 3.1.3 presents the pattern of betacyanin and betaxanthin accumulation as expressed per g fresh weight (A) and per culture (B) as well as the change in the proportion of pigmented cells (C) throughout the culture cycle. As can be seen from

Fig. 3.1.3(A) the amount of betacyanin and betaxanthin per g. f.wt. had increased slightly by day two, indicating that the onset of betalain synthesis started during the lag phase probably prior to, or immediately after, the start of cell division. However, between day 5 and day 9 the amount of pigments per unit f.wt. had decreased to reach

65 Figure 3.1.2 The separation of betacyanin (a) and betaxanthin (b) from a cell extract

(time 0), in 80% methanol, of Beta vulgaris on TLC plate (A), and the corresponding visible spectrum (B) of the separated betacyanin (a) and betaxanthin (b).

Solvent Front

(a)

0.20

0.18 535

0.16 472 0.14

0.12 C-) C (a) ca - 0.10 0 (I) 0 < 0.08

0.06

0.04 (b)

0.02

0.00 400 450 500 550 600 650 Wave length (nm) Figure 3.1.3 The pattern of betacyanin and betaxanthin accumulation throughout the growth cycle of a Beta vulgaris cell culture, (A) content of betacyanin (measured at 535 nm) and betaxanthin (measured at 472 nm) per g. fresh weight, (B) total betacyanin and betaxanthin per culture, (C) the percentage of pigmented cells. Each value is a mean of 3 replicates ± se.

(A) 1.6

1.41

!1.2 0' 10 -C--- B.cyanin 0.8 B.xanthin

0.6

0 0.4

0.2 I 0 2 4 6 810121416182022 Time from subculture (d)

101 I

a 8

I C a) - B.cyanin E B.xanthin .2' 4 0. 0 02

01. IIU'IIUIIUU I 0 2 4 6 8 10 12 14 16 18 20 22 Time from subculture (d)

LO 60

a, 50 E .2' 0.

301. •u-,I•II•I'I•u • u • I o 2 4 6 810121416182022 Time from subculture (d)

67 a value which was approximately equal to the starting value. Between day 9 and 13 there was further slight decrease in pigment content followed by an increase from day

13 up to day 15. Subsequently the betacyanin level decreased and the betaxanthin remained constant until the end of the culture period. The amount of betacyanin and betaxanthin accumulated per culture increased from day 0 until day 15, then levelled off. The betacyanin per culture decreased sharply between day 17 and the end of the experiment, by day 21 the pigments had begun to leach out into the medium and the cultures had turned black. The proportion of pigmented cells more or less paralleled the accumulation of betalain per g. f. wt. during the culture cycle.

Overall the results show that the pattern of growth of suspension cultures of

Beta vulgaris includes a lag phase which lasts for 2 days followed by a phase of active cell division and growth followed by a stationary phase which was reached around day 15. Both betacyanin and betaxanthin were produced by the culture and the onset of betalain synthesis occurred during the lag phase, probably prior to, or immediately after, cell division had started. However, the accumulation of pigments was synchronised with growth and the maximum level was attained when growth had reached a maximum. The changes in the proportion of pigmented cells throughout the growth cycle closely followed the changes in betalain content per g. f.wt.

Having found that the accumulation of betalains correlates closely with growth, it might be expected that culture conditions which modify growth would also affect betalain accumulation. In the next section an attempt was made to optimise the conditions of culture for optimum growth and also to enhance betalain production.

68 3.2 EFFECT OF CULTURE CONDITIONS ON GROWTH AND BETALAIN

ACCUMULATION IN SUSPENSION CULTURES OF Beta vulgaris

There are number of articles in the literature reporting the crucial effect of environmental factors on growth and secondary metabolism in cell and tissue cultures.

These factors include light, temperature and composition of the culture medium. The effects of various components of the medium on cell growth and the production of secondary metabolites have been reviewed by Kato et al. (1977), Dougall (1980),

Fujita et al. (1981), Mantell and Smith (1983), De-Eknamkul and Ellis (1985),

Yeoman and Yeoman (1996). However, it is well known that the nutrient and growth regulator requirements to enable maximum growth or product formation in cultured plant cells differ from species to species and from product to product. In addition, a medium sufficient for growth is not always sufficient for optimum production of the metabolite of interest, therefore it is important to determine the optimum composition of the medium for each case.

In the following set of experiments, the effects of various concentrations of several components of the medium including major nutrients (nitrogen, phosphorus and sucrose), growth regulators (2,4-D and kinetin), and betalain precursors (tyrosine and DOPA) supplementation were investigated, on growth and betalain production in an attempt to optimise culture yield.

69 3.2.1 Effect of Manipulation of Major Nutrients on Growth and Betalain

Production

Nitrogen, phosphorus, and sucrose are major nutrients in the medium of cell

and tissue cultures and manipulation of these compounds can influence not only the

growth but also secondary metabolite production (see Knobloch and Berlin 1980,

Sakuta et al., 1986, 1987, Hirose et al., 1990, Kitamura etal., 1991, and Norton et al.,

1991). In this subsection, therefore, the effect of various concentrations of each of

these nutrients was examined on the growth and betalain accumulation.

3.2.1.1 Effect of the Concentration of Inorganic Nitrogen in the Medium on

Growth and Betalain Accumulation

Nitrogen is an essential nutrient element in the medium of tissue cultures and

plays an important role not only in growth, but also in secondary metabolism (Dougall

1980). Generally, it is added to the medium in a form of nitrate NO 3 and/or

ammonium N114+. However, the form in which nitrogen is provided and the

concentration supplied have a critical effect on the production of secondary metabolites. The role of this element in secondary metabolism has been investigated in relation to growth in many different systems. In some systems, an inverse relationship was observed between growth and metabolite production and here limitation of nitrogen resulted in an enhanced production of secondary products

(Amorim et al., 1977, Knobloch and Berlin 1980, Yamakawa et al., 1983, Lindsey

1985). In contrast, in other systems secondary product accumulation was increased by

70 raising the total amount of nitrogen in the medium. For example, the formation of

shikonin derivatives in cell cultures of Lithospermum eythrorhizon increased with

increasing nitrogen in the medium within the range 67 to 104 mM, and decreased at

nitrogen concentrations higher than 134 mM (Mizukami et al., 1977). In addition, in

Phytolacca americana, betacyanin accumulation per cell increased as the total

nitrogen concentration rose within the range 0-40 mM (Sakuta et al., 1987). The aim

of this experiment was to investigate the effect of various concentrations of inorganic

nitrogen (i-N) on the growth and betalain accumulation on Beta culture.

Four different media (Gainborg's 135) with various concentrations of nitrogen

were prepared as described in 2.1.2.2. The level of nitrogen in the media was altered

by the addition of different amounts of (NH 4 ) 2 SO4 and KNO3 keeping the same ratio

of NH4 :NO3 (1:11) originally found in the standard 135 medium. The final

concentrations of inorganic nitrogen (i-N) in the prepared media were (14.5 mM, 29

mM (control), 58 mM, and 116 mM). After preparation, the media were poured into

250 ml Erlenmeyer flasks (50 ml per flask) and autoclaved. Each flask was then

inoculated with 1 g (wet weight) of cells obtained from well mixed 13 d. old cultures.

All flasks were then placed on an orbital shaker and incubated under the conditions

described in 2.1.4. At regular intervals of 5 days, 3 flasks of each treatment were

harvested for the determination of fresh weight (see 2.2.1) and betalain content (see

2.3 and 2.4).

Fig. 3.2.1 shows the pattern of growth, in terms of fresh weight of cultures in media with different concentrations of nitrogen. As can be seen from the control

71 Figure 3.2.1 Changes in fresh weight of suspension cultures of Beta vulgaris grown for

20 days in media with various concentrations of inorganic nitrogen (i-N). Each value is a mean of 3 replicates ± Se.

12 —0-- 14.5mMi-N

10 -0-- 29.0mM i-N

58.0mM i-N

116 mMi-N

0 5 10 15 20 25

Time (days)

72 medium values (i-N 29 mM) little increase in fresh weight was attained within the

first 5 days, as this period includes the lag phase. After day 5 the fresh weight

increased more rapidly and linearly until day 15 at which a 9.4 fold increase had been

achieved. Subsequently the culture fresh weight decreased slightly until the end of the

experiment. On media with a nitrogen (i-N) concentration at either 14.5 mM or 58

mM the fresh weight increase was closely similar to the control. On the medium with a nitrogen concentration of 116 mM the fresh weight of cultures was significantly lower than the control on days 10, 15, and 20.

The amounts of betacyanin accumulated per g. fiwt. under the different treatments are shown in Fig. 3.2.2 (A) from which it can be seen that, in the control medium (i-N = 29 mM) the production of betacyanin per g. f.wt. was more or less constant over the growth period. In the different treatments, betacyanin accumulation was not significantly different from the control. However, per culture (Fig. 3.2.2 (B)) betacyanin accumulation was similar to the control at nitrogen concentrations of (i-N

= 14.5 mM) and (i-N = 58 mM) whereas it was significantly lower than the control on day 15 and 20 at a nitrogen concentration of (i-N = 116 mM).

Fig. 3.2.3 presents the amounts of betaxanthin accumulated throughout the culture period, under treatments, as expressed per g. fresh weight (A) and per culture

(B). As shown by the control values (i-N = 29 mM) the production of betaxanthin per g. fresh weight was almost constant during the growth period until day 15 after which it decreased. In the treatments betaxanthin accumulation was not significantly different from the control. The total betaxanthin accumulated per culture (Fig. 3.2.3

Figure 3.2.2 Changes in the betacyanin content per g. fresh weight (A) and per culture

(B), measured at 535 nm over 20 days in cell cultures of Beta vulgaris grown in media

with various concentrations of inorganic nitrogen (i-N). Each value is a mean of 3

replicates ± Se.

1.4

- 1.2

C) D 14.5 MM i-N

S 29.0mM i-N - 1.0 CO —0-- 58.0mM i-N CO -•--- 116 MM CD 0.8

0.6 I • • • •• I 0 5 10 15 20 25 Time (days)

10. I

C.) - D-- 14.5mMi-N

U- 6 C S 29.0mM i-N C CO >-. —0-- 58.0mM i-N

ci) •-O- -- 116 mMi-N

0-f 0 5 10 15 20 25 Time (days)

Figure 3.2.3 Changes in the betaxanthin content per g. fresh weight (A) and per

culture (B), measured at 472 nm, over 20 days in cell cultures of Beta vulgaris grown

in media with different concentrations of inorganic nitrogen (i-N). Each value is a

mean of 3 replicates ± Se.

1.4

1.2

C) -- 1.0 —•0-- 14.5mMi-N C S 29.0mM i-N

0.8 --- 58.0mM i-N

—O— 116 mMi-N

8 0 .6

0.4 I • • • • I 0 5 10 15 20 25 Time (days)

10. I

—•O--- 14.5mMi-N C 6 5 29.0mM i-N C —a--- 58.0mM i-N

a) lop 116 mM i-N 02 0

0 - I 0 5 10 15 20 25 Time (days)

75 (B)) was also not significantly different from the control.

The overall results show that decreasing the concentration of inorganic nitrogen to half (i-N = 14.5) of that originally present in B5 medium, or increasing it up to the double (i-N = 58 mM) the level, had no significant effect on either growth or betalain production, however, elevating its concentration up to (116 mM) resulted in a reduction in the growth of culture, in terms of fresh weight. However, the production of betacyanin and betaxanthin, on a per g. f.wt. or per culture basis, was not significantly influenced.

In the next experiment, the effects of various concentrations of phosphorus, another major nutrient, was examined on growth and betalain production.

3.2.1.2 Effect of the Concentration of Inorganic Phosphorus in the Medium on

Growth and Betalain Accumulation

The results of the previous experiments showed that altering the concentration of inorganic nitrogen in the medium did not enhance either growth or betalain production. In this experiment the effects of various concentrations of phosphorus was studied. Phosphorus is an essential element in the tissue culture media and the effects of this element on growth and secondary metabolism in cell and tissue cultures have been investigated by a number of researchers for example Merillon et al., 1983,

Knobloch and Berlin 1981, 1983, Lindsey 1985, Yamamoto et al., 1989, Hirose et al.,

1990 and Deliu et al., 1992. With regard to betalain production, the elimination of phosphate from the medium results in a more intensively pigmented callus of Beta

76 vulgaris (Constable and Nassif-Makki 1971). In contrast, omitting phosphate from the medium of cell cultures of Chenopodium rubrum L. (Berlin et al., 1986) caused a significant decrease in betacyanin production and completely suppressed pigment synthesis in Phytolacca americana cell cultures and this inhibition was reversible by the addition of phosphate (Hirose et al., 1990). Sakuta et al. (1986) reported that betacyanin production and cell number were increased by raising the phosphate concentration within the range 0-1.25 mM in Phytolacca americana cell culture. In

Beta vulgaris cell cultures (Monroy et al., 1994) showed that phosphate was rapidly consumed and its absence from the medium could be correlated with a betalain increase. Therefore, this experiment was carried out in order to determine the optimum concentration of phosphorus for growth and betalain production.

Media (Gamborg's 135) with different concentrations of phosphorus were prepared as described in 2.1.2.2. Phosphate (NaH2PO4.21-120) was added to the media at various concentrations varying from 0.5 up to 4 times the standard level of B5 medium to attain final concentrations of inorganic phosphorus (i-P) as follows (0.55 mM, 1.10 mM (control), 2.2 mM and 4.4 mM). After preparation, the media were poured into 250 ml Erlenmeyer flasks (50 ml per flask) and autoclaved. Each flask was then inoculated with 1 g (wet weight) of cells obtained from well mixed 13 d. old cultures. All cultures were then incubated on an orbital shaker under the conditions described in 2.1.4. At regular intervals (5 days) three flasks for each treatment were harvested and the fresh weight (see 2.2. 1) and betalain content (see 2.3 and 2.4) were determined.

77 Results presented in Fig. 3.2.4 show the changes in fresh weight of cultures grown at different phosphate concentrations. In the control (i-P = 1.1 mM) little increase in culture fresh weight was observed within the first 5 days, as this period includes the lag phase. After 5 days the fresh weight increased rapidly and linearly to attain a 9 fold increase by day 15. After that it remained almost constant. In the treatment where the phosphorus level was reduced to half the control (i-P = 0.55 mM), the increase in culture fresh weight was similar to the control within the first 10 days but was significantly lower than the control after 10 days until the end of experiment.

In treatments in which the phosphorus concentration was increased up to 2.2 mM or

4.4 mM, the increases in the fresh weight were not significantly different from the control.

Fig. 3.2.5 (A) presents the amounts of betacyanin accumulated per g. f.wt. over the culture period at different concentrations of phosphorus from which it can be seen that in the control (i-P 1.1 mM) the betacyanin content of the cultures decreased slightly between day 5 and 10 and then remained almost constant. In the test treatments betacyanin production over the culture period was not significantly different from the control, except that at high concentrations of phosphorus (i-P 2.2 mM, and 4.4 mM) cultures were turning brown/black in colour from day 15 showing death of the cells. This was probably caused by the high concentration of Na ions in the phosphate salt (NaH2PO4.21-120). The amounts of betacyanin accumulated per culture (Fig. 3.2.5 (B)) in the treatments were also not significantly different from that accumulated by the control, apart from a lethal effect which appeared after day 15 at i-

P of 2.2 and 4.4 mM.

78 Figure 3.2.4 Changes in the fresh weight of cell cultures of Beta vulgaris grown for 20 days in media with different concentrations of inorganic phosphate (i-P). Each value is a mean of 3 replicates ± se.

-0-- 0.55 MM i-P

• 1.10mMi-P 10 a 2.20mM i-P

-'O'- 4.40 mM i-P 8 0)

0 4

0 0 5 10 15 20 25 Time (days)

Figure 3.2.5 Changes in betacyanin content per g. fresh weight (A) and per culture

(B), as measured at 535 nm over the culture period in Beta vulgaris cultures grown in media with various concentrations of inorganic phosphorus (i-P). Each value is a mean of 3 replicates ± Se.

1.1

1.0

0.9 0) 0.8 0 055mM i-p • 1.10mMi-P caC >.0.7 —Co•-- 2.20mM i-P 0 a) -•---'-- 4.40mM i-P a) 0.6 a 0 0.5

0.41 0 5 10 15 20 25 Time (days)

0) = 6

0 —Ci-- 0.55 mM i-P C • 1.10mMi-P >CU 0 ---- 2.20mM i-P Co a) ------O------4.40mM i-P

0-I. 0 5 10 15 20 25 Time (days)

80 The changes in betaxanthin content per g. fresh weight over the growth period

in cultures grown at different concentrations of phosphorus are shown in Fig. 3.2.6

(A). As the figure shows, in the control cultures (i-P 1.1 mM) betaxanthin production over the growth period was more or less constant. On day 5 in all of the treatments betaxanthin content per g. fresh weight appeared to be higher than the control but was not statistically significant. However, after day 5 betaxanthin accumulation in all treatments was closely similar to the control until the end of experiment in the treatment of i-P 0.55 mM and up to day 15 in cultures with i-P 2.2 mM and i-P 4.4 mM, after which cultures turned brown/black in colour. On a per culture basis (Fig.

3.2.6 (B)) bataxanthin production was also not affected by any of the treatments, apart from the browning effect which appeared after day 15 at i-P of 2.2 mM and 4.4 mM.

The overall results show that decreasing the concentration of inorganic phosphorus in the medium to 0.55 mM caused a reduction in the growth of cultures but did not significantly affect the production of betalain pigments. In contrast, increasing the concentration of phosphorus, up to 2.2 mM or 4.4 mM had no significant effect on either the growth or betalain production within the first 15 days.

After that the treatments had a lethal effect.

In the following experiment the effect of various concentrations of sucrose, another important component of the medium, on the growth and betalain production was examined.

81 Figure 3.2.6 Changes in betaxanthin content per g. fresh weight (A) and per culture

(B), as measured at 472 nm, over the culture period in Beta vulgaris cultures grown in media with various concentrations of inorganic phosphorus (i-P). Each value is a mean of 3 replicates ± Se.

1.1

1.0

::: -0-- 0.55 MM i-P • 1.10mMi-P

ca 0.7 - 0-- 2.20 MM i-P

-0-- 4.40mM i-P 0.6

0 0.5

0.4 0 5 10 15 20 25 Time(days)

II,]

0 0 0.55 mM i-P C • 1.10 MM i-P C Ca —a— 2.20mM i-P a) •-0-- 4.40mM i-P

0-I 0 5 10 15 20 25 Time (days)

82 3.2.1.3 Effect of Sucrose Concentration on Growth and Betalain Accumulation

In the previous two experiments attempts to stimulate betalain production by manipulating the concentration of nitrogen or phosphate in the medium were not successful. In this experiment attempts were made to enhance the production of betalain by raising the concentration of sucrose. Sucrose is the main source of carbon in tissue culture media, besides its ability to support growth, it has also been shown to affect the production of secondary metabolites. There are a number of references in the literature which show that higher yields of secondary metabolites can be attained by increasing the level of sucrose in the medium, for example, Kitamura et al. (1991) showed that a high concentration of sucrose (7-10%) improved the growth and alkaloid production in root cultures of Duboisia myoporoides and Duboisia leichhardtii, Wysokinska and Swiatek (1991) also observed that increasing the sucrose concentration up to 7% increased the growth and iridoid synthesis in cell culture of Penstemon serrulatus. A similar observation was made by Norton et al.

(1991) on rubber production in tissue cultures of Guayule. In the present experiment the effects of various concentrations of sucrose (2% (control), 3%, 6% and 9%) on the growth and betalain accumulation were examined over a period of 25 days.

Four media of Gamborg's B5 were prepared as described in 2.1.2.1 and sucrose was added at the following concentrations 2% (control), 3%, 6% and 9%: The media were then poured into 250 ml Erlenmeyer flasks (50 ml per flask) and autoclaved. Each flask was then inoculated with 1 g (wet weight) of cells obtained from well mixed 13d. old cultures. The flasks were then placed on an orbital shaker

83 and incubated for 25 days under the standard conditions described in 2.1.4., 3 flasks

were sampled every 5 days for each treatment to determine fresh weight (see 2.2.1)

and betalain content (see 2.3 and 2.4).

Data presented in Fig. 3.2.7 show the changes in fresh weight of cultures grown in the media supplemented with increasing concentrations of sucrose. From which it can be seen that cultures grown in the control medium (2% sucrose) barely increased in fresh weight within the first 5 days which includes the lag phase. After day 5 the fresh weight increased rapidly and approximately doubled every 5 days until day 15. It then remained constant until day 20 after which it decreased up to the end of experiment. In cultures grown with the test concentrations of sucrose (3%, 6% and

9%) the increase in culture fresh weight throughout the culture period was similar to the control until day 15. After that it continued up to day 20 in the case of 3% and 6%, and up to day 25 in the case of 9% suggesting that nutrients were still available and that 2% sucrose limited the growth in the medium.

Fig. 3.2.8 (A) presents the amounts of betacyanin per g. f.wt. accumulated throughout the incubation period in cultures grown at different concentrations of sucrose. From which it can be seen that the production of betacyanin in the control was almost constant over 15 days. Then it declined and after day 20 the cultures turned black and pigment measurement was not available by day 25. In cultures grown at 3% sucrose betacyanin production was similar to the control for the first 10 days but after that it increased and by day 15 the amount of betacyanin produced per g. f.wt. was higher than in the control by ca 45%. After that it remained almost

84 Figure 3.2.7 Changes in the fresh weight of suspension cultures of Beta vulgaris grown for 25 days in media with different concentrations of sucrose. Each value is a mean of 3 replicates ± se.

—0-- 2% suc. 12 __• 3%suc.

10 a 6%suc.

- —0—• 9% suc. 8

0 -f 0 5 10 15 20 25 30

Time (days)

Figure 3.2.8 Changes in the betacyanin content per g. fresh weight (A) and per culture

(B), as measured at 535 run, over 25 days in cell cultures of Beta vulgaris grown in media

with different concentrations of sucrose. Each value is a mean of 3 replicates ± Se.

1.8

1.6

1.4

CD 1.2 —0--- 2% sue.

. 3% sue. CO

—0-- 6% sue.

—c•— 9% sue.

0.2 u .i. 0 5 10 15 20 25 30 Time (days)

20 ci) 16

—a--- 2% sue. . 12 C • 3% sue. CIS >.. 0 —a--- 6% sue. CIS 8 ci) —O 9% sue.

0 -I. 0 5 10 15 20 25 30 Time (days)

86 constant until the end of experiment. With 6% sucrose, betacyanin accumulation was similar to the control and was constant until day 20 after which it decreased. At 9% sucrose, the betacyanin content of cultures was not significantly lower than that of the control cultures on day 5 and 10, but by day 15 the betacyanin content was significantly less than in the control by ca 52%. However, after day 15 the betacyanin content increased until day 25 matching the levels accumulated by the control between day 0 to 15. It would appear that within the first 15 days, a high concentration of sucrose (9%) depressed betacyanin production but after day 15 when the level of sucrose in the medium had decreased, as a result of the consumption by growing cells, the inhibitory effect was relaxed.

The accumulation of betacyanin per culture over the growth period under the different treatments is shown in Fig. 3.2.8 (B). As can be seen from the control values there was little increase in betacyanin content over the first five days. After that, the amount of betacyanin approximately doubled every 5 days until day 15 at which time a maximum was achieved. After day 20 pigment leached into the medium and the culture turned brown/black in colour. The results were similar for 3% of sucrose where the production of betacyanin was similar to the control within the first 10 days.

After that, pigment accumulation increased, over the control, and by day 15 the amount of betacyanin produced had exceeded the control by ca 45%. Subsequently it remained more or less constant up to the end of experiment. In 6% sucrose betacyanin accumulation was similar to the control, however, it continued until day 20 then decreased. 1119% sucrose betacyanin production was lower than the control within the

87 first 15 days. After that it increased linearly until day 25 when it matched that produced in 3%.

The data presented in Fig. 3.2.9 (A) show the amounts of betaxanthin produced per g. fresh weight throughout the culture period. In the control culture (2% sucrose) the production of betaxanthin was more or less constant over the growth cycle. At 3% betaxanthin accumulation followed pattern similar to the control within the first 15 days. After that, however, it increased and by day 25 the amount of betaxanthin was much higher by ca 80%. In 6% of sucrose, the betaxanthin contents on day 5 and 10 were also similar to the control but on day 15 and 20 they were significantly higher than the control. In 9% sucrose, the production of betaxanthin decreased from the onset of subculture until day 15. After that it increased and by day

25 the level of betaxanthin achieved was similar to that produced by the control cultures on day 10.

Fig. 3.2.9 (B) shows the pattern of betaxanthin accumulation per culture over the growth period. As can be seen the amount of betaxanthin in the control increased only slightly by day 5 but subsequently increased rapidly until day 15. Then it remained stable up to day 20 after which cultures died. In 3% sucrose, the betaxanthin content was similar to the control within the first 15 days, after that, however, it increased linearly until the end of experiment (day 25) by which time the amount of betaxanthin was ca 2.3 that produced by the control on day 15. In 6% sucrose, the production of betaxanthin was closely similar to that in 3% sucrose over 20 days of growth after which it decreased, however, by day 20 the amount of betaxanthin

88 Figure 3.2.9 Changes in the betaxanthin content per g. fresh weight (A) and per culture

(B), as measured at 472 nm, over 25 days in cell cultures of Beta vulgaris grown in media with different concentrations of sucrose. Each value is a mean of 3 replicates ± Se.

1.8

1.4

C) • 2% C -c 1.0 —0-- 3% C Cu • 6%suc.

ci) —0-- 9% suc. -o 0.6 0 0

0.2 - f 0 5 10 15 20 25 30 Time (days)

C) 16

0 • 2%suc. C 12 -C —0-•-- 3%suc. Cx Cu -•-- 6%suc. U) Cu ci) —0- 9%suc. -o 04 0

0 - f 0 5 10 15 20. 25 30 Time (days)

89 produced in 6% was not significantly different from the control. In 9% sucrose betaxanthin accumulation was significantly lower than the control within the first 15 days. After that, however, it increased until the end of culture period (day 25) and reached a value similar to that achieved on day 20 in 3% sucrose.

Overall the results show that increasing the sucrose concentration in the medium from 2% up to 3%, 6% or 9% did not affect the rate of growth, in terms of fresh weight but extended the period of growth. With regard to betalain accumulation,

3% sucrose enhanced the production of betacyanin and betaxanthin, however, the stimulatory effect appeared after day 10 in the case of betacyanin and after day 15 in the case of betaxanthin. At a sucrose concentration of 6% betalain accumulation was not significantly different from the control for the first 15 days. On day 20 the betaxanthin but not the betacyanin content per g. f.wt. was slightly higher than the control, and in 9% sucrose betalain accumulation was inhibited within the first 15 days after which the inhibitory effect was overcome.

Results from this subsection showed that betalain production could not be enhanced by altering the concentration of nitrogen or phosphorus in the medium but it could be stimulated by increasing the concentration of sucrose up to (3%).

In the following subsection the effect of growth regulators supplementation on the growth and betalain production was investigated.

90 3.2.2 Effect of Growth Regulators Supplementation on Growth and Betalain

Production

Plant growth regulators, particularly auxins and cytokinins are important components in the medium of cell and tissue cultures. The type and concentration as well as the balance of auxin and cytokinins are critical in determining growth and secondary metabolites production (see El-Bahr et al., 1989, Panda et al., 1992, and

Choi et al., 1994). However, requirement for these phytohormones varies from product to product and from one culture to another. In this subsection, experiments were carried out in order to optimise the level of 2,4-D and kinetin in the medium for growth and betalain production.

3.2.2.1 Effect of Different Concentrations of 2,4-D on Growth and Betalain

Production

The Auxin 2,4-D "dichiorophenoxyacetic acid" has been reported to influence the production of betacyanin in culture systems of different species producing this metabolite (Sakuta et al., 1986, Komamine et al., 1989 and Hirose et al., 1990). In callus cultures of Beta vulgaris Constable and Nassif-Makki (197 1) have reported that

1 mgT' of 2,4-D inhibited the formation of betacyanin while removal of this auxin from the medium, resulted in the reappearance of the pigment. Therefore, in the following set of experiments, the effect of various concentrations of 2,4-D on the growth and betalain production was investigated.

91 3.2.2.1.1 Effect of 2,4-D at concentrations ranging from 0 to 0.2 mg.F'

In this experiment the auxin 2,4-D was either omitted from the medium or added at various concentrations lower or higher than that present in the standard growth medium in order to find out the appropriate concentration for the improved production of betalain.

Six media (Gamborg's 135) supplemented with different concentrations of 2,4-

D (0.02 (control), 0, 0.002, 0.01, 0.04, 0.2 mgi') and 0.1 mg.F' of kinetin were prepared as described in 2.1.2.1. The media were poured into 250 ml Erlenmeyer flasks (50 ml per flask) and autoclaved. Flasks were then inoculated with cells obtained from well mixed 13 d. old cultures (1 g. wet weight per flask). All cultures were then incubated on an orbital shaker under the conditions described in 2.1.4. After

10 days, 5 cultures for each treatment were harvested for the determination of fresh weight (see 2.2. 1) and betalain content (see 2.2 and 2.4).

Results in Fig. 3.2.10 present the effects of different concentrations of 2,4-D on the growth of cultures, in terms of fresh weight, as measured 10 days after subculture, from which it can be seen that the omission of 2,4-D from the medium or decreasing the concentration down to 0.002 mg.l' resulted in an increase in the fresh weight of the culture compared to the control (2,4-D 0.02 mg.r'). In contrast, increasing the concentration of 2,4-D in the medium up to 0.2 mg.1' caused a reduction in the fresh weight of cultures. Other treatments (2,4-D 0.01 and 0.04 mg.F

')had no significant effect.

92 Figure 3.2.10 Effect of 2,4D on the growth of cell cultures of Beta vulgaris.

Measurements of the fresh weight were made 10 days after subculture. Each value is a mean of 3 replicates ± Se.

iI cont. 0 0.002 0.01 0.04 0.2 2,4D concentration mg /I

93 The amounts of betacyanin and betaxanthin produced by cultures in different concentrations of 2,4-D, are presented in Fig. 3.2.11, expressed per g. fresh weight

(A) and per culture (B). From the results it can be seen that on a per g. f.wt. basis betacyanin and betaxanthin contents were slightly enhanced by decreasing the concentration of 2,4-D in the medium to 0.01 mg.r'. At 0.002 mg.1' 2,4-D betacyanin content was not significantly affected while betaxanthin content was slightly enhanced. Removing the 2,4-D from the medium, however, resulted in a significant increase in betacyanin whereas betaxanthin remained unaffected. Under the conditions of high concentrations of 2,4-D (0.04 and 0.2 mgI') betacyanin production was inhibited but betaxanthin was not. On a per culture basis, betacyanin accumulation was stimulated at concentrations of 0 and 0.002 mg.1' 2,4-D and inhibited at (0.2 mg.1-1 2,4-D) whereas, at 0.01 and 0.04 mgS' 2,4-D it was not significantly affected.

Betaxanthin production per culture, was also enhanced at reduced concentrations of

2,4-D (0-0.01 mg.r') but not significantly affected by increased concentrations (0.04 and 0.2 mg.r').

The overall results indicate that low concentrations of 2,4-D are preferred for better growth and production of betacyanin and betaxanthin. The best production of betacyanin was observed in cultures grown in the medium in which 2,4-D was deleted whereas the best production of betaxanthin took place in cultures grown in the medium with 0.002 mg.l' 2,4-D. However, as the culture seemed to be very sensitive to low concentrations of 2,4-D, the tiny amount of 2,4-D which might be carried over from the previous culture with cells of the inoculum, might have a considerable effect.

Therefore, it was decided in the following experiment to examine the effect of 2,4-D

Figure 3.2.11 Effect of 2,413 on betacyanin (A 535 nm) and betaxanthin (A 472 nm) content per g. fresh weight (A) and per culture (B). Each value is a mean of 3 replicates ± Se.

2.0

i1.5

0) O B.cyanin . 1.0 Cc B.xanthin Cu 2

0.0 cont. 0 0.002 0.01 0.04 0.2 2,4 D concentration mg/I

10 2?

B.cyanin cc B.xanthin

cont. 0 0.002 0.01 0.04 0.2 2,4 0 concentration mg/I

95 throughout several passages. However, as low concentrations of 2,4-D were found to be more suitable for the growth and pigment production, 2,4-D at concentrations ranging from 0 to 0.002 mg.r' were examined in the next experiment.

3.2.2.1.2 Effect of 2,4-D at concentrations ranging from 0 to 0.002 mg.1 1 on growth and betalain production over 3 passages

It was observed from the previous experiment that growth and betalain production were significantly enhanced when the auxin 2,4-D was removed from the medium or used at low concentration (0.002 mg.r'). Therefore, the aim of this experiment was to examine the effect of 2,4-D when used at low concentrations ranging from 0 to 0.002 mgT' on growth and betalain production. To test the possible effect of auxin carried over with the cells of the inoculum, the experiment was run for three passages and results were recorded at each passage.

Media used in this experiment were prepared as described in 2.1.2.1 but supplied with the following concentrations of 2,4-D 0, 0.00002, 0.0002, 0.002, 0.02

(control) mg.l' and 0.1 mg.1' of kinetin. The media were then poured to 250 ml

Erlenmeyer flasks, 50 ml per flask, and then autoclaved. Each flask was then inoculated with ca 1 g, wet weight, of cells obtained from well mixed 13 d. old cultures. The cultures were then incubated on an orbital shaker and maintained under the conditions described in 2.1.4 for three passages. At each passage 5 cultures for each treatment were harvested at day 10 from subculture and the fresh weight and

96 betalain content were determined according to the method described in 2.2.1 and (2.3 and 2.4) respectively.

The appearance of 10 d. old cultures of all treatments after three subcultures is shown in Fig. 3.2.12.

Table 3.2.1 presents the mean growth rates of cultures grown at each of the

2,4-D concentrations over three passages. As can be seen from the table in the first passage the growth rate of cultures was higher than the control at the concentrations of

0 and 0.00002 mg.r' 2,4-D and similar to the control at other concentrations of 2,4-D.

However, in the second passage, the mean growth rate of cultures was reduced in all treatments, especially at 0.0002, 0.00002, and 0 mg.r' 2,4-D. In the third passage, a further reduction in the mean growth rate of cultures was observed and in the medium without 2,4-D no growth was observed indicating that by this passage no auxin had been carried over with the inoculum cells.

Fig. 3.2.13 presents the results for betacyanin and betaxanthin per g. fresh weight over three passages of 10 d. old cultures under different treatments. As the results shows, in the first passage (A) the betacyanin content of cultures grown with

0.002 and 0.0002 mgf 1 2,4-D was not significantly different from the control (0.02 mg.r' 2,4-D). Whereas, it was greater than the control at 0.00002 mgI' 2,4-D and at

0 2,4-D. In contrast, in the second passage (B), the betacyanin content was significantly higher than the control in all treatments. And by the third passage (C) a further increase in betacyanin content over the control was attained with all treatments. At this passage, the amounts of betacyanin produced by the cultures had

015 Figure 3.2.12 The appearance of 10 d. old cell cultures of Beta vulgaris maintained for three passages in media with low concentrations of 2,41). A, B, C, D and E are cultures grown with 2,413 at 0.02 (control), 0.002, 0.0002, 0.00002 and 0 mg.l.

, ~ U1

[pr

0 C Li]

3 cm

98 Table 3.2.1 The mean growth rate of Beta vulgaris cell cultures maintained for 3 passages in media with different low concentrations of 2,4-D. Each value is a mean of three replicates ± Se.

Mean growth rate (data x102)

Concentration of First passage Second passage Third passage 2,4-D (mg.l')

0.02 (control) 6.80 7.37 6.57

±0.10 ±0.11 ±0.10

0.002 7.57 6.74 4.18

± 0.40 ± 0.22 ± 0.18

0.0002 7.58 3.18 0.52

±0.15 ±0.07 ±0.24

0.00002 7.77 2.60 0.36

±0.07 ±0.17 ±0.16

0 7.74 2.8 0

±0.09 ±0.12

99 Figure 3.2.13 The content of betacyanin (A 535) and betaxanthin (A472) per g. fresh weight in 10 d. old cell cultures of Beta vulgaris grown for three passages in media with low concentrations of 2,413 (0 - 0.02 mg.F'). Each value is a mean of 5 replicates ± se.

(A) I St passage 2.5

2.0

1.5 O B.cyanin C Ca • 1.0

o 0.5

0.0 cont. 0.002 0.0002 0.00002 0 2,40 concentration mg/I

2 nd passage 2.5

2.0

0 1.5 O B.cyanin C • 1.0

o 0.5

cont. 0.002 0.0002 0.00002 0 2,4 D concentration mg/I

3 rd passage 2.5 * 2.0 1.5 B.cyanin C cc • Exanthin . 1.0 a o 0.5

0.0 cont. 0.002 0.0002 0.00002 0 2.4 D concentration mg/i

100 increased by ca 165% at 0.002 mg.1' 2,4-D, 111% at 0.0002 mg.r' 2,4-D, 93% at

0.00002 mg.1' 2,4-D and 100% at 0 2,4-D as compared to the control. Turning to betaxanthin, the Fig. (3.2.13) shows that in the first passage (A) betaxanthin content per g. fresh weight was not significantly different from the control with 2,4-D of

0.002 mgI' whereas it had increased under all the other treatments. In the second passage (B) a substantial increase in betaxanthin content had occurred in all treatments, compared to the control. In the third passage (C), the betaxanthin content of cultures was higher than the control by ca 101% at 0.002 mg.i' 2,4-D, 58% at

0.0002 mgI' 2,4-D 41% at 0.00002 mgI' and 54% at 0 2,4-D.

The total betacyanin and betaxanthin produced per culture throughout the three passages in all the treatments are presented in Fig 3.1.14. As can be seen from these results, in the first passage (A) the amounts of both betacyanin and betaxanthin increased in all treatments. In the second passage (B), betacyanin and betaxanthin content increased at 0.002 2,4-D and decreased at lower concentrations of 2,4-

D. In the third passage (C), betacyanin content increased by 26% at 0.002 mgI' 2,4-D while betaxanthin content was not significantly different from the control. At other treatments, however, betacyanin and betaxanthin content decreased compared to the control (betacyanin content was less than the control by 54% at 0.0002 mgI', 61% at

0.00002 mgf', and 65% at 0 2,4-D, and betaxanthin content was less than the control by 65% at 0.0002 mgI', 71% at 0.00002 mg.1 -1 and 73% at 0 2,4-D.

Overall, the results of this experiment show that auxin carried over with the inoculum cells at subculture has a considerable effect that must be taken into account

101

Figure 3.2.14 The amounts of betacyanin (A 535) and betaxanthin (A472) produced per culture in 10 d. old suspension cultures of Beta vulgaris grown for three passages in media with low concentrations of 2,413 (0 - 0.02 mg.i'). Each value is a mean of 5 replicates ± se.

1 St passage 16

TD 12

O B.cyanin C8 •

0 cont 0.002 0.0002 0.00002 0 2,4 0 concentration mg/i

2 nd passage 14

0 I- O• B.cyanin

cont. 0.002 0.0002 0.00002 0 2,4 0 concentration mg/I

3 rd passage 10

8 LD

B.cyanin

cc 4 • 0 a 0 2

MIJ cont. 0.002 0.0002 0.00002 0 2,4Dconcentration mg/I

102 when the effect of 2,4-D is examined. Indeed more than three passages are required to ensure that all of the residual auxin is removed. In addition, based on the results obtained from the third passage, 0.002 mg.r' of 2,4-D caused a reduction in the growth of cultures, in terms of fresh weight, while stimulated the production of both betalains on a per g. f.wt. basis while enhanced the production of betacyanin but not betaxanthin on a per culture basis.

However, as the 0.002 mg.i' 2,4-D treatment gave the best production of betacyanin but not betaxanthin per culture, among those tested, and as this treatment caused some reduction in the growth of cultures compared to the control, the next experiment examined the effect of 2,4-D at concentrations ranging from 0.002 mgf'

up to 0.01 mg.1 1 , in an attempt to search for the best concentration of 2,4-D which enhance both betalains production and support better growth.

3.2.2.1.3 Effect of 2,4-D at concentrations ranging from 0.002 to 0.01 mg.1'

In the previous experiment it was shown that betacyanin production per culture was stimulated in cultures grown on a medium with a 2,4-D concentration of

0.002 mg.l', however, betaxanthin production was not significantly affected and the fresh weight of cultures was markedly reduced, after 3 subcultures at this level of 2,4-

D. The aim of this experiment was to examine the effect of 2,4-D on the growth and betalain production when used at concentrations ranging from 0.002 up to 0.01 mg.F' in an attempt to search for the proper concentration of 2,4-D which enhance both betalains and support good growth.

103 Six media (Gamborg's B5) were prepared as described in 2.1.2.1 and 2,4-D was supplemented at the following concentrations (0.002, 0.004, 0.006, 0.008, 0.01 and 0.02 mg.r' (control)) and kinetin applied at 0.1 mg.1'. The media were poured into 250 ml Erlenmeyer flasks (50 ml per flasks) and autoclaved. The flasks were then inoculated with cells obtained from well mixed 13 d. old cultures (1 g. wet weight, per flask). The cultures were then incubated on an orbital shaker under the conditions described in 2.1.4. To eliminate the auxin carried over with the inoculum, cultures were maintained for each treatment for 5 passages and subcultured every 14 days. At the fifth passage 3 flasks for each treatment were harvested on day 10 and 15 and the fresh weight of cultures (see 2.2.1) and betalain content (see 2.3 and 2.4) were determined.

The effect of each concentration of 2,4-D on the fresh weight of cultures is presented in Fig. 3.2.15, as estimated at two stages of growth, day 10 and 15, from which it can be seen that on day 10 the fresh weight of cultures was increased with

2,4-D at 0.008 mg.F' and decreased at 0.002 mg.r' whereas it was not significantly influenced at other concentrations, as compared to the control (P = 0.03). On day 15 the fresh weight of cultures grown under all treatments was quite similar to the control. However, it is worthy to note here that limitation of nutrients, resulting from consumption by growing cells, at this stage (day 15) may restrict the increase in fresh weight of cultures with a higher initial growth rate that may be caused by the treatments.

104

Figure 3.2.15 The growth of Beta vulgaris cell cultures on days 10 and 15 from subculture after maintenance for five passages in media with different concentrations of

2,413 ranging from 0.002 to 0.02 mg.r'. Each value is a mean of 3 replicates ± Se.

- 10 • 0.020mg/i

• 0.010mg/I

• 0.008mg/i

D 0.006 mg/I

o 0.004mg/I

• 0.002 mg/i 2

0 10 15

Time (days)

105 Fig. 3.2.16 presents the amount of betacyanin produced by cultures under

different treatments, as expressed per g. fresh weight (A) and per culture (B), at two

stages of growth, day 10 and 15. As shown by the results betacyanin content of

cultures per g. fresh weight was not significantly influenced by 0.01, 0.008 and 0.006

mg.1 on day 10 whereas it was significantly increased with 2,4-D at 0.004, and 0.002

mg.l' as compared to the control. By day 15 the betacyanin content per g. fresh

weight had decreased with 2,4-D of 0.01 mg.r' while it had increased in the other

treatments which had enhanced the production of betacyanin equally. On a per culture

basis (B) betacyanin content of cultures grown at all concentrations of 2,4-D was not

significantly different from the control on day 10 whereas on day 15 it had

significantly increased at 0.008, 0.006 and 0.004 mgI' compared to the control. The highest increase (ca 57%) occurred at 0.006 mg.1 -1 2,4-D, however, according to

statistical analysis there is no significant difference between these three treatments

(0.008, 0.006, and 0.004 mgI' 2,4-D) in their stimulatory effect. Other treatments, on the other hand, had no significant effect when compared with the control.

Turning to betaxanthin, Fig. 3.1.17 (A) and (B) shows that on day 10 the production of betaxanthin per g f.wt. was slightly increased at 2,4-D of 0.002-0.008 mg.l', however, analysis of variance showed that this increase was not significant (P

= 0.1). When the pigment content was expressed per culture, betaxanthin production was increased with 2,4-D at 0.004-0.1 mg.r', again this increase was not significant (P

= 0. 1), as the standard error was high within the treatments. On day 15 the treatments of 0.008, 0.006 and 0.004 mg.1' 2,4-D significantly increased the content of betaxanthin per g. fresh weight and per culture. The highest increase in betaxanthin

106

Figure 3.2.16 The amounts of betacyanin (A 535 nm) produced per g. fresh weight (A) and per culture (B) as measured on day 10 and 15 during subculture in suspension cultures of

Beta vulgaris after maintenance for five passages in media with different concentrations of 2,413 ranging from 0.002 to 0.02 mgi'. Each value is a mean of 3 replicates ± se.

2.0

1.5 • 0.020 mg/i • 0.010mg/i I to • 0.008 mg/i Ca 0.006 mg/i o 0.004mg/i 0.5 0.002 mg/i

me 10 15 Time (days)

a) 12 • 0.020mg/I 0 • 0.010mg/i C • 0,008mg/i >cc C) EZ 0.006 mg/i 0) o 0.004mg/i 0.002 mg/i 0

0 10 15 Time (days)

107 Figure 3.2.17 The amounts of betaxanthin (A 472 Mn) produced per g. fresh weight (A) and per culture (B) as measured on day 10 and 15 during subculture in suspension cultures of Beta vulgaris after maintenance for five passages in media with different concentrations of 2,4-D ranging from 0.002 to 0.02 mg.l. Each value is a mean of 3 replicates ± Se.

(A) 2.0

1.5 • 0020mg/i 0) • 0.010mg/i C .0 1.0 D 0.008mg/i C Ca O 0.006mg/i ca 0.004mg/i a) o 0.5 0.002 mg/i a B Al

DIF 10 15 Time (days)

(B) 16

a) 75 12 • 0.020 mg/I • 0.010mg/i ii B 0.008mg/i 0.006mg/i o 0.004mg/I I: 13 0.002 mg/i

10 15 Time (days)

108 production was attained at 0.006 mg.1' 2,4-D, at which betaxanthin content was

higher than the control by ca 36% on per g. fresh weight basis and by ca 57% on a per

culture basis. However, the increases in betaxanthin production at 0.008 and 0.004

mgi' 2,4-D were not significantly lower than that at 0.006 mg.r'.

Overall the results showed that decreasing the concentration of 2,4-D in the

medium to 0.008, 0.006 or 0.004 mg.1' resulted in an enhanced production of

betacyanin and betaxanthin although it did not significantly affect the growth. This

stimulation was significant only at the later stage of growth (15 d.) and it was not

proportional to the concentration. The highest production of pigments was attained

with 0.006 mg.1 2,4-D, however, statistically the treatments (0.008, 0.006 and 0.004

mg.i' 2,4-D) were equally stimulatory on pigment production. At 0.002 mg.r' 2,4-D

betacyanin content per g. fresh weight was enhanced at the early stage of growth (10

d.) while the fresh weight of culture was reduced. However, this effect was

diminished at the later stage of growth (15 d.), as compared to the control.

Results from 3.2.2.1 showed that the production of betalain in cultures could be enhanced by decreasing the concentrations of 2,4-D in the medium, in the following experiments, the effect of various concentrations of kinetin in the medium was examined.

109 3.2.2.2 Effect of Various Concentrations of Kinetin on Growth and Betalain

Production

Kinetin has been reported to stimulate the production of betalain in the dark

(see Colomas 1975, Piattelli 1981, Bianco-Colomas and Hugues 1990). Recently,

Rudat and Goring (1995) reported that increasing the concentration of kinetin in the medium, from 0.1 mg.1' to 5 mg.r' caused a substantial increase in betacyanin content of cell cultures of Chenopodium album grown in white light. The aim of this experiment was to examine the effect of various concentrations of kinetin in the medium on growth and betalain production.

Five media (Gamborg's B5) were prepared as described in 2.1.2.1 but with the following concentrations of kinetin 0.01, 0.05, 0.1 (control), 0.5 and 1.0 mg.l'. 2,4-D was supplied to all media at the standard concentration (0.02 mg.1 1). Erlenmeyer flasks (250 ml) each containing 50 ml of medium were inoculated with 1 g. (wet weight) of cells obtained from 13 d. old suspension cultures (see 2.1.4). The cultures were then placed on an orbital shaker and incubated under the conditions described in

2.1.4 and remained for three passages, in order to remove the residual kinetin or 2,4-D carried over with the inoculum cells. At the third passage, 5 flasks for each treatment were harvested at day 13 for analysis. The fresh weight of cells was determined as described in 2.2.1 and betalain content was extracted and determined as indicated in

2.3 and 2.4 respectively.

The effect of the different concentrations of kinetin on the growth of cultures,

(fresh weight) is presented in Fig 3.2.18, measured 13 d. after subculture. As can be

110 Figure 3.2.18 Effect of different concentrations of kinetin on the growth of Beta vulgaris cell cultures, as measured in 13 d. old of cultures. Each value is a mean of 5 replicates ± se.

[.1 0)

., 4

Cont. 0.01 0.05 0.5 1.0 Kin'etin concentration mg/I seen from the results, high concentrations of kinetin (0.5, 1.0 mg.r') resulted in a reduction in the fresh weight of cultures compared to the control (0.1 mg.1 -5. In contrast, low concentrations (0.05 or 0.01 mgI') had no effect on fresh weight.

Fig. 3.2.19 presents the amount of betacyanin and betaxanthin produced by 13 d. old cultures grown at different concentrations of kinetin, as expressed per g. fresh weight (A) and per culture (B). As the results show 0.01, 0.05, and 0.5 mgI' kinetin had no significant effect on betacyanin and betaxanthin production, on per g. fresh gf weight or per culture base, however, at 1 kinetin a remarkable reduction in both betalain content, per g. fresh weight and per culture, was observed in comparison to the control.

Overall the results of this experiment show that kinetin at a concentration of 1 mg.l' inhibited both growth and betalain production. Lower concentrations, within the range tested, had no significant effect.

Results from this subsection (3.2.2) showed that the production of betalain could be enhanced by decreasing the concentration of 2,4-D in the medium but it could not be increased by altering the concentration of kinetin, within the range of concentrations tested. The stimulation in betalain production which occurred at low concentration of 2,4-D seems to have resulted from a specific effect of 2,4-D and not from a change in the ratio of 2,4-D to kinetin. For example, betalain production was significantly enhanced when 2,4-D and kinetin were applied to the medium at concentrations of 0.002, and 0.1 mgI' respectively, where the ratio of 2,4-D to kinetin was 1150 (see Fig. 3.2.12(C)), whereas betalain accumulation was inhibited by

112

Figure 3.2.19 The content of betacyanin (A 535 nrn) and betaxanthin (A 472 nrn) of 13 d. old suspension cultures of Beta vulgaris cell cultures, grown at different concentrations of kinetin as expressed per g. f.wt. (A) and per culture (B). Each value is a mean of 5 replicates ± Se.

1.8 1.6 1.4 !1.2 1.0 O B.cyanin 0.8 B.xanthin 0.6 00.4 0.2 0.0 Cont. 0.01 0.05 0.5 1.0 Kinetin concentration mg/I

B.cyanin B.xanthin

0 0 2

0 Cont. 0.01 0.05 0.5 1.0 Kinetin concentration mg/I

113 the treatment at which the concentrations of 2,4-D and kinetin in the medium were of

0.02, and 1 mg.11 respectively, despite having the same ratio of 2,4-D to kinetin

(1150) (see Fig. 3.2.19).

In the following subsection further attempts to increase the yield of betalain in cultures were made by the application of betalain precursors to the medium.

3.2.3 Effect of Precursor Feeding on Growth and Betalain Production

There are many reports in the literature demonstrating the value of supplying plant tissue cultures with specific metabolic precursors as a means to enhance the accumulation of certain desired secondary metabolites in vitro (see Yeoman et al.,

1980, Fowler 1983, Mantell and Smith 1983, Sugimoto et al., 1994, Yeoman and

Yeoman 1996).

In this subsection supplements of tyrosine or DOPA, compounds implicated in betalain biosynthesis (see Piattelli 1976, 1981, Steglich and Strack 1990, and Hirano and Komamine 1994) were examined for their ability to increase the production of betalains in cultures of Beta vulgaris.

3.2.3.1 Effect of Supplementation of Tyrosine on Growth and Betalain

Production

The aim of this experiment was to investigate whether the application of tyrosine could enhance the production of betalain in cell cultures of Beta vulgaris.

114 Cultures were initiated by inoculating 1 g (wet weight)of cells, obtained from

13d. old well mixed cultures, into 250 ml Erlenmeyer flasks containing 50 ml of the standard growth medium (see 2.1.4) supplemented with tyrosine. Prior to the initiation of cultures tyrosine was applied to the flasks by the addition of 0.1 ml of a solution of tyrosine, sterilised by filtration through a membrane filter with a pore size of 0.2 jim held in a filtration unit. Tyrosine was applied at various concentrations to give final concentrations in the flasks of 0. 1, 0.2 and 0.5 mM. In the control medium 0.1 ml of sterile distilled water was added instead of tyrosine solution. All cultures were then placed on an orbital shaker and incubated under the conditions described in 2.1.4.

Three flasks for each treatment were collected on day 10 and 15 and cells were harvested for analysis. The fresh weight of cultures was determined as described in

2.2.1 and betalain content was extracted and determined according to the methods described in 2.3 and 2.4 respectively.

The effect of tyrosine on the growth of cultures, in terms of fresh weight, is presented in Fig 3.1.20 as measured at two stages during the growth cycle (day 10 and day 15). As shown by the results supplementation of tyrosine, at a range of concentrations, had no significant effect on the growth of cultures, in terms of fresh weight.

Fig. 3.2.21 presents the amounts of betacyanin produced per g. fresh weight

(A) and per culture (B) in cultures fed with tyrosine as measured on day 10 and 15. As can be seen from these results, the production of betacyanin on a per g. fresh weight, and per culture basis in cultures supplied with tyrosine, was not significantly different

115

Figure 3.2.20 Effect of tyrosine supplementation on the growth of Beta vulgaris cell cultures, as measured on day 10 and 15. Each value is a mean of 3 replicates ± Se.

174

M, • 0.0 mM Tyr. U) • 0.1 mMTyr. • 0.2 mM Tyr. 4 0 0.5 mM Tyr.

10 15 Time (days)

116

Figure 3.2.21 The content of betacyanin (A535 rim) per g. f.wt. (A) and per culture

(B), as measured on day 10 and 15, in cell cultures of Beta vulgaris supplied with various concentrations of tyrosine. Each value is a mean of 3 replicates ± se.

0.8

0.6

• 0.0 mM Tyr.

0.4 • 0.lmMTyr. cc • O2mMTyr.

cc 0.5 mM Tyr.

0.0 10 15 Time (days)

6 0 -S • 0.OmMTyr. • O.lmMTyr. cc 4 >s 0 • O2mMTyr. cc D 0.5 mM Tyr. .0 0 0

0 10 15 Time (days)

117 from that of tyrosine free cultures (control) on day 10 or on day 15. Similarly the content of betaxanthin as expressed per g. fresh weight or per culture (Fig. 3.2.22 (A) and (B)) was also not significantly affected by the treatments at either of the two sampling times (day 10 and 15).

The result of this experiment showed that supplementation with tyrosine did not stimulate the accumulation of betalain. Therefore in the following experiment the addition of DOPA, another compound implicated in betalain synthesis, was examined in an attempt to enhance the production of betalain.

3.2.3.2 Effect of Supplementation of DOPA on Growth and Betalain Production

In the previous experiment attempts to enhance the production of betalain by feeding cultures with tyrosine were not successful. In this experiment attempts were made to increase the yield of betalain by supplying cultures with various concentrations of DOPA (3,4 dihydroxyphenylalanine). This putative precursor for the pigment, was reported to stimulate the production of betalain in Amaranthus tricolor seedlings (Elliot 1983) and Portulaca grandflora flowers (Rink and Böhm

1985).

Gamborg's B5 medium was prepared as described in 2.1.4 and then poured into 250 ml Erlenmeyer flasks (50 ml per flask). After autoclaving the flasks were supplemented with DOPA by injecting 0.1 ml of a filter sterilised solution of DOPA.

The DOPA solution was applied at various concentrations to give final concentrations

118 Figure 3.2.22 The content of betaxanthin (A 472 Mn) per g. f.wt. (A) and per culture

(B), as measured on day 10 and 15, in cell cultures of Beta vulgaris supplied with various concentrations of tyrosine. Each value is a mean of 3 replicates ± Se.

(A) 1.0

0.8

'-0.6 • 0.OmMTyr. C • 0.lmUTyr.

0.4 • O2mMTyr. 0.5 mM Tyr.

0.2

0.0 ' 10 15 Time (days) (B) 8

2 • 0.0 mM Tyr. • 0.lmMTyr. • O2mMTyr. 0.5 mM Tyr. 21

15 Time (days)

119 in the flasks of 0. 1, 0.2 and 0.5 mM. Each flask was then inoculated with 1 g of cells, obtained from well mixed 13 d. old cultures. All cultures were then placed on an orbital shaker and incubated under the conditions described in 2.1.4. Three flasks for each treatment were collected on day 10 and 15 and the content of each flask was filtered and cells were used for the analysis. The fresh weight was determined as described in 2.2.1 and betalain content was extracted and determined according to the methods described in 2.3 and 2.4 respectively.

Fig. 3.1.23 presents the effect of DOPA supplementation on the fresh weight of cultures as measured at two stages of growth (day 10 and 15). As can be seen from the results, the fresh weight of cultures supplied with DOPA over a range of concentrations, was not significantly different from the control at day 10 or day 15

The amount of betacyanin produced per g. fresh weight and per culture (Fig.

3.1.24 (A) and (B)) in the treatments was also closely similar to the control on day 10 and not significantly lower than the control on day 15.

Similarly the production of betaxanthin per g. fresh weight and per culture

(Fig 3.1.25 (A) and (B)) were also non significantly affected by the DOPA.

120

Figure 3.2.23 Effect of DOPA supplementation on the growth of Beta vulgaris cell cultures, as measured on day 10 and 15 during subculture. Each value is a mean of 3 replicates ± Se.

• 0.0 mM DOPA (1) 0.1 mM DOPA CD 6 • I- 1- • 0.2 mM DOPA 2 4 0.5 mM DOPA

is 10 15 Time (days)

121

Figure 3.2.24 The content of betacyanin (A 535 nm) per g. f.wt. (A) and per culture

(B), as measured on day 10 and 15 during subculture, in cell cultures of Beta vulgaris supplied with various concentrations of DOPA. Each value is a mean of 3 replicates ±

Se.

1.0

j0.8

0) 0.6 • 0.0 mM DOPA • 0.ImMDOPA ( >.. O2mMDOPA 0.4 • 9 0.5mMDOPA

0 MON, 0.0 -'-- 10 15 Time (days)

2 8

C-) - 6 • 0.0 mM DOPA • 0.1 mM DOPA • 0.2mMDOPA o 0.5 mM DOPA 0 0

ork 15 Time (days)

122 Figure 3.2.25 The content of betaxanthin (A472 nrn) per g. f.wt. (A) and per culture

(B), as measured on day 10 and 15 during subculture, in cell cultures of Beta vulgaris

supplied with various concentrations of DOPA. Each value is a mean of 3 replicates ±

Se.

1.0

0.8

-.- 0.6 • 0.0mM DOPA C • 0.1mMDOPA 0.4 • 0.5mMDOPA

8 0.2

10 15 Time (days)

I-

C., • 0.0 mM DOPA C • C • 0.2 MM DOPA U 0.5mMDOPA

0 2

0 10 15 Time (days)

123 Summary of the results in 3.2

Altering the concentration of inorganic nitrogen (i-N) in the medium did not increase either the growth, in terms of the fresh weight or the content of betalains of

Beta vulgaris cell cultures, however, the reduction in the fresh weight of cultures occurred at i-N 116 mM was not associated with a decrease in pigment production on per g. fresh weight basis.

Increasing the concentration of inorganic phosphorus (i-P) above that present in

Gamborg's 135 medium did not significantly influence either growth or production of betalain, in the cell cultures. In contrast, reducing the concentration of i-P in the medium to 0.55 mM caused a reduction in the growth of cultures, in terms of fresh weight, while betalain accumulation was not significantly affected.

Elevating the concentration of sucrose in the medium from 2% to 3% significantly increased the production of both betalains, however, the increase in pigment content per g. f.wt. appeared after day 10 in the case of betacyanin and after day 15 in case of betaxanthin. This treatment, in contrast, did not stimulate the growth of the cultures but did extend the duration of growth. On the other hand, at 9% sucrose the accumulation of betalains was inhibited, however, the inhibitory effect was overcome after 15 days. Meanwhile the growth of culture was not affected by the treatment but it was prolonged up to the end of the experiment.

A low concentration of 2,4-D was shown to stimulate the production of betalains, nevertheless, the yield of pigment was not proportional to the auxin concentration.

124 The best yield of betacyanin and betaxanthin per culture was obtained at 0.006 mg.1 1

2,4-D. At this concentration, growth of the cultures was not significantly affected.

Altering the concentration of kinetin in the medium did not increase either growth or production of betalains.

Results from experiments at which the concentration of 2,4-D or kinetin was manipulated, suggest that the stimulation of betalain production that occurred at low concentrations of 2,4-D, resulted from a specific effect of 2,4-D concentration but not from a change in the balance between 2,4-D and kinetin.

Administration of either tyrosine or DOPA at concentrations of 0.1, 0.2, or 0.5 mM did not significantly influence either growth or betalain production.

Overall the results in this section show that the production of betalain in cell cultures of Beta vulgaris can be improved by the manipulation of the culture medium.

The conditions of culture influence both the growth and betalain accumulation, however, modification in pigment production was not always correlated with paralleled changes in the growth of cultures and the optimum conditions for product formation may be different from those which promote optimum growth. In the following section the production of betalains was investigated in relation to cellular heterogeneity.

125 3.3 INVESTIGATION INTO BETALAIN PRODUCTION IN RELATION TO

CELLULAR HETEROGENEITY

It is clear from microscopic observations that the cellular populations of Beta vulgaris cultures are highly heterogeneous with regard to pigmentation. Differences between cells were observed not only in the intensity of pigmentation, but also in colour. Cells could be arranged into four groups, according to colour, violet, orange, yellow, and white (non-pigmented). In the following series of experiments the production of betalains was investigated in relation to cellular heterogeneity, with regard to the colour phenotype of cells, of cultures of Beta vulgaris. The aims of this study were:

To establish lines from single cells of different colours (violet, orange, yellow and white), this would presumably result in clones with different productivities of betacyanin and/or betaxanthin and might enable characterisation of the pigment produced by each type of cells.

To develop high yielding systems by cloning single deeply pigmented cells with high pigment productivity.

To study the effect of culture manipulation on pigment production in relation to cellular heterogeneity.

This study should give some information about the control of cellular heterogeneity in cultures of Beta vulgaris and will enable a better understanding of the possible ways in which higher yield of betalains might be attained.

126 3.3.1 Single Cell Cloning and Characterisation of the Derived Cell Lines

3.3.1.1 Establishment and characterisation of cell lines

Four different cell lines (violet, orange, yellow and white) were selected, using a single cell plating technique (see 2.1.5). The cell lines were characterised, with regard to appearance of the cell culture, growth, betalain content, betalain related compounds content, appearance of the cellular population and the proportion of pigmented cells. The experiment was performed three months after the first establishment of single cell clones.

Cells from 14 day old suspension cultures of each of the selected cell lines and the mother culture were inoculated into 250 ml Erlenmeyer flasks (Ca 1 g. f.wt. of cells per flask) containing 50 ml of standard growth medium (see 2.1.2.1). Cultures were incubated under the conditions described in 2.1.4. The growth of individual cell lines was followed by determining the fresh weight of samples taken on days 0, 3, 6,

9, 12, 15, 18 and 21 and the other parameters were measured on samples taken on day

14 of the culture cycle.

The appearance of the selected cell lines and the mother culture is shown in

Fig. 3.3.1.

I. Growth of selected cell line cultures and the mother culture

As shown in Fig. 3.3.2, the patterns of fresh weight increase, of all cell

127 Figure 3.3.1 The appearance of 14 day old suspension cultures of (A) the mother culture, (B) violet cell line, (C) orange cell line, (D) yellow cell line, and (E) white cell line.

128 Figure 3.3.2 Characterisation of growth, in terms of fresh weight, of the mother culture (MC), violet cell line (V), orange cell line (0), yellow cell line (Y), and white cell line (W). Each value is a mean of 3 replicates ± se.

-0---- MC 10 -0•--- V Cl) 8 a) -s-- 0

6 0 -0--- V

4 --h- W

0 5 10 15 20 25

Time from subculture (d)

129 lines were similar. However, the growth rate of the yellow and white cell lines was less than the other cell lines. All cell lines passed through a lag phase for about 3 days after which the fresh weight increased rapidly up to day 15. Overall the increase in fresh weight of the yellow and white cell lines was less than that of the violet, orange and mother cultures. After day 15 some increase was observed in the fresh weight of the violet and yellow cell lines up to day 18 after which it decreased until the end of the experiment. Fresh weight, either decreased or remained constant for the other cell lines from day 15 until the end of the culture period.

II. Analysis of betalains produced by selected cell line cultures and the mother culture

Fig. 3.3.3 shows the visible spectrum of 80% methanol extracts, extracted as described in 2.6.3, of 14 day old cultures of each of the selected cell lines, as well as the mother culture. The visible spectra of the violet and orange cell lines are similar to that of the mother culture with two peaks, one at 472 nm and the other at 535 nm, showing that the two cell lines produced both betacyanin and betaxanthin. However, the proportion of betacyanin to betaxanthin produced by the violet and the orange cell lines appears to be different. The visible spectrum of the yellow cell line extract (Fig.

3.3.3 (D)) shows that betaxanthin but not betacyanin was produced as indicated by the peak observed at 472 nm. The visible spectrum of the white cell line extract shows that neither betacyanin nor betaxanthin were produced by this cell line.

130 Figure 3.3.3 The visible spectrum of cell extracts in 80% methanol of (A) mother culture, (B) violet cell line, (C) orange cell line, (D) yellow cell line, and (E) white cell line.

(A) (B) 0.9 472 2.50 0.8

0.7 2.00

0 0.6 U 0.5 1.50

0.4 .1.00 0.3

0.2 0.50 0.1

0.001 400 460 520 580 640 700 400 460 520 580 640 700 Wave length (nm) Wave length (nm)

I C o 0.3 U C 0.8 0

0.6 0.2

0.4 0.1 0.2

0 01 400 460 520 580 640 700 400 460 520 580 640 700 Wave length (nm) Wave length (nm) (E) 0.6. 464

0.5

0.4 U C 0 0.3

0.2

0.1

0 400 460 520 580 640 700 Wave length (nm

131 TLC analysis (see 2.5) of the cell extracts shown together with standard solutions of betacyanin (betanin/isobetanin) and betaxanthin (miraxanthin V) (Fig.

3.3.4) confirmed that the mother culture, violet cell line and orange cell line produced both betacyanin and betaxanthin, the yellow cell line produced only betaxanthin, and the white cell line produced neither of the pigments.

An accurate qualitative and quantitative analysis of betalains produced by the different cell lines was carried out using high performance liquid chromatography

(HPLC) according to the method described in 2.6.

Fig. 3.3.5 shows the HPLC chromatograms monitored at 535 nm of the mother culture (A), and the selected cell lines [violet (B), orange (C), yellow (D), and white

(E)]. The peaks a, b, and c were identified as betanin, isobetanin and isobetanidin respectively. Other peaks (d, e, f, and g) are unknown compounds. With regard to the identified peaks, it can be seen that although the mother culture produces betacyanin only as betanin, the violet cell line produced betacyanins as betanin, isobetanin, and isobetanidin and, the orange cell line produced betacyanin as betanin and isobetanin.

However, betanin was the main betacyanin derivative produced by both cell lines, the violet and the orange. No betacyanin could be detected in either the yellow or the white cell lines.

Fig. 3.3.6 presents the HPLC chromatograms monitored at 472 nm of the mother culture (A) and the selected cell lines [violet (B), orange (C), yellow (D) and

132 Figure 3.3.4 The TLC separation of betalains, in 80% methanol, extracts of selected cell lines together with the mother culture. MC = mother culture, V = violet cell line,

0 = orange cell line, Y = yellow cell line, W = white cell line, CS. = betacyanin standard and XS. = miraxanthin V standard.

Solvent Front

0000

obo 0

MC V. 0 Y W CS XS

133 Figure 3.3.5 HPLC chromatograms, monitored at 535 nm showing the betacyanins

produced by (A) mother culture, (B) violet cell line, (C) orange cell line, (D) yellow

cell line, and (E) white cell line. Pigment was extracted and analysed as described in

2.6 [a = Betanin, b = Isobetanin, c = Isobetanidin].

a 100 100

90 90

80 Be

70 70

80 60

80 50

40 40

30 30

20 20

10 10

0 0

0.00 5.00 10.88 15.00 20.00 25.00 30. 0.00 5.80 10.80 15.08 4W. 08 4 t3. 88 30

100 100

90 90

Be Be

70 70

60 60

50 58

8) C) 40 40

30 38 0 r1 20 20 0 p.- I-! 10 18

8.00 5.00 10.00 15.180 L8.08 zt,.Uva 318. 0.00 5.08 118.08 I.18O .

100

0.00 5.00 18.00 15.08 48.08 4.18W 318. Retention Time (mm) 134 Figure 3.3.6 HPLC chromatograms monitored at 472 nm showing betaxanthin

produced by (A) mother culture, (B) violet cell line, (C) orange cell line, (d) yellow

cell line, and (E) white cell line. Pigment was extracted and analysed as described in

2.6 [a = Betanin, b = Isobetanin, c = Miraxanthin V].

a lee (A) SO- 90

80. Be

70 70

so- 60

SO- 50

40— 40

30- 30

to- I0

000 500 10.00 15.00 20.00 25.00 30

100 100

90 90

80 Be

70 70

60 60

50 50

40 40 0) C.) 30 30

0 20 20 0 ze 10 10 0 0

0.00 5.00 10.00 15.00 20.00 25.00 30, 0.00 5.08 10. too V. V. '.= (E) 90-

80.

so-

so_

40 -

30 -

20_

0.00 5.00 10.00 IS.00 20.00 25.00 30

Retention Time (mm) ic white (E)]. Several peaks appeared at this wave length in each chromatogram.

However, the major peaks labelled as a, b, and c could be identified. Peaks a and b were identified as betanin and isobetanin respectively since they were found to absorb light at 472 nm by ca 52% of that absorbed at 535 run. Peak c was identified as miraxanthin V. The figure shows that miraxanthin V was produced by all cell lines except the white one. However, in the yellow cell line it was produced at very low amount.

The amounts of betacyanins and miraxanthin V produced by each cell line are presented in Table 3.3.1 from which it can be seen that the violet cell line was best for betacyanin production. The total betacyanin produced by this cell line was ca 30 times that produced by the mother culture. 95% of these betacyanins were present as betanin, 4.5% as isobetanin, and 0.3% as isobetanidin. In contrast, the orange cell line produced a much lower amount of betacyanin, ca 7% of the betacyanin produced by the violet cell line. Nevertheless, the orange cell line produced more betacyanin, almost twice as much as the mother culture of which 94% was present as betanin and

6% as isobetanin. There were no significant differences between the amounts of miraxanthin V present between the violet cell line, the orange cell line, and the mother culture. The yellow cell lines, however, produced a very low amount of miraxanthin

V. No betacyanin or miraxanthin V was detected in the white cell line.

III. Analysis of betalain related compounds

As indicated previously in 3.2.1.2 the selected cell lines varied qualitatively

136 Table 3.3.1 Betalain content, in mM.g', of 14 day old cultures of each of the selected cell lines as well as the mother culture. Each value is a mean of 4 replicates ± se.

mother violet cell orange cell yellow cell white cell

culture line line line line

Betanin 0.14 3.98* 0.27* nd nd

± 0.02 ± 0.43 ± 0.02

Isobetanin nd 0.19 0.02 nd nd

±0.03 ±0.01

Isobetanidin nd 0.012 nd nd nd

± 0.004

Total 0.14 4.18* 0.29* nd nd

betacyanins ± 0.02 ± 0.46 ± 0.02

Miraxanthin V 0.66 0.86 1.06 0.02* nd

±0.17 ±0.20 ±0.18 ±0.01

* = significant at P< 0.05 nd = not detected

137 and quantitatively with regard to betalain content. The violet cell line predominantly produced a large amount of betacyanin, the orange cell line produced predominantly miraxanthin V, the yellow cell line produced a very small amount of miraxanthin V and the white cell line did not produce either betacyanin or miraxanthin V. In addition to this analysis, it was also decided to measure the amounts of some betalain related compounds in the cell extracts of these different cell lines, in an attempt to characterise the biochemical status of the different cell lines. The levels of tyrosine,

DOPA, dopamine, and betalamic acid which are involved in the biosynthesis of the betalains were measured using HPLC analysis according to the methods described in

2.6. The amounts of these compounds are presented in Table 3.3.2 from which it can be seen that the tyrosine content in the orange and yellow cell lines was significantly higher than that of the mother culture whereas tyrosine content of the violet and white cell lines was similar to that of the mother culture. DOPA was not detected in any of the culture extracts. However, dopamine was found in appreciable amounts in cell extracts of all the cell lines but the differences in amounts between cell lines were not significant except for the yellow cell line which was significantly lower than the others. Betalamic acid was also present in small amounts in extracts of the violet, orange and mother cultures but was not detectable in extracts of the yellow and white cell lines.

IV. Physical appearance of cells and proportions of pigmented cells in the different cell lines

Fig. 3.3.7 shows the appearance of cells from 14 day old cultures of the selected cell lines together with the mother culture. As shown by the photographs

138 Table 3.3.2 The content of betalain related compounds, in of 14 day old cultures of each of the selected cell lines as well as the mother culture. Each value is a mean of 4 replicates ± Se.

mother violet cell orange cell yellow cell white cell

culture line line line line

Tyrosine 16.40 13.26 32.32* 27.08* 17.23

± 0.87 ± 1.94 ± 3.16 ± 5.96 ± 3.28

Dopa nd nd nd nd nd

Dopamine 104.5 71.92 106.4 49.60* 64.16

±24.71 ±4.26 ± 12.79 ± 11.15 ± 12.25

Betalamic acid 0.06 0.06 0.01 nd nd

± 0.02 ± 0.02 ± 0.02

* = significant at P< 0.05

139

Figure 3.3.7 The appearance of cell populations from 14 day old cultures of, (A)

mother culture, (B) violet cell line, (C) orange cell line, (D) yellow cell line, and (E)

white cell line.

A B f .41

• 4 a

:.', 'r I p_• V. .. 4" fy

/ , r— S. __..0 S •' ss51J Ok \ / • . .

. .. .>'.-. L kr ø

40 gm 4VT !j

-- 'S

.. •i'3i&. .:\-Jr - :• - r . -' 5••__• II.* there were no major differences between the shapes of cells of the selected cell lines, but the pigment colours were different. The white line contained no pigmented cells.

The data presented in Table 3.3.3a show the proportion of pigmented cells present in each population as well as the percentage of coloured cells of each pigment type.

Table 3.3.3b presents the same data after arcsine transformation to show the standard error of the mean. As shown from the data, the proportions of pigmented cells of the violet, orange and mother cultures were similar. The yellow cell line contained a much lower proportion of pigmented cells and the white cell line as expected contained no pigmented cells. However, the composition of the population of pigmented cells, by pigment type, was significantly different between the cell lines. In the mother culture the pigmented cell population consisted of a mixture of violet, orange, and yellow cells in approximately equal proportions. The violet cell line consisted mainly of violet cells with a few orange cells (12.5% of the pigmented cell population). In the orange cell line, the pigmented cell population consisted mainly of orange and yellow cells, in approximately equal proportions, with a few violet cells

(ca 6% of the pigmented cell population). In the yellow cell line, the pigmented cell population consisted only of yellow and orange cells in equal proportions.

Overall, results from 3.3.1 show that with the use of a single cell cloning technique, various cell lines (violet, orange, yellow and white), having the same type of colour of the parent cells, could be obtained. These cell lines exhibited remarkable differences in their pigment content (betacyanin and/or betaxanthin) which was associated with observable differences in the composition of the cellular population

141 Table 3.3.3a The proportion (percentage) of pigmented cells of 14 day old cultures of the mother culture and each of the selected cell lines. Each value is a mean of 3 replicates.

pigmented cells coloured cells as % of total

as % of total violet cells orange cells yellow cells

mother culture 31.47 12.80 8.37 10.30

violet cell line 35.00 30.63 4.37 -

orange cell line 31.00 1.90 14.37 15.07

yellow cell line 8.88 - 3.93 4.97

white cell line

Table 3.3.3b The arcsine values of the data presented in Table 3.2.3a showing the standard error of the mean after transformation of the data.

pigmented cells coloured cells as % of total

as % of total Violet cells Orange cells Yellow cells

mother culture 34.11 ± 0.83 21.08±1.22 16.35 ±2.98 18.68 ±0.50

violet cell line 36.13 ± 3.87 33.47 ± 3.15 11.66 ± 2.11 -

orange cell line 33.74 ± 2.69 6.32 ± 3.41 22.22 ± 1.27 22.53 ± 3.04

yellow cell line 17.28 ± 0.93 - 11.43 ± 0.43 12.79 ± 1.04

white cell line

142 with regard to pigmentation. However, it is not known whether these cell lines are stable or would undergo further changes with sub-culture, with regard to betalain production. In the following experiment an attempt was made to discover whether these cell lines were stable over a period of non-selective subculturing.

3.3.1.2 Investigation of the Stability of the Selected Cell Lines

In the previous experiment four cell lines, with different pigment characteristics, were obtained by cloning single cells of different colours. However, as variation and instability in culture systems is a very common phenomenon, with regard to secondary metabolite production and other aspects (see Meins and Binns

1977, Skirvin 1978, Zenk et al., 1977, Dougall et al., 1980 and Deus-Neumann and

Zenk 1984) it was decided to examine the degree of stability in the cell lines developed in this study, paying particular attention to betalain production and pigmentation characteristics of each cellular population. Measurements were made three times over a period of 15 months. The first analysis was performed 3 months after establishment of the single cell clones, the second after 9 months, and the third after 15 months. Cultures were grown under the conditions described in 2.1.4 and samples were collected at day 14 of each culture cycle.

I. Stability of selected cell lines with regard to betalain content

Table 3.3.4 presents the betalain content of 14 day old cultures of each of the selected cell lines measured at three different times, the first was performed 3 months after establishment of single cells, the second after 9 months, and the third at 15 months. The results show that betacyanin content of the violet cell line decreased

143 Table 3.3.4 Betalain content, in of 14 day old cultures of each of the selected cell lines as measured at three different times over a period of 15 months. The first analysis was performed 3 months after establishment of cell clones, the second after 9 months and the third at 15 months.

Violet cell line Orange cell line Yellow cell line White cell line

Betanin 3.89 ± 0.44 0.27 ± 0.02 nd nd

First Isobetanin 0.19 ± 0.03 0.02 ± 0.01 nd nd

analysis Isobetanidin 0.01 ± 0.004 nd nd nd

Total betacyanins 4.10 ± 0.46 0.29 ± 0.02 - -

MiraxanthinV 0.86 ± 0.20 1.06 ± 0.18 0.02 ± 0.01 nd

Betanin 0.55 ± 0.06 0.02 ± 0.003 0.002 ± 0.001 nd

Second Isobetanin 0.02 ± 0.01 nd nd nd

analysis Isobetanidin nd nd nd nd

Total betacyanins 0.57 ± 0.06 0.02 ± 0.003 0.002 ± 0.001 -

Miraxanthin V nd 0.03 ± 0.006 0.02 ± 0.01 nd

Betanin 2.23 ± 0.10 nd 0.06 ± 0.01 nd

Third Isobetanin 0.06 ± 0.004 nd nd nd

analysis Isobetanidin nd nd nd nd

Total betacyanins 2.29 ± 0.10 - 0.06 ± 0.01 nd

Miraxanthin V 0.12 ± 0.02 0.03 ± 0.01 0.07 ± 0.01 nd

nd = not detected

144 significantly over the test period. This reduction in the amount of betacyanin at 9 months was greater than at 15 months and reflected the fluctuation in betacyanin content observable from one subculture to another. In addition, at all three sampling times betanin was the main betacyanin and represented ca 95% - 97% of the total. The miraxanthin V produced by the violet cell line also decreased sharply over the test period from a high value at 3 months to a very low amount, at 15 months. Moreover, at 9 months it could not be detected.

In the orange cell line betacyanin (which was present as betanin and isobetanin, which formed ca 94% and 6% of the total betacyanin respectively) and miraxanthin V, decreased sharply over the experimental period. After 9 months, a sudden reduction in pigment production resulted in only 8.3% of betacyanin and 2.7% of miraxanthin V compared to the first analysis. At 15 months betacyanins were not detectable and miraxanthin V was present at a level similar to that measured at the second sampling time.

In the yellow cell line, although betacyanin was not detectable, after 9 months traces of betacyanin as betanin were present. After 15 months a remarkable increase in betacyanin content was observed. There was no difference in the amount of miraxanthin V measured at the first and second analysis, whereas, by the third analysis, it had increased by ca 3.7 fold compared to the amount at the first analysis.

The fluctuation in betalain production by this cell line was obvious from visual observation of the cultures.

145 The white cell line did not produce either betacyanin or miraxanthin V over the experimental period. However, after 15 months it was unable to survive in liquid culture although it could be maintained as callus.

II. Stability of selected cell lines with regard to the proportion of pigmented cells

In the previous analysis, the variability of the selected cell lines with regard to betalain production was examined over a period of 15 months. In this analysis the proportion (percentage) of pigmented cells in each cell culture line at each sampling time was also estimated, in order to find out whether variation in betalain production in these cell lines was related to changes in the proportion of pigmented cells.

The results presented in Table 3.3.5a show the percentage of pigmented cells of each cell line at 3, 9, and 15 months as specified previously in 3.3.2. Table 3.3.5b presents the same data after arcsine transformation.

Statistical analysis of the data show that in the violet cell line there was no significant difference in the proportion of total pigmented cells and the proportion of violet cells at the three sampling times. However, orange cells were not detected at 9 months but reappeared at 15 months at a proportion that was not significantly different from that estimated at 3 months. No yellow cells were observed at any of the three sampling times.

In the orange cell line the percentage of pigmented cells decreased sharply

146 Table 3.3.5a The proportion (percentage) of pigmented cells of 14 day old cultures of each of the selected cell lines at three different times over a period of 15 months. The first analysis was performed after 3 months, the second after 9 months, and the third at

15 months.

pigmented coloured cells as % of total

cells as %

of total Violet cells Orange cells Yellow cells

violet cell line 35.00 30.63 4.37 -

First orange cell line 31.33 1.90 14.37 15.07

analysis yellow cell line 8.90 - 3.93 4.97

white cell line

violet cell line 32.93 32.93 - -

second orange cell line 7.30 3.63 3.47 0.20

analysis yellow cell line 8.76 - 4.53 4.23

white cell line

violet cell line 43.9 42.4 1.51

Third orange cell line 4.20 1.67 2.53 -

analysis yellow cell line 9.20 0.30 6.57 2.33

white cell line

147 Table 3.3.5b The arcsine values of the data presented in Table 3.3.5a showing the standard error of the mean for the transformed data.

pigmented coloured cells as % of total

cells as %

of total Violet cells Orange cells Yellow cells

violet cell line 36.13 ±3.87 33.47 ± 3.15 11.66 ± 2.11 -

First orange cell line 33.74 ± 2.69 6.32 ± 3.41 22.22 ± 1.27 22.53 ± 3.04

analysis yellow cell line 17.28 ± 0.93 - 11.43 ± 0.43 12.79 ± 1.04

white cell line

violet cell line 35.02 ± 0.55 35.02 ± 0.55 - -

Second orange cell line 15.63 ± 0.91 10.61 ± 2.07 10.7 ± 0.5 2.06 ±1.08

analysis yellow cell line 17.20 ± 0.56 - 12.25 ± 0.74 11.87 ± 0.13

white cell line

violet cell line 41.49 ± 0.83 40.62 ± 0.79 7.02 ± 0.27 -

Third orange cell line 11.75 ± 0.97 7.33 ± 0.83 8.82 ± 1.79 -

analysis yellow cell line 17.64 ± 0.47 3.10 ± 0.31 15.92 ± 0.89 8.56 ± 1.41

white cell line

148 after 9 months (see Table 3.3.5a) and at 15 months was only 13.4% of that at 3 months. In contrast, the proportion of violet cells remained steady throughout the experimental period whereas the proportion of orange cells decreased sharply after 9 months, and then remained stable. The proportion of yellow cells had decreased sharply at 9 months and disappeared completely by 15 months.

In the yellow cell line the proportion of pigmented cells was similar at the three sampling times. A few violet cells were observable only at 15 months. The proportion of orange and yellow cells was similar at 3 and 9 months. However, at 15 months there was a slight increase in the proportion of orange cells. The proportion of yellow cells was similar at 3 and 9 months and not significantly lower at 15 months.

In the white cell line no pigmented cells were observed at any of the sampling times.

The results presented in 3.3.12 show that the productivity of the selected cell lines, with regard to betalain production, changes over the investigation period of 15 months. In the violet cell line betacyanin content was reduced by 50%. However, this decline did not correlate with a reduction in the proportion of total pigmented cells or in the proportion of violet cells. In addition, the miraxanthin V content also reduced sharply. Although miraxanthin V was only detectable whenever the orange cells are present, the amount was not correlated with the proportion of orange cells. In the orange cell line betalain content decreased sharply over the test period and that

149 decrease was associated with a sharp reduction in the proportion of the orange and yellow cells. The yellow cell line always produced low amounts of betalain. But there was a fluctuation in betalain content which was associated with a change in the proportion of orange and violet cells. The white cell line did not produce betalains and there were no pigmented cells.

Overall the results from 3.3.1 show that the cell plating technique can be used successfully for the isolation of various cell lines with different productivities, for the accumulation of betacyanin and/or betaxanthin. These clones also showed differences in the composition of the cellular population with regard to cell pigmentation.

Although the cell lines underwent some change over the term of non selective subculturing, the violet cell line remained more or less stable showing high productivity of betacyanin (mainly betanin). This clone also displayed a high proportion of violet cells over 15 months, in contrast, the yellow cell line remained with low productivity of betalains and low proportion of pigmented cells. These two cell lines, therefore, were chosen for further study on the relationship between the production of betalain and cellular heterogeneity under modified culture conditions.

3.3.2 Effect of Culture Conditions on Betalain Production in the Violet Cell Line

It was shown in 3.3.1.2 that the violet cell line was capable of producing large amount of betacyanin with smaller amounts of betaxanthin and the pigmented cell population of this cell line contained mainly violet cells, at relatively high proportion

(35-41%) with few orange cells. The aim of this series of experiments was to

150 investigate the effect of culture conditions on betalain production focusing on the relationship between production of pigments and cellular heterogeneity.

3.3.2.1 Effect of 2,4-D on growth and betalain production in cell cultures of the violet cell line

It was shown previously (see 3.2.2.1.3) that reducing the concentration of 2,4-

D in the medium from 0.02 mg.r' to 0.006 mg.1' enhanced the production of betalain in suspension cultures of Beta vulgaris (non selected cultures). The aim of this experiment was to study the effect of this low concentration of 2,4-D (0.006 mg.1 -1) on the growth, betalain production and proportion of pigmented cells, in the selected violet cell line.

Two media were used (Gamborg's 135) which differed only in their 2,4-D concentration, one at 0.02 mg.r' (control) (see 2.1.2.1) and the other at 0.006 mg.F'.

Each of these media was poured into 250 ml Erlenmeyer flasks, 50 ml per flask which were autoclaved, cooled and inoculated with cells obtained from well mixed 13 day old cultures, ca 1 g. wet weight cells per flask. The flasks were then placed on an orbital shaker and incubated under the conditions described in 2.1.4. The cultures were maintained under these conditions for 5 passages. At the fifth passage they were harvested at 14 days and analysed. Culture fresh and dry weights were determined as described in 2.2.1 and 2.2.2. The contents of Betalain and betalain related compounds were determined using HPLC (see 2.6), and the proportion of pigmented cells measured as indicated in 2.2.6. The results are presented in Tables 3.3.6 and 3.3.7.

151 The results presented in Table 3.3.6 show that reducing the 2,4-D level in the medium from 0.02 mgI' to 0.006 mg.r' caused a decrease in the growth of cultures as shown by a significant reduction in both fresh and dry weights. The low 2,4-D concentration (0.006 mgI'), however, had no effect on the total betacyanin content of cultures although isobetacyanin increased slightly but was present in a very low amount. On the other hand, miraxanthin V content was higher (3.9 fold) at the lower

2,4-D concentration. Dopamine content of the cultures grown at 0.006 mg.1' 2,4-D was also higher (2 fold). In contrast lowering 2,4-D had no effect on the Tyrosine and betalamic acid contents of the cultures.

The proportions of pigmented cells (Table 3.2.7), total pigmented cells violet and orange cells, in the cultures were not affected by the 2,4-D concentration.

The results show that 2,4-D at 0.006 mg.r' did not affect the production of betacyanin in cell cultures of the selected violet cell line whereas it caused a significant increase in the production of miraxanthin V. This increase was associated with a significant increase in dopamine content. The proportions of pigmented cells, however, were not affected by the treatment. In the next experiment the effect of sucrose concentration on the production of betalains in the violet cell line was examined.

152 Table 3.3.6 The effect of 2,4-D on the growth, betalain content, and betalain related compounds content of 14 day old cultures of the violet cell line. Each value is a mean of 3 replicates ± Se.

2,4-D level in mg.1'

0.02 0.006

Culture fresh wt. 8.37 5•73* (g) ±0.18 ±0.06

Culture dry wt. 0.48 0.39* (g) ±0.01 ±0.01

Betanin 2.19 2.08

(mM.g dSl ' ) ± 0.09 ± 0.05

Isobetanin 0.06 0.10*

(mM.g d.SSI. ' ) ± 0.004 ± 0.01

Total betacyanin 2.26 2.18 (MM-g&) ± 0.10 ± 0.05

MiraxanthinV 0.12 0.47* (MM-g&) ± 0.02 ± 0.01

Tyrosine 8.66 11.40

(mM.g d ' ) ± 0.91 ± 0.27

Dopamine 65.34 114.83* (MM. g.1) ± 2.4 ± 4.0

Betalamic acid 0.05 0.07 (mM.g') ± 0.01 ± 0.002

* = Significant at P <0.05

153 Table 3.3.7 Effect of 2,4-D on the proportion of pigmented cells of 14 day old cultures of the violet cell line. The data are presented as percentage, arcsine values of the percentage, and standard error of the mean after transforming the percentages into arcsine values. Each value is a mean of 3 replicates ± Se.

2,4-D 0.02 mg.r' 2,4-D 0.006 mg.i' different coloured different coloured total cells total cells pigmen- pigmen- ted cells violet orange ted cells violet orange cells cells cells cells Percentage 43.87 42.37 1.5 42.20 40.81 1.39

Arcsine value of 41.47 40.62 7.023 40.44 39.73 6.71 the percentage Se. of the transform- ± 0.80 ± 0.79 ± 0.27 ±1.93 ± 2.0 ± 0.78 ed Dercentae

154 3.3.2.2 Effect of sucrose on growth and betalain production in cell cultures of the violet cell line

It was shown in 3.2.1.3 that increasing sucrose concentration in the medium from 2% up to 3%, resulted in an enhanced production of betalains in Beta vulgaris cell cultures. In this experiment, a comparison was made between the effects of 2% and 3% sucrose on the growth and betalain production in cell cultures of the violet cell line.

Two media were used (Gamborg's 135) which differed only in their sucrose concentration. One with a standard amount of sucrose (2%) (see 2.1.2.1) and the other with 3% sucrose. Each of these media was poured into 250 ml Erlenmeyer flasks, 50 ml medium per flask, and then sterilised. After cooling the flasks were inoculated with cells obtained from a well mixed 14 day old culture, ca 1 g. wet weight cells per flask.

Cultures were then incubated on a rotary shaker under conditions described in 2.1.4.

On day 14, cultures were collected and analysed. Culture fresh and dry weight were determined as described in 2.2.1 and 2.2.2, the content of betalain and betalain related compounds was analysed by HPLC as described in 2.6, and the proportion of pigmented cells was determined as described in 2.2.6. Results are presented in Table

3.3.8.

Table 3.3.8 shows that 3% sucrose did not significantly affect the growth of cultures as shown by fresh and dry weight. On betacyanin production, sucrose at 3%

155

Table 3.3.8 The effect of sucrose on the growth, betalain content, betalain related

compound content and the proportion of pigmented cells of 14 day old cultures of the

violet cell line. Each value is a mean of 3 replicates ± Se.

2% 3%

sucrose sucrose

Culture fresh wt. 8.77 9.98 (g) ± 0.45 ± 0.34

Culture dry wt. 0.65 0.71 (g) ± 0.01 ± 0.02

Betanin 0.60 1.24 (mM.9. 1 ) ± 0.08 ± 0.06

Isobetanin 0.02 0.02 (mM.g ' ) ±0.02 ±0.01

Total betacyanin 0.62 1.27* (mM.9 1) ± 0.09 ± 0.05

Miraxanthin V nd nd

Tyrosine 10.45 12.55 (mM.g') ± 1.10 ± 0.73

Dopamine 73.70 52.28* (MM- g) ± 0.65 ± 2.20

Betalamic acid 0.08 0.16 (mM.gd ') ± 0.01 ± 0.03

% pigmented cells 30.27 31.17

arcsin value of percentage data 33.40 33.92 ±0.45 ±1.18

* = significant at 0.05 nd = not detected

156 caused a significant increase in betanin content. The cultures grown on 3% sucrose produced almost twice the amount of betanin compared to the control with 2% sucrose. In contrast, isobetanin production was not affected by increasing the sucrose level from 2% to 3%. Miraxanthin V was not detectable in any of the cultures. The tyrosine and betalamic acid content of the cultures was also not affected by the increase in sucrose concentration. However, dopamine content was significantly lower in cultures grown on 3% sucrose (control). The proportion of pigmented cells, which were all violet, was similar on both 2 and 3% sucrose.

The results show that sucrose at 3% enhanced the betanin content and this enhancement was not associated with an increase in the proportion of pigmented cells.

In addition, this treatment caused a reduction in dopamine content of the cultures. In the following experiment, the effect of light, a factor known to have a marked influence on betalain formation, was studied.

3.3.2.3 Effect of light on growth and betalain production in cell cultures of the selected violet cell line

It has been reported in the literature that light plays an important role in betalain biosynthesis. In some reports it has been shown that light is an essential requirement, e.g. in Amaranthus caudatus seedlings (Köhler, 1972), in Portulaca grandflora cell cultures (Liebish and Böhm 198 1) and callus (Kishima et al., 1991), in suspension cells of Chenopodium rubrum (Berlin et al., 1986), in Beta vulgaris cell cultures (Girod and Zryd 1985), and in Amaranthus tricolor suspension cultures

(Bianco-Colomas and Hugues 1990). Whereas, in other cases reports showed that

157 betalain synthesis can take place in the dark but is increased by light, e.g. in seedlings

of some species of the Centrospermae (Wohipart and Mabry 1968), in seedlings of

Amarathus tricolor and Celosiaplumosa (Giudici De Nicola et al., 1972, 1973). It has

also shown with callus of Portulaca grandflora (Endress et al., 1984) and

Chenopodium rubrum (Berlin et al., 1986) that betacyanin formation was much higher

in the dark than in the light. From these conflicting reports it would appear that light

can affect betalain synthesis differently in a range of species and sometimes

differently between plant material of the same species. In the following experiments

the effect of light on the growth and betalain accumulation in the violet cell line was

investigated.

3.3.2.3.1 Effect of light and dark treatments on growth and betalain production

The aim of this experiment was to investigate the effect of light on betalain

production in cell cultures of the violet cell line.

250 ml Erlenmeyer flasks each containing 50 ml of standard growth medium

(see 2.1.2.1) were inoculated with pooled cells obtained from well mixed 13 day old

cultures, ca 1 g wet wt. cells per flask. The flasks were then divided into two groups

one group was placed in a black box covered tightly with two layers of aluminium

foil, to prevent any leakage of light, and the other group was incubated at an

irradiance of 15 ±1 tmol.m 2.s', supplied by Thorn natural light fluorescent tubes

(Thom EMI UK.). Both groups were incubated at 25 ±1 °C on an orbital shaker,

158 rotating at 86 rpm. After 14 days, the cultures were harvested and analysed. The appearance of cultures grown in the dark and those grown in the light is shown in Fig.

3.3.8 The fresh and dry weights of cultures were determined as described in 2.2.1 and

2.2.2, the content of betalain and betalain related compounds was determined using

HPLC (see 2.6), and the proportion of pigmented cells was estimated as indicated in

2.2.6. Results are presented in Tables 3.3.9 and 3.3.10.

As shown from the data presented in Table (3.3.9) the fresh weight of the cultures grown in the dark was less than that of cultures grown in the light. However, there was no significant difference in the dry weight of the cultures grown in the dark or in the light. The betanin and isobetanin content of the cultures grown in the light was much higher (Ca 2x) than that of cultures grown in the dark. Miraxanthin V content was also higher in the illuminated cultures but was only present in small amounts. The tyrosine and dopamine content of cultures grown in the dark and those grown in the light were not significantly different. Betalamic acid was not detectable in any of the cultures examined, however, it was previously detected in other experiments but in very small amounts and may have been lost during sample preparation. The proportions of pigmented cells (Table 3.2.10), total pigmented cells

(violet cells and orange cells) of cultures were not affected by light.

The results show that although betalain content was higher in light grown cultures, there was no significant difference in the proportion of pigmented cells of cultures grown in light or dark indicating that the pigmented cells were capable of

159 Figure 3.3.8 The appearance of 14 day old cultures of the selected violet cell line (A) grown in the light and (B) in the dark.

IF i A B

160 Table 3.3.9 Effect of light on growth, betalain content, and content of betalain related compounds of 14 day old cultures of the violet cell line. Each value is a mean of 3 replicates ±se.

Light Dark

Culture fresh wt. 9.07 7.69 (g) ± 0.07 ± 0.21

Culture dry wt. 0.47 0.43 (g) ± 0.03 ± 0.01

Betanin 1.47 0.66*

(mM.g d ' ) ± 0.07 ± 0.05

Isobetanin 0.05 0.03 k

(mM.g d ,') ± 0.01 ± 0.002

Total betacyanin 1.52 0.68*

(mM.9 d 1 ) ± 0.08 ± 0.06

MiraxanthinV 0.09 0.01*

(mM.g d 1 ) ± 0.02 ± 0.01

Tyrosine 6.77 6.15 (mm-g1. ') ± 0.45 ± 0.69

Dopamine 139.20 116.50 (MM. ± 9.20 ± 7.50

Betalamic acid nd nd

(mM.g d ' )

* = Significance at 0.05 nd = Not detectable

161 Table 3.3.10 Effect of light on the proportion of pigmented cells of 14 day old cultures of the violet cell line. The data are presented as percentage, arcsine values of the percentage, and standard error of the mean after transforming the percentages into arcsine values. Each value is a mean of 3 replicates ±se.

light dark different coloured different coloured total cells total cells pigmen- pigmen- ted cells violet orange ted cells violet orange cells cells cells cells Percentage 43.13 41.83 1.31 41.26 38.88 2.38

Arcsine value of 41.03 40.30 6.53 39.99 38.67 5.19 the percentage se. of the transform- ± 0.71 ± 0.88 ± 0.74 ±1.40 ± 0.02 ± 5.0 ed Dercentae

162 accumulating betalain in the dark and that light promoted pigment accumulation in these cells. In the following experiment the effect of light intensity on the growth and betalain production was examined in an attempt to discover the optimum irradiance level for pigment production.

3.3.2.3.2 Effect of irradiation level on growth and betalain content in cell cultures of the violet cell line

In the previous experiment (3.3.2.3.1) white light at an intensity of 15 ±1 p.mol.m 2.s') had a promotive effect on betalain content. The aim of this experiment was to examine the effect of various intensities of light on the growth and betalain content in cell cultures of the violet cell line.

250 ml Erlenmeyer flasks, each containing 50 ml of standard growth medium

(see 2.1.2.1) were inoculated with cells obtained from well mixed 13 day old cultures, ca 1 g wet weight cells per flask. The cultures were then divided into four groups and 1 incubated at four different irradiation levels (7±1, 15±1, 35±1, and 70±1 tmoI.m 2s )

These were achieved by placing the cultures at varying distances from the light source. The illumination was supplied by Thom natural light fluorescent tubes (Thorn

EMI UK). The light intensity was measured using a LI-COR steady state photometer model LI-1600. The cultures were incubated at 25 ±1 °C on an orbital shaker, rotating at 86 rpm. After 14 days the cultures were harvested and analysed. Culture fresh and

163 dry weights were determined as described in 2.2.1 and 2.2.2, and betalain content was measured using HPLC (see 2.6). The results are presented in Table 3.3.11.

The results presented in Table (3.2.11) show that the fresh weights of cultures subjected to an irradiance level of 7 ±1 .tmol.m 2.s' were slightly lower than that of cultures subjected to higher irradiance levels. However, there was no significant difference in dry weight. Betanin and isobetanin content of the cultures grown under the examined irradiance levels were also not significantly different. Miraxanthin V was not detected in any of the treatments including the control.

The results show that the levels of irradiance, within the range examined, had a similar effect on betalain production. This suggests that the minimum level of irradiance needed to stimulate betalain formation in the culture is 7 ±1 tmol.m 2.s' or lower.

Overall the results from this sub-section show that pigment yield in cultures of the violet cell line may be increased by modifying the culture conditions. However, changes to the amount of betalains accumulated under different culture conditions do not correlate with the proportions of pigmented cells.

It is assumed that the proportion of pigmented cells in cultures of this cell line is at its maximum level, which is probably genetically controlled and therefore cannot be increased under the conditions employed to enhance pigment production. In the following sub-section the production of betalain in the yellow cell line, was

164 Table 3.3.11 Effect of irradiance intensity on the growth and betalain production in

14 day old cultures of the violet cell line. Each value is a mean of 3 replicates ± Se.

Irradiance intensity in tmol.m 2.s'

7±1 15±1 35±1 70±1

Culture fresh wt. 6.09 7.84 7.39 8.00 (g) ± 0.35 ± 0.56 ± 0.13 ± 0.34

Culture dry wt. 0.39 0.45 0.44 0.43 (g) ± 0.03 ± 0.03 ± 0.01 ± 0.003

Betanin 0.49 0.66 0.81 0.62 (mM.g d ) ± 0.02 ± 0.12 ± 0.03 ± 0.03

Isobetanin 0.04 0.04 0.06 0.05 (mM.g dl ) ± 0.004 ± 0.01 ± 0.004 ± 0.001

Total betacyanin 0.53 0.70 0.86 0.66 (mM.g dl j ± 0.03 ±0.13 ± 0.04 ± 0.03

Miraxanthin V nd nd nd nd (mM.g dl j

nd = Not detectable

165 investigated under manipulated conditions. This cell line was shown to have a low proportion of pigmented cells. Assuming that the proportion of the productive cells in this cell line is below the maximum, it would be expected that under the conditions that promote greater production of pigment, the proportion of pigmented cells may be increased. This hypothesis was examined in the following sub-section.

3.3.3 Effect of Culture Conditions on Betalain Production in the Yellow Cell Line

It was shown previously in 3.3.1.2 that the yellow cell line produced very small amounts of betalain (mainly miraxanthin V and occasionally betanin) and contained a low proportion of pigmented cells. In the following experiments, attempts were made to increase the production of betalain in this cell line by modifying some of the culture conditions and to discover whether changes in betalain content could be correlated with the proportion of pigmented cells.

3.3.3.1 Effect of 2,4-D on growth and betalain production in cultures of the yellow cell line

Previously it had been shown in (3.2.2.1.3) that reducing the 2,4-D concentration in the medium from 0.02 mg.F' to 0.006 mg.1' enhanced the production of betalains in the original cell culture of Beta vulgaris. Therefore, it might be expected that lowering the 2,4-D concentration to 0.006 mg.r' might promote betalain content in the yellow cell line.

166 Two media (Gamborg's 135) were used which differed only in 2,4-D concentration one at 0.02 mg.r' (control), and the other at 0.006 mg.r'. The media were poured into 250 ml Erlenmeyer flasks, 50 ml per flask and then autoclaved.

After cooling each flask was inoculated with ca 1 g. wet wt. of cells obtained from well mixed 13 day old cultures. The cultures were then placed on an orbital shaker and incubated under the conditions described in 2.1.4 and maintained for 5 passages.

At the fifth passage, the cultures were harvested at 14 days and analysed. The growth of cultures was determined by measuring the fresh and dry weights (see 2.2.1 and

2.2.2), the amounts of betalain and betalain related compounds were determined by

HPLC analysis (see 2.6), and the proportion of pigmented cells was estimated as described in 2.2.6. The results are presented in Tables 3.3.12 and 3.3.13 and Fig.

3.3.9.

The results in Table (3.3.12) show that reducing the 2,4-D level in the medium from 0.02 to 0.006 mg.i' caused a significant reduction in the growth of cultures as shown by the fresh and dry weight values. A low concentration of 2,4-D (0.006 mg.l

'), significantly enhanced the production of betanin (5-fold) but had no effect on the miraxanthin V content. However, from the HPLC chromatogram monitored at 472 nm, (see Fig. 3.3.8) it can be seen that 2,4-D at the lower concentration induced the appearance of several peaks of unknown compounds, which might be betaxanthins other than miraxanthin V. Tyrosine and dopamine content of the cultures grown at

0.006 mgI' 2,4-D was lower than that of the control cultures. In contrast, a trace amount of betalamic acid was present at the lower 2,4-D concentration but was not

167 Table 3.3.12 Effect of 2,4-D on the growth, betalain content, and betalain related compounds content of 14 day old cultures of the yellow cell line. Each value is a mean of 3 replicates ±se.

Level of 2,4-D in mg.l'

0.02 0.006

Culture fresh wt. 8.36 5•73* (g) ±0.17 ±0.06

Culture dry wt. 0.48 039* (g) ±0.01 ±0.01

Betanin 0.02 0.10* (mM.g d ') ± 0.01 ± 0.01

Miraxanthin V 0.04 0.04 (MM .gd) ± 0.003 ± 0.004

Tyrosine 29.40 20.47*

(mM.g 1 ) ± 0.50 ± 0.27

Dopamine 55.24 14.47* (MM. g') ± 1.70 ± 2.20

Betalamic acid nd 0.02 (mM.g d ') ± 0.01

* = Significance at P <0.05 nd = Not detectable

168 Table 3.3.13 Effect of 2,4-D on the proportion of pigmented cells of 14 day old cultures of the yellow cell line. The data are presented as percentages, arcsine values of the percentages, and standard errors of the mean after transformation of the percentages. Each value is a mean of 3 replicates ±se.

2,4-D 0.006 mg.r' 2,4-D 0.006 mgT'

Total Different coloured cells Total Different coloured cells pigmen- Violet Orange Yellow pigmen- Violet Orange Yellow ted cells cells cells cells ted cells cells cells cells Percentage 9.2 0.3 6.57 2.33 25.63 0.8 6.77 18.0

Arcsine value of 17.64 3.1 14.75 8.56 30.39 5.12 15.06 25.07 the percentage Se. of the transfor- ±0.47 ±0.31 ± 1.3 ± 1.4 ±1.1 ±0.19 ± 0.48 ± 1.2 med percentage

169 Figure 3.3.9 An HPLC chromatogram monitored at 472 nm obtained of a cell extract

(see 2.6.3) of 14 d. old cultures of the yellow cell line grown in media with 2,4D at

either 0.02 mg.1' (cont.) (A) or at 0.006 mg.l'(B). Peaks (a) and (b) = Betanin and

miraxanthin V, respectively.

100

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

Retention Time (mm)

170 detectable in the controls. The proportion of pigmented cells (Table 3.3.13) of cultures grown in the medium with 2,4-D at 0.006 mg.r' was higher than the controls. This increase, however, was accompanied by an increase in the proportion of yellow and violet cells but not orange cells.

Overall, 2,4-D at 0.006 mg.r' stimulated the production of betanin but not miraxanthin V. It also induced the production of several unknown compounds that have an appreciable absorption at 472 nm. The proportion of total pigmented cells was increased at 0.006 mg.r' 2,4-D, however, this increase was due to a rise in the proportion of yellow and violet cells but not orange cells. In addition, 2,4-D at the lower concentration caused a significant reduction in growth, tyrosine, and dopamine content, but enhanced, slightly, the amount of betalamic acid. As the increase in betanin at 0.006 mg.r' 2,4-D was coupled with an increase in betalamic acid. It could be that betalamic acid is limiting the production of betalain. Therefore, in the next

experiment the effect of betalamic acid added to the medium on betalain production

was investigated.

3.3.3.2 Effect of added betalamic acid on growth and betalain production in

cultures of the yellow cell line

In the previous experiment (3.3.3.1) it was shown that the enhancement in

betanin production, occurred at 0.006 mg.1 1 2,4-D, and was associated with an

increase in the content of betalamic acid. The aim of this experiment, therefore, was to

171 examine the effect of added betalamic acid both on betalain production and the proportion of pigmented cells.

Betalamic acid was prepared according to the method described in 2.6.5.3. The final concentration of the preparation was determined as described in 2.6.6. The preparation was divided into 3 parts and each was diluted appropriately to produce three different concentrations (ca 1, 2, and 4 mlvi). Each fraction was then sterilised by filtration through a 0.2 p.m nylon membrane filter (Whatman) held in a Millipore filter unit (Millipore, UK Ltd, Watford) attached to a syringe.

250 ml Erlenmeyer flasks each containing 50 ml of standard growth medium

(see 2.1.2.1) were divided into four groups. One group used as a control, was left free from added betalamic acid, the other three groups were supplemented with the three different concentrations of betalamic acid prepared previously. Each flask was injected with 100 p.l of the betalamic acid solution, using a sterile syringe. The final concentrations in the flasks were ca 2,4, and 8 j.tM. The flasks were shaken and then inoculated with cells obtained from 13 day old well mixed cultures, ca 1 g. wet wt. of cells per flask. The flasks were incubated on an orbital shaker under the conditions described in 2.1.4. After 14 days the cultures were harvested and analysed. Culture fresh and dry weights were determined as described in 2.2.1 and 2.2.2. Betalain

content was determined using HPLC analysis (see 2.6). Results are presented in

Tables 3.3.14 and 3.3.15.

The results in Table 3.3.14 shows that betalamic acid, at concentrations of 2, 4,

172 Table 3.3.14 Effect of different concentrations of betalamic acid on growth, betalain content, and content of betalain related compounds of 14 day old cultures of the yellow cell line. Each value is a mean of 3 replicates ± Se.

Concentration of added betalamic acid

Control 2 .LM 4 iM 8 iM

Culture fresh wt. 6.62 5.76 6.21 6.05 (g) ± 0.26 ± 0.18 ± 0.41 ± 0.32

Culture dry wt. 0.51 0.46 0.45 0.40* (g) ± 0.01 ± 0.002 ± 0.02 ± 0.04

Betanin nd nd nd nd

* Miraxanthin V 0.03 0.01 nd nd

(MM .g d. ' ) ± 0.001 ± 0.004

Tyrosine 22.21 18.75 20.21 15.39

(mM.g dl ') ± 1.52 ± 0.95 ± 1.75 ± 2.09

Dopamine 80.67 84.30 83.35 71.10

(mM.g dl ' ) ±3.43 ±10.10 ±3.40 ±10.80

Betalamic acid nd nd nd nd

(mM.g dl ')

* = Significance at P <0.05 nd = Not detectable

173 3.3.15 Effect of added betalaniic acid on the proportion of pigmented cells of 14 day old cultures of the yellow cell line. The data are presented as percentages, arcsine values of the percentages, and standard errors of the mean after transforming the percentages into arcsine values. Each value is a mean of 3 replicates ± Se.

Concentration of added betalamic acid Cont. 2tM 4j.tM 8j.tM Total Different Total Different Total Different Total Different pigmen- coloured cells pigmen- coloured cells pigmen- coloured pigmen- coloured cells ted cells ted cells ted cells cells ted cells Org. Yel. Org. Yel. Org. Yel. Org. Yel. Percentage 8.83 4.67 4.17 7.52 3.5 4.07 4.2 - 4.2 4.37 - 4.37

Arcsine value 16.95 12.44 11.77 15.96 10.76 11.63 11.82 - 11.82 12.04 - 12.04 of the percentage Se of the ±0.3 ±0.72 ±0.33 ±0.4 ±0.56 ±0.21 ±0.25 - ± ±0.53 - ±0.53 transformed percentage _

174 or 8 pM, did not have a significant effect on fresh weight. The dry weight of the

cultures was also not affected by these treatments apart from 8 jiM which caused a

small reduction in dry weight. The content of Miraxanthin V was decreased by the

addition of betalamic acid and at the treatments of 4, and 8 tM became undetectable.

Betanin was not detected either in the control or the treated cultures, however, as

previously mentioned the production of betalain in this cell line was shown to

fluctuate from subculture to another. Tyrosine and dopamine contents were not

significantly affected by these treatments. The proportion of pigmented cells (Table

3.3.15) was also not significantly influenced by the addition of betalamic acid under

the treatments. However, the orange cells have completely disappeared when

betalamic acid was added at concentrations of 4, and 8 jiM. However, it is possible

that the added betalamic acid had been decomposed in the medium before it was used

by cells. And so the apparent reduction in miraxanthin V production was caused by

the products of betalamic acid breakdown.

The results show that the addition of betalamic acid to the culture medium

depressed the production of miraxanthin V. and at high concentrations of betalamic

acid when miraxanthin V became undetectable, the orange cells could not be

observed.

Results from this subsection showed that in the yellow cell line, changes in the

production of betalain under modified culture conditions may correlate with changes

in the proportion of pigmented cells.

175 Summary of the results in 3.3

Using the technique of single cell cloning, four cell lines (violet, orange, yellow and white were obtained from the original culture of Beta vulgaris (mother culture). The cell lines exhibited qualitative and quantitative differences with regard to betalain production.

Betanin and miraxanthin V were the main betalains produced by the original culture and the derived cell lines.

The composition of the cellular population was different in the different cell lines the white cell line contained no pigmented cells while other pigmented lines contained limited proportions of pigmented cells, not exceeding that found in the original culture. The pigmented cell populations consisted mainly of violet cells, in the violet line, and of orange and yellow cells in the orange and yellow lines.

Changes in the capacity of the cell lines (pigmented cell lines) to produce betalains were observed during the period of non-selective subculturing. However, the violet cell line retained a high betacyanin content while the yellow cell line showed a reduced ability to produce betalains. The orange cell line lost the capacity to produce both betalains after 9 months of clone establishment and remained so until the end of the period of study.

176 Modification to culture conditions brought about changes in the betalain content of the violet and yellow cell lines. However, modification to pigment production was not correlated with changes in the proportions of pigmented cells in the violet cell line but was correlated in the yellow cell line.

The production of miraxanthin V in the violet cell line was associated with the presence of orange cells and the production of betanin in the yellow cell line was correlated with the presence of violet cells.

Reducing the concentration of 2,4-D in the medium from 0.02 mg.1 -1 to 0.006 mg.1 1

stimulated the production of miraxanthin V but not betanin in the violet cell line whereas, it enhanced the production of betanin but not miraxanthin V in the yellow

cell line. However, in the yellow cell line 2,4-D at 0.006 mg.i' induced the production

of several unknown compounds of appreciable absorption at 472 nm and this was

associated with a remarkable increase in the proportion of yellow cells.

Addition of betalamic acid to the medium depressed the production of miraxanthin

V. At concentrations of 4 and 8 jtM, miraxanthin V accumulation was completely

suppressed and this was associated with the disappearance of orange cells, while the

proportion of yellow cells remained unaffected.

Increasing the sucrose level in the medium from 2% to 3% increased the production

of betanin in the violet cell line.

177 10. Light is not a compulsory requirement for betalain formation in the violet cell line.

However, it had a stimulatory effect on pigment production. The level of irradiance necessary to express such a promotive effect was 7 ±1 tmol.m 2.s' or lower.

Overall, results obtained from this section showed that the violet cell line is characterised by high betacyanin production (mainly betanin). Changes in betacyanin content of this cell line, as a result of culture variability or by modification to culture conditions, does not appear to result from changes in the proportion of pigmented cells but from a change in the capacity of cells to produce or store the pigment possibly as a result from changes within the pigment pathway.

In the following section, the highly productive violet cell line was chosen to investigate the possible role of tyrosine hydroxylase, an enzyme known to be involved in betalain biosynthesis, on betalain yield.

178 3.4 INVESTIGATION INTO THE POSSIBLE ROLE OF TYROSINE

HYDROXYLASE ACTIVITY ON BETALAIN PRODUCTION

Tyrosine hydroxylase (tyrosinase) is the enzyme which catalyses the conversion of tyrosine to L-DOPA (dihyroxyphenylalanine) (Letts and Chase, 1974,

Wichers et al., 1983, 1984, and Muller et al., 1993, 1994). It has been suggested that this hydroxylation is the entry point to the pathway leading to betalain synthesis (see

Endress, 1977, 1979, Böbm and Rink, 1988, and Muller et al., 1993). Using data obtained from feeding experiments with labelled tyrosine and DOPA, Zryd et al.

(1982) and Hirano and Komamine (1994) concluded that the hydroxylation of tyrosine and the formation of DOPA is a regulatory step in betacyanin biosynthesis.

Accordingly, in this section attempts have been made to relate tyrosine hydroxylase activity to the biosynthesis of betalains.

In 3.3.1 it was shown that the selected white and violet cell lines were two distinctive cell lines in which betacyanin biosynthesis was either blocked (in the white cell line) or activated to produce abundant amounts of the pigment (in the violet cell line). In this section (3.4), the activity of tyrosine hydroxylase in these two cell lines was determined in order to determine whether this enzyme plays a key role in betalain synthesis. In addition the activity of the enzyme was also determined in cell cultures of the violet cell line grown under conditions in which betacyanin production was either enhanced (3% sucrose) (3.3.2.2) or reduced (in the dark) (3.3.2.3).

179 3.4.1 The Activity of Tyrosine Hydroxylase in Cell Cultures of the White and

Violet Cell Lines

It was shown in 3.3.1 that the violet cell line produced large amounts of betacyanin, and the white cell line, in contrast, did not produce any betalain. The aim of this experiment was to examine the activity of tyrosine hydroxylase in these two cell lines in order to determine whether the activity of this enzyme is related to the accumulation of betalains.

Suspension cultures of the white and violet cell lines were grown in

Gamborg's B5 medium under the conditions described in 2.1.4. After 14 days the cells were harvested and divided into two portions. One was used for the determination of betalain content (using HPLC see 2.6) and the other was used for the enzyme assay. Tyrosine hydroxylase was extracted and assayed according to the methods described in 2.9.1 and 2.9.3 respectively. The protein content of the enzyme extract was determined as described in 2.9.3. Table 3.4.1 presents the activity of tyrosine hydroxylase and the content of betalain in 14 day old cell cultures of the white and the violet cell lines. The results show that the activity of the enzyme in the violet cell line, which produced large amount of betacyanin was ca 59% higher than that of the white cell line, which produced no betalain. When the activity was expressed as units per mg. protein (specific activity); a similar increase (ca 60%) in specific activity was observed for the violet cell line.

180 Table 3.4.1 Tyrosine hydroxylase activity and betalain content in 14 day old cell cultures of the violet and white cell lines. Each value is a mean of 3 replicates ± se.

Violet cell line White cell line

Activity of enzyme (U) 27.21* 17.01 ± 2.0 ± 0.29 Specific Activity (U.mg' protein) 498.0* 313.08 ± 37.0 ±2.5 Betacyanin content (mM.g.d.wt.') 2.26 nd 0.01 Betaxanthin content 0.12 nd (mM.g.d.wt.') ± 0.02

nd = not detectable * = Significant at p < 0.05 U = Unit of enzyme

The results suggest that tyrosine hydroxylase plays an important role in betacyanin biosynthesis. However, as the white cell line exhibited some activity of the enzyme although it produced no pigment, this would suggest that in a non-betacyanin producing cell line(white cell line) tyrosine hydroxylase at the site of betacyanin synthesis was inactive. Or it had a low activity and so the small amount of DOPA formed was preferentially incorporated into other competing pathways. Alternatively, it may be that another enzyme participates in regulating the synthesis of betalain in the white cell line.

In the following experiment, the activity of the enzyme in the violet cell line was examined under conditions in which betacyanin production was enhanced in order to discover whether there was any correlation between the activity of tyrosine hydroxylase, and the final yield of pigment.

181 3.4.2 The Activity of Tyrosine Hydroxylase in Cell Cultures of the Violet Cell

Line Grown at Two Different Concentrations of Sucrose

It was shown previously in 3.3.2.2 that betacyanin production was stimulated in cultures grown in medium with 3% sucrose. The aim of this experiment was to compare the activity of tyrosine hydroxylase in cultures grown in a medium with 2% sucrose and those grown within 3% sucrose in order to determine whether the stimulation in pigment production was accompanied by an increase in the activity of tyrosine hydroxylase.

Suspension cultures of the violet cell line were grown in Gamborg's B5 medium containing either 2% or 3% sucrose (see 2.1.2.1) under the conditions specified in 2.1.4. At day 14 of subculture, the cultures were harvested and cells were examined for betalain content, as measured by HPLC (see 2.6) and tyrosine hydroxylase activity. The activity of enzyme was assayed according to the methods described in 2.9.1 and 2.9.3 respectively. The protein content of the enzyme extract was determined as described in 2.9.2. Table 3.4.2 presents the activity of tyrosine hydroxylase and betalain content of cell cultures grown in either 2% or 3% sucrose.

As can be seen from the results from cell cultures grown in 3% sucrose where betacyanin content was ca 100% higher than those grown in 2% sucrose, the activity of the enzyme was also higher but with only ca 30%. However, when activity of the enzyme is expressed as specific activity, the increase is ca 61%.

182 Table 3.4.2 Tyrosine hydroxylase activity and betalain content in 14 day old cell cultures of the violet cell line grown in either 2% or 3% sucrose. Each value is a mean of 3 replicates ± se.

2% sucrose 3% sucrose

Activity of enzyme (U) 13.43 17.44* ±038 ±0.35

Specific Activity (U.mg' protein) 161.5 260.3* ±7.9 ±21.0

Betacyanin content (mM.g.d.wt.') 0.62 1.27* ±0.09 ± 0.05

Betaxanthin content nd nd (mM.g.d.wt.')

nd = not detectable

* = Significant at p = 0.05

U = Unit of enzyme

The results indicate that the increase in amount of pigment was partially correlated with an increase in tyrosine hydroxylase activity. However, the increase in the enzyme activity was not quantitatively correlated with the increase in amount of pigment.

183 In the following experiment, the activity of tyrosine hydroxylase was examined under culture conditions where betacyanin production was reduced (in the dark).

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

It was shown previously in 3.3.2.3 that betacyanin production in the dark was markedly inhibited. In this experiment the activity of tyrosine hydroxylase was determined and compared in light and dark grown cultures in order to find out whether the reduction in the amount of betacyanin produced in the dark could be correlated with a reduction in the activity of tyrosine hydroxylase.

Suspension cultures of the violet cell line were grown in Gamborg's B5 medium (see 2.1.2.1) and maintained either in the light or in the dark (see 2.1.4), for

14 days then cells were harvested and examined for betacyanin content, measured by

I-IPLC (see 2.6) and for tyrosine hyroxylase activity. The enzyme was extracted and assayed according to the methods described in 2.9.1 and 2.9.3 respectively. The protein content of the enzyme extract was determined as described in 2.9.2.

Table 3.4.3 presents the activity of tyrosine hydroxylase and betalain content of cell cultures grown in the light and in the dark, from which it can be seen that in dark grown cultures the betacyanin content was ca 55% lower than in the light grown

184 cultures. The activity of enzyme in the dark grown cultures was also lower ca 29%, when the activity was expressed per g. protein (specific activity) it was lower with ca

35.7% as compared to light grown cultures.

The results show that a decrease in betalain production in the dark was accompanied by a reduction in tyrosine hydroxylase activity. However, the reduction in enzyme activity did not parallel the decrease in the amount of pigment.

Table 3.4.3 Tyrosine hydroxylase activity and betalain content in 14 day old cell cultures of the violet cell line grown either in the light or in the dark. Each value is a mean of 3 replicates ± Se.

Light Dark

Activity of enzyme (U) 24.1 17.1 * ±1.0 ± 0.34

Specific Activity (U.mg' protein) 448.1 288* ±37.0 ±7.3

Betacyanin content (mM.g.d.wt.') 1.52 0.68* ± 0.08 ± 0.06

Betaxanthin content 0.09 0.01 *

(mM.g.d.wt. 1 ) ± 0.02 ±0.01

* = Significant atp=O.05

U = Unit of enzyme

185 Summary of the results in 3.4

Tyrosine hydroxylase activity was much higher (Ca 59%) in the betalain producing cell line (violet cell line) than in the non betalain producing cell line (white cell line).

The enhancement in betacyanin production in 3% sucrose was correlated with a stimulation in tyrosine hydroxylase activity. However, the increase in enzyme activity was not quantitatively related to the increase in pigment content.

The reduction in betacyanin production which occurred in the dark was associated with a decrease in the activity of tyrosine hydroxylase. However, again the decline in enzyme activity did not parallel the reduction in pigment content.

Overall the results suggest that tyrosine hydroxylase plays a regulatory role in betalain biosynthesis, however, other factors may determine the extent of betalain biosynthesis.

186 CHAPTER FOUR

DISCUSSION

187 Betalains, the red violet (betacyanin) and orange yellow (betaxanthin) pigments are accumulated in most members of the Centrospermae (Piattelli 1981).

The accumulation of both betacyanin and betaxanthin has also been observed in cultured cells of a variety of species from the Centrospermae e.g. Phytolacca americana (Sakuta et al., 1986), Chenopodium rubrum (Berlin et al., 1986),

Portulaca grandflora and Beta vulgaris (Constable 1967, Weller and Lasure 1981).

However, the production of these pigments occurs at a lower level than in the intact plant. In the present investigation, the accumulation of betacyanin and betaxanthin in cell cultures of Beta vulgaris was investigated. In this discussion, four aspects of betalain production are considered. The first section, (4.1) considers the relationship between growth and betalain accumulation. In the second (4.2) the factors

(environmental factors) affecting growth and betalain production are examined and in the third (4.3) the production of betalain is considered in relation to cellular

heterogeneity. Finally (4.4) the possible role of tyrosine hydroxylase in the regulation

of betalain synthesis is discussed.

4.1 GROWTH AND BETALAIN PRODUCTION

Most secondary metabolites in higher plants accumulate in differentiated and

non-proliferating tissue and cells at specific stages of growth. In cell and tissue

cultures the accumulation of secondary products generally takes place at the stationary

phase of the growth cycle after cell division has ceased. This inverse relationship

between growth and secondary product formation has been reported by several

188 investigators (Kaul and Staba 1969, Davies 1972, Yeoman et al., 1980, Hall and

Yeoman 1986a, Hirose et al., 1990, Sakuta et al., 1994). In the present study the accumulation of betacyanin and betaxanthin was correlated with growth (see Fig. 4.1) and maximum production of the pigments was achieved when growth had reached a maximum. Moreover, as the increase in cell number slowed down the increase in pigment accumulation, per culture, also slowed down. This reinforces the putative positive correlation between growth and betalain accumulation and is in agreement with those obtained from studies on betacyanin production in other species, for example, by Sakuta et al. (1986) (Phytolacca americana) and Hirose et al. (1990)

(Chenopodium rubrum) where product accumulation was maximal during the active phase of the growth cycle. Similar observations have also been reported in culture systems producing secondary metabolites other than betalain, such as volatile oils in

Ruta graveolens (Corduan and Reinhard 1972), podophyllotoxin in Podophyllum peltatum (Kadkade 1982), sapogenin in Agave wightii (Sharma and Khanna 1980) and berberine in Thalictrum rugosum (Kim et al., 1991). According to Hirano and

Komamine (1994) and Sakuta et al. (1994) betacyanin synthesis and accumulation in

Phytolacca americana was correlated not only with DNA synthesis, but also with progression through the cell cycle. In the present study there was a slight increase in the pigment content, per g. f. wt. on day 2 (see Fig. 3.1.3(A)) end of lag phase. Two explanations for this observation can be made. First, the synthesis of betalain occurred with the onset of cell division but prior to the full elongation of the cells. Secondly, the synthesis of the pigment launched immediately prior to the start of cell division.

189 Figure 4.1 A diagram showing the relationship between growth and betalain accumulation in a suspension culture of Beta vulgaris. (a), shows the correlation between cell number increase and pigment amount, r = 0.95 for betacyanin and 0.97 for betaxanthin. (b), shows the correlation between culture fresh weight and betalain amount, r = 0.92 for betacyanin and 0.95 for betaxanthin [betacyanin content was measured at 535 nm, and betaxanthin at 472 nm].

(A) 10

0 8 0 w

0 C 0 0 B.cyanin Cd 00 • B. xanthin 0 0 0

0 0 0 0 0

0 I I • I • I • I • I 0 2 4 6 8 10 12 Cell no./m1x10 5

(B) 10

0 8 0 C)

o6 C 0 0 B.cyanin CCS 0 0 O B.xanthin 0 0 0 0 0 0 2 • 0 0 0 0

I • I • I • 0 2 4 6 8 10

Culture fresh wt. (g)

190 In conclusion, the results presented here show that betacyanin and betaxanthin accumulation are closely related to growth and that cell division and the onset of pigment synthesis are closely linked. It is still uncertain whether the onset of pigment synthesis occur prior to or immediately after the start of cell division.

4.2 FACTORS REGULATING BETALAIN PRODUCTION

4.2.1 Effect of Sucrose Concentration on Growth and Betalain Accumulation

Sucrose is the most popular carbon source for the culture of plant cells and tissues. The effects of sucrose concentration on cell proliferation and secondary metabolite production have been investigated by several workers and a positive correlation has been reported between sucrose concentration and the production of secondary metabolites in a number of systems. For example, the production of lignin in cultured sycamore cells (Carceller et al., 1971), for solasodine production in callus cultures of Solanum nigrum (Bhatt et al., 1983), and for anthocyanin production in

Vitis suspension cells (Yamakawa et al., 1983). The positive effects of sucrose have been related to the nutritional role of this compound as a carbon source. However, there are also reports of an optimum concentration of sucrose for secondary metabolite production e.g. 8% sucrose for the accumulation of alkaloids in dark grown suspension cultures of Catharanthus roseus (Knobloch and Berlin 1980), and 5% sucrose for shikonin production in Lithospermum erythrorhizon callus cultures

(Mizukami et al., 1977). The optimum sucrose concentration for metabolite production is not always optimum for growth of the culture. In some instances, a

191 higher concentration of sucrose stimulated product formation as well as growth

(Kitamura et al., 1991, Norton et al., 1991, Wysokinska and Swiatek 1991, and

Dupraz et al., 1994). Whereas in other cases the enhancement of secondary metabolite production caused by a high level of sucrose was associated with a reduction in the amount of cell biomass (Nigra et al., 1990, Cormier et al., 1990, Do and Cormier

1990, 1991b, Rudge and Morris 1986). Zenk et al. (1975) reported that in Morinda suspension cultures the optimal concentration of sucrose for anthraquinone production was independent of the optimal concentration for growth.

In the present study (see 3.2.1.3) increasing the sucrose concentration in the medium from 2% to 3%, 6% or 9% had no effect on the growth rate of cultures but it prolonged the period of growth and postponed the start of the stationary phase indicating that the concentration of sucrose (2%) used in the standard growth medium was a limiting factor for the duration of growth in these cultures. The delay in senescence of cells, with an increased level of sucrose in the medium was also observed by McCarthy et al., 1980, Merillon et al., 1984 and Arias-Castro et al.,

1993b.

On the other hand, the accumulation of betalains per culture was significantly affected by 3% and 9% sucrose. The results presented here show that 3% sucrose was the optimum concentration for betacyanin and betaxanthin. A similar observation was made by Sakuta et al. (1987) for betacyanin production in Phytolacca americana which was maximal at 88 mM (ca 3%) on a per cell basis. Wohlport and Black (1973)

192 showed that a certain minimal sucrose level was necessary for betacyanin production in leaf disks of Beta vulgaris and that sucrose above this level has relatively little effect on betacyanin production.

However, in the present study, the stimulatory effect of 3% sucrose on betalain production did not appear until several days, after 10 days in the case of betacyanin, and after 15 days for betaxanthin. Two different explanations can be offered to explain this delay in response. First, the effects of sucrose on pigment synthesis might be indirect and affect other processes coupled to betalain biosynthesis which are related to the stage in the growth cycle. Second, this delay might be related to sucrose assimilation (preferential uptake of the products of sucrose hydrolysis, glucose and fructose). Here the results of Arias-Castro et al. (1993b) who reported that the presence of a high concentration of sucrose (8%) in the culture medium enhanced biphasic growth, with two different growth rates, in suspension cultures of

Glycyrrhiza glabra are relevant. The first growth phase corresponded with the period when glucose was taken up into the cells more rapidly than fructose (from 0 to 14 d.) and the second, when fructose was the only source of carbon (between day 14 and 21).

However, Drapeau et al. (1986) reported that if glucose became depleted from the medium the switch to the utilisation of fructose occurs smoothly with no apparent effect on the growth rate. Therefore, in the present study it is possible that pigment production was stimulated when glucose was depleted from the medium and fructose was then taken up. In other words, fructose at a higher concentration than the control might stimulate the production of the pigment either directly or indirectly.

193 Although the accumulation of betacyanin and betaxanthin in Beta vulgaris cell cultures was shown to be closely correlated with growth, the stimulation of betalain production which occurred with 3% sucrose was not associated with an increase in culture growth although the growth period was prolonged (up to day 20). It is noteworthy that even on day 20 cell biomass was not significantly different from the maximum attained on day 15 with 2% sucrose. This would suggest that 3% sucrose had a specific effect on pigment synthesis that was independent of growth. This observation is inconsistent with those of Kitamura et al. (1991), Wysokinska and

Swiatek (1991), and Norton et al. (1991) who showed that stimulation in product formation by high concentrations of sucrose was associated with enhanced growth.

However, there was an alteration to the relationship between growth and formononetin production in cell cultures of Glycyrrhiza glabra associated with a modification to the culture conditions (Arias-Castro etal., 1993a).

In 3.3.2.2 the effect of 3% sucrose on growth and betacyanin production was investigated in a selected cell line characterised by high productivity of betacyanin

(violet cell line). In that cell line, sucrose at 3% stimulated the production of betacyanin by 106% but not growth. This increase in the betacyanin content was not associated with an increase in the proportion of pigmented cells but from an increase in the yield of pigment in the individual pigmented cells (all or some). Results obtained from 3.4.2 showed that the activity of tyrosine hydroxylase was higher in cultures grown in 3% sucrose than those grown in 2% sucrose (Ca 30% when the activity was expressed as U of enzyme, and ca 61% when the activity expressed as

194 U.mg' protein, specific activity). This enzyme catalyses the conversion of tyrosine into DOPA (dihydroxyphenylalanine) (Lefts and Chase 1974, Wichers et al., 1983,

1984 and Muller et al., 1993, 1994), probable entry point to the biosynthetic pathway leading to betalain (see Endress, 1977, 1979, and Böhm and Rink 1988 and MUller et al., 1993). So it would seem that the increase in activity of this enzyme may be part of the mechanism by which sucrose promotes the production of betalain. In addition, the content of dopamine (see Table 3.3.8) of cultures grown with 3% sucrose was less than that of cultures grown in 2% sucrose. This may suggest that with 3% sucrose decarboxylation of DOPA to form dopamine (see Smith 1980, and Nemec 1995) might be inhibited and so, increasing the availability of DOPA, for betacyanin synthesis (see Endress et al., 1984).

The inhibition in betalain accumulation with 9% sucrose (see Fig. 3.2.8 and

Fig. 3.2.9) was relaxed after 15 days in subculture. This probably occurred when the level of sucrose in the medium was reduced, as a result of consumption by the growing cells, to a level which enabled the cells to produce higher amounts of pigments.

As the inhibition of pigment accumulation by 9% sucrose was not associated with a reduction in culture fresh weight, it would appear that the suppression in pigment production did not result from the osmotic effect of a high concentration of sucrose on growth, but from a specific influence of sucrose. The inhibition of

195 secondary product formation (ubiquinone) by high concentrations of sucrose has also been reported by Ikeda et al. (1976) in tobacco cell cultures.

In conclusion, it seems clear that 3% sucrose is the optimum concentration, within the treatments studied, for betacyanin and betaxanthin production, and that sucrose influences the production of betalains differently from growth.

4.2.2 Effect of 2,4-D on Growth and Betalain Production

In Beta vulgaris cell cultures omitting 2,4-D from the medium or reducing the concentration to 0.002 mg.1' or below resulted in a reduction in the growth of cultures

(see Table 3.2. 1) and enhancement of betalain production (see 3.2.13 (C)). These results are in agreement with those previously reported for other species by Zenk et al.

(1975), Sufford et al. (1985) and Arias-Castro et al. (1993b) who showed that 2,4-D supports good growth but inhibits secondary product formation. The inhibitory effect of 2,4-D on a wide range of secondary products such as pigments (Tabata et al., 1972 and 1974), alkaloids (Furuya et al., 1971 and Tabata et al., 1971) and phenolics

(Sugano et al., 1975, and Sugano and Ogawa 1981) is widely reported. Moreover, in some studies the exclusion of 2,4-D from the medium was essential for metabolite synthesis e.g. betalain synthesis in beet root callus (Constable and Nasif Makki 1971) and anthocyanin synthesis in carrot cell cultures (Ozeki and Komamine 1981). On the other hand, high concentrations of 2,4-D were found to promote secondary metabolite accumulation in other systems e.g. alkaloid production in Datura stramonium L.

196 where cultures were induced by 1 mg.r' 2,4-D (El-Bahr et al., 1989). An optimum concentration of 2,4-D for high production of secondary products has also been reported by some workers. For example, Ladd et al. (1992) found that the highest

accumulation of alkaloid was attained in calli grown on 106 M 2,4-D. Sakuta et al.

(1991) reported that in Phytolacca americana 5 mM 2,4-D could elevate betacyanin content (per cell) up to 252% compared to the control (2,4-D free). These different responses to 2,4-D are probably due to the different physiological states of the

different species or plant tissues.

In an attempt to search for the optimum concentration of 2,4-D for betalain

production in the present study, it was shown that 0.006 mg.i' 2,4-D was optimum for

betacyanin and betaxanthin production in Beta vulgaris culture (see Figs. 3.2.16(B)

and 3.2.17(B)). However, when 2,4-D at this concentration (0.006 mg.l') was

supplied to selected cell lines (violet and yellow) which were derived from the

original culture of Beta vulgaris, different responses were observed. In the violet cell

line betaxanthin but not betacyanin was enhanced (see Table 3.3.6), whereas in the

yellow line betacyanin was increased but betaxanthin was not, as compared to the

control (see Table 3.3.12). These results indicate that the response to 2,4-D, at certain

concentration, is tissue specific.

An interesting observation from this study with 2,4-D is that when the auxin

was excluded from the medium, betalain content per g. fresh weight was increased

markedly (see Fig. 3.2.13(C)), despite the complete suppression of growth (see Table

197 3.2.1). This contrasts with the finding previously reported which showed a positive correlation between betalain accumulation and growth. It suggests that the effect of

2,4-D on betalain synthesis is independent of growth. A similar result was obtained by

Fernandes-Ferreira et al. (1992) who suggested that phytohormones may influence triterpenol biosynthesis independently of the increase in biomass as the optimal concentration of growth regulators to promote metabolite production were different from that which produced optimal biomass yield. It is also worthy to note that cultures grown with 2,4-D at 0.002 mg.r' or lower (see 3.2.13(C)) accumulated similar amounts of betalain (per g. f. wt.) but displayed markedly different rates of growth.

All of these results reinforce the idea that 2,4-D has a specific effect on betalain

synthesis, which can be different from its effect on growth.

The mechanism by which low concentration of 2,4-D stimulate the production

of betalain is not known. However, Sakuta et al. (1991) showed that at 5 mM 2,4-D

betacyanin synthesis in Phytolacca americana was stimulated and free tyrosine

content of cells was also increased. Based on the result obtained from tracer

experiments using labelled tyrosine they concluded that the increase in betacyanin

accumulation, caused by 2,4-D resulted from an increase in tyrosine supply and

activation in the biosynthesis of betacyanin from tyrosine. Obata-Sasamoto and

Komanine (1983) reported that low concentrations of 2,4-D (0-0.025 mg.l') was

favoured for the accumulation of DOPA in Stizolobium callus. And at high

concentrations of 2,4-D the synthesis of DOPA was suppressed and tyrosine was

directed towards protein synthesis (Obata-Sasamoto et al., 1981). Wichers et al. 1993

198 also reported that in the presence of 10 mg.1' of 2,4-D DOPA accumulation was suppressed in Mucuna pruriens. Therefore, it might be suggested here that at low concentration of 2,4-D the synthesis of betalain may be stimulated by increasing the availability of precursor (e.g. tyrosine and DOPA) or/and by activation of enzymes of the pigment pathway.

4.2.3 Effect of Light on Growth and Betalain Production

Light has been shown to be an important regulator of betalain formation in cell cultures and plants producing these compounds. However, the role of light in betalain synthesis is not clear and there are many contrasting observations in the literature. For instance, intensively pigmented cell cultures of Beta vulgaris (Girod and Zryd 1987) and Portulaca grand/lora (Böbm et al., 1991, Kishima et al., 1991) became colourless when maintained in the dark. It has also been shown that light is essential requirement for betalain synthesis in Amaranthus caudatus seedlings (Köhler 1972).

In contrast, callus cultures of Portulaca grandflora accumulated a much higher level of betacyanin in the dark than in the light (Endress et al., 1984) and callus cultures of

Chenopodium rubrum also accumulated higher amounts of betacyanins in the dark than in the light. However, when maintained as liquid cultures in the dark betacyanin level became markedly reduced as compared with the light grown cultures (Berlin et al., 1986). Synthesis of betalains in the dark was also observed in seedlings of

Amaranthus tricolor and Celosia plumosa, however, in these cases pigment production was enhanced by the light. In seedlings of Amaranthus tricolor seedlings the stimulating effect of light on betacyanin biosynthesis could be replaced by the

199 application of cytokinins (Colomas 1975, Stobart and Kinsman 1977, Piattelli 1981,

Bianco-Colomas 1986). A synergistic effect of light and kinetin on betacyanin production was also observed in cultured tissue of the same species (Bianco-colomas and Hugues 1990). The application of kinetin or exposure of dark grown culture to fluorescent light resulted in increased betacyanin production, and when the light and kinetin treatments were repeated in the presence of 5,6 dichloro-1--D

Ribofuranosylbenzimidazol (DRB, a specific inhibitor of the physiological responses promoted by cytokinin), a pronounced inhibition of the kinetin-dependent betacyanin synthesis was observed. However, the light-induced synthesis remained unaffected by the presence of DRB (Bianco-colomas et al., 1988). These results led the author to suggest a possible independent action of the two inducing factors on betacyanin biosynthesis. Recently, Rudat and Goring (1995) reported that kinetin enhanced the betacyanin formation in cell cultures of Chenopodium album grown in white light but it could not induce betacyanin in darkness.

In the present study (see Table 3.3.9), the accumulation of betacyanin (betanin and isobetanin) and betaxanthin (miraxanthin V) was much lower in dark grown cultures than in illuminated cultures. In contrast, the growth, in terms of dry weight, of cultures grown in the dark and those grown in the light was similar. Similar observations were made earlier by Berlin et al. (1986) with a Chenopodium rubrum cell culture where betacyanin production in the dark was markedly reduced while growth remained unaffected, when compared to cultures grown in the light. Data presented here (Table 3.3.10) show that the proportions of pigmented cells in cultures

200 grown in the dark and those grown in the light were similar. This would indicate that individual, pigmented cells, were capable of accumulating betalains in the dark, however, pigment production was stimulated by light, in other words, light was beneficial but not an absolute requirement for betalain synthesis. This result contrasts with those reported by Leathers et al. (1992) who demonstrated that light was a prerequisite for betalain formation in suspension cultures of Beta vulgaris (var.

Bikores Monogerm). These conflicting findings indicate that the effect of light on betalain biosynthesis is not only species dependent but also variety dependent.

The quality and quantity of light can influence the production of betalain, nevertheless, their effects are not fully understood (see for review Piattelli 1981,

Leathers et al., 1992, and Rudat and Goring 1995). In the present study, it has been shown that white light from 7 to 70 ± 1 iMol.m 2.s' has a similar effect on growth and betacyanin production. This would indicate that the minimum light intensity needed to stimulate pigment production is 7 ± 1 tMol.m 2.s' or lower.

The mechanism by which light influences betalain accumulation is not fully understood. Based on the findings obtained from radioactive feeding experiments, it was suggested that light influences the reactions leading to the formation of the dihydropyridin moiety of betalains (French et al., 1973, Giudici de nicola et al.,

1974). French et al. (1973, 1974) further suggested that photo control is exerted on at least two parts of the biosynthetic pathway, one between tyrosine and DOPA, and a second between DOPA and betacyanin (amaranthin). In fact DOPA production was

201 strongly stimulated by light in Mucuna pruriens cell cultures (Huizing and Wichers

1985).

Results obtained in the present study (see Table 3.4.3) are in agreement with this postulation as the activity of tyrosine hydroxylase, the enzyme which catalyses the conversion of tyrosine into DOPA, was higher in light grown cultures than dark grown cultures. However, the increase in enzyme activity which occurred in the light was not paralleled with an increase in betalain production. This would suggest that light has activated not only the formation of DOPA but may also have influenced other reactions involved in pigment biosynthesis. The effect of light on the activity of hydroxylation was previously reported by Griffith and Conn (1973). Moreover, the production of DOPA was strongly stimulated by light in Mucuna pruriens cell cultures (Huizing and Wichers 1985, Chattopadhyay et al., 1994). The fact that the enhancement in miraxanthin V production, observed in the present study, occurred in the light was greater than the stimulation in betacyanin accumulation (see Table 3.3.9) suggests that light may affect not only the reactions leading to the formation of betalamie acid (pigment chromophore) but also the reactions involved in the step between the pigment moiety and miraxanthin V formation. However, in order to obtain a better understanding of the role of light in betalain biosynthesis further investigations with radioactive tracers and enzyme studies are required.

202 4.3 CELLULAR HETEROGENEITY AND BETALAIN PRODUCTION

4.3.1 Betalain Production in Selected Cell Lines Derived from Single Cells of

Different Colours

Cell line selection has been used effectively to increase the yield of cultured cells and to produce systems with specific desired characteristics (see Zenk 1978,

Yamamoto and Mizuguchi 1982, Yamada and Fujita 1983, Collin and Dix 1990,

Kishima et al., 1991, and Sakamoto et al., 1994). In the present study (see 3.3.1) various cell lines (violet, orange, yellow and white) were derived from the original culture (mother culture) of Beta vulgaris by cloning single cells of different colours.

The cell lines were characterised by their ability to accumulate betacyanin and/or betaxanthin to different extents and the different composition of the cell population, with regard to pigmentation.

Both the violet and orange cell lines (see Table 3.3.1) produced both betacyanin and betaxanthin, nevertheless, the ratio of these two pigments was different in the two lines (the ratio of betacyanin to betaxanthin was ca 5:1 in the violet cell line and ca 1:4 in the orange cell line). Both cell lines produced more betacyanin than the mother culture. In addition, betacyanin was presented as betamin

(95%), isobetanin (4.5%) and isobetanidin (0.3%) in the violet cell line and as betanin

(94%) and isobetanin (8%) in the orange cell line. While betanin was the only betacyanin produced in the mother culture. However, as the production of betanin in both cell lines (violet and orange) was superior to the other betacyanins and as

203 isobetanin and isobetanidin represented only at very low proportion of the total betacyanin, it is assumed that these two pigments were produced by the mother culture but in very small amounts which were not detectable with the methods employed. In contrast, betaxanthin content (present as miraxanthin V) in the violet and orange cell lines, was similar to that of the mother culture.

It is interesting that the cloned cell populations consisted of a mixture of cells of different colours (see Table 3.3.3a), although these cell lines were cloned from single pigmented cells. The violet and orange cell lines consisted of ca one third pigmented cells, violet and orange in the violet cell line, and violet, orange, and yellow in the orange line and ca two thirds colourless cells. The proportion of pigmented cells in these cloned lines (violet and orange lines) was similar to that present in the mother culture, although, the cloned lines produced more betacyanin than the mother culture. In addition, the increased proportion of violet cells observed in the violet cell line was not paralleled with an increased production of betacyanin compared to the mother culture (an increase in the proportion of violet cells of 100% was accompanied by an increase in betacyanin of 2885%). Moreover, the proportion of violet cells in the orange cell line, was much lower than that present in the mother culture although the betacyanin content was higher. All of these data indicate that the high production of betacyanin observed in these cell lines (violet and orange) resulted from an increase in the capacity of the pigmented cells, all or some, to produce pigment rather than an increase in the proportion of cells synthesising this metabolite.

The strict control of the proportion of pigmented cells in a tissue culture of

Catharanthus roseus has been reported previously by Hall and Yeoman (1986b). A

204 relevant question to be raised at this point is whether the betacyanin was produced by the violet cells and the betaxanthin by the orange cells or whether both pigments were produced by each cell type (violet and orange). With the data obtained from this study it is not possible to give a clear answer as these two cell lines; violet and orange both produced both betacyanin and betaxanthin, and both contained violet and orange cells.

From the ratios of betacyanin to betaxanthin and violet to the orange cells in each cell line (violet and orange) (see Table 4.1) it may be concluded that betacyanin was produced by the violet cells and betaxanthin by the orange cells in these lines. If this were not so each pigment would be produced predominantly by the corresponding coloured cells. Clearly, the use of repeated clonal selection, to develop systems which produce either the yellow or the violet betalain pigments would be commercially useful and save the cost of separation of the two pigments which is necessary when the beetroot is used as a source of the pigments.

Table 4.1 The ratio of betacyanin to betaxanthin and the violet to the orange cells in each of cell line (violet and orange).

Violet cell line Orange cell line Approx. ratio Betacyanin! betaxanthin 5:1 1:4 Approx. ratio of violet cells / orange cells 7:1 1:8

In the yellow cell line developed in the present study (see Table 3.3.1) only miraxanthin V was detected and the pigmented cell population (see Table 3.3.3) included yellow cells as well as orange cells. This would give further confirmation

205 that the orange cells produce betaxarithin but not betacyanin. However, it should be noted that this cell line produced very low amounts of pigment and this was associated with a low proportion of pigmented cells (9%).

Another interesting observation made in this study was that the white

(colourless) cell line, produced neither betacyanin nor betaxanthin. Here, the entire cellular population consisted of non pigmented cells. In contrast, other coloured cell lines (violet, orange and yellow) contained pigmented cells, although the larger part of the population, was non- pigmented. This would suggest that the non-pigmented cells might have an important role in supporting the pigmented cells by supplying metabolites which are utilised by the coloured cells for pigment synthesis. Indeed,

Colijn et al. (1981) found that the purple parts of Petunia hybrida callus were unable to survive, for more than 2 weeks, when transferred to fresh medium (MS) unless about one third of the transferred piece of callus consisted of white cells, and so he suggested that the non-pigmented cells were essential for growth of the callus. In conclusion, the maximum limit of the proportion of cells producing the metabolite, seems to be a property of the tissue and is necessary to retain the balance in metabolism between the cells in a given tissue.

4.3.2 Instability of the Selected Cell Lines with Regard to Betalain Production

It has long been recognised that plant cell and tissue cultures are not stable.

Spontaneous changes in the characteristics of cultures are known to occur from time to time (see Duncan 1997). A number of morphological and physiological changes

206 have been reported for cultured cells e.g. variability in organogenesis (Skirvin 1978,

Kochevenko and Ratushnyak 1995, and Barotti et al., 1995), loss in exogenous growth factor requirement (Meins and Binns 1977) and variation in secondary metabolite production (Zenk et al., 1977, Dougall et al., 1980, and Kunakh 1994).

In the present study (see 3.3.1.2), instability, with regard to betalain production, was observed in the selected cell lines (violet, orange and yellow) over a long period of non-selective subculturing (15 months). The violet cell line which accumulated both betacyanin and miraxanthin V, 3 months from the first establishing of the single cell clones, lost the ability to produce miraxanthin V for some time and then regained it. In this cell line, the absence of miraxanthin V was associated with the disappearance of the orange cells, and the reappearance of this betaxanthin was coupled with the reappearance of the orange cells. This observation reinforces the postulation previously made that betaxanthin is produced by the orange cells. In addition, the production of betacyanin in the violet cell line was sharply reduced for some time but then increased again. However, this variation in the betacyanin productivity of the culture did not result from a qualitative and quantitative change in the proportion of pigmented cells but from a change in the productivity of the existing pigmented cells (some or all). Such spontaneous reversible changes in the ability of plant cell cultures to produce secondary metabolites has been reported by Dougall et al. (1980) for anthocyanin production in cell cultures of wild carrot. The fact that the violet cell line always produced a high amount of betacyanin, despite the changes to betacyanin production, suggests that its ability to produce a high yield of betacyanin is

207 an inheritable characteristic and the variation in the yield probably results from epigenetic changes that are limited by the genetic potential of the cells. A similar observation was made earlier by Dougall et al. (1980) for anthocyanin production in wild carrot cultures where they concluded that the changes in the ability of cells to accumulate anthocyanin does not involve a qualitative change in the genetic information of the cells (a mutation).

In the orange cell line, a sharp reduction in betacyanin and betaxanthin production was observed over the period of study (15 months), and by the end of this period no betacyanin was detectable and only very low amounts of miraxanthin V were produced. This was associated with a reduction in the proportion of the pigmented cells (violet, orange and yellow). This reduction does not seem to be associated with a lower rate of division of the pigmented cells, as the change occurred suddenly, 8 months after the first establishment of the clone. Two explanations might be made to account for this observation, one is that the apparent reduction in the proportion of pigmented cells was due to the inability of the observer to count cells with a very low level of pigment content. The other is that, the reduced proportion of pigmented cells is due to an altered characteristic of the cell line which has probably resulted from a mutation.

In the yellow cell line fluctuations in pigment production were observed

during long term subculture. This cell line produced only miraxanthin V, 3 months

after establishment of the clone, eventually produced some betanin. Moreover, the

208 amount of miraxanthin V produced by this cell line had also increased after 15 months. However, only small amounts of both pigments were produced by this cell line. Once again this result suggests that the ability of cells to produce the pigment is an heritable characteristic and controlled by the genetics of the cells. As the proportion of pigmented cells in this cell line was always very small, the control of pigment production in this culture seems to be implemented by a reduction in the proportion of pigmented cells which have a low capacity to accumulate the pigment. It is worthy to note here that the appearance of betanin in this cell line was associated with the appearance of violet cells. This would reinforce the conclusion made previously in 4.3.1 that betacyanin is produced by the violet cells.

The white (colourless) cell line remained non-pigmented during the period of study, however, 15 months after first establishment of the clone it lost its ability to survive as a liquid culture. This event is further evidence of instability of cultured systems. However, instability and variability of cultured cells are very common phenomena, but mechanisms by which such changes occur are not yet fully understood. It is suggested that these changes are of epigenetically origin; e.g. mutable genes or transposable elements, as the gene is not permanently altered, these changes which are limited by the genetic potential of the cells, are stable, heritable and reversible (Meins 1983 and Borkowaska 1995). However, the association of chromosomal variability with the variation in the morphological and physiological characteristics of cells in callus and suspension cultures is also well known (Partanen

1963, Larkin and Scowcroft 1983, Lee and Phillips, 1988 and Handro et al., 1993).

209 According to Zorinyants et al. (1995) long term, in vitro, culturing of plant cells can cause variation in the number and structure of chromosomes. Deletion, insertion, translocations, telocentric chromosomes isochromosomes and dicentrics and their derivatives were all observed in cultured cells, and chromosome modifications in vitro do not correspond to in vivo variation, this difference is caused by differences in the mechanisms of adaptation to the environment at the level of the cell and the whole organism.

Finally, despite the relative instability of the cell lines developed in this study, repeated selection can be used as an effective method to improve and stabilise the yield of such systems. Indeed such techniques have been used to establish high yielding stable systems e.g. for biotin production in Lavandula vera (Watanabe 1982) and for anthocyanin production in Aralia carodata (Sakamoto et al., 1994). Moreover, maintenance of the cell line in a proper medium may help to stabilise the productivity of the system. Thus Yamamoto et al. (1989) could obtain a stable cell line from

Euphorbia millii yielding large amount of anthocyanin (32 mg/l/day) by selection and maintenance on a modification of Gamborg's B5 medium.

4.3.3 Betalain Production in the Violet and Yellow Cell Lines under Modified

Conditions

The effect of culture conditions on the production of betalain and the proportion of pigmented cells was investigated in the violet and the yellow cell lines

210 as two distinct systems, the first was characterised by high productivity of pigment and a relatively high proportion of pigmented cells (violet line) and the second was characterised with low productivity of betalain and a low proportion of pigmented cells. In the violet cell line modification of culture conditions (see 3.3.2) brought changes in the production of betalain but did not effect the proportion of pigmented cells. This would suggest that the limit to the proportion of pigmented cell is not controlled by environmental factors. In contrast, in the yellow cell line, changes in betalain accumulation caused by manipulating the culture conditions (see 3.3.3) were associated with changes in the proportion of pigmented cells. This indicates that the conditions of cultures have influenced the proportion of pigmented cells. However, even under conditions which promote the pigment production the yellow cell line only produced small amounts of pigment and contained only a low proportion of pigmented cells. These results suggest that the proportion of productive cells, with regard to metabolite production, is a property of the culture which seems to be genetically controlled. Cultures containing productive cells at proportion below the maximum limit may undergo some increase in the proportion of such cells but within a limited extent. Hall and Yeoman (1986b) have suggested, from their study on anthocyanin accumulation in Catharanthus roseus, that the maximum size of the productive cell population is fixed and an inherent property of the culture, under a given set of conditions.

Another interesting observation worthy of note is that in the yellow cell line grown in the medium with 0.006 mg.r' of 2,4-D, a remarkable increase in the

211 proportion of yellow cells was associated with the appearance and increase in the amounts of certain unknown compounds, while miraxanthin V content and the proportion of orange cells remained unchanged. This would suggest that miraxanthin

V was produced by the orange cells but not the yellow cells.

4.4 Tyrosine Hydroxylase Activity and Betalain Production

The hydroxylation of tyrosine and the formation of 3,4 dihydroxyphenylalanine (DOPA) has been suggested to be the entry point to the biosynthetic pathway leading to the formation of betalain (see Endress 1977, 1979,

Böhm and Rink 1988, and Miller et al., 1993). Based on the data obtained from radioactive feeding experiments using labelled tyrosine and DOPA, Zryd et al. (1982) and Hirano and Komamine (1994) concluded that this step (formation DOPA) is a regulatory step in betacyanin biosynthesis. Indeed, stimulation of betalain biosynthesis through the administration of DOPA has been reported by French et al.

(1974) in Amaranthus, Elliott (1983) in Amaranthus tricolor seedlings, and Rink and

Böhm (1985) in Portulaca grandflora flowers The enzyme responsible for the conversion of tyrosine into DOPA, is tyrosine hydroxylase (tyrosinase) (see Lefts and

Chase 1974, Wichers et al., 1983, 1984, and Muller et al., 1993, 1994). In the present study, the relationship between the activity of tyrosine hydroxylase and the final yield of betacyanin in cultures was investigated in certain cases 1. In two different cell lines one producing no pigment and the other accumulating a substantial amount of betacyanin, 2. In cultures grown under conditions that stimulated betacyanin

212 production (3% sucrose), 3. In cultures grown in the dark a condition in which betacyanin production was inhibited.

Results obtained (see 3.4. 1) show that the activity (specific activity) of tyrosine hydroxylase was much higher (ca 60%) in the betalain producing cell line

(violet cell line) than in the non-betalain producing cell line (white cell line), suggesting that the absence of pigment in the white cell line could be linked to a restriction in the supply of DOPA for pigment synthesis. However, this result is not clear cut as the enzyme expressed some activity in the white cell line although it was incapable of producing any betalain. This may be because this enzyme at the site of betalain biosynthesis is either inactive or have a low activity so that the small amount of DOPA formed is preferentially incorporated into other competing pathways.

Another possibility is that other enzymes participate in regulating the synthesis of betalain in this cell line. Zryd et al. (1982) found from their study with two cell lines

(red and white) of Beta vulgaris that DOPA was largely deficient in the white cell line. In addition, based on the data they obtained from feeding experiments with labelled tyrosine and DOPA they concluded that the block in betacyanin synthesis in the white cell line occurs at the step of DOPA synthesis from tyrosine. They also concluded that the metabolism of tyrosine and DOPA is compartmented. Bokern et al.

(1991) also reported that in cultures of Chenopoduim rubrum, appreciable amount of

DOPA was accumulated in cell lines producing large amounts of betacyanin, while it

was undetectable in cell lines producing low amounts of betacyanin.

213 From the data presented here (3.4.2) it was shown that in cultures grown with

3% sucrose where betacyanin was markedly increased (Ca 100%), the activity of tyrosine hydroxylase was also enhanced (Ca 30%) compared to the control cultures.

This would suggest that this enzyme might play a regulatory role in betacyanin synthesis. However, as the stimulation in activity of the enzyme was not parallel to the increase in pigment production, it would seem that tyrosinase might not be the only enzyme playing a regulatory role in betacyanin production. In addition dark- grown cultures (3.4.3) in which betalain accumulation was reduced also showed a reduction in tyrosine hydroxylase activity compared to the light grown control.

Nevertheless, the reduction in betalain production was not paralleled to the decrease in enzyme activity. Again suggesting that tyrosine hydroxylase may play an important role in regulating the biosynthesis of betacyanin in Beta vulgaris cell cultures.

However, other factors may determine the extent of betalain biosynthesis.

Conclusions

The production of betacyanin and betaxanthin in cell cultures of Beta vulgaris is closely related to growth.

Modification of culture conditions may influence the production of betalains differently from growth.

214 The production of betalains was stimulated with 3% sucrose, 0.006 mg.F' 2,4-D or light.

Selection techniques can be used as an effective method in developing high yielding systems which predominantly produce either the violet or the yellow betalains.

Betanin and miraxanthin V are the main betalains produced by Beta vulgaris cell cultures, the original culture and derived cell lines. Betanin is produced by the violet cells, and miraxanthin V is produced by the orange cells. The yellow cells may contain betaxanthins other than miraxanthin V or other yellow compounds.

The maximum proportion of pigmented cells is a property of the culture which is limited and heritable.

The proportion of pigmented cells has little effect in determining the yield of cultures, while the capacity of these cells to synthesise the pigment may play the most important role in governing the culture 'productivity'.

The ability of cells to produce large or low amount of pigment is an heritable characteristic which may be subject to variation but to a limited extent.

Tyrosine hydroxylase plays a regulatory role in betalain biosynthesis. However, other factors may determine the extent of betalain biosynthesis.

215 FUTURE WORK:

It has been shown that culture conditions influence growth and product accumulation. In particular, increase in pigment production could be attained with 3% sucrose or 0.006 mg.r' 2,4-D. So, an investigation into the effect of these two treatments in combinations is relevant. In general, a more detailed study on the effect of cultural factors (quality and quantity) including macro and micro-nutrients, growth regulators, and light might optimise the culture yield.

It is clear that developing cell lines which produce either betacyanin or betaxanthin can be established using the selection technique. Stabilisation and improvement to the yield of such cell lines needs further investigation particularly the nature of culture variability and the relationship between such changes and culture environment.

It was shown that with 0.006 mg.1 1 2,4-D the proportion of the yellow cells was increased and that was coupled with the appearance of several unknown compounds,

so identification of these compounds whether they are betaxanthin or other

metabolites is worthy.

Betalain pigments always accumulate inside the cell vacuole and never leak into the

medium. Developing methods that induce the leakage of pigments, could lead to an

enhanced production of the metabolite by preventing possible feed back inhibition

caused by elevated intracellular concentration of the product. This could be achieved

by cell permeabilisation, in either a continuous or intermittent process, using methods

which either increase membrane fluidity or which induce holes to form in cell

216 membranes e.g. anaesthetics detergents, organic solvents (see Brodelius et al., 1979), altering the NaIKICa ionic balance, sonication (see Hunter and Kilby 1988, Kilby and

Hunter 1990). Obviously an essential feature of any such method would be that the cell viability and metabolism would have to remain relatively unaffected by the treatment applied.

5. Investigation into the biosynthesis of the betalains and its regulation by studying the flux of metabolites down the pathway leading to the formation of the pigment.

This could be achieved through radioactive feeding of precursors e.g. tyrosine and

DOPA and studies on the enzyme activities. It was shown in the present study that pigment production was stimulated by 3% sucrose, low concentrations of 2,4-D, or light. Feeding cultures, grown under these conditions, with label precursors and subsequent measurements of the radioactivity in the betalain would show whether the pigment synthesis was stimulated and at which step(s) the activation in the pathway has occurred. By further study on the enzymes activity it would be possible to determine whether the stimulation in pigment production resulted from an increase in the activity of enzymes or from increased availability of precursors or both.

Application of labelled precursors to the white cell line developed in this study, which was incapable of producing betalains, and subsequent measurement of labelled intermediates of betalain pathway, would show at which point the block in the pathway of pigment occurs.

217 CHAPTER FIVE

REFERENCES

218 ALFERMANN, A. W., BERGMANN, W. FIGUR, C. HELMBOLD, U. SCHWANTAG, D. SCHULTER, I. and REINHARD, E. (1983). Biotransformation of 3-methyl digitoxin to n-methyl digoxin by cell cultures of Digitalis lanata. In Plant Biotechnology, (eds.) S. H. Mantel! and H. Smith, pp. 67-74. Cambridge University Press, Cambridge.

AMORIM, H., DOUGALL, D. and SHARP, W. (1977). The effect of carbohydrate and nitrogen concentration and phenol synthesis in Paul's Scarlet Rose cells grown in tissue culture. Physiologia Plantarum, 39: 91-95.

ARIAS-CASTRO, C., SCRAGG, A. H. and RODRIGUEZ-MENDIOLA, M. A. (1993a). The effect of cultural conditions on the accumulation of formonoetin by suspension cultures of Glycyrrhiza glabra. Plant Cell, Tissue and Organ Culture, 34 (1):63-70.

ARIAS-CASTRO, C., SCRAGG, A. H., STAFFORD, A. and RODRIGUEZ- MENDIOLA, M. (1993b). Growth characteristics of Glycyrrhiza glabra cell suspension cultures. Plant Cell, Tissue and Organ Culture, 34: 77-82.

ARNOW, L. E. (1937). Colorimetric determination of the components of 3,4 dihydroxyphenylalanine-tyrosine mixtures. Journal of Biological Chemistry, 118: 531-537.

BALLICA, R., RYU, D. and KADO, C. (1993). Tropane alkaloid production in Datura stramonium suspension cultures: Elicitor and precursor effects. Biotechnology and Bioengineering, 41(11): 1075-1081.

BAUERNFEIND, J. (1981). Natural food colours. In Carotenoids as Colorants and Vitamin A Precursors (Technological and Nutritional Applications), (ed.) J. Bauernfeind, pp. 1-45. Academic Press.

BAROTTI, S., FAMBRINI, M., PUGLIESI, C. and LENZI, A. (1995). Genetic variability in plants regenerated from in vitro culture of sunflower (Helianthus annuus L.). Plant Breeding, 114(3): 275-276.

BERLIN, J., SIEG, S., STRACK, D., BOKERN, M., and HARMS, H. (1986). Production of betalains by suspension cultures of Chenopodium rubrum L. Plant Cell, Tissue and Organ Culture, 5: 163-174.

BHATT, P., BHATT, D. and SUSSEX, I. (1983). Studies on some factors affecting solasodine contents in tissue cultures of Solanum nigrum. Physiologia Plantarum, 57:159-162.

BIANCO-COLOMAS, J. (1986). Etude de la regulation de la biosynthèse des bétalaines chez quelques plantules d'Amarantacées: comparison des effects de la lumière et des cytokinines. France: University of Nice; thesis.

219 BIANCO-COLOMAS, J and HUGUES, M. (1990). Establishment and characterisation of a betacyanin producing cell line of Amaranthus tricolor: indusive effect of light cytokinin. Journal of Plant Physiology., 136: 734-739.

BIANCO-COLOMAS, J., LENOL, C., and BULARD, C. (1988). Use of 5,6 - dichioro- 1 -13-D-ribofuranosylbenzamidazole to distinguish light stimulation from cytikinin stimulation of amaranthin synthesis in Amaranthus tricolor. Plant Growth Regulators, 7: 19-27.

BILYK, A. (1981). Thin-layer chromatographic separation of Beet pigments. Journal of Food Science, 46: 298-299.

BOHM, H. and RINK, E. (1988). Betalains. In Cell Culture and Somatic Cell Genetic of Plants, (eds.) F. Constabel & I. Vasil, vol. 5, pp. 449-463. Academic press Inc.

BOHM, H., BOHM, L., and RINK, E. (1991). Establishment and characterisation of a betaxanthin producing cell culture from Portulaca grandflora. Plant Cell, Tissue and Organ Culture, 26: 75-82.

BOKERN, M., WRAY, V. and STRACK, D. (1991). Accumulation of phenolic acid conjugates and betacyanins, and changes in the activities of enzymes involved in feruloylgiucose metabolism in cell-suspension cultures of Chenopodium rubrum L. Planta, 184:261-270.

BORKOWASKA, B. (1995). Variability in in vitro cultures. Wiadomosci Botaniczna, 39(3-4): 53-57.

BOYD, M. G. (1991). Studies on carotenoid production in cultures of Bixa orellana. Ph.D Thesis, University of Edinburgh.

BRADFORD, M. M. (1976). A rapid and sensitive method for quantification of microgram quantities of protein utilising the principle of protein -Dye Binding. Analytical Biochemistry, 72: 248-254.

BRODELIUS, P., DEUS, B., MOSBACH, K. and ZENK, M. (1979). Immobilised plant cells for the production and transformation of natural products. FEBS Letters, 103: 93-97.

BRODELIUS, P. (1990). Transport and accumulation of secondary metabolites. In Progress in Plant Cellular and Molecular Biology, (eds.) H. Nijkamp, L. Van der Plas and J.Van Aartrijk, pp. 567-576. Kluwer, Netherland.

BUSCH, L. (1991). Biotechnology: consumer concerns about risks and values. Food Technology, April:96-101.

BUTENKO, R. G. (1968). Plant Tissue Culture and Plant Morphogenesis, (ed.) M. Chailakhyan, p. 291. Israel Program for Scientific Translations Ltd, Jerusalem.

220 CARCELLER, M., DAVEY, M. R., FOWLER, M. W. and STREET, H. E. (1971). The influence of sucrose, 2,4-D and kinetin on the growth, fine structure and lignin content of cultured sycamore cells. Protoplasma, 73: 367-385.

CHANG, C., KIMLER, L., and MABRY, T. (1974). Biogenesis of betalamic acid. Phytochemistry, 13: 2771-2775.

CHATTOPADHYAY, S., DATTA, S. and MAHATO, S. (1994). Production of L- DOPA from cell suspension culture of Mucuna pruriens f pruriens. Plant Cell Reports, 13: 5 19-522.

CHOI, K. T., AN, I. 0 and PARK, J. C. (1994). Ginsenoside synthesis in Panax ginseng tissue culture. Fiziologiya Rastenii (Moscow), 41(6): 89 1-895.

COLIJN, C., JONSSON, L., SCHRAM, A. and KOOL, A. (1981). Synthesis of malvidin and petunidin in pigmented tissue cultures of Petunia hybrida. Protopiasma, 107: 63-68.

COLLIN, H. A. (1987). Determinants of yield of secondary products in plant tissue cultures. In Advances in Botanical Research, vol. 13, (ed.) J. A. Callow, pp. 145-187. Academic Press.

COLLIN, H., and DIX, P. (1990). Culture systems and selection procedures. In Plant Cell Line Selection, (ed.) P. Dix, pp. 3-18. VCH Publishers.

COLOMAS, J. (1975). Role des cotyledons dans l'induction de la biosynthèse d'amarantine chez des plantules d'Amaranthus tricolor L. var. biocolor ruber Hort. C.R. Acad. Sci., 280:1249-1252.

CONFORTI-FROES-N., VARELLA-GARCLA-M., and MANZATO-A-J. (1990). In vitro evaluation of betanin mutagenicity. Cytologia (Tokyo), 55(2): 203-208.

CONSTABEL, F. (1967). Pigmentbildung in Kalluskulturen aus Beta-Ruben. Naturwissenschaften, 54: 175-176.

CONSTABEL, F. and HAALA, G. (1968). Recherches sur la formation de pigments dans les tissues de betterave fourragere cultives in vitro. Coil. Nationaux C.NR.S., 223-229.

CONSTABEL, F. and NASSIF-MAKKI, H. (1971). Betalainbildung in Beta- Calluskulturen. Ber. Dtch. Bot. Ges., 84: 629-636.

CORDUAN, G. and REINHARD, E. (1972). Synthesis of volatile oils in tissue cultures of Ruta graveolens. Phytochamistry, 11: 917-922.

221 CORMIER, F., CREVIER, H. A. and DO, C. B. (1990). Effects of sucrose concentration on the accumulation of anthocyanins in grape (Vitis vinfera) cell suspension. Canadian Journal of Botany, 68: 1822-1825.

CURTIN, M. E. (1983). Harvesting profitable products from plant tissue culture. Biotechnology, 1: 649-657.

CVIKROVA, M., HRUBCOVA, M., MERVAY, L. and POSPISIL, F. (1988). Changes in the content of phenolic substances during the growth of Nicotiana tabacum cell suspension culture. Biologia Plantarum, 30: 185-192.

DAVIES, M. E. (1972). Polyphenol synthesis in cell suspension cultures of Pauls's Scarlet rose. Planta (Ben.), 104: 50-65.

DAWES, E. (1957). Photometric analysis. In Quantitative Problems in Biochemistry, (ed.) E. Dawes, pp. 126-127. E. & S. Livingstone Ltd., Edinburgh and London.

DE-EKNAMKUL, W. and ELLIS, B. (1985). Effects of macronutrients on growth and rosmarinic acid formation in cell suspension cultures of Anchusa officinalis. Plant Cell Reports, 4: 46-49.

DELIU, C., NICOARA, A. MUNTEANU-DELIU, C., BERCEA, V., TIRNOVEANU, D. and KEUL, M. (1992). Some aspects of the metabolism of Berberis parvfolia cell suspensions. Studia Universitatis Babes-Bolyai Biologia, 37: 29-36.

DEMEYER, K. and DEJAEGERE, R. (1992). Effect of the nitrogen form used in the growth medium (nitrate, ammonium) on alkaloid production in Datura stramonium L. Plant and Soil, 147(1): 79-86.

DEUS-NEUMANN B. and ZENK, M. (1984). Instability of indole alkaloid production in Catharanthus roseus cell suspension cultures. Planta Medica, 50: 427- 431.

DIXON, R., JENNINGS, A., DAVIES, L., GERRISH, C. and MURPHY, D. (1989). Elicitor-active components from French bean hypocotyls. Physiological and Molecular Plant Pathology, 34: 99-115.

DO, C. B. and CORMIER, F. (1990). Accumulation of anthocyanins enhanced by a high osmotic potential in grape (Vitis vinfera L.) cell suspensions. Plant Cell Report, 9: 143-146.

DO, C. and CORMIER, F. (1991a). Effects of low nitrate and high sugar concentrations on anthocyanin content and composition of grape (Vitis vinfera L.) cell suspension. Plant Cell Reports, 9: 500-504.

222 DO, C. and CORMIER, F. (1991b). Accumulation of peonidin 3-glucoside enhanced by osmotic stress in grape (Vitis vinfera) cell suspension. Plant Cell, Tissue and Organ Culture, 24: 49-54.

DOLLER, G., ALFERMANN, A. and REINHARD, E. (1976). Production von indolalkaloiden in callus-kulturen von Catharanthus roseus. Planta Medica, 30:14- 20.

DOPP, H., MAURER, S., SASAKI, A. and MUSSO, H. (1982). Die konstitution der musca-aurine. Liebigs Annalen der Chemie, 254-264.

DOUGALL, D. K. (1980). Nutrition and metabolism. In Plant Tissue Culture as a Source of Biochemicals, (ed.) E. J. Staba, pp. 21-58. CRC Press Boca Raton, FL., ISBN 0-8493-5557-5.

DOUGALL, D., JOHNSON J. and WHITTEN, G. (1980). A clonal analysis of anthocyanin accumulation by cell cultures of wild carrot. Planta, 149: 292-297.

DRAPEAU, D., BLANCH, H. W. and WILKE, C. R. (1986). Growth kinetics of Discorea deltoidea and Catharanthus roseus in batch culture. Biotechnology and Bioengineering, 28: 1555-1563.

DUNCAN, R. (1997). Tissue culture-induced variation and crop improvement. In Advances in Agronomy, vol. 58, (ed.) D. Sparks, pp. 201-240. Academic Press Inc, California, USA..

DUPRAZ, 3., CHRISTEN, P. and KAPENTANIDIS, I. (1994). Tropane alkaloids is transformed roots of Datura quercfolia. Planta Medica, 60(2): 158-162.

EILERT, V., KURZ, W., and CONSTABEL, F. (1987). Alkaloid accumulation in plant cell cultures upon treatment with elicitors. In Plant Tissue and Cell Culture, (eds.) C. E. Green, D. A. Somers, W. P. Hacket, and D. D. Biesboer, pp. 213-219. A. R. Liss Inc, New York.

EILERT, U. (1989). Elicitor induction of secondary metabolism in dedifferentiated and differentiated in vitro systems of Ruta graveolens. In Primary and Secondary Metabolism of Plant Cell Cultures II, (ed.) W. Kurz, pp. 219-228. Springer-Verlag.

EL-BAHR, M. K., HUSSEiN, M. S. and MOUSRY, H. A. (1989). Effect of some growth regulators on the growth and alkaloid production of Datura stramonium L. cultured in vitro. Egyptian Journal of Botany, 32(1-2): 53-62.

ELKIND, Y., EDWARDS, R., MAVANDAD, M., HEDRICK, S., RIBAK, 0., DIXON, R. and LAMB, C. (1990). Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene. Proceedings of the National Academy of Sciences, USA, 87: 9057-9061.

223 ELLIOTT, D. C. (1983). The pathway of betalains synthesis: effect of cytokinin on enzymic oxidation and hydroxylation of tyrosine in Amaranthus tricolor seedlings. Physiologia Plantarum., 59: 428-437.

ENDRESS, R. (1976). Betacyan-Akkumulation in Kallus von Portulaca grandflora 2+ var. JR unter dem Einfluss von Phytohormonen und Cu -ionen auf unterschiedlichen grundmedien. Biochemie und Physiologie der Pflanzen, 169: 87-98.

ENDRESS, R. (1977). Enfluss moglicher phosphodiesterase inhibitoren und CAMP auf die betacyan-akkumulation. Phytochemistry, 17: 1549-1554.

ENDRESS, R. (1979). Mogliche beteliligung einer phenylalanin hydroxylase und einer Tyrosinase bei der betacyan akkumulation in Portulaca kallus. Biochemie und Physiologie der Pflanzen, 174: 17-25.

ENDRESS, R., JAGER, A. and KRIES, W. (1984). Catecholamine biosynthesis dependent on the dark in betacyanin-forming Portulaca callus. Journal of Plant Physiology, 115: 291-295.

FERNANDES-FERREIRA, M., NOVAIS, J. M., and PAlS M. S. S. (1992). Hormonal control of titerpenols synthesis in Euphorbia caracias calli. Bioresorurce Technology, 39(1): 31-38.

FISCHER, N. and DREIDING, A. (1972). Biosynthesis of betalains: on the cleavage of the aromatic ring during the enzymatic transformation of DOPA into betalamic acid. Helvetica Chimica Acta, 55: 649-659.

FIVAZ, J., ZAIKO, M., HINZ, U. and ZRID, J. (1994). The evolution of betalain biosynthesis in plant and fungi. Experientia, 50(abstracts): A45.

FOWLER, M. W. (1983). Commercial applications and economic aspects of mass plant cell culture. In Mantel! and Smith (1983), pp. 3-38.

FOWLER, M. W. (1987). Process possibilities for plant-cell cultures. In Plant and Animal Cells: Process Possibilities, (eds.) C. Webb and F. Mavituna, pp. 21-32. Ellis Horwood Ltd.

FOWLER, M. W. (1988). Problems in commercial exploitation of plant cell cultures. In Application of Plant Cell and Tissue Culture (CIBA Foundation Symposium 137), pp. 239-253. Wiley, Chichester.

FOWLER, M. W., CRESSWELL, R. C. and STAFFORD, A. M. (1990). An economic and technical assessment of the use of plant cell cultures for natural product synthesis on an industrial scale. In Bioactive Compounds from Plants, Ciba foundation symposium 154, (eds.) D. J. Chadwick and J. Marsh, pp. 157-174. Wiley Interscience Publications.

224 FRANCIS, F. (1987). Lesser-known food colorants. Food Technology, April: 62-68.

FRENCH, C. J., PECKET, R. C. and SMITH, H. (1974), Effect of exogenous DOPA and tyrosine on amaranthin synthesis and pigment type in Amaranthus. Phytochemistry, 13: 1505-1511.

FRENCH, C. J., PECKET, R. C. and SMITH, H. (1973). Effect of light and exogenously applied precursors on amaranthin synthesis in Amaranthus caudatus. Phytochemistry, 12: 2887-2891.

FUJITA, Y., HARA, Y., OGINO, T. and SUGA, C. (1981). Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhison I, Effects of nitrogen source on the production of shinkonin derivatives. Plant Cell Report, 1: 59- 60.

FUJITA, Y. (1988). Industrial production of shikonin and berberine. In Application of Plant Cell and Tissue Culture, Ciba foundation symposium 137, (eds.) G. Bock and J. Marsh, pp. 228-23 5. J. Wiley & Sons.

FUNK, C., GUGLER, K. and BRODELIUS, P.(1987). Increased secondary product formation in plant cell suspension cultures after treatment with a yeast carbohydrate preparation (elicitor). Phytochemistry, 26: 401-405.

FURUYA, T., KOJIMA, H. and SYONO, K. (1971). Regulation of nicotine biosynthesis by auxins in tobacco callus tissue. Phytochemistry, 10: 1529-1532.

GAFF, D. F. and OKONG'O-OGOLA, 0. (1971). The use of nonpermeating pigments for testing the survival of cells. Journal of Experimental Botany, 22(72): 756-758.

GAMBORG, 0. L., MILLER, R. A. and OJIMA, K. (1968). Nutrient requirements of suspension cultures of soy bean root cells. Experimental Cell Research, 50: 151-158.

GIROD, P. A. and ZR'D, J. P. (1991). Secondary metabolism in cultured red beet (Beta vulgaris L.) cells: differential regulation of betaxanthin and betacyanin biosynthesis. Plant Cell, Tissue and Organ Culture, 25: 1-12.

GIROD, P. A. and ZRID, J. P. (1987). Clonal variability and light induction of betalain synthesis in red beet cell culture. Plant Cell Reports, 6: 27-30.

GIROD, P. A. and ZRID, J. P. (1985). Regulation of the synthesis of betalains in red beet (Beta vulgaris ssp) cells in culture. Experientia, 41: 782.

GIUDICI DE NICOLA, M., PIATTELLI, M., CASTROGIOVANNI, V. and AMICO, V. (1972). The effects of light and kinetin on amaranthus synthesis in relation to phytochrome. Phytochemistry, 11: 1011-1017.

225 GIUDICI DE NICOLA, M., PIATTELLI, M. and AMICO, V. (1973). Photocontrol of betaxanthin synthesis in Celosia plumosa. Phytochemistry, 12: 353-357.

GIUDICI DE NICOLA, M., AMICO, S. and PIATTELLI, M. (1974) Effect of light on betalain formation. Phytochemistry, 13: 439-442.

GOLDENBERG, N. (1977). Colours - do we need them. In Why Additives? The Safety of Foods, (ed.) The British Nutrition Foundation, pp. 22-24. Forbes Publications, London.

GOMEZ, K. A. and GOMEZ, A. A. (1984). Statistical procedures for Agricultural Research, 2nd edition, pp. 7-13, 188-205 and 357-369. A Wiley Inter-Science Puplication.

GRIFFITH, T. and CONN, E. (1973). Biosynthesis of 3,4-Dihydroxyphenylalanine in Viciafaba. Phytochemistry, 12: 1651-1656.

HALL, R. D. and YEOMAN, M. M. (1986a). Factors determining anthocyanin yield in cell cultures of Catharanthus roseus (L.). G. New Phytologist, 103: 33-43.

HALL, R. D. and YEOMAN, M. M. (1986b). Temporal and spatial heterogeneity in accumulation of anthocyanins in cell cultures of Catharanthus roseus (L) G. Don. Journal of Experimental Botany, 37(174): 48-60.

HALL, R. D. and YEOMAN, M. M. (1987). Intercellular and intercultural heterogeneity in secondary metabolites accumulation in cultures of Catharanthus roseus following cell line selection. Journal of Experimental Botany, 38: 1391-1398.

HALL, R. D. and YEOMAN, M. M. (1991). The influence of intracellular pools of phenylalanine derivatives upon the synthesis of capsaicin by immobilised cell cultures of the chilli pepper, Capsicumfrutescens. Planta, 185: 72-80.

HANDRO, W., FERREIRA, C. and FLOH, I. (1993). Chromosomal variability and growth rate in cell suspension cultures of Stevia rebaudiana (Bert.) Bertoni. Plant Science, 93:169-176.

HILTON, M. G. and RHODES, M. J. (1994). The effect of varying levels of Gamborg's B5 salts and temperature on the accumulation of starch and hyoscyamine in batch cultures of transformed roots of Datura stramonium. Plant Cell, Tissue and Organ Culture, 38: 45-5 1.

HINZ, U., SCHMIDT, I. and ZRYD, J. (1995). Similarity of fungal estradiol- dioxygenases from betalain and muscaflavin producing species. Experientia, 51(Abstr.): A16.

226 HIRANO, H. and KOMAMINE, A. (1994). Correlation of betacyanin synthesis with cell division in cell suspension cultures of Phytolacca americana. Physiologia Plantarum, 90: 239-245.

HIROSE, M., YAMAKAWA, T., KODAMA, T. and KOMAMINE, A. (1990). Accumulation of betacyanin in Phytolacca americana cells and of anthocyanin in Vitis sp. cells in relation to cell division in suspension cultures. Plant Cell Physiology, 31(2):267-271.

HOBBS, M. C. and YEOMAN, M. M.(1991). Effect of light on alkaloid accumulation in cell cultures of Nicotiana sp. Journal of Experimental Botany, 22: 1371-1378.

HOLDEN, M., HOLDEN, P. and YEOMAN, M. (1988a). Elicitation of cell cultures. In Manipulating Secondary Metabolism in Culture, (eds.) R. Robins and M. Rhodes, pp. 57-65. Cambridge University Press.

HOLDEN, P., HOLDEN, M. and YEOMAN, M. (1988b). The effects of fungal elicitation on secondary metabolism in cell cultures of Capsicum frutescens. In Manipulating Secondary Metabolism in Culture, (eds.) R. Robins and M. Rhodes, pp. 67-72. Cambridge University Press.

HUNTER, C. S. and KILBY, N. J. (1988). Electropermeabilisation and ultrasonic techniques for harvesting secondary metabolites from plant cells in vitro. In Manipulating Metabolism in Culture, (eds.) R. J. Robins and M. J. C. Rhodes, pp. 285-289. Cambridge University Press, Cambridge, UK.

HUIZING, H. J. and WICHERS, H. J. (1985). Innovations in Biotechnology, (eds.) EH. Houwink and RR. Van der Meer, pp. 217-228. Elsevier Science Publishers By, Amsterdam.

IKEDA, T., MATSUMOTO, T. and NOGUCHI, M. (1976). Effects of nutritional factors on the formation of ubiquinone by tobacco plant cells in suspension culture, Agricultural and Biological Chemistry, 40: 1765-1770.

IMPELLIZZERI, G. and PIATTELLI, M. (1972). Biosynthesis of betalains: formation of indicaxanthin in Opuntiaficus-indicus fruits. Phytochemistry, 12: 2499- 2502.

ISHIKURA, N., WATANABE, Y. and TERAMOTO, S. (1989). The formation of flavonoids in cell suspension cultures of Prunus yedoensis Matsum. Botanical Magazine Tokyo, 102(1068): 547-560.

JAAFAR, M. S. (1992). Studies on the biosynthesis of betalains in cell cultures of Beta vulgaris. M Phil. Thesis, University of Edinburgh.

JUNG, K. H., KWAK, S. S., CHOI, C. Y. and LIU, J. R. (1994). Development of two stage culture process by optimisation of inorganic salts for improving catharanthine

227 production in hairy root cultures of Catharanthus roseus. Journal of Fermentation and Bioengineering, 77(1): 57-61.

KADKADE, P. G. (1982). Growth and podophyllotoxin production in callus tissues of Podophyllum peltatum. Plant Science Letters, 25: 107-115.

KATO, A., FUKASAWA, A. SHIMIZU, Y. SOH, Y., and NAGAI, S. (1977). 2+ Requirements of P043 , NO3 , S02 , K and Ca for the growth of tobacco cells in suspension culture. Journal of Fermentation Technology, 55(3): 207-212.

KAUL, B. and STABA, E. J. (1969). Dioscorea tissue cultures. I. Biosynthesis and isolation of diosgenin from Dioscorea deltoidea callus and suspension cells. Lloydia, 31(92): 171-179.

KEEN, N., PARTRIDGE, J. and ZAKI, A. (1972). Pathogen-induced elicitor of a chemical defense mechanism in soybean mono-genically resistant to Phytophtora megasperma var. Sojae. Phytopathology, 62: 768.

KILBY, N. and HUNTER, C. (1990). Repeated harvest of vacuole-located secondary product from in vitro grown plant cells using 1.02 Mhz ultrasound. Applied Microbiology and Biotechnology, 448-451.

KIMLER, L., LARSON, R., MESSENGER, L., MOORE, J. and MABRY, T. (1971). Betalamic acid, a new naturally occurring pigment. Journal of Chemical Society Communications, 1329-1330.

KIM, D., PEDERSEN, H. and CHIN, C. (1988). Effects of light on berberine production in cell suspension cultures of Thalictrum rugosum. Biotechnology Letters, 10(10): 709-712.

KIM, D. I., PEDERSON, H. and CHIN, C. K. (1991). Development of process strategies for berberine production in plant cell suspension cultures. Journal of Biotechnology, 21(3): 201-208.

K1NOSHITA, Y., YAMAMOTO, S., HAYASE, S., SAKATA, K., KATAYAMA, A. and YAMADA, Y. (1990). Large scale production and industrial use of anthocyanin in cultured Euphorbia millii cells. The International Congress on Plant Tissue and Cell Culture, June 24-29, Amsterdam, Abstract VII th.

KISHIMA, Y., NOZAKI, K., AKASHI, R. and ADACHI, T. (1991). Light-inducible pigmentation in Portulaca callus: selection of a high betalain producing cell line. Plant Cell Reports, 10(6-7): 304-307.

KISHIMA, Y., SHIMAYA, A. and ADACHI, T. (1995). Evidence that blue light induces betalain pigmentation in Portulaca callus. Plant Cell, Tissue and Organ Culture, 43: 67-70.

228 KITAMURA, Y., SUGIMOTO, Y., SAMEJIMA, T., HAYASHIDA, K. and MIURA, H. (1991). Growth and alkaloid production in Duboisia myoporoides and Duboisia leichhardtii root cultures. Chemical and Pharmaceutical Bulletin, 39(5): 1263-1266.

KLAUI, H. and BAUERNFEIND, J. (1981). Carotenoids as food colours. In Carotenoids as Colorants and Vitamin A Precursors (Technological and Nutritional Applications), (ed.) J. Bauernfeind, pp. 47-317. Academic Press.

KNEWSTUBB, C. and HENRY, B. (1988). Natural colours - a challenge and an opportunity. Food Technology International - Europe 1988. Sterling Publications Ltd.

KNOBLOCH, K. H. and BERLIN, J. (1980). Influence of medium composition on the formation of secondary compounds in cell suspension cultures of Catharanthus roseus (L) G. Don. Zeitshrift Fur Naturforschung Section C. Biosciences, 35: 551- 556.

KNOBLOCH, K. H. and BERLIN, J. (1981). Phosphate mediated regulation of Cinnamoyl putrescine biosynthesis in cell suspension cultures of Nicotiana tabacum. Planta Medica, 42: 167-172.

KNOBLOCH, K. H., BAST, G. and BERLIN, J. (1982). Medium and light-induced formation of serpentine and anthocyanins in cell suspension cultures of Catharanthus roseus. Phytochemistry, 21: 591-594.

KNOBLOCH, K. H. and BERLIN, J. (1983). Influence of phosphate on formation of the indole alkaloids and phenolic compounds in cells of suspension cultures of Catharanthus roseus I. comparison of enzyme activities and product accumulation. Plant Cell, Tissue and Organ Culture, 2: 333-340.

KOCHEVENKO, A. and RATUSHNYAK, Y. (1995). The protoplast culture and somaclonal variability of species from a series Juglandifolia (Solanaceae). Dopovidi Natsional 'noyi Akademiyi Nauk Ukrayiny, 2: 114-116.

KOHLER, K. (1972). Photocontrol of betacyanin synthesis in Amaranthus caudatus seedlings in the presence of kinetin. Phytochemistry, 11: 133-137.

KOMAMINE, A., SAKUTA, M., HIROSE, M., HIRANO, H., TAKAGI, T., KAKEGAWA, K., and OZEKI, Y. (1989). Regulation of secondary metabolism in relation to growth and differentiation. In Primary and Secondary Metabolism of Plant Cell Cultures, II, (ed.) W. G. Kurz, pp. 49-52. Springer-Verlag, Berlin, Heidelberg, Germany.

KUNAKH, V. A. (1994). Genome variability and accumulation of indoline alkaloids in Rauwolfia serpentina Benth. cell culture. Biopolimery i Kletka, 10(1): 3-30.

KUTNEY, J., CHOI, L., KOLODZIEJCZYK, P., SLEIGH, S., STUART, K., WORTH, B., KURZ, W., CHATSON, K. and CONSTABEL, F. (1981). Alkaloid

229 production in Catharanthus roseus cell cultures. VII. Effect of parameter changes and catabolism studies on cell line PRL No. 953. Helvetica Chimica Acta, 64:1837-1843.

LADD, M., RUBLOU, A. and QU1NTERO, A. (1992). The effects of media conditions on Datura innoxia callus and alkaloids production. Phyton (Buenos Aires), 53(l):43-46.

LARKIN, P. and SCOWCROFT, W. (1983). Somaclonal variation and crop improvement. In Genetic Engineering of Plants, (eds.) T. Kosuge, C. Meredith and A. I-Iollaender, pp. 289-314. Plenum Press, New York.

LEATHERS, R., DAVIN, C. and ZRID, J. (1992). Betalain producing cell cultures of Beta vulgaris L. var. bikores monogerm (Red Beet). In Vitro Cell. Dev. Biol., 28b: 39-45.

LEE, M. and PHILLIPS, R. (1988). The chromosomal basis of somaclonal variation. Annu. Rev. Plant Physiol., Plant Molecular Biology., 39: 413-438.

LETTS, D. and CHASE T. JR. (1974). Chemical modification of mushroom Tyrosinase for stabilisation to reaction in activation. Adv. Exp. Med. Biol., 42: 317- 328.

LIEBISCH, H. and BOHM, H. (1981). Untersuchungen zur Physiologie der Betalainbildung in Zellkulturen von Portulaca grandflora. Pharmazie,36: 218.

LINDSEY, K. (1985). Manipulation, by nutrient limitation of the biosynthetic activity of immunised cells of Capsicumfrutescens Mill. cv. annuum. In Plant Biotechnology, SEB Seminar Series, 18, (eds.) S. H. Mantell and H. Smith, Cambridge University Press.

LINDSEY, K. (1986). The production of secondary metabolites by immobilised plant cells. In Secondary Metabolism in Plant Cell Cultures, (eds.) P. Morris, A. H. Scragg, A. Stafford and M. W. Fowler, pp. 143-155. Cambridge University Press.

LINDSEY, K. and YEOMAN, M. M. (1983). The relationship between growth rate, differentiation and alkaloid accumulation in cell cultures. Journal of Experimental Botany, 34:1055-1065.

LINDSEY, K. and YEOMAN, M. M. (1984). The synthetic potential of immobilised cells of Capsicumfrutescens Mill.cv.annum. Planta, 162: 495-501.

LINDSEY, K. and YEOMAN, M. (1985). Dynamics of plant cell cultures. In Cell Culture and Somatic Cell Genetics of Plants, vol. 2, (ed.) I. K. Vasil, pp. 61-101. Academic Press.

LUCKNER, M. (1990). Secondary metabolism. In Microorganisms, Plants, and Animals, third edition, p. 563, Springer-Verlag, New York.

230 MABRY, T. J. (1980). Betalains. In Encyclopedia of Plant Products, (eds.) E. A. Bell and B. V. Charlwoo, pp. 5 13-533. Springer Verlag, New York.

MANTELL, S. H. and SMITH, H. (1983). Cultural factors that influence secondary metabolite accumulation in plant cell and tissue cultures. in Plant Biotechnology, (eds.) S. Mantell and H. Smith, pp. 75-108. Cambridge University Press.

MATILE, P. (1990). The toxic compartment of plant cells. In Progress in Plant Cellular and Molecular Biology, (eds.) H. Nijkamp, L. Van der Plas and J.Van Aartrijk, pp. 557-566. Kluwer, Netherland.

McCARTHY, J. J., RATCLIFFE, D. and STREET, H. E. (1980). The effect of nutrient medium composition on the growth cycle of Catharanthus roseus G. Done cells grown in batch culture. Journal of Experimental Botany, 31: 1315-1325.

ME1NS, F. (1983). Heritable variation in plant cell culture. Annual Review of Plant Physiology, 34: 327-346.

MEINS, F. JR. and BD'INS, A. (1977). Epigenetic variation of cultured somatic cells: evidence for gradual changes in the requirement for factors promoting cell division. Proceedings of the National Academic of Sciences USA, 74: 2928-2932.

MERILLON, J. M., CHENIEUX, J. C. and RIDEAU, M. (1983). Time course of growth, evolution of sugar-nitrogen metabolism and accumulation of alkaloids in a cell suspension of Catharanthus roseus. Planta Medica, 47: 169-176.

MERILLON, J. M., RIDEAU, M. and CHENIEUX, J. C. (1984). Influence of sucrose on levels of ajamilicine, serpentine and tryptamine in Catharanthus roseus cells in vitro. Planta Medica, 48: 497-501.

MINIAB INC. (1994). Minitab Reference Manual: Release 10 for Windows. Minitab Inc, USA.

MIYASAKA, H., NASU, M., YAMAMOTO, T. and YONEDA, K. (1985). Production of ferruginol by cell suspension cultures of Salvia miltiorrhiza. Phytochemistry, 24: 1931-1933.

MIZUKAMI, H., KONOSHIMA, M. and TABATA, M. (1977). Effect of nutritional factors on shikonin derivative formation in Lithospermum callus cultures. Phytochemistry, 16: 1183-1186.

MOL, J., DE LANGE, P., OOSTDAM, A. and VAN DER PLAS, L. (1990). Use of genetic engineering to improve yields in cell cultures, e.g. (anti)sense DNA technology. In Progress in Plant Cellular and Molecular Biology, (eds.) H. Nijkamp, L. van der Plas and J. Aartrijk, pp. 712-716. Kluwer Academic Publishers.

231 MONROY, M. R., APARICIO, A. J., ORTIZ, G. D. and JIMENEZ, G. S. (1994). Effect of carbon source on cell growth and betalain production in cell suspension culture of Beta vulgaris. Biotechnology Letters, 16(8): 853-858.

MORRIS, P. (1986). Regulation of product synthesis in cell cultures of Catharanthus roseus. Effect of culture temperature. Plant Cell Reports, 5: 427-429.

MULLER, L., HINZ, U. and ZRID, J. (1993). Betalain biosynthesis: the role of tyrosine hydroxylase. Experientia, 49(abstracts): A38.

MULLER, L., H1NZ, U. and ZRD, J. (1994). Characterisation of a tyrosinase from Amanita muscaria involved in betalain biosynthesis. Experientia, 50(abstracts): A44.

NAKAJIMA, H., SONOMOTO, K., SATO, F., ICHIMURA, K., YAMADA, Y. and TANAKA, A. (1989). Enhancement of pigment productivity of immobilised cultured Lavandula vera cells by limitation of nitrogen sources. Journal of Fermentation and Bioengineering, 67(4): 306-308.

NAPOLI, C., LEMIEUX, C. and JORGENSEN, R. (1990). Introduction of a chimeric chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes. In trans. The Plant Cell, 2: 279-289.

NEMEC, X., WEISSOVA, K., KOVACS, P., KAKONIOVA, D. and LISKOVA, D. (1995). Decarboxylation of L-Tyrosine and L-DOPA by immobilised cells of Papaver somnferum. Phytochemistry, 38(4): 859-860.

NILSSON, T. (1970). Studies into the pigments in beetroot (Beta vulgaris L. ssp vulgaris var rubra L.). LantbrukshOgskolans Annaler, 36: 183.

NIGRA, H. M., ALVAREZ, M. A. and GIULIETTI, A. M. (1990). Effect of carbon and nitrogen sources on growth and solasodine production in batch suspension cultures of Solanum eleagnfolium Cay. Plant Cell, Tissue and Organ Culture, 21: 55- 60.

NOGUCHI, H. and SANKAWA, U. (1982). Formation of geomichrysone by tissue cultures of Cassia torosa: induction of secondary metabolism in the lag phase. Phytochemistry, 21: 319-323.

NORTON, R. A., RADIN, D. N. and RODRIGUEZ, E. (1991). Environmental and chemical effects on growth, resin and rubber production in guayule tissue cultures. Phytochemistry, 30(8): 2615-2618.

OBATA-SASAMOTO, H. NISHI, N. and KOMAMINE A. (1981). Mechanism of suppression of DOPA-accumulation in a callus culture of Stizolobium hassjoo. Plant Cell Physiology., 22, 827-83 5.

232 OBATA-SASAMOTO, H. and KOMAMINE A. (1983). Effect of culture conditions on DOPA accumulation in a callus culture of Stizolobium hassjoo. Planta Medica, 49: 120-123.

OBRENOVIC, S. (1990). Effect of hypocotyl excision on red light-mediated betacyanin formation in amaranthus seedlings. Annals of Botany, 66: 245-248.

OHTA, S. and YATAZAWA, M. (1978). Effect of light on nicotine production in tobacco tissue culture. Agricultural and Biological Chemistry. 42: 873-877.

OZEKI, Y. and KOMAM1NE, A. (1981). Induction of anthocyanin synthesis in relation to embryogenesis in a carrot suspension culture: correlation of metabolic differentiation with morphological differentiation. Physiologia Plantarum, 53: 570- 577.

PANDA, A., MISHRA, S. and BISARIA, V. (1992). Alkaloid production by plant cell suspension cultures of Holarrhena antidysenterica: I. Effect of major nutrients. Biotechnology and Bioengineering, 39(10): 1043-1051.

PARTANEN, C. (1963). Plant tissue culture in relation to developmental cytology. International Review of Cytology, 15: 213-243.

PASCH, J. H., VON ELBE, J. H., and SELL, R. D. (1975). Betalains as colorants in dairy products. Journal of Milk Food Technology, 38(1): 25.

PASCH, J. H. and VON ELBE, J. H. (1978). Sensory evaluation of betalain and concentrated beet juice. Journal of Food Science, 43(5): 1624-1625.

PAYNE, G. (1991). Root cultures. In Plant Cell and Tissue Culture in Liquid Systems, (eds.) G. Payne, V. Bringi, C. Prince and M. Shuler, pp. 225-280. Hanser Publishers, Munich, New York, Vienna and Barcelona.

PHILLIPS, R. and HENSHAW, G. (1977). The regulation of synthesis of phenolics in stationary phase cell cultures of Acer pseudoplatanus L. Journal of Experimental Botany, 28: 785-794.

PHILLIPSON, J. D. (1990). Plants as sources of valuable products. In Secondary Products from Plant Tissue Culture, (eds.) B. V. Charlwood and M. J. Rhodes, pp. 1- 21. Clarendon Press, Oxford.

PIATTELLI, M. (1976). Betalains. In Chemistry and Biochemistry of Plant Pigments, vol. 1, pp. 560-596. Academic Press, London.

PIATTELLI, M. (1981). The betalains: structure, biosynthesis, and chemical taxonomy. In The Biochemistry of Plants, vol. 7, (ed.) E. E. Conn, pp. 557-575. Academic Press, New York.

233 REINERT, J. and YEOMAN, M. M. (1982). Plant Cell and Tissue Culture, p. 201. Springer-Verlag, Berlin.

REINHARD, E., KREIS, W., BARTHLEN, U. and HELMBOLD, U. (1989). Semicontinuous cultivation of Digitalis lanata cells: production of -methyldigoxin in a 300-1 airlift bioreactor. Biotechnology and Bioengineering, 34: 502-508.

RINK, E. and BOHM, H. (1985). Changed betaxanthin pattern in violet flowers of Portulaca grandfloraafter feeding of DOPA. Phytochemistry, 24: 1475-1477.

RUDAT, A. and GORING, H. (1995). Induction of betacyanin formation in cell cultures of Chenopodium album under UV-light irradiation. Journal of Experimental Botany, 46(282): 129-134.

RUDGE, K. and MORRIS, P. (1986). The effect of osmotic stress on growth and alkaloid accumulation in Catharanthus roseus. In Secondary Metabolism in Plant Cell Cultures, (ed.) P. Morris, pp. 75-81. Cambridge University Press.

SAKAMOTO K., IIDA K., SAWAMURA K., HAJIRO K., ASADA Y., YOSHIKAWA T. and FURUYA T. (1994). Anthocyanin production in cultured cells of Aralia cordata Thunb. Plant Cell, Tissue and Organ Culture, 36: 21-26.

SAKUTA, M., HIRANO, H. and KOMAM1NE A. (1991). Stimulation by 2,4- Dichiorophenoxyacetic acid of betacyanin accumulation in suspension cultures of Phytolacca americana. Physiologia Plantarum,83: 154-158.

SAKUTA, M., TAKAGI, T. and KOMAMINE, A. (1987). Effect of sucrose on betacyanin accumulation and growth in suspension cultures of Phytolacca americana. Physiologia Plantarum, 71: 455-485.

SAKUTA, M., TAKAGI, T. and KOMAMINE, A. (1986). Growth related accumulation of betacyanin in suspension cultures of Phytolacca americana L. Journal of Plant Physiology, 125: 337-343.

SAKUTA, M., HIRANO, H., KAKEGAWA, K., SUDA, J., HIROSE, M., W.JOY IV, R., SUGIYAMA, M. and KOMAMINE, A. (1994). Regulatory mechanisms of biosynthesis of betacyanin and anthocyanin in relation to cell division activity in suspension cultures. Plant Cell, Tissue and Organ Culture, 38: 176-179.

SATO, F. and YAMADA, Y. (1984). High berberine-producing cultures of Coptis japonica cells. Phytochemistry, 23: 281-285.

SCHLATMANN, J., NUVTILA, A., VAN GULIK, W., TEN HOOPEN, H., VERPOORTE, R. and HEININEN, J. (1993). Scaleup of ajmalicine production by plant cell cultures of Catharanthus roseus . Biotechnology and Bioengineering, 41: 253-262.

234 SCHOTTE, H and LIEBISCH, H. (1985). Alkaloids derived from tyrosine and phenylalanine. In Biochemistry of Alkaloids, (eds.) K. Mothes, H. SchUtte and M. Luckner, pp. 188-271. VEB Deutcher Verlag der Wissenschaften, Berlin.

SHARMA, 0. P. and KHANNA, P. (1980). Studies on steroidal sapogenin from tissue cultures of Agave wightii. Lloydia, 43(4): 459-462.

SKIRV1N, R. (1978). Natural and induced variation in tissue culture. Euphytica, 27: 241-266.

SMITH, T. (1980). Plant Amines. In Secondary Plant Products, Encyclopedia of Plant Physiology, New Series 8, (eds.) E. A. Bell and B. V. Charlwood, pp. 433-460. Springer Verlag, Berlin.

SMOLOV, A. P. and OLEINIKOVA, T. A. (1994). The influence of phosphate on growth of hetero- and mixotrophic soya (Glycine max) callus. Izvestiya Akademii Nauk Seriya Biologicheskaya (Moscow), 0(3): 479-482.

SOKAL, R. R. and ROHLF, F. J. (1981). The arcsine transformation. In Biometry: The principles and practice of statistics in biological research, 2nd edition, pp. 427- 429. W. H. Freeman and Company, New York.

SONGSTAD, D. D., DE LUCA, V., BRISSON, N., KURZ, W. G. and NESSLER, C. L. (1990). High levels of tryptamine accumulation in transgenic tobacco expressing tryptophan decarboxylase. Plant Physiology, 94: 1410-1413.

SONGSTAD, D. D., KURZ, W. G. and NESSLER, C. L. (1991). Tyramine accumulation in Nicotiana tabacum transformed with a chimeric tryptophan decarboxylase gene. Phytochemistry, 30: 3245-3246.

SOSNOVA, V. (1970). Reproduction of sugar beet mosaic and tobacco mosaic viruses in anthocyanized beet plants. Biologia Plantarum, 12: 424-427.

STEGLICH, W. and STRACK, D. (1990). Betalains. In The Alkaloids, vol. 39, (ed.) A. Brossi, pp. 1-62. Academic Press Inc., New York.

STENLID, G. (1976). Physiological effects of betalains upon higher plants. Phytochemistry, 15: 661-663.

STICKLAND, R. and SUNDERLAND, N. (1972). Photocontrol of growth, and anthocyanin and chlorogenic acid production in cultured callus tissues of Haplopappus gracilis. Annals of Botany, 36: 671-685.

STOBART, A. K. and KINSMAN, L. T. (1977). The hormonal control of betacyanine synthesis in Amaranthus caudatus. Phytochemistry, 16: 1137-1142.

235 STREET, H. E. (1977). Cell (suspension) cultures techniques, iii Plant Tissue and Cell Culture, Botanical Monograph vol. II, 2nd Edition, (ed.) H. Street, pp. 97-102. Blackwell Scientific Publications.

SUFFORD, A., SMITH, L. and FOWLER, M. (1985). Regulation and product synthesis in cells cultures of Catharanthus roseus, (L) G Don. Plant Cell, Tissue and Organ Culture, 4: 83-94.

SUGANO, N., IWATA, R. and NISHI, A. (1975). Formation of phenolic acid in carrot cells in suspension cultures. Phytochemistry, 14: 1205-1207.

SUGANO, N. and OGAWA, Y. (1981). Effect of 2,4-dichlorophenoxyacetic acid on the activity of o-methyltransferase in carrot cell culture. Phytochemistry, 20: 617-619.

SUGIMOTO, Y., YOSHIDA, A., UCHIDA, S. and YAMADA, Y. (1994). Dauricine production in cultured roots of Menispermum dauricum. Phytochemistry, 36(3): 679- 683.

SUNDQVIST, C., BJORN, L. 0. and VIRGIN, H. I., (1980). Factors in chioroplast differentiation. In Chloroplasts, (ed.) J. Reinert, pp. 201-224.

TABATA, M., YAMAMOTO, H., HIRAOKA, N., MARUMOTO, Y. and KONOSHIMA, M. (1971). Regulation of nicotine production in tobacco tissue culture by plant growth regulators. Phytochemistry, 10: 723-729.

TABATA, M., YAMAMOTO, H., HIRAOKA, N. and KONOSHIMA, M. (1972). Organisation and alkaloid production in tissue cultures of Scopolia parvflora. Phytochemistry, 11: 949-955.

TABATA, M., MIZUKAMI, H., HIRAOKA, N. and KONOSHIMA, M. (1974). Pigment formation in callus cultures of Lithospermum erythrorhizon. Phytochemistry, 13: 927-932.

TABATA, M., OGINO, T. YOSHIOKA, K. YOSHIKAWA, N. and HIRAOKA, N. (1978). Selection of cell lines with higher yield of secondary products. In Frontiers of Plant Tissue Culture, (ed.) T. A. Thrope, pp. 313-322. University of Calgary, Calgary.

TAMURA, H., FUJIWARA, M., TAKAHASHI, K. and SUGISAWA, H. (1994). Regulation of the productivity of anthocyanins and rosmarinic acid from cultured cells of Perilla frutescence var crispa (Labiatae). Kagawa Daigaku Nogakubu Gakujutsu Hokoku, 46(2): 135-140.

TERRADAS, F. and WYLER, H. (1991). 2,3-secodopa, the biosynthetic intermediates generated from L-DOPA by an enzyme system extracted from the Fly Agaric, Amanita muscaria L., and their spontaneous conversion to muscaflavin and betalamic acid, respectively, and betalains. Helvetica Chimica Acta, 74: 124-140.

236 TIMBERLAKE, C. and HENRY, B. (1986). Plant pigments as natural food colours. Endeavour, 10: 31-36.

TOWERS, G. H. and YAMAMOTO, E. (1985). Interactions of cinnamic acid and its derivatives with light. In Annual Proceedings of the Phytochemical Society of Europe, vol. 25: The Biochemistry of Phenolics, (eds.) C F. van Sumere and P. J. Lea, pp. 271-287. Clarendon Press, Oxford.

TREZZINI, G. F. and ZRYD, J. P. (1991). Characterisation of some natural and semi- synthetic betaxanthins. Phytochemistry, 30(6): 1901-1903.

VAN DER KROL, A., LENTING, P., VEENSTRA, J., VAN DER MEER, I., KOES, R., GERATS, A., MOL, J. and STUITJE, A. (1988). An antisense chalcone synthase gene in transgenic plants inhibits flower pigmentation. Nature, 333: 866-869.

VAN DER KROL, A., MUR, L., BELD, M., MOL, J. and STUITJE, A. (1990). Flavonoid genes in Petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. The Plant Cell, 2: 291-299.

VAZQUEZ-FLOTA, F., MORENO-VALENZUELA, 0. MIRANDA-HAM, M. L., COELLO-COELLO, J. and LOYOLA-VARGAS, V. M. (1994). Catharanthin and ajmalicine synthesis in Catharanthus roseus hairy root cultures: Medium optimisation and elicitation. Plant Cell, Tissue and Organ Culture, 38(2-3): 273-279.

VILLARREAL, L., ROJAS, G., MECKES, M. and NICASIO, P. (1993). Studies on Mimosa tenuflora callus culture: Interaction of kinetin and 2,4-dichlorophenoxyacetic acid in initiation and growth. Biotechnology Letters, 15(7): 721-726.

VON ELBE, J. H. and MAING, I. Y. (1973). Betalains as possible food colorants of meat substitutes. Cereal Science Today, 18(9): 263.

VON ELBE, J. H., KLEMENT, J. T., AMUNDSON, C. H., CASSENS, R. G. and LINDSAY, R. C. (1974). Evaluation of betalain pigments as sausage colorants. Journal of Food Science, 39: 128.

WATANABE, K., YANO, S. and YAMADA, Y. (1982). The selection of cultured plant cell lines producing high levels of biotin. Phytochemistry, 21(3): 513-5 16.

WELLER, T. A. and LASURE, L. L. (1981). Betalains in beet root tissue culture. Journal of Food Science, 47: 162-163.

WESTPHAL, K (1990). Large-scale production of new biologically active compounds in plant-cell cultures. In Progress in Plant Cellular and Molecular Biology, (eds.) H. Nijkamp, L. Van der Plas and J.Van Aartrijk, pp. 601-608. Kluwer, Netherland.

237 WICHERS, H. J., MALINGRE, TH. M., and HUIZING, H. J. (1983). The effect of some environmental factors on the production of L-Dopa by alginate-entrapped cells of Mucuna pruriens. Planta, 158: 482-486.

WICHERS, H. J., PEETSMA, G. J., MALINGRE, TH. M., and HUIZING, H. J. (1984). Purification and properties of phenol oxidase, derived from suspension cultures of Mucunapruriens. Planta, 162: 334-341.

WICHERS, H. J., MALINGRE, T. M., and HUIZING, H. J. (1985). Induction of phenoloxidase in cell suspension cultures of Mucuna pruriens L. Effects on accumulation of L-3 ,4-dihydroxyphenylalanine and biotransformation capacity. Planta, 165:264-268.

WICHERS, H., VISSER, J., HUIZING, H. and PRAS, N. (1993). Occurrence of L- DOPA and dopamine in plants and cell cultures of Mucuna pruriens and effects of 2,4-D and NaCl on these compounds. Plant Cell, Tissue and Organ Culture, 33: 259- 264.

WINK, M. (1990). Physiology of secondary product formation in plants. In Secondary Products from Plant Tissue Culture, (eds.) B. V. Charlwood and M. J. Rhodes, pp. 23- 41. Clarendon Press, Oxford.

WOHLPART, A. and MABRY, T. (1968). On the light requirement for betalain biogenesis. Plant Physiology, 43: 457-459.

WOHLPART, A. and BLACK, S. M. (1973). Accumulation of betalain in disks of Beta vulgaris leaves. Phytochemistry, 12: 1325-1329.

WOLTERS, B. and EILERT, U. (1983). Elicitoren-Auslöser der Akkumulation von Pflanzenstoffen. Deutsche Apotheker Zeitung, 123: 659-667.

WYSOKINSKA, H. and SWIATEK, L. (1991). Production of iridoid glucosides in cell suspension cultures of Penstemon serrulatus: effects of nutritional factors. Plant Science, 76: 249-258.

YAMADA, Y. and FUJTTA Y. (1983). Production of useful compounds in culture. In Handbook of Plant Cell Culture, vol. 1, (eds.) D. Evans, W. Sharp, P. Ammirato and Y. Yamada, pp. 717-728. Macmillan Publishing Company.

YAMAKAWA, T., OHTSUKA, FL, ONOMICHI, K., KODAMA, T. and MINODA, Y. (1982). Production of anthocyanin pigments by grape cell cultures. In Plant Tissue Culture 1982, (ed.) A. Fujiwara, pp. 273-274. Japanese Association for Plant Tissue Culture, Tokyo.

YAMAKAWA, T., KATO, S., ISHIDA, K., KODAMA, T. and M1NODA, Y. (1983). Production of anthocyanins by Vits cells in suspension culture. Agricultural and Biological Chemistry, 47: 2185-2191.

238 YAMAMOTO, Y. and MIZUGUCHI, R. (1982). Selection of a high and stable pigment-producing strain in cultured Euphorbia millii cells. Theoretical and Applied Genetics, 61: 113-116.

YAMAMOTO, Y., K1NOSHITA, Y., WATANABE, S. and YAMADA, Y. (1989). Anthocyanin production in suspension cultures of high-producing cells of Euphoria millii. Agricultural and Biological Chemistry, 53(2): 417-423.

YEOMAN, M. M. (1986). Use of immobilised plant cells for the production of food flavours and colours. Biotechnology in the Food Industry, 23-28.

YEOMAN, M. M. and FORCHE, E. (1980). Cell proliferation and growth in callus cultures. International Review of Cytology, Supplement hA, 1-24.

YEOMAN, M. M., MIEDZYBRODZKA, M. B., LINDSEY, K. and McLAUCHLAN, W. R. (1980). The synthetic potential of cultured cells. In Plant Cell Cultures: Results and Perspectives, (eds.) F. Sala, B. Parisi, P. Cella and 0. Ciferri, pp. 327-343. Elsevier North-Holland Biochemical Press.

YEOMAN, M. M., HOLDEN, M. A., CORCHET, P., HOLDEN, P. R., GOY, J. G. and HOBBS, M. C. (1990). Exploitation of disorganised plant cultures for the production of secondary metabolites. In Secondary Products from Plant Tissue Culture, (eds.) B. V. Chariwood and M. J. Rhodes, pp. 139-166. Clarendon Press, Oxford.

YEOMAN, M. M. and YEOMAN, C. L. (1996). Tansley Review No. 90: manipulating secondary metabolism in cultured plant cells. New Phytologist, 134: 553-569.

ZENK, M. (1978). The impact of plant cell culture on industry. In Frontiers of Plant Cell Tissue Culture, (ed.) T. Thrope, pp. 1-13, The International Association for Plant Tissue Culture, University of Calgary, Calgary.

ZENK, M. H., EL-SHAGI, H., ARENS, H., STOCKIGT, J., WElLER, E. W. and DEUS, B. (1977). Formation of indole alkaloids serpentine and ajmalicine in cell suspension cultures of Catharanthus roseus. In Plant Tissue Culture and its Biotechnological Application, (eds.) W. Barz, E. Reinhard and M. Zenk, pp. 27-43. Springer-Verlag, Berlin, Heidelberg, and New York.

ZENK, M. H., EL-SHAGI, H. and SCHULTE, U. (1975). Anthraquinone production by cell suspension cultures of Morinda citrfolia. Planta Medica Supplement, 79-101.

ZORINYANTS, S. E., NOSOV, A. V., BADAEVA, E. D., SMOLENSKAYA, I. N. and BADAEV, N. S. (1995). Cytogenetic analysis of a long-term Triticum timopheevii (Zhuk) Zhuk. cell suspension culture. Plant Breeding, 114(3): 2 19-225.

239 ZRYD, J. P., BAUER, J., WYLER, H., and LAVANCHY, P. (1982). Pigment biosynthesis and precursor metabolism in red beet semi-continuous cell suspension cultures. In Proceedings of the 5th International Congress of Plant Tissue and Cell Culture: Plant Tissue Culture, (ed.) A. Fujiwara, pp. 387-388. The Japanese Association for Plant Tissue Culture.

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