MICROPROPAGATION OF GLORIOSA SPECIES AND THEIR POTENTIAL AS A SOURCE OF COLCHICINE
A thesis submitted for the degree of Doctor of Philosophy
by
J ayasiri Ranatunga
School of Biochemistry and Molecular Genetics The University of New South Wales December, 1994 CERTIFICATE OF ORIGINALITY
I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree or diploma of a university or other institute of higher learning, except where due acknowledgement is made in the text.
(Signed) CONTENTS
SUMMARY i
ACKNOWLEDGEMENTS iii
ABBREVIATIONS iv
CHAPTER 1 - INTRODUCTION 1
1. 1 Genus Gloriosa 1
1. 2 Plant Tissue Culture 6
1. 2.1 Botanical Aspects of In Vitro Propagation 9
1.2.2 Methods of In Vitro Propagation 9
1.2.2.1 Callusing 9
1.2.2.2 Adventitious Bud Fonnation 10
1.2.2.3 Axillary Bud Fonnation 11
1. 2. 3 General In Vitro Procedure for Micropropagation 11
1.2.4 In Vitro Propagation of Monocotyledonous Plants 13
1.2.5 In Vitro Propagation of Gloriosa species 17
1. 3 Secondary Metabolites 19 1. 3. 1 Colchicine 26 1. 3. 1. 1 Occurrence in Plants 28
1. 3. 1. 2 Isolation, Purification and Quantitation of Colchicine From Plants 29
1.3.1.3 Biosynthesis of colchicine 31
1. 4 Project Aims 34
CHAPTER 2 - MATERIALS AND METHODS 35
2.1 Plant Materials 35
2.2 Chemicals 35
2.3 Sterile Technique 37
2.4 Aseptic Seed Gennination 37
2.5 Aseptic Tuber Sprouting 37 2.6 Callus Initiation 38
2.7 Proliferation and Maintenance of Callus 38
2.8 Root Cultures 39
2.9 Growth Rate of Callus 39
2.10 Initiation of Shoots from Tuber Explants 39
2.11 Initiation of Shoots from Shoot Bases 40
2.12 Initiation of Shoots from Root-Coleoptile Axis of Seedlings 41
2.13 Initiation of Shoots from Callus 42
2.14 Maintenance of Stock Cultures 42
2.15 Multiplication of Shoots 42
2.16 Replacement of NAA by IAA in Shoot Multiplication Medium 43
2.17 Alteration of Ammonium and Nitrate Concentration in Shoot Multiplication Medium 43
2.18 Alteration of Sucrose Concentration in Shoot Multiplication Medium 44
2.19 Development of Roots and Tubers 44
2.20 Effect of Sucrose on Development of Roots and Tubers 44
2.21 Effect of ABA and GA3 on Development of Roots and Tubers 45
2.22 Deflasking and Transferring to Soil 45
2.23 Micropropagation Growth Measurements 45
2.24 Statistical Analysis of Data 46
2.25 Extraction of Plant Tissues 46
2.26 Partial Purification of Colchicine 46
2.27 HPLC Analysis of Extracts 47
2.28 Mass Spectrometry 47
2.29 Thin Layer Chromatography 47
2.30 Counting of Radioactivity 48
2.31 Dry Weight Determination 48
2.32 Addition of Putative Colchicine Precursors in Liquid Shoot Multiplication Medium 48 2.33 Distribution of Colchicine in Mature Gloriosa simplex Tissues 48
2.34 Screening of High Yielding Cell Lines for Production of Colchicine and Conservation through Plant Regeneration 49
CHAPTER 3 - RESULTS - CALLUS 50
3. 1 Callus Cultures Derived From Mature Tuber Explants 50
3. 2 Callus Cultures Derived From Mature Seedling Ex plants 52
3. 3 Proliferation and Maintenance of Undifferentiated Callus 55
3.3.1 G. superba 55
3.3.2 G. simplex 57
3.4 Proliferation and Maintenance of Differentiated Callus 57
CHAPTER 4 - RESULTS - INITIATION OF SHOOTS 59
4.0 Introduction 59
4.1 Initiation of Shoots 60
4.1.1 Initiation of Shoots From Tuber Explants 60
4.1. 2 Initiation of Shoots From Shoot Base Explants 67
4.1.3 Initiation of Shoots From Root-Coleoptile Axes of Seedlings 71
4.2 Tuber Initiation From Tuber Explants 75
4.3 Tuber Initiation from Root-Coleoptile Axis Explants 80
CHAPTER 5 - RESULTS - SHOOT AND TUBER MULTIPLICATION 83
5. 0 Shoot and Tuber Multiplication 83
5. 1 G. rothschildiana 83
5. 1.1 Multiplication of Cultures Derived From Tuber Explants 83
5. 1. 2 The Effect of Altered Inorganic Nitrogen Concentration and Composition on Shoot and Tuber Multiplication 88
5. 1. 3 The Effect of Altered Sucrose Concentration on Shoot and Tuber Multiplication 91
5 .1.4 Multiplication of Cultures Derived From Shoot Base Explants 91 5 .1. 5 Multiplication of Cultures Derived From Root-Coleoptile Axes of Seedlings 92
5. 2 G. superba 94
5. 2.1 Multiplication of Cultures Derived From Tuber Explants 94
5.2.2 Multiplication of Cultures Derived From Shoot Base Ex plants 95
5.2.3 Multiplication of Culturres Derived From Root-Coleoptile Axes of Seedlings 96
5. 3. G. simplex 98
5.3.1 Multiplication of Cultures Derived From Tuber Explants 98
5.3.2 Multiplication of Cultures Derived From Shoot Base Explants 99
5.3.3 Multiplication of Cultures Derived From Root-Coleoptile Axis of Seedlings 100
5.3.4 Multiplication of Shoots Derived From Undifferentiated Callus 100
5 .4 G. verschuurii 102
5.4.1 Multiplication of Cultures Derived From Tuber Explants 102
5.5 G. richmondensis 103 5.5.1 Multiplication of Cultures Derived from Tuber Explants 103
CHAPTER 6 - RESULTS - ROOT AND TUBER DEVELOPMENT 104
6.0 Root and Tuber Development 104
6.1 Root and Tuber Development of Gloriosa species 104
6.1.1 The Effect of Sucrose Concentration on Root andTuber Development 106
6.1.2 The Effect of ABA and GA3 on Root and Tuber Development of G. rothschildiana 110
CHAPTER 7 - RESULTS - ESTABLISHMENT IN SOIL 111
7. 0 Establishment in Soil 111
7. 1 Survival Rates of In Vitro Generated Gloriosa Species in Soil 111
7. 2 Yield of Tubers After the First Growth Cycle under Glasshouse Conditions 112 7. 3 Yield of Tubers After Second and Third Growth Cycles under Glasshouse Conditions 113
7.4 The Effect of Potting Medium on Tuber Growth of G. superba. 115
7 .5 Micropropagation Protocol For Gloriosa Species 116
CHAPTER 8 - RESULTS - QUANTITATION OF COLCHICINE:DEVELOPMENT OF METHODS 118 8. 0 Extraction, Purification and Quantitation of Colchicine from Gloriosa 118 8. 1 Extraction and Purification from Plant Tissue 118
8. 2 Quantitation of Colchicine 123
8. 3 Purity of the Internal Standard ([3H]-Colchicine) 123
CHAPTER 9 - RESULTS - QUANTITATION OF COLCHICINE IN IN VITRO AND IN VIVO TISSUES 126
9.0 Colchicine Content of in viva and in vitro Tissues of Gloriosa species and in viva Tissues of Sandersonia aurantiaca 126 t 9 .1 Colchicine Con4ent of in viva Tissue 126
9 .1.1 Colchicine Content of Glasshouse Grown Gloriosa and Sandersonia aurantiaca 126
9 .1. 2 Distribution of Colchicine within a Mature G. simplex Vine and a Tuber 128
9.2 Colchicine Content of In Vitro Cultures 130
9.2.1 Colchicine Content of Shoot Initiation Cultures (Stage I) 130
9.2.2 Colchicine Content of Shoot Multiplication Cultures (Stage II) 131 d 9. 2. 3 Manipulation of Multiplication Medium for Increaset_Yield of Colchicine From G. rothschildiana Shoot Cultures 137
9.2.4 The Effect of Putative Colchicine Precursors on the Colchicine Content of Shoots and Tubers of G. superba 140
9. 2. 5 Colchicine Content of Root and Tuber Development Cultures (Stage III) 141
9. 3 Colchicine From In Vitro Generated Plants When Grown Under Glasshouse Conditions 145 9. 4 Colchicine from Undifferentiated and Redifferentiated Callus 14 7
9 .4.1 Colchicine Concentration of G. rothschildiana and G. simplex Differentiated Callus 147
9.4.2 Colchicine Concentrations of G. simplex Undifferentiated Cells 148
9. 5 A Protocol to Screen and Recover Cell Lines Highly Productive for Colchicine 149
CHAPTER 10 - DISCUSSION 151 Initiation and Maintenance of Callus Cultures 151
Micropropagation 154
Colchicine 168 Concluding Remarks 175
REFERENCES 177
APPENDICES 192 (i)
SUMMARY
Gloriosa species are increasing in popularity in the cut flower industry and they are also a potential source of the phannaceutical, colchicine. However their propagation on a commercial scale is hampered by the slow rate of vegetative multiplication.
Furthermore, little information is available on the distribution of colchicine in the genus, except G. superba. This thesis describes studies on the in vitro micropropagation of
G. rothschildiana, G. superba, G. simplex, G. verschuurii and the cultivar
G. richmondensis. In addition colchicine was quantitated from in vivo and in vitro
generated tissues of the above species, growing under identical conditions.
A successful protocol for the rapid micropropagation of these species was
developed which combined good shoot initiation, a high rate of multiplication and tuber
initiation, followed by root and tuber development. Plantlets could then be directly
transferred to soil without acclimatization.
The effects of different explants, various auxins and cytokinins, and basal
media on shoot and tuber initiation and subsequent multiplication were also determined.
Juvenile tuber tissues made the best explants and Woody Plant medium was superior to
that of Murashige and Skoog,. The best plant growth regulator (PGR) combination was
10 µM NAA and either 20µM or 40 µM BAP for shoot initiation and either 10 µM IAA
and 40 µM BAP or 1 µM IAA and 20 µM BAP for multiplication. Root and tuber
development was best in the absence of plant growth regulators. The in vitro cloned
plants grew normally and produced flowers during the third growth cycle in soil.
Increasing the sucrose concentration in root and tuber development medium
from 2% to 4%, doubled the number of tubers per replicate for G. superba, while that
for G. rothschildiana increased about 25%. Tuber weight per replicate doubled for both
species. The optimum concentration of nitrogen was 30mM at a 1 :2 ratio of
NH4+/NO3-, for both shoot and tuber multiplication of G. rothschildiana.
Abscisic acid or gibberellic acid decrease the number of tubers and roots per replicate for G. rothschildiana when present in root and tuber development medium. (ii)
Depending on the species, 150-6000 tubers ready for planting out, could be obtained from a single tuber in 48 weeks, compared with 2-4 tubers by conventional propagation,
Tissues were extracted in lM HCl, the extract cleaned up using C-18 solid phase extraction catridges, and the colchicine quantitated by reverse phase HPLC.
3H-colchicine used as an internal standerd. The effects of different media and plant growth regulators on the colchicine content at different stages of micropropagation were investigated. For example, G. rothschildiana accumulated 11mg colchicine/jar in eight weeks when a four-shoot clump was subcultured onto Woody Plant medium containing
4% sucrose and no plant growth regulators. For G. superba under identical conditions it was 18mg of colchicine per jar. Like shoot and tuber multiplication, colchicine synthesis was best in medium containing NI4+ and NO3- in a 1:2 ratio.
Feeding putative colchicine precursors was investigated as a means of increasing the colchicine content of G. superba shoot cultures. Only Tyrosine increased the colchicine concentration in the shoots, but decreased it in the tubers. Both cinnamic acid and coumaric acid increased the colchicine concentration of tubers above the control.
Present study also introduced a new approach aimed at preserving colchicine producing cell lines derived from undifferentiated callus. Tissue derived from
G. simplex tuber explants was taken through callus, plant regeneration and soil establishment.
The colchicine content of different organs of the Gloriosa species and
Sandersonia aurantiaca plants was determined. Shoots and tubers from in vitro generated Gloriosa, contained more colchicine than the glass house grown in vivo tissues. Tissue from S. aurantiaca had less colchicine than those from the Gloriosa.
The data on quantitation of colchicine from in vivo and in vitro tissues of
Gloriosa highlight the potential of this genus to be an alternative source of colchicine and in particular to produce this colchicine by tissue culture of the plants. (iii)
ACKNOWLEDGEMENTS
I am indebted to my supervisors Dr. I.J. Mcfarlane and Professor B.V.
Milborrow for their supervision, invaluable advice and encouragement throughout this project.
I would like to thank Professor W. O'Sullivan, Dean, Faculty of Biological and
Behavioural Sciences and Professor I. Dawes, Head, School of Biochemistry and
Molecular Genetics, for allowing me to undertake post graduate studies in the School of
Biochemistry and Molecular Genetics.
I wish to express my thanks to the Commonwealth Government of Australia for financial assistance and the staff members of the Australian International Assistance
Bureau (AIDAB) for their continued support.
I am grateful to the Government of Sri Lanka and the Ceylon Institute of
Scientific and Industrial Research (CISIR) for granting me the study leave to undertake this project.
My grateful thanks to V. Premajayantha, Statistician, Faculty of Management,
University of Western Sydney and Padmasiri K. Perera, University of New South Wales for their assistance with the statistical analysis.
I wish to acknowledge the help of the Royal Botanic• Gardens, Sydney, for plant identification and Dr. Sumedha Dharmarathna for her assistance in light microscopy.
My thanks to the fellow lab members for their cooperation throughout and my special thanks to Nina Artanti for her help on many occasions.
My greatest thanks to my mother and father for their well wishes and encouragement throughout the years.
Finally I thank my wife Ruki for her support and encouragement, and my daughter Y amesha who missed my attention during the study period. (iv)
ABBREVIATIONS
2,4-D 2,4-Dichlorophenoxyacetic acid ABA Abscisic acid ANOVA Analysis of Variance B5 Gamborg B5 basal medium BAP 6-Benzylaminopurine D.F Degree of freedom DMRT Duncan's Multiple Range Test dpm Disintegrations per minute DW Dry weight EtOH Ethanol FW Fresh weight GA3 Gibberellic acid HPLC High performance liquid chromatography IAA Indole-3-acetic acid IBA Indole-3-butyric acid Kinetin 6-Furfury laminopurine MS Murashige and Skoog basal medium NAA ~-Naphthaleneacetic acid PGR Plant growth regulator POPOP 1,4-Bis[2-(5-phenyloxazolyl)]benzene PPO 2,5-Diphenyloxazole Pr Probability Rf Ratio of travel of compound to travel of solvent front rpm Rotations per minute SAS Statistical Analysis System (SAS Institute, Cary, N.C., USA) SH Schenk and Hildebrandt basal medium SPE Solid phase extraction TLC Thin layer chromatography UNE#B de Fossard's inorganic nutrient medium UV Ultra violet WP Woody plant basal medium Some of the results of this investigation were presented at the following meetings:
Ranatunga, J., Mcfarlane, I.J. and Milborrow, B.V. (1990) In Vitro Propagation of Gloriosa Species. Proceedings of the Australian Biochemical Society, Vol. 22, SP80.
Ranatunga, J., Mcfarlane, I.J. and Milborrow, B.V. (1991) In Vitro Propagation of Gloriosa Species. Proceedings of the N Australian Branch Meeting, International Association for Plant Tissue Culture, p. 25.
Ranatunga, J., Milborrow, B.V. and Mcfarlane, I.J. (1993) Colchicine Content of Tissue Cultured Gloriosa Species. RACI Natural Products Group, 2nd 1 Day Symposium. 1
CHAPTER 1
INTRODUCTION
1 . 1 The Genus Gloriosa
The genus Gloriosa L., belongs to the tribe Uvularieae of the family Liliaceae
(Lugade and Hegde, 1992). The name derives from the Latin Gloriosus, meaning splendid. The flowers are magnificient and conspicuous, with colours ranging from red, orange and yellow to purple. The plants are deciduous climbing vines and are natives of tropical and southern Africa, Madagascar, India, Sri Lanka, China, and South-East Asia.
They are commonly known as Climbing Lily, Flame Lily or Glory Lily. Everett (1981) lists five species; namely G. rothschildiana, G. superba, G. simplex, G. verschuurii and
G. carsonii.
G. rothschildiana grows to a height of 2.5m. The leaves are broadly-lanceolate to
ovate-lanceolate, 125 to 175mm long by 38 to 50mm wide (Photograph 1. 1). The
flowers face downwards with strongly reflexed petals up to 80mm in length and with
smooth margins. They are bright red and yellow at first, but gradually change over 2-3
weeks to ruby or garnet (Photograph 1.2). It blooms earlier than G. superba in
temperate areas (Everett, 1981).
G. superba, grows to a height of 2m or sometimes more. The long-lanceolate to
narrowly ovate-lanceolate leaves are 110-130mm long and up to 25mm wide
(Photograph 1.1 ). Its flowers have crimped petals which are yellow when they open, but soon change to orange-brown or red (Photograph 1.2).
G. simplex (syn. G. virescens, G. plantii)(Everett, 1981) blooms in summer in temparate climates and grows to a height of 1 to 1.3m. The leaves are 80mm-100mm
long by 25-30mm wide (Photograph 1. 1). Its flowers have "spathulate petals" about
50mm long, with slightly wavy margins. They are deep orange and yellow or a clear yellow when grown in the shade (Photograph 1.2). 2
G. verschuurii is found mostly in East Africa and is a compact plant up to 1.5m
tall, with leaves wider than those of G. rothschildiana. The flower stallc is short and the
petals broad, reflexed, undulated and deep crimson with yellow bases and margins.
G. richmondensis, an apparent cultivar of G. superba (T.C. Chambers personal
communication) has similar features to G. superba, although it has broader leaves
(Photograph 1.1) and flower petals (Photograph 1.2) compared with those of
G. superba.
A W FABER CASlELL "323"
Photograph 1.1 Leaves of Gloriosa species and the cultivar, G. richmondensis. (a) G. rothschildiana (b) G. superba (c) G. simplex (d) G. richmondensis
G. carsonii grows to a height of 0.9m. The leaves are 100-125mm long. The
flower petals are about 65mm long, with recurved and crisped margins. The wine
purple petals are edged with yellow toward the base (Bailey, 1963; Everett, 1981 ). 3
Photograph 1.2 Flowers of Gloriosa species and the cultivar, G. richmondensis. (a) G. rothschildiana (b) G. superba (c) G. simplex (d) G. richmondensis 4
The tubers of Gloriosa are distinctive, long, brittle white structures which are usually in the form of a V or L shape (Photograph 1.3). Occasionally there are three prongs. New shoots develop from the prominent end of the arm (Everett, 1981).
/,,, Ill II,, I' Ill '"I' I "''""I"' 'l"''l""\llll\1111\1111\1'111""1""1""1'' ,""""':"'"""l"i' ,11,111 Lio U / 1'3 iio 00 tr7 l8 U9 2io 12i2Z3Z<.!i,11ie.h.le•ao
l • H 1...lll[U l · ' ~
Photograph 1.3 Typical 'V' or 'L' shape of Gloriosa tubers. The arrow points to a growing tip of a tuber.
Horticulturally the genus is well suited for growing permanently outdoors in warm climates and the gorgeous flowering vines have been suggested as flower borders, fences, and grouping in spaces at the front of shrubberies. They also grow well in large containers, as hedges or in glasshouses. The Gloriosa flower has been a popular subject for poetry and for artists because of its unique shape and attractive colours. Gloriosa flowers are excellent for cutting because of their striking colours and appearance and their long vase life (Everett, 1981; Finnie and van Staden, 1989).
Countries with a strong demand for Gloriosa cut flowers include Australia, Japan,
Thailand, Hong Kong and the United States (Cooper, 1993). According to Cooper, demand in Australia exceeds the local supply. As indicated by Custers and Bergervoet
(1994), the Netherlands has established an annual production of 3.5 million flowers.
Further, they mention that a market for Gloriosa as a pot plant has been established recently, with 75 000 sold in 1992. 5
The species have a strong tradition of medicinal use in the indigenous medicine of both Africa and Asia. The powdered tubers of G. simplex have been used for the treatment of impotency and barrenness by the Zulu (Bryant, 1909). Redgrove (1930) reports that the root stock is poisonous to the pig and some small animals and is dangerous to cattle in large amounts. In India and Sri Lanka flowers of G. superba are used in religious ceremonies and the root material in promoting labour pains and in procuring abortion. Watt and Breyer-Brandwijk (1962) cite a number of folk uses of tubers in Sri Lanka and India. In India it has been used as a remedy for snake and scorpion bites (Chopra, 1933). The material is popularly believed throughout India to be highly poisonous and is used to some extent for suicide and to induce abortions. In a number of cases it has caused poisoning from accidental overdosing in the course of its use in traditional medicine (Badhwar, 1946).
The tubers, leaves and seeds of G. superba (Thakur et al., 1975) and G. simplex
(Ntahomvukiye et al., 1984) have been reported to contain the alkaloid colchicine.
Medically colchicine is used to provide effective pain relief in acute gouty arthritis
(Sartorelli and Creasey, 1969; Flower et al., 1985) and, together with its derivative colchiceine, is of interest for its protective effect against acute liver damage (Marisabel et al., 1989). Recently Tiegs et al., (1992) demonstrated the ability of colchicine to
prevent lipopolysaccharide induced hepatitis. In cell biology it is a useful research
chemical; for example, in generating polyploid plants (Mathur et al., 1987; Alemanno
and Guiderdoni, 1994).
Since the discovery of colchicine in some species of Gloriosa, a number of
researchers have drawn attention to the potential of this genus as a commercial source of
the alkaloid (Narain and Khoshoo, 1967; Sarin et al., 1974; Finnie and van Staden,
1991). The main commercial source of colchicine remains the autumn crocus
(Colchicum autunmale L) (Wildman and Pursey, 1968), but the colchicine content of
G. superba is comparable to or higher than that in Colchicum species (Finnie and van Staden, 1991). 6
As interest in Gloriosa as a cut flower and a phannaceutical raw material increases, the expansion of cultivation is an immediate need. However propagation of
Gloriosa on a commercial scale is hampered by its slow rate of multiplication.
Conventionally, propagation is by annual binary multiplication of the tubers or from seeds. Tubers require several seasons growth to achieve a sufficient size to produce a
vigorous and healthy plant with a large number of blooms.
Propagation from seeds takes even longer and, of couse, does not necessarily
give plants true to type. Relatively slow vegetative techniques, therefore, are required to ensure high level fidelity of a new cultivar or of a superior individual. The limited
supplies of planting materials is a major factor restricting the expansion of Gloriosa
cultivation. Material is collected from natural habitats at the present time and large scale
collection may adversely effect a fragile ecosystem. The availability of large quantities
of Gloriosa plants with desired characteristics, such as flower excellence or a high
colchicine content, would help to alleviate the problems now experienced by both the
horticultural and pharmaceutical industries.
If plants cannot be produced cheaply and easily, then the plant will have little
commercial value (Dawson, 1968). The commercial exploitation by the cut flower or
pharmaceutical industry, therefore, requires the development of a rapid propagation
system for Gloriosa.
1 . 2 Plant Tissue Culture
Plant tissue culture broadly refers to the cultivation of all plant parts, whether a single cell, a tissue or an organ, under aseptic conditions (Biondi and Thorpe, 1981).
Debergh and Read (1993) stated that micropropagation is true to type propagation of a selected genotype using plant tissue culture techniques, usually without dedifferentiation. Bhojwani and Razdan (1983) stated that multiplication of genetically identical copies of a cultivar by asexual reproduction is clonal propagation and a plant population derived from a single individual by asexual reproduction constitutes a clone. Micropropagation via plant tissue culture provides a useful tool for rapid vegetative propagation of plants 7 when normal reproduction is very slow or not possible (Hiraoka and Tomita, 1990).
This technology has been successfully applied to the commercial production of many ornamental. plants (Pierik and Post, 1975; Slabbert et al., 1993), vegetative crops
(Hasegava et al., 1973) and forest trees (Warrag et al., 1989; Chang and Donald, 1992).
Vochting (1878) obtained very luxuriant callus with explants from Brassica
species and he reported that the development of plant fragments was characterised by
polarity. In 1902 Goebel confirmed Vochting's results and suggested the notion of
totipotency, ie. that any one cell could give rise to a whole organism. The experiments
carried out by the German botanist Haberland in 1902 resolved many outstanding
problems in plant tissue culture such as the failure to promote division of isolated cells
to give cell aggregates in cell cultures.
A new approach to plant tissue culture was conceived simultaneously in Germany
by Kotte (1922) and in the United States by Robbins (1922), who showed that
meristematic tissues taken from root tips or buds could be used to establish in vitro
cultures. White (1934) opened the door to root culture by obtaining cultures of tomato ,(, roots which grew indefinitely. Although Robbins (1922) and White (1933) attempted
to establish bud cultures, success occured only when Loo (1945) obtained cultures of
Asparagus and Dodder stem tips. Ball (1946), considered to be the father of
micropropagation, identified the exact part of a shoot meristem that was able to give rise
to a whole plant.
Curtis and Nichol (1948) began the first in vitro cultivation of orchid seeds. La
Rue (1947) initiated endosperm cultures of Zea mays which grew and could be
subcultured. This was the first successful attempt to cultivate monocotyledonous tissue
continuously.
White's ( 1937) discovery of the importance of the B vitamins and the recognition
of the role of indole-3-acetic acid stimulated a great development in plant tissue culture.
The work of Miller et al., (1956) led to the discovery of kinetin. The bud promoting
properties of kinetin suggested a broad interaction of this substance with auxin (Skoog
and Miller, 1957). This realisation led to the classic experiments which showed that 8 root and bud initiation were conditioned by a balance between auxin and kinetin, the fonner substance inducing root fonnation and the latter bud initiation.
Research on tissue culture was considerably enhanced by improvements in the mineral element content of culture media. Although the first mineral solutions were chosen empirically, in practice they gave satisfactory results. They were not optimal and soon Hildebrandt et al., (1946) proposed a new medium containing a higher concentration of Na2S04 than previously used. Murashige and Skoog (1962) proposed a solution that was far more concentrated than the previously used fonnulae. Other popular media include Gamborg's B-5 (Gamborg et al., 1968), Schenk and Hildebrandt
(1972) and Woody Plant (McCown and Lloyd, 1981). Often these were very successful on hitherto refractory species.
A major advance in clonal propagation is attributed to Morel (1960) who introduced the technique of meristem culture. His method found almost immeil(iiate commercial use, and a new market-oriented propagation technique became available that was within the economic reach of the average person (Debergh and Maene, 1983).
Rapid clonal propagation using tissue culture techniques has been reported for many ornamental monocotyledonous plants, eg. Gladiolus (Ziv, 1989); Iris (Meyer et al.,1975); Hyacinthus (Pierik and Post, 1975) and Lilium (Novak and Petru, 1981).
The technique has been used successfully to overcome problems in the commercial propagation of many plant species. For example, problems in acclimatization of micropropagated Narcissus shoots (Seabrook, 1990) and garlic shoots (Matsubara and
Chen, 1989), were solved by inducing the formation of bulbils or corms prior to transplanting shoot clump cultures into a non-sterile environment.
Wang and Hu (1982) and Ng (1988) comment that culture-derived bulbs, tubers or conns can be stored conveniently, are easily distributed, and can be used at any time of the year. Furthermore John et al., (1993) suggested that microtubers, originating from plant tissues in vitro, would be better for intemationMal germplasm exchange because they are more tolerant of variations in light and temperature, and are less prone to damage, than cultured shoots. Ng (1988) found that Dioscorea microtubers could be 9
stored for long periods without losing their viability and that establishment of plants in
the field from microtubers was easy and inexpensive.
1. 2 .1 Botanical Aspects of In Vitro Propagation
New growth of plants is usually derived from meristems, juvenile tissues made up of cells which have not yet been programmed for differentiation. Meristematic cells
are found at the tips of stems and roots, in the leaf axils, in stems as cambium and
occasionally on leaf margins. Axillary meristems are usually formed in the axil of every
leaf and have the potential of reproducing the main shoot. Meristematic tissues have the
potential to differentiate into leaves, stems, roots and other organs. Conversely, some
differentiated cells (usually parenchymatous cells) have the capacity to revert to a
meristematic or dedifferentiated state and then initiating the growth of new shoots, buds,
roots or leaves from unusual locations, a phenomenon referred to as adventitious
growth. Callus has generally proved to be unsuitable for in vitro propagation as it tends
to accumulate chromosomal abnormalities and lose the capacity to regenerate whole
plants. For long term multiplication and storage of a selected genotype, cultures are
required that combine the minimum loss of totipotency with a maximum of genetic
stability. Techniques that exploit the natural mechanism of axillary and adventitious
shoot formation seem most likely to achieve this.
1. 2. 2 Method of In Vitro Propagation
Three approaches have been followed to achieve in vitro shoot multiplication;
callusing, adventitious bud formation and axillary bud formation.
1.2.2.1 Callusing
The potential of plant cells to multiply indefinitely in culture as callus and their
totipotent nature permits a very rapid multiplication of individuals of many plant
species (Bhojwani and Razdan, 1983). Differentiation of plants from callus
may occur via shoot-root formation or somatic embryogenesis. Although 10
micropropagation through callus often achieves the highest rate of shoot
multiplication, Murashige (1974) suggested that it should be avoided as it
causes chromosomal changes, for example, formation of polyploid and
aneuploid plants (Takatori et al., 1968). Nevertheless, regeneration of plantlets
from callus can be an important technique, utilised in developing new
germplasm. Examples include somatic hybridisation by protoplast fusion
(Power et al., 1970), mutagenesis and selection of desired strains (Erikson
1967), the screening of cell lines (Constable et al., 1981) and callus derived
from plants that produce desired secondary metabolites. The use of callus
cultures for the production of secondary metabolites is discussed later in this
thesis.
1.2.2.2 Adventitious Bud Formation
Many plant species produce adventitious shoots in vitro, under the influence of
appropriate combinations of growth regulators. Hussey (1975) states that many
of the monocotyledons, which have specialised storage organs, have a strong
tendency to produce adventitious buds. Finnie and van Staden (1989) claimed
that this mode of regeneration is usually as genetically stable as axillary
meristem propagation. According to Queralt et al., (1991) the process of
adventitious plantlet formation on excised tissue is of prime importance for the
in vitro propagation of flower-bulb crops. In many plants adventitious shoots
appear to be genetically homogenous, especially if they are initiated on
comparatively young tissue. However it is important that frequent observations
of in vitro generated plants are made to determine if there are any off-type
plantlets. The monitoring of growth performance should continue after transfer
to soil. 11
1.2.2.3 Axillary Bud Formation The outgrowth of axillary shoots depends on the supply of cytokinin to the
meristem in many plants. Hussey (1983) showed that shoot tips cultured on a
medium which was free from growth regulators, typically developed into a
single shoot with strong apical dominance. When cultured on medium
containing a suitable cytokinin, axillary shoots developed prematurely and were
followed by secondary, tertiary, etc. shoots in a proliferating cluster. Once
such a cluster had developed sufficiently it could be divided up into smaller
clumps of shoots or separate shoots which formed similar clusters when
subcultured on fresh medium. This process may be continued indefinitely. The
rate of multiplication of shoots varies widely with species and is affected by the
composition of the nutrient medium. Cultures for axillary multiplication can be
started either from excised shoot tips or from in vitro adventitious shoots. In
bulbs and corms the axillary meristematic sites are in the basal plate (which
represents the stem) (Hussey, 1976a).
1. 2. 3 General In Vitro Procedure for Micropropagation
General micropropagation procedures comprise
(a) establishment of aseptic cultures and multiplication of shoots
(b) transfer of shoots to media, the composition of which induces root
formation and
(c) planting out into soil in the glasshouse or in the field.
In a somewhat formalised manner, Murashige (1974) subdivided micropropagation into four stages, although Yeoman (1986) feels that these rigid distinctions have limited usefulness, because the exact sequence of events will vary with the type of plant. De
Fossard (1985) summarised Murashige's stages as follows. 12
Stage 1: Establishment of Aseptic Cultures (Initiation) This begins with the selection of explant materials from conventionally
grown plants with the desired characters or phenotype and their
disinfestation. Secondly, culture media and incubation conditions which
will induce explant materials to grow need to be determined. This will
provide an early indication of the suitability of a particular medium and
plant growth regulator combination.
Stage 2: Multiplication of Shoots The main objective here is multiplication of propagula. In many cases
conditions in Stage 1 will be the basis of a multiplication medium,
although modification of the medium for enhanced multiplication is
desirable. Stage 2 cultures also provide disease-free reserve stocks. The
power of in vitro propagation lies in the ease with which materials can be
recycled through this multiplication stage (Wetherell, 1982). Stage 3: Root Formation and Preparation for Planting-Out
In this stage, shoots are transferred to a medium that will induce root
formation. This usually needs to be determined empirically. What is
required ultimately is the formation of a root and shoot system that will be
strong enough to withstand the transition from an axenic in vitro
environment to soil and glasshouse conditions with the usual microflora of benign and infectious agents. Stage 4: Acclimatisation (Planting-out)
The acclimatisation of many plants generated in vitro has been difficult
and often low survival rates have been obtained, eg. Garlic (Osawa and
Sugawara, 1980), Jojoba (Rost and Hinchee, 1980) and Narcissus (Seabrook, 1990). A critical part of acclimatisation is the formation of fully-functional roots in a "potting mixture" (de Fossard, 1985).
Wetherell (1982) pointed out that plantlets grown in vitro are usually more 13
susceptible to damage by high light intensity, water stress and disease.
To minimise these problems, the sterilisation of potting mix, the use of
mist sprayers with heavy shading for the first few weeks, and the gradual
lowering of humidity have been adapted for many species. When
possible, in vitro formation of tubers, corms or bulbils has proved to be a
successful solution to the acclimatisation problem (Hosoki and Asahira,
1980; Chow et al., 1992). Apart from improved survival rate, tubers,
corms or bulbils generated in vitro can be conveniently stored, distributed
and used at any time of the year (Estrada et al., 1986; Ng, 1988). For
example, in the Netherlands, 16 million Lily bulblets were produced by
micropropagation in 1989 (Pierik, 1990).
1. 2. 4 In Vitro Propagation of Monocotyledonous Plants
Monocotyledonous plants are more difficult to propagate in vitro than dicotyledonous plants. Partenan (1963) and Carter et al., (1967) have commented on this apparent refractory nature. Nevertheless, many monocotyledons have been successfully cultured in vitro. This includes members of the Gramineae such as oats
(Carter et al., 1967) and rice (Zimny and Lorz, 1986), as well as some bulbous and related species. Examples are Lilium (Sheriden, 1968), Asparagus (Wilmar and
Hellendoom, 1968), Gladiolus (Ziv, 1989) and Allium (van der Valk et al., 1992).
Hussey (1975) worked on selected species of some families of monocotyledons, including the Liliaceae, and found that plantlets could be induced directly on stem tissue in nine species, on ovary tissue in five species and on leaf tissue in four species without intervening callus. He could also induce adventitious plants on either bulb or corm pieces from 10 species and achieved regeneration of plantlets from callus with seven species, including members of the Liliaceae. However, he found that plantlet induction frequency using the same type of explants varied according to the species.
The ability of an explant to develop buds will depend on a number of factors,
such as age, vigour and growing conditions of the parent plant and the season of 14 harvest. Takayama and Misawa (1980) reported that the physiological state of Lilium auratum explants was critical in determining the mophogenetic response in vitro.
Niederwieser and van Staden (1990) showed that young tissues of Lachenalia species formed more buds compared to older tissues.
The chemical composition of the nutrient medium was an important determining factor in the initiation and development of shoots in vitro. The most widely used inorganic nutrient mixture is the formulation of Murashige and Skoog (1962), originally developed for tobacco callus, but many other well known media have been developed.
Many monocotyledons, including species of the family Liliaceae, have been successfully cultured in a wide range of media. Most experiments have studied the effects of mineral nutrients, especially the NOT and NH4+, the sucrose concentration, and growth regulators on shoot formation and tuberisation in vitro. Bhojwani and
Razdan, ( 1983) thought that the organic nutrients of MS medium were generally adequate for micropropagation of most species. Asoken et al., (1983) found that a reduction in the amount of inorganic nitrogen, from 60mM to 15mM, was optimal for callus growth and adventitious plant regeneration of Dioscorea species.
There have been only limited investigations into the nutrition of monocotyledonous plants (Bach et al., 1992a). However, increased interest in tuberization in vitro has resulted in the reinvestigation of mineral nutrition. Asahira and
Nitsch (1968) found that in the presence of kinetin, shoot growth of Dioscorea batatas was promoted in the presence of NO3- and NH4+, but microtuber formation was delayed in the absence of NH4+. Mantel et al., (1989) found that in the absence of
NH4 + ions, D. alata had fewer new shoots, but increased microtuber induction. In contrast, microtuber induction in cultures of D. bulbifera was unaffected by the presence or absence of NH4 +. There is some evidence in the literature for an effect of the
NO3-: Nif4+ ratio on shoot and tuber formation. For example, Nagakubo et al., (1993) showed that varying the NO3- and NH4+ ratio from 2: 1 to 16: 1 increased
Allium sativum shoot regeneration frequency from 47% to 91 %. Leblay et al., (1991) 15 found that NO3- and NH4+ ratios favouring nitrate, increased adventitious shoot fonnation in some Rosaceous species.
There are many reports about the effect of exogenous sugars on growth and development of bulbous monocotyledons in vitro (Takayama and Misawa 1982; Mantell and Hugo 1989; Bach et al., 1992b and Chow et al., 1992). The general pattern is that increased sucrose concentration results in an increased number of tubers as well as an increase in mean fresh weight, but the requirements for tuberizati.on appear to be species specific. Mantell and Hugo (1989) found that optimal micro-tuberization of Dioscorea alata shoot cultures occured on full strength MS medium with 2 % sucrose, while
D. bulbifera needed 4% sucrose under the same conditions.
Narcissus produced few bulbils in shoot proliferation medium which contained
3% sucrose. When the sucrose concentration was increased to 6% or 9%, the percentage of shoots forming bulbils was 49% and 71 % respectively (Chow et al.,
1992). They also found that bulbil size was markedly increased by the elevated sucrose concentration. Nagakubo et al., (1993) found that increasing the sucrose concentration from 6% to 12% promoted bulbing of garlic in vitro. Aartrijk and van der Linde (1986) also found an effect of sucrose concentration on bulbing of some Amaryllidacea species.
Heath and Hollies (1965) reported that the greater the concentration of sucrose, the greater the tendency towards bulbing of onion. They also found that non-rooted onion plants gave a higher bulbing ratio at all concentrations of sucrose compared to rooted plants.
In a survey of the literature, Hussey (1980) found that BAP was the most useful, reliable and cheapest cytokinin for promoting axillary branching and suggested that it should be tested first for a new micropropagation system. Earlier he showed (Hussey,
1976a) that BAP caused branching of single in vitro shoots of some families of monocotyledons including the Liliaceae. He found that Liliaceae species required a much higher concentration of BAP (2-8 mg/Lor 9µM-36µM) to promote branching when compared to other families of monocotyledons; eg. Iridaceae (0.12-2 mg/L). In
Lilium, distortion of shoots was evident at 16 mg /L, but only severe at 32 mg/L. Very 16 little callus was produced, even at the highest BAP concentration. Root formation was inhibited in all species by concentrations of BAP that caused branching (Hussey 1976a).
However by successive reduction of the BAP concentration over a number of subcultures, normal rooted plantlets could be obtained. In these experiments he used a cytokinin only (viz BAP) but he suggested that the addition of auxin at appropriate concentrations could be beneficial, especially with plants of the Liliaceae and
Amaryllidaceae. Many studies have been conducted on the effect of plant growth regulators
(PGRs) on the induction of tubers in shoot cultures in vitro in many families of
monocotyledons. Examples include Dioscoreaceae (Ng, 1988), Iridaceae (Bruyn and
Ferreira, 1992), Amaryllidaceae (Chow et al., 1992; ) and Liliaceae (Somani et al.,
1989; Finnie and van Staden, 1989; Dabrowski et al., 1992; Custers and Bergervoet,
1994).
Takayama and Misawa (1979) observed that Lilium auratum bulblet formation
occurred spontaneously. However, Stimart and Ascher (1978) found that the addition
of auxins, even at low concentrations (0.03 mg/Lor 0. lµM) significantly increased the
number of bulblets from Lilium longiflorum. Similarly Hackett (1969) increased the
formation of Lilium bulblets by incorporating IAA into the medium. Novak and Petru
(1981) showed that NAA also had a stimulating effect on bulblet development of a
Lilium hybrid. Further, they showed that increased BAP concentration also resulted in
an increased number of bulblets, but simultaneously decreased root formation and callus proliferation.
There is, of course, evidence that auxins induce the formation of roots, but
Novak and Petru (1981) found that bulblets removed from primary explants of Lilium
produced roots when transferred onto auxin free medium. Nagakubo et al., (1993) also
had similar results when garlic shoots were taken from proliferation medium and transferred to auxin free Linsmaier and Skoog medium. In these instances it is possible that auxin in the tissue was carried over into the other medium. Mantell and Hugo 17
(1989) reported that kinetin stimulated the induction of small tubers in some Dioscorea species. The stimulatory effect of abscisic acid (ABA) on tuberization has been shown by many authors (Klerk et al., 1992; Wareing,1982; Ziv,1989). According to Klerk et al.,
(1992), ABA is the key factor in bulb formation of Lily. Wareing (1982) concluded that
ABA appears to be involved in tuber formation of potato. Gibberellic (GA3) also influenced tuberization (Vreugdenhil and Struik, 1989;
Wareing, 1982; Ziv, 1989). Although the role of GA3 is not clear, paclobutrazole, an inhibitor of GA synthesis, blocked organogenesis, but the effect was reversed by simultaneous addition of GA 4+7 (Vreugdenhil and Struik, 1989; Wareing, 1982; Ziv,
1989). In addition it has been reported that GA3 has an effect on root formation. For example GA3 inhibited root formation of Bryophyllum tubiflorum (Nanda and Jain,
1972).
The literature reviewed describes the factors which are known to influence the micropropagation of monocotyledon plants, including Liliaceous ornamentals. The development of successful micropropagation systems for commercial exploitation requires the testing of a number of PGRs in various combinations and concentrations, on different types of explants, at different stages of maturity, and in different media. Apart from these, other factors such as the total nitrogen content, the NI-4+ : N03- ratio, and the sucrose concentration also can be varied to increase propagation yield.
After a successful protocol for micropropagation has been established, it is then necessary to evaluate the results of transfer to soil, at least until flowering and fruit setting. In addition it is desirable to carry out sufficient replication to allow statistical validation.
1. 2. 5 In vitro Propagation Of Gloriosa Species.
There are three reports on the propagation of Gloriosa species in vitro. Two of these (Finnie and van Staden, 1989; Somani et al.,1989) appeared shortly after this work began and are limited in scope and deal with G. superba only. The third (Custers 18 and Bergervoet, 1994) appeared in the final stages of the preparation of this thesis and deals with the offspring from reciprocal crossings between the G. rothschildiana clone
'Philips Duphar' and new accessions of G. rothschildiana, and G. rothschildiana x
G. superba hybrids.
Somani et al., (1989) used explants with root and leaf primodia from sprouted tubers and achieved an 80-to 100-fold annual asexual multiplication rate for G. superba compared to the 2-to 4-fold increase by conventional vegetative propagation. They found that tubers which developed on multiple shoots were "very small" when compared to those which developed on single shoots in vitro. Their study was limited to low concentrations (4.5-20µM) of kinetin in MS medium. They did not test the effect of auxins, combinations of auxin and cytokinin, or other media.
Finnie and van Staden (1989), could only produce multiple shoots on explants derived from mature tubers when using lµM NAA and lOµM kinetin in MS medium.
Although they used both BAP and kinetin as cytokinins, the range of concentrations was relatively narrow (0-5mg/L or 0-20µM). Similarly the auxin (NAA) concentration was from 0-5mg/L or 0-20µM and they too only used MS medium. Although both of these reports showed the potential for shoot multiplication and tuber formation for G. superba, the value of their experiments was limited because of the lack of quantitative data and statistical analysis. This makes it difficult to assess the commercial potential of their protocol. Further both of these groups used only MS medium and the range of growth regulators tested was very narrow. Somani et al., (1989) used only kinetin and the treatment differences were not tested statistically nor was the number of explants used in each experiment given. A successful micropropagation system must also be tested in the field. Finnie and van Staden (1989) only state that tubers formed in vitro could be potted out without necessitating a hardening off period. Somani et al., ( 1989) reported a 100% survival, after two months, of rooted shoots when transferred to a sand-soil-compost
( 1: 1: 1) mixture. It is not clear from their data if these rooted shoots had tubers attached.
Custers and Bergervoet (1994) produced multiple shoots from explants derived from G. superba and hybrids of G. superba x G. rothschildiana sprouted tubers and 19 tuber tips. Their shoot multiplication protocol involves applying alternately a high p (10mg/L) and low (lmg/L) concentration of B.Af_ combined with O.lmg/L NAA or IAA.
They used the same medium supplemented with 0.3mg/L NAA and no cytokinin to achieve rooting of in vitro generated shoots. They achieved 4-7 fold shoot multiplication in an 18 week cycle. The cytokinin was limited to BAP in the range 0-lOmg/L and they only used MS medium. Although they used NAA and IAA, the concentrations tested {he.- were O.lmg/L and 0.3mg/L. Similar to/previous two reports (Somani et al., 1989;
Finnie and van Staden, 1989), Custers and Bergervoet (1994) also describe the presence of attached tubers on shoots during the multiplication stage. However none describe attempts to increase tuber yield nor are quantitative data given for tuberisation.
1 . 3 Secondary Metabolites
Plants synthesize an impressive array of compounds which are believed to deter predators, attract pollinators, or combat infectious pathogens (Reinstein, 1985;
Whitaker et al., 1986). These natural products are often valuable commercial commodities and include flavours, fragrances, pigments, cosmetics, agrochemicals, antimicrobials and pharmaceuticals (Farnsworth and Morris, 1976). Most of the pharmacologically active compounds have complex structures and cannot be synthesised chemically at a competitive price. Synthesis by plants therefore will continue to be the source of these compounds for commercial purposes. Most plants from which important pharmaceuticals are isolated are grown in large scale plantations, although a few are still collected from natural habitats. As with all field-grown products, the general problem of crop failure exists. Increasing human population and farming activities have destroyed much of the natural environment and this has decreased the supply of medicinal plants.
Political and socio-economic conditions, seasonal influences, pests and diseases can also disrupt supplies of medicinal plants (Tabata, 1977). An additional problem is that the geographical area where the plants have to be grown for climatic reasons are distant from the major markets. 20
For almost two decades plant biotechnologists have been investigating the use of plant cell cultures as a source of these valuable compounds in order to overcome the problems and restrictions that are inherent in plant production in the field. The production of metabolites by plant cell culture could provide a useful alternative, as many of the above problems would be alleviated. High quality control and a flexible response to the demands of the market are other arguments for factory-type production (Berlin,
1988). Since plant cells are totipotent, it has long been argued that the genetic information for natural product synthesis will be present in an isolated cell. Therefore, it was hoped that cultured cells would be able to produce natural products similar to those of the plant grown in vivo. A wide range of natural products have been successfully isolated from cell cultures, but in only a small number of examples, has accumulation of the desired product been comparable to that of the parent plant (Fowler, 1983).
A variety of reasons have been suggested to explain the loss of ability to accumulate natural products in dedifferentiated plant tissue cultures. The most widely accepted is the lack of expression in non-specialised cells of genes that control the essential steps in the biosynthetic pathway. The differentiation of unorganised tissue often leads to the partial or complete restoration of the capability to accumulate natural products. Furthermore, organ-specific differentiation enables biomass production, and hence carbon flux, to be channelled into those tissues of the plant that actually express both their biosynthetic and accumulative potential of the target compounds. For example, results from experiments with undifferentiated callus and cell suspension cultures of Artemisia annua have been disappointing with respect to artemisinin production, with only traces of this compound present (Nair et al.,1986). However, shoot cultures contained a higher artemisinin concentration than callus or cell suspension cultures (Woerdenbeg et al., 1993). Martinez and Staba (1988) concluded from their results that a certain degree of differentiation of the cultures is a prerequisite for production of artemisinin in vitro. Further examples giving a higher yield in organised tissues compared with unorganised tissues are root cultures of Dubosia leichhardtii for 21 scopolamine production (Endo and Yamada, 1985) and shoot tip cultures and root cultures of Coleus forskohlii for forskohli production (Sen et al., 1992). Further,
Digitalis lanata cells produce barely detectable amounts of digoxin (0.06mg% dry weight), but leaf cultures and dark grown root cultures produced much larger amounts
(9mg% dry weight and 1.9mg% dry weight respectively)(Lui and Staba 1979). Violon et al., (1984) found that undifferentiated callus of Centranthus ruber had a low production of monoterpenes, but once organogenesis had been induced, greater production of monoterpenes (3mg% dry weight) occured.
Unorganised tissues have the advantage of adapting to fermentor culture better than organised tissues (Staba, 1985), but the latter are genetically more stable. Recently
Asaka et al., (1994) suggested that saponin production by regenerated plantlets of Panax ginseng is due to tissue differentiation, rather than culture conditions.
Multiple shoot cultures of medicinal plants in vitro could be a good source of known and new pharmacologically active plant constituents (Heble, 1985). Roja et al.,
(1987) found that in vitro multiple shoot cultures of Rauvolfia serpentina produced comparable levels of alkaloids (0.88% dry weight) to that in roots and leaves of intact plants (1.28% dry weight and 0.63% dry weight respectively).
Staba ( 1985) has pointed out the importance of the stability of the genotype, the desirability of uniform growth of cells and the need for a proper economic feasibility study in order to achieve the production of natural products economically. Yeoman et al., (1982) claimed that the evidence supported the concept of maximal natural product synthesis in whole plants and cultures, when the growth rate decreased and differentiation of cells into roots, shoots, etc. increased. Shoots can now be grown in large capacity stirred-tank or air-lift bioreactors and those conditions are claimed to give up to four-times the dry matter of comparable cell cultures (Heble, 1985).
In recent years much research has been devoted to developing methods for
optimising the production of natural products by plant tissue culture. The choice of plant
material and culture conditions offer a wide spectrum of possibilities. 22
1 . Screening and Selection Based on Explant Material
Controversy over the significance of the explant continues, but it appears that the highest producing tissue from the highest producing plant is best (Staba, 1985).
Using Catharanthus roseus as a model, Heijden et al., (1989) summarised the choices available when considering explant material to be screened for a desired secondary product (Table 1.1).
Table 1.1 Factors to be considered when selecting explant material for screening of desired secondary metabolites.
Different species where possible Different cultivars Different individuals belonging to the same species or cultiver Different cell lines: -initiated from identical plant parts -initiated from different plant parts --derived from the same cell lines (somaclonal variations) Different levels of differentiation -plant -seedlings -shoot or root cultures --callus -suspension cultures Physiological state -heterotrophy or autotrophy -prototrophic or growth regulator dependent Altered genetic status _Ti-plasmid transformation -introduction of RNA or DNA by other techniques
2. Screening and Selection Based on Culture Conditions
There is now considerable evidence that demonstrates the importance of medium
optimisation and environmental regulation for the highest production of metabolites.
For example, anthraquinones (Zenk et al., 1975), shikonins (Fujita et al., 1981) and
artemisinin (Woerdenbag et al., 1993). Some of the cultural conditions that have been
manipulated are listed in Table 1.2.
In general, the best growth medium will seldom be the best production medium.
Berlin (1988) suggested that a good production medium must give both growth and
productivity. Therefore its composition must be carefully balanced between growth
and the best specific production rate for optimal results. 23
Table 1.2 Cultural conditions which have been employed to increase the production of secondary metabolites. A. Manipulation of the internal culture environment.
a) Composition of the medium ---<=arbohydrate source -mineral salts -vitamins -gelling agents b) Plant growth regulators
c) Addition of precursors
B . Manipulation of the external culture environment.
a) Light (periodicity, intensity and quality)
b) Temperature c) Agitation of medium C. Time over which culture was exposed to particular growth conditions.
The major constituent of the medium is the carbohydrate (sucrose) which serves as a carbon and energy source and makes an important contribution to the osmotic potential. Altered sucrose concentrations effect anthraquinone synthesis by Marinda citrifolia cells (Zenk et al., 1975), digitoxin formation from Digitalis purpurea shoot cultures (Hagimori et al., 1982b) and indole alkaloids formation from Catharanthus roseus suspension cultures (Knobloch et al., 1982).
Schulte et al., (1984) found that cultures of Rubia fruticosa produced maximal
levels of anthraquinones in a medium that initially contained 12% sucrose and that the
response was species specific. Carew and Krueger (1977) found that raising the sucrose
concentration resulted in decreased production of indole alkaloids from C. roseus suspention cultures. Okazaki et al. , (1982) found that decreasing sucrose from 3% to
1% (w/v), increased synthesis of scopoletin and scopolin in Nicotiana tabacum cultures, IY\Ok:S 11'\ole~ from 0.07µ11/g fresh weight to 0.1 lµlf/g fresh weight. Merillon et al., (1984) showed
that increasing the sucrose concentration from 2% to 6%, increased ajmalicine and serpentine 24
accumulation in C. roseus tissue culture nine and 4-fold respectively. Endo and Yamada
(1985) found that the nicotine content in root cultures of Duboisia leichhardtii in B-5 medium and D. myoporoides supplemented with 5% sucrose was three times higher than that in 3% (0.9% dry weight).
At present it is generally accepted that sucrose concentrations will improve alkaloid yields although this may be due in part to an increased growth of cells. The mechanisms by which carbohydrate influences secondary metabolite production are unknown, but are presumably complex, because the pathways of carbohydrate metabolism are influenced by other nutritional factors (Jessup and Fowler, 1976).
When optimising the yield of product in tissue culture it is important to manipulate the form and concentration of certain mineral nutrient elements. Endo and
Yamada (1985) also found that the alkaloid content varied with the basal medium. For example, Duboisia leichhardtii root cultures produced 1.8 times more nicotine in
Linsmaier-Skoog medium (LS) than in B-5 medium. The major difference between
these media is the mineral composition which presumably can influence the production of
secondary metabolites. A wide range of nitrogen sources, both organic and inorganic,
have been used in nutrient media, although in most it is usually provided as a mixture of NO3- and NH4+ions. Both the ratio of these two nitrogen sources and the total level of
nitrogen can affect the production of secondary metabolites (Kirby et al., 1987; Hagimori
et al., 1982b). b Hagimori et al., (1982) increased the digitoxin content in shoot forming cultures
of Digitalis purpurea by reducing the total nitrogen concentration in MS medium by one
third. They reasoned that because MS medium was developed for the rapid growth of
tobacco cells, the high concentration of nitrogen may be excessive for the production of
some secondary metabolites.
The ratio between the amount of nitrate-N and ammonium-N is characteristic for each culture medium. For MS and WP medium the nitrate/ammonium ratio is 2; for B-5
medium it is 12.5 and for SH it is 10. When used as the sole source of nitrogen, 25
ammonium-N could not support growth of C. roseus (Zenk et al., 1977), Digitalis purpurea (Hagimori et al., 1982b) and Nicotiana tabacum (Matsumoto et al., 1971)
although addition of both ions resulted in a rapid increase in biomass. Matsumoto et al.,
(1971) suggested that in medium containing Nl4Cl as the sole nitrogen source, the fall
in pH due to uptake of NI4+ ions causes an inhibition of growth.
Fowler (1986) also suggested that pH changes when ammonium nitrogen is used
as a sole nitrogen source may have profound effects on the pattern of secondary
metabolite synthesis. It is difficult, therefore, to be sure whether or not the effect noted with NH4+ is due to the nature and concentration of the nitrogen source or to a change in
pH.
There is also considerable evidence to show that secondary metabolite synthesis
is influenced by the type and concentration of PGRs. For example, in cultures of
Marinda citrifolia, a thirty-fold stimulation of anthraquinone synthesis was observed
when 2,4-D was replaced by NAA (Zenk et al., 1977). Similarly, IBA was more
effective than 2,4-D in stimulating the production of colchicine in Colchicum autumnale
suspention cultures (Hayashi et al., 1988).
In as much as the degree of shoot-differentiation in shoot-forming cultures of
Digitalis purpurea appears to determine the digitoxin content of the cells (Hagimore et al.,
1982a), plant growth regulators would be expected to affect the digitoxin content greatly
but the effect could be indirect. Similarly BAP induced the highest yield of alkaloids in C. roseus suspension cultures (Zenk et al., 1977). GA3 added to shoot cultures of
Artemisia annua enhanced the production of artemisnin, although growth remained
unaffected (Woerdenbeg et al., 1993). However at higher concentrations of GA3, growth and alkaloid production were decreased.
Addition of appropriate precursors or related compounds to the culture medium is also an important consideration, especially if the precursor is inexpensive compared to
the desired product. This approch has been used successfully in a number of plant cell
cultures. Zenk et al., (1977) reported a three-fold stimulation of alkaloid biosynthesis
when L-tryptophan was fed to root cultures of one strain of C. roseus. They further 26 showed that the response was cultivar dependent. Alkaloid synthesis in another strain
was strongly inhibited at all L-tryptophan concentrations tested. Benjamin et al., (1987)
showed that the precursors of tropane alkaloids marginally increased the alkaloid
synthesis in shoot cultures of Atropa belladona. Hagimori et al., (1983) improved the
digitoxin content per flask to 180% of that control shoot-forming cultures of Digitalis purpurea with addition of progesterone at 0.lmg/lOOmL medium.
A major limitation to precursor feeding is lack of knowledge of the biosynthetic
pathways for many secondary metabolites (Fowler, 1983). When single step
biotransformations are considered, conversions can be very efficient. Furthermore
precursor feeding experiments may be complicated by the precursors not being taken up,
not transported to the synthetic machinery in the cell or it may be toxic even at low
concentrations.
1. 3. 1 Colchicine
Colchicine, N-(5,6,7 ,9-tetrahydro-1,2,3, 10-tetramethoxy-9-oxobezo [a] heptalen-7-yl)
(s)-acetamide is an odourless and pale yellow powder which darkens on exposure to
light. The structure (Figure 1.1) was established by chemical degradation (Wildman,
1960) and confirmed by total synthesis (Wildman and Pursey, 1968; Capraro and Brossi, 1984).
It is a neutral alkaloid with useful biological properties (Herbert et al., 1990). It
has been used for centuries for the symptomatic relief of gouty arthritis (Thompson,
1985) and even in current medicine, it is the drug of choice for the treatment of the pain
of gout (Hayashi et al., 1988; Muzaffar and Brossi, 1991). It is believed to function by
indirectly inhibiting the deposition of urate crystals in inflamed joints (Thompson, 1985).
Zemer et al., (1974) demonstrated the efficacy of colchicine in the treatment of Familial
Mediteranean Fever and recently this drug has been introduced into clinical practice
(Muzaffar and Brossi, 1991). Kershenobich et al., (1979) mention that colchicine is
beneficial in the treatment of liver cirrhosis. According to Tiegs et al., ( 1992) colchicine
prevents lipopolysaccharide induced hepatitis by counteracting tumor necrosis factor 27
(TNF) toxicity. Demecolcine, a related alkaloid has been reported to have antineoplastic activity (Hayashi et al., 1988).
There is renewed interest in the use of colchicine derivatives for cancerous conditions (Evans et al., 1981). Its direct use as an antitumor agent is restricted because of its high toxicity to mammals, but the less toxic desacetylcolchicine, is being investigated for the treatment of advanced malignancies (NCI Investigational Drugs,
1988). Thiocolchicoside (3-0-glucosylthiocolchicine) is used as a myorelaxant and analgesic (Solet et al., 1993).
5
;, .. NHCOCH3 2 H
12
0
Figure 1.1 Colchicine
Chemical synthesis of colchicine results in the two enantiomeric isomers, only one of which is biologically active. The American Food and Drug Administration
recently ruled that only the active enantiomer can be marketed. Biologically produced
colchicine contains only the active form. Colchicine is use<4 not only as a therapeutic
agent, but in biological and agricultural practice to induce polyploidy (Jr et al., 1991).
Colchicine is being used in the area of plant breeding to induce male sterility to make the
first filial generation by hybridization (Alemanno and Guiderdoni, 1994).
Colchicine binds to the protein tubulin, and disrupts the formation of
microtubules. This stops plant and animal cell division in vivo resulting in the arrest of
mitosis in the metaphase. In plant cells this mitotic action leads to failure of spindle 28 formation, and inhibition of cytokinesis after the division of the chromosomes. This results in polyploidy or doubling of the number of chromosomes. If the drug is removed, the nucleus divides normally producing plants in which the 4n number of chromosomes is maintained indefinitely. It has been used to produce polyploidy of many economically important plant species, eg. Rauvolfia serpentina (Mathur et al.,
1987); Citrus sinensis (Jr et al., 1991); Oryza sativa (Alemanno and Guiderdoni, 1994).
Colchicine is an extremly toxic compound. A single dose of 3mg has caused the death of an adult human, but the minimum fatal dose is probably nearer 8 mg (Watt and
Breyer-Brandwijk, 1962). Colchicine poisoning of cattle by corms of Colchicum autumnale has been reported (Yoneda et al., 1984). In addition, poisoning while under medical supervision for the treatment of malignancy or gout has also been reported (Carr,
1965).
1 . 3. 1. 1 Occurrence In Plants
Colchicine has been detected in many species in the family Liliaceae, especially the genera Colchicum (Wildman,1960; Tojo et al., (1990), Merendera (Potesilova et al.,
1968; Sarg et al., 1989), Gloriosa (Kaul et al.,1911; Finnie and van Staden, 1991); lphigenia (Wildman and Pursey, 1968; Sharma et al., 1986) and Sandersonia (Wildman
and Pursey, 1968; Finnie and van Staden, 1991). Within the genus Gloriosa the alkaloid
has been rigorously identified only in G. superba. For example from flowers (Kaul et
al., 1977), and tubers, leaves and seeds (Finnie and van Staden, 1991). Ntahomuukiye
et al., (1984) detected colchicine from seeds, leaves and tubers of G. simplex. At
present the major commercial source of colchicine is Colchicum autumnale. In 1980,
180 tonns of seeds provided by 130 million Colchicum plants were utilized by the
European pharmaceutical industry for colchicine production (Bellet and Gaignault, 1985).
There is much evidence for the effect of various factors on the colchicine content
of plants. Thakur et al., (1975) reported that dry tubers of G. superba cultivated in
European hot houses, contained up to 0.2% of colchicine, while similar material 29 collected from the tropical region of Africa and India contained only 0.02%. A variation of colchicine content within the plant was also been reported. For examples, Husek et al., (1990) found a variable amount of colchicine in the corms, leaves, seeds and flowers of Colchicum turcicum, and Finnie and van Staden (1991) found variations in the colchicine content of different parts of G. superba plants.
The colchicine content of G. superba and Sandersonia aurantiaca varied with the time of harvest (Finnie and van Staden 1991). Hamersley (1950) recommended the use of one-year old corms of C. autumnale for maximum colchicine content. According to him the physiological age was critical and he found that the corms of plants harvested just prior to flowering contained the largest amounts of colchicine. Wildman (1960) found that as the mother C. autumnale corm senesced, the colchicine content decreased while it increased in the daughter corms. Finnie and van Staden (1991) found that
Gloriosa tubers of all ages had approximately the same colchicine content, although it was highest at the beginning of the growing season.
Thakur et al., (1975) have criticized the majority of earliar papers published on the detection and isolation of alkaloids from the various species of Gloriosa. Most lack information about the time and location of collection. In some, the quantitative differences in alkaloid content can be accounted for by inadequate plant identification and variations in the isolation procedures (Thakur et al., 1975).
1. 3 .1. 2 Isolation, Purification and Quantitation of Colchicine From Plants
Numerous analytical techniques have been published for the isolation and assay of colchicine from biological materials. Most of these are time consuming multi step procedures unsuitable to the extraction of a large number of small samples. Sharma et al., (1986) defatted Iphigenia stellata seeds with petroleum ether (60-80C 0 ), extracted with 80% ethanol and then partitioned the neutral phenols into CHCl3 and colchicine isolated from a CHCl3-petroleum ether mixture. Dvorackova et al., (1984) extracted finely ground G. superba tubers and seeds with methanol. The methanolic extract was concentrated in vacuo, diluted with water and acidified to pH 4 with citric acid, extracted 30
the with ether, then with CHCl3 to obtai°"neutral phenolic fraction. The neutral phenolic I fraction was analysed by column chromatography on At2O3 for colchicine. Husek et al., (1990) Soxhlet extracted powdered plant material of Colchicum turcicum with MeOH. The residue after evaporation was taken up in 0.1 % H2SO4 and successively partitioned with diethyl ether and CHCl3. The CHCl3 frac.tion was analysed by column chromatography on AliO3 for colchicine. Kaul et al., (1977)
Soxhlet extracted air dried flowers of G. superba with methanol for 16 hours and the concentrate was taken up in H2o and extracted with ether followed by CHCl3. The aqueous solution was basified (NaOH) and extracted with CHCl3 again. This fraction contained the colchicine and related compounds. Methods to quantitate colchicine include gravimetric (British Pharmacopoeia,
1973); colorimetric (Clarke, 1969; British Pharmacopoeia, 1980); spectrophotometric
(Karawya and Diab, 1975); fluorimetric (Arai and Okuyania, 1975; Lukic et al., 1989), volumetric (Karawya and Diab, 1975) chromotographic (Hunter and Klaassen,1975; Husek et al., 1990) and immunoassay (Ertel et al., 1976; Scherrmann et al., 1980;
Poulev et al., 1994).
Most of these methods are designed to measure the total alkaloid content and hence are not specific for colchicine. Furthermore they involve multiple extraction and purification procedures and poorly defined quantitation. HPLC analysis has been used successfully for the determination of colchicine and its separation from related compounds (Jarvie et al., 1979; Lhermitte et al., 1985; Thompson, 1985; Lo and
Krause, 1987; Sarg et al., 1989). The majority of published HPLC methods are directed at pharmaceutical preparations and are generally unsuitable for quantitative determination of colchicine from crude plant extracts. The existing methods for HPLC detection of
colchicine in plant extracts lack simplicity, precision, sensitivity and specificity and therefore improved procedures are needed. For example, Sutlupinar et al., (1988) analysed extracts of Colchicum cilicicum using a reverse phase Cts column with methanol:water:triethylamine (48:51:0.08v/v) as the mobile phase. They identified tropolone alkaloids by their retention time and quantitated them with the aid of external 31 calibration curves and N-fonnyl-N-deacetylcolchicine and 4-dimethylaminobenzaldehyde as internal standards. Their isolation procedure involved time consuming percolation through alumina and TLC on silica gel glass plates.
Rusek et al., (1990) and Finnie and van Staden (1991) used gradient HPLC elution which requires re-equilibration after each sample. Such methods· are less satisfactory for the analysis of a large number of samples. Hayashi et al., (1988) used a simplified method for the quantitation of colchicine from C. autumnale callus. Their method involved extraction of fresh callus tissue with EtOH at 80°C for 30min. After clarification by centrifugation the supernatant was analysed for colchicine by HPLC with
UV detection. Sarg et al., (1989) separated colchicine from related compounds isocratically on a reverse-phase C18 column using MeOH:H2O (60:40). They measured the colchicine by comparision with an external calibration curve within the range of 0.2-
l .5µg colchicine per injection. Every procedure in the isolation of a cellular metabolite causes losses so that measurements are minima. The use of a suitable internal standard allows correction for losses incurred during purification.
The existing classical extraction and purification methods for colchicine are time consuming, less reproducible for small sample sizes and require large volumes of organic solvents. Heigden et al., (1989) recommended solid phase extraction (SPE) in combination with an isocratic HPLC system for routine analysis of alkaloids, when a large number of samples have to be examined. Solid phase extraction methods have
been successful in the detennination of many other alkaloids such as the indole alkaloids. Lhennitte et al., (1985) used a C18 Sep-Pak cartridge in their extraction procedure when
detennining colchicine in human urine samples. No reports have been published using
SPE for the isolation of colchicine from plant materials.
1. 3. 1. 3 Biosynthesis of Colchicine +,·v The biosynthe-pathway of colchicine as cited in Yoshida et al., (1988) is shown
in Figure 1.2. There is good evidence to show that ring A and carbon atoms 5, 6 and 7 32 are derived from the phenylalanine - cinnamic acid pathway (Leete and Nemeth, 1960;
Herbert, 1980), with methionine providing the methoxyl groups (Leete, 1963; Battersby et al., 1964). Ring C is built by ring expansion of tyrosine (Battersby et al., 1964).
According to Battersby and Herbert (1962), androcymbine is the first intermediate in the pathway with the tropolone ring. Demecolcine was found to be metabolised· through demethylation and acetylation to colchicine (Barker et al., 1967). Robinson (1968) showed that trans-cinnamic acid and p-coumaric acid were derived from phenylalanine, and that tyramine was formed from tyrosine. Colchicine content vary with species, physiological state of the plant, different organs and even different parts of the same organs. For the industrial utilization of Gloriosa species for the production of colchicine it is desirable to investigate all of those factors.
In order to overcome the problems such as difficulties in regular supply of authentic plant materials, climatic changes which influence the yield of plant secondary metabolites, the application of plant tissue culture technology for the production of secondary metabolites has attracted considerable research findings in recent years.
However the low yield of product is a major drawback to the commercial production of useful secondary metabolites from plant tissue and cell cultures. It is accepted that genetic variation arises spontaneously in cell or tissue culture. It has been suggested therefore that cell or tissue culture steps should be used directly to increase the genetic variation in plant species. It would appear that organized cultures provide the best available experimental system in which to study the many biochemical and physiological aspects of production of secondary metabolites. Moreover, once the ability to increase the rate of biomass production with organ cultures has been achieved, it may be appropriate to utilize these organized cultures for the production of selected high value secondary metabolites.
In many cases multiple shoot cultures have the ability to produce desired products in amounts similar to or higher than those of the field grown plants and are genetically more stable than undifferentiated callus. The productivity of cultures is greatly influenced by the culture conditions. Among the strategies which have been successfully 33 employed to increase secondary metabolites in organ cultures are selection of high yielding species, manipulation of the mineral composition of the medium , alteration of the sucrose concentration and the feeding of putative precursors of the desired compounds. There are no reports in the literature on the colchicine content of other
Gloriosa species or the establishment of in vitro cultures for the production of colchicine.
NH1 ~,~H_JH I - COOH Phenylalanine rnrn• • Cinnamic acid
H~-H=cH ~ "-COOH p·Coumanc acid NH 2 NH, I
H~H --l H~H2~ ! ~21 - ~ ' I ' I COOH ' I ', I Ty!Oline Tyrunine , I \J I I I t
...... NMe
, , , ., MeO M "' Androcymbine
/ MeO Demecolcine
MeO Colchic:ine
Figure 1.2 Proposed biosynthetic pathway for colchicine in Colchicine autumnalae (From Yoshida et al., 1988). 34
1. 4 Project Aims
In order to improve the micropropagation of Gloriosa species for the cut flower industry as well as for the establishment of shoot cultures as a source of colchicine for the pharmaceutical industry, the objectives of the project were; 1. To determine suitable conditions for the micropropagation of
G.rothschildiana, G. superba, G. simplex, G. verschuurii and the cultivar,
G. richmondensis. 2. To investigate quantitatively the contribution of species, explant type, growth regulator type and concentration, composition of the media and the interactions between some of these variables 3. To monitor adaptation to growth in soil through the growth until flowering occurs.
4. To quantitate colchicine in both in vitro and in vivo grown plant material.
In addition to determine the colchicine content of Sandersonia aurantiaca
when grown under identical glasshouse conditions to the Gloriosa. 35
CHAPTER 2 MATERIALS AND METHODS
MATERIALS 2 . 1 Plant Material
Seeds or tubers of Gloriosa species and Sandersonia aurantiaca were either donated or obtained from commercial suppliers (Table 2.1 ). Voucher specimens were deposited in the University of NSW Herbarium.
Table 2.1 The sources of plant materials
Species Material Supplier Voucher No. G. rothschildiana Seeds and Tubers Gloriosa Nursery, VIC.
G. superba Seeds CISIR, Colombo, 21181 Sri Lanka G. simplex Seeds and Tubers Randwick, NSW 21182 ex hortico plants G. verschuurii Seeds Chiltern Seeds Ulverston, UK G. richmondensis Seeds Reichenbach Seeds 21694 Gibson, New Zealand S. aurantiaca Tubers Bryan Tonkin, Kalorama VIC.
2.2 Chemicals
Abscisic acid, 6-benzylaminopurine, colchicine, p-coumaric acid,
2,4-dichlorophenoxyacetic acid, gibberellic acid, indole-3-acetic acid, kinetin, a. naphthalene acetic acid, nicotinic acid, PPO, POPOP, pyridoxine.HCI, thiamine.HCI and Tween 80 were purchased from Sigma Chemical Co., USA. Myo-inositol was purchased from Calbiochem, USA Glycine, cinnamic acid, 36
L-phenylalanine)tyrosine and tyramine.HCl were from Merck, Germany. Fe-EDTA was
purchased from Ajax Chemicals, Sydney. Agar (No 3 grade) was purchased from Davis
Gelatine (Australia), Sydney, Australia.
[Ring-A-4-3H]colchicine (8.4Ci/mmol) was obtained from the Radiochemical Centre,
Amersham, UK. Marvo-Linn household bleach (5% w/v available chlorine) and Hortico
Bordeaux fungicide powder (125g/kg copper present as copper hydroxide) were
purchased from a local supermarket. All other chemicals used were AR grade unless
otherwise stated. Methanol was redistilled before use. Milli-Q water was used
throughout. 37
METHODS 2. 3 Sterile Techniques All sterile manipulations were carried out in a laminar air flow cabinet. Glassware and media were sterilized by autoclaving at 121 ·c with steam pressure at 103 kPa for 20 minutes. If necessary, filter sterilization of solutions used 0.2 micron
Acrodisc 13 sterile single use filters (Gelman Sciences Inc, Sydney).
2.4 Aseptic Seed Germination
After removal of dried flesh, seeds were disinfested by immersion in aqueous
70% (v/v) EtOH for 3 minutes, followed by bleach solution (2% w/v available chlorine;
0.01 % (v/v) Tween 80) for 18 minutes. They were then rinsed with three sequential washes in sterile H2O. Seeds were placed on lOmL quarter strength UNE#B medium
(de Fossard, 1981), solidified with 1% (w/v) agar contained in 28mL glass McCartney bottles and incubated under continuous cool white fluorescent light (4.5 watts/m2) at 25°C.
2.5 Aseptic Tuber Sprouting
Tubers were gently scrubbed with a soft nylon brush under running tap water (15 min), pre-soaked in 0.2% HgCli (w/v) for 4 minutes, and then rinsed thoroughly with distilled water. The terminal end (2 cm) with the dormant growing tip was excised and the cut surface sealed by dipping in molten paraffin wax. The tuber explants were disinfested by immersion in aqueous 70% (v/v) EtOH for 3 minutes, followed by bleach solution (2% w/v available chlorine; 0.01 % (v/v) Tween 80) for 10 minutes. They were then rinsed with three sequential washes in sterile H2o. The paraffin wax was cut away and the explant placed on 50mL UNE#B medium (de Fossard, 1981) solidified with 1%
(w/v) agar contained in 250mL polycarbonate jars (Disposable Products Pty Ltd, South
Australia). Cultures were incubated at 25°C with continuous cool white fluorescent 38 light (4.5 watts/m2) and allowed to sprout and reach the 4-6 leaf stage. Some were maintained under the same conditions until new daughter tubers formed (8 weeks).
2. 6 Callus Initiation Sterile seedlings (Section 2.4) raised on UNE#B medium to the 4-6 leaf stage, and tubers harvested from mature G. rothschildiana, G. superba, and G. simplex plants at the flowering stage were used for callus establishment.
Seedlings were separated into leaf and stem or leaf sheath removed from seedlings
(Photograph 2.1 ). The stem was trimmed into 5mm lengths and the leaf and leaf sheath
cut into 5 x 5 mm sections. The sterile tuber tips were cut into 1 x 1 cm pieces. Four
different solidified (1 % w/v agar) basal media, viz. Murashige and Skoog (MS) (1962),
Gamborg's B5 (1968), Schenk and Hildebrandt (SH) (1972) and Woody Plant (WP)
(Mccowan and Lloyd, 1981) were used. With each basal medium all possible ('II combinations of a 3 x 3 matrix of NAA and kinetin (1, 10, and 100 µ•) and a 5 x 5
matrix of 2,4-D and kinetin (0, 0.25, 2.5, 5, 10 mg/L) (0, 1, 11, 23, 45µM
respectively) were used. Basal media without growth regulators served as controls.
Five pieces of each tissue type were placed on medium in the one petri dish, with 4 or 5
replicates of each growth regulator treatment. Petri dishes were sealed with a double + layer of strtthed Parafilm.
Incubation was under low intensity continuous cool white fluorescent light at
25°C. After 4 weeks the effect of each treatment on callus initiation and growth was
evaluated by visual observation of the response.
2 . 7 Proliferation and Maintenance of Callus
Based on callus initiation rates, appearance and texture, a small number of
treatments were selected for further evaluation with respect to maintenance and
proliferation of the callus. The best callus (either undifferentiated or partly differentiated)
was transferred to the respective fresh medium (40mL) contained in lOOmL glass jars 39
and incubated at 25°C with continuous cool white fluorescent light (4.5 watts/m2) and subcultured at 30 day intervals.
2.8 Root Cultures
Root cultures were established by innoculating rhizogenic callus pieces from callus initiation grids into appropriate liquid media (50mL) contained in 250mL conical flasks sealed with a double layer of aluminium foil. Cultures were grown on a gyratory shaker (140rpm) at 25°C under low intensity continuous cool white fluorescent light and subcultured every 4 weeks.
2.9 Growth Rate of Callus
The growth rate of undifferentiated or rhyzogenic callus was calculated as a fold increase per subculture which was defined as;
Growth rate = weight of callus at subculture (fold increase) initial weight of callus
2. 10 Initiation of Shoots from Tuber Explants
Either daughter tubers produced from aseptically grown Gloriosa plants (Section
2.5) or daughter tubers collected from field grown plants were used as explant sources in these experiments. Daughter tubers grown in vitro were cut longitudinally into four sections (1 x 1cm) (Photograph 2.1). The dormant terminal end (2cm) of disinfested field grown tubers (Photograph 2.1) were split twice longitudinally to give four identical sections. The quadrants from one tuber were inoculated into a lOOmL glass jar containing 40mL of medium, with five replicates for each treatment used.
G. rothschildiana, G. superba and G. simplex were cultured on solidified (1 % w/v agar) MS and WP media. With each medium a 4 x 4 matrix of NAA (0, 1, 10,
1OOµM) and kine tin (0, 1, 10, 1OOµM) and a 4 x 5 matrix of NAA (0, 1, 10, 20µM) and
BAP (0, 10, 20, 40, 60µM) was used. 40
G. verschuurii and the cultivar G. richmondensis were cultured on solidified (1 % w/v agar ) WP medium. lOµM NAA and 40µM BAP and lµM NAA and l00µM kinetin combinations were used for each. The cultures were incubated under continuous cool white fluorescent light (4.5 wattfm2) at 25°C.
2.11 Initiation of Shoots from Shoot Bases
Juvenile shoots (4cm length) were separated 1cm above the terminal base from aseptically sprouted mature tubers (Section 2.5) or from shoot cultures derived in vitro from mature tuber explants (Section 2.10 ). Each shoot was split longitudinally once and sectioned transversally into 1cm pieces (Photograph 2.1 ). The four sections closest to the shoot base were used as explant material.
A 1>
Photograph 2.1 Different types of explant (except root-coleoptile axies tissues) used for experiments (a) Four longitudinal sections from a daughter tuber (b) Longitudinally split shoot bases from sprouted tubers (c) Leaf sheath tissues from seedlings (d) Terminal sections from mature tubers (split once) 41
G. rothschildiana was cultured on 40mL solidified (1 % w/v agar) MS and WP media in lO0mL glass jars. With each medium a 4 x 5 matrix of NAA ( 0, 1, 10,
20µM), and BAP ( 0, 1, 10, 20, 60 µM) was used. Five explants were used in each treatment and each treannent was replicated 5 times. Cultures were incubated under cool white fluorescent light (4.5 watts/m2) at 25°C.
G. superba and G. simplex were cultured on 40mL solidified (1 % w/v agar) MS and WP media in lO0mL glass jars. With each medium a 4 x 4 matrix of NAA ( 0, 1,
10, l00µM) and kinetin ( 0, 1, 10, lO0µM) and a 4 x 5 matrix of NAA ( 0, 1, 10,
20µM) and BAP ( 0, 10, 20, 40, 60µM) were used. Five or six explants were used in each treannent and each treatment was replicated 5 or 8 times.
2. 12 Initiation of Shoots from Root-Coleoptile Axes of Seedlings
The axis tissue (5mm) either side of the root-coleoptile junction (Photograph 2.2) from 3 week old aseptic seedlings (Section 2.4) was separated and placed onto MS and
WP medium (40mL) in l00mL glass jars.
With each medium a 4 x 4 matrix of NAA ( 0, 1, 10, lO0µM) and kinetin ( 0, 1,
10, l00µM), and a 4 x 5 matrix of NAA ( 0, 1, 10, 20 µM) and BAP (0, 10, 20, 40,
60µM) was used. Five explants were used in each treatment and each treatment was replicated 8 times.
Photograph 2.2 The arrow points to the root-coleoptile axis of a three weeks old G. superba seedling. 42
2. 13 Initiation of Shoots from Callus O<\ Callus (0.2g), derived from tuber explants, and one month from inoculation~WP medium with 2,4-D [ 22.6µM ( 5mg/l)] and kinetin [ 1.16 µM (0.25mg/L) ], was transferred to WP medium (40mL) containing lµM NAA and lO0µM kinetin in lO0mL glass jars and incubated under continuous high intensity flourescent light at 25°C. The experiment was set up with 8 replicates.
2.14 Maintenance of Stock Cultures
Four-shoot clumps were taken from the media achieving the best growth performance for each type of initial explant. These were transferred onto fresh medium
(40rnL) contained in lO0mL glass jars and cultured under continuous high intensity fluorescent light at 25°C. These cultures were maintained as stock cultures by subculture at eight-week intervals. For experiments, stock cultures were transferred to the new media, 4 weeks after they were last subcultured.
2.15 Multiplication of Shoots
G. rothschildiana shoots (Section 2.14) were cultured on 40rnL solidified ce5 ( 1% w/v agar) MS or WP media on one or more of the following rnatriJ or part thereof.
A 4 x 5 matrix of NAA ( 0, 1, 10, 20µM) and BAP ( 0, 10, 20, 40, 60µM), a 4 x 5 matrix of IAA ( 0, 1, 10 , 20µM) and BAP ( 0, 10, 20, 40, 60µM) or a 4 x 4 matrix of
NAA (0,1,10,lO0µM) and kinetin (0, 1, 10, lOOµM). G. superba and G. simplex shoots (Section 2.14) were cultured on 40rnL solidified (1 %w/v agar) MS medium and/or WP medium. The following auxin and cytokinin combinations were used with each medium; NAA (lµM) and BAP (40µM); IAA (l0µM) and BAP (40µM); NAA (lµM) and Kinetin (l00µM).
G. verschuurii and the cultivar G. richmondensis shoots (Section 2.14) were cultured on 40rnL solidified ( 1% w/v agar) WP medium. The following auxin and 43 cytokinin combinations were used; NAA (lµM) and BAP (40µM); IAA (l0µM) and
BAP (40µM); NAA (lµM) and kinetin (lOOµM).
In all of the above experiments, a four-shoot clump was used for a replicate and each treatment was replicated five times. The cultures were maintained under continuous high intensity fluorescent light at 25°C for 8 weeks.
2. 16 Replacement of NAA by IAA in Shoot Multiplication Medium
In selected multiplication media containing NAA, the NAA was replaced by the same concentration of IAA. Stock shoots maintained on WP medium supplemented with
NAA:kinetin (lµM: lOOµM) were used. A four-shoot clump was transferred to each fresh medium (40mL) contained in lOOmL glass jars. Each treatment was replicated five times and maintained for eight weeks. A similar set of cultures which retained NAA as the auxin, served as a control. Cultures were maintained under continuous high intensity fluorescent light at 25°C for eight weeks.
2.17 Alteration of Ammonium and Nitrate Concentrations in Shoot Multiplication Medium Nitrogen free stock solutions of WP medium were prepared and modification of
the NH4+ and NO3- concentrations were done using Nl4Cl and NaNO3. When both
NH4+ and NO3- were present, Nl4NO3 was also used. The potassium concentration
was maintained by replacing KNO3 with KCL Stock shoot cultures originating from
tuber explants were maintained on WP medium supplemented with NAA (l0µM) and
BAP (40µm). Four week old, four-shoot clumps were harvested and cultured on the
nitrogen modified WP medium (40mL) contained in lOOmL glass jars. Twelve
treatments of nitrogen comprising three levels of total nitrogen (15mM, 30mM, and
60mM) were incorporated in nitrogen free WP medium supplemented with lOµM NAA
and 40µM BAP, in the following NH4+/NO3- ratios; (0/15, 5/10, 10/5, 15/0); (0/30,
10/20, 20/10,30/0); and (0/60; 20/40; 40/20; 60/0). A single clump was used for each
jar, with four replicates for each treatment. The cultures were incubated for 8 weeks under continuous high intensity fluorescent light at 25°C. 44
2.18 Alteration of Sucrose Concentration in Shoot Multiplication Medium Four-week old four-shoot clumps derived from a G. rothschildiana tuber explant, and cultured on WP medium (2% sucrose) supplemented with lOµM NAA and
40µM BAP were used as stock materials for this experiment. The clumps were transferred onto 40mL solidified (1 % w/v agar) WP medium supplemented with 2% or
4% sucrose (w/v) and IAA:BAP (lµM:20µM). Each sucrose treatment was replicated 8 times and a single four-shoot clump was used in each replicate. The cultures were incubated for 8 weeks under continuous high intensity fluorescent light at 25°C.
2.19 Development of Roots and Tubers
Shoot clumps originating from tuber explants (with or without tuber buds and
roots) were transferred from multiplication media after 8 weeks onto growth regulator
free WP medium (50mL) contained in polycarbonate jars (150mm height x 65mm dia.)
(Disposable Products Pty. Ltd., South Australia) and cultured for 6 to 8 weeks under
continuous high intensity fluorescent light at 25°C. G. rothschildiana, G. superba,
G. simplex and G. richmondensis each had 10-20 replicates. G. verschuurii had five
replicates.
2.20 Effect of Sucrose on Development of Roots and Tubers
Eight-week old, four-shoot clumps (with or without tuber buds and roots) were
harvested from shoot mutliplication medium of G. rothschildiana and G. superba and
transferred to WP medium (50mL) without PGRs _,. :n 500mL polycarbonate jars,
supplemented with 0, 1, 2, 3, 4 and 5% (w/v) sucrose. Five replicates were used for
each treatment and each replicate contained a single four-shoot clump. The cultures were
incubated for 8 weeks under continuous high intensity fluorescent light at 25°C. 45
2.21 The Effect of ABA and GA3 on Development of Roots and Tubers
Four-week old, four-shoot clumps (with or without tuber buds) were harvested from WP medium supplemented with lµM IAA and 20µM BAP and transferred to a nitrogen modified WP medium [Nf4+ (15mM) and NO3- (5mM)] and sucrose (4%,w/v
) supplemented with ABA and GA3 as follows: 0.5µM ABA and absence of GA3, lµM
ABA and absence of GA3, lµM GA3 and absence of ABA, lOµM GA3 and absence of
ABA, and lµM ABA and lµM GA3. The absence of both GA3 and ABA served as the control in this experiment. Five replicates were used for each treatment and each replicate contained a single four-shoot clump. The cultures were incubated for 8 weeks under continuous high intensity fluorescent light at 25°C.
2.22 Deflasking and Transferring Plantlets to Soil
Propagules with a well developed root system were harvested from root and tuber development medium (Section 2.19) after 8 weeks. Alternately shoot clumps that were developing roots and tubers on shoot multiplication medium (Section 2.15) were harvested after 6 weeks. The root zone was thoroughly washed with running tap water to remove agar residues and plantlets were potted in either a non sterile peat:sand
(3: 1, v/v) mixture or a non sterile commercial all-purpose potting mix (Hortico Australia
Pty. Ltd, Victoria) contained in plastic pots (130mm dia). Plants were maintained under ambient glass house conditions and fertilized with a slow release fertilizer (N:P:K; 3:1:2) every four weeks. Plants were grown through several seasons until flowering and fruit setting occurred.
2. 2 3 Micropropagation Growth Measurements
Growth measurements were performed at each stage of micropropagation. The number of new shoots or tubers, their length and weight were recorded using destructive methods after eight weeks growth. 46
The growth of plants in soil was monitored for phenotypic abnormalities. Their survival rate was calculated 2 months after potting into soil. At the end of each growing season, the number of tubers, their length and weight were also recorded.
2. 2 4 Statistical Analysis of Data The data from experiments were subjected to statistical analysis and treatment differences were determined by the Analysis of Variance (ANOVA) using the Statistical
Analysis System (SAS) computer programme package, VAX/VMS version V6.0 (SAS
Institute, Cary, N.C., USA). On the basis of the F value in the ANOVA table, the differences among the treatment means were considered to be significantly different if the value of the probability (Pr) < 0.05. When applicable, treatment means were compared and ranked by Duncan's Multiple Range Test (DMRT), at the 5% significance level.
2. 2 5 Extraction of Plant Tissue
Plant tissue (ea. 5g) was ground using a mortar and pestle with a 2- to 5-fold volume (mL/g fresh weight) of IM HCl under minimum light at room temperature.
[Ring-A-4-3H]colchicine (ea. 10,000 dpm/g fresh weight) was added as an internal standard prior to grinding. The brei was centrifuged (3000g x 5min) and 2.5 to 5mL of supernatant transferred to Eppendorf tubes and further centrifuged at 12000 rpm for 8 minutes. The pellet was discarded and duplicate aliquots of the supernatant taken for scintillation counting (Section 2.30).
2.26 Partial Purification of Colchicine
A C-18 Sep-Pak (Waters Associates, Sydney) was activated by washing with
MeOH (l0mL), followed by H2o (l0mL). A supernatant sample (2.5 to 5mL) was loaded and the Sep-Pak eluted with 5mL each of the following step gradient: 0%, 20%, 30%, 50% and 100% [acetonitrile: MeOH (3 : 1,v/v)] in H2o (v/v). The eluates were evaporated in a gentle stream of air at room temperature under minimum light, and dried 47 in vacua over silica gel. The residue was dissolved in lmL of H2o and duplicate aliquots were taken for scintillation counting (Section 2.30).
2. 2 7 HPLC Analysis of Extracts
HPLC analysis was performed using a Waters Associates HPLC system incorporating a Model 6000A and Model M-45 solvent delivery systems, Model U6K injector and Model 450 variable wavelength detector. Separations were performed in a
10µ Partisil ODS-2, 25cm x 4.6mm ID stainless steel column (Alltech Associates, Inc.
Sydney). The elution was performed isocratically with acetonitrile: water (50: 50, v/v), at a flow rate of lmL/min. The effluent was monitored at 353 nm and peak measurements were performed using a Spectra-Physics Minigrator®. Suitable standards
( 1.5- lOµg per injection) were also assayed. Colchicine peaks were collected and the eluates evaporated. The residue was dissolved in lmL of H2o and duplicate aliquots were taken for scintillation counting (Section 2.30).
2. 2 8 Mass Spectrometry
Low resolution solid probe electron impact mass spectrometry on purified colchicine samples (Section 2.27) utilised a VG AUTOSPEC Q with VG Opus software and a V AXstation 2000 computer. Positive ion electron impact was used with filament emission energy of 70e V and a source temperature of 220°C. The direct insertion probe was heated from 50-200°C. The resolution was set at 1000 and scans for 50-600 daltons at 1 sec per decade.
2. 2 9 Thin Layer Chromatography of Colchicine
TLC was carried out using Merck 0.1mm silica gel 60F254 plastic backed TLC plates. Development used CHCl3:diethylamine (98 : 2, v/v). Colchicine was detected under short-wave UV light. 48
2.30 Counting of Radioactivity Radioactivity of samples was determined by adding lOmL of 10% aqueous (v/v)
Triton-Toluene scintillation fluid (Greene et al., 1968) to samples and counting with a
Packard 1900 TR Liquid Scintillation Analyzer. The duplicate samples were counted to less than 1% error.
2. 31 Dry Weight Determination
Independent samples of tissue were air dried at 75°C to constant weight.
2. 3 2 Addition of Putative Colchicine Precursors in Liquid Shoot Multiplication Medium.
The ability of putative colchicine precursors to increase the accumulation of colchicine in shoot cultures of G. superba was determined by supplementing multiplication medium with phenylalanine, tyrosine, phenylalanine plus tyrosine, p-coumaric acid, cinnamic acid and tyramine-HCl (final concentration lmM).
Supplements were adjusted to pH 5.2 and filter sterilized. The liquid shoot multiplication medium (50mL) (modified WP medium containing 4% sucrose, 20mM
NH4+ and 40mM NO3- and lµM IAA and 20µM BAP) in 250mL conical flasks was inoculated with four-shoot clumps (with developing tubers), taken from solidified shoot multiplication medium one month after subculture. A single four-shoot clump was transferred to each flask. Cultures were grown on a gyratory shaker (140rpm) at 25"C under continuous cool white flourescent light (4.5 watts/m2). Duplicate cultures were established for each treatment and precursor-free cultures served as controls. Cultures were harvested after two months. Tissues were thoroughly washed, separated into shoots and tubers and analysed for colchicine (Sections 2.25, 2.26 and 2.27).
2. 3 3 Distribution of Colchicine in Mature Gloriosa simplex Tissues
A mature plant of G. simplex grown under glasshouse conditions was harvested just after flowering during the summer. The above-ground portion of the plant was sectioned into 10cm segments and the tuber into 2cm segments. Each segment was individually analysed for colchicine (Section 2.25, 2.26 and 2.27). 49
2. 3 4 Screening of High Yielding Cell Lines for Production of Colchicine and Conservation Through Plant Regeneration.
Callus derived from tuber tissues of G. simplex on WP medium with 22.6µM
2,4-D was harvested after its third passage and transferred to WP medium supplemented with lµM NAA and lOOµM kinetin to regenerate plantlets (Section 2.13). The tubers derived from the regenerated plantlets where transferred to root and tuber development media (WP medium with no PGR) and harvested after 8 weeks. Individual tubers were split longitudinally once and half of each tuber analysed for colchicine (Section 2.25,
2.26 and 2.27) while the other half was cultured in shoot initiation medium (Section
2.10) to conserve the cell line through clonal propagation. 50
CHAPTER 3 RESULTS
3. 1 Callus Cultures Derived From Mature Tuber Explants
The influence of some PGR combinations and concentrations and three different basal media, on callus formation from tuber explants was determined for the four species and one cultivar of Gloriosa. A qualitative estimate of callus formation for each treatment was made after a four week incubation period. The detailed results of one such experiment using a 5 x 5 matrix of 2,4-D and kinetin with tuber explants of G. superba on WP medium are shown in Table 3.1.
Table 3.1 Effect of 2,4-D and kinetin on callus induction from mature tuber explants of G. superba. Results are the mean of 4 or 5 replicates.
Do D1 D2 D3 D4 (0) (1) (11) (23) (45) ~Kinetm Ko(0) - + +++ +++-! - (0) (35) (40) (60) (0)
- ++ +++ ++ - Kl (1) (0) (28) (65) (45) (0)
K2 (11) - ++ ++ ++ ++ (0) (24) (60) (30) (40)
K3 (23) - ++ ++ + ++ (0) (15) (24) (20) (28)
K4(45) - ++ ++ + ++ (0) (12) (20) (16) (28) - no change to explant + swollen explant ++ callusing on either 1 or 2 cut surfaces +++ callusing on all cut surfaces ++++ callusing over the whole surface of explant Values in parentheses are % of ex plants that responded to treatment.
They show that the PGR combination D3.Ko [2,4-D (23µM) and kinetin (0µM)] gave the best callus initiation with a 60% response to the treatment. D2Ko [ 2,4-D
(1 lµM) and kinetin (0µM)] and D2K1 [ 2,4-D (1 lµM) and kinetin(lµM)] also gave 51 good callus growth and a high response to treatment. Neither explant response nor callus formation occurred in the absence of 2,4-D. When considering both the amount of callus formed and the% response to treatment, the combinations D3Ko, D2K1 and D2Ko in WP medium can be recommended for callus establishment of G. superba from mature tuber explants (Photograph 3.la). Formation of undifferentiated callus only occured when 2,4-D was the auxin used. When NAA was used, the initial callus redifferentiated after 3-4 weeks, forming a large number of root-like structures ("rooted callus")
(Photograph 3. lb). A range of 2,4-D and kinetin PGR combinations produced undifferentiated callus from G. simplex tuber explants (Table 3.2). Although the D1K3 combination initially resulted in undifferentiated callus formation, it quickly redifferentiated forming shoots.
Table 3.2 Successful PGR matrix treatments for callus induction from tuber explants of Gloriosa species
Species Matrix Succesful PGR Type of Callus Experiment Treatment(s)* G. rothschildiana WPDK5x5 D2Ko, D2K1, D3Ko undifferentiated D3K1
WPDK5X5 D2Ko, D2K1,D3Ko undifferentiated MSDK5X5 D2Ko, D3Ko undifferentiated G. superba WPNK3X3 XM rooted callus MS NK3X3 HM, HH, HX rooted callus
D2Ko, D2K1, D2K3 undifferentiated WPDK5X5 D3Ko, D3K1, D3K3 unfifferentiated D1K3 differentiated forming shoots G. simplex SH NK 3 X3 MH, HH, XH rooted callus WPNK3X3 MH,HX rooted callus MS NK3 X3 MH, HX, XH rooted callus * MH-lµM NAA + lOµM kinetin MX-lµM NAA + lOOµM kinetin HM-lOµM NAA + lµM kinetin HH-lOµM NAA + lOµM kinetin HX-lOµM NAA + lOOµM kinetin XM-lOOµM NAA + lµM kinetin XX-lOOµM NAA + lOOµM kinetin
* For 2,4-D and kinetin (DK) compositions see Table 3.1. 52
The best callus initiation media for G. rothschiliana were D2Ko, D2K1, D3Ko and
D3K1 in WP medium. In all the species tested, callus formed using all NAA and kinetin combinations redifferentiated forming roots.
Photograph 3.1 Callus derived from G. superba tuber explants (a) Undifferentiated callus initiated on WP medium with 1 lµM 2,4-D and lµM kinetin after six weeks culture. (b) Differrentiated callus forming root-like structures (rooted callus) on WP medium with lOµM NAA and lµM kinetin after eight weeks culture.
3. 2 Callus Cultures Derived From Seedling Explants
The effect of some PGR combinations and concentrations and four different basal media on callus formation from seedling explants was determined for the four species and one cultivar of Gloriosa. Again a qualititative estimate of callus formation for each treatment was made after a four week incubation period. G. rothschildiana, G. superba, and G. simplex were tested on each medium with a 3 x 3 NAA/kinetin matrix.
G. verschuurii and the cultivar, G. richmondensis were tested on MS and WP media only.
In addition G. superba and G. simplex were tested with a 5 x 5 matrix of 2,4-D and kinetin on WP medium. A summary of the most successful PGR treatments for callus formation are shown in Table 3.3.
All species including the cultivar G. richmondensis failed to form good undifferentiated callus under these growth conditions. The derived callus rapidly 53 differentiated, fmming a large number of roots, even with 2,4-D as the auxin. The
"rooted callus" adapted well to their respective liquid medium and were successfully maintained for several years (Photograph 3.2).
Table 3.3 Successful PGR matrix treatments for callus induction from seedling derived explants (leaf and stem tissues) of Gloriosa species.
Species Matrix Successful PGR Type of Exoeriment Treatment(s)t Callus SH NK 3X3 MH, HM, HH, HX, XH rooted callus WPNK 3X3 MM,XM rooted callus G. rothschildiana MSNK 3X3 MH, HM, HH, HX, XH rooted callus B-5 NK 3 X3 XM,XH rooted callus
SH NK 3 X3 XH, HH, HM, MH rooted callus WPNK 3X3 MM,HH rooted callus G. superba MSNK 3X3 MH,HM,HH rooted callus B-5 NK 3 X3 HM, XM, XH rooted callus WPDK 5 X5 D2K1, D2K2, D3K2 rooted callus
SH NK 3X3 HM, HH, HX, MH rooted callus XM,XH rooted callus G. simplex WPNK 3X3 HH, HX, XM rooted callus MSNK 3X3 HM,HH,XM rooted callus B-5 NK 3·x3 HX, XM, XH rooted callus WPDK 5 x5 D2K2, D3K2 rooted callus
G. verschuurii MS NK 3 X 3 MH, XM, XH rooted callus WPNK 3X3 MM,XM rooted callus
G. richmon ensis MS NK 3 x 3 MH, HH, XH rooted callus WPNK 3X3 HH, XM rooted callus t MM-lµM NAA + lµM kinetin MH-lµM NAA + lOµM kinetin MX-lµM NAA + lOOµM kinetin HM-lOµM NAA + lµM kinetin HH-lOµM NAA + lOµM kinetin HX-lOµM NAA + lOOµM kinetin XM-lOOµM NAA + lµM kinetin XH-lOOµM NAA + lOµM kinetin XX-lOOµM NAA + lOOµM kinetin For 2,4-D and kinetin (DK) compositions see Table 3.1.
In contrast, leaf sheath from seedlings of G. superba and G. simplex on WP medium supplemented with lOOµM NAA and lOµM kinetin resulted in undifferentiated friable callus (Table 3.4) and (Photograph 3.3). 54
Photograph 3.2 G. superba rooted callus derived from leaf and stem explants proliferating in liquid SH medium supplemented with lOOµM NAA and lOµM kinetin
However, when on MS medium, the
initial undifferentiated callus
redifferentiated after 3-4 weeks into a
large number of roots.
G. rothschildiana G. verschuurii and
the cultivar; G. richmondensis failed to
produce callus from leaf sheath under
the treatments tested (Table 3.4).
Fhotograph 3.3 Undifferentiated callus derived from G. simplex leaf sheath explant on WP medium with lOOµM NAA and lµM kinetin after eight weeks culture 55
Table 3.4 PGR matrix treatments for callus induction from leaf sheath explants of Gloriosa species.
Species Matrix Successful Type of Callus Experiment PGR Treatment(s)* WPNK3X3 XM Undifferentiated callus (friable) G. superba MSNK3X3 XM Rooted callus WPNK3X3 XM Undifferentiated callus (friable) G. simplex MSNK3X3 HM,XM Undifferentiated callus soon differentiated into roots G. rothschildiana WPNK3X3 none MS NK3X3 none G. verschuurii WPNK3X3 none MSNK3X3 none G. richmondensis WPNK3X3 none MSNK3x3 none * NK-NAA and kinetin combination XM-lOOµM NAA + lµM kinetin HM-lOµM NAA + lµM kinetin
3. 3 Proliferation and Maintenance of Undifferentiated Callus 3.3.1 G. superba
Undifferentiated callus initiated from mature tuber explants of G. superba was placed on selected best 16 PGR treatments (based on the results of 5x5 matrix experiment, Section 3.1) of 2,4-D and kinetin on WP medium to select a good medium
for callus proliferation (Section 2.7). The results are shown in Table 3.5 and are the mean of duplicate determinations.
The highest growth rate (3.1 fold increase) occurred with lµM 2,4-D and llµM
kinetin (D1K2). Several other combinations gave at least a doubling of the initial fresh
weight (Figure 3.2) Callus cultures were routinely maintained on D1K2 medium. 56
Table 3.5 Proliferation of G. superba callus derived from tuber explants after 30 days growth.
PGR Growth rate PGR Growth rate Combination (fold increase) Combination (fold increase) D1Ko 1.4 D3K0 1.2 D1K1 2.5 D3K2 2.0 D1K2 3.1 D3K3 1.6 D1K3 2.4 D3K4 1.2 D2Ko 1.3 D4K2 1.5 D2K1 2.3 D4K3 1.8 D2K2 1.7 D4K4 1.9
2.5------, 4------, b a
,..._ 2 .c ~ 3 c 0 5 -0= ::; 1.5 _§ 2.5------, 2-r------, C d 2- :c :c 1.5 - C = -0 -0 _§ {\( _§ ... 1.5- ... "' "' ... "...... "... 1- l:::\:::l:::\:::j:::\:::j:::\:::l::: ~:::_l:::~:::_l:::l:::_l:::~:::_l:::l:::_ - -".. ..u ~-l:\.\:l.l:l.l:l.l: ".. ..u -=·-= -=-C -"Cl -"Cl ==' 00=='- 0.5- i:.;---e~ i:.;-..... ,:: l::i::l::l::l::!::1::1::::: •.l:i.:_:.:_•.:_:. !:_l::!:_l::•:_!::•:_i::•:. l:_l::[:_\::l:_l::l:_l::l:_ \:l:l:•:1:•:1:1::=•: o...u:.;.:.;,..:...:.L...1a.:..;,'-'-'J.....J.:.:.;-,;.;L-L..:...:,:-'--'--._,,___._.::::::::: o--..--,--,,--~---r--,--,-- D3Ko D3K1 D3K2 D3K3 D31<4 D4Ko D4K1 D4K2 D4K3 D41<4 PGR Combination PGR Combination Figure 3.2 Effect of various concentrations of kinetin when combined with (a) lµM 2,4-D (b) llµM 2,4-D (c) 23µM 2,4-D and (d) 45µM 2,4-D on proliferation of callus derived from mature tuber explants of G. superba on WP medium, after eight weeks culture. * For PGR compositions see Table 3.1 57 3.3.2 G. simplex The results in Table 3.6 show the proliferation rate of G. simplex undifferentiated callus initiated from leaf sheath explant of seedlings. The results indicate that in WP medium with NAA(lOOµM) and kinetin (lOµM), (WP NK XH medium treatment) the callus achieved 2.2 fold increase/month. Table 3.6 Proliferation of undifferentiated callus derived from leaf sheath explants of G. simplex on WP medium supplemented with NAA (lOOµM) and kinetin (l0µM) after 30 days culture. ± sd Culture Growth rate Replicate (fold increase) 1 2.3 2 2.5 3 2.0 4 2.3 5 2.2 6 1.8 Mean 2.2±0.2 3. 4 Proliferation and Maintenance of Differentiated Callus The results presented in Fig 3.3 shows the growth rates of differentiated (rooted ed callus) callus of 3 species and one cultivar of Gloriosa when maintain-, in SH liquid cultures. The medium was supplemented with lµM NAA and lOµM kinetin. The highest growth rate was observed for G. rothschildiana (4.4) followed by G. superba (4.0), G. richmondensis (3.2) and G. simplex (3.0). 58 6------, 5 - T T {{(( T T :':::::::=:::=:: !_ll_ll_ll_l_l_l!_II_I -:-:-:-:-:-:-:-: -:-:-:-:-:-:-:-: 1 1111111111111111 1111111111111111 111111:111:1111 111111111111111 0 ...... ,., ...... __ ...... ,-- ...... _...._ ...... _,-... 1 2 3 4 Species* Figure 3.3 Growth rates of differentiated callus (rooted callus) in liquid SH mediwn supplemented with NAA (lµM) and kinetin (lOµM). The results are the mean of 5 replicates. * (1) G. rothschildiana (2) G. superba (3) G. simplex (4) G. richmondensis 59 CHAPTER 4 RESULTS MICROPROPAGATION 4. 0 Introduction The successful propagation in vitro of important medicinal and ornamental Liliaceous plants have been reported for several species. The application of tissue culture technology to the rapid propagation of Liliaceous bulbs, corms or tubers has greatly increased for many practical reasons (as previously discussed). Two reports on the micropropagation of Gloriosa superba were published shortly after this work began (Finnie and van Staden 1989; Somani et al., 1989). The results of these studies were not encouraging with respect to routine production of a large number of plantlets or tubers for commercial purposes. A third report (Custers and Bergervoet, 1994) was published on micropropagation of G. superba and hybrids of G. superba x G. rothschildiana during the preparation of this thesis. No further reports on the micropropagation of other Gloriosa species have been published. A number of these species are commercially available in Australia, with a growing market, particularly for G. rothschildiana as a cut flower. The aim of this work was to develop an in vitro procedure suitable for the rapid production of plantlets and tubers of the Gloriosa species available in Australia. These were G. rothschildiana, G. superba, G. simplex, G. verschuurii and the apparent G. superba cultivar, G. richmondensis. 4 . 1 Initiation of Cultures The establishment of axenic cultures and initiation of multiple shooting is crucial for the success of in vitro multiplication. Experience has shown that this process is affected by many factors such as disinfestation procedures, the species chosen, type of 60 explant, physiological state of the explant, composition of the medium, and the type and concentration of plant growth regulators (PGR). In this chapter the influence of species and type of explant on two standard media containing different types and concentrations of auxin and cytokinin were tested for their ability to initiate shoots and tubers from Gloriosa explants. 4. 1 . 1 Initiation of Shoots from Tuber Explants Tuber explants were cultured on MS and WP media, with NAA and kinetin in a 4 x 4 matrix experiment and NAA and BAP in a 5 x 4 matrix experiment (Section 2.10). The results of the experiments with NAA and kinetin are shown in Table 4.1 and the Analysis of Variance for each species is shown in Appendices la, 1b and le. Analysis of Variance was performed using the SAS programme with the number of shoots per er explant against the PGR treatments stving as the dependent variable. On the basis of the F value in the ANOV A table, the differences among the treatment means were considered to be significantly different if the value of probability (Pr) < 0.05,. The treatment means were compared using Duncan's Multiple Range Test (DMRT) at the 5% significance level. The Analysis of Variance results show that the PGR, the medium and their interaction had a highly significant effect (Pr < 0.05) on shoot initiation from tuber explants, for the three species (Appendices la-le). Analysis using the Duncan's Multiple Range Test (DMRT) over all the data for each medium found that WP medium was better than MS medium (5% level) for shoot initiation (Table 4.2). Figures 4.la- 4.lf depict the results as dose response curves for each species and medium. The concentrations of NAA and kinetin giving the best shoot initiation were lµM and lOOµM respectively for all species on both media. The maximum number of shoots per explant after eight weeks was 2.0, 1.8 and 1.5 for G. rothschildiana, G. superba and G. simplex respectively, with WP medium (Table 4.land Figures 4.lb, 4.ld and 4.lf). 61 Table 4.1 The effect of NAA and kinetin on shoot initiation from tuber explants of G. rothschildiana, G. superba and G. simplex on MS and WP media after eight weeks culture. Each value is the mean of 20 explants. In the two columns under each species, means followed by a common letter are not significantly different at the 5% level. No. of Shoots Per Tuber Explant PGR (µM) G. rothschildiana G. suoerba G. simplex NAA Kinetin MS WP MS WP MS WP 0 0 0.25 i 0.2 i 0.25 hi 0.2 hi 0.25 efg 0.13 efg 0 1 0.25 i 0.2 i 0.3 ghi 0.2 ghi 0.45 d-g 0.25 efg 0 10 0.35 hi 0.4 ghi 0.56 fg 0.45 gh 0.35 d-g 0.4 d-g 0 100 0.4 ghi 0.6 e-i 0.6 efg 0.95 d 0.44 d-g 0.7 cd 1 0 0.35 hi 0.45 g-i 0.5 gh 0.4 ghi 0.21 efg 0.2 fg 1 1 0.8 d-g 1.0 def 0.8 def 1.4 b 0.25 efg 0.25 efg 1 10 1.0 de 1.4 be 1.35 b 1.55 ab 0.50 def 1.0 be 1 100 1.6 ab 1.95 a 1.5 b 1.8 a 1.00 be 1.5 a 10 0 0.35 hi 0.6 e-i 0.5 gh 0.5 gh 0.25 efg 0.2 fg 10 1 0.8 d-g 0.75 e-h 0.9 d 1.3 be 0.40 d-g 0.3 efg 10 10 1.0 de 1.2 cd 0.9 d 1.05 cd 0.60 de 0.6 de 10 100 1.4 be 1.6 ab 1.3 be 1.0 d 0.44 dg 1.2 ab 100 0 0.4 g-i 0.55 f-i 0.2 hi 0.15 i 0.15 fg 0.1 g 100 1 0.6 e-i 0.8 d-g 0.4 ghi 0.45 gh 0.25 e-g 0.3 efg 100 10 0.8 d-g 0.8 d-g 1.0 d 0.85 de 0.37 d-g 0.3 efg 100 100 1.0 de 1.2 cd 0.2 hi 0.55 fg 0.25 efg 0.19 fg Table 4.2 The effect of the medium on the mean number of shoots per tuber explant after eight weeks culture on different combinations and concentrations of NAA and kinetin. In a column, means followed by a common letter are not significantly different at the 5% level. Mean No. of Shoots per Tuber Explant Medium G. rothschildiana G. superba G. simplex MS 0.73 b 0.7 b 0.37 b WP 0.86 a 0.8 a 0.49 a Increasing the NAA concentration above lµM, significantly reduced the number of shoots initiated, particularly at lOµM and l00µM kinetin (Figures 4. la-4. lf ). At 0µM and lµM kinetin, increasing concentrations of NAA had less effect, and the frequency of shoot initiation was low (Figures 4.1 a-4. lf). The rate of shoot initiation was increased with increased level of kinetin up to lO0µM, when the medium was combined with a low level (lµM) of NAA. 62 2-r-----.....------2-r------,A...... _G. rothschildiana G. rothschildiana on MS 3 b I ,, .. I ',,, C .. on WP ea C I,...... ,,. I A., e,1.5· I ,,. .! 1.5 I ,, ,' ...... A. Q. ,' o.... ',, I ,, ~ I ,' ...... j I ',, ',, 0 ~ I : ·-a., 'A 0 I ',, .. 0 I ,, .. , .. "' -5i 1- / ,0------0...... A 0 I ,, °'·· ...... I ,' ...... -="' I ,' ....· •••·•· .. 0 ... I ,' .-·· ...... 41A,, ...... ~ / ,.<...... o···· .. ·········~ ...... 0 .. ::·o I ,' •.-· v .cI.I I,' ...·· ···o .. A/_..., s o.5 I , .• jo.5 , .. z= E ~... l ~ z= 0 .....--,r------,r------,..------....J 0 .....- ...... ---....-----.------..1 0 1 10 100 0 10 100 NAA (µM) NAA (µM) 2 ,------2,------G. superha on MS ..... d G. superha on WP ..C C A, .! / \ .. , \ ~ 1.5- C ea 1.5 / ,0. \ ~ I I .. \ c. I • C) •••~ ...\ .. = ~ ,, ,' l ; \·····-o g I I : -~ • -= ~.. 0 I / 1 : ~\. "' l - // •. ... 0 I. : ! 0 --~.. . -="' ,,, l: ',,~\...... "O .. ... ,' .l ,,•. .cI.I 0 E o.5 - .. ,' i '~ = .cI.I 0.5 Of:1 __...... ~0 z E ! z= ! 0 ----,,r-----,,------,,----,-.....J 0 .....--,---- .....------_J 0 l 10 100 0 10 100 NAA (µM) NAA (µM) 1.25 .. G. simplex C on MS 2-r------ea e .. f G. simplex c. l • C ~ on WP I.I ,A. I \ .! ... , \ "' I \ ~ 1.5 - 0 I \ 0 / \ 0.75 - I \ ~ -= I \ .. "' , \ 0 ... / \ 0 0 I \ .Q, -= I ---·'t •• "' 1 - .. 0.5 • I a•• \ ' ... .cI.I 0 Q. ·::oe-· .-·· 't: , ·-. "", .. E o· ··· ..I.I = ···· ...... o .c z 0.25 - S o.5 - ~ z= 0----,,,----,7,---,,r----..,-_J 0 .....--,r------,..-----,----,--- 0 I 10 100 0 1 10 JOO NAA (µM) NAA (µM) -0- Kinetin (0 µM) --·-0···- Kinetin (10 µM) ...... ()...... Kinetin (lµM) ----6---- Kinetin (100 µM) Figures 4.la-4.lf The effect of NAA and kinetin on shoot initiation from tuber explants of G. rothschildiana, G. superba and G. simplex on MS and WP media after eight weeks culture. 63 The results of the experiments with NAA and BAP are shown in Table 4.3 and the Analysis of Variance for each species is shown in Appendices 2a, 2b and 2c. Analysis of Variance of the effect of NAA and BAP for G. rothschildiana (Appendix 2a) G. superba (Appendix 2b) and G. simplex (Appendix 2c) showed that the number of shoots produced varied significantly with the medium used and PGR treatment. Table 4.3 The effect of NAA and BAP on shoot initiation from tuber explants of G. rothschildiana, G. superba and G. simplex on MS and WP media after eight weeks culture. Each value is the mean of 20 explants. For the two columns under each species, means followed by a common letter are not significantly different at the 5% level. No.of Shoots oer Exolant PGR (uM) G. rothschildiana G. suverba G. simvlex NAA BAP MS WP MS WP MS WP 0 0 0.30 00 0.25 00 0.2 n 0.1 n 0.25 ii 0.3 hii 0 10 0.5 11-Q 0.5 11-Q 0.25 mn 0.35 Imn 0.4 l!-i 0.25 ii 0 20 1.0 i-o 1.6 re 0.7 hi 0.7 hi 0.5 f-i 0.4 l!-i 0 40 0.8 i-o 1.6 re 0.8 l!-k 1.0 e-i 0.6 e-i 0.6 e-i 0 60 0.75 i-D 1.55 re 0.65 i-m 0.9 f-i 0.75 d-1! 0.7 e-h 1 0 0.35 00 0.55 m-o 0.3 lmn 0.3 Imn 0.3 hii 0.25 ii 1 10 1.05 f-k 0.9 h-n 0.7 hi 0.6 i-n 0.6 e-i 0.4 l!-i 1 20 1.6 re 2.1 abc I.I e-i 1.05 e-i 0.9 Qr 0.65 e-i 1 40 1.4 M 2.1 abc 1.4 b-e 1.55 a-d I.I bed I.I bed 1 60 0.95 h-l 1.2 e-i 0.95 f-i 1.0 e-i 0.6 e-i 0.7 e-h 10 0 0.65 k-o 0.6 l-a 0.3 lmn 0.4 k-n 0.3 hii 0.4 l!-i 10 10 1.25 e-h 1.6 e 0.6 i-n 0.7 hi 0.5 f-i 0.5 f-i 10 20 2.1 ab 2.45 a 1.05 e-i 1.15 d-b 0.9 c-f 1.0 ere 10 40 1.8 bed 1.7 ai 1.8 ab 1.9 a 1.4 ab 1.6 a 10 60 I.I f-i 1.6 re 1.3 c-f 1.4 b-e 1.0 Qr 1.2 be 20 0 0.5 Il-Q 0.45 000 0.4 k-n 0.36 k-n 0.2 i 0.3 hii 20 10 1.0 f-l 1.0 f-l 0.9 f-i 1.0 e-i 0.4 l!-i 0.35 l!-i 20 20 1.45 M 2.2 ab 1.0 e-i 1.1 e-i 0.7 e-h 0.55 f-i 20 40 1.4 d-1! 1.6 re 1.3 c-f 1.6 abc 1.0 ere 1.2 be 20 60 1.0 f-l 1.4 d-1! 0.65 i-m 1.2 C-1! 0.9 c-f 0.9 c-f Figures 4.2a to 4.2f depict the results as dose response curves for each species and medium. 64 2.5...------, rothschildiana on WP G. rothschildiana on MS a G. b 0, ....P...... = 2.5 .. -a.= 2 ./ •• •• Q -a." 0-···4·-.._ ~ ~ 2 I / I \ ', •._ / ,,.____ \ QI I ' l \' \ ~ i .,.,· I \,...... ~ : / --- ...... 0 1.5 / / .A--:-::~~-.. 0 / p \ 0 ., ,,,,·r '-- /' .c P .,.. :,,,' ._\ ju I/ : . , ..."' ./,/ ,,o ..."' i / / 0 1 -~ 0 //,A ... ,,,'7 ... QI QI 6,.//, .0 .0 8 s ::s 0.5 ~J' ::s 0.5 o/ z z Ii. o..l....--.----r---,r---.---~- 0 0 10 20 40 60 0 10 20 40 60 BAP (µM) BAP (µM) 2-.------, 2...------, ...... = = -a." 1.5 .!! 1.5 ~ =- QI ~ -.l!l ~ 0 ... 0 0 .c .c0 ..."' ..."' 0 0 ...:; ... .0 ~ 0.5 S 0.5 s z= z= 0 ...... ---.------.------,----,-~ 0 10 20 40 60 0 10 20 40 60 BAP (µM) BAP (µM) 1.5 -,------, 2...------, G. simplex on WP f ..o ... = =1.5 " c." ./· ...... ~l ~:; QI ,/ A ••• t) 3 -..."' lb'-, 0 ... 0 0 ,.: ,r;-.... ··, .c0 .c ···a "' .,.,., 11.,· ·· ...... , "' ... / '\, ... 0 ...O 0.5 ...... ,.... ,1,i ,, QI :; .0 .ci ...--·· -~ .0 0.5 0-...... · ..o-::..---- s s ,;~- z= z= 0 ...... ---.----.-----.------.---....-- 0 0 10 20 40 60 0 10 20 40 60 BAP (µM) BAP (µM) ---0-- NAA(OµM) ...... ,0-...... NAA(lµM) ····0··· NAA(lOµM) ·---tJ.-- NAA(20µM) Figures 4.2a-4.2f The effect of NAA and BAP on shoot initiation from tuber explants of G. rothschildiana, G. superba and G. simplex on MS and WP media after eight weeks culture. 65 The optimum combination of NAA and BAP giving the best shoot initiation from G. rothschildiana tuber explants was lOµM NAA and 20µM BAP (Figures 4.2a -4.2b and Photograph 4.1). 11~ 1\ll~ll''t 'l"t Photograph 4.1 Shoot initiation from G. rothschildiana tuber explants on WP medium containing lOµM NAA and 20µM BAP after eight weeks culture In contrast, G. superba and G. simplex explants gave the best shoot initiation with lOµM NAA and 40µM BAP for both MS and WP media (Table 4.3 and Figures 4.2c-4.2d and 4.2e-4.2f respectively). The maximum number of shoots per explant after eight weeks culture was 2.5, 1.9 and 1.6 for G. rothschildiana, G. superba and G. simplex respectively, when using WP medium (Table 4.3 and Figures 4.2a-4.2f). The results of the DMRT over all the data for each medium found that WP medium was better than MS medium (Pr < 0.05) for G. rothschildiana and G. superba shoot initiation (Table 4.4 and Appendices 2a-2c). However, for G. simplex, there was no significant difference in the number of shoots when cultured on the two media (Table 4.4). The best individual PGR combination for G. simplex, viz. lOµM NAA and 40µM BAP, gave 66 1.6 shoots per explant on WP medium and 1.4 shoots per explant on MS medium (Table 4.3). Table 4.4 The effect of medium on the mean number of shoots per tuber explant after eight weeks culture on different combinations and concentrations of NAA and BAP. In a given column, means followed by a common letter are not significantly different at the 5% level. Mean No. of Shoots per Explant Medium G. rothschildiana G. suverba G. simvlex MS 1.05 b 0.82 b O•b6 a WP 1.38 a 0.93 a O•bb a 2"'T'------, -0--- MS -= = ...... ·O········ WP -a 1.5 ~ Q,I :l -0 0 .c .. rll ...... -0 ...... a.. Q,I (S ...··· .c c o.5 z= 0 ---...... ------,,----,----...... --- 1:10 1 :20 1:40 1:60 NAA:BAP(µM) Figure 4.3 The effect of medium on G. superba shoot initiation from tuber explants at different ratios of NAA and BAP for lµM NAA. Results of the Analysis of Variance (Appendix 2b) showed that there was no significant interaction between media and PGR treatments (Pr> 0.05). The results also indicate that despite the medium used 40µM BAP was the optimum for shoot initiation from tuber explants of G. superba (Figure 4.3). The shoot initiation abilities of G. verschuurii and G. richmondensis were tested only with two different PGR treatments; lOµM NAA and 40µM BAP and lµM NAA and lOOµM kinetin on WP medium due to unavailability of sufficient explant materials (Section 2.10). The results showed that both G. verschuurii and G. richmondensis 67 achieved more shoots per tuber explant with NANBAP combination, 1.8 and 1.6 shoots per explant respectively. With NANkinetin PGR treatment those initiated only 1.2 and 0.8 shoots per explant respectively. 4. 1. 2 Initiation of Shoots from Shoot Base Explants Explants from the proximal ends of juvenile shoots of G. rothschildiana, G. superba and G. simplex were cultured on MS and WP media with NAA and kinetin and/or NAA and BAP (Section 2.11). The results of the experiments with NAA and kinetin are shown in Table 4.5 and the Analysis of Variance for G. superba and G. simplex are shown in Appendices 3a and 3b respectively. Table 4.5 The effect of NAA and kinetin on shoot initiation from shoot bases of G. superba and G. simplex on MS and WP media after eight weeks culture. Each value is the mean of at least 25 explants. In a column, means followed by a common letter are not significantly different at the 5% level. No. of Shoots per Explant PGR (µM) G. rothschildiana G. suDerba G. simolex NAA kinetin MS WP MS WP MS WP 0 0 - - 0 e 0 e 0.05 cde 0 e 1 0 - - 0 e 0 e 0.07 b-e 0 e 10 0 - - 0 e 0 e 0 e 0 e 100 0 - - 0 e 0 e 0 e 0 e 0 1 - - 0 e 0 e 0 e 0 e 1 1 - - 0 e 0 e 0 e 0 e 10 1 - - 0 e 0 e 0 e 0 e 100 1 - - 0 e 0 e 0.02 de 0 e 0 10 - - 0 e 0.10 c-f 0 e 0 e 1 10 - - 0.05 f 0.15 cd 0.05 cde 0.10 bed 10 10 - - 0.12 cde 0.15 cd 0 e 0.05 cde 100 10 - - 0.10 c-f 0.05 ef 0.1 b-e 0.05 cde 0 100 - - 0.05 ef 0.25 ab 0 e 0 e 1 100 0.35 0.4 0.17 be 0.30 a 0.12 a-e 0.20 a 10 100 - - 0.25 ab 0.32 a 0.15 ab 0.12 ab 100 100 - - 0.05 def 0.10 c-f 0 e 0 e (-) Experiment not performed The results show that the response from the shoot base explant was limited in both MS and WP media, especially at low concentrations of kinetin. 68 G. superba only produced shoots if the kinetin was~ lOµM. However at lOOµM NAA, shoot initiation decreased significantly (5% level) specially when NAA was combined with lOOµM kinetin. A similar trend was also observed for G. simplex (Table 4.5). Nevertheless the Analysis of Variance showed that shoot initiation of G. superba varied significantly depending on the PGR treatment, the medium used, and the interaction between the medium and PGR treatment (M x PGR)(Appendix 3a). Although the PGRs had a significant effect on the shoot initiation rate of G. simplex, neither the medium (M) nor M x PGR interaction had a significant effect (Appendix 3b). DMRT analysis over all the data for each medfom showed that for G. superba, WP medium gave a significantly (5% level) higher rate of shoot initiation (0.09 shoots per explant) compared to MS medium (0.05 shoots per explant)(Appendix 3a; Duncan grouping). However, there was no significant difference between these media for the mean value of shoot initiation from G. simplex (Appendix 3b; Duncan grouping). The maximum number of shoots per explant for both G. superba (0.3) and G. simplex (0.2) was with WP medium, supplemented with lµM NAA and lOOµM kinetin (Table 4.5 and Photograph 4.2). This PGR treatment with MS and WP medium gave 0.35 and 0.4 shoots per explant respectively, for G. rothschildiana (Table 4.5). This combination was identical to that for tuber explants (Table 4.1) with these three species. However tuber explants gave more shoots per explant. In general the response of the shoot initiation from shoot bas_e explants was poor fr\~ with NAA and kinetin despite the medium. However it was interest• to note that when using NAA and BAP PGR combination these explants showed significantly higher shoot initiation capacity and the results of these experiments are shown in Table 4.6 and the Analysis of Variance for G. rothschildiana, G. superba and G. simplex are shown in Appendices 4a, 4b and 4c respectively. Figures 4.4a-4.4f depict the results as dose response curves for each species and medium used. 69 Photograph 4.2 Shoot initiation from G. superba shoot base explants on WP medium with lµM NAA and lO0µM kinetin after eight weeks culture. Table 4.6 The effect of NAA and BAP on shoot initiation from shoot base explants of G. rothschildiana, G. superba and G. simplex on MS and WP media after eight weeks culture. Each value is the mean of at least 25 explants. In the two columns under each species, means followed by a common letter are not significantly different at the 5% level. No. of Shoots per Exolant PGR (µM) G. rothschildiana G. suverba G. simplex NAA BAP MS WP MS WP MS WP 0 0 00 k 0.0 k 0 .0 k 0 i 0 i 0 i 0 10 0.25 b-k 0.2 ik 0.006 i 0 i 0 i 0 i 0 20 0.5 fgb 0.6 efg 0 i 0.12 bi 0 i 0 i 0 40 0.6 efg 0.6 efg 0 i 0 i 0 i 0 i 0 60 0.4 gbi 0.7 ef 0 i 0 i 0 i 0 i 1 0 0.1 jk 0.0 k 0 i 0 i 0 .15 f-i 0 i 1 10 0.8 re 0.6 efg 0.04 gbi 0.02 gbi 0.13 gbi 0.05 bi 1 20 1.2 be 1.8 a 0.13 f 0 .16 ef 0.25 e-b 0.22 fgb 1 40 1.2 be 1.4 b 0.24 C 0.20 re 0.45 ere 0.3 efg 1 60 0.6 efg 0.8 re 0.05 ghi 0.04 ghi 0.13 ghi 0.12 ghi 10 0 0 k 0.0 k 0 i 0 i 0 i 0 i 10 10 0.2 ijk 0.2 ijk 0.02 gbi 0.02 ghi 0.2 f-i 0.1 ghi 10 20 0.6 efg 1.0 ai 0.2 ere 0.31 b 0.45 ere 0.25 e-h 10 40 0.8 re 1.4 b 0.24 C 0.64 a 0.95 a 0.67 b 0 10 60 0.8 re 1.0 ai 0.07 0 0.04 ghi 0.55 be 0.35 def 20 0 0 k 0 k 0 I 0 i 0 i 0 i 20 10 0.4 ghi 0.3 bij 0.12 f 0.04 ghi 0.2 f-i 0.03 hi 20 20 0.3 hij 0.8 re 0.12 f 0.07 g 0.3 5 M 0.17 f-i 20 40 0.5 fgh 0.6 efg 0.14 f 0.07 g 0.58 be 0.42 ere 0 20 60 0.4 ghi 0.4 ghi 0.06 0 0.04 ghi 0.5 bai 0.25 e-h 70 1.5-.------. G. rothschildiana on MS 2'T"------,G. rothschildiana on WP a .. b ~ ...... -0 .. =~ ;Q··········· ... =~ c. 1.5 c...... \ ~ / ),~ ~ ~ ~ .."' / ,,,' -~-~,, 0 .."' .o--~··:...--o 0 :" ,' \ ', 0 tl 0 l ,, ... -="' / p' \ .... ''o -="' / ,a·_.. \~ 'o I : \ ... :i ,: , J!J,...... ~ 0 0.5 l ,' ... Q,I 1. /,' ...... ' . .c 1t# ~, Q,I ./ ,' .c i ,, S 0.5 ...... I s l ,, = = / ,, z ....··· r z o/ ....·",,' l "" 0 I 0 20 40 60 0 10 20 40 60 BAP (µM) BAP (µM) 0.8 ------0.25 ------, G. superba on MS ,•? \ G. superba on WP , . ' C d ,,' / \ C 0.2 o· i \ , : \ =~ 0.6 ~ ,• / \ c. c. ~ ~ '' :: '.,, Q,I Q,I I : ) ~ ~ 0.15 ' ~ .. ' ~ ..0 0 : o' ...... 6 i 0.4 0 ,' : ,,,, ', ~ _g -= b,---r-ttr \ t, "' ' "' I I I \. \I ' "'" 0.1 I I I \ \I ...0 ' 0 I I I \ \I '' ... ,' ,' l: '\\', ... Q,I ,' ,,;..: ~Q j 0.2 .c s S 0.05 / i = z= ,i z I I o .J..--{~---,--41----£1----£]-...I 0 10 20 40 60 0 10 20 40 60 BAP (µM) BAP (µM) 0.8 ------G. simplex on MS 0 G. simplex on WP '. ' . e .. '' '. = ,' \ ~ ,!= 0.75 ' .. c. ,' ~ g., \ Q,I ~ . . . ' ~ ~ ' .' ,•'. ,,A-...... 8 0 -= .=8 0.5 ,' ,,' ...... & "' "' ,0 ~,, --~ ...0 ... ,' ,, ..·· ··.. 0 ...... ,,',,.4 ..····· ··... Q,I Q,I , , ..· ··. .c .C 0.25 ,,,, 1:i ··. s s = = o-...... ;.r.':;,:: ..... -···· \ ...o z z ,,,' 0 10 20 0 10 20 40 60 40 60 HAP (µM) BAP (µM) ---er-- NAA (0 µM) ...... -<>-...... NAA (lµM) ····O-··· NAA (10 µM) ·---t,.-- NAA (20 µM) Figures 4.4a-4.4f The effect of NAA and BAP on shoot initiation from G. rothschildiana, G. superba and G. simplex shoot base explants on MS and WP media after eight weeks culture. 71 The Analysis of Variance of the data show that shoot initiation from G. rothschildiana and G. simplex explants was significantly influenced (Pr< 0.05) by the PGR treatment, the basal medium and the media x PGR interaction, tested at the 5% level. However shoot initiation from G. superba shoot base explants did not differ between the two media, although it was influenced by the PGR treatments and the media interaction (Table 4.7 and Appendix 4b). Table 4. 7 The effect of medium on the mean number of shoots per shoot base explant after eight weeks culture. In a column, means followed by a common letter are not significantly different at the 5% level. Mean No. of Shoots oer Shoot Base Explant Medium G. rothschildiana G. superba G. simplex MS 0.48 b 0.072 a b 0·~4- WP 0.62 a 0.070 a a 0. ,s The maximum number of shoots (1.8/explant) for G. rothschildiana was achieved with lµM NAA and 20µM BAP in WP. medium. The maximum number of shoots (,0.64/explant) for G. superba was achieved with lOµM NAA and 40µM BAP, also in WP medium. The latter PGR combination was found to be the best for G. simplex (0.95/explant), but in MS medium rather than WP medium (Table 4.6). The presence of BAP was the most important factor affecting shoot initiation, because no shoots were produced at all concentrations of NAA in the absence of BAP (Table 4.6). However at each BAP concentration, the addition of NAA generally stimulated shoot initiation. 4. 1 . 3 Initiation of Shoots from Root-Coleoptile Axes of Seedlings Explants taken from the root-coleoptile axes of seedlings of G. rothschildiana, G. superba and G. simplex were cultured on MS and WP media with NAA and kinetin, and NAA and BAP (Section 2.12). The results of the experiments with NAA and 72 kinetin are shown in Table 4.8 and the Analysis of Variance for each species are shown in Appendices 5a, 5b and 5c. Table 4.8 The effect of NAA and kinetin on shoot initiation from root-coleoptile axes explants of G. rothschildiana, G. superba and G. simplex on MS and WP media after eight weeks culture. Each value is the mean of 40 explants. In the two columns under each species means followed by a common letter are not significantly different when tested at the 5% level. No.of Shoots per Root-Coleoptile Axis Explant PGR(µM) G. rothschildiana G. suverba G. simplex NAA Kinetin MS WP MS WP MS WP 0 0 0.20 g 0.52 cde 0.23 f-i 0.20 g-i 0.53 e-h 1.00 ar 1 0 0.30 fg 0.60 cd 0.17 ii 0.19 g-i 0.61 e-h 1.00 ar 10 0 0.30 fg 0.52 cde 0.17 ij 0.20 g-i 0.50 e-h 1.00 ar 100 0 0.25 g 0.40 e-g 0.18 ii 0.10 j 0.30 g-h 0.80 d-g 0 1 0.30 fg 0.60 cd 0.17 ij 0.25 f-j 0.50 e-h 0.95 de 1 1 0.37 efg 0.60 cd 0.30 f-j 0.30 f-i 0.90 d-h 0.87 d-f 10 1 0.25 g 0.50 de 0.17 ii 0.20 g-i 0.60 e-h 1.00 ar 100 1 0.25 g 0.40 efg 0.20 g-i 0.20 g-j 0.40 f-h 0.90 de 0 10 0.45 def 0.70 be 0.47 d-g 0.45 d-i 0.60 e-h 1.50 be 1 10 0.60 cd 0.70 be 0.46 d-h 0.35 ii 1.60 ab 1.50 be 10 10 0.40 efg 0.55 cde 0.50 def 0.30 f-i 1.00 gh 1.20 ar 100 10 0.50 de 0.50 de 0.26 f-j 0.30 f-j 0.20 h 0.75 d-h 0 100 0.60 cd 1.10 a 0.70 ocd 0.70 ocd 1.00 ar 1.00 ar 1 100 0.80 b 1.10 a 0.95 b 2.10 a 1.70 ab 2.00 a 10 100 0.55 cde 0.50 de 0.80 be 0.95 b 0.87 d-f 1.00 ar 100 100 0.40 efg 0.42 d-g 0.58 ar 0.45 d-i 0.40 fgh 0.68 e-h The Analysis of Variance of the data show that the number of shoots initiated from root-coleoptile axis explants was significantly influenced by the NAA and kinetin treatments, and the medium used. In addition, significant interaction between the medium and the PGR treatment (media x PGR) was also observed on G. rothschildiana~ OM.~ G. superba W shoot initiation when tested at the 5% level (Appendices 5a and 5b ). No significant interaction was observed on G. simplex shoot initiation (~r ~ 0.05) (Appendix 5c). Addition of more than lµM NAA had no effect or significantly reduced shoot initiation at all concentrations of kinetin tested in either medium (Table 4.8). This was true for each species tested. A combination of lµM NAA and lOOµM kinetin in WP medium gave the highest number of shoots per explant (Photograph 4.3). These were 1.1, 2.1 and 2.0 for G. rothschildiana, G. superba and G. simplex respectively. 73 Further analysis of the pooled data by the Duncan's Multiple Range Test (DMRT) show that the overall mean values for the number of shoots per explant in WP medium was significantly higher than MS medium for the three species tested (Table 4.9 and Appendices 5a, 5b and 5c). Photograph 4.3 Shoot initiation from G. superba root-coleoptile axis explants on WP medium supplemented with lµM NAA and lO0µM kinetin after eight weeks culture. Table 4.9 The effect of medium on the mean number of shoots per root-coleoptile explant after eight weeks culture. In a column, means followed by a common letter are not significantly different at the 5% level. Mean Number of Shoots per Root-Coleoptile Explant Medium G. rothschildiana G. superba G. simplex MS 0.41 b 0.39 b 0.7 b WP 0.61 a 0.45 a 1.1 a The results of the experiments with NAA and BAP are shown in Table 4.10 and the Analysis of Variance of the data are shown in Appendices 6a and 6b. 74 Only the influence of lOµM NAA and 40µM BAP was tested for shoot initiation ability of root-coleoptile explants of G. rothschildiana due to unavailability of sufficient seedlings for other treatments. Table 4.10 The effect of NAA and BAP on shoot initiation from root-coleoptile axes explants of G. superba and G. simplex on MS and WP media after eight weeks culture. Each value is the mean of 40 explants. In the two columns under each species means followed by a common letter are not significantly different when tested at the 5% level. No. of Shoots per Root-Coleoptile explant PGR (µM) G. rothschildiana G. suoerba G. simplex NAA BAP MS WP MS WP MS WP 0 0 - - 0.25 hi 0.25 hi 0.15 d-f 0.20 c-f 0 10 - - 0.30 hi 0.35 gh 0.15 d-f 0.20 c-f 0 20 - - 0.62 a-d 0.75 a 0.20 c-f 0.25 b-e 0 40 - - 0.45 fg 0.52 c-f 0.20 c-f 0.20 c-f 0 60 - - -- 0.20 c-f 0.20 c-f 1 0 - - 0.22 hi 0.30 hi 0.17 c-f 0.20 c-f 1 10 -- 0.45 fg 0.45 fg 0.20 c-f 0.25 b-e 1 20 -- 0.55 c-f 0.65 abc 0.25 b-e 0.35 ab 1 40 -- 0.70 ab 0.65 abc 0.25 b-e 0.30 be 1 60 --- - 0.20 c-f 0.25 b-e 10 0 - - 0.17 i 0.30 hi 0.10 f 0.12 ef 10 10 - - 0.65abc 0.70 ab 0.25 b-e 0.25 b-e 10 20 -- 0.45 fg 0.55 c-f 0.30 be 0.33 ab 10 40 0.16 0.28 0.50 def 0.60 b-e 0.30 be 0.45 a 10 60 - - - - 0.20 c-f 0.27 bed 20 0 - - 0.22 hi 0.20 i 0.07 f 0.10 f 20 10 - - 0.50 def 0.60 b-e 0.10 f 0.15 def 20 20 - - 0.65 abc 0.70 ab 0.15 def 0.20 c-f 20 40 - - 0.35 gh 0.47 efg 0.17 c-f 0.25 b-e 20 60 - - - - 0.1 f 0.15 def (-) Experiment not performed. The Analysis of Variance results show that the PGR and the medium significantly (Pr < 0.05) influenced shoot initiation from G. superba and G. simplex explants. However, they also show that there was no significant influence by the interaction of the media with the PGR treatment (Appendices 6a and 6b ). Analysis of the pooled data for each medium using the DMRT found that WP medium was superior to MS medium for both species (Table 4.11 and Appendices 6a and 6b ). 75 Table 4.11 The effect of the medium on the mean number of shoots per root-coleoptile explant of G. superba and G. simplex. In a column, means followed by a different letter are significantly different at the 5% level. Mean No of Shoots per Explant Medium G. superba G. simplex MS 0.43 b 0.18 b WP 0.50 a 0.24 a The maximum number of shoots per explant for G. superba was 0.75 on WP medium containing 20µM BAP only (Table 4.10). For G. simplex it was 0.45, also on 0 WP medium, but containing 10 µM NAA and 40 µM BAP. ~~n MS and WP media only one treatment was tested for G. rothschildiana, viz l0µM NAA and 40µM BAP which gave 0.28 shoots per explant. 4. 2 Tuber Initiation From Tuber Explants During the experiments to initiate primary shoots on the various tuber explants it was noticed that many cultures contained tuber buds at the base of shoots (Photograph 4.4). The number of these (greater than 3mm in diameter) were recorded and in some cases the weight also. The results for tuber initiation with NAA and kinetin are shown in Table 4.12 and the Analysis of Variance of the data are shown in Appendices 7a, 7b and 7c. Photograph 4.4 Developing tuber buds of G. rothschildiana at the base of shoots grown on WP medium with lµM NAA after eight weeks culture. 76 Table 4.12 The effect of NAA and kinetin on tuber initiation from primary cultures of tuber explants of G. rothschildiana, G. superba and G. simplex on MS and WP media after eight weeks culture. Each value is the mean of 20 explants. In two columns under each species, means followed by a common letter are not significantly different at the 5% level. No. of Tuber Buds per Tuber Explant PGR (µM) G. rothschildiana G. superba G. simplex NAA Kinetin MS WP MS WP MS WP 0 0 0.15 ij 0.25 ghi 0.13 M 0.2 b-f 0.2 gh 0.20 gh 0 1 0.40 d-i 0.50 b-g 015 e-f 0.25 b-e 0.15 hi 0.25 e-h 0 10 0.40 M 0.60 a-e 0.18 e-f 0.25 b-e 0.15 hi 0.20 gh 0 100 0.50 b-g 0.65 a-d 0.15 e-f 0.10 M 0.20 gh 0.10 i 1 0 0.40 d-i 0.50 b-g 0.30 bed 0.30 bed 0.20 gh 0.30 ef 1 1 0.60 a~ 0.60 a-e 0.40 ab 0.50 a 0.30 e-h 0.35 or 1 10 0.70 a~ 0.80 a 0.50 a 0.50 a 0.40 al 0.55 be 1 100 0.65 a-d 0.75 ab 0.30 bed 0.40 ab 0.60 be 0.90 a 10 0 0.35 e-i 0.40 d-i 0.15 e-f 0.25 b-e 0.10 i 0.10 i 10 1 0.40 d-i 0.45 e-h 0.20 b-f 0.20 b-f 0.30 ef 0.15 hi 10 10 0.50 b-g 0.70 abc 0.20 b-f 0.35 abc 0.40 al 0.40 al 10 100 0.55 a-f 0.50 b-g 0.20 b-f 0.25 b-e 0.50 e 0.70 b 100 0 0.10 j 0.15 ij 0.00 f 0.10 M 0.05 j 0.10 i 100 1 0.15 ij 0.15 ij 0.10 M 0.1 M 0.05 j 0.10 i 100 10 0.30 f-i 0.25 g-j 0.10 M 0.15 c-f 0.20 gh 0.15 hi 100 100 0.40 d-i 0.20 hi 000 f 0.15 c-f 0.30 ef 0.05 j Analysis of Variance and Duncan's Multiple Range Test show that across all the data WP medium was better than MS medium for G. superba, but there were no significant differences between the two media for G. rothschildiana and G. simplex (Table 4.13 and Appendices 7a, 7b and 7c). Table 4.13 The effect of medium on tuber initiation from primary cultures of tuber explants after eight weeks culture. In a column, means followed by a common letter are not significantly different at the 5% level. Mean Number of Tuber Buds per Explant Medium G. rothschildiana G. superba G. simplex MS 0.41 a 0.19 b 0.27 a WP 0.46 a 0.25 a 0.35 a The highest number of tuber buds per explant was 0.8 and 0.9 for G. rothschildiana and G. simplex respectively both on WP medium. G. rothschildiana achieved its best tuber initiation with lµM NAA and lOµM kinetin while G. simplex with the same concentration of NAAybut with 1OOµM kinetin. 77 G. superba initiated 0.5 tuber buds per explant at its best and interestingly, G. superba showed no significant difference between the two media at the best combination of NAA and kinetin (lµM and lOµM respectively; Table 4.12), while the Anova of the pooled data showed that the PGR treatment (NAA and kinetin), and the medium had a significant effect on tuberization from primary cultures. However there was no significant interaction between the medium and the PGR treatment (media x PGR) when tested at the 5% level (Appendices 7a, 7b and 7c). The results on tuber initiation with NAA and BAP are shown in Table 4.14 and the Analysis of Variance of the data are shown in Appendices 8a and 8b. Maximum tuber initiation (0.85 tuber buds/explant) was achieved by G. rothschildiana on WP medium with l0µM NAA and 20µM BAP. For G. simplex it was 0.5 tuber buds /explant, also in WP, but either with lOµM NAA and 40 µM BAP or 20µM NAA and 40µM BAP. However taken overall the data there was no significant difference between media for this species (Table 4.15). The Analysis of Variance results show that there was a significant effect of medium and PGR treatment on tuberization for both speci~s. However the medium x PGR interaction was not significant for tuber initiation of either G. rothschildiana or G. simplex (Appendices 8a and 8b). 78 Table 4.14 The effect of NAA and BAP on tuber initiation from primary cultures of tuber explants of G. rothschildiana and G. simplex on MS and WP media after eight weeks culture. Each value is the mean of 20 explants. In the two columns under the each species, means followed by a common letter are not significantly different at the 5% level. No. of Tuber Buds per Tuber Explant PGR (µM) G. rothschildiana G. simplex NAA BAP MS WP MS WP 0 0 0.15 ii 0.25 g-j 0.20 cde 0.15 b-e 0 10 0.05 j 0.25 g-i 0.20 cde 0.25 b-e 0 20 0.20 hii 0.30 f-i 0.25 b-e 0.25 b-e 0 40 0.35 e-i 0.35 e-i 0.20 cde 0.20 cde 0 60 0.25 g-j 0.30 f-i 0.10 e 0.10 e 1 0 0.55 c-f 0.50 c-g 0.20 cde 0.20 cde 1 10 0.55 c-f 0.65 a-d 0.30 a-e 0.35 a-d 1 20 0.60 b-e 0.70 abc 0.40 abc 0.40 abc 1 40 0.50 c-g 0.45 c-h 0.30 a-e 0.30 a-e 1 60 0.45 c-h 0.45 c-h 0.15 de 0.10 e 10 0 0.40 d-i 0.70 abc 0.20 cde 0.25 b-e 10 10 0.55 c-f 0.50 c-g 0.35 a-d 0.25 b-e 10 20 0.55 c-f 0.85 a 0.40 abc 0.40 abc 10 40 0.55 c-f 0.80 ab 0.45 ab 0.50 a 10 60 0.35 e-1 0.45 c-h 0.30 a-e 0.20 cde 20 0 0.25 g-i 0.40 d-i 0.25 b-e 0.35 a-d 20 10 0.25 g-j 0.45 c-h 0.20 cde 0.30 a-e 20 20 0.35 e-i 0.45 c-h 0.30 a-e 0.35 a-d 20 40 0.45 c-h 0.45 c-h 0.45 ab 0.50 a 20 60 0.50 c-g 0.35 e-i 0.20 cde 0.30 a-e Table: 4.15 The effect of medium on tuber initiation from primary cultures of tuber explants after eight weeks culture. In a column, means followed by a common letter are not significantly different at the 5% level. Mean Number of Tuber Buds per Explant Medium G. rothschildiana G. simplex MS 0.39 b 0.27 a WP 0.48 a 0.28 a In the latter experiment with NAA and BAP, the tuber buds were excised and weighed. The results are shown in Table 4.16 and the Analysis of Variance of the data are shown in Appendices 9a and 9b. DMRT analysis of the complete data set with respect to the two media is shown in Table 4.17. 79 Table 4.16 The effect of NAA and BAP on the weight of tubers initiated from primary cultures of tuber explants of G. rothschildiana and G. simplex on MS and WP media after eight weeks culture. Each value is the mean of 20 explants. In the two columns under each species, means followed by a common letter are not significantly different at the 5% level. Weight of Tubers oer Replicate (mg) PGR (LIM) G. rothschildiana G. simplex NAA BAP MS WP MS WP 0 0 138 kl 280 e-k 150 a-f 84 d-h 0 10 40 1 199 ijk 152 a-f 120 b-h 0 20 280 e-k 300 e-i 120 b-h 160 abe 0 40 400 cde 350 d-h 72 e-h 128 a-h 0 60 208 h-k 392 c-f 56 fgh 44 h 1 0 380 c-g 380 c:.g 104 c-h 152 a-f 1 10 400 cde 400 cde 120 b-h 216 a 1 20 450 bed 350 d-h 208 ab 208 ab 1 40 550 b 414 b-e 102 c-h 130 a-h 1 60 238 g-k 400 cde 84 d-h 48 gh 10 0 420 b-e 400 cde 90 c-h 140 a-h 10 10 500 be 500 be 96 c-h 110 c-h 10 20 680 a 520 be 102 c-h 148 a-f 10 40 480 bed 450 bed 180 a-d 160 a-e 10 60 520 be 380 c-g 144 a-g 96 c-h 20 0 280 e-k 300 e-1 144 a-g 120 b-h 20 10 450 bed 350 d-h 140 a-h 120 b-h 20 20 340 d-i 400 cde 96 c-h 112 c-h 20 40 280 e-k 350 d-g 183 abc 180 a-d 20 60 160 ikl 250 i-k 64 e-h 60 fgh Table 4.17 The effect of medium on the mean weight of tubers per replicate from primary cultures of tuber explants after eight weeks cultures. In a column, means followed by a common letter are not significantly different at the 5% level. Mean weight of Tuber Buds per Replicate (mg) Medium G. rothschildiana G. simplex MS 368 a 120 a WP 360 a 127 a The results show that the mean weight of tubers from G. rothschildiana tuber explants was greater than that from G. simplex on either medium. However the choice of medium did not significantly affect the mean weight of tubers for a given species (Table 4.17). No PGR and medium interaction was observed on tuber weight when tested at the 5% level. The mean of the tuber weights per replicate were not affected by the medium. However a significant interaction was observed between the medium and 80 PGR treatment on G. rothschildiana tuber weight (Appendix 9a) and it was non significant for G. simplex (Appendix 9b). 4 . 3 Tuber Initiation from Root-Coleoptile Axis Explants Like the tuber explants, it was noticed that root-coleoptile axis explants also had a propensity to initiate tuber buds in primary culture. The number of these (greater than 3mm diameter) were recorded for G. rothschildiana and G. simplex after eight weeks culture on NAA and kinetin. The results are shown in Table 4.18 and a summary of the Analysis of Variance of the data is shown in Appendices 10a and 10b. Table 4.18 The effect of NAA and kinetin on tuber initiation from primary cultures of root-coleoptile axes explants of G. rothschildiana and G. simplex on MS and WP media after eight weeks culture. Each value is the mean of 40 explants. In the two columns under each species, means followed by a common letter are not significantly different at the 5% level. No. of Tuber Buds per Explant PGR t(µ,M) G. rothschildiana G. simplex NAA kinetin MS WP MS WP 0 0 0.18 f 0.57 abc 0.32 b-g 0.44 a-e 0 1 0.18 f 0.40 cde 0.20 d-g 0.18 efg 0 10 0.50 bed 0.40 cde 0.25 c-g 0.25 c-g 0 100 0.40 cde 0.50 bed 0.12 g 0.15 fg 1 0 0.20 f 0.57 abc 0.35 a-g 0.50 abc 1 1 0.35 def 0.50 bed 0.50 abe 0.56 ab 1 10 0.70 a 0.66 ab 0.50 abc 0.40 a-f 1 100 0.40 cde 0.57 abc 0.50 abc 0.40 a-f 10 0 0.18 f 0.40 cde 0.25 c-g 0.20 d-g 10 1 0.25 ef 0.40 cde 0.50 abc 0.30 c-g 10 10 0.70 a 0.57 abc 0.60 a 0.40 a-f 10 100 0.50 bed 0.30 ef 0.44 a-e 0.30 c-g 100 0 0.18 f 0.30 ef 0.30 c-g 0.35 a-g 100 1 0.20 f 0.30 ef 0.45 a-d 0.40 a-f 100 10 0.30 ef 0.40 cde 0.44 a-e 0.40 a-f 100 100 0.30 ef 0.30 ef 0.35 a-g 0.19 e-g The Analysis of Variance for G. rothschildiana show that tuber initiation is significantly (Pr< 0.05) influenced by the PGR combinations, the medium as well as the interaction of the medium x PGR treatment (Appendix 10a). The medium, and PGR combination also significantly affected tuber initiation of G. simplex, although the interaction of the medium x PGR was not significant at the 5% level (Appendix 10b). 81 The results of the DMRT on the mean number of tuber buds per explant showed a significant difference between MS and WP media for G. rothschildiana, but not for G. simplex (Table 4.19). Table: 4.19 The effect of medium on tuber initiation from primary cultures of root coleoptile axis explant after eight weeks culture. In a column, means followed by a common letter are not significantly different at the 5% level. Mean No of Tubers per Explant Medium G. rothschildiana G. simplex MS 0.34 b 0.38 a WP 0.45 a 0.34 a Both G. rothschildiana and G. simplex root-coleoptile axis explants readily initiated tuber buds, even in the absence of PGR. Although primary cultures of tuber explant also readily initiated tuber buds under these culture conditions the frequency was d low compar~to that of root-coleoptile primary cultures (see for example Figure 4.5). 0.8 -r------, C .!- g. ~ 0.6- ~ "O .c= ~ 0.4- .c= - ii T.·:::l:::_::::_ i Ol-u uI nll 0 --,,--,.,...,...... ,....., ...... ,...... I I I I I I I I I I I 0 ..... 000 ..... 0 0 0 ..... 0 0 0 0 0 ...... 0 oO~~~ ...... 0 0 0 ..... - - .....0 0 ...... 0 -0 0 ...... 0 - -0 0 ..... - - 0 NAA : Kinetin (µM) Figure 4.5 The effect of NAA and kinetin on tuber initiation from G. rothschildiana root-coleoptile axis explants on MS medium after eight weeks culture. The values are the mean of 40 explants. 82 In general WP medium gave a significantly larger number of tuber buds than MS medium with both species (Table 4.19). Tuber bud formation was strongly dependant on kinetin, being maximal at 10µ-Mi\.ddition of NM (1 or lOµM) resulted in a further increase in the number of tuber buds per explant at lOµM kinetin. The increased NAA concentration even up to lOOµM had no effect on the rate of tuber initiation if the medium was absence of kinetin (Figure 4.5). G. simplex responded a little differently to the NM and kinetin combinations, depending on the medium used. On MS medium the response pattern was similar to G. rothschildiana, but on WP medium the maximum number of tuber buds (0.56 per explant) was with NM and kinetin each at lµM. 83 CHAPTER 5 RESULTS 5. 0 Shoot and Tuber Multiplication The main objective of Stage II of micropropagation is the multiplication of cultures by successive re-inoculations. In many cases the conditions in Stage I (Initiation) will be the basis of multiplication medium, but further modification should increase the multiplication rate. The results of experiments to enhance the multiplication of shoots from Gloriosa previously initiated in vitro are describe~ in this chapter. 5 .1 G. rothschildiana 5. 1 . 1 Multiplication of Cultures Derived from Tuber Explants Stock cultures (Section 2.14), consisting of a four-shoot clump and growing on WP medium were subcultured onto fresh medium as described in Sections 2.15 and 2.16. The choices of WP medium instead of MS medium and BAP instead of kinetin were based on the results of the experiments performed to determine their abilities to initiate shoots (Section 4 ). The results of the effect of NAA or IAA with BAP on shoot multiplication and length are shown in Table 5.1 and the Analysis of Variance of the data, are shown in Appendix lla and llb. Figures 5.1 and 5.2 depict the multiplication results as a dose response curve for each auxin. 84 Table 5.1 The effect of NAA or IAA with BAP on multiplication and length of shoots derived from tuber explants of G. rothschildiana after eight weeks culture on WP medium. Values are the mean of five replicates. In the two columns under each growth parameter, means followed by a common letter are not significantly different at the 5% level. PGR(µM) No. of New Shoots per Mean Length of a Shoot Reolicate (mm) Auxin BAP NAA IAA NAA IAA 0 0 2.0 k 2.0 k 85 a 82 a 0 10 11.0 f-k 10.0 f-k 54 cde 47 e 0 20 17.6 b-h 17.0 b-h 55 cde 52 cde 0 40 21.2 a-d 21.0 a-d 59 cde 59 cde 0 60 18.4 b-h 18.4 b-h 57 cde 57 cde 1 0 2.0 k 10.0 f-k 70 a-d 85 a 1 10 25.8 abc 18.4 b-h 60 b-e 66 a-e 1 20 27.2 ab 29.2 a 55 cde 60 b-e 1 40 20.0 a-g 21.4 a-d 65 a-e 70 a-d 1 60 15.4 d-h 17.6 b-h 50 de 48 e 10 0 2.6 ik 4.2 ik 65 a-e 62 h-e 10 10 12.0 d-k 11.2 d-k 70 a-d 58 b-e 10 20 18.4 b-h 18.8 b-g 65 a-e 65 b-e 10 40 18.0 b-h 20.8 abc 65 a-e 72 abc 10 60 13.6 d-i 13.2 d-i 45 e 56 e 20 0 3.0 ik 10.4 f-k 65 a-e 55 cde 20 10 8.0 iik 16.0 c-h 60 b-e 58 cde 20 20 14.4 d-h 20.2 a-f 50 de 55 cde 20 40 12.2 d-i 19.6 b-g 52 cde 62 b-e 20 60 12.6 d-i 9.5 g-k 46 e 51 cde 30 30 Ill ...... Ill (j " (j =a. 25 =" 25 Ill c. .. ..Ill ~... ~ 0 20 0 20 -=0 0 "' -="' ~ 15 15 Ill ~ ...= =Ill O 10 ...0 10 ..Ill .. .&I Ill .&I s 5 = E 5 z z= 0 0 0 20 40 60 80 0 20 40 60 80 BAP (µM) BAP (µM) --0-- NAA(~ ····0···· NAA(lOµM) --0-- !AA (0 µM) ····0···· !AA (l0µM) ·--.. 0--.. NAA(lµM) --6-· NAA(20µM) ·--0- !AA (lµM) ·--6-- !AA (20µM) Figure 5.1 The effect of BAP Figure 5.2 The effect of BAP and NAA on shoot multiplication of and IAA on shoot multiplication of G. rothschildiana after eight weeks G. rothschildiana after eight weeks culture on WP medium. culture on WP medium. 85 Maximum shoot multiplication was achieved with 20µM BAP with either lµM NAA or IAA. This was 27 and 29 new shoots per replicate, respectively with each replicate beginning as a four-shoot clump. The Analysis of Variance results show that shoot multiplication was strongly affected by the BAP concentration with either NAA or IAA (Pr< 0.05). However the auxin used as well as the auxin x cytokinin interaction also affected shoot multiplication (Appendix I la). The results of the Duncan's Multiple Range Test (DMRT) show that shoot multiplication of G. rothschildiana was significantly better with IAA than NAA (Table 5.2). Table 5.2 The effect of auxin on the mean number of shoots per replicate after eight week culture on WP medium. 1\,(i,COrding to the Duncan's Multiple Range Test, means followed by a common letter are~gnificantly different at the 5% level. Treatment Mean No.of New Shoots Duncan Grouping per Replicate NAA 13.3 b IAA 16.6 a The results of the effect of NAA or IAA with BAP on the length of shoots are also shown in Table 5.1. The largest shoot length occurred at the lowest multiplication rate, viz. in the absence of PGRs. This possibly reflects competition for nutrients by individual shoots in the denser shoot cultures (Photograph 5.1). As might be expected therefore, the Analysis of Variance results show that NAA or IAA in combination with BAP had a signifcant effect on the length of shoots (Pr< 0.05) (Appendix 1 lb). However, there was no significant difference between the two auxins (Pr> 0.05) (Appendix 1 lb). This was further supported by the Duncan's Multiple Range Test of the data set (Table 5.3). 86 Photograph 5.1 Eight week-old dens shoot cultures of G. rothschildiana (a) grown on WP solid medium and (b) grown in WP liquid medium, both supplemented with lOµM NAA and 20µM BAP. Table 5.3 The effect of auxin on the mean length of shoots after eight weeks culture on WP medium. According to the Duncan's Multiple Range Test, means followed by a common letter are not significantly different at the 5% level. Treatment Mean Length per Shoot (mm) Duncan Grouping NAA 61 a IAA 60 a In the process of shoot multiplication tuber initiation is continued and the size and weight of tubers are also increased. The effect of NAA or IAA with BAP on number and the weight of tubers derived from multiplying shoots of G. rothschildiana after eight weeks culture on WP medium was examined. The results of the effect of NAA or IAA with BAP on number and weight of tubers are shown in Table 5.4 and the Analysis of Variance is shown in Appendices 12a and 12b. 87 Table 5.4 The effect of NAA or IAA with BAP on number and weight of tubers derived from multiplying shoots of G. rothschildiana after eight weeks culture on WP medium. Values are the means of five replicates. In the two columns under each growth parameter, means followed by a common letter are not significantly different at the 5% level. PGR (µM) No. of Tubers per Replicate Weight of Tubers per Replicate (mg) AuxinBAP NAA IAA NAA IAA 0 0 2.0 fgh 1.8 h 180 abc 177 abc 0 10 2.4 fg 2.7 fg 135 be 137 be 0 20 2.6 fg 2.8 efg 150 abc 155 abc 0 40 3.6 de 3.7 d 163 abc 164 abc 0 60 2.6 fg 2.6 fg 168 abc 156 abc 1 0 3.0 efg 5.2 cd 140 be 150 abc 1 10 3.6 de 7.6 b 160 abc 200 abc 1 20 4.8 cd 11.2 a 180 abc 154 abc 1 40 4.0 cd 7.6 b 180 abc 175 abc 1 60 2.6 fg 5.8 C 180 abc 160 abc 10 0 2.6 fg 3.8 d 120 C 170 abc 10 10 3.4 ef 3.6 de 150 abc 160 abc 10 20 3.8 d 7.2 b 160 abc 160 abc 10 40 3.2 ef 7.2 b 170 abc 178 abc 10 60 2.0 fgh 2.2 fg 180 abc 182 abc 20 0 1.6 h 3.6 de 120 C 138 abc 20 10 2.2 fg 7.8 b 180 abc 210 a 20 20 2.6 fg 8.0 b 140 be 160 abc 20 40 2.4 fg 6.6 be 140 be 150 abc 20 60 2.0 fgh 3.2 ef 120 C 197 ab The maximum tuber number was 4.8 per replicate with lµM NAA and 20µM BAP or 11 per replicate with lµM IAA and 20µM BAP. The Analysis of Variance results show that IAA was more effective than NAA (Pr < 0.05), at all concentrations of BAP tested (Appendix 12a). For the highest number of tubers lµM IAA and 20µM BAP was the best treatment (Photograph 5.2). DMRT analysis of the mean weight of tubers harvested found no significant p difference at the 5% level (;r > 0.05) due to the auxin used (Appendix 12b). 88 ''.!;/""''Trrn::r"'"~{:!,''f'11:r'''''~:'ri1~1;,,,,,i"'''t'P''t'l'''t'l'''t'\111i:1i1~,,,,~~1~1~1111~,1,,~ - - - I AA BA P \AA B AP I ~ I 4 f"r I ,,.,1 I Photograph 5.2 The effect of different concentrations of BAP combined with lµM IAA on shoot and tuber multiplication of G. rothschildiana after eight weeks culture on WP medium. 5. 1 . 2 The Effect of Altered Inorganic Nitrogen Concentration and Composition on Shoot and Tuber Multiplication. Nitrogen is one of the most important mineral nutrients for in vitro shoot multiplication as well as tuberization of many monocotyledonous plants (previously discussed). There are many evidence for supporting that the total nitrogen as well as the ratio of NH4+ and NO3- as important factors to be experimented for a plant species.to achieve optimum shoot and tuber multiplication (Section 1). In this work the effect of three concentrations of inorganic nitrogen (15, 30 and 60mM), provided by various combinations of NH4+ and NO3-, was tested on shoot and tuber multiplication as described in Sections 2.17, 2.23 and 2.24. The results are shown in Table 5.5 and the Analysis of Variance of the data is shown in Appendix 13. 89 Table 5.5 The effect of inorganic nitrogen concentration and composition on shoot and tuber multiplication of G. rothschildiana after eight weeks culture on WP medium supplemented with lµM NAA and 20µM BAP. Values are the mean of four replicates. In the column under each growth parameter, means followed by a common letter are not significantly different at the 5% level. Nitrogen Composition No. of New Shoots No. of Tubers NH4+ NQ3- per Replicate per Replicate (mM) (mM) 0 15 20.7 b 5.2 ab 0 30 26.2 a 7.0 ab 0 60 13.5 C 3.0 ab 5 10 23.5 b 5.5 ab 10 5 14.0 C 6.0 ab 10 20 27.0 a 5.2 ab 15 0 0 e 0 e 20 10 16.7 d 3.7 ab 20 40 20.0 b 8.0 a 30 0 0 e 0 -t,e 40 20 9.2 d 0 •e 60 0 0 e 0 -.e The results show that the effect of inorganic nitrogen on shoot and tuber < multiplication are significant when tested at the 5% level (Pr• 0.0001 and 0.04 respectively) (Appendix 13). The cultures failed to grow on NH4+ as the sole nitrogen source (Table 5.5 and Photograph 5.3a). When NQ3- was used as sole nitrogen source, growth and multiplication was observed (Table 5.5 and Photograph 5.3b). Maximum shoot and tuber multiplication occurred at 30mM total nitrogen given as either lOmM NH4+ and 20mM NO3-, or 30mM NO3- only (Photograph 5.3c). There was no significant difference in the number of new shoots for these two treatments (Table 5.5). When the 30mM total nitrogen was provided as 20mM NH4+ and lOmM NO3- there were relatively fewer shoots and tubers (Table 5.5). Although the number of tubers per replicate differed according to the treatment, DMRT ranking of the results showed that there was no significant difference between most of the treatments (Table 5.5). 90 Photograph 5.3a The effect of (1)15mM, (2) 30mM and (3) 60mM NH4+ as the sole nitrogen source on shoot multiplication of G. rothschildiana after eight weeks culture on WP medium supplemented with lµM NAA and 20µM BAP. Photograph 5.3b The effect of (1)15mM, (2) 30mM and (3) 60mM NO3- as the sole nitrogen source on shoot multiplication of G. rothschildiana after eight weeks culture on WP medium supplemented with lµM NAA and 20µM BAP. Photograph 5.3c Shoot cultures provided with (a) l0mM NH4+ + 20mM NO3- or (b) 30mM NO3- after eight weeks culture on WP medium supplemented with lµM NAA and 20µM BAP 91 5.1.3 The Effect of Altered Sucrose Concentration on Shoot and Tuber Multiplication. It has been shown that sucrose is an important factor in the development of tubers in shoot cultures of many monocotyledons including some Liliaceous plants, and increased sucrose concentration results in • increased tuberization in many plant species (Section 1). However for other plant species, such as Dioscorea alata increased sucrose inhibited microtuber induction and reduced their mean fresh weight (Mantell and Hugo, 1989). Therefore the effect of an increased sucrose concentration on shoot multiplication and tuber development (number of tubers and weight of tubers per replicate) was tested as described in Sections 2.18, 2.23 and 2.24. The results are shown in Table 5.6 and the Analysis of Variance of the data is shown in Appendix 14. Table 5.6 The effect of increased sucrose (4%) on shoot and tuber multiplication of G. rothschildiana after eight weeks culture on WP medium supplemented with lµM IAA and 20µM BAP. Across two columns under 2% and 4% sucrose concentrations, means followed by a common letter are not significantly different at the 5% level. Parameter per Replicate 2% Sucrose 4% Sucrose No. of new shoots 20 a 16 b No. of tubers 7.6 a 5.7 b Weight of tubers (mg) 180 a 196 a They show that when the sucrose concentration was increased from 2% to 4% a significant decrease in the number of new shoots and tubers occurred (Table 5.6). Although the mean weight of the tubers rose on the higher sucrose concentration, the increase was not significant at the 5% level (Table 5.6 and Appendix 14). 5.1.4 Multiplication of Cultures Derived from Shoot Base Explants The stock cultures (Section 2.14), consisting of a four-shoot clump and growing on WP medium were subcultured onto fresh medium as described in Section 2.15. The best PGR treatments for shoot initiation from shoot-base explants of G. rothschildiana 92 were selected and incorporated into both MS and WP media. Shoot cultures on either MS or WP media supplemented with lOµM NAA and 40µM BAP were preincubated for four weeks and shoot clumps (with four shoots) (>3cm) cultured on these selected media as described in Section (2.15). The results are shown in Table 5.7 and the Analysis of Variance of the data is shown in Appendix 15. Although there were differences in the number of new shoots per replicate, the statistical analysis of the data found no significant difference (Pr> 0.1625) at the 5% level between the PGR and medium treatments (Appendix 15). Table 5.7 The effect of selected media on shoot multiplication of shoot cultures derived from G. rothschildiana shoot base explants after eight weeks culture. Values are the mean of five replicates. Means followed by a common letter are not significantly different at the 5% level. Media Composition No.of New Shoots per Replicate MS (1 0µM IAA + 40µM BAP) 15.2 ab WP ( 10µM IAA + 40µM BAP) 16.8 a MS (l0µM NAA + 40µM BAP) 12.0 b-e WP (l0µM NAA + 40uM BAP) 13.6 abc MS (luM NAA + lOOµM kinetin) 11.2 b-f WP (lµM NAA + lO0uM kinetin) 11.0 b-f MS (lOµM NAA + lOOµM kinetin) 12.0 b-e WP (l0µM NAA + lOOµM kinetin) 11.2 b 5. 1 . 5 Multiplication of Cultures Derived from Root-Coleoptile Axis Explants Stock cultures (Section 2.14) growing on WP medium were subcultured onto fresh medium as described in Section 2.15. The results of the effect of selected NAA and kinetin treatments are shown in Table 5.8 and Figure 5.3.The Analysis of Variance of the data is shown in Appendix 16. The Analysis of Variance results showed that NAA and kinetin treatments significantly affected shoot multiplication 93 (Pr > 0.0001), but there were no significant differences observed in the number of tubers per replicate for the same PGR treatments (Table 5.8 and Figure 5.4). Table 5.8 The effect of NAA and kinetin on shoot and tuber multiplication of shoot cultures derived from the root-coleoptile axis explants of G. rothschildiana after eight weeks culture on WP medium. Values are the means of five replicates. In a column, means followed by a common letter are not significantly different at the 5% level. Treatment PGR (µM) Number of New Number of Tubers Number NAA Kinetin Shoots per Replicate per Replicate 1 0 0 1.2 d 2.0 a 2 1 0 1.6 cd 1.6 a 3 1 1 2.8 C 1.6 a 4 1 10 5.2 b 2.0 a 5 1 100 7.6 a 2.0 a 6 0 100 6.0 b 2.0 a 7 10 100 6.0 b 1.6 a 8 100 100 2.4 cd 1.2 a ~ ~ s------~ ~ ·--a ~ .!:! ~ 6- ~ [] Number of new shoots /replicate .,Q= - E3 Number of tubers / replicate 1 2 3 4 5 6 7 8 NAA:kinetin Treatment * Figure 5.4 The effect of NAA and kinetin on shoot and tuber multiplication of shoot cultures derived from the root-coleoptile axis explants of G. rothschildiana after eight weeks culture on WP medium. Values are the means of 5 replicates. * For the composition of each NAA:kinetin treatment see Table 5.8. 94 5.2 G. superba 5. 2 .1 Multiplication of Cultures Derived from Tuber Explants Stock cultures (Section 2.14) growing on either MS or WP medium were subcultured onto six different multiplication media as described in Section 2.15. Data were collected and analysed as described in Sections 2.23 and 2.24. The results are shown in Table 5.9 and the Analysis of Variance of the data is shown in Appendix 17. Maximum shoot multiplication (21 per replicate) occurred on the WP medium supplemented with lOµM IAA and 40µM BAP. In general, WP medium gave a better growth performance, except for the NAA/BAP combinations where there was no significant difference between the two media (Table 5.9). Table 5.9 The effect of multiplication media on the growth and multiplication of shoots and tubers II derived from tuber explants G. superba after eight weeks culture. Values are the mean of five replicates. In a column, means followed by a common letter are not significantly different at the 5% level. No. of Length Number Weight of Multiplication New per of Tubers Tubers per medium Shoots per Shoot per Replicate Replicate (cm) Replicate (mg) MS (lOµM NAA + 40µM BAP) 9.4 C 3.2 C 1.0 C 554 C WP ( lOµM NAA + 40µM BAP) 11.8 C 3.7 ab 2.0 C 480 C MS (l0µM IAA + 40µM BAP) 17.0 b 3.1 C 2.2 C 704 ab WP (1 0µM IAA + 40µM BAP) 21.4 a 3.5 be 3.8 a 564 C MS (lµM NAA + lOOµM kinetin) 11.7 C 3.5 be 1.8 C 920 a WP (lµM NAA + lO0µM kinetin) 18.8 ab 4.0 a 3.0 b 494 C The maximum mean length of shoots (4 cm) was also on WP medium, but with lµM NAA and lO0µM kinetin. Culture on MS medium tended to produce shorter shoots than culture on WP medium at the same PGR concentrations. The maximum number of tubers per replicate (3.8) occurred on WP medium supplemented with lOµM IAA and 40µM BAP. However the maximum mean weight of tubers (920mg per replicate) was on MS medium (Table 5.9). This was similar to the results for G. simplex (Table 5.12) and again may reflect the higher nitrogen content of MS medium. 95 5.2.2 Multiplication of Cultures Derived from Shoot Base Explants Stock cultures (Section 2.14) growing on WP medium were subcultured onto eight multiplication media as described in Section 2.15. Data was collected and analysed as described in Sections 2.23 and 2.24. The results are shown in Table 5.10 and Figure 5.5 and the Analysis of Variance of the data is shown in Appendix 18. Table 5.10 The effect of PGR treatments on multiplication of shoots from shoot base explants of G. superba after eight weeks culture on MS and WP media. Values are the mean of five replicates. Means followed by a common letter are not significantly different at the 5% level. Multiplication Medium No. of New Shoots per Replicate MS (lOµM NAA + 40µM BAP) (No. 1) 8.8 d WP (lOµM NAA + 40µM BAP) (No. 1) 12 ab MS (lOµM IAA + 40µM BAP) (No. 2) 11 be WP (lOµM IAA + 40µM BAP) (No. 2) 15 a MS (lµM NAA + lOOµM kinetin) (No. 3) 7.6 C WP (lµM NAA + lOOµM kinetin) (No. 3) 9.2 bd MS (lOµM NAA + lOOµM kinetin) (No. 4) 4.4 e WP (lOµM NAA + lOOµM kinetin) (No. 4) 7 C WP medium was significantly better for shoot multiplication at each PGR combination and at lOµM IAA and 40µM BAP gave the maximum multiplication rate (15.0 per replicate)(Table 5.10 and Figure 5.4). The results clearly show the superiority of IAA as the auxin and BAP as the cytokinin, on either medium for multiplication of G. superba shoot base explants (Figure 5.5). 96 20------, Q,I D MS -=CJ ·- [3 WP l 15 ~ ....rll 0 0 .c rll 10 ~ Q,I c...= 0 ... 5 Q,I .ce z= 1 2 3 4 Multiplication media Figure 5.5 The effect of PGR treatments on multiplication of shoots derived from shoot base explants after eight weeks culture on MS and WP media. Media compositions are as given in Table 5.10. 5. 2. 3 Multiplication of Cultures Derived from Root-Coleoptile Axes Explants Stock cultures (Section 2.14) growing on WP medium were subcultured onto fresh WP medium as described in Section 2.15. PGRs as shown in Table 5.11 were used in this experiment and the choices of medium and PGRs were based on the results of the experiments performed to determine their abilities to initiate shoots (Section 4). Data was collected and analysed as described in Sections 2.23 and 2.24. The results are shown in Table 5.11 and Figure 5.6, and Analysis of Variance of the data is shown in Appendix 19. 97 Table 5.11 The effect of NAA and kinetin on multiplication of shoots derived from root-coleoptile axes explants of G. superba after eight weeks culture on WP medium. Values are the mean of five replicates. Means followed by a common letter are not significantly different at the 5% level. PGR(µM) No. of New Shoots per NAA Kinetin Replicate 0 0 1 C 0 10 2.8 C 0 100 3.0 C 1 10 7.2 b 1 100 10.2 a 10 100 6.8 b 12-.------, 0:0 0:10 0:100 1:10 1:100 10:100 NAA:Kinetin (µM) Figure 5.6 The effect of NAA and kinetin on multiplication of shoots derived from root-coleoptile axis explants of G. superba after eight weeks culture on WP medium. The results show that both NAA and kinetin had a significant effect on shoot multiplication. The best PGR combination was found to be lµM NAA and lOOµM kinetin, which gave a ten-fold increase in the multiplication rate when compared with the control. 98 5. 3 G. simplex 5. 3 .1 Multiplication of Cultures Derived From Tuber Explants Stock cultures (Section 2.14) consisting of a four-shoot clump growing on WP medium were subcultured onto fresh medium as described in Section 2.15. A limited number of variations in the PGRs in MS and WP media were tested and the results are shown in Table 5.12 and the Analysis of Variance of the data are shown in Appendix 20. Table 5.12 The effect of PGRs and medium on multiplication of shoots and tubers and tuber weight from shoots of G. simplex after eight weeks culture. Values are the mean of five replicates. In a column, means followed by a common letter are not significantly different at the 5% level. Basal No. of No. of Weight of PGR Combination Medium New Tubers per Tubers per Shoots per Replicate Replicate (mg) Replicate lOµM NAA + 40µM BAP MS 8.0 e 2.2 C 480 be lOµM NAA + 40µM BAP WP 9.4 d 3.6 b 360 C lOµM IAA + 40µM BAP MS 12 b 3.8 b 740 a lOµM IAA + 40µM BAP WP 15 a 5.2 a 560 b lµM NAA + lOOµM Kinetin MS 11 d 3.8 •b 530 be lµM NAA + lOOµM Kinetin WP 12 b 4.8 ab 420 be The Analysis of Variance of results show that all dependant variables were significantly influenced by the different treatments (Appendix 20). WP medium gave greater shoot multiplication than MS medium at the same auxin and cytokinin combination (Table 5.12). This was also true for the number of tubers per replicate, except the NAA and kinetin combination (Table 5.12). In contrast, greater tuber weight per replicate (740mg) was achieved in MS medium. An important difference between MS and WP is the higher nitrogen content (60mM and 15mM, respectively) and this may account for the larger weight of tissue formed. For shoot and tuber multiplication, the best combination of PGRs was found to be lOµM IAA and 40µM BAP. This also gave the best weight of tubers per replicate (Table 5.12). 99 5.3.2 Multiplication of Cultures Derived from Shoot Base Explants The stock cultures maintained on MS and WP media supplemented with PGRs as shown in Table 5.13 were used in this experiment (Sections 2.14 and 2.15). The results are shown in Table 5.13 and the Analysis of Variance of the data is shown in Appendix 21. Table 5.13 Comparison of shoot multiplication of shoot cultures derived from G. simplex shoot base explants. Values are the mean of five replicates. Means followed by a common letter are not significantly different at the 5% level. Media Composition No. of New Shoots per Replicate MS (l0µM IAA + 40µM BAP) 10.4 b WP (1 0µM IAA + 40µM BAP) 13.2 a MS (lOuM NAA + 40µM BAP) 8.0 C WP (l0uM NAA + 40µM BAP) 7.2 C MS (lµM NAA + lOOµM kinetin) 6.8 d WP (lµM NAA + lOOµM kinetin) 3.6 d MS (1 0µM NAA + lO0µM kinetin) 4.4 d WP (l0µM NAA + lOOµM kinetin) 5.2 d There were significant differences between the media when tested at the 5% level (Appendix 21). The best shoot multiplication (13.2 per replicate) was achieved on WP medium, supplemented with lOµM IAA and 40µM BAP (Table 5.13). The NAA and kinetin combinations proved to be relatively ineffective on both media (Table 5.13). 100 5. 3. 3 Multiplication of Cultures Derived from Root-Coleoptile Axes Explants Six different treatments of NAA and kinetin in WP medium were compared to determine the most suitable combination for multiplication of shoots derived from root coleoptile axis explants (Sections 2.15, 2.23 and 2.24). The results are shown in Table 5.14 and the Analysis of Variance of the data is shown in Appendix 22. The statistical analysis of the data showed that the different treatments had a significant effect on shoot multiplication (Pr < 0.05)(Appendix 22). The best combination of NAA and kinetin was found to be lµM and lO0µM, respectively (6.4 new shoots/replicate) (Table 5.14). Table 5.14 The effect of NAA and kinetin on multiplication of shoot cultures derived from root-coleoptile axis explants of G. simplex after eight weeks culture on WP medium. Values are the mean of five replicates. Means followed by a common letter are not significantly different at the 5% level. PGR (µM) No. of New Shoots per NAA Kinetin Replicate 0 0 0.6 d 0 10 2.0 cd 0 100 2.0 cd 1 10 4.0 b 1 100 6.4 a 10 100 3.2 be 5. 3. 4 Multiplication of Shoots Derived from Undifferentiated Callus Undifferentiated callus obtained from mature tuber explants was maintained on WP medium supplemented with 2,4-D (23µM) and kinetin (lµM). When transferred to WP medium supplemented with lµM NAA and lO0µM kinetin it began to form shoot primordia (Photograph 5.4a). The results shown in Table 5.15 were from an experiment performed to determine the effect of other auxin and cytokinin combinations on the multiplication of the shoot primodia which had already been initiated on the above medium. The Analysis of Variance of the data for this experiment is presented in Appendix 23 and they show that the ability of callus to regenerate shoots was 101 significantly affected by the composition of the media (Pr < 0.05) when tested at the 5% level (Appendix 23). The maximum number of shoots from lcm3 callus was 34 after 16 weeks and it was on WP medium supplemented with 1OµM IAA and 40µM BAP (Photograph 5.4b and Table 5.15). This combination was statistically better than the other combinations on WP medium. Photograph 5.4a Shoot formation Photograph 5.4b Multiplication from G. simplex callus when grown of shoots from G. simplex callus on WP medium supplemented with when transferred to WP medium lµM NAA and lOOµM kinetin for 16 supplemented with lOµM IAA and weeks. 40µM BAP after eight weeks culture. Table 5.15 The effect of different PGR treatments on shoot regeneration from callus of G. simplex after 16 weeks culture on MS and WP media. Values are the mean of five replicates. In a column, means followed by a common letter are not significantly different at the 5% level. Media Composition No.of Shoots per Replicate MS (lOµM NAA + 40µM BAP) 21 C WP (lOµM NAA + 40µM BAP) 25 be WP (lOµM IAA + 40µM BAP) 34 a WP (lµM NAA + lOOµM kinetin) 29 be 102 5. 4 G. verschuurii 5. 4. 1 Multiplication of Cultures Derived From Tuber Explants Stock cultures (Section 2.14) growing on WP medium were subcultured onto three multiplication media as described in Section 2.15. Selection of medium and PGR treatments were based on the results of the experiments performed for G. rothschildiana, G. superba and G. simplex in a range of PGR treatments. Selected three combinations (Table 5.16) in general performed better shoot and tuber multiplication for other species. Data were collected and analysed as described in Sections 2.23 and 2.24. The results are shown in Table 5.16 and the Analysis of Variance of the data is shown in Appendix 24. Table 5.16 The effect of PGR treatments on shoot and tuber multiplication and tuber weight, of shoots derived from tuber explants of G. verschuurii after eight weeks culture on WP medium. Values are the mean of five replicates. In a column, means followed by a common letter are not significantly different at the 5% level. No. of New No. of Tubers Weight of PGR Treatment Shoots per per Replicate Tubers per Replicate Replicate (mg) lOuM NAA + 40uM BAP 8.0 b 2.6 C 477 b lOµM IAA + 40uM BAP 10.8 a 4.6 a 818 a lµM NAA + lOOuM kinetin 7.8 b 3.4 b 506 b Maximum shoot multiplication (11 new shootsper replicate), tuber multiplication (4.6 per replicate), and mean fre'6h weight of tubers (818mg per replicate) all occurred with lOµM IAA and 40µM BAP. The other media had significantly lower rates of shoot and tuber multiplication and mean tuber weight (Table 5.16 and Appendix 24). 103 5. 5 G. richmondensis 5.5.1 Multiplication of Cultures Derived From Tuber Explants Stock cultures (Section 2.14) growing on WP medium were subcultured onto three multiplication media as described in Section 2.15. Selection of medium and PGR treatments for this experiment were based on the results of the experiments performed for G. rothschildiana, G. superba and G. simplex in a range of PGR treatments. Selected three combinations (Table 5.17) in general performed better shoot and tuber multiplication for other species tested. Data were collected and analysed as described in Sections 2.23 and 2.24. The results are shown in Table 5.17 and the Analysis of Variance of the data is shown in Appendix 25. Table 5.17 The effect of PGR treatments on shoot and tuber multiplication, and tuber weight, of shoots derived from tuber explants of G. richmondensis after eight weeks culture on WP medium. Values are the mean of five replicates. In a column, means followed by a common letter are not significantly different at the 5% level. No. of No. of Weight of PGR Treatment New Shoots Tubers per Tubers per per Replicate Replicate Replicate (mg) lOµM NAA + 40µM BAP 11.6 C 3.2 b 432 a lOµM IAA + 40µM BAP 18.6 a 4.4 a 500 a 1uM NAA + 1OOµM kinetin 12.6 b 2.0 C 402 a Maximum shoot multiplication (19 new shoots per replicate), tuber multiplication (4.4 per replicate) and mean weight of tubers (500mg per replicate) all occurred with with l0µM IAA and 40µM BAP, although there was no significant differencekrespect to mean tuber weight (Table 5.17). 104 CHAPTER 6 RESULTS 6. 0 Root and Tuber Development The main objective in Stage ID of micropropagation is manipulation of the medium to promote the growth of tubers and root system which is strong enough to withstand the transition from the in vitro conditions to soil and glass house growth. Tuberization has e. proved to be a successful alternative strategy that ow-comes the difficulties of in vitro to ex vitro growth. or- The results presented in this chapter faro a series of experiments aimed at production of either plantlets with attached tubers and a well developed root system or of tubers suitable for storage. In addition, the results from the measurement of growth parameters during tuber development are presented. 6. 1 Root and Tuber Development of Gloriosa Species Stock cultures were randomly taken from multiplication cultures at the eighth week and subcultured onto WP medium without PGRs and contained in 150mm (height) x 65mm (diameter) polycarbonate jars (Photograph 6.1). There were 10-20 replicates (40 to 80 shoots) for each species, except for G. verschuurii where only five replicates were used. The experiments were performed as described in Section 2.19 and the cultures were harvested after eight weeks. Data were collected and analysed as described in section 2.23 and 2.24. The results are shown in Table 6.1. 105 Photograph 6.1 G. superba single shoot, after two weeks culture on WP medium contained in polycarbonate jars for root and tuber development Table 6.1 Growth parameters of tubers harvested from WP medium after eight weeks culture. Values are the mean of 10-20 replicates except for G. verschuurii where five replicates were used. ± SO No of Tuber Length of a Total Weight Species Tubers per Weight Tuber (mm) of Tubers per Replicate (g) Replicate (g) G. rothschildiana 6.4 ± 2.2 0.24 ± 0.12 8.3 ± 2.4 1.51 ± 0.25 G. superba 3.0 ± 1.0 0.52 ± 0.24 5.4 ± 3.5 1.56 ± 0.28 G. simplex 4.2 ± 1.4 0.38 ± 0.18 5.2 ± 2.8 1.57 ± 0.25 G. verschuurii 4.0 ± 1.2 0.40 ± 0.24 3.8 ± 1.5 1.60 ± 0.28 G. richmondensis 4.0 ± 0.8 0.32 ± 0.12 4.6 ± 1.8 1.30 ± 0.12 106 At the end of the culture period each replicate had a well developed root system, with > 10 roots per plantlet that were > 5cm in length (Photograph 6.2). Photograph 6.2 G. superba plantlets with an attached tuber and well developed root system after eight weeks culture on WP medium. Although there were variations in the growth parameters among the species (Table 6.1), no significant variation was found in the total weight of tubers per replicate. This suggests that within a replicate there may be competition between the individual tubers for a nutrient that was a limiting factor in tuber growth. 6. 1. 1 The Effect of Sucrose Concentration on Root and Tuber Development The effect of sucrose concentration on the growth and development of tubers on PGR-free WP medium was performed as described in Section 2.20. Data were collected and analysed as described in Sections 2.23 and 2.24. The results are shown in Table 6.2 and Figure 6.1. The Analysis of Variance of the data is shown in Appendices 26a, 26b and 26c. 107 Table 6.2 The effect of sucrose concentration on the growth of tubers on WP medium after eight weeks culture. Values are the mean of five replicates. Within a column, means followed by a common letter are not significantly different at the 5% level. Total Weight of No. of Tubers per Tuber Length (mm) Tubers per Replicate Replicate ( ) Sucrose (%,w/v) Species 1 Species 2 Species 1 Species 2 Species 1 Species 2 0 0.0 d 1.7 C 0 d 1.4 C 0.0 C 0.60 C 1 4.5 C 1.9 C 6.4 C 5.9 b 1.9 ab 0.86 C 2 6.3 b 3.0 be 8.3 be 5.4 b 1.6 b 1.6 b 3 7.4 ab 4.2 b 11.0 a 12.0 a 2.6 ab 2.2 b 4 7.8 a 6.6 a 9.2 ab 12.0 a 3.0 a 3.4 a 5 6.6 ab 4.0 b 9.4 ab 14.0 a 2.4 ab 2.0 b Species 1: G. rothschildiana Species 2: G. superba The results show that the growth of tubers was significantly affected by the sucrose concentration (Pr < 0.05) when tested at the 5% level (Appendices 26a, 26b and 26c). In addition the interaction between a species and the sucrose concentration was found to be significant at the 5% level (Appendix 26a, 26b and 26c). However there e was no significant difference between the two spfeies for either the pooled mean length of tubers or the pooled mean weight of tubers (Appendices 26b and 26c). There were significant differences within species between the different sucrose concentrations according to the DMRT (Table 6.2). For both species, 4% sucrose clearly gave the best response. 108 8 The maximum number of tubers per a Q, replicate, for G. rothschildiana (7 .8) and ~ ,_ 6 G. superba (6.6) was achieved with 4% "',_ ~ .=i sucrose in the medium(Photographs 6.3a = 4 ...;, ,_ ~ and 6.3b). Increasing the sucrose .=is 2 .. z= concentration to 4% from the 2% ':':'.:'.: ~:·:~:·: :,,·_:,_,,_:_:,_:_:,,.::_:,,:,, .. ff ... normally found in WP medium, doubled 0 0 2 4 15 the number of tubers per replicate for i G. superba, while that for ._.s "',_ ~ .=i G. rothschildiana increased about 25%. -= 10 ...;, Both species doubled the weight of .c ell -C tubers per replicate when the sucrose was ~ 5 C :::;"~ doubled from 2% to 4%. The length of tubers showed no significant change 0 2 4 4-r------, between 3% and 5% sucrose for both C species (Table 6.2). Neveretheless, at all concentrations of sucrose tested good :§ "',_ formation of roots was observed :ii 2- .=i = (Photograph 6.3a) . ...;, 0 G. rothschildiana ITill G. superba Figure 6.1 The effect of sucrose concentration on the growth of tubers from G. rothschildiana and G. superba shoot cultures on WP medium after eight weeks culture. Values are the mean of five replicates. (a) Number of tubers per replicate (b) Average length of a tuber (c) Weight of tubers per replicate 109 Photograph 6.3a The effect of sucrose concentrations on the growth of roots and tubers of G. superba after eight weeks culture on WP medium without PGRs. [(a) 0%; (b) 1%; (c) 2%; (d) 3%; (e) 4% and (f) 5%] Photograph 6.3b G. superba plantlets after eight weeks growth of shoots on WP medium supplemented with 4% sucrose and without PGRs. 110 6. 1. 2 The Effect of ABA and GA3 on Root and Tuber Development of G. rothschildiana. Experiment was performed and data were analysed according to the method described in Section 2.21 and 2.24. The results are shown in Table 6.3 and the Analysis of Variance of the data is shown in Appendix 27. Table 6.3 The effect of ABA and GA3 on the growth of roots and tubers of G. rothschildiana after eight weeks culture on WP medium. Values are the means of five replicates. Means followed by a common letter within a column are not significantly different at the 5% level. No. of Tuber Weight of No. of Mean Treatments Treatment Tubers Length Tubers per Roots per Length of Number per (cm) Replicate Replicate a Root Replicate (mg) (cm) Control* 1 6.8 a 0.76 a 1110 a 8.8 a 3.2 a ABA (0.5µM) 2 5.4 b 0.50 be 491 b 1.0 b 1.4 C ABA(lµM) 3 3.4 cd 0.6 b 472 b 0 b 0 C GA3 (lµM) 4 4.0 C 0.56 be 551 b 1.0 b 1.9 ab GA3 (l0µM) 5 3.0 e 0.47 be 320 b 0 b 0 C ABA(lµM)+ 6 3.2 e 0.42 C 312 b 0 b 0 C GA3 (lµM) * WP medium without ABA and GA3. The Analysis of Variance results show that growth of tubers and roots was significantly affected by the ABA and GA3 treatments (Appendix 27). They show that the number of tubers, their weight and their size was significantly decreased when the medium contained either ABA or GA3 (Table 6.3). Similar results were observed for the number of roots per replicate and the mean length of the roots (Table 6.3). 111 CHAPTER 7 RESULTS 7 . 0 Establishment In Soil Establishment of propagules in soil (Stage IV of the micropropagation) is one of the critical factors that determine the success of micropropagation. The results presented in this chapter are from experiments aimed at determining the ability of Gloriosa propagules or tubers to adapt to ex vitro growth conditions until flowering and fruit setting. 7. 1 Survival Rates of In vitro Generated Gloriosa Species in Soil Establishment and maintenance in soil were as described in Section 2.22. After two months the number of viable shoot clumps or number of tubers that had sprouted were recorded. The results are shown in Table 7 .1. Table 7.1 The survival of micropropagated plantlets or tubers of Gloriosa species eight weeks after transfer to soil (peat:sand 3: 1). Values are the mean of 30-40 replicates except for G. verschuurii which are the mean of 10 replicates. Propagule Survival ( % ) In vitro Propagules G.rothschil- G. superba G. simplex G. verchuurii G.richmon- Transferred to Soil diana densis Shoot clumps from multiplication 86 75 80 nd nd medium Shoot clumps from root and tuber >95 >95 >95 >95 >95 development medium Tubers from root and tuber development 100* 100* 100* 100* 100* medium nd= not done. * % of sprouting after 2-3 weeks They show that the survival of shoot clumps from root and tuber development medium (Stage Ill) was higher than those from multiplication medium (Stage Il)(Table 7.1). Mature tubers from root and tuber development medium (ie. those with completely 112 senescent shoots), sprouted and developed into viable plants with 100% success (Photograph 7.1). Photograph 7.1 G. rothschildiana plant derived from sprouted tuber when tubers harvested from root and tuber development medium was transferred to soil. 1..0~e Either freshly harvested mature tubers or those that had been stored at ambient temperature for up to 12 months exhibited 100% sprouting. Thorough soil irrigation was needed during the initial stages of sprouting. No plants flowered during the first growth cycle in soil. 7. 2 Yield of Tubers After the First Growth Cycle Under Glasshouse Conditions Six months after transfer to soil, established plants began to senesce. When this was completed, the dormant tubers were harvested and their number and weight recorded. The results are shown in Table 7.2 and Photographs 7.2a and 7.2b. 113 Table 7.2 Growth measurements of Gloriosa tubers after the first growth cycle in soil. Values are the mean of 20-30 replicates except for G. verschuurii which are the mean of 10 replicates. ± SD Species No.of Tuber Tuber Tuber Tubers per Weight (g) Weight per Length* Pot Pot (g) (cm) G. rothschildiana 6.5 ± 1.2 4.8 ± 1.2 31.3 ± 3.5 5.8/2.5 G. superba 6.3 ± 1.5 3.9 ± 0.8 24.4 ± 6.2 5.3/2.9 G. simplex 5.0 ± 1.2 3.1 ± 0.8 15.9 ± 4.5 4.3/2.1 G. verschuurii 4.0 ± 1.0 3.6 ± 1.0 14.4 ± 3.5 5/1 G. richmondensis 6.0 ± 1.4 2.8 ± 0.5 16.8 ± 4.4 3/2 * The two figures are the length of each wing of the "V" shaped tubers. The best yield of tubers was from G. rothschildiana (31.3g) and G. superba (24.4g). 7. 3 Yield of Tubers After the Second and Third Growth Cycles Under Glasshouse Conditions. Growth measurements were made after the second growth cycle (12 months after deflasking) and the third growth cycle (18 months after deflasking). The results are shown in Table 7 .3 and 7.4. There was a large increase in the weight and size of the tubers between the first and second six month growth cycle. For example, the mean tuber weight for G. rothschildiana increased from 4.8g to 15.5g, and the tuber size from a total length of 8.3cm to 11cm. Similarly there was rapid growth of the tubers during the third growth cycle with the G. rothschildiana mean tuber weight increasing to 50g and total tuber length to 20cm (Table 7.4). Flowering and seed set occurred during the third growth cycle, with a frequency of about 75%. 114 Photograph 7.2 a G. superba tubers harvested during the first growth cycle in soil, before plants had begun to senesce. Photograph 7.2b G. superba tubers harvested at the end of the first growth cycle in soil after senescence of the aerial pans. 115 Table 7.3 Growth measurements of Gloriosa tubers after the second growth cycle in soil. Values are the mean of 20-30 replicates except for G. verschuurii which are the mean of 10 replicates. Species Mean Weight per Tuber (g) Tuber Length (cm) G. rothschildiana 15.5 ± 4.2 8/3 G. superba 12.6 ± 3.0 6/4 G. simplex 8.0 ± 2.4 6/2 G. verschuurii 10.8 ± 4.2 6/3 G. richmondensis 8.4 ± 2.8 4/2 Table 7.4 Growth measurements of Gloriosa tubers after the third growth cycle in soil. Values are the mean of at least 10 replicates. Species Mean Weight per Tuber (g) Tuber Length (cm) G. rothschildiana 50± 10 12/8 G. superba 38 ± 12 14/10 G. simplex 26 ±08 8/4 G. verschuurii nd G. richmondensis 30±06 10/8 (nd)- Data not yet recorded with sufficient number of replicates. 7. 4 The Effect of Potting Medium on Tuber Growth of G. superba Shoot clumps derived from the root-coleoptile axis were harvested from multiplication medium (WP medium supplemented with lµM NAA and lOOµM kinetin) and transferred to either peat:sand (3:1), or a commerical potting mix (Hortico) as described in Section 2.22. The results are shown in Table 7 .5. The data were analysed and the treatments ranked using the DMRT. The results of the Analysis of Variance are shown in Appendix 28. The results show that there was no significant difference between the two potting media (Table 7 .5). 116 Table 7.5 The effect of potting medium on growth parameters of G. superba tubers after the first six month growth cycle. Values are the mean of 10 replicates. In a column, means followed by a common letter are not significantly different at the 5% level. Potting mix Mean No.of Weight of Tubers Mean Length of Tubers Per Pot Per Pot (g) Tuber (cm) Peat:sand (3: 1) 6.4 a 34 a 10.0 a Commercial mix 5.2 a 30 a 9.8 a 7. 5 Continuous Micropropagation Protocol for Gloriosa Species A comparison of the multiplication rates for each stage of micropropagation on the best media when using tuber explants is presented in Table 7.6. The multiplication rate varied with the species. Four explants taken from a single tuber of G. rothschildiana could potentially produce 6000 plantlets in 48 weeks including an eight week soil establishment period. If stage IV is replaced by another 8 week multiplication cycle then 40,000 plantlets with microtubers could be produced in 48 weeks. It may be possible to adapt these to large bioreactors for biomass production and hence the industrial production of colchicine. At the end of the soil establishment period, the number of vigourous tubers of G. superba is about 800 compared to the 1-2 produced by conventional propagation. The species variation in annual multiplication is mainly due to the difference in multiplication rates during Stage II. However the multiplication rates also varied with the type of explant used. For example, when the explant was root coleoptile axis tissues, the annual multiplication of G. superba was higher than that of G. rothschildiana (calculations not shown). Table 7.6 Description of continuous micropropagation protocol for Gloriosa species and their rate of multiplication at each stage. Data is for a tuber explant on WP medium. Rate of Multiplication Stage Time Medium G. rothschildiana G. superba G. simplex G. verschuurii G. richmondensis (weeks) I. Initiation lOµM NAA + 40µM BAP 1.9 1.6 1.8 1.6 8 (lOµM NAA+20µM BAP)* 2.5 II. Multiplication IOµM IAA +40µM BAP 5.3 3.8 2.7 4.6 8 (5.33) (3.83) (2.73) (4.63) (8x3)¥ (lµMIAA +20µM BAP)+ 7.3 (7.33) III. Root and tuber 8 PGR free 1.6 0.75 1 1 1 development IV. Establishment 8 Peat : Sand (3: 1 v/v) 0.95 0.95 0.95 0.95 0.95 in soil Theoretical No. of plantlets surviving from a single tuber 1478 201 83 37 148 explant per 48 weeks.<][ Multiplication rate /tuber/annum § 5912 804 332 148 592 * lOµM NAA and 20µM BAP PGR combination for G. rothschildiana shoot initiation. + lµM IAA and 20µM BAP PGR combination for G. rothschildiana. multiplication. ¥ Three multiplication cycles. --...J 118 CHAPTER 8 RESULTS 8 . 0 Extraction, Purification and Quantitation of Colchicine From Gloriosa. 8 . 1 Extraction and Purification from Plant Tissue Initially fresh plant tissue (5g) spiked with [3H]-colchicine, was homogenized in either water, lOmM phosphate buffer (pH 7) or MeOH and the colchicine extracted as described in Section 2.25. Recovery of [3H]-colchicine was typically > 90% for all extraction solvents. Aliquots were analysed by TLC (Section 2.29) and HPLC (Section 2.27) and typical results are shown in Figures 8.1 and 8.2 respectively. Good resolution of colchicine on TLC was achieved, but a partial overlap with another solute was evident in the HPLC trace, interfering with quantitation. Although containing fewer peaks, HPLC of CHCl3 extracts still contained the interfering contaminant. Solvent .~root C c::::::, C) <:=> 0 __ , ···: ,. ,--, .. .. -- .- , c::» c::::> .--·-•' c:::, ;=:, Origin 1 2 3 Figure 8.1 TLC of colchicine standard (1), hot water extract of G. superba leaves (2) and the colchicine containing fraction from C-18 Sep-Pak filtrates (3) derived from sample 2. Chromatographic conditions are given in Section 2.29. 119 0.1 . ("' . ·------· --·---- - ·-- - Colchicine QJ ~ C _g 0.05 I. 0 r'1 ,Q < ! - -- . ------+-~+--. o 2 4 6 8 10 Time (mins) Figure 8.2 HPLC trace of a hot water extract of G. rothschildiana tubers_ Chromatographic conditions are given in Section 2-27. Rt for colchicine was 6.5 min. 120 To simplify the isolation procedure by eliminating the labour-intensive CHCl3 extraction step, C-18 Sep-Pak extraction was investigated. This proved very successful, with all the colchicine in a [3H]-colchicine spiked aqueous samples being retained on the cartridge matrix. After washing, > 80% of the [3H]-colchicine could be eluted with aqueous 30% AcCN/MeOH (3:1, v/v) (Table 8.1), giving a significantly 'cleaner' fraction (Figures 8.1 and 8.3). However, the HPLC trace again showed the peak that partially overlapped that of colchicine. Baseline separation of these two peaks by HPLC was not achieved, either with variations in solvent composition or the use of gradient elutions. Table 8.1 Recovery of [3H]-colchicine eluted from a C-18 Sec-Pak solid phase extraction catridge loaded with an extract from G. superba leaves. Chromatographic conditions are given in Section 2.26. Fraction Fraction Eluate Recovery of No. Colchicine (%) (a) Sample load 0 (b) H2O wash 0 (c) 20% AcCN: MeOH (3:1) 2 (d) 30% II II 90 (e) 50% II II 4 (f) 100% II II 1 Homogenisation in lM HCl followed by the solid phase extraction protocol effectively eliminated the contaminating peak. The results of TLC and HPLC analysis of the fraction eluted with aqueous 30% AcCN : MeOH (3: 1, v/v) are shown in Figures 8.4 and 8.5. This procedure was subsequently used for isolation and partial purification of the colchicine found in Gloriosa tissues. 121 0.5 0.7 0.5 Colchicine standard (b) (c) -E 0.4 0.4 C: ~ II) ~ '-' 0.3 0.3 QJ 0.35 c..i C: ~ .Q a. 0.2 0 0.2 r,i ,Q ~ 0.1 0.1 o.o.,__ ___ 0.0 ----- 0.0 6.5 13 0 6.5 0 6.5 13 0.5 0.5 0.5 (d) (e) (f) 0.4 -~ 0.4 0.4 '-' 0.3 0.3 0.3 0.2 0.2 I 0.1 0.1 i. rI L 0.1 0.0-i----i ....__ __,; 0.0 ~ 0.0-1.._ ____ ....___ 1 0 6.5 13 0 6.5 13 O 6:s 13 Time (mins) Time (mins) Time (mins) solid Figure 8.3 HPLC traces of fractions (see Table 8.1) eluted from a C-18 Sep-Pak phase extraction cartridge loaded with an extract from G. superba leaves. HPLC conditions are given in Section 2.27. Rt of colchicine was 6.5 min. § HPLC trace of sample load fraction is not shown 122 Solvent Front 0 0 0 . . . ~··· .. ~ .. :._ .,"'· .... , 1...... e::::, Origin 1 2 3 Figure 8.4 TLC of colchicine standard ( 1), and the colchicine containing fraction eluted from C-18 Sep-Pak catrigdes of a hot water extract (2), and a lM HCl extract (3) of G. superba leaves. Chromatagraphic conditions are given in Section 2.29. - (a) (b) =5 f"') II) -f"') Time (mins) Time (mins) Figure 8.5 HPLC traces of the colchicine containing fraction eluted from C-18 Sep-Pak cartridges of a hot water extract (a), and a lM HCl extract of G. superba leaves (b). Chromatographic conditions are given in Section 2.27. 123 8. 2 Quantitation of Colchicine Standard curves for colchicine were constructed by plotting the peak area against the amount of colchicine injected. The injection volumes ranged from 7 .5µ1-50µ1. The standard curve for extracts from shoots, tubers and glasshouse grown plants is shown in Figure 8.6. For the analysis of the callus extracts, the HPLC detector sensitivity was increased and a corresponding standard curve used (100ng-2000ng; graph not shown). Both standard curves were linear over the range of colchicine concentrations tested. The precision and reproducibility of the values was verified by repeated injections of standard at different dilutions and injection volume. 200 ------.y = - 0.24343 + 17.811x R"2 = 1.000 0 +----.--.....----...-...... -..- ...... -..--.-----.--~ 0 2 4 6 8 1 0 1 2 Colchicine (µg) Figure 8.6 Standard curve for colchicine. Values are the mean of three injections. 8. 3 Purity of The Internal Standard ([3H]-Colchicine) The purity of the [3H]-colchicine was determined by HPLC and TLC methods. A colchicine solution was spiked with [3H]-colchicine and aliquots chromatographed as described in Section 2.27. The eluate from HPLC was collected every 20 sec and the radioactivity determined. The purity of the [3H]-colchicine was calculated as the fraction 124 of the radioactivity injected that corresponded to the colchicine mass peak. An aliquot of the spiked colchicine solution was chromatographed by TLC as described in Section 2.29. The silica gel corresponding to the colchicine carrier was scraped off and the radioactivity determined. The purity of the [3H]-colchicine was calculated as the fraction of dpm present in the band against the total dpm chromatographed. The purity of the [3H]-colchicine was 75% using HPLC and 80% by TLC. Because HPLC was used to quantitate the colchicine present in tissue extracts, its purity was taken to be 75% for the calculations in this work. 8 . 4 Mass Spectrometry The HPLC colchicine fractions from leaf, tuber, and seed HCl extracts of each Gloriosa species and S. aurantiaca were pooled from multiple injections and the solvent removed in vacua. The white amorphous powder was analysed by solid probe electron impact mass spectrometry. Some representative traces are shown in Figures 8.7b and 8.7c along with that of a colchicine standard (Figure 8.7a). Both the colchicine standard and the tissue samples gave identical mass spectrums, with a strong molecular ion (M+) at m/z 399. The fragmentation patterns were also identical confirming that the HPLC fractions were essentially pure colchicine. In addition the fragmentation pattern was similar to that obtained by Finnie and van Staden, (1991) for G. superba and S. aurantiaca extracts. 125 r _.... 1 00 3 2 5 . 9ES 95 a 5 . 6ES 90 5 . 3E5 85 5 . lES BO 4 . 8ES 75 4 . SES 70 4 . 2E5 65 3 . 9E5 3 99 60 3 . 6E5 55 3 . 3ES 5 0 3 . 0E5 45 2 . 7E5 40 2 97 2 . 4ES 35 2 . lES 2 81 3 71 30 l . BES 25 l . SES 2 0 l . 2ES 15 8 . 9E4 3 40 10 5 . 9E4 3 . OE4 0 . OEO '~~~~ -~~J 400 .. ,so·~-. 5 0 m/ z. 100 3 2 7. 9E4 95 b 7 . SE4 90 7 . 1E4 85 6 . 7E4 BO 6 . 3E4 75 6 . OE4 70 5 . 6E4 65 399 5 . 2E4 60 4 . 8E4 55 4 . 4E 4 50 .... . 4 . OE4 45 -~- 3 . 6E 4 40 =Q,I 2 97 3 . 2E4 35 2 . BE4 30 37 1 -= 2 81 2 . 4E4 ""'" 25 Q,I 2 . 0E4 2 0 > l . 6E4 356 ·- 15 l.2E4 10 2s , 269 I -= I 7 . 9E3 ~ c:i:: 2 4 . 0E3 .~ ul,.,uhlijr.,,~ijl1~Jul11 1Jijl' ., l,1_JJ L.~ L,"'~L.~ 1·1: ... ,. lr, .. J .. ,~ .. ,.. ., 1, .. , 0 . OE O o 2io 2!o 230 2<10 2so 7ro' 2~ 2 o 290 300 3io 320 3:io 3 o 350 360 31 0 380 390 4 o , ------m/ z 2 6 5 . 3ES rl::· 5 . IES C 90 4 . BES 85 4 . SES 80 4 . 3E5 75 4 . OES 70 3 . ? ES 3 99 65 3 12 3 . SES 60 I 3 . 2E5 55 2 . 9ES 50 2 . 7E5 45 3~6 2 . 4ES 40 2. !ES 35 I . 9E5 2 42 30 2 13 l . 6E5 1 . JES 1 . lES B. OE4 5 . 3E 4 2 . 7E 4 1 1~~ 1/.1,,;1:;!;JJ 1,- •. , ..; 0 . OEO l1~r. 0 ;, 4 0 J m/ z. [__l!.::..::._:______1~;;-· .. '.~: 1tr~:l.,~L~ ~1! - - . - - m/z Figure 8.7 Electron impact mass spectra of colchicine standard (a) and those isolated from G. rothschildiana tuber tissue (b) and G. ve rschuurii leaves (c). 126 CHAPTER 9 RESULTS 9. O Colchicine Content of In Vivo and In Vitro Tissues of Gloriosa Species and In Vivo Tissues of Sandersonia aurantiaca The importance of colchicine and the potential of plant tissue culture for the production of plant secondary metabolites has been discussed previously. A method for rapid micropropagation and tuber production of Gloriosa species was developed in this work and the results have been presented earlier in this thesis. A method for the quantitation of colchicine from tissues of Gloriosa was developed and used to determine the colchicine content of in viva and in vitro generated Gloriosa tissues. The results of these experiments are described in this chapter. 9. 1 Colchicine Content of In Vivo Tissues 9. 1 . 1 Colchicine Content of Glasshouse Grown Gloriosa and Sandersonia aurantiaca Plants of the five Gloriosa species and S. aurantiaca were grown under identical conditions in a glasshouse and analysed for colchicine at different times in the growth cycle. The results are shown in Table 9.1 and Figure 9.1. The seedlings had a relatively high concentration of colchicine, but it was low in the stems and shoots of more mature plants. During each growth cycle the mother tuber regressed as new daughter tubers developed. The colchicine content appears to parallel this reproductive habit and decline in the parent tuber as it increases in the offspring. At the end of a growth cycle, the main site of colchicine accumulation was the seeds and daughter tubers (Figure 9.1). However during the first growth cycle, when flowering does not occur, the tubers were the main site of colchicine accumulation. N -..} - glasshouse. nd nd nd nd nd nd 1.6 5.0 a 0.58 0.59 0.24 0.13 in aurantiaca S. grown nd nd nd nd nd nd nd nd 0.4 5.4 3.3 4.8 aurantiaca richmondensi,r, G. DW) Sandersonia nd nd nd nd nd nd nd nd nd (mg/g 3.3 5.1 and 0.26 verschuurii G. Content species Gloriosa nd nd nd nd 7.8 8.4 9.4 4.8 2.0 Colchicine 7.8 0.51 0.57 five superba the G. for cycle 1.7 1.9 6.7 8.3 8.7 3.6 3.3 2.7 7.7 0.46 0.63 0.37 simplex growth G. the in times nd nd nd nd 2.8 7.2 5.3 2.9 4.8 6.8 0.34 0.69 rothschildiana G. different determinations. at duplicate content of flowering) (at mean seedlings) week) week) months) stem Colchicine the flowering) (2 Tissue done tubers flowering) (4th (8th (whole) tubers* of (from are (at 9.1 and (at not coat = Values Source Table SeedlinJ?;s Shoots Tubers Flowers Roots Mother Daughter Pericarp Seed Pericarp Tubers Seeds nd 128 10------, D seeds - IEJ tubers at flowering Q~ 7.5 l!I shoots at flowering ~ -e~ 5 -Cl-' = ~ ·-.c: ·-~ -u0 2.5 2 3 4 5 6 Species Figure 9.1 Colchicine content of various tissues of Gloriosa species and Sandersonia aurantiac;a. 1 G. rothschildiana 2 G. superba 3 G. simplex 4 G. verschuurii 5 G. richmondensis 6 S. aurantiaca Indeed, the colchicine concentration in these tubers was higher than the seeds from the same plant produced in the next growth cycle. The colchicine content of the dried fruit (pericarp) was low compared to that of the seeds themselves in the two species tested (Table 9.1). 9. 1. 2 Distribution of Colchicine Within a Mature G. simplex Vine and a Tuber A G. simplex vine was harvested just after flowering and sections analysed for their colchicine content (Section 2.33). The results are shown in Figures 9.2 and 9.3. About 75% of the colchicine in the aerial part of the plant was concentrated in the apical 10cm of the plant, with a decreasing gradient from the apex to the base. However, the section closest to the tuber showed a small increase in colchicine. This could be due to inadvertent excision of tuber material at the stem/tuber junction or diffusion from the much higher concentration in the tuber (Figure 9.3). The colchicine content of the sectioned tuber 129 also decreased from the meristematic tissue at the distal end of the tuber arms to the basal plate in the "V" shaped tubers (Figure 9.3). - ~ 0.75- Cll ~e -~ 0.5 - C ·-:a~ ~ 0 U 0.25 - 0 _____...... J·...... :·:·:.,...·:·:· ..... :]_ .._. ·-·. ·..... · .. _ ...... _.__....· ..... · ...... ·..... · ·- I I I I I top 10 cm 2 nd 10 cm 3 rd 10 cm 4 th 10 cm 5 th 10 cm Section of the Shoot Figure 9.2 Distribution of colchicine within the aerial part of a G. simplex vine just after flowering. 5~------. 4- 3- ~ C 2- ·-~ ·-.c ~ 0 u 1 - !l\llilil:111 :•:•:-:-:-:-:- ::::::::::::: ...... 0 -----.----""'1.t""-...... __ ...... ,;.;...,...... "-' Distal 2 cm Next 2 cm Next 2 cm Next 2 cm Proximal Section of the Tuber Figure 9.3 Distribution of colchicine in a mature tuber of G. simplex just after flowering. 130 9. 2 Colchicine Content of In Vitro Cultures Tissue was harvested at every stage of micropropagation and its colchicine content y determined. The aim of this stu•was to assess the capacity of each species to accumulate colchicine and how modification of the culture conditions might affect this accumulation. 9. 2 .1 Colchicine Content of Shoot Initiation Cultures (Stage I). The colchicine content of pooled shoots or tubers harvested from shoot initiation cultures (4 x 4 matrix of NAA/kinetin) at the eighth week when using tuber tissue as explant material (Section 4.1.1) are shown in Figures 9.4 and 9.5. Shoot and tuber tissues were separated before colchicine determination. 10~------. I:] MSmedium - WPmedium Q~ 7.5- ~ ~~ ~e 5- -~ = ·-CJ ·-.c CJ u0 2.5- ~i ·:·:·:·:·:·: ::::!!::::.::::;:; 0 - ...... ~l""""'------1-- ...... ~~- .... G. rothschildiana G. superba G. simplex Species Figure 9.4 The colchicine content of pooled shoots of Gloriosa harvested from a NAA and kinetin 4 x 4 matrix on MS and WP media at the eighth week from initiation. Values are the mean of duplicate isolations from 5g of the pooled matrix tissue. 131 15------, (] MSmedium BJ WPmedium -~ Q 10- c.o -ec.o -QI = ·-CJ 5- ·--=CJ -u0 0 ...L..Ji...:...... ,...... iL...... """""'1~-i...... i ...... ~i,;,;.;,;,;..-1..1 G. rothschildiana G. suverba G. simplex Species Figure 9.5 The colchicine content of pooled tubers of Gloriosa harvested from a NAA and kinetin 4 x 4 matrix on MS and WP media at the eighth week from initiation. Values are the mean of duplicate isolations from 5g of the pooled matrix tissue. For each species, the colchicine content of tubers was greater than shoots. There was little difference between the two media with respect to tuber colchicine, but both G. rothschildiana and G. superba shoots had a significantly greater concentration of colchicine when grown on WP medium. 9. 2. 2 Colchicine Content of Shoot Multiplication Cultures (Stage II) The colchicine content of the separated shoot and tuber tissue from the five species after eight weeks on two of the best media for multiplication, are shown in Table 9.2. The colchicine content of whole tissues (shoots and tubers) of G. rothschildiana harvested from different multiplication medium after two, four and eight weeks of culture are shown in Table 9.3 and Figure 9.6. The colchicine content after four weeks varied from 2.7mg/gDW to 9.7mg/gDW and after eight weeks from 3.9mg/gDW to lOmg/gDW depending on the multiplication medium (Table 9.3). 132 Table 9.2 Colchicine content of shoots and tubers from each species harvested from two multiplication media after eight weeks culture. Values are the mean of duplicate isolations from 5g of pooled tissue harvested from five replicates. Colchicine Content (mg/gDW) Tissue Plant Multiplication Basal Shoots Tubers Medium Medium G. rothschildiana 3 WP 1.6 9.9 5 WP 2.2 13 G. simplex 3 WP 2.6 12 5 WP 4.0 17 G. superba 3 WP 4.0 12 5 WP nd nd G. verschuurii 3 WP nd nd 5 WP 0.9 7.6 G. richmondensis 3 WP nd nd 5 WP 2.8 5.1 nd = not detected # 3 = lOµM NAA + 40µM BAP incorporated in WP medium # 5 = lµM IAA + 20µM BAP incorporated in WP medium Table 9.3 Colchicine content of whole tissues of G. rothschildiana harvested from different multiplication media after two, four and eight weeks of culture. Values are the mean of duplicate isolations from 5g of pooled tissue harvested from five replicates. Multiplication Basal Medium Time of Harvest Colchicine Content Medium (weeks) (mg/g DW) 0 3.8 1 WP 4 2.7 8 3.9 0 3.8 2 MS 4 4.1 8 4.4 0 3.8 3 WP 4 4.4 8 6.2 0 3.8 4 WP 4 8.2 8 9.0 0 3.8 5 WP 2 8.0 4 9.7 8 10 #1- NAA, lµM; Kine tin, 1OOµM incoporated in WP medium #2- NAA, lOµM; BAP, 40µM incoporated in MS medium #3- NAA, lOµM; BAP,40µM incoporated in WP medium #4- IAA, lOµM; BAP, 40µM incoporated in WP medium #5- IAA, lµM; BAP, 20µM incoporated in WP medium 133 Interestingly, most of the colchicine accumulated in the first four weeks of growth, with relatively little increase thereafter. Indeed in medium 5, from which a two week sample was taken the colchicine concentration reached 80% of its maximum value at the second week. (Figure 9.6). 10 D lnoculum @] 2nd week -~ Q 7 .5 4th week CJ:) II CJ:) - 8th week ..._,= 5 • ~ ·~= ·-.c (,J u-0 2 .5 0 1 2 3 4 5 Multiplication medium Figure 9.6 Colchicine content of whole tissues of G. rothschildiana harvested from different multiplication media after two, four and eight weeks of culture. Compositions of multiplication media are given below. # 1 - NAA, lµM; Kinetin, lOOµM incoporated in WP medium #2- NAA, lOµM ; BAP, 40µM incoporated in MS medium #3- NAA, lOµM; BAP,40µM incoporated in WP medium #4- IAA, l0µM; BAP, 40µM incoporated in WP medium #5- IAA, lµM; BAP, 20µM incoporated in WP medium The colchicine content of the separated shoot and tuber tissue of G. rothschildiana harvested from different multiplication media after eight weeks culture are shown in Table 9.4. 134 Table 9.4 Colchicine content of shoots and tubers of G. rothschildiana harvested from different multiplication media after eight weeks culture. Values are the mean of duplicate isolations from 5g of pooled tissue harvested from five replicates. Colchicine Content (mg/g DW) Medium Basal Medium Shoots Tubers 1 WP 2.2 7.6 2 MS 1.7 10 3 WP 1.6 9.9 4 WP 1.9 12 # 1- NAA, lµM; Kinetin, lOOµM incorporated in WP medium # 2 - NAA, lOµM; BAP, 40µM incorporated in MS medium # 3 - NAA, lOµM; BAP, 40µM incorporated in WP medium # 4 - IAA, lOµM; BAP, 40µM incorporated in WP medium Comparing Table 9.3 with Tables 9.2 and 9.4 it is clear that most of the colchicine is present in the tubers, with much less in the shoots. Similarly comparing Table 9.4 with Table 9 .1 it appears that G. rothschildiana tissues generated in vitro can have a significantly higher colchicine concentration than glasshouse grown material. The combination of IAA and BAP in WP medium was particularly effective in shoot and tuber multiplication (Table 5.1 and Table 5.4 respectively). Therefore the effect of these growth regulators on colchicine accumulation in G. rothschildiana shoot cultures was more thoroughly examined. Tissue was harvested after four and eight weeks culture and analysed for colchicine. The results are shown in Table 9.5 and Figure 9.7a and 9.7b. They show that at each IAA concentration there was a significant effect of BAP on the colchicine concentration, with an optimum at 20µM BAP. 135 Table 9.5 The effect of IAA and BAP in WP medium on the growth and colchicine accumulation of G. rothschildiana shoot cultures. Data for tissue weight are the mean of five replicates and that for colchicine concentration are the mean of duplicate isolations from 5g of pooled tissue harvested from five replicates. PGR Colchicine (mg/g OW) Total Tissue Total Treatment DWat8th Colchicine Experiment IAA:BAP 4th Week 8th Week week at 8th week Number (µM) (wreplicate) (mwreplicate) 1 1: 0 0.86 5.1 0.18 0.92 2 1:10 6.3 7.8 0.34 2.7 3 1:20 9.7 11.0 0.38 4.2 4 1:40 2.5 3.1 0.35 1.1 5 1:60 1.1 3.6 0.26 0.94 6 20:0 3.3 4.9 0.17 0.83 7 20:10 4.8 6.2 0.37 2.3 8 20:20 9.3 9.1 0.28 2.5 9 20:40 2.2 3.0 0.12 0.36 10 20:60 1.7 3.9 0.19 0.74 This results is pleasing because lµM IAA and 20µM BAP also strongly promotes multiplication of shoots and tubers. The results also strongly suggest that there was a direct interaction between the PGR treatments and the synthesis of colchicine. The effect could also be due to a difference in the number of tubers per shoot for any particular PGR treatment. For example, if the tuber to shoot ratio is high, the contribution of tubers is greater and therefore the colchicine concentration will be higher because tubers contain more colchicine than shoots. However the temporal data suggests that the colchicine is synthesised early when tuberization would be less. 136 15 ...... ------, [] week4 lµM IAA IE] week 8 10 1=l) -e1=l) --~ = ·-(,J ~ 5 Q u 0 10 20 40 60 [BAP] (µM) Figure 9.7a The effect of different concentrations of BAP combined with lµM IAA on colchicine concentration of shoot cultures of G. rothschildiana after four and eight weeks culture on WP medium. Values are the mean of duplicate isolations from 5g of pooled tissue harvested from five replicates. 10 ...... ------, IAA [) week4 20µM Ii] week8 -~ Q 7.5 1=l) -1=l)e 5 --~ = ·-(,J .c ·-(,J 8 2.5 0 10 20 40 60 [BAP] (µM) Figure 9.7b The effect of different concentration of BAP when combined with on colchicine concentration of shoot cultures of G. rothschildiana after four and eight weeks culture on WP medium. Values are the mean of duplicate isolations from 5g of pooled tissue harvested from five replicates. 137 9.2.3 Manipulation of Multiplication Medium for Increased Yield of Colchicine from G. rothschildiana Shoot Cultures. WP medium supplemented with lµM IAA and 20µM BAP gave the highest colchicine concentration (1 lmg/gDW) and amount (4 mg/replicate) and tissue yield (0.38g OW/replicate) after eight weeks culture (Table 9.5). The nitrogen and sucrose in the medium were modified as described in Sections 2.17 and 2.18 to see if this would increase the yield of colchicine. Previously it was found that increased sucrose concentration from 2% to 4% (w/v) in this medium unaffected tuber yield per replicate although number of shoots and tuber per replicate were decreased significantly (Table 5.6). The results of the sucrose experiment are shown in Table 9.6. The colchicine in shoots increased four-fold when the sucrose concentration was raised to 4%, although there was a 20% decrease in dry matter (Table 9.6). The elevated sucrose concentration also significantly increased the colchicine concentration of tubers, but with a small increase in dry matter reflecting the slight increased tuber yield(Table 9.6). Table 9.6 The effect of sucrose on colchicine concentration and biomass of shoots and tubers of G. rothschildiana after eight weeks culture on WP medium. Data for tissue weights are the mean of five replicates and that for colchicine concentration are the mean of duplicate isolation from 5g of pooled tissue harvested from five replicates. Sucrose Colchicine Tissue Dry Weight Total Colchicine Concentration (mg 1g DW) (g/replicate) (mg)/replicate (g/lOOmL) Shoots Tubers Shoots Tubers from whole tissue 2 2.1 12 0.47 0.25 4.0 4 8.3 19 0.36 0.28 8.2 These remarkable increases resulted in a doubling of the colchicine yield per replicate. Experiments testing the effect of nitrogen involved varying the ratio of NI4+ to N03-, while keeping the total nitrogen at either 15mM, 30mM or 60mM, as described in Section 2.17. The results of these experiments are shown in Table 9. 7 and Figures 9 .10 and 9 .11. 138 Table 9.7 The effect of Niti+ and NO3- on colchicine concentration of G. rothschildiana shoot and tuber tissues after eight weeks culture on WP medium. Values are the mean of duplicate isolations from 2g-5g of pooled tissue harvested from five replicates. Nitrogen Composition Colchicine Concentration (mM) (mg/~ DW) Niti+ NO3- Shoots Tubers 0 15 1.7 6.7 5 10 3.1 9.6 10 5 1.4 l.Of 15 0 0.7 0 0 30 1.6 6.4 10 20 3.1 9.9 20 10 0.54 6.1 30 0 0.25 0 0 60 0.21 9.3 20 40 1.6 11.0 40 20 0.25 0 60 0 0 0 When NH4+ was used as the sole source of nitrogen, no tuber formation was observed (Table 5.5). Consequently little or no colchicine was detected (Table 9.7). Nevertheless the Niti+fNQ3- treatments used in this experiment had a marked effect on the colchicine concentration of both shoots and tubers. Interestingly, the highest colchicine concentration was achieved in both shoots and tubers when the Niti+:NO3- ratio was 1:2, irrespective of the total nitrogen concentration (Table 9.7). It is worth noting that 20mM NH4+ : 40mM NO3- is that found in MS medium and 5mM NH4+ : lOmM NO3- in WP medium. Yet there is little difference in the tuber colchicine concentration between these two media. Nevertheless WP medium was significantly better for multiplication of shoots and tubers at these PGR concentration (lµM IAA and 20µM BAP) (Table 5.1 and 5.4). In general other combinations of Niti+ and NO3- gave significantly less colchicine. 139 ·-~ .c: O.s ~ u0 a Fig. 9.10 The effect of NJLi+ and N03- on colchicine concentration of G. rothschildiana shoot tissues after eight weeks culture on WP medium. Values are the mean of duplicate isolations from 2g-5g of pooled tissue harvested from five replicates. A= N03- only B = NJLi+ : N03-, 1:2 C = NJLi+: N03-, 2: 1 D = NH4+ only 140 lZ ,-._ ~ lO Q e.o ~ -e.o .._,,E 6 Figure 9.11 The effect of NI4+ and N03- on colchicine concentration of G. rothschildiana tuber tissues after eight weeks culture on WP medium. Values are the mean of duplicate isolations from 2g-5g of pooled tissue harvested from five replicates. A= N0 3- only B = NI4+: N0 3-, 1:2 C = NI-4+: N03-, 2: 1 D = NI-4+ only 9. 2. 4 The Effect of Putative Colchicine Precursors on the Colchicine Content of Shoots and Tubers of G. superba . Putative precursors were added to liquid medium as described in Section 2.31. The results of these feeding experiments are shown in Table 9.8 and the results of Analysis of X Variance are shown in Appendi@"# t29J. The Analysis of Variance of the data found that the precursor treatments had a significant (Pr < 0.05) effect on the colchicine content. With shoots, only tyrosine gave an increase in colchicine concentration (3.5mg/g DW) compared with the control (2.3 mg/g OW). All other precursor treatments resulted in a significant decrease in the colchicine concentration (Table 9.8). 141 Table 9.8 The effect of putative colchicine precursors on the colchicine content of G. superba shoot cultures after eight weeks growth on a modified WP medium (4% sucrose, 20mM NH4+, 40 mM NO3-, lµM IAA and 20µM BAP). Values are the mean of duplicate isolations from 2g-5g of pooled tissue harvested from duplicate cultures. In a column means followed by a common letter are not significantly different at the 5% level. Treatments Colchicine Content (mg/g DW) Precursor (lmM) Shoots Tubers Control 2.30 b 4.8 C Phenylalanine 0.94 cd 3.1 e Tyrosine 3.5 a 3.2 e p-Coumaric acid 1.7 C 8.2 a Cinnamic acid 0.92 d 6.4 b Tyramine-HCl 1.0 C 4.3 d Phenylalanine+ Tyrosine 1.5 C 3.1 e In contrast, with tubers, tyrosine decreased the colchicine concentration by 33% and cinnamic acid increased it by 33% (Table 9.8). The best stimulation occurred with p-Coumaric acid which almost doubled the colchicine content by the end of the eight weeks growth. It should be noted that these cultures are growing in agitated liquid media and that direct comparison with solid media is not possible. Thus the colchicine content of tubers in the control is less than that for the nearest solid media (Table 9.2). 9. 2. 5 Colchicine Content of Root and Tuber Development Cultures (Stage 111) The main objective of Stage III of Gloriosa micropropagation was to produce well developed plantlets with good-sized tubers and a well functioning root system. Root and tuber development medium consisted of WP medium containing 2% sucrose and no PGRs. The colchicine concentration of tissues associated with root and tuber growth during the 142 development stage was detennined for each of the Gloriosa species. In addition the ability of altered sucrose concentrations to increase the production of colchicine was examined. Shoots with or without tubers were harvested from WP medium (lµM NAA and lOOµM kinetin) and transferred to PGR-free WP medium as described in Section 2.19. Shoots and tubers were harvested at the 4th and 8th weeks and the colchicine quantitated (Sections 2.25 to 2.30). The results are shown in Table 9.9. G. rothschildiana plants in culture contained their maximum colchicine concentration after four weeks in both stems and tubers. For the other species, the maximum occurred in tubers after eight weeks culture. The decline in colchicine concentration in G. rothschildiana between the fourth and eighth week of culture may be due to the rapid growth of tubers during this period. At the eighth week some of the stems were senescing, while others still appeared to be functional. These were analysed separately for colchicine and the senescing stems found to have a very low colchicine concentration. This could be due to colchicine degradation or transport to the growing tubers and functional leaves. Roots had very small quantities of colchicine compared to the other tissues (Table 9 .9). Table 9.9 The colchicine content of shoot and tuber tissues at different physiological ages of Gloriosa species in Stage III of micropropagation. The colchicine concentrations are the mean of duplicate isolations from 5g of pooled tissue harvested from five replicates. Colchicine content (m_g/gDW) Source of Tissue Plant Age Shoots Tubers Roots (weeks) G. rothschildiana 4 6.1 13.8 nd 8 2.0 (0.5*) 7.6 0.4 G. simplex 4 nd nd 0.3 8 nd 6.4 nd G. superba 4 5.0 9.6 nd 8 2.6 (0.8*) 11.3 0.6 G. verschuurii 4 nd nd 8 nd 8.8 nd G. richmondensis 4 nd nd . . 8 nd 4.5 nd (*) Colch1cme content of senescmg leaves found on the lower part of the stem . (nd)-not done 143 Because sucrose altered the colchicine concentration of tissues growing on multiplication medium (Table 9.6), the effect of sucrose on colchicine concentration of G. rothschildiana and G. superba tissue cultured on PGR-free WP medium was also determined. The results are shown in Table 9.10 and Figures 9.12a and 9.12b. The higher colchicine concentration of G. rothschildiana (8.5mg/gDW) was achieved with 4% sucrose while that for G. superba (15mg/gDW) was at 5% sucrose (Table 9.10 and Figures 9.12a and 9.12b). However both species achieved their maximum total amount of colchicine per jar at 4% sucrose (Table 9.10). Table 9.10 The effect of sucrose on colchicine concentration and biomass of tubers of G. rothschildiana and G. superba after eight weeks culture on WP medium in the absence of PGRs. Data for tuber dry weight are the mean of five replicates and that for colchicine concentration are the mean of duplicate isolations from 5g of pooled tissue harvested from five replicates. G. rothschildiana G. superba Sucrose Colchicine Tuber Total Colchicine Tuber Total (g/lOOmL) (mg/gDW) Dry Weight colchicine (mg/g DW) Dry Weight Colchicine (g/iar) (mg/iar) (g/iar) (mg/iar) 0 1.7 0.07 0.13 2.2 0.07 0.16 1 6.5 0.46 3.0 8.8 0.36 3.2 2 7.6 0.72 5.5 11.3 0.91 10.2 3 8.0 1.1 9.0 11.6 1.1 12.8 4 8.5 1.3 11.0 13.0 1.4 18.3 5 5.3 1.1 5.9 15.0 1.1 16.1 144 12.5 ------, [] mg/gDW 10 - IE) mg/jar ... 0 ~ 7.5- Q t e 5 - -~ =CJ ·-:S 2.5 - CJ u0 0 1 2 3 4 5 Sucrose concentration (g/l00mL) Figure 9.12a The effect of sucrose on colchicine yield from G. rothschildiana after eight weeks culture on PGR-free WPmedium. ~ 20------, ~ .:! [] mg/gDW -a t ffi] mg/replicate =iie 15- ... 0 ~:-:-:-- Q~ 10- C.() =iie 5- -~ = CJ I ·-:a CJ 0 rn ;:::; u 0 ....1.1,=-f,-..1.:.-F;;;;.a.J.:.:.:-iF>=LJ.:.-fi:i;;;;;;;a...&..;.;.~;;a..i;..:..:.+;;;;.;;;;;u 0 1 2 3 4 5 Sucrose concentration (g/l00mL) Figure 9.12b The effect of sucrose on colchicine yield from G. superba after eight weeks culture on PGR-free WPmedium. 145 9. 3 Colchicine from In vitro Generated Plants When Grown Under Glasshouse Conditions Plantlets with tubers and a well developed root system, in vitro generated tubers after storage, or shoot clumps with developing tubers and roots were transferred to soil and maintained under glasshouse conditions (Section 2.22). Growth performance was monitored until the flowering stage. Different parts of the plant were analysed for colchicine near the end of the first growth cycle. The results are shown in Table 9 .11. Flowers had a low colchicine concentration which was similar to that from wild-type plants (Table 9.1). Table 9.11 Colchicine concentration of tubers just before flowering, and flowers produced from in vitro generated plants when grown under glasshouse conditions. colchicine content are the mean of duplicate isolations from I 0g of pooled tissue from tubers harvested from 10-20 pots or 5g of pooled tissue from randomly selected 10 flowers. Colchicine Content (mg/g DW) Source of Tissue Plant Tubers Flowers G. rothschildiana 3.4 0.58 G. superba 9.5 0.59 G. simplex 10.0 0.83 G. verschuurii 5.4* nd G. richmondensis 4.3 nd * Mature tubers but not of a sufficient size to produce flowers nd = not done A comparison of the colchicine concentration of tubers from in vitro and in vivo propagated plants grown under glasshouse conditions is shown in Table 9.12 and Figure 9.13. 146 Table 9.12 Comparison of colchicine contents of in vitro and in vivo generated tissues of Gloriosa species. In vitro tissues were from stage I of the micropropagation. Colchicine (mg/g DW) G. rothschildiana G. surierba G. simplex Tubers Shoots Tubers Shoots Tubers Shoots In vivo 4.8 0.34 8.4 0.51 8.7 0.46 In vitro (WP) 11.5 9.2 10.7 8.7 9.4 4.4 In vitro(MS) 11.7 7.3 12.1 5.0 8.2 4.2 * Other species were not included as their initiation cultures were limited only to selected PGR treatments. The results comparing here were from pooled tissues from identical set of grids. 12.5 D. Wild Type -~ Q 10 - Em Micropropagated ~- 1:,1) -s1:,1) - 7.5 - 2 3 4 5 Species Figure 9.13 Colchicine concentration of tubers derived from in vivo and in vitro propagated Gloriosa when grown under glasshouse conditions. * 1 G. rothschildiana 2. G. superba 3. G. simplex 4.G. verschuurii 5. G. richmondensis 147 There were only small differences in the colchicine concentration between the two sources of tubers, suggesting that the ability to produce secondary metabolites has not been affected by the transfer through in vitro conditions. 9.4 Colchicine from Undifferentiated and Redifferentiated Callus Two types of callus were established from Gloriosa species (Section 3). Stable and friable undifferentiated callus was obtained only from G. simplex leaf sheath explants. Tissues taken from other parts of this plant and other plants also formed callus on many media, but all of these quickly (within 1-2 weeks) redifferentiated forming a large number of root-like structures, which in this thesis have been called "rooted callus". 9. 4 .1 Colchicine Concentration of G. rothschildiana and G. simplex Differentiated Callus The colchicine concentrations of rooted callus from G. rothschildiana and G. simplex when proliferated on three media supplemented with a variety of auxin and cytokinin concentrations are shown in Table 9.13. Table 9.13 The colchicine content of G. rothschildiana and G. simplex rooted callus after eight weeks culture. Values are the mean of duplicate isolations from 5g of pooled tissue harvested from five replicates. Colchicine Content (mg/g OW) Media and PGR Comoosition G. rothschildiana G. simvlex MS lOµM NAA; 40µM BAP 0.20 0.08 WP lOµM NAA; 40µM BAP 0.08 nd MS lOµM NAA; lu.M Kinetin 0.18 nd MS lOu.M NAA; lOuM Kinetin 0.15 0.04 SH l0uM NAA; lOOµM Kinetin 0.07 nd SH lOOµM NAA; l0uM Kinetin 0.12 nd MS lµM IAA 20µM BAP* 0.13 nd SH lµM NAA; lOµM Kinetin* 0.09 0.05 * Liquid medium nd= wasn't done 148 The most notable feature is the very low concentration of colchicine in the callus tissue, particularly when compared to differentiated tissue (shoots and tubers). Because of the low concentrations, it is difficult to know if there are significant differences between treatments, although MS medium, with its higher nitrogen concentration, appears to increase the amount of colchicine present. The results of another experiment using various concentrations of IAA and BAP in WP medium for G. superba showed that rooted callus gave a colchicine content that range from 0.03mg/g DW to O.OCJ mg/g DW. 9.4.2 Colchicine Concentration of G. simplex Undifferentiated Callus The colchicine content of undifferentiated callus from G. simplex derived from leaf sheath explants on MS, WP and B-5 media supplemented with either NAA or IBA and kinetin at the rate of lOOµM and lµM respectively are shown in Table 9.14. c.oJl1A5 Table 9.14 The colchicine content of G. simplex undifferentiatedt_after eight weeks culture. Values are the mean of duplicate isolations from 5g of pooled tissue harvested from five replicates. Media and PGR Composition Colchicine content (m DW) MS 100 MNAA; 1 M kinetin Trace• WP 100 NAA; 1 kinetin Tracetjt B-5 100 NAA; 1 M kinetin 0.03 MS 100 MIBA; 1 M kinetin Trace\l, WP 100 M IBA; 1 M kinetin Trace• B-5 100 IBA; 1 kinetin 0.02 149 ed G. simplex undifferentiated callus showed only tracefs of colchicine when proliferat* on MS or WP medium. However on B-5 medium with the same PGR composition it produced just detectable amount of colchicine. 9 . 5 A Protocol to Screen and Recover Cell lines Highly Productive For Colchicine It is generally accepted that genetic variation (somaclonal variation) can arise spontaneously in all types of plant cell cultures. The conservation of these mutated cultures may provide the basis for the establishment of highly productive lines for natural product synthesis. However for the recovery of useful traits, it is important to have a suitable protocol to screen the cultures and then conserve them. A protocol was devised and tested so that if spontaneous variations in a culture's synthetic capacity could be detected, the cell line could be conserved as differentiated tissue. Callus was induced from a single tuber of G. simplex (Section 2.6). Shoots were initiated on independant pieces of callus (Section 2.13) and subsequently tubers (Section 2.19). Fifteen tubers were taken and each tuber was longitudinally split into two sections. One section of each tuber was analysed for colchicine and the other was cultured on shoot initiation medium followed by multiplication medium, to preserve the cell line through plant regenaration. All fifteen cell lines were conserved through regenerating plants and some were transferred to soil (Section 2.22). The colchicine concentration of the individual tubers is shown in Figure 9.14. 150 6-.------, 5- -~ Q 4- 3 - =~ ·-CJ ·--5 f::::_l=:::_l=:::_ T==_i::i==. u0 : 1 ··=,... ":""=.== T==::_i=====_i==::_ 1 - 1:: :=: rn :=: li 11 Q I I I I I I I I I I I I I I I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Tuber number (cell line) Figure 9.14 Colchicine content of G. simplex tubers produced via redifferentiation of callus. Values are the mean of duplicate isolation of 1 to 2g of tissues from longitudinally split tubers. They show that the tubers derived from a population of callus cells were heterogeneous with respect to their colchicine concentration. The initial callus showed only traces of colchicine, but upon redifferentiation, those cell lines demonstrated some capacity for colchicine synthesis. However the cell line with the best colchicine concentration (No. 12) was still available for further study. 151 CHAPTER 10 DISCUSSION Initiation and Maintenance of Callus Cultures Until comparatively recently monocotyledonous plants have been regarded as difficult to culture in vitro. Hussey (1975) working on several genera of Liliaceae found that the effect of auxin and its concentration on callus formation differed widely. The results of the present study also found variations in callusing among species of the genus Gloriosa. Furthermore the response varied for different explants taken from the same plant.Explants from mature tuber explants of G. rothschildiana, G. superba and G. simplex easily formed undifferentiated callus when cultured on either MS or WP medium supplemented with 2,4-D and kinetin at various concentrations and combinations (Chapter 3). It was also found that other components in the medium influenced callusing. G. superba callused on both MS and WP medium, but G. simplex and G. rothschildiana achieved better growth on WP medium when 2,4-D was replaced by equimolar NAA. The undifferentiated callus soon redifferentiated forming a large number of root-like structures on both MS and WP medium. Only rooted callus was obtained with all media and PGR combinations when using either root, stem or leaf explants from young seedlings. However when carefully separated leaf sheath explants of G. superba and G. simplex were cultured on WP medium supplemented with NAA and kinetin, good friable callus was obtained which could be maintained indefinitely in the undifferentiated state. Finnie and van Staden (1989) also describe the callusing of G. superba tissue. They obtained undifferentiated callus from embryos, and tuber explants with 2,4-D and kinetin on MS medium. However it is not clear if they used leaf sheath tissue as an explant. They also found that on removal of the callus from 2,4-D, multiple root and/or tuber formation occurred and claimed that the tubers were similar in morphology to that of the wild 152 type. The present study found these root-like structures to be more like unorganised roots, rather than tubers. True tubers were formed in vitro either at the base of multiple shoots or occasionally on the surface of tuber explants. When these tubers were cultured, either on fresh media or transferred to soil, sprouting occurred. In contrast, the root-like bodies failed to sprout either in vitro or in the soil. Tubers from the base of shoots produced multiple shoots when sectioned and cultured on shoot initiation media, whereas sectioned root-like bodies failed to produce even a single shoot. Finnie and van Staden (1989) do not appear to have tried sprouting these structures or transfering them to soil. Light microscope observations of hand sectioned tissues also found that these root like structures were more comparable with roots than tubers. When clumps of rooted callus were transferred to liquid medium, they rapidly multiplied, increasing the length of the root like structures up to 20 cm. In contrast, when true tubers were transferred to liquid medium they neither multiplied nor elongated. The only observation was sprouting after four to six weeks. The colchicine content of the rooted callus was similar to that of true roots and much lower than that of tubers. Based on this morphological, physiological and biochemical evidence, the root-like structures were clearly unorganised roots or rooted callus and not tubers. However, Hussey (1975) also found redifferentiation of callus from monocotyledons, including Liliaceae plant tissue, particularly when the medium had a relatively low concentration of auxin. Continued high proliferation of undifferentiated callus derived from tuber explants of G. superba (3 fold increase/month) was observed with 2,4-D and kinetin in WP medium. Once callus had been initiated with the necessary high concentration of 2,4-D, excellent proliferation could be maintained with a lower concentration. SH medium proved to be the best for proliferation of the rooted-callus. The callus formed from tuber explants was granular in texture and friable in structure, while that formed from the leaf sheath of seedlings was soft and friable. Finnie and van 153 Staden (1989) reported that undifferentiated and rooted callus exuded a clear "gel" at the interface between the medium and callus. This was also observed here except for undifferentiated callus from leaf sheath explants. No attempt was made to characterise this substance. This study also provides evidence for a species specific variation in callus initiation and growth. The establishment of differentiated and undifferentiated callus from Gloriosa species, will be useful for biochemical and physiological studies, and phenotypic improvement through somatic hybridization and somaclonal variations. Establishment of e callus is a prerequisi~in the production of secondary metabolites from cell cultures. The use of plant cell cultures has contributed significantly to the progress in the enzymology of alkaloid biosynthesis (Zenk, 1980). Biomass with a rapid growth was established when root-like structures from redifferentiated callus, were transferred to liquid medium. It is interesting to note that this vigorous elongation of root-like structures occurred without formation of laterals. The ability to grow in agitated liquid medium, its stability and fast growth thus providing large biomass may be a useful feature in utilising this in vitro material in large-scale bioreactors for the production of secondary metabolites. Limited attempts to establish cell suspention cultures from each species were unsuccessful. 154 Micropropagation In this project some of the factors that might effect in vitro organogenesis of Gloriosa were investigated. These were the species, explant source, basal medium and plant growth regulator combinations. Explants were from mature plants, aseptically grown seedlings or in vitro generated tubers. A summary of the results from the best shoot iniation media are shown in Table 10.1. Table 10.1 Best media treatments from matrix experiments for shoot initiation from different explants of Gloriosa species. Source of Medium PGR Number of Shoots per Explant per 8 Weeks Explant Treannent (uM) G. rothschildiana G. suverba G. simvlex Root/coleoptile WP NAA/Kin (1:100) 1.1 2.1 2.0 axis WP NANBAP (0:20) 0.75 WP NANBAP (10:40) 0.28 0.45 WP NAA/Kin (1:100) 0.4 0.3 0.2 Shoot base WP NANBAP (1:20) 1.8 WP NANBAP (10:40) 0.42 MS NANBAP (10:40) 0.95 WP NAA/Kin (1:100) 2.0 1.8 1.5 Tuber WP NANBAP (10:20) 2.5 WP NANBAP (10:40) 1.9 1.6 A summary of the statistical analysis of the data is shown in Table 10.2 and they clearly show that there was a significant influence by species, explant type, medium and p PGR treatments (fr< 0.05), and their interactions except species x medium. Nevertheless a s few generalisation[about culture treatments for shoot initiation can be made. For example, most explants grew well on WP medium and all tuber explants performed best on this medium with NAA and BAP. Furthermore,meristematic tissue, eg. tuber tips and root coleoptile axes, were much more regenerative. In summary, each species needed to be 155 individually optimised with respect to explant type, medium and PGR combination for shoot initiation. Table 10.2 Summary of the analysis of variance of shoot initiation from explants of Gloriosa species under the influence of selected PGR treatments in MS and WP media after eight weeks culture. Source of Variance D.F. Mean Square F-Value Pr>F Model 19 3.24 34.55 0.0001 Error 160 0.09 Species (Sp) 2 0.4025 4.47 0.0230 Explant (Ex) 2 17.49 186.4 0.0001 Medium (M) 1 1.59 16.32 0.0001 PGR 1 0.4218 4.49 0.0204 SpxEx 4 1.26 13.45 0.0001 SpxM 2 0.0942 1.00 0.3686 Sp x PGR 2 0.5797 6.18 0.0026 ExxM 2 0.6078 6.48 0.0022 ExxPGR 2 8.69 92.68 0.0001 MxPGR 1 0.4152 4.48 0.0370 This is the first study presenting such a comprehensive comparison of in vitro organogenesis between Gloriosa species. Wernicke and Brettel (1980) found that young and meristematic explant tissues of Sorghum bicolor produced regenerative cultures, whereas mature and differentiated explants failed to show such a response. Bruyn et al., (1992) e working on Amaryllis bllladona showed that shoot bud initiation was higher when using young scape tissue than mature tissues. Niederwieser and van Staden (1990) showed the influence of genotype and the endogenous cytokinin level on adventitious bud formation in seven Lachinalia species. Further they found that the cytokinin level in young leaf tissues was higher than in older tissue. In experiments using six species of Lili um, Niimi ( 1986) found a gradient in regeneration potential within an organ as a result of different 156 developmental stages. The differential response of explants to culture conditions could be due to different levels of endogenous hormones within different explants. During the past decade, it has become clear that the hormonal regulation of plant growth and development not only depends on the levels of growth regulators, but also on the sensitivity of the organ to the growth regulators. Hormone receptors are thought to be an important determinant of this tissue sensitivity (Vreugdenhil and Struike, 1989). The abundance of putative hormone receptors can be modulated by external factors (Waltan and Ray, 1981) and can vary during cell development (Vreugdenhil and Struike, 1989). The earlier reports on Gloriosa micropropagation used only MS medium with no suggestion of having tested others (Finnie and van Staden, 1989; Somani et al., 1989; Custers and Bergevoet, 1994). Dougall (1981) reported that reduced nitrogen was essential for organogenesis in several tissue culture systems. It is worth noting that the nitrogen content of WP medium is about a quarter that of MS medium. WP medium was superior to MS medium for shoot initiation and multiplication and this may reflect a requirement for lower nitrogen concentrations for Gloriosa tissues. Root bud formation is known to be stimulated by high nitrogen levels in several plant species (Peterson, 1975). Interestingly, when shoots were cultured on MS medium, some root-like callus developed at the base of shoots. In this study an extended range of auxins and cytokinins was tested in a series of v..)e.('€. matrix experiments and the results a subjected to statistical analysis to assess the significance of treatment differences. The shoot initiation capacity of various types of Gloriosa explants was found to be affected by the growth regulator status of the medium (Section 4). Optimum shoot initiation from root/coleoptile axis tissue was achieved with lµM NAA and 100 µM kinetin in WP medium for all five species of Gloriosa tested. However the optimum for tuber explants and shoot base explants was with various NANBAP combinations, depending on the species. 157 In general Gloriosa responded to high concentrations of cytokinin. For example the optimum BAP varied from 20-40µM, while for kinetin it was as high as lOOµM. Finnie and van Staden (1989) found 2.5 mg/L (1 lµM) BAP or kinetin to be the best for multiple shoot formation from G. superba tuber explants on MS medium. At 5mg/L kinetin or BAP (with 0-5 mg/L NAA) they only obtained single plantlets or callus. Somani et al., (1989) reported that kinetin at 2-4 mg/L (10-20µM) produced multiple shoots on MS medium from sprouted in vitro developed tubers. 1 mg/L kinetin only gave single shoots. They did not test higher concentrations nor other cytokinins. However, Custers and Bergervoet (1994) also found that a high concentration of BAP (10mg/L; 45µM) was required for maximal proliferation of multiple shoots from tuber tips of various G. rothschildiana and G. superba hybrids and cultivars on MS medium. Concentrations higher than 10mg/L were not tested. According to Hussey (1976a) the addition of auxin at appropriate concentrations may well modify the cytokinin concentration required, especially in the Liliaceae. Furthermore he also stated that some species within the Liliaceae required much higher concentrations of BAP to promote organogenesis and that they could tolerate up to 16 mg/L (70µM) BAP without morphological distortion. The results of Custers and Bergervoet ( 1994) and those reported here also support Hussey's findings (1976a). No distortion was observed with BAP concentrations up to 60 µM. The effect of growth regulators on organogenesis of Liliaceous plants in vitro has been studied by many authors. In some cases there was no effect of cytokinins on the regeneration capacity of primary explants and they may even inhibit• differentiation (Aartrijk and Blom-Bamhoom, 1979). Others found that there was a stimulating effect of cytokinins on the regeneration of bulbils and shoots (Takayama and Misawa, 1982). The addition of auxins, even at low concentrations, can significantly increase the number of bulblets obtained in vitro (Stimart and Ascher, 1978). For Gloriosa it was found in this wa..~ work that addition of both auxin and cytokini~ needed for better shoot initiation, multiplication and tuberization from primary explants. 158 These studies also aimed to determine the effect of various combinations and concentrations of auxins and cytokinins in different media on the tuberization of Gloriosa species cultured in vitro. The rate of tuber initiation in Stage I varied with the type of explant used. It occurred consistently with root/coleoptile axis and tuber explants, but very few tuber buds were observed with shoot base explants. There was rooted callus at the base of initiated shoots, especially when using MS medium, which may inhibit tuber initiation. Both G. rothschildiana and G. simplex initiated tuber buds in the absence of PGRs on WP e medium. However tuber initiation was strongly stimulated by addition of auxin~ pecially in the presence of cytokinin. In all cases WP medium gave higher rates of tuber initiation when compared with MS medium, except for the root/coleoptile axis tissues of G. rothschildiana which favoured MS medium. However Somani et al., (1989) and Finnie and van Staden (1989) only used MS medium for tuberization of G. superba. Similarly Custers and Bergevoet (1994) also used only MS medium for tuberization of G. superba and hybrids of G. rothschildiana x G. superba. Basal medium did not significantly effect the weight of tubers, but statistical analysis found that the medium and PGR interaction had a significant effect on the mean weight of tubers per replicate. A comparison of shoot and tuber multiplication at the end of the multiplication period (Stage II) for the best multiplication media are presented in Table 10.3. Although the highest number of tubers was for G. rothschildiana, the mean weight of tubers was the least for this species on the same medium. Perhaps high tuber number and large tuber size are mutually exclusive, because a medium component is limiting, for example, the sucrose. The multiplication of shoots and tubers was also dependant on a specific auxin and cytokinin interaction. This was shown when NAA was replaced by equimolar IAA. Obato-Sasamoto and Suzuki (1979) observed an increased IAA concentration in the stolon of potato shortly before tuber initiation. 159 Table 10.3 Comparison of (a) shoot multiplication (b) tuberisation and (c) tuber weight per replicate of different species of Gloriosa when cultured on selected multiplication media. Values followed by a common letter across the three columns are not significantly different at the 5% level. No. of New Shoots per Re Jlicate Species Medium 1 Medium 2 Medium 3 G. rothschildiana 12.2 ce 19.6 a 9.8 de G. superba 11.8 ce 21.4 a 18.8 ab G. simplex 9.4 de 15.0 be 12.2 ce G. verschuurii 8.0 e 10.8 ce 7.8 e G. richmondensis 11.6 ce 18.8 ab 12.6 cd No. of Tubers per Replicate Species Medium 1 Medium 2 Medium 3 G. rothschildiana 2.4 fg 6.6 a 3.4 cg G. superba 2.0 g 3.8 cf 3.0 cg G. simplex 3.6 cf 5.2 b 4.8 be G. verschuurii 2.6 fg 4.6 bd 3.4 cg G. richmondensis 3.2 dg 4.4 be 2.0 g Weirht of Tubers Per Replicate Species Medium 1 Medium 2 Medium3 G. rothschildiana 140 e 208 de 128 e G. superba 480 be 564 b 494 be G. simplex 360 cd 480 be 420 be G. verschuurii 477 be 818 a 506 be G. richmondensis 432 be 419 be 402 be Medium 1 -WP medium supplemented with lOµM NAA and 40µM BAP Medium 2 -WP medium supplemented with 10µM IAA and 40µM BAP Medium 3 -WP medium supplemented with lµM NAA and l00µM kinetin Working with Narcissus species, Chow et al., (1992) reported that BAP strongly inhibited bulbil formation and size and they suggested that the concurrent BAP inhibition of rooting was reducing the uptake of nutrients and thus limiting the supply of 160 assimilates to tubers. In contrast, in Gloriosa BAP stimulated tuber initiation especially with added auxin. BAP strongly inhibited rooting, but inhibition of rooting did not effect tuberization. BAP had very little effect on shoot length except at 60µM when it was significantly reduced. The optimum time for subbing of shoot cultures was at the eighth week. If multiplication cultures were maintained for longer a decrease in shoot multiplication and spontaneous rooting was observed. Shoot tissues probably do not produce cytokinins as effectively as root tissue (Kuiper et al., 1989) and there is no cytokinin supply from shoots to roots (Henson and Wareing, 1976). Forsyth and van Staden (1981) suggested that root tips are the main site of cytokinin synthesis and if cytokinins are available to plantlets they may not produce roots. Thus when the medium is exhausted with respect to cytokinin, plantlets will produce roots in order to fulfil their internal requirements for cytokinin. This suggestion is supported by the results with Gloriosa shoots which produced roots on aged medium or when transferred to PGR free medium. The best corm production of Gladiolus tristis was achieved when the sucrose concentration was increased from 3% to 6% or 9% (Bruyn and Ferreira, 1992). Increased sucrose concentration (3% to 9% ) also increased the frequency of Narcissus jonquilla bulbing from 9% to 71% (Chow et al., 1992). Heath and Hollies (1965) found that the greater the concentration of sucrose, the greater the frequency of bulbing in onion. Further they showed that non-rooted onion plants gave higher bulbing at all concentrations of sucrose, compared with rooted plants. Increased sucrose concentration in the root and tuber development medium (Stage 111), resulted in a higher number of tubers as well as a higher mean weight of tubers per replicate. Rooting occurred at all concentrations of sucrose in the PGR-free medium, including zero sucrose. The major form of nitrogen taken up by plants is N03-, but there is evidence that the growth rate can be increased when NI4+ is supplied simultaneously (Pilbeam and Kirkby, 161 1992). There have been only limited investigations into the nutrition of monocotyledonous plants (Bach et al., 1992a) and this is the first investigation to determine the effect of medium nitrogen on organogenesis of Gloriosa. It was found that the inorganic nitrogen makeup of the medium significantly affected shoot and tuber multiplication and tuber weight. Shoots of G. rothschildiana failed to multiply or form tubers when NI-4+ was used as the sole nitrogen source. In contrast, when NO3- was used as the sole nitrogen source, good shoot and tuber multiplication occurred. In agreement with these results numerous studies have shown that N14+ as a sole nitrogen source was deleterious to the growth of many higher plants in vivo (Bennette et al., 1964) and in vitro (Scotte et al., 1982; Mantell and Hugo 1989). It was found that a combination of NH4+1No3- in a 1:2 ratio at 30mM was the best for shoot and tuber multiplication of G. rothschildiana. This is probably the reason for the higher shoot and tuber multiplication of m G. rothschildiana on WP medium which contains 15JM nitrogen when compared to MS medium (60mM), although the ratio of NI4+/NO3- is 1:2 in both media. Leblay et al., (1991) showed that NH4+/NO3- ratios favourable to nitrate resulted in increased shoot regeneration in Rosaceous species. There is evidence that NI-4+ inhibits the assimilation of NO3- in plants (Radin 1975; Mackown et al., 1982). Generally the uptake of NO3- is accompanied either by the uptake of a monovalent cation or the release of an OH- equivalent k into the growth medium (Pilbeam and ~rkby, 1992). The uptake of NH4+ is accompanied by the release of a monovalent anion. Since nitrogen containing ions are taken up in large amounts, these differences in uptake of the two nitrogen forms are thought to be responsible for the alkalization or acidification of the rhizosphere if the balance of NI4+ and NO3- is incorrect. This then is thought to depress mineral ion uptake and so reduce the rate of growth (Findenegg et al., 1989). Plant tissue culture media are generally unbuffered and a proper balance of NI-4+ and NO3- will serve to counteract their individual effects on medium pH Okazawa and Chapman ( 1962) suggested that tuberization of potato might be regulated by changes in the balance between a tuber forming stimulus and natural gibberellin inhibitors 162 in the plant. They also suggested that when there is no active growing point to form giberellins, tuberization occurred early. Micropropagation methods for four Gloriosa species and one cultivar were developed in this project. The conditions were defined for routine production of plantlets or tubers and these were successfully transferred to soil, followed by flowering and fruit setting. The procedure was divided into four stages and the conditions for each stage were manipulated separately. These stages were as follows. Stage I Shoot and tuber initiation from primary explants. Stage II Shoot and tuber multiplication on defined media. Stage III Development of plantlets with a well developed root system and larger tubers.~ Stage IV Transfer of propagules to soil and ex vitro conditions. A procedure for the commercial production of either Gloriosa plantlets with tubers or t ,ciormantf bers for storage is outlined in Figure 10.1. Explants were chosen systematically considering the source of tissue and its physiological age. Success was achieved using juvenile or meristematic explant tissue, either from field grown mature plants, aseptically sprouted tuber tips or aseptically grown seedlings. Compared to mature explant tissue, the juvenile tissue had a higher regeneration and proliferation capacity. Early difficulties in disinfesting tubers were overcome by taking only the terminal 3cm section of the tuber and so avoiding the basal plate which had root debris and an uneven surface. This was the area where contamination most frequently originated. Somani et al., (1989) reported that 50% of their G. superba tubers became contaminated. Custers and Bergervoet (1994) report 39- 50% depending on storage. Finnie and van Staden (1989) made no comment. The contamination frequency here was 5%. 163 PARENT PLANT Disinfestation EXPLANT Differentiation STAGE I SHOOT AND TUBER INITIATION (8 weeks) shoots (1-3 cm) STAGE II SHOOT AND TUBER MULTIPLICATION~------,, 6-8 weeks shoots (> 3 cm) STAGE III DEVELOPMENT OF THE ROOT SYSTEM AND LARGER TUBERS 8-10 weeks STAGE IV TUBERS FOR -----1._~ POTTING IN SOIi~ STORAGE l PRODUCTION OF FLOWERS AND TUBERS Figure 10.1 Schematic diagram of a procedure for routinJ micropropagation of Gloriosa species 164 In these experiments attempts were made to regenerate shoots, tubers and roots from the various types of explants keeping the incidence of callusing to a minimum, since callus tissue can introduce variability and it tends to lose regenerative capacity (Torrey 1967). In addition sufficient replicates were taken and experiments were conducted in proper experimental designs when appropriate to perform statistical analysis. The results clearly show the influence of species, explant type, medium and PGR treatment and their interactions on the micropropagation of Gloriosa species. In particular it was found that shoot initiation was crucial but once initiated, a range of media and PGR treatments could be used for shoot and tuber multiplication. The proliferating shoot cultures were maintained for 1-2 years by repeated subculture and the regenerative response of all species remained constant and repeatable. Murashige (1974) included an extra stage in the traditional micropropagation protocol with the objective of better preparing plantlets for re-establishment in soil, by developing the root system and improving the photosynthetic capacity. However Deberge and Maene (1981) argued that because the rooting process in vitro was at least 35% of the total cost of an in vitro produced plant, it was better to avoid in vitro rooting if possible. In the present study shoot clumps with attached tubers could be harvested from Stage nf multiplication medium after 6-8 weeks and transferred directly to soil with a high survival rate and therefore a separate stage for rooting was not needed. Presumably the presence of tubers, which can survive well under ex vitro conditions, or the presence of pre-formed root primordia contributed to the high survival rate. However a Stage III was included in the micropropagation protocol for several reasons. Firstly, plantlets harvested from Stage III cultures could be easily separated from shoot clumps and had better uniformity when compared to plantlets harvested from multiplication medium (Stage II). Secondly, the survival of the plant when transferred from Stage III to soil was much higher (>95%) than those transferred from Stage II media (75%-86% ). This is presumably due to the well developed root system and larger tubers present at the base of the shoots. Thirdly, leaf area 165 for photosynthesis was higher for plants taken from Stage III because the plantlets were grown in taller jars than those used for shoot multiplication. However, perhaps the most important reason for growing plantlets through Stage III of the micropopagation was for the production of tubers, either for storage and later use or for industrial utilization. Somani et al., (1989) also achieved in vitro tuberization of G. superba and_ they mentioned that the tubers were very small. No size was given and precociously sprouted early, perhaps due to over crowding and continuous exposure to kinetin. They did not achieve dormant tubers suitable for storage purposes. According to Bailey (1963) success of Gloriosa cultivation depends on having large tubers. Inclusion of Stage III here produced larger tubers having a short dormancy period. After eight weeks growth in multiplication .l medium, abundant root prfmordia and nascent tubers were observed at the base of shoots. The strategy then was to restrict further multiplication of the shoots and so reduce competition for nutrients between the potential roots and tubers, and the aerial parts of the plant. This was achieved when the cultures were transferred to PGR-free medium. Furthermore increasing the sucrose concentration to 4% significantly increased tuber weight, length and the number of tubers per replicate. nee Maintaining cultures for, longer than eight weeks in Stage III, led to senesc~of the aerial parts and dormant tubers. These tubers could be stored for more than a year at room temperature without loss of viability, and yet only a week after harvest they could be 0 sprouted in soil if t1}toughly irrigated. The control of dormancy is important in micropropagation of bulbilous crops (Hussey , 1982). During the multiplication phase dormancy is undesriable, since it may reduce the propagation ability. Gerrits et al., (1992) suggested that the final cycle before transfer to soil should be used to produce bulblets and not shoots, and these bulblets should be dormant to prevent precocious sprouting in vitro. Nevertheless the dormancy should not be so long that it unnecessarily lengthens the growth cycle. 166 The micropropagation protocol recently described by Custers and Bergervoet (1994) for G. rothschildiana hybrids have five steps from shoot initiation to transfer of plantlets to soil. During this multi-step procedure they used four different PGR combinations and possibly two concentrations of sucrose (3% and 4% ). Their protocol needs a total of 30 weeks to prepare plantlets for transfer to soil: Stage I initiaion, 4 weeks; Stage II multiplication, 10 weeks; Stage Illa root development, 8 weeks; Stage Illb root and tuber growth, 8 weeks. They achieved a four-to seven fold multiplication every 18 weeks. These plantlets then needed a further 8 weeks maturation (Stage Illb above) before they could be transferred to soil. Thus they could produce a maximum of about 350 well developed plantlets per 58 weeks from a single tuber explant. These had a 70% success rate on transfer to soil. Direct comparison between Custers and Bergervoets' results and those presented here needs care because of the genotypic differences in the plant material used and some differences in the culture conditions, notably the light intensity and photoperiod. However there are clear differences between the two micropropagation protocols. Firstly WP medium was superior to MS. Secondly only two PGR treatments were required (one for initiation and the other for multiplication) and thirdly the total in vitro period was 24 weeks from shoot initiation to plantlets .ready to be transferred to soil. A multiplication rate of 7- fold also was observed here, but{only'" 8 weeks. It is the shorter times for each stage in this protocol that gives it such an advantage over that of Custers and Bergervoet. In 48 weeks a single tuber of G. rothschildiana could produced about 11, 000 plantlets. The reasons for their low multiplication rate may be due to several reasons. They used tuber tips from mature tubers while in the present study tuber explants came from juvenile tubers. During the shoot multiplication stage they stimulated root development by alternately applying high and low concentrations of BAP, avoiding auxins in both multiplication media. The inclusion of this additional step to promote rooting, resulted in the 18 week long multiplication period. Then they promoted tuberization with NAA (0.3 mg/L) only in the medium. Heath and Hollies (1965) found that non-rooted onion plants gave 167 more bulbs in vitro than rooted plants. The developing roots will require nutrients from the medium and this could decrease the supply of nutrients for multiplying shoots. Also early rooting could inhibit shoot multiplication. The present method multiplied both shoots and tubers during Stage II, and combined lµM IAA with 20µM BAP. This combination increased the number of tubers /replicate from 3 to 11 (Table 5.4) and conversly the absence of auxin in the multiplication medium by Custers and Bergervoet has suppressed tuberization. Debergh and Maene (1981) stated that exogenous auxins are normally required for root initiation, but not for root elongation and indeed they may inhibit root elongation. Interestingly Custers and Bergervoet avoided auxin at the initial stage of rooting but included it when root elongation was taking place. Here, when shoot clumps with or without tubers (and after with no visible roots) were transferred to PGR free root and tuber development medium, they readily formed a root system within 2-3 weeks, which was well elongated after 4-6 weeks. The presence of auxin in the multiplication medium may be the key factor for root and tuber initiation and its omission results in decreased tissue viability. Custers and Bergervoet (1994) found that shoots harvested from rooting.medium (4 weeks with NAA) did not successfully transfer to soil. Transplantation was more successful (70%) after a further 4 weeks culture, when plants had formed tubers. Perhaps the tubers had not achieved a sufficient size or vigour to tolerate ex vitro conditions. Their method promoted tuberization during Stage IV and the maximum age of tubers was 8 weeks. The current protocol overcame this problem by promoting tuberization during the multiplication period. Initiation of tubers took place in Stage I, multiplication in stage II and rapid growth in Stage III. Thus when plants were harvested from Stage III the average physiological age of the tubers was about 16 weeks. Survival was 100% ex vitro. Plantlet regeneration from tuber explant callus of G. simplex and G. superba was achieved. However regeneration from callus may not be a desirable method of clonal 168 propagation of Gloriosa, because of the possibility of somaclonal variation. On the other hand, regeneration of plantlets from callus is a technique which could be utilized to develop modified germplasm with enhanced horticultural traits or increased accumulation of colchicine. The protocol developed here reduces the subculturing steps and hence the cost of commercial micropropagation of Gloriosa plantlets. The availability of an in vitro tuber production system has great potential in horticultural practice. For example. the initiation of tubers attached to plantlets minimises the acclimatization step. In addition the technique allows a faster rate of tuber production and a shortening of the time taken for the development of a tuber having a sufficient size to produce flowers. In vitro generated tubers may be useful as an industrial source of colchicine. Colchicine Although not especially novel, a simple, sensitive and rapid method for the determination of colchicine in plant tissues was developed. Most of the existing methods were gravimetric or colourimetric and designed to measure total alkaloid content. Other disadvantages included time consuming steps, less reproducibility for small sample sizes, and the demand for large volumes of organic solvents. These disadvantages were largely overcome by developing suitable extraction, purification and quantitation methods for routine analysis of colchicine from in vitro and in vivo Gloriosa tissues. The steps involved a simple homogenisation of tissue, solid phase extraction (SPE) and concentration, and detection and quantitation of colchicine by isocratic reverse phase HPLC. The adoption of SPE was crucial to the success of the procedure, especially for tissues which contained a small amount• of colchicine, for example callus cultures. The SPE was rapid and efficient, and the colchicine fraction could be directly analysed by HPLC. This is the first use of SPE for the purification of colchicine from plant extracts. Heijden et al., (1989) recommended SPE for the isolation of alkaloids in plant cell cultures. 169 [3H]-colchicine was used as an internal standard to correct for losses during all steps of colchicine quantitation. The HPLC analysis could detect and quantitiate lOOng of colchicine. Therefore under the isolation conditions used here, the minimum colchicine concentration that could be quantitated was 0.6µg/g fresh weight of tissue (0.00006% ). The colchicine concentration of in vivo plants varied with the stage of plant development, the type of organ and the species. Seeds and tubers were found to be rich in colchicine in all the Gloriosa species as well as Sandersonia aurantiaca. The colchicine content of G. superba has been reported by many authors (Kaul et al., 1977; Finnie and van Staden, 1991) using a variety of methodologies. One report on the quantitiative analysis of colchicine in G. simplex was found (Ntahomvukiye et al., 1984). The work in this thesis reports for the first time some quantitative data for tissues of G. rothschildiana, G. verschuurii and the cultivar G. richmondensis (Table 9.1). The colchicine content of G. superba seeds (0.78%) was comparable with that reported (0.61 ± 0.2%) by Finnie and van Staden (1991). However the result for seeds of G. simplex (0.77%) was higher than that reported (0.37%) by Ntahomvukiye etal., (1984). The colchicine content of tubers varied with the stage of development. Tubers produced during the first growth cycle of seedlings were small. After flowering and fruit setting, the mother tuber senesces and the colchicine content decreases. During maturation of daughter tubers the colchicine gradually increases reaching its maximum at the flowering stage. Finnie and van Staden (1991) suggested that breakdown of the alkaloid is responsible for the decreased concentration in senescing tubers. Alternately the increasing level in daughter tubers may be due to translocation of colchicine from the senescing mother tubers after flowering. The colchicine content of G. superba tubers was comparable with that reported by Finnie and van Staden (1991). They analysed plant material grown under controlled environment conditions. The plant parts analysed here were taken from glasshouse grown material during the summer. It is interesting to note that despite the 170 different sources of Gloriosa superba, there was a comparable amount of colchicine at the same physiological age. However most of the published data on the colchicine content of G. superba tubers covers a wide range. Thakur et al., (1975) blamed incorrect identification of the plant species, the different times of collection and improper processing of plant material. Each species used here was identified, grown under identical conditions and the colchicine content compared at similar physiological ages. Mature leaves and stems contained very low levels of colchicine as did Sandersonia. Leaves and stem of 2-month old Gloriosa seedlings had a higher concentration of colchicine often approaching that of the seeds. The colchicine content of flowers also was found to be very low. Ntahomvykiye et al., (1984) reported ea. I% in G. simplex flowers while here it was found to be ea. 0.06%. Finnie and van Staden reported 0.1 % for G. superba flowers and other reports show that in general, flowers are low in colchicine. The high value reported by Ntahomvukiye et al., (1984) may be due to the inclusion of developing capsules containing immature seeds. The majority of the colchicine found in mature vines of G. simplex and S. aurantiaea was concentrated at the apex of the vine. This suggests that tender tissues of the plants are a good site for either synthesis and/or accumulation of colchicine. Similarly tubers of G. simplex had their highest concentration of colchicine in the meristematic terminal sections with a decreased amount toward the central basal plate. In 1980, the colchicine requirement of the European pharmaceutical industry was 180 tonnes of seeds provided by the fruits from 130 million Colehieum plants (Bellet and Gaignault, 1985). There was difficulty in collecting this amount of plant material and Colehieum was in short supply. Because the colchicine content of G. superba and Colehicum autumnale tuber and corms are similar (0.9% and 0.62% respectively), Bellet and Gaignault (1985) suggested the commercial viability of Gloriosa as a source of colchicine. Similarly Finnie and van Staden (1991) also reported the potential of G. superba as a commercial source of 171 colchicine, when they compared the colchicine content of G. superba , S. aurantiaca, Littonia modesta and Androcymbium melanthioides. The results of the present study, which compares four species of Gloriosa and S. aurantiaca, also demonstrates the potential of seeds, tubers and shoots of Gloriosa species as a commercial source of colchicine. Although the colchicine content of Sandersonia seeds was low, a comparatively higher amount was found in tubers. A major constraint in the industrial utilization of Gloriosa for colchicine production has been its slow rate of propagation. The complete development of a G. superba tuber takes 5-6 years (Bellet and Gaignault, 1985). This is the first report on quantitation of colchicine from cultured tissues of Gloriosa species. The undifferentiated callus of G. superba, and G. rothschildiana showed only traces of colchicine. However undifferentiated callus derived from the leaf sheath of G. simplex produced a low, but detectable level of colchicine. Hayashi et al., (1988) reported the detection of colchicine in callus established from flower shoots of Colchicum autumnale, but the amount too was very low (0.03-0.04mg/g fresh weight). The decrease in secondary metabolite synthesis in callus cultures is frequently observed in plant species. Little information exists which explains this phenomenon, although, many reasons have been put forward (Wink, 1987). The response has been correlated with hormonal, nutritional or organizational characters (Zenk, 1978). The differentiated callus (rooted callus) produced here contained a comparatively higher amount of colchicine (Table 9.13), supporting the idea that regulation of colchicine biosynthesis is a facet of organogenesis. The ability to produce colchicine in differentiated callus may be of practical importance as the callus could be established in liquid shake cultures. Analysis of colchicine in shoots and tubers at the different stages of shoot culture demonstrates clearly the increased synthetic capacity of differentiated tissue. Interestingly the in vitro organised tissue contained more• colchicine than glasshouse grown in vivo tissue. Although there were large differences between the in vivo tissues, this was not so for the in vitro tissues 172 (Table 9 .12). This suggests that colchicine synthesis was influenced by the in vitro conditions, perhaps by the high nitrogen conditions. There was a remarkable increase in accumulated colchicine in in vitro shoots compared to in vivo. The colchicine content of tissues harvested from multiplication media also varied with the maturity of the tissue. Leaving shoot cultures without subbing more than eight weeks resulted in leaf senescence and a rapid decline in leaf colchicine. Increasing sucrose from 2% to 4% resulted in an increase in colchicine accumulation in shoots and tubers both in multiplication stage (Table 9.6) and root and tuber development stage (Table 9.10). Total production of colchicine also increased, but shoot multiplication decreased by 20%. This suggests the need to develop two separate media, one for shoot multiplication and the other for colchicine production. The production medium would not be suitable for subculturing, although it would give rise to a modest increase in shoot number. a.+ There are many reports that show thf increased sucrose concentrations result in increased alkaloid production (Hagimori et al., 1982b; Schulte et al., 1984; Endo and Yamada, 1985). Woerdenbag et al., (1993) found that increasing the sucrose concentration up to 4% in Artemisia annua shoot cultures strongly inhibited nitrate uptake from the medium. Hayashi et al., (1988) showed that 3% sucrose was optimal for colchicine synthesis by Colchicum autumnale suspension cultures in MS medium. In this study G. rothschildiana shoot cultures achieved their highest colchicine content at 4% sucrose on solid WP medium and G. superba at 5% sucrose on the same medium (Table 9.10). The difference may be due to either genera specificity)medium composition or the physical state of the medium (solid/liquid). Colchicine biosynthesis in shoot cultures of G. rothschildiana was influenced by the nitrogen status of the medium. Hayashi et al., (1988) made similar observations on colchicine production by C. autumnale suspension cultures. They found colchicine formation was strongly inhibited with either NH4+ or NO3- as sole nitrogen source. In G. 173 rothschildiana shoot cultures, either NH4+ or NO3- as a sole nitrogen source decreased colchicine synthesis. The inhibitory effect of NH4+ was due to inhibition of growth (Table 9.7). G. rothschildiana shoot cultures favoured a 1:2 ratio of NH4+fNQ3- in the medium for maximum colchicine production. Interestingly the maximum colchicine concentration was 30mM total nitrogen for shoots, but 60mM total nitrogen for tubers. This suggests that the total nitrogen in the medium was not only influencing colchicine production, but its partitioning between different organs of the plant. The incorporation of lmM colchicine precursors in shoot and tuber multiplication medium altered the amount and distribution of colchicine in tissues of G. superba (Table 9.8). For example, tyrosine increased colchicine concentration by 33% in shoots, but decreased it in tubers. In contrast, cinnamic acid and p-coumaric acid decreased colchicine in shoots, but increased it in tubers with p-coumaric doubling the colchicine concentration. Other precursor treatments, phenylalanine and tyramine, resulted in a significant decrease in colchicine concentration. Growth was largely unaffected by the presence of the exogenous chemicals. Yoshida et al., (1988) found a similar type of response to putative precursors by suspension cultured C. autumnale cells. They found that the addition of phenylalanine or whereo_.5 tyrosine, alone or in combination, did not increase the concentration of colchicineJ tyramine and p-coumaric acid increased it. The reason for the stimulation of colchicine synthesis in G. superba tissues, by the exogenous precursors is not known, but it does suggest that some endogenous substrates may be limiting. Species specific colchicine synthesis was observed (Table 9 .9) in tissues harvested from Stage III cultures. The colchicine content of the senescing aerial portion gradually decreased from week two to week eight. The concurrent increase in G. superba tubers may be evidence for its translocation from shoots to tubers. The colchicine concentration of G. rothschildiana tubers decreased with maturity but an increase ) 174 in total colchicine per tuber was observed. Presumably this is due to a higher rate of "solid" deposition than colchicine accumulation. The decreased colchicine content with maturity was well compensated for by the increased dry matter yield of tubers, which resulted in more colchicine per jar. Like shoot multiplication, the colchicine content of tubers as well as the total yield of colchicine per jar could be further increased by increasing the sucrose concentration of the root and tuber development medium (Table 9.10). In particular, G. superba (whole tissue) produced 18.3 mg per jar at 4% (w/v) sucrose in the medium (Table 9.10). These findings are of considerable practical importance for the commercial production of colchicine. Variations in the colchicine content of in vitro generated shoots and tubers of Gloriosa species were found at all stages of micropropagation, as well as with the advancing incubation in each stage. The colchicine level was influenced by various in vitro conditions and in most cases the in vitro generated tubers and shoots produced more colchicine than ex vitro grown tubers and shoots. The comparison of the colchicine content of wild-type tubers with in vitro generated potted plants, when grown under identical in vivo conditions showed that there were only slight differences in the colchicine content. This suggests that the method of micropropagation developed here for Gloriosa species, not only produced plants true to type phenotypically, but also conserved the level of colchicine in the clones. However the higher level of colchicine found under in vitro conditions could not be maintained when plantlets were transferred to soil. After transfer#f to soil the colchicine level was quite similar to glasshouse grown. This may be due to competition or limitation of nutrients or other differences in the environmental factors, such as temperature, illuminination, relative humidity, etc. An important difference between the in vivo and in vitro system is the gaseous environment. The high concentration of some gases in closed containers may affect the physiology and metabolism of the plant (Wiebers et al., 1990). 175 The technique of tissue culture was used successfully to recover and re-establish productive G. simplex cell lines from the callus tissues. This was developed as a two step procedure, which included regeneration of plantlets from callus tissues derived from tuber explants, followed by determination of colchicine in each plantlet and conservation of each cell line through micropropagation. The selected plants could then be micropropagated indefinitely. The colchicine content of the regenerated plants varied. The variations show the heterogeneous biosynthetic character of callus. The ability to produce colchicine in plantlets recovered from callus shows the totipotency of the cells. This technique could be used to select high colchicine bearing G. simplex plantlets. As such this may be a useful technique in genetic improvement of Gloriosa species for improved secondary metabolite production. CONCLUDING REMARKS The development of a micropropagation protocol that gives a high rate of multiplication for several species of Gloriosa enables the mass production of plantlets with attached tubers and a well developed root system that survive well on transfer to soil. This is a significant improvement for the commercial propagation of Gloriosa at low cost. The ability to produce in vitro tubers has many advantages. In vitro generated tubers are more tolerant of variations in light and temparature and are less prone to damage than cultured shoots. They can be stored with minimal lose of viability and establishment of plants in the field from tubers is relatively easy. Besides these advantages the tubers could be used as a raw material for the production of colchicine. The culture of multiple shoots from tuber or shoot base explants gives clonal propagation of Gloriosa plants with known and desirable characteristics such as flower excellence or improved colchicine content. Multiple shoots from root-coleoptile axis 176 explants may be useful in conventional breeding programmes to multiply new seedlings derived from different crosses. Plant regeneration from callus is a useful' technique to preserve desired characters arising from somaclonal variation. The availability of a rapid micropropagation protocol for several species of Gloriosa, together with the quantitative data on the colchicine content of these species will further encourage studies into the biotechnological application of this genus for colchicine hou,.t production. This would provide high quality raw materials throug• the year)unaffected by environmental and political factors. 177 REFERENCES Aartrijk, J. van and Blom-Bamhoom, G.J. (1979). Some influence of naphtylacetic acid on the differentiation of meristems of Lilium speciosum in vitro. Acta Hort. 91, 269-279. Aartruk, J. van and van Der Linde, P.C.G (1986). In vitro propagation of flower bulb crops. 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Mean F-Value Pr> F Square Model 15 0.8381 14.21 0.0001 MS medium Error 64 0.0589 PGR (sub treatment) 15 0.8381 14.21 0.0001 Model 15 1.2347 13.01 0.0001 WPmedium Error 64 0.0943 PGR (sub treatment) 15 1.2347 13.01 0.0001 Model 31 1.023 13.3 0.0001 MSandWP Error 128 0.0769 pooled Media 1 0.6250 8.12 0.0051 PGR 15 2.007 26.08 0.0001 MediaxPGR 15 0.143 3.2 0.0002 Treatments Mean no. of shoots per explant Duncan grouping MS 0.73 b WP 0.86 a Appendix lb Anova of the effect of NAA and kinetin on shoot initiation from tuber explants of G. superba on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 15 0.8950 24.12 0.0001 MS medium Error 64 0.0371 PGR (sub treatment) 15 0.8950 24.12 0.0001 Model 15 1.2162 27.49 0.0001 WPmedium Error 64 0.0442 PGR (sub treatment) 15 1.2162 27.49 0.0001 Model 31 1.0417 25.63 0.0001 MSandWP Error 128 0.0406 pooled Media 1 0.4515 11.11 0.0011 PGR 15 1.9207 47.25 0.0001 MediaxPGR 15 0.1434 3.53 0.0001 Treatments Mean no. of shoots per explant Duncan grouping MS 0.70 b WP 0.81 a 193 Appendix le Anova of the effect of NAA and kinetin on shoot initiation from tuber explants of G. simplex on MS and WP media after eight weeks culture. Treatments Source of variance D.F Mean F-Value Pr> F Square Model 15 0.3197 7.72 0.0001 MS medium Error 64 0.0414 PGR (sub treatment) 15 0.3197 7.72 0.0001 Model 15 0.8460 17.95 0.0001 WPmedium Error 64 0.0471 PGR (sub treatment) 15 0.8460 17.95 0.0001 Model 31 0.5809 13.13 0.0001 MS andWP Error 128 0.0442 pooled Media 1 0.5221 11.8 0.0008 PGR 15 1.0227 23.11 0.0001 MediaxPGR 15 0.1430 3.23 0.0002 Treatments Mean no. of shoots per explant Duncan grouping MS 0.37 b WP 0.49 a Appendix 2a Anova of the effect of NAA and BAP on shoot initiation from tuber explants of G. rothschildiana on MS and WP media after eight weeks culture. Treatments Source of variance D.F. Mean F-value Pr> F square Model 19 19.1468 16.87 0.0001 MS medium Error 80 1.1350 PGR (sub treatment) 19 19.1468 16.87 0.0001 Model 19 21.2926 38.37 0.0001 WPmedium Error 80 0.5550 PGR (sub treatment) 19 21.2926 38.37 0.0001 Model 39 26.81 20.16 0.0001 MS andWP Error 160 1.33 pooled Media 1 87.12 65.5 0.0001 PGR 19 46.15 34.7 0.0001 MediaxPGR 19 4.29 3.2 0.0001 Treatments Mean no. of shoots per explant Duncan grouping MS 1.05 b WP 1.38 a 194 Appendix 2b Anova of the effect of NAA and BAP on shoot initiation from tuber explants of G. superba on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 19 0.9216 11.77 0.0001 MS medium Error 80 0.0783 PGR (sub treatment) 19 0.9216 11.77 0.0001 Model 19 1.1282 12.24 0.0001 WPmedium Error 80 0.0921 PGR (sub treatment) 19 1.1282 12.24 0.0001 Model 39 1.0144 11.9 0.0001 MSandWP Error 160 0.0852 pooled Media 1 0.6166 7.24 0.0079 PGR 19 1.9939 23.39 0.0001 MediaxPGR 19 0.0559 0.66 0.8565 Treatments Mean no. of shoots per explant Duncan grouping MS 0.82 b WP 0.93 a Appendix 2c Anova of the effect of NAA and BAP on shoot initiation from tuber explants of G. simplex on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 19 11.6671 12.21 0.0001 MS medium Error 80 0.9554 PGR (sub treatment) 19 11.6671 12.21 0.0001 Model 19 8.4900 6.74 0.0001 WPmedium Error 80 1.260 PGR (sub treatment) 19 8.49 6.74 0.0001 Model 39 9.82 8.87 0.0001 MSandWP Error 160 1.107 pooled Media 1 0.0 0.0 1.0000 PGR 19 19.416 17.53 0.0001 MediaxPGR 19 0.7408 0.67 0.8453 Treatments Mean no. of shoots per explant Duncan grouping MS 0-hb a WP O•hb a 195 Appendix 3a Anova of the effect of NAA and kinetin on shoot initiation from shoot baseexplants of G. superba on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 15 0.0466 9.01 0.0001 MS medium Error 112 0.0051 · PGR (sub treatment) 15 0.0466 9.01 0.0001 Model 15 0.1059 12.43 0.0001 WPmedium Error 112 0.0085 PGR (sub treatment) 15 0.1059 12.43 0.0001 Model 31 0.0770 11.24 0.0001 MS andWP Error 224 0.0068 pooled Media 1 0.0976 14.25 0.0002 PGR 15 0.1349 19.70 0.0001 MediaxPGR 15 0.0176 2.58 0.0014 Treatments Mean no. of shoots per explant Duncan grouping MS 0.05 b WP 0.09 a Appendix 3b Anova of the effect of NAA and kinetin on shoot initiation from shoot base explants of G. simplex on MS and WP media after eight weeks culture. Treatments Source of variance D.F. Mean F-Value Pr> F Square Model 15 0.0141 8.42 0.0001 MS medium Error 64 0.0061 PGR (sub treatment) 15 0.0141 8.42 0.0001 Model 15 0.0201 4.44 0.0001 WPmedium Error 64 0.0045 PGR (sub treatment) 15 0.0201 4.44 0.0001 Model 31 0.0167 5.41 0.0001 MS andWP Error 128 0.0031 pooled Media 1 0.0052 1.70 0.1941 PGR 15 0.0302 9.75 0.0001 MediaxPGR 15 0.0041 1.33 0.1934 Treatments Mean no. of shoots per explant Duncan grouping MS 0.032 a WP 0.035 a 196 Appendix 4a Anova of the effect of NAA and BAP on shoot initiation from shoot base explants of G. rothschildiana on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 19 10.13 16.89 0.0001 MS medium Error 80 0.13 PGR (sub treatment) 19 10.13 16.89 0.0001 Model 19 21.29 38.37 0.0001 WPmedium Error 80 0.55 PGR (sub treatment) 19 21.29 38.37 0.0001 Model 39 15.697 27.18 0.0001 MSandWP Error 160 0.577 pooled Media 1 15.125 26.19 0.0001 PGR 19 29.131 50.44 0.0001 MediaxPGR 19 2.293 3.97 0.0001 Treatments Mean no. of shoots oer explant Duncan grouping MS 0.48 b WP 0.62 a Appendix 4b Anova of the effect of NAA and BAP on shoot initiation from shoot base explants of G. superba on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 19 0.0666 65.43 0.0001 MS medium Error 80 0.0010 PGR (sub treatment) 19 0.0666 65.43 0.0001 Model 19 0.0328 25.79 0.0001 WPmedium Error 80 0.0012 PGR (sub treatment) 19 0.0328 25.79 0.0001 Model 39 0.0484 42.3 0.0001 MSandWP Error 160 0.0011 pooled Media 1 0.0001 0.16 0.6905 PGR 19 0.0913 79.78 0.0001 MediaxPGR 19 0.0080 7.04 0.0001 Treatments Mean no. of shoots per explant Duncan grouping MS 0.072 a WP 0.070 a 197 Appendex 4c Anova of the effect of NAA and BAP on shoot initiation from shoot base explants of G. simplex on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 19 13.9413 16.0 0.0001 MS medium Error 140 0.8715 PGR (sub treatment) 19 13.9413 16.0 0.0001 Model 19 6.7236 10.67 0.0001 WPmedium Error 140 0.6303 PGR (sub treatment) 19 6.723 10.67 0.0001 Model 39 10.5207 14.01 0.0001 MS andWP Error 280 0.7509 pooled Media 1 17.672 23.53 0.0001 PGR 19 19.754 26.31 0.0001 MediaxPGR 19 0.9101 1.21 0.2465 Treatments Mean no. of shoots per explant Duncan grouping - , MS 0·~4 b WP o .. 1'5 a Appendix Sa Anova of the effect of NAA and kinetin on shoot initiation from root/coleoptile axis explants of G. rothschildiana in MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 15 0.2099 11.79 0.0001 MS medium Error 112 0.0178 PGR (sub treatment) 15 0.2099 11.79 0.0001 Model 15 0.3591 11.79 0.0001 WPmedium Error 112 0.0314 PGR (sub treatment) 15 0.3591 11.79 0.0001 Model 31 0.3554 14.42 0.0001 MSandWP Error 224 0.0246 pooled Media 1 2.4806 100.66 0.0001 PGR 15 0.4938 20.04 0.0001 MediaxPGR 15 0.0752 3.06 0.0002 Treatments Mean no. of shoots per explant Duncan grouping MS 0.41 b WP 0.61 a 198 Appendix Sb Anova of the effect of NAA and kinetin on shoot initiation from root/coleoptile axis explants of G. superba on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 15 0.8227 8.72 0.0001 MS medium Error 64 0.0948 PGR (sub treatment) 15 0.8227 8.72 0.0001 Model 15 0.1464 34.25 0.0001 WPmedium Error 64 0.0042 PGR (sub treatment) 15 0.1464 34.25 0.0001 Model 31 5.4772 15.83 0.0001 MSandWP Error 128 0.3460 pooled Media 1 81.54 253.66 0.0001 PGR 15 4.42 12.78 0.0001 MediaxPGR 15 1.53 4.42 0.0001 Treatments Mean no. of shoots per explant Duncan grouping MS 0.39 b WP 0.45 a Appendix Sc Anova of the effect of NAA and kinetin on shoot initiation from root-coleoptile axis explants of G. simplex on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 15 0.8106 9.77 0.0001 MS medium Error 64 0.0829 PGR (sub treatment) 15 0.8106 9.77 0.0001 Model 15 0.5439 3.88 0.0001 WPmedium Error 64 0.1401 PGR (sub treatment) 15 0.5439 3.88 0.0001 Model 31 0.8437 7.58 0.0001 MS andWP Error 128 0.1113 pooled Media 1 5.8360 52.44 0.0001 PGR 15 1.2390 11.14 0.0001 MediaxPGR 15 0.1151 L03 0.4252 Treatments Mean no. of shoots per explant Duncan grouping MS 0.70 b WP 1.10 a 199 Appendix 6a Anova of the effect of NAA and BAP on shoot initiation from root-coleoptile axis explants of G. superba in MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 15 0.1518 13.93 0.0001 MS medium Error 64 0.0109 PGR (sub treatment) 15 0.1518 13.93 0.0001 Model 15 0.1464 34.25 0.0001 WPmedium Error 64 0.0042 PGR (sub treatment) 15 0.1464 34.25 0.0001 Model 31 0.150 19.71 0.0001 MSandWP Error 128 0.0076 pooled Media 1 0.1822 23.93 0.0001 PGR 15 0.2818 37.01 0.0001 MediaxPGR 15 0.0074 0.98 0.4791 Treatments Mean no. of shoots per explant Duncan grouping MS 0.43 b WP 0.50 a Appendix 6b Anova of the effect of NAA and BAP on shoot initiation from root-coleoptile axis explants of G. simplex on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 19 0.0275 2.40 0.0019 MS medium Error 140 0.0114 PGR (sub treatment) 19 0.0275 2.40 0.0001 Model 15 0.0554 5.59 0.0001 WPmedium Error 140 0.0099 PGR (sub treatment) 19 0.0554 5.59 0.0001 Model 39 0.0469 4.39 0.0001 MSandWP Error 280 0.0106 pooled Media 1 0.2531 23.66 0.0001 PGR 19 0.0777 7.27 0.0001 MediaxPGR 15 0.0052 0.49 0.9659 Treatments Mean no. of shoots per explant Duncan grouping MS 0.18 b WP 0.24 a 200 Appendix 7a Anova of the effect of NAA and kinetin on tuber initiation from tuber explants of G. rothschildiana on MS and WP media after eight weeks culture Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 15 0.1441 4.17 0.0001 MS medium Error 64 0.0346 PGR (sub treatment) 15 0.1441 4.17 0.0001 Model 15 0.2295 6.91 0.0001 WPmedium Error 64 0.0332 PGR (sub treatment) 15 0.2295 6.91 0.0001 Model 31 0.1844 5.44 0.0001 Error 128 0.0339 MSandWP Media 1 0.1128 3.33 0.0050 pooled PGR 15 0.3483 10.27 0.0001 MediaxPGR 15 0.0253 0.75 0.7308 Treatments Mean no. of tubers per explant Duncan grouping MS 0.41 a WP 0.46 a Appendix 7b Anova of the effect of NAA and kinetin on tuber initiation from tuber explants of G. superba on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr>F Square Model 15 1.3878 5.12 0.0001 MS medium Error 64 0.2710 PGR (sub treatment) 15 1.3878 5.12 0.0001 Model 15 1.4398 5.05 0.0001 WPmedium Error 64 0.2849 PGR (sub treatment) 15 1.4398 5.05 0.0001 Model 31 1.4320 5.15 0.0001 MSandWP Error 128 0.2779 pooled Media 1 1.9691 7.08 0.0089 PGR 15 2.6216 9.43 0.0001 MediaxPGR 15 0.1272 0.46 0.9569 Treatments Mean no. of tubers per explant Duncan grouping MS 0.19 b WP 0.25 a 201 Appendix 7c Anova of the effect of NAA and kinetin on tuber initiation from tuber explants of G. simplex on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr>F Square Model 15 0.1199 3.40 0.0003 MS medium Error 64 0.0353 PGR (sub treatment) 15 0.1199 3.40 0.0003 Model 15 0.2940 2.39 0.0085 WPmedium Error 64 0.1228 PGR (sub treatment) 15 0.2940 2.39 0.0085 Model 31 0.2072 2.63 0.0001 MSandWP Error 128 0.0787 pooled Media 1 0.2149 2.63 0.1010 PGR 15 0.3495 4.44 0.0001 MediaxPGR 15 0.0643 0.82 0.6573 Treatments Mean no. of tubers per explant Duncan grouping MS 0.27 a WP 0.35 a Appendix Sa Anova of the effect of NAA and BAP on tuber initiation from tuber explants of G. rothschildiana on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 19 2.005 4.18 0.0001 MS medium Error 80 0.48 PGR (sub treatment) 19 2.005 4.18 0.0001 Model 19 2.45 5.98 0.0001 WPmedium Error 80 0.41 PGR (sub treatment) 19 2.45 5.98 0.0001 Model 39 2.328 5.23 0.0001 MSandWP Error 160 0.445 pooled Media 1 6.125 13.76 0.0003 PGR 19 3.857 8.67 0.0001 MediaxPGR 19 0.598 1.35 0.1623 Treatments Mean no. of tubers per explant Duncan grouping MS 0.39 b WP 0.48 a 202 Appendix Sb Anova of the effect of NAA and BAP on tuber initiation from tuber explants of G. simplex on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 19 0.0492 2.22 0.0073 MS medium Error 80 0.0221 PGR (sub treatment) 19 0.0492 2.22 0.0073 Model 19 0.0646 2.91 0.0005 WPmedium Error 80 0.0221 PGR (sub treatment) 19 0.0646 2.91 0.0005 Model 39 0.0557 2.51 0.0001 MS andWP Error 160 0.0221 pooled Media 1 0.0112 0.51 0.4774 PGR 19 0.1049 4.73 0.0001 MediaxPGR 19 0.0089 0.40 0.9882 Treatments Mean no. of tubers per explant Duncan grouping MS 0.27 a WP 0.28 a Appendix 9a Anova of the effect of NAA and BAP on the weight of tubers initiated per tuber explant of G. rothschildiana on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-value pr> F Square Model 19 123886 10.68 0.0001 MS medium Error 60 11602 PGR (sub treatment) 19 123886 10.68 0.0001 Model 19 30289 4.41 0.0001 WPmedium Error 60 6861 PGR (sub treatment) 19 30289 4.41 0.0001 Model 39 75205 8.15 0.0001 MSandWP Error 160 9231 pooled Media 1 3655 0.40 0.5301 PGR 19 120784 13.08 0.0001 MediaxPGR 19 33392 3.62 0.0001 Treatments Mean weight of tubers per Duncan grouping explant (mg) MS 368 a WP 360 a 203 Appendix 9b Anova of the effect of NAA and BAP on the weight of tubers initiated per tuber explant of G. simplex on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr>F Square Model 19 22240 6.95 0.0003 MS medium Error 80 3199 PGR (sub treatment) 19 22240 6.95 0.0003 Model 19 11001 2.45 0.0029 WPmedium Error 80 4481 PGR (sub treatment) 19 11001 2.45 0.0029 Model 39 0.2072 2.54 0.0001 MS andWP Error 160 3774 pooled Media 1 2080 0.55 0.4589 PGR 19 15334 4.06 0.0001 MediaxPGR 19 4239 1.12 0.3326 Treatments Mean weight of tubers per explant (mg) Duncan grouping MS 120 a WP 127 a Appendix 10a Anova of the effect of NAA and kinetin on tuber initiation from primary cultures of root-coleoptile axis explants of G. rothschildiana on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr>F Square Model 15 0.2543 13.07 0.0001 MS medium Error 112 0.0194 PGR (sub treatment) 15 0.2543 13.07 0.0001 Model 15 0.1147 4.23 0.0001 WPmedium Error 112 0.0271 PGR (sub treatment) 15 0.1147 4.23 0.0001 Model 31 0.2008 8.62 0.0001 MS andWP Error 224 0.0233 pooled Media 1 0.6909 29.66 0.0001 PGR 15 0.2577 11.06 0.0001 MediaxPGR 15 0.1113 4.78 0.0001 Treatments Mean no. of tubers per explant Duncan grouping MS 0.34 b WP 0.45 a 204 Appendix 10b Anova of the effect of NAA and kinetin on tuber initiation from primary cultures of root-coleoptile axis explants of G. simplex on MS and WP media after eight weeks culture. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 15 0.0813 2.91 0.0016 MS medium Error 64 0.0279 PGR (sub treatment) 15 0.0813 2.91 0.0016 Model 15 0.0656 2.97 0.0013 WPmedium Error 64 0.0220 PGR (sub treatment) 15 0.0656 2.97 0.0013 Model 31 0.0734 2.93 0.0897 MSandWP Error 128 0.0250 pooled Media 1 0.0732 2.93 0.0002 PGR 15 0.1233 4.93 0.0001 MediaxPGR 15 0.0235 0.94 0.5207 Treatments Mean no. of tubers per explant Duncan grouping MS 0.38 a WP 0.34 a Appendix lla Anova of the effect of NAA or IAA with BAP on multiplication of shoots derived from tuber explants of G. rothschildiana after eight weeks culture on WP medium. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 19 248.8 4.18 0.0001 NAA Error 80 12.38 BAP (sub treatment) 19 248.8 4.18 0.0001 Model 19 168.76 5.98 0.0280 IAA Error 80 89.86 BAP (sub treatment) 19 168.76 5.98 0.0280 Model 39 270.35 5.23 0.0001 NAA/IAA Error 160 50.63 pooled NAA/IAA 1 542.87 13.76 0.0013 BAP 19 371.06 8~67 0.0001 Auxin x Cvtokinin 19 46.5 1.35 0.0001 Treatments Mean no. of new shoots per replicate Duncan grouping NAA 13.3 b IAA 16.6 a 205 Appendix llb Anova of the effect of NAA or IAA with BAP on average length of shoots derived from tuber explants of G. rothschildiana after eight weeks culture on WP medium. Treatments Source of Variance D.F. Mean F-Value Pr> F Square Model 19 488.21 4.36 0.0001 NAA Error 80 112.0 BAP 19 488.2 4.36 0.0001 Model 19 463.61 2.30 0.0055 IAA Error 80 201.46 BAP 19 463.61 2.30 0.0055 Model 39 465.43 2.98 0.0001 NAA/IAA Error 160 156.16 pooled NANIAA 1 067.64 0.43 0.5120 BAP 19 676.10 4.33 0.0001 Auxin x Cvtokinin 19 275.71 1.77 0.0312 Treatments Mean length of shoots (mm) Duncan grouping NAA 61 a IAA 60 a Appendix 12a Anova of the effect of NAA or IAA with BAP on number of tubers derived from multiplying shoots of G. rothschildiana after eight weeks culture on WP medium. Treatment Source of D.F. Mean F-Value Pr>F Variance Square Model 19 3.28 3.63 0.0001 NAA Error 80 0.90 BAP 19 3.28 3.63 Model 19 15.18 11.05 0.0001 IAA Error 80 1.35 BAP 19 15.10 11.05 0.0001 Model 39 350.6 1.52 0.0392 Pooled Error 160 231.20 NAAandIAA Auxin 1 929.48 4.02 0.0001 Cytokinine 19 427.70 1.85 0.0440 auxin x 19 832.32 3.6 0.0001 Cvtokinine Treatment No. of tubers per replicate Duncan grouping NAA 2.85 b IAA 5.21 a 206 Appendix 12b Anova of the effect of NAA or IAA with BAP on weight of tubers derived from multiplying shoots of G. rothschildiana after eight weeks culture on WP medium. Treatment Source of D.F. Mean F-Value Pr>F Variance Square Model 19 2342.4 1.44 0.1313 NAA Error 80 1623.8 BAP 19 2342.4 1.44 0.1313 Model 19 2485.5 1.47 0.1209 IAA Error 80 1691.8 BAP 19 2485.5 1.47 0.1209 Model 39 2411.32 1.45 0.0566 Pooled Error 160 1657.41 NAAandIAA Auxin 1 2310.67 1.39 0.2395 Cytokinine 19 2787.92 1.68 0.0443 auxin x 19 2040.02 1.23 0.2393 Cytokinine Treatment Weight of tubers per replicate Duncan grouping NAA 158 a IAA 165 a Appendix 13 Anova of the effect of nitrogen concentration and composition on multiplication of shoots and tubers derived from tuber explants of G. rothschildiana after eight weeks culture on WP medium. Dependent Variable: No. of new shoots Jer replicate Treatment Source D.F. Mean F-Value Pr>F Sauare Model 11 193.23 108.67 0.0001 NH4+/N03- Error 35 1.77 Treatment 11 193.23 108.67 0.0001 Dependent Variable: No. of tubers per replicate Treatment Source D.F. Mean F-Value Pr>F Square NH4+/NQ3- Model 11 49.12 2.92 0.0412 Error 35 16.80 Treatment 11 49.12 2.92 0.0412 207 Appendix 14 Anova of the effect of elevated sucrose concentration (from 2% to 4%) on multiplication of shoots and tubers from shoots derived from tuber explants of G. rothschildiana after eight weeks culture on WP medium. Dependent Variable: No. of new shoots :,er replicate Treatment Source D.F. Mean F-Value Pr>F Square Sucrose Model 1 40.0 7.02 0.0293 Error 8 5.7 Treatment 1 40.0 7.02 0.0293 Dependent Variable: No. of tubers per replicate Treatment Source D.F. Mean F-Value Pr>F Square Sucrose Model 1 12.1 7.81 0.0239 Error 8 1.55 Treatment 1 12.1 7.81 0.0239 Dependent variable: Weight of tubers per replicate Treatment Source D.F. Mean F-Value Pr>F Square Sucrose Model 1 608.4 0.62 0.455 Error 8 988.4 Treatment 1 608.4 0.62 0.455 Appendix 15 Anova of the effect of various media on multiplication of shoots derived from shoot base explants of G. rothschildiana after eight weeks culture. Dependent variable: (No. of new shoots per replicate) Treatment source DF Mean F-value Pr>F square Model 7 22.85 1.63 0.1625 Media Error 32 14.01 Treatment 7 22.85 1.63 0.1625 208 Appendix 16 Anova of the effect of NAA and kinetin on shoot and tuber multiplication of cultures derived from root-coleoptile axis explants of G. rothschildiana after eight weeks culture on WP medium. Dependent Variable: No. of new shoots oer replicate Treatment Source D.F. Mean F-Value Pr>F Square Model 7 28.5 24.01 0.0001 NANkinetin Error 32 1.18 Treatment 7 28.5 24.01 0.0001 Dependent variable: No. of tubers per re Jlicate Treatment Source D.F. Mean F-Value Pr>F Square Model 7 0.4428 0.69 0.6760 NANkinetin Error 32 0.6375 Treatment 7 0.4428 0.69 0.6760 Appendix 17 Anova of the data from the effect of different PGR treatments on shoot and tuber multiplication, length of shoots and tuber weight on multiplying shoots derived from tuber explants of G. superba after eight weeks culture on MS or WP media. Dependent variable: No. of new shoots :>er replicate Treatment Source D.F. Mean F-Value Pr>F Square Multiplication Model 5 109.3 24.01 0.0001 media Error 23 4.5 Treatment 5 109.3 24.01 0.0001 Dependent variable: Mean length of a shoot Treatment Source D.F. Mean F-Value Pr>F Square multiplication Model 5 0.528 5.3 0.0022 medium Error 23 0.099 Treatment 5 0.528 5.3 0.0022 Dependent variable: No. of tubers per reolicate Treatment source D.F. Mean F-Value Pr>F Sauare Multiplication Model 5 13773 0.98 0.452 medium Error 23 14081 Treatment 5 13773 0.98 0.452 Dependent variable: Mean weight of tubers per replicate Treatment Source D.F. Mean F-Value Pr>F Square multiplication Model 5 166135 4.18 0.0076 medium Error 23 39746 Treatment 5 166135 4.18 0.0076 209 Appendix 18 Anova of the data from the effect of different PGR treatments on shoot multiplication of multiplying shoots derived from shoot base explants of G. superba after eight weeks culture on MS and WP media. Dependent variable: No. of new shoots oer replicate Treatment Source D.F. Mean F-Value Pr>F Square multiplication Model 7 55.7 7.51 0.0001 media Error 32 7.42 Treatment 7 55.7 7.51 0.0001 Appendix 19 Anova of the data from the effect of NAA and kinetin on shoot multiplication of multiplying shoots derived from root-coleoptile explants of G. superba after eight weeks culture on MS and WP media. Dependent variable: No. of new shoots oer replicate Treatment Source D.F. Mean F-Value Pr>F Square multiplication Model 5 54.0 12.61 0.0001 media Error 24 4.28 Treatment 5 54.0 12.61 0.0001 Appendix 20 Anova of the effect of PGR treatments on multiplication of shoots and tubers and tuber weight from tuber explants derived shoots of G. simplex after eight weeks culture on WP medium. Dependent Variable: No. of new shoots oer replicate Treatment Source D.F. Mean F-Value Pr>F Square Multiplication Model 5 29.6 3.56 0.0151 medium Error 24 8.3 Treatment 5 29.6 3.56 0.0151 Dependent variable: No. of tubers per reolicate Treatment Source D.F. Mean F-Value Pr>F Square Multiplication Model 5 5.5 4.85 0.0033 medium Error 24 1.13 Treatment 5 5.5 4.85 0.0033 Dependent variable: Weight of tubers per replicate Treatment Source D.F. Mean F-Value Pr>F Square Multiplication Model 5 87150 5.49 0.0017 medium Error 24 15875 Treatment 5 87150 5.49 0.0017 210 Appendix 21 Anova of the effect of various media on multiplication of shoots derived from shoot base explants of G. simplex after eight weeks culture. Dependent Variable: No. of New Shoots per replicate Treatment Source D.F. Mean F-Value Pr>F Square Model 7 47.94 16.18 0.0001 Media Error 32 2.96 Treatment 7 47.94 16.18 0.0001 Appendix 22 Anova of the data from the effect of NAA and kinetin on multiplication of shoot cultures derived from root-coleoptile axis explants of G. simplex after eight weeks culture on WP medium. Dependent variable: No. of new shoots Jer replicate Treatment Source D.F. Mean F-Value Pr>F Square PGR Model 5 20.35 13.13 0.0001 treatment Error 24 1.55 Treatment 5 20.35 13.3 0.0001 Appendix 23 Anova of the data from the effect of different PGR treatments on shoot regeneration from G. simplex callus after 16 weeks culture on MS and WP media. Dependent variable: No. of shoots per replicate Treatment Source D.F. Mean F-Value Pr>F Square multiplication Model 3 150.0 5.78 0.0071 media Error 16 25.95 Treatment 3 150.0 5.78 0.0071 211 Appendix 24 Anova of the data from the effect of multiplication media on shoot and tuber multiplication, and tuber weight of shoots derived from tuber explants of G. verschuurii after eight weeks culture on WP medium. Dependent variable: No. of new shoots oer replicate Treatment source D.F. Mean F-value Pr>F Square Multiplication Model 2 14.06 3.15 0.0795 medium Error 12 4.46 Treatment 2 14.06 3.15 0.0795 Dependent variable: No. of tubers per replicate Treatment Source D.F. Mean F-Value Pr>F Square Multiplication Model 2 5.06 6.33 0.0133 medium Error 12 0.80 Treatment 2 5.06 6.33 0.0133 Dependent variable: Mean weight of tubers per replicate Treatment Source D.F. Mean F-value Pr>F Square Multiplication Model 2 178475 4.19 0.0417 medium Error 12 42607 Treatment 2 178475 4.19 0.0417 Appendix 25 Anova of the data from the effect of multiplication media on shoot and tuber multiplication, shoots derived from tuber explants of G. richmondensis after eight weeks culture on WP medium. Dependent variable: No. of new shoots oer replicate Treatment source D.F. Mean F-Value Pr>F Square Multiplication Model 2 71.66 5.75 0.0177 medium Error 12 12.46 Treatment 2 71.66 5.75 0.0177 Dependent Variable: No. of tubers per replicate Treatment Source D.F. Mean F-Value Pr>F Square Multiplication Model 2 7.2 6.17 0.0144 medium Error 12 1.16 Treatment 2 7.2 6.17 0.0144 212 Appendix 26a Anova of the effect of sucrose concentration on G. rothschildiana and G. superba tuber multiplication after eight weeks culture on WP medium without PGRs. Dependent variable: Mean no. of tubers per replicate Treatment Source D.F. Mean F-Value Pr>F Square Model 5 41.74 50.67 0.0001 G. rothschildiana Error 24 0.823 Sucrose(%) 5 41.74 50.67 0.0001 Model 5 15.10 11.06 0.0001 G. superba Error 24 1.36 Sucrose(%) 5 15.10 11.06 0.0001 Model 11 29.6 27.19 0.0001 G. rothschildiana and Error 48 1.08 G. superba (pooled) Species 1 41.52 38.13 0.0001 Sucrose(%) 5 46.5 42.7 0.0001 Species x 5 10.35 9.5 0.0001 Sucrose(%) ean no. of tubers Duncan 5.3 a 3.6 b Appendix 26b Anova of the effect of sucrose concentration on G. rothschildiana and G. superba tuber length after eight weeks culture on WP medium without PGRs. Dependent variable: Mean length of a tuber Treatment Source D.F. Mean F-Value Pr>F Sauare Model 5 76.72 30.89 0.0001 G. rothschildiana Error 24 2.48 Sucrose(%) 5 76.62 30.89 0.0001 Model 5 123.12 53.28 0.0001 G. superba Error 24 2.3 Sucrose(%) 5 123.12 53.28 0.0001 Model 11 93.2 38.88 0.0001 G. rothschildiana and Error 48 2.3 G. superba (pooled) Species 1 26.6 11.13 0.0017 Sucrose(%) 5 184.1 76.82 0.0001 Species x 5 15.57 6.49 0.0001 Sucrose(%) Mean len th of a tuber (mm) Duncan G. rothschil iana 7.0 G. su erba 9.0 a 213 Appendix 26c Anova of the effect of sucrose concentration on total weight of tubers from G. rothschildiana and G. superba after eight weeks culture on WP medium without PGRs. Dependent variable: Mean weight of tubers per replicate Treatment Source D.F. Mean F-Value Pr>F Suuare Model 5 5.51 9.51 0.0001 G. rothschildiana Error 24 0.58 Sucrose(%) 5 5.51 9.51 0.0001 Model 5 4.47 18.46 0.0001 G. superba Error 24 0.24 Sucrose(%) 5 4.47 18.46 0.0001 Model 11 4.54 10.95 0.0001 G. rothschildiana and error 48 0.41 G. superba (pooled) Species 1 0.04 0.11 0.7338 Sucrose(%) 5 8.77 21.16 0.0001 Species x Sucrose(%) 5 1.20 2.92 0.0231 Species Mean weight of tubers Duncan grouping per replicate (g) G. rothschildiana 1.9 a G. superba 1.8 a 214 Appendix 27 Anova of the effect of ABA and GA3 on tuber and root development of shoots of G. rothschildiana derived from tuber explants after eight weeks culture on WP medium supplemented with 4% sucrose. Dependent variable: No. of tubers per re Jlicate Treatment Source D.F. Mean F-Value Pr>F Square ABAandGA3 Model 5 56.3 15.01 0.0001 Error 24 18.0 Treatment 5 56.3 15.01 0.0001 Dependent variable: Mean length of tubers Treatment Source D.F. Mean F-Value Pr>F Square ABAandGA3 Model 5 0.367 5.57 0.0015 Error 24 0.316 Treatment 5 0.367 5.57 0.0015 Dependent variable: No. of roots per replicate Treatment Source D.F. Mean F-Value Pr>F Square ABAandGA3 Model 5 60.0 46.75 0.0001 Error 24 1.28 Treatment 5 60.0 46.75 0.0001 Dependent variable: Mean length of roots Treatment Source D.F. Mean F-Value Pr>F Square ABAandGA3 Model 5 8.79 8.26 0.0001 Error 24 1.06 Treatment 5 8.79 8.26 0.0001 215 Appendix 28 Anova of the effect of potting mix on growth parameters of G. superba tubers after the first six month growth cycle. Dependent variable: No. of tubers per re Dlicate Treatment Source D.F. Mean F-Value Pr>F Square Potting mix Model 1 7.2 0.65 0.4313 Error 18 200.0 Treatment 1 7.2 0.65 0.4313 Dependent variable: Wei~ht of tubers per replicate Treatment Source D.F. Mean F-Value Pr>F Square Potting mix Model 1 64.08 0.36 0.5577 Error 18 179.54 Treatment 1 64.08 0.36 0.5577 Dependent variable: Length of a tubers Treatment Source D.F. Mean F-Value Pr>F Square Potting mix Model 1 0.1711 0.01 0.9111 Error 18 Treatment 1 0.1711 0.01 0.9111 Appendix 29 Anova of the effect of colchicine precursors on colchicine contents of G. superba shoots and tubers after eight week culture in modified WP liquid medium (4% sucrose, 20mM NI4+, 40mM NO3-) with lµM IAA and 20µM BAP. Dependent variable: Colchicine content of shoots Treatment Source D.F. Mean F-Value Pr>F Square Precursors Model 6 472489 25.10 0.0001 Error 21 18820 Treatment 6 472489 25.10 0.0001 Dependent variable: Colchicine content of tubers Treatment Source D.F. Mean F-Value Pr>F Square Precursors Model 6 13143302 310.14 0.0001 Error 21 42378 Treatment 6 13143302 310.14 0.0001