In Vitro Culture of Red Clover (Trifolium Pratense L
IN VITRO CULTURE OF RED CLOVER (TRIFOLIUM PRATENSE L. ) AND
EVALUATION OF REGENERATED PLANTS
by
HONG WANG
A THESIS SUBMITTED IN PARTIAL FULFILMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in
THE FACULTY OF GRADUATE STUDIES
Plant Science
We accept this thesis as conforming
to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA
October, 1985
© HONG WANG, 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced
degree at the University of British Columbia, I agree that the Library shall make it
freely available for reference and study. I further agree that permission for extensive
copying of this thesis for scholarly purposes may be granted by the head of my
department or by his or her representatives. It is understood that copying or
publication of this thesis for financial gain shall not be allowed without my written
permission.
Department of
The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3
DE-6(3/81) Abstract
Red clover (Trifolium pratense L.) cvs 'Altaswede'
(2n=2X=14) and 'Norseman' (2n=4X=28) were used in the present study to investigate tissue culture initiation, plant regeneration and the occurrence of somaclonal variation. Hypocotyl explants of aseptic seedlings were
inoculated into L2 medium containing 0.06 mg/1 Picloram and
0.1 mg/1 benzyladenine for callus induction. Calli were usually induced after two weeks of culture. Callus
induction frequency was 60% to 85% of the explants cultured with 'Altaswede' showing a slightly higher frequency than
'Norseman'. Satisfactory results were obtained under dark
or light conditions using either test tubes or petri plates,
as culture vessels.
After callus induction, an experiment was conducted to
regulate shoot induction by subculturing the calli on L2
medium containing 0.01 mg/1 2,4-dichlorophenoxy acetic acid
and 2 mg/1 adenine (LSE) and on B5 medium containing 2 mg/1
naphthalene acetic acid and 2 mg/1 adenine, media which have
been reported to be shoot-supportive. However, both media
failed to initiate shoots under the present experimental
conditions. Further tests confirmed that LSE medium did not
induce shoots from these calli and that callus growth on LSE
medium steadily deteriorated over several subcultures.
Subsequently, various media were tested with an emphasis on
different combinations of growth regulators. Root
differentiation from these calli was frequently observed.
i i Shoots were initiated from some calli when they were transferred from SCP medium to media containing naphthalene acetic acid and kinetin. Embryogenic callus of one genotype was selected and maintained on LSP medium, leading to the
regeneration of numerous plants. Supplementation with arginine, glutamic acid and casein hydrolysate did not show a significant effect on callus growth and differentiation.
The source of callus influenced rates of growth and the
occurrence of differentiation. Usually 'Norseman' calli
grew faster and produced more roots than 'Altaswede' calli,
while shoots were induced only from 'Altaswede' calli.
Although 'Norseman' had more shoot tips induced to produce
multiple shoots, the multiple shoot number per culture of
'Altaswede' was higher than that of 'Norseman'. Shoot tip
cultures were also established to induce multiple shoots and
to regenerate plants via root organogenesis.
Regenerants from initial multiple shoots (RG1),
multiple shoots after two subcultures (RG2), three-month
calli (RG3) and one-year calli (RG4) were evaluated for
chromosome number stability, morphology and several
biochemical traits. When 'Altaswede' plants were analysed
for chromosome number, RG1 and RG3 plants were normal, while
one RG2 plant and 23% of 119 RG4 plants had tetraploid
chromosome numbers. Regenerated plants were quite stable
regarding their isozyme patterns of malate dehydrogenase,
6-phosphogluconate dehydrogenase, phosphoglucose isomerase,
phosphoglucomutase and shikimate dehydrogenase and their nodule leghaemoglobin profiles. Morphologically, the leaflet length to width ratio of RG1, RG2 and RG3 plants of
'Altaswede' showed significantly more variation than control plants (P<0.01), while RG4 plants of 'Altaswede' and RG1 and
RG2 plants of 'Norseman' were not different from control plants. It is suggested that the absence of detectable differences in the RG4 'Altaswede' plants was a consequence of their origin from one original genotype. Variability and stability of regenerated plants are discussed.
iv Abbreviations for Growth Regulators and Media
Auxins
2,4-D: 2,4-dichlorophenoxy acetic acid
IAA: indole-3-acetic acid
NAA: naphthalene acetic acid
PIC: Picloram (4-amino-3,5,6-trichloropiclinic acid)
Cytokinins
ADE: adenine (6-amino purine)
BA: benzyladenine
2iP: (2-isopentenyl) adenine
KIN: kinetin
Media
L2: Phillips and Collins (1979a)
LSE: L2 medium containing 0.01 mg/1 2,4-D and 2 mg/1 ADE
LSP: L2 meidum containing 0.002 mg/1 PIC and 0.2 mg/1 BA
SCP: L2 medium containing 2 mg/1 2,4-D, 2 mg/1 BA and 2 mg/1
ADE.
v Table of Contents
Chapter Page
Abstract ii
Abbreviations for Growth Regulators and Media iii
Table of Contents vi
List of Tables x
List of Figures xii
Aknowledgement xiv
1. INTRODUCTION 1
2. LITERATURE REVIEW 4
2.1 Tissue Culture in Red Clover 4
2.2 Somaclonal Variation .7
2.2.1 Variation among Regenerated Plants of Several Crop Species .8
2.2.1.1 Sugar Cane 8
2.2.1.2 Potato .9
2.2.1.3 Tobacco 11
2.2.1.4 Rice and Wheat 12
2.2.2 Origin of and Factors Affecting Somaclonal Variation 13
2.2.3 Genetic Basis 17
3. MATERIALS AND METHODS 22
3.1 General Culture Methodology 22
3.1.1 Media 22
3.1.2 Explants 24
3.1.3 Culture Conditions 25
3.2 Specific Culture Methodology 26
3.2.1 Callus Induction 26
vi 3.2.2 Callus Maintenance and Plant Regeneration :
3.2.2.1 Primary Test of Two Media :
3.2.2.2 Callus Growth on LSE Medium :
3.2.2.3 2,4-D and BA :
3.2.2.4 PIC and BA :
3.2.2.5 NAA and KIN :
3.2.2.6 NAA and BA in Presence of ADE ...
3.2.2.7 Callus Differentiation on Four Test Media
3.2.2.8 Callus Growth at High or Low Auxin to Cytokinin Ratios
3.2.2.9 Callus Growth on SCP Medium
3.2.2.10 Light Effect on Calli Growing on SCP Medium i
3.2.2.11 Selection and Maintenance of Embryogenic Callus 36
3.2.2.12 Growth of Embryogenic and Non-embryogenic Calli
3.2.3 Shoot Tip Culture
3.2.4 Rooting of Shoots ' 38
3.2.5 Scanning Electron Microscopy
3.3 Growth and Analysis of Regenerated Plants
3.3.1 Plant Transfer and Growth in Greenhouse ..
3.3.2 Chromosome Analysis
3.3.3 Leghaemoglobin Analysis
3.3.4 Isozyme Analysis
3.3.5 Morphological Analysis
RESULTS
4.1 Callus Induction
vii 4.1.1 Callus Induction Frequency 47
4.1.2 Genotypic Effect on Callus Induction 49
4.1.3 Observations on Callus Induction 49
4.2 Callus Maintenance and Plant Regeneration 52
4.2.1 Primary Test of Two Media 52
4.2.2 Callus Growth on LSE Medium 53
4.2.3 2,4-D and BA 61
4.2.4 PIC and BA 61
4.2.5 NAA and KIN 64
4.2.6 NAA and BA in Presence of ADE 73
4.2.7 Callus Differentiation on Four Media 75
4.2.8 Callus Growth and Auxin to Cytokinin Ratio 75
4.2.9 Callus Growth on SCP Medium 79
4.2.10 Light Effect on Calli Growing on SCP Medium 84
4.2.11 Observations on Embryogenesis 84
4.2.12 Selection and Maintenance of Embryogenic Calli 87
4.2.13 Growth of Embryogenic and Non-embryogenic Calli 91
4.2.14 Summary of Experiments on Callus Growth
and Differentiation 94
4.2.15 Plant Regeneration from Callus 95
4.3 Shoot Tip Culture 96
4.3.1 Multiple Shoot Induction 96
4.3.2 Multiple Shoot Propagation 96
4.4 Growth and Analysis of Regenerated Plants ...... 100
4.4.1 Plant Transfer and Survival 101
viii 4.4.2 Chromosome Number Stability 103
4.4.3 Isozyme Analysis 106
4.4.4 Nodule Formation and Leghaemoglobin Profile 114
4.4.5 Plant Morphology 114
5. DISCUSSION 121
5.1 Callus Induction 121
5.2 Callus Maintenance and Plant Regeneration 122
5.2.1 Primary Test of Two Media ...122
5.2.2 Callus Growth on LSE Medium 123
5.2.3 Callus Growth on SCP Medium 125
5.2.4 Plant Regeneration: Organogenesis and Embryogenesis 126 .
5.2.5 Genotype Effect ' 133
5.3 Shoot Tip Culture 136
5.4 Regenerated Plants 136
6. Summary 143
LITERATURE CITED 146 •
APPENDIX 1 158
ix List of Tables
Table Page
1. Treatment combinations used to test the effect of PIC and BA on plant regeneration. 31
2. Callus induction frequency for two cultivars of red clover on L2 medium. 48
3. Callus induction from three explants derived from the same seedling. 50
4. Callus growth on LSE medium. 53
5. Response of calli on LSE medium: initial and subculture of Test 1 recorded after four and five weeks of culture, respectively. 55
6. Response of calli from LSE medium subcultured onto four media. 59
7. Response of calli on L2(D0.01A5) medium. 60
8. Response of calli on media containing 2,4-D and BA recorded after three weeks of culture. 62
9. Response of 'Altaswede' calli on media containing PIC and BA in presence and absence of arginine. 63
10. Response of calli on media with added casein hydrolysate and glutamic acid. 65
11. Response of calli of two cultivars on media with various combinations of PIC and BA. 67
12. Response of 'Norseman' calli from LSE medium to various combinations of NAA and KIN. 68
13. Response of calli from SCP medium to combinations of NAA and KIN. 70
14. Response of 'Norseman' calli to combinations of NAA and KIN. 72
15. Response of 'Norseman' calli on media containing various levels of NAA in combination with BA and ADE. 74
x 16. Differentiation of 'Norseman' calli from L2(D2B2A5) medium on four media. 76
17. Response of 'Norseman' calli at high (11) or low (0.1) auxin to cytokinin ratio. 77
18. Recovery frequency of senescing calli after transfer to SCP medium. 80
19. Light effect on growth and differentiation of 'Altaswede' calli on SCP medium. 85
20. Embryo development and plantlet formation from embryogenic calli subcultured onto LSP medium. 89
21. Multiple shoot induction from shoot tips of red clover on L2(P0.003B2) mediium. 97
22. Comparison of two cultivars of red clover for multiple shoot induction. 97
23. Multiple shoot production of two cultivars of red clover. 99
24. Survival rate of plants transferred to greenhouse after different treatments. 102
25. Chromosome numbers of 'Altaswede' plants regenerated through different ways. 104
26. Leaflet number in control (seed-derived) and regenerated plants of red clover. 116
27. Means and variances of leaflet length/width ratios for different populations of red clover. 118
28. Comparisons of regenerated plants and control plants for means of leaflet length/width ratio. 119
29. Comparisons of regenerated and control plants for the variances of leaflet length/width ratio. 119
xi List of Figures
Figure Page
1. Callus induction and genotype effect. 'Norseman' hypocotyl explants on L2 medium. 51
2. Response of 'Altaswede' calli on LSE medium after two weeks of culture. 56
3. Response of 'Altaswede' calli on LSE medium after five weeks of culture. 57
4. Response of 'Altaswede' calli on L2 medium containing 0.005 mg/1 PIC, 1 mg/1 BA and 1 g/1 casein hydrolysate after five weeks of culture. 66
5. Shoot formation from 'Altaswede' callus on L2(N0.05K2) medium. 71
6. Response of 'Norseman' calli on media with different auxin to cytokinin ratios after
five weeks of culture. 78
7. Recovery of senescing calli on SCP medium. 81
8. Callus maintenance on SCP medium. 82
9. Embryogenic callus of 'Altaswede'. 86 10. Somatic embryo development from embryogenic callus of 'Altaswede'. 88
11. Selection of callus capable of embryogenisis. 90
12. Embryogenic callus arising from regenerated plantlet at the point of root origin. 92
13. Embryogenic and non-embryogenic calli of 'Altaswede' 93
14. Multiple shoot production in red clover. 98
15. Karyotype of normal root tip of red clover cv. 'Altaswede' (2n=2X=14). 105
16. Root tip chromosome numbers of a diploid/tetraploid RG4 plant of 'Altaswede'. 107
xii 17. Zymogram of phosphoglucose isomerase from RG3 plants of red clover cv. 'Altaswede'. 110
18. Zymogram of phosphoglucomutase from calli of red clover cv. 'Altaswede'. 111
19. Zymogram of malate dehydrogenase from RG3 plants of red clover cv. 'Altaswede'. 112
20. Zymogram of 6-phospholguconate dehydrogenase from RG3 plants of red clover cv. 'Altaswede'. 113
21. Cellulose acetate electrophoresis of leghaemoglobin of red clover cv. 'Altaswede'. 115
xi i i Aknowledgement
I am most grateful to Dr. F.B.Holl, my supervisor, for his direction, encouragement and help throughout the period of my studies, and to the members of my thesis committee,
Drs. A.D.M.Glass, C.R.Norton and V.C.Runeckles for their reviewing the draft and very useful comments.
Sincere thanks are extended to Ms. Mei Sun for her help in the isozyme analysis and to Mr. L.J.Veto for his guidance and assistance with the SEM photos of cultured materials. Thanks also go to Drs. R.R.Smith, E.Rosenberg and K.Cole for their advice with respect to methods for cytogenetic analysis.
I want to express my appreciation to the faculty, staff and students of the Department of Plant Science who provided
help and friendship during my studies at UBC.
I am also grateful for the financial support of the
Ministry of Education of the People's Republic of China and
the continuing encouragement of my family and friends in
China. I thank them for their commitments.
xi v 1. INTRODUCTION
Legume seed and forage species are the second most important food and feed crops after cereals in world agriculture. The present and future utility of plant cell and tissue culture for the improvement of these crops depends upon efficient plant regeneration from the cultured materials. Unfortunately legume species are often difficult to regenerate from cell and callus cultures. It has been suggested that these species, especially the seed legumes, are equally or perhaps even more difficult to manipulate in vitro than grasses (Conger, 1981). However, much progress has been made recently in the achievement of plant regeneration in forage legumes such as alfalfa (Medicaqo sativa L.) (McCoy and Walker, 1984) and red clover
(Trifolium pratense L.) (Phillips and Collins, 1983).
Red clover is an important hay crop with good feed value (Taylor, 1973; Taylor and Smith, 1979). It is also excellent for use as a rotation crop due to- its soil
improving properties. A red clover crop may return to the
soil between 125 and 200 kg of nitrogen per ha. annually, thus greatly reducing the need for and cost of nitrogen
fertilizer for subsequent crops.
Plant regeneration from suspension and callus cultures of non-meristematic tissues (e.g. hypocotyl) of red clover was reported previously by Phillips and Collins (1979a;
1980). However, regeneration was dependent on the genotype
used and the frequency of regeneration from hypocotyl callus
1 2 was low (Phillips and Collins, 1979a). Therefore, plant regeneration could be a problem when using another cultivar, or a larger number of regenerated plants are required for study.
It is believed that somaclonal variation may represent a novel source of variation that can be exploited in plant breeding programs (Larkin and Scowcroft, 1981). Previous work on the tissue culture of red clover focussed on the development of ijn vitro methods. Regenerated plants were observed to be normal both morphologically and cytogenetically (Beach and Smith, 1979; Phillips and
Collins, 1979a). Information is lacking as to whether abnormalities are a common phenomenon in regenerated red clover plants.
Most reports concerned with the changes induced via tissue culture have been centered on quantitative characters. Because these characters are variable and easily affected by environmental factors, somaclonal variation is confounded by environmental variation. It is also more difficult to assess the frequency of variation for quantitative characters. Irvine (1984), using five sugar cane (Saccharum officinarum) clones with distinctive markers, observed only a low frequency of stable variation.
The experiments conducted here, using red clover as the experimental material, were designed for two main purposes:
(1) to investigate the regulation of callus culture derived
from hypocotyl segments and plant regeneration from the cultures and (2) to evaluate the regenerated plants for somaclonal variation in the characters analysed. 2. LITERATURE REVIEW
2.1 Tissue Culture in Red Clover
Niizek and Kita (1973) initiated the tissue culture of red clover using anthers as explants to induce callus. The basal media of Miller (1961) and Bourgin and Nitsch (1967) modified with various combinations of IAA, NAA, BA and GA were evaluated for callus induction. Only on medium with 17
MM IAA and 0.6 MM BA added was callus induced. No morphogenesis occurred. Following this report other
investigators (Ahloowalia, 1976; Ranga Rao, 1976) described callus cultures initiated from different tissues. These
reports described little evidence for morphogenesis and no plants were regenerated.
Beach and Smith (1979) cultured seedling hypocotyls and ovaries. Callus formed when 2,4-D was present in combination with auxin and KIN. In the absence of 2,4-D
little or no callus resulted. Hypocotyl explants produced more callus than ovary tissue. Callus formation was most
successful on B5 medium (Gamborg et al., 1968) or a modified
B5 basal medium containing 10 mg/1 thiamine, 11 uM NAA and
10 uM each of 2,4-D and KIN. Callus production was poor
when B5 was substituted by the mineral salts of Blaydes
(1966) or Murashige and Skoog (1962). After four weeks
calli were transferred to another medium for shoot-bud
differentiation. Gamborg's B5 with 20 mg/1 thiamine, 10 uM
NAA and 15 MM ADE was the most effective combination. Beach
4 5 and Smith (1979) also observed that it was the white friable callus with isolated green segments, rather than uniformly green callus, that differentiated into plants. KIN, 2iP and
BA failed to induce shoot differentiation and the addition of coconut milk did not enhance callus formation.
More intensive investigations have been conducted by
Phillips and Collins (1979a; 1980; 1981). They (1979a)
first evaluated the basal nutrient requirements for callus cultures induced from hypocotyl segments. Visual evaluation of cultures produced using more than 125 media led to the development of a modified medium, referred to as L2. This medium was shown to be more supportive of red clover genotypes in callus culture than other media tested. PIC was used as the auxin source, as it was shown to be more efficient in action than 2,4-D (Collins et al., 1978).
Callus was induced on L2 medium containing 0.06 mg/1
PIC and 0.1 mg/1 BA, a combination which had resulted from
the evaluation of several regulators including IAA, NAA,
PIC, 2,4-D, BA and KIN (Phillips and Collins, 1979a). Plant
regeneration was achieved when calli were subcultured onto
other media. Generally meristem-derived calli were more
capable of regenerating plants than non-meristem derived
calli. The frequency of genotypes that gave regeneration
for the former was 30% to 80% but for the latter it was only
1% (Phillips and Collins, 1979a). For regeneration from
non-meristem derived calli, a 2,4-D-containing medium (in
combination with other auxins) was more effective than the 6
PIC + BA combination.
Plant regeneration was also achieved from cell
suspension culture (Phillips and Collins, 1980). Explants
of hypocotyl and epicotyl sections of seedlings were put
into liquid L2 medium containing 0.06 mg/1 PIC and 0.1 mg/1
BA to initiate cell suspension cultures. Callus recovery
and bud initiation resulted after a small amount of
suspension culture was inoculated on agar media. Both the
kind and concentration of auxin were critical for bud
initiation and the medium (designated LSE) containing 0.01
mg/1 2,4-D and 2.0 mg/1 ADE was most effective. A second
medium (LSP), which contains 0.001 mg/1 PIC and 0.2 mg/1 BA
was found to be effective for shoot development from buds.
Shoot tips could be proliferated on a medium containing
0.003 mg/1 PIC and 0.5 mg/1 BA (Parrot and Collins, 1983).
In other experiments by Campbell and Tomes (1983) BA was
superior to 2iP and KIN, with maximum shoot number at the
concentration of 2.0 mg/1 BA.
Other ijn vitro experiments have been conducted to
develop methods of special use. Phillips and Collins
(1979b) established the procedures for meristem culture to
obtain virus-symptom free plants. Embryo culture techniques
were employed to rescue immature hybrid embryos (Evans,
1962; Collins et al., 1981; Phillips et al., 1982) and
methods for long-term cold storage of shoot cultures have
been developed by Cheyne and Dale (1980). 7
2.2 Somaclonal Variation
The phenomenon of variability among regenerated plants from cell and tissue cultures was reported more than a decade ago (Sacristan and Melchers, 1969; Morel, 1971).
However, it is only in the last few years that much attention has been paid to it. In a review article Larkin and Scowcroft (1981) used the term "somaclonal variation" to describe the variation observed among regenerated plants as a consequence of tissue culture. Since the recognition of the importance of this phenomenon in the application of tissue culture to plant breeding, numerous papers have been published addressing the problem. Somaclonal variation has been reported for many species investigated, although
stability of character expression in regenerated plants has been observed in other experiments (Larkin and Scowcroft,
1981; 1983a; Lorz, 1983; Scowcroft and Larkin, 1983; Reisch,
1983; Evans et al., 1984). Wherever the objective is to
obtain plants identical to parental type for propagation,
somaclonal variation is a hazard. When the objective is to
select variants as source material for plant improvement,
somaclonal variation represents a novel source of variation, at least in terms of the method by which variation is
generated.
A tissue culture cycle refers to the procedure which
involves the establishment of a more or less
dedifferentiated cell or tissue culture from diverse sources
(multicellular explants, isolated single cells or 8
protoplasts), on defined media, proliferation for a number of cell generations and the final regeneration of plants.
The regenerated plants may exhibit variation in such aspects as morphology, yield, disease resistance and biochemical characters. Although there are numerous proposals concerning genetic and cytogenetic abnormalities which could be responsible for the observed phenotypic variation of regenerants, the origin of these abnormalities is not yet clearly understood. In some cases the observed variation is transmissable through sexual reproduction (Reisch, 1983).
In this thesis representative examples of some species and the generalities of this phenomenon will be reviewed.
2.2.1 Variation among Regenerated Plants of Several Crop
Species
2.2.1.1 Sugar Cane
One interesting and important aspect of somaclonal
variation in sugar cane has been the appearance of
disease-resistant regenerants from tissue cultures of
susceptible cultivars. Following reported variation in
morphological, cytogenetic and isozyme traits (Heinz
and Mee, 1969; Heinz, 1973), efforts were made to seek
resistance to Fiji disease (a leafhopper transmitted
virus) and downy mildew (Sclerospora sacchari) (cf.
Heinz et al., 1977). A predominant shift toward
increased resistance was observed for both diseases
even though the variety used for initiation of tissue 9 culture was apparently resistant. Extensive variation among sugar cane somaclones to several diseases was confirmed (e.g. Heinz et al., 1977; Larkin and
Scowcroft, 1981; 1983b). Interestingly, plants regenerated from cultivar Q101, which was susceptible to eyespot disease (Helminthosporium sp.), shifted significantly to the resistance side of the Q101 reaction, whereas all 52 somaclones from resistant cultivar Q47 remained resistant (Larkin and Scowcroft,
1981). Subsequently, Larkin and Scowcroft (1983b)
initiated cultures from a sugar cane which was highly
susceptible to eyespot disease and from which they were able to isolate somaclones with varying degrees of
tolerance. Evaluation of subsequent vegetative
generations indicated that at least some of the
variation could be retained through vegetative
propagation.
2.2.1.2 Potato
Potato "is another species where somaclonal
variation has been extensively demonstrated. Shepard
et al. (1980) regenerated plants from leaf protoplasts
of 'Russet Burbank'. Among 10,000 somaclones screened
significant and stable variation was found for tuber
morphology, plant growth habit, photoperiod response,
maturity date and yield. In another study (Secor and
Shepard, 1981) 65 selected clones of 'Russet Burbank'
from leaf protoplasts were analysed for 35 characters. 10
Significant variation was found for 22 of the characters and all somaclones differed from 'Russet
Burbank' in one or more characters. Variation ranged
from a minimum of one character in three somaclones to a maximum of 17 characters for one somaclone. A
similar result was obtained by Thomas et al. (1982);
all the 23 protoplast-derived plants differed from each
other in one or more of the 10 morphological characters
analysed. Only one somaclone of the parental type was
recovered. Morphological variation and chromosome
instability have also been reported in other studies
(Karp et al., 1982; Ramulu et al., 1983; Creissen and
Karp, 1985). Regenerated plants were screened for
resistance to early blight (Alternaria solani) and late
blight (Phytophthora infestans) (Bidney and Shepard,
1981). Five out of 500 somaclones were more resistant
to early blight than the parent; 20 out of 800 were
more resistant to late blight. This altered expression
was retained through several vegetative generations.
In contrast, Wenzel et al. (1979) observed remarkable
uniformity among the regenerated plants from potato
protoplast cultures. The discrepancy between the
results of Shepard et al. (1980) and Wenzel et al.
(1979) may be attributed to genotypic difference,
ploidy level (Shepard: 4X; Wenzel: 2X); sample size
(Shepard: 10,000; Wenzel: 211) and tissue source of
protoplasts (Shepard: leaf mesophyll; Wenzel: stem tip 11 culture). Thomas et al. (1982) using the same technique as Wenzel et al. (1979) observed variation among the protoplast-derived regenerated plants of a tetraploid cultivar of potato.
2.2.1.3 Tobacco
In tobacco a number of reports have described variability in plants derived from i_n vitro culture.
Evidence for somaclonal variation was reported in work by Burk and Matzinger (1976). These authors self-fertilized an inbred variety 'Coker' for 15 generations following its release as a pure line, to ensure a high degree of inbreeding. Selfed progeny of tissue culture-derived lines were analysed with respect to characters such as yield, grade index, days to
flowering and total alkaloids. Significant variability was shown for all ten characters investigated. A more detailed analysis of regenerants was carried out by
Burk and Chaplin (1980) using cultures initiated from a hybrid, heterozygous for two loci affecting chlorophyll
synthesis and leaf color. Genetic analysis of progeny of somaclonal variants indicated that the frequency of mutation was 3.5% and 3.6% at each locus respectively among 1,666 plants. In a recent study on plants
regenerated from mesophyll protoplasts heterozygous for
the sulfur locus (Su/su), 79 out of 2,156 morphogenic colonies were identified to be non-parental and about
75% of the 79 colonies were claimed to be somaclonal 12 variants because they produced regenerated plants of different genotypes (Lorz and Scowcroft, 1983). Among
the selfed regenerated plants 37% (33/96) had
segregation ratios which deviated significantly from
the expected 1:2:1 ratio.
2.2.1.4 Rice and Wheat
Oono (1978) and Kucherenko (1979) studied the
inheritance and variations of traits of rice (Oryza
sativa L.) somaclones. Results showed that rather high
frequencies of variation occurred for traits like plant
height and tillering in progeny of somaclones. An
intensive study of variation in rice somaclones and
their progenies has been carried out (Sun et al., 1981
cited in Sun et al., 1983; Zhao et al., 1982). When
the inheritance of and variations for some traits of
more than 2,000 plants were investigated in the second
and third generations of regenerated plants, only 24%
of the rice lines were normal for all the traits
studied (Sun et al., 1983). The frequency of variation
for different traits varied from 11.5 to 39.5%. A
dwarf mutant which was controlled by a recessive gene
was identified. Trends were also observed for plants
to be shorter, to produce more productive tillers and
for 1000-grain weight to be reduced (Sun et al., 1983).
More recent experiments described the identification of
promising breeding lines in yield, maturity and height
(Zhao et al., 1984). Variation in grain protein 13
content has also been described among progenies of
dihaploids regenerated from anther callus cultures
(Schaeffer et al., 1984).
In wheat (Triticum aestivum L.) using immature
embryos as explants improved efficiency of plant
regeneration (Bhojwani and Hayward, 1977; O'Hara and
Street, 1978; Karp and Maddock, 1984; Larkin et al.,
1984). Somaclonal variation has been reported for
wheat in several cases, but the analysis was mainly
on regenerated plants (e.g. Lupi, 1981; Yurkova et
al., 1982; Karp and Maddock, 1984). Recently more
intensive work by Larkin et al. (1984) reported
variant characters among 142 regenerants of 'Yaqui 50E'
which included height, awns, tiller number, grain
color, heading date, waxiness, glume color, gliadin
protein and a-amylase regulation and the occurrence of
both dominant and recessive mutations. They claimed
that chromosome loss or addition were not evident as
the primary source of variation.
2.2.2 Origin of and Factors Affecting Somaclonal Variation
It is important both for academic interests and for practical use to determine whether the observed genetic variation simply reflects the variability pre-existent in somatic cells before the culture cycle or is a consequence of the culture process. Lorz and Scowcroft (1983) regenerated plants from protoplasts of Su/su heterozygotes 14 of Nicotiana tobaccum and, using leaf color analysis, estimated that the majority (92.2-98.4%) of colonies were parental type, but 1.4-6.0% were culture-induced variants and only 0.1-1.8% pre-existing variants. Thomas et al.
(1982) found that considerable variation existed for ten morphological characters among regenerated plants of potato from the same callus originated from a single cell of a tetraploid cultivar, demonstrating clearly that the variation was cell-culture induced. However the occurrence of somaclonal variation may depend on the genotype and explant used, length of culture period or other iji vitro conditions.
It has been well documented that changes in karyotypic
structure are increased with the length of culture period
(Bayliss, 1980; Reisch, 1983). Accordingly, plants
regenerated from long-term cultures tend to have more
abnormalities. Flower and leaf abnormalities were commonly
found among tobacco plants regenerated from long-term (up to
28 months) cultures, while they were not observed among the
plants from short-term (1-5 months) cultures (Syono and
Furuya, 1972). McCoy et al. (1982) observed that plants
showing cytogenetic abnormalities derived from tissue
cultures of oat increased from 12% to 48% and 49% to 88% in
two lines, respectively, when plants were regenerated after
four and twelve months in cell culture. Chrysanthemum
morifolium plants, regenerated from leaf callus, maintained
for nine years showed altered leaf shape, smaller and 15 abnormally shaped flowers and excessive lateral shoot growth as compared with plants regenerated from callus in culture
for only one year (Sutter and Langhans, 1981).
One or more components in the medium under culture conditions may act as mutagens to induce variation in cultured cells. Phytohormones, especially synthetic auxins, may be the most probable candidate (Lorz, 1983). Shepard
(1981) observed differences in the frequency of variants after regenerating plants from potato protoplasts using different concentrations of NAA and 2,4-D in the medium.
Few reports are available on this topic, partly because
there is no good system with which to work. Deambrogio and
Dale (1980) regenerated plants from tissue cultures of
barley (Hordeum vulgare) using 1, 2, 3 and 4 mg/1 2,4-D.
Variation in such traits as albinism, leaf shape, and tiller
fertility happened only at 4 mg/1 2,4-D. Plants were
regenerated on two different media (NK and I) from cultures
which originated from a single stock callus of Haworthia
setata and eight characters were analysed (Ogihara, 1981).
Chromosome analysis revealed that NK medium tended to
regenerate more tetraploid plants, but fewer plants with
translocations than the I medium.
The relationship between occurrence of variants and
genetic background of material was indicated in some
publications. First, there is an obvious difference among
species concerning the level of variability. McCoy et al.
(1982) reported a high frequency of chromosome abnormalities 16 in oat (Avena sativa L.) in one study, while McCoy and
Phillips (1982) observed a high degree of chromosome stability, although the techniques employed were similar in both cases. Second, differences among cultivars of the same species have been clearly demonstrated. In sugar cane, variants were found among a total of 4,600 regenerated plants (Liu and Chen, 1976). For clone F156 the frequency of morphological change was 34.0% while for clone 146 it was
1.8%. In potato considerable variation was observed among the protoplast-derived plants of tetraploid cultivars while no variation was observed among the regenerated plants of a diploid cultivar evaluated using similar procedures (Wenzel,
1979; Shepard et al.. , 1 980; Thomas et al., 1982). It was suggested that this difference was due to differences in genetic background rather than ploidy level of the starting materials (Lorz, 1983). In an analysis of cell-derived plants of Oryza sativa L. more than 2,000 plants were
investigated (Sun et al., 1983). The percentages of polyploids for nine varieties of the 'Hsien' (indica) group were 0-13.3%, but no polyploid was detected in the nine varieties of the 'Keng' (japonica) group. Differences were also found in protoplast-derived potato plants of two
tetraploid cultivars (Karp et al, 1982). The cytogenetic analysis of plants from tissue cultures of two Avena sativa
lines revealed that the line 'Tippecanoe' had twice as many cytological alterations as the line 'Lodi' (McCoy et al.,
1982). 17
Explant source could also have a significant effect on somaclonal variation. Plants regenerated from callus of four types of explants of pineapple (Ananas comosus L.) were altered according to the explant source (Wakasa, 1979).
Plants from the slip, crown and axillary buds showed changes only in spine characters, but plants from syncarp included variants in leaf color, spine, wax and foliage density. Van
Harten and Broertjes (1981) showed that morphological variations were observed in 12.3% of plants from leaflet disks, but in 50.3% of plants from rachis- and petiole-derived callus.
Although it is generally believed that plants regenerated via embryogenesis produce fewer variants than those via organogenesis, direct comparisons between the two regeneration pathways are very rare. Somatic embryogenesis has been achieved in many cereal species and obvious phenotypic and cytogenetic variants have not been found among the regenerated plants (Vasil and Vasil, 1981; Vasil et al., 1982). The stability of regenerated plants was reconfirmed in a recent study (Swedlund and Vasil, 1985).
However, chromosome number and structure changes were observed in plants regenerated via embryogenesis from
Triticum aestivum L. cultures (Karp and Maddock, 1984).
2.2.3 Genetic Basis
Understanding the genetic basis of somaclonal variation
is important to facilitate its practical use for plant 18 improvement. Based upon existing experimental knowledge somaclonal variation may be attributed to (1) chromosome changes (number and structure); (2) gene alterations; (3) cytoplasmic DNA changes and (4) other kinds of heritable change such as transposible elements.
Chromosome variability of regenerated plants has been well documented (D'Amato, 1977; 1978; Sunderland, 1977;
Bayliss, 1980; Larkin and Scowcroft, 1981; Krikorian et al.,
1983) . Ploidy changes are the most frequently observed chromosomal abnormality. Prat (1983) regenerated 172 plants
from mesophyll protoplast culture of two lines of Nicotiana
sylvestris . Seventy two regenerated plants were diploid
like the original lines, while the remainder were polyploid; no aneuploids were recovered. From haploid protoplasts of
N. sylvestris Faccioti and Pilet (1979) regenerated plants which were essentially diploids. Ploidy changes were also
readily observed in potato and tobacco (Karp et al., 1982;
1984) .
Aneuploids have been recovered among regenerated plants
in many cases where chromosome abnormality occurs, although
the frequency is usually lower than euploids (Faccioti and
Pilet, 1979; Karp et al., 1984). Analysis of plants derived
from cultured immature embryos of four wheat cultivars
(Triticum aestivum 2n=6X=42) showed that 29% of the 192
plants examined were aneuploids ranging in chromosome number
from 38 to 45 (Karp and Maddock, 1984). 19
In addition to chromosome number changes chromosomal rearrangements such as deletions, translocations and inversions may also be expected. Translocations have been observed among regenerated plants of ryegrass (Lolium sp.)
(Ahloowalia, 1976), potato (Shepard, 1982), oat (McCoy et al., 1982), Haworthia setata (Ogihara, 1981) and wheat X rye hybrids (X Triticosecale Wittmack) (Lapitan et al., 1984).
In work with large chromosomes of Haworthia setata, Ogihara
(1981) was able to demonstrate translocations and deletions.
More new karyotypes were detected in regenerated plants than in cloned calli. Rearrangements were confirmed to be reciprocal translocations by the observation of quadrivalents in metaphase I. Chromosome bridges and segments, or segments alone, were interpreted as evidence of paracentric inversions and sub-chromatid aberrations.
Plants regenerated from callus cultures of hybrids of
Hordeum vulqareX H. jubatum showed enhanced freqency of bivalents and multivalents and they were presumed to be the result of either translocation or the deletion of a pairing suppressor (Orton, 1980).
Increases in recombination rate have been observed on tomato (Sibi et al., 1984). Phenotypically distinguishable recessive marker genes were selected in the 'Monalbo'
homozygous background. Cotyledons of individual F2 hybrid seedlings were used as ir\ vitro tissue culture material, while the rest of the plant was grown as a control.
Recombination rates of the selfed progenies from regenerated 20 plants and matched controls were compared. For two loci
'bs-ms32' on chromosome I increases of 6.07 to 6.91 map
units were observed and for the 'aa-d' region of chromosome
II, the increases ranged from 4.92 to 6.04 map units.
Cytoplasmic genetic changes may also be responsible for
observed variation. The resistance of corn to leaf blight
and cultivar sensitivity to a host toxin of Helminthosporium
maydis race T is tightly associated with Texas male sterile
(cms-T) cytoplasmic genotypes in seed-derived plants.
Gengenbach et al. (1977) selected for resistance to toxin
in vitro and regenerated resistant plants with the aim of
recovering resistant, cms-T breeding lines. However,
resistance was associated with a concomitant reversion to
male fertility.
Gengenbach et al. (1981) observed mitochondrial DNA
variation in T-cytoplasmic male sterile maize plants
regenerated from tissue culture and selected for resistance
to Helminthosporium maydis race T. Restriction enzyme
analysis of mitochondrial DNA showed differences between
different regenerated plants and also differences when
compared to T or N cytoplasm maize plants. A specific type
of transposition event in maize mitochondrial DNA was shown
to be correlated to a change in callus morphology (Chourey
and Kemble, 1982). The observed DNA variation was limited
to two plasmid-like DNA components called SI and S2. When
DNA analysis was performed on doubled haploid plants of
Nicotiana sylvestris produced from pollen culture, heritable 21 quantitative and qualitative changes were observed in the nuclear DNA (De Paepe et al., 1983). Doubled haploid plants contained, on average, increasing amounts of total DNA and increasing amounts of highly repeated sequences. More research at the molecular level will be essential for a better understanding of the genetic basis of somaclonal variation. 3. MATERIALS AND METHODS
3.1 General Culture Methodology
3.1.1 Media
In this study an established medium L2 (Phillips and
Collins, 1979a) was used except in one test in which B5
(Gamborg et al., 1968) (Appendix 1) was used. Composition
of basal L2 medium is as follows (mg/1): NH4N03, 1,000;
KN03, 2,100; KH2PO„, 325; NaH2P0„-H20, 85; CaCl2.2H20, 600;
MgS0«-7H20, 435; FeSO««7H20 (EDTA), 25; H3B03, 5; KI, 1;
MnSO„'H20, 15; ZnSO«-7H20, 5; CuSO«'5H20, 0.1; Na2MoO„2H20,
0.4; CoCl2-6H20, 0.1; Thiamine, 2.0; Pyridoxine-HCl, 0.5;
Myo-inositol, 250; sucrose, 25,000. Different growth
regulators and concentrations were added in various tests
depending on the culture objectives. Growth regulators used
were: cytokinins: BA, ADE and KIN; auxins: 2,4-D, IAA, NAA
and PIC.
Stock solutions were made for (1) macro-nutrients
(NH„N03, KNO3, KH2POft, NaH2PO„-H20 and MgS0fl-7H20) X10; (2)
CaCl2-H20, X20; (3) FeSO«-7H20 (EDTA) X200; (4)
micro-nutrients (H3B03, MnSOft«H20, ZnS0i,'7H20, CuS0««5H20,
Na2MoO(,-2H20 and CoCl2.6H20) X400; (5) KI X1000; and (6)
thiamine and pyridoxine X1000. In preparing FeSOa*7H20
stock solution, 5.00 g FeSOa«7H20 were first dissolved in
hot distilled water and then 7.45 g Na2EDTA were added. The
volume was brought up to 500 ml, resulting in a solution of
22 23
5 mg/1 FeSOa«7H20. The CaCl2«2H20 stock solution was made separately to prevent precipitation in the stock basal salts. Thiamine and pyridoxine were prepared as aqueous solut ions.
Stock solutions were also prepared for each of the growth regulators used: BA, 1 mg/ml; ADE, 1 or 2 mg/ml; KIN,
1 mg/ml; IAA, 1 mg/ml, NAA, 0.2 mg/ml; 2,4-D, 0.5 mg/ml; and
PIC, 0.06 mg/ml. BA and ADE were first dissolved in a small amount of 1N KOH and distilled water was added to the appropriate volume. KIN was first dissolved in 1N HC1 and subsequently diluted with water. The auxins 2,4-D, NAA and
PIC were dissolved in a small amount of "ethanol prior to the addition of water. Stock solutions were stored in colored bottles or bottles wrapped with tinfoil at 4°C except for the IAA stock solution which was stored frozen.
Media were prepared by adding appropriate volumes of stock solutions of salts, vitamins and growth regulators to a large volume of distilled water. Sugar was then added and the media were solidified with 8 g/1 agar (Difco Bacto) unless otherwise specified. The pH of media was adjusted to
5.8 with IN KOH before adding agar and media were autoclaved at 121°C, 1.0 kg/cm2 for 15 min. (20 min when a container held more than 1,000 ml of medium).
Media in these experiments are specified by the abbreviations of basal media and the initials of growth regulators. For example, L2(P0.06B0.2) refers to L2 medium containing 0.06 mg/1 PIC and 0.2 mg/1 BA. The letter before 24 the brackets represents a basal medium. L2 (Phillips and
Collins, 1979a) and B5 (Gamborg et al., 1968) refer to two basal media respectively. Within the brackets letters symbolize the growth regulators, usually an auxin and a cytokinin. Symbols for growth regulators were: 2,4-D (D);
IAA (I); NAA (N); PIC (P); ADE (A); BA (B); and KIN (K).
Each regulator symbol is followed by a number which indicates the concentration used in mg/1. Several media which were used frequently have been assigned special abbreviations. All these media contain L2 basal salts.
Growth regulators used in these media were as follows: (1)
L2: containing 0.06 mg/1 PIC and 0.1 mg/1 BA; (2) LSE: containing 0.01 mg/1 2,4-D and 2 mg/1 ADE; (3) LSP: containing 0.002 mg/1 PIC and 0.2 mg/1 BA and (4) SCP: containing 2 mg/1 2,4-D, 2 mg/1 BA and 2 mg/1 ADE. L2, LSE and LSP have been specified by Phillips and Collins (1979a;
1980).
3.1.2 Explants
Trifolium pratense L. cv 'Altaswede' and cv 'Norseman' were used. The former is a diploid cultivar and the latter a tetraploid cultivar. 'Altaswede' was obtained from
Richardson Seed Company Ltd and 'Norseman' from Charles
Sharpe & Company Ltd.
Seeds were germinated in half-strength L2 basal medium with 5 g/1 sucrose and 8 g/1 agar. The medium was dispensed into 25 X 150 mm tissue culture tubes, each receiving 20 ml 25 volume. Seeds were sterilized in 75% ethanol for 5 min., then in 20% commercial bleach (1.0% sodium hypochlorite in
final concentration) for 20 min., and rinsed in sterile distilled water at least three times. Six to eight
sterilized seeds were put into test tubes to germinate and were incubated for 8-10 days in a growth chamber at 25°C
with a photoperiod of 16 hours (200 izEm" 2sec" 1 ) . Seedling
hypocotyls were cut aseptically with a scalpel into 3-4 mm
segments for callus induction and 3-4 mm shoot tips were
utilized for shoot tip cultures. All experimental manipulations of explants were carried out under aseptic
conditions. Transfers were facilitated using a laminar air
flow cabinet .(Environmental Air Control Co.).
3.1.3 Culture Conditions
Two kinds of culture vessels were used in these
experiments. When test tubes (25 X 150 mm) were used, bulk
medium with all components added and pH adjusted to.5.8 was
put into a microwave oven to melt the agar. Once the agar
had melted the medium was dispensed into the test tubes.
Tubes were capped with plastic closures (Bellco Biological
Glassware & Equipment). The subculture interval was usually
four weeks.
When petri plates were used, the bulk medium was poured
into the plates (30 ml/plate) in the laminar air flow
cabinet when medium temperature had cooled to approximately
45°C after autoclaving and allowed to set. Following 26 explant transfer the plates were wrapped with parafilm to prevent contamination and reduce moisture loss. The
subculture interval for plate cultures was also four weeks.
Six growth chambers were used during the experiments.
Therefore, different experiments may have different light
intensities. They will be specified in descriptions of methods for individual experiments. Light was provided by
cool-white fluorescent tubes. The light/dark period was
16/8 hours unless otherwise indicated. The temperature in
these chambers was 25±1°C.
3.2 Specific Culture Methodology
3.2.1 Callus Induction
Hypocotyl segments 3-4 mm in length were transferred
onto L2(PO.06BO.1) medium for callus induction. Cultures
were maintained at 25°C in growth chambers. Callus
induction was evaluated visually two weeks later.
In batch one, test tubes were used as culture vessels,
Cultures were incubated in a growth chamber with light
intensity of 500 yEm"2sec"1. In batch two, sterile petri
dishes were used containing explants from two seedlings with
each having three to five explants. These petri dishes were
put into a growth chamber at a light intensity of 50
nEm~2sec"1.
Five batches of cultures were established to induce
callus from hypocotyl explants. Test tubes were used in 27 three batches and petri plates in the other two. Each test tube contained three explants from one seedling.
Observations were made in two of these three batchess to determine the genotype effect on callus induction i.e. whether any relation exsits between the three explants for callus induction.
3.2.2 Callus Maintenance and Plant Regeneration
The following observations were made to evaluate callus
growth and differentiation on various media:
(1) green-spotted callus (GSC): callus with isolated green
regions containing meristem-like structures distributed
on the callus. It has been reported that these
structures could be an indicator of regeneration (Kim
and Jang, 1984). Calli were scored using this
structure to predict their potential for
differentiation.
(2) shoot callus (SC): callus with one or more shoots,
indicating shoot differentiation.
(3) root callus (RC): callus with one or more roots.
(4) brown callus: callus with brown color. This kind of
callus did not grow well.
(5) dead or necrotic callus: callus which was dark brown and
sometimes hard in texture.
(6) growth: In some experiments callus was graded for their
relative growth rate indicated by plus signs (more plus
signs meaning faster growth). 28
3.2.2.1 Primary Test of Two Media
After successful callus induction, attempts were made to achieve plant regeneration from callus
cultures. Two media were first tried: (1) B5 medium containing 20 mg/1 thiamine, 2 mg/1 NAA and 2 mg/1 ADE
(=B5(N2A2)) and (2) L2 medium containing 0.01 mg/1 2,4-D and 2 mg/1 ADE (=LSE). These media have been reported to be capable of inducing plant regeneration (Beach and
Smith, 1979; Phillips and Collins, 1980). Calli were induced on L2 medium and transferred to plates of one of the above media. Each plate was inoculated with three pieces of callus. Cultures were maintained in a chamber with light intensity of 900 uEm_2sec"1. The cultures were evaluated two weeks later for differentiation and growth. Some calli were transferred onto fresh media of both types and incubated for four more weeks before evaluation. Fifty calli of each cultivar which had been cultured on LSE for seven weeks were further subcultured on LSE medium to determine the effect of subsequent subcultures.
3.2.2.2 Callus Growth on LSE Medium
Test 1: The LSE medium failed to stimulate shoot
induction in the previous experiment (Section 3.2.2.1).
It was possible that this absence of success could be attributed to (1) a small callus sample size and/or (2)
some minor environmental condition which did not meet
the requirements for shoot induction. In another 29 experiment, 1452 calli of 'Altaswede' and 1556 calli of
'Norseman' were transferred from L2 to LSE medium on
August 3 and 4, 1984, and cultured for four weeks.
Subsequently, 984 'Altaswede' calli and 568 'Norseman' calli were subcultured onto fresh LSE medium. Various light treatments were applied to the cultures: (1)
Light intensity varied from 70 to 900 juEirr2 sec ~ 1 . This gradation was accomplished by stacking ten culture plates in one column in two chambers with maximum light
intensities of 500 or 900 /uEirr2 sec ~1; (2) Photoperiods of 16 and 24 hours were used for 'Altaswede' calli and
16 hours for 'Norseman' calli.
Test 2: It was also possible that calli growing on
LSE medium required tranfer to another medium for shoot
emergence because of the absence of some factor in LSE medium necessary for shoot development. To test this hypothesis, calli were first initiated from hypocotyl
explants. They were then transferred onto LSE medium
for two or three weeks. These calli were subcultured
onto the following four media: (1) LSP medium, which
has been claimed to be supportive of shoot development
(Phillips and Collins, 1980); (2) L2(P0.002B0.2 and
half strength of salts) medium, to evaluate the effect
of reduced concentrations of basal salts on callus
differentiation; (3) L2(NO.05KO.5) medium and (4)
medium 4, which had only L2 basal medium without growth
regulators added. Calli were cultured in plates with 30
24 hour photoperiod and 100 MEm_2sec"1 for four weeks.
Test 3: In this test, different callus sources were used. They had been cultured on L2(D2B2) medium.
Callus from primary cultures was transferred onto a medium having the same composition as LSE except containing 5 mg/1 ADE instead of 2 mg/1 ADE. Calli were cultured in test tubes at 400 AtEm~2sec~1 and
16-hour photoperiod for three weeks.
Because LSE medium failed to support shoot
induction in any experiment, further work was carried out to determine the medium composition which would be
supportive of shoot induction.
3.2.2.3 2,4-D and BA
In this test different levels of 2,4-D and BA were evaluated. Two batches of culture were used. The
treatments for the first batch were: 2 and 4 mg/1 BA combined with 2 mg/1 2,4-D. The four treatments for
the second batch were 0.01, 0.1, 2.0 and 10.0 mg/1
2,4-D combined with 2 mg/1 BA and additional 5 mg/1 ADE
was incorporated into the medium containing 2 mg/1
2,4-D and 2 mg/1 BA.
3.2.2.4 PIC and BA
Three tests were carried out. The treatments in
the three tests are listed in Table 1. For
embryogenesis of callus, reduced nitrogen, e.g. amino
acids, has been shown to be a major factor (Raghavan, 31
Table 1. Treatment combinations used to test the effect of PIC and BA on plant regeneration (concentration in mg/1).
Other Test No Treatment No PIC BA Additives1
1 0.001 0.2 1740 Arg 2 0.0005 1 1740 Arg 3 0.001 1 1740 Arg 4 0.005 1 1740 Arg 5 0.01 1 1740 Arg 6 0.05 1 1740 Arg 7 0.0005 1 8 0.001 1 9 0.005 1 10 0.01 1 1 1 0.05 1
1 0.005 1 100 CH 2 0.005 1 500 CH 3 0.005 1 1000 CH 4 0.005 1 5000 CH 5 0.005 1 147 GA
1 0.002 0.2 2 0.005 0.2 3 0.01 0.2 4 0.002 1 5 0.005 1 6 0.01 1
^rg = Arginine; CH = Casein Hydrolysate; GA = Glutamic Acid 1740 mg/1 arginine = lOrnM. 32
1976; Wetherall and Dougall, 1976; Kohlenbach, 1978).
In alfalfa both quality and number of embryos were
stimulated by adding amino acids (Stuart and
Strickland, 1984a). In this experiment arginine,
glutamic acid and casein hydrolysate were added to some
of the media tested.
Test 1: This test was established to investigate
the effect of various concentrations of PIC and BA on
callus growth in presence or absence of arginine. Only
'Altaswede' calli were used. Calli were previously
cultured on L2(D0.01A2) medium. They were transferred
onto test media in plates and incubated at 400
iiEm~2sec~1 with a 16 photoperiod at 25°C. Cultures
were evaluated three weeks later.
Test 2: Another test was established to determine
the effect of casein hydrolysate and glutamic acid
inclusion in medium on callus growth. Aqueous
solutions were prepared using these substances and
their pH adjusted to 5.8. After filter sterilization,
solutions were added to the medium which was cooling
down after autoclaving to give the desired final
concentration. Transferred calli from LSE medium were
incubated with a 16-hour photoperiod at 280 juEm^sec"1 i and 25°C for four weeks.
Test 3: In this test 'Altaswede' calli were
previously cultured on L2(D2B2N2) medium. For
'Norseman' calli of three sources were used in the 33 test: (1) L2(D2B2N2) medium, (2) L2(D2B0.5A2) medium and (3) L2(PO.002B0.2) medium. Each plate had two calli incubated with a 24-hour photoperiod at 200
AiEm"2sec_1 and 25°C. Cultures were evaluated four weeks after the transfer.
3.2.2.5 NAA and KIN
Test 1: In this test 'Norseman' calli which had been cultured on LSE medium for one month were transferred onto media with various concentrations of
NAA (0.001, 0.01, 0.1, 1.0 and 5.0 mg/1) combined with
1 mg/1 KIN. Plates containing the cultures were incubated with a 16-hour photoperiod at 440 juEirr2 sec ~ 1 and 25°C for three weeks.
Test 2: Yellow-green calli of cv 'Altaswede' growing on SCP medium were used for this test. NAA was tested at the following concentrations (mg/1): 0.01,
0.05 and 0.1. Concentrations of KIN were 0.5 or 2.0 mg/1. Calli were transferred onto various medium combinations and cultured with a 24-hour photoperiod at
250 MEm~2sec"1 and 25°C for three weeks.
Test 3: 'Norseman' calli in this test were derived from three sources for each treatment: (1) 14 calli from L2(D2A2) medium, (2) 14-20 calli from
B5(D2N2K2)medium and (3) 16 calli from L2(D2B0.5A2) medium. Cultures were incubated with a 24-hour photoperiod at 250 MEm~2sec~1 and 25°C for four weeks. 34
3.2.2.6 NAA and BA in Presence of ADE
The test was designed to determine the effect of various concentrations of NAA in combination with BA and ADE on callus differentiation. 'Norseman' calli from LSE medium were used. Different levels of NAA (0,
0.001, 0.01 and o.1 mg/1) and BA (0 and 0.2 mg/1) were used with 2 mg/1 ADE in all tests. Calli in test tubes were incubated with a 16-hour photoperiod at 250
/uEm~2sec"1 and 25°C for four weeks.
3.2.2.7 Callus Differentiation on Four Test Media
'Norseman' calli, previously cultured on
L2(D2B2A5) medium, were transferred onto several media:
(1) L2(B0.2A2), (2) L2(DO.01B0.2A2), (3) (NO.01B0.2A2) and (4) L2 medium with half stregth of basal salts and vitamins containing 0.2 mg/1 IAA and 2 mg/1 nicotinic acid. Each large callus was divided into four pieces with each piece put into one of four media contained in
test tubes. All the cultures were incubated with a
16-hour photoperiod at 500 MEm~2sec"1 and 25°C.
Response was recorded after three weeks of culture.
3.2.2.8 Callus Growth at High or Low Auxin to Cytokinin
Ratios
'Norseman' calli from LSE medium were transferred
onto the following two media (1) L2(D11K1) and (2)
L2(D1K10). The first medium had a high auxin to
cytokinin ratio (11), while the second one had a very 35 low ratio (0.1). Calli were cultured with a 24-hour photoperiod at 200 MEm"2sec"1 and 25°C for five weeks.
3.2.2.9 Callus Growth on SCP Medium
In experiments when calli were transferred onto a medium which did not promote efficient callus growth
(e.g. on LSE medium), it would be useful to restore
such calli to a normal vigorous growth pattern. This
test was established to determine the effect of SCP medium as a vehicle for restoring senescing calli.
Calli were previously cultured on LSE medium for
various time periods: (1) 'Altaswede' calli and (2)
'Norseman' calli, both of which had been cultured on
LSE medium for 55 days (At this time the calli had
stopped growing and were showing brown color. Some
calli were dead); (3) 'Altaswede' calli which had been
cultured on LSE medium for 42 days (Some of calli had
senesced, but the cultures were generally better than
those of (1) and (2)); (4) 'Norseman' calli which had
been cultured on LSE medium for 37 days and showed
slight browning (Some calli showed a little growth);
(5) 'Altaswede' calli induced on L2 medium were first
transferred onto LSE medium for 23 days and then
subcultured to L2(P0.01B0.2) medium (At this time these
calli turned yellow brown or dark brown). Calli from
the above sources were transferred onto SCP medium (1
mg/1 2,4-D) in plates. Cultures were incubated at 25°C
and under 16-hour photoperiod with light intensities of 36
250 luEm" 2sec" 1 for callus sources (1), (4) and (5)and
700 MEm"2sec"1 for callus sources (2) and (3). Growth and appearance were recorded after four weeks of culture.
Calli on SCP medium were continuously subcultured on fresh SCP medium for one year.
3.2.2.10 Light Effect on Calli Growing on SCP Medium
This test was established to investigate the effect of light intensity on callus differentiation on
SCP medium in attempts to achieve plant regeneration.
'Altaswede' calli , which had been cultured on LSE medium for one month , were transferred onto SCP medium
in plates and incubated with a 16-hour photoperiod and
at 25°C for 40 days. Different light intensities
(70-450 MEm~2sec"1) were obtained by stacking plates as
14 columns, each having eight to ten plates. Each
plate contained three to four calli.
3.2.2.11 Selection and Maintenance of Embryogenic
Callus
After plantlet regeneration was achieved on two
media containing NAA and KIN, embryogenic calli of
these two treatments were transferred and then
subcultured on LSP medium. Three calli from these
cultures were further subcultured on LSP medium for
embryo proliferation and plant regeneration.
Eventually calli from one callus line (one genotype) 37 was retained. Calli which were capable of embryogenesis were thus obtained. These embryogenic calli were subcultured on LSP medium. Test, tubes containing these calli were incubated with a 16-hour photoperiod at 500 MEm"2sec"1 and 25°C. They were evaluated after four weeks of culture, for embryo development and plantlet production.
3.2.2.12 Growth of Embryogenic and Non-embryogenic
Calli
Calli may be identified on the basis of the occurrence of embryogenesis, i.e. embryogenic and non-embryogenic. This experiment attempted to determine the difference between the two kinds of calli when they were cultured on different media.
Embryogenic calli of 'Altaswede' were obtained from the cultures in which embryogenesis had been observed.
Non-embryogenic calli were from another callus culture of 'Altaswede'. All calli were cultured on the following ten media: (1) L2(NO.01 BO.1), (2)
L2(N0.05B0.1), (3) L2(NO.1 BO.1), (4) L2(NO.01 BO.5), (5)
L2(N0.05B0.5), (6) L2(PO.001 BO.2), (7) L2(N2A2), (8) L2 basal containing 2 g/1 yeast extract and 10 g/1 agar,
(9) L2 basal medium containing 600 mg/1 glutamine and
3% sucrose and (10) L2(N0.5K0.5). Non-embryogenic calli had 19 tubes for each treatment and embryogenic calli, two tubes per treatment. Cultures were incubated at 480 MEm"2sec"1. 38
3.2.3 Shoot Tip Culture
Shoot tips 3-4 mm in length were used as explants for multiple shoot induction. Shoot tips were first cultured on
L2 medium. Three weeks later they were transferred onto
L2(PO.003B2) medium 'for multiple shoot induction. Each test tube contained one shoot tip. Thirty tubes in one rack were treated as a group and groups were randomly assigned.
Cultures were evaluated four weeks later for multiple shoot induction frequency and the number of shoots produced per shoot tip based on group.
Multiple shoots were subcultured twice onto L2 medium containing 0.002-0.003 mg/1 PIC and 1 mg/1 BA for propagation at a subculture interval of one month. Shoots were isolated with a scalpel from the multiple shoot rosette. Only well-formed shoots were utilized.
These cultures were incubated with a 16-hour photoperiod at 200 MEnr2sec~1 and 25°C.
3.2.4 Rooting of Shoots
Shoots obtained from shoot tip cultures or from embryogenic calli were transferred to rooting medium consisting of half strength salts and vitamins of L2 medium and containing 1 mg/1 nicotinic acid, 2.5 mg/1
3-aminopyridine, 0.2 mg/1 IAA, 10 g/1 sucrose and 8 g/1 agar
(Phillips and Collins, 1979a). Cultures were maintained
under a 16-hour photoperiod and 250 /zEnr2 sec ~1. They were
ready to be transferred to pots after one month of growth. 39
3.2.5 Scanning Electron Microscopy
Samples from embryogenic cultures were initially fixed in 4% glutaraldehyde (primary fixative) followed by 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for at least one hour (secondary fixative).
Following fixation, samples were dehydrated through a graded series of ethanol 30%, 50%, 70%, 85% and 95% for 5-10 minutes each and finally to absolute ethanol with two changes of 30 minutes each.
The dehydrated specimens were transferred to a critical point drying apparatus (CPD; Parr 4770). Specimens were placed in the drying chamber with sufficient alcohol to prevent air-drying prior to critical point drying. The cover of the specimen chamber was secured and liquid carbon dioxide admitted to the chamber from an external tank. The inlet valve was kept open and chamber pressure increased to
820 p.s.i. Three to four minutes later the exhaust valve was opened for three to four minutes. After three minutes the exhaust valve was closed. The exhaust valve was alternately opened and closed every three minutes for a total of 14 times. Then the exhaust valve and the liquid carbon dioxide inlet valves were closed. Hot water was poured around the specimen chamber, heating it to 44°C, sufficient to achieve the critical point of carbon dioxide i.e. 31°C. During the heating process the pressure in the chamber rose above 1,600 p.s.i. (pound per square inch).
After a steady pressure of 1,400 p.s.i. at 44°C had been 40 stabilized, the exhaust valve was slowly opened and the pressure was released to 0 p.s.i. while maintaining the specimen chamber at 44-45°C.
The dried specimens were mounted on specimen stubs, transferred to a vacuum evaporator and coated using a
"Nanotech Semprep 2" sputter coater. Coated specimens were viewed under a scanning electron microscope (Cambridge
Stereoscan 250, Cambridge Instrument Co.) and pictures were taken on Polaroid Type 55 film.
3.3 Growth and Analysis of Regenerated Plants
3.3.1 Plant Transfer and Growth in Greenhouse
A mixture of soil, sand and vermiculite (1:1:1 by volume) was used to fill 6 X 6 cm pots and also 50 X 32 cm flats into which the pots were placed. Each flat held 28 pots. The flats were wrapped with a piece of brown paper and autoclaved at 121°C, 1 kg/cm2 for 20 minutes and allowed to cool for at least one day prior to receiving plants.
Soil in individual pots was inoculated with commercial
Rhizobium trifolii (Nitragin Co. Ltd) prior to plant transfer. Plants to be transferred were usually cultured on rooting medium for one month. They were removed from the test tubes using forceps and rinsed free of agar in lukewarm sterilized water. Plants were transferred into pots with the root tip in a downward position. 41
Transplanted plants were transferred to the greenhouse.
High humidity was provided for the first two weeks by covering flats with pieces of Saran Wrap (or transparent plastic film). During this period they were watered frequently. The plants were exposed to normal greenhouse conditions after two weeks. One week after plants were transplanted they were watered with soluble fertilizer
(15-15-18). All subsequent fertilization was carried out using a N-less nutrient solution applied approximately once a week to facilitate root nodule development.
An experiment was initiated to determine the effect of different treatments on the survival of regenerated plantlets. Shoots of 'Altaswede' induced from cultures were rooted in vitro for six weeks. Plants transferred were treated as follows: (1) Transferred plants were left under ambient laboratory conditions for the first week and then transferred to the greenhouse (Plants were covered with
Saran Wrap for the first two weeks); (2) Plants were treated as in (1) except they were placed in greenhouse for the entire period; (3) Plants were moved directly to the greenhouse, but were not covered with Saran Wrap.
Sufficient water for growth was supplied during this period.
Evaluation of survival was made four weeks later in all treatments. 42
3.3.2 Chromosome Analysis
Root tips of regenerated and control plants
(seed-derived) were collected individually from fresh, thick roots. Preparation and staining methods were provided by
R.R.Smith (Univ. of Wisconsin, Madison, personal communication) with minor modifications. Root tips were pretreated in 0.003 M 8-hydroxyquinoline at 4°C for 4-6 hours. They were then rinsed with distilled water and fixed in ethanol - acetic acid (3:1 v/v) at room temperature
(25°C) overnight. Usually fixation was followed immediately by hydrolysis, but the material could be stored in 70% ethanol after this step. Root tips were hydrolysed in concentrated HCl (ethanol : hydrochloric acid = 1:1 v/v) for
45-60 sec. and then rinsed with 70% ethanol before staining with modified carbol fuchsin (Kao, 1976). Root tips were squashed and observation of cells made on a Zeiss light microscope. For each plant, two root tips were squashed and the chromosome number of at least two cells were examined.
When abnormal chromosome numbers were observed additional
root tips and cells were examined.
3.3.3 Leghaemoglobin Analysis
All extractions and sample preparation were carried out on ice at 0-4°C. Nodules (about 20) from each plant were collected into a microcentrifuge tube. They were ground with a plastic pestle in the tube after adding a small drop
of extraction buffer containing 0.1 M Na/K phosphate and 1 43 mM EDTA at pH 7.4. The tubes were centrifuged in a microcentrifuge for three minutes at 15,600 X g.
Leghaemoglobin components were analyzed using the cellulose acetate electrophoresis (CAE) method of Holl et al. (1983)
(Gelman, Sepra-Tek electrophoresis system). Cellulose acetate electrophoresis strips (Sepraphore III) were pre-soaked in buffer containing (g/1): Tris
(Tris-(hydroxymethyl)aminomethane): 20.72; EDTA: 0.15; glycine: 3.53 and sucrose: 15.0. Following sample application the strip was run at 300 V for 40-50 minutes at
room temperature. After electrophoresis the strip was
stained for protein in Ponceau S (100 mg/ml in 7.5% aqueous
trichloroacetic acid) for ten minutes and cleared in 5% acetic acid. Strips from several runs could be further
stained specifically for haem proteins using the
o-dianisidine method described by Holl et al. (1983).
3.3.4 Isozyme Analysis
In these experiments qualitative changes were evaluated
for the following five enzymes: malate dehydrogenase (MDH),
6-phosphogluconate dehydrogenase (6-PGDH), phosphoglucose
isomerase (PGI), phosphoglucomutase (PGM) and shikimate
deyhdrogenase (SKDH). The morpholine-citrate buffer system
of Clayton and Tretiak (1972) was used. Buffer composition
was as follows: electrode buffer: 0.04 M citric acid
(anhydrous) with pH adjusted to 6.1 with
N-(3-aminopropyl)-morpholine; gel buffer: 0.002 M citric 44 acid (anhydrous) with the pH adjusted to 6.4. Starch (49 g, lot No. 392) and sucrose (35 g) were placed in one flask and
18 ml gel buffer and 332 ml water into another. About 1/3 of the buffer solution was added to the starch/sugar mixture while the remainder of the buffer solution was heated in an oven to boiling. The boiling buffer solution was then added to the starch mixture with shaking to achieve an even mixture. The solution was degassed and was poured onto gel formers, shaken slightly to give a smooth surface. Gels were wrapped with Saran Wrap and kept at room temperature overnight. They were cooled down to 4°C prior to electrophoresis.
Two small young leaflets from each plant and two drops of cold extraction buffer (Helenurm, 1983) were added to each well of a sample grinding pad, placed on ice to prevent isozyme denaturation. The material was homogenized with a glass pestle. The crude extract was taken up directly into filter paper wicks precut to approximately 3 mm wide. Wicks were inserted sequentially into 30 slots at the cathodal end of the gel. A wick containing dye (red food dye) was inserted in one side slot.
The gel was still wrapped with Saran Wrap but approximately 2 cm of both ends were exposed. This wrapping protected the gel from dessication and contamination. The ends of gel were joined with the electrode buffer via cellulose sponges. The gel was run at 300 V, 50-60 mA at
3-4°C. Enzyme proteins moved anodally at a rate determined 45 by their size, shape and charge.
The staining solutions were prepared according to the receipes of Valliejos (1983). Following electrophoretic separation, starch gels were sliced into 5-6 slices.
Staining solutions were poured into individual staining boxes and resulting slices were separated and introduced into the appropriate stain. Boxes were incubated in darkness at 37°C. After gels were well-stained, they were fixed in 4% methanol and 10% acetic acid.
3.3.5 Morphological Analysis
Normally each leaf except the first leaf of red clover is composed of three leaflets. Changes in leaflet number were evaluated for both regenerated and seed-derived plants.
The leaflet length to width ratio was chosen as another morphological trait to look at in regenerated plants. To limit variation by other factors, two further conditions were met when measurements were taken: (1) Only the middle of three leaflets was measured and (2) leaflets measured were in a restricted range of length; approximately 25 mm ranging from 23 to 27 mm for 'Altaswede' and 30 mm ranging
from 28 to 32 mm for 'Norseman'. Leaflets were randomly chosen and measured for length and width. Ratios were calculated from the observed data. Mean, variance and standard deviation were obtained for each of different
"populations" of regenerated and control plants. The mean and variance of each population were compared with those of 46 the proper control. For the comparison of variance, F values were calculated by following equation: F = o\/o\ and were tested for significance. For the comparison of means from regenerated and control plants, the u test was performed. <7_ _ can be obtained by following equation (Ma, x! -x2 1978):
2 o = /a /n, + al/n2 X 1 X 2 and then u = (x,-x2)/a x 1 x 2
The significance of difference between means is thus testable. 4. RESULTS
4.1 Callus Induction
4.1.1 Callus Induction Frequency
The data for callus induction frequency are shown in
Table 2. 'Altaswede' in the second batch showed a higher callus induction frequency than 'Norseman'. For 'Norseman' the frequency of induction was constant from the first to the second batch. However, 'Altaswede' showed an increased
frequency in Batch 2. The data suggest that 'Altaswede' explants may perform better at low light intensity or when grown in petri dish culture. An increase in callus
induction frequency for 'Altaswede' was also observed when
light intensity decreased in another batch of culture. A
total of five batches of cultures were initiated for callus
induction. Induction frequencies were recorded after two weeks incubation. In one batch of cultures, 50 tubes which
did not have callus when examined after two weeks of culture produced calli in four cultures when re-examined 30 days
after the explant transfer. Therefore, callus induction
frequencies may be slightly higher if measurements are made
after a longer period of initial culture.
It was also apparent that the callus induction
frequency of 'Norseman' was greatly affected when a thin
layer of water on the agar medium did not evaporate in test
tubes wrapped with parafilm while 'Altaswede' was less
47 48
Table 2. Callus induction frequency for two cultivars of red clover on L2 medium.
Batch No 1 2
Cultivar Altaswede Norseman Altaswede Norseman
No explants cultured 288 174 272 1246
No explants producing 192 1 12 236 816 callus
Frequency (%) 66.7 64.4 86. 7 65.5
2X2 Contingency x2 Analysis
2 1 Batch 1 Altaswede vs Norseman X = 0 .023 ns
Batch 2 Altaswede vs Norseman X2 = 7 .954 ** (P<0.01)
Combined Altaswede vs Norseman X2 = 4 .030 * (P<0.05) data
1 ns: not significant; . *:significant; **: highly signi f icant. 49 affected by the same experimental conditions.
4.1.2 Genotypic Effect on Callus Induction
Tubes with callus induced in the two batches were
grouped into 1, 2 or 3-callus tubes, depending upon the
number of explants for which callus was evident. The
results are shown in Table 3. It is clear from the data
that the three explants from one genotype behaved similarly.
If one explant produced callus the other two in the same
tube also showed a similar response. Calli originating from
different genotypes could look different (e.g. in color)
whereas the three calli from one genotype were
phenotypically similar. The possibility that this
similarity among three explants from one individual was
simply a response to the uniform environment in the test
tube was eliminated by later experiments, which showed that
explants from one individual produced calli while explants
from another genotype in the same culture vessel did not
(Figure 1).
4.1.3 Observations on Callus Induction
After the initial transfer, some explants retained a
fresh appearance and did not shrivel, or turn brown. After
3-4 days such explants swelled and callus could develop from
the swollen portion. During this process the green color of
the initial explant disappeared. The green color loss
commenced at the periphery of the explant, leaving the 50
Table 3. Callus induction for three explants coming from the same seedling in two batches of culture. Data for two culture batches have been combined.
Culture Cultivar
Altaswede (%) Norseman (%)
No. of tubes cultured 266 167
No. of tubes with 197 (100) 114 (100) callus
3-callus tube 180 (90.4) 109 (95.6)
2-callus tube 12 (6.1) 3 (2.6)
1-callus tube 5 (2.5) 2 (1.8) 51
Figure 1. Callus induction and genotype effect. 'Norseman' hypocotyl explants on L2 medium. Explants from one individual (upper half of plate) produced calli, while explants from a second genotype did not after two weeks culture. 52 center green. At day 7-8 some explants showed early callus development, followed by continuing division and callus formation after two weeks. Explants, which withered after initial transfer, did not produce callus. Other explants showed slight browning with some swelling; these explants could develop further but seldom produced vigorous callus growth. Calli formed under dark condition were white while those induced in the light usually showed some green coloration.
4.2 Callus Maintenance and Plant Regeneration
4.2.1 Primary Test of Two Media
Approximately 85 calli of each cultivar were cultured on each of the two media. In neither case was shoot induction or bud initiation observed when recorded after two
weeks of culture. Although calli grew faster on B5(N2A2) medium, more green-spotted calli were observed on LSE medium
at this time. Calli on B5 were whiter than those on LSE
medium. Some root formation was observed on B5 medium.
After 17 days some calli in both cases were transferred onto fresh media. In these subcultures many of the greenish regions became brown and the number of green-spotted calli decreased.
Callus response on LSE medium is shown in Table 4.
More green-spotted calli were observed in cultures from
'Norseman' than 'Altaswede' after two weeks of culture.
Then calli turned darker and after the subculture the number 53
Table 4. Callus growth on LSE medium. Data for the initial culture and subculture are shown. The first culture was measured after two weeks of culture and the subculture was measured four weeks after the transfer.
Culture Cultivar NC GSC (%) SC RC
1 Altaswede 86 21 (24.4) 0 0
1 Norseman 88 40 (45.5) 0 0
2 Altaswede 56 2 ( 3.6) 0 2
2 Norseman 59 10 (16.9) 0 0
NC=Number of Calli; GSC=Green -Spotted Calli; SC=Shoot
Calli; RC=Root Calli 54 of green-spotted calli was sharply decreased. From each cultivar 50 calli were cultured on LSE for a further three weeks. During this period no obvious growth of the calli was observed. All the green spots disappeared and one callus of 'Altaswede' produced some new roots. Upon
termination of the test, calli had been growing on LSE medium for about two months. Continuous subculturing of callus onto LSE medium did not initiate bud or shoot differentiation, as well as being associated with poorer
callus growth.
4.2.2 Callus Growth on LSE Medium
Test 1: Results of the initial and the first subculture
are presented in Table 5. None of these applied conditions
had promotional effect on shoot induction. After calli were
transferred from L2 medium to LSE medium, growth and good
color was maintained for a short period. Then the color of
callus changed from fresh yellow-green to brown or
yellow-brown. Green spots disappeared and calli stopped
growing. Some calli produced roots during the initial
culture (Figure 2). After the calli were subcultured, they
continued to show an unhealthy growth response; more calli
senesced; no fresh roots were produced and previously formed
roots turned brown (Figure 3). At the end of the culture
period approximately 95% of calli of both cultivars were
senesced or dead. Therefore, the failure of LSE to induce
shoots in the present case is less likely related to light 55
Table 5. Response of calli on LSE medium: initial culture and subculture of Test 1 recorded after four and five weeks of culture, respectively.
Culture Cultivar Light NC SC RC
intensity
MEm"2 sec"1
Initial Altaswede 100-900 1452 0 1 3
Subculture Altaswede 100-900 984 0 0
Initial Norseman 70-450 1 556 0 1 5
Subculture Norseman 70-450 568 0 0
NC=Number of Calli; SC= Shoot Calli; RC=Root Calli 56
Figure 2. Response of 'Altaswede' calli on LSE medium after two weeks of culture. Calli were browning, green spots began to disappear and roots were induced during this culture period. Figure 3. Response of 'Altaswede' calli on LSE medium after five weeks of culture. Calli showed an unhealty growth response. (a) Calli became brown and stopped growing (upper), (b) No fresh roots were produced and previously formed roots turned brown (lower). 58 condition or sample size of callus.
Test 2: No shoot was ever formed on the four media
(Table 6). As demonstrated by Phillips and Collins (1980) and other results of the present study, LSP medium is
supportive of embryo development. In addition L2(NO.05K0.5) was shown to induce shoots in the tests with NAA and KIN.
The failure to induce shoot formation on the media used in
this test suggested that no embryos had been formed on LSE medium, otherwise they should be able to develop into shoots
on these media. On L2(P0.002B0.2), calli on full strength
basal salts appeared healthier than those on half strength
solutions; fewer calli showed brown color. Roots formed
only from calli on the medium without added growth
regulators. However, adding regulators tended to keep
green-spotted calli longer.
Test 3: As shown in Table 7, results were similar to
these obtained through previous tests. Calli of 'Norseman'
were not as watery as they had been before their transfer
onto this medium.
Results from these tests demonstrated that LSE medium
did not induce shoot production under current experimental
conditions nor was it a good medium for callus maintenance.
Calli on this medium showed a.similar response throughout
these tests, browning, senescence and ultimately death.
This response pattern strengthened with the time of callus
on this medium. 59
Table 6. Response of calli from LSE medium subcultured onto four media (concentration in mg/1).
Medium 1 2 3 4
PIC 0.002 0.002 no growth
BA 0.2 0.2 regulators
NAA 0.05
KIN 0.5
SALTS full half full full
Results
NC 42 36 39 33
RC 0 0 0 2
GSC 2 1 0 0
BC 3 6 5 6
NC=Number of Calli; RC=Root Calli; GSC=Green-Spotted
Calli; BC=Blackened Calli 60
Table 7. Response of calli on L2(D0.01A5) medium,
Cultivar Altaswede Norseman
NC 99 99
GSC 3 9
RC 1 0
Color brown or black slightly brown
Rate of Growth ++ +++
NC=Number of Calli; GSC=Green-Spotted Calli; RC=Root Calli.
++, +++: More plus signs indicate faster relative . callus growth. 61
4.2.3 2,4-D and BA
Results from Batch one are presented in Table 8. Calli on these two media were very similar for a single cultivar.
Obvious differences existed between calli of the two cultivars. 'Norseman' generally had more green-spotted calli than 'Altaswede' and 'Norseman' calli grew faster than
'Altaswede' calli. It was observed that two leaf-like
structures formed on L2(D2B2) medium, a better sign for
shoot differentiation. When recorded after four weeks of
culture, some calli of both cultivars had turned brown and
the number of green-spotted calli decreased. One of the two
leaf-like structures had also become brown, while one
remained green.
For Batch two, 20 to 25 calli of 'Altaswede' were
cultured for each treatment. No shoots were induced on any
of these media. However, green-spotted calli were observed
on L2(D2B2A5) medium. Calli on this medium grew faster.
Only one callus on medium with 2 mg/1 2,4-D had root
differentiation. Data recorded after three weeks and four
weeks of culture showed the same trend.
4.2.4 PIC and BA
Test 1: Results in Table 9 indicated that PIC at the
concentrations tested did not induce calli to produce shoots
or roots, 10 mM arginine added to the medium also had no
effect on shoot or root induction and in the presence of
arginine, high levels of PIC induced more green-spotted (52
Table 8. Response of calli on media containing 2,4-D and BA recorded after three weeks of culture (concentration in mg/1).
Growth Medium regulator 1 2 3 4
2,4-D 2 2 2 2
BA 2 2 4 4
Cultivar Altaswede Norseman Altaswede Norseman
NC 50 50 50 36
GSC 3 16 4 17
Morphoge- 0 two 0 0 ' nesi s leaf-like structures
Growth Rate ++ +++ ++ +++
NC=Number of Calli; GSC=Green-Spotted Calli. ++, +++: More plus signs indicate faster relative callus growth. 63
Table 9. Response of 'Altaswede' calli on media containing PIC and BA in presence and absence of arginine (1740 mg/1) (growth regulator concentration in mg/1).
Treatment PIC BA Arg NC RC SC GSC
1 0.001 0.2 + 14 0 0 0
2 0.0005 1 + 16 0 0 0
3 0.001 1 + 14 0 0 0
4 0.005 1 + 14 0 0 0
5 0.01 1 + 14 0 0 2
6 0.05 1 + 18 0 0 7
7 0.0005 1 - 20 0 0 1
8 0.001 1 - 21 0 0 1
9 0.005 1 - 20 0 0 2
10 0.01 1 - 22 0 0 1
1 1 0.05 1 - 20 0 0 2
NC=Number of Calli; RC=Root Calli; SC=Shoot Calli; GSC=Green-Spotted Calli 64 calli.
Test 2: The results presented in Table 10 showed that adding glutamic acid and casein hydrolysate to the medium had no effect on shoot induction, nor did it increase root induction. More green-spotted calli appeared at low levels of casein hydrolysate, but the small sample size prohibited a conclusive interpretation. Calli growing on these media were browner than those on media without these additives
(Figure 4). Those calli of 'Norseman' on medium of 5,000 mg/1 casein hydrolysate became black.
The results were shown in Table 11. No shoots were
induced using various combinations of PIC and BA. In contrast to the results of Test 1, roots formed at all combinations indicating that callus source is an important
factor in determining root formation. More green-spotted calli and roots formed with 0.005 mg/1 PIC at both BA concentrations with one exception i.e. 'Altaswede' calli on
L2(P0.005BO.2) medium. More rooted calli and green-spotted calli were produced from 'Norseman'.
Some calli of 'Altaswede' from L2 were also transferred
onto L2(P0.002B0.2) and L2(P0.02B0.2) media. Neither shoots
nor roots were induced.
4.2.5 NAA and KIN
Test 1: Results (Table 12) showed that no shoots were
induced when calli from LSE were subcultured onto media
containing NAA and KIN. Roots and green-spotted calli were 65
Table 10. Response of calli on media with added casein hydrolysate and glutamic acid.1
Cultivar CH or GA (mg/1) NC RC SC
Altaswede 100 CH 10 0 0
500 CH 1 1 0 0
1000 CH 10 0 0
5000 CH 10 0 0
147 GA 10 0 0
Norseman 100 CH 12 0 0
500 CH 12 0 0
. 1000 CH 12 0 0
5000 CH 10 0 0
147 GA 12 1 0
NC=Number of Calli ; RC=Root Calli; SC=Shoot Calli; CH=Casein Hytrolysate; GA=Glutamic Acid.
1A11 media contained 0.005mg/l PIC and 1 mg/1 BA. 66
Figure 4. Response of 'Altaswede' calli on L2 medium containing 0.005 mg/1 PIC, 1 mg/1 BA and 1 g/1 casein hydrolysate after five weeks of culture. Calli showed slightly brown coloration. 67
Table 11. Response of calli of two cultivars on media with various combinations of PIC and BA (concentration in mg/1).
Cultivar PIC BA NC RC (%) GSC (%)
Altaswede 0.,00 2 0.. 2 1 4 4 (29) 8 (57)
0..00 5 0.. 2 14 2 (24) 9 (64)
0.,0 1 0.. 2 13 0 3 (20)
0..002 ' 1 1 4 0 5 (36)
0..00 5 1 14 4 (29) 1 1 (79)
0..0 1 1 14 4 (29) 7 (50)
Norseman 0..00 2 0.. 2 46 18 (39) 33 (72)
0..00 5 0., 2 44 21 (48) 36 (82)
0..0 1 0.. 2 44 8 (18) 36 (82)
0,.00 2 1 42 6 (14) 27 (64)
0..00 5 1 42 7 (17) 33 (79)
0..0 1 1 46 6 (13) 34 (74)
NC=Number of Calli; RC=Root Calli; GSC=Green-Spotted Calli 68
Table 12. Response of 'Norseman' calli from LSE medium to various combinations of NAA and KIN (concentration in mg/1).
NAA KIN NC RC GSC
0.001 1 16 3 6
0.01 1 20 1 3
0.1 1 22 0 1
1.0 1 20 0 2
5.0 1 20 0 3
NC=Number of Calli; RC=Root Calli; GSC=Green-Spotted Calli 69 produced when the NAA concentration was low (0.001 and 0.01 mg/1).
Test 2: Shoots were induced on two media in this
experiment (Table 13). They originated from the upper part
of calli. Several shoots arose from a single callus. Some
shoots had well-formed cotyledons and roots, suggesting that
they were from embryos (Figure 5). Both treatments which
induced shoots contained 0.05 mg/1 NAA. In contrast, more
rooted calli were produced when the NAA concentration was
low (0.01 mg/1). Many roots originated from one callus, the
number ranging from several to several dozens. Most of them
originated from the top part of callus. Few roots originated
from callus-medium interface. All these calli were
yellow-green in color.
Test 3: It was shown (Table 14) that root formation was
maximum when NAA concentration was 0.1-0.2 mg/1 at both KIN
concentrations and minimum when the auxinrcytokinin ratio
was highest (1.0). The maximum number of green-spotted
calli appeared at 0.1 mg/1 NAA when KIN concentration was
0.5 mg/1, but at 1.0 mg/1 NAA when KIN concentration was 2.0
mg/1. Fewer green-spotted calli resulted when both NAA and
KIN concentrations were low.
The three tests above were aimed at studying the effect
of NAA and KIN on callus growth and differentiation.
Although root formation occurred in all tests, shoots were
induced only in one case. This suggests that: (1)
embryogenesis had been triggered on SCP medium before calli 70
Table 13. Response of calli from SCP medium to combinations
of NAA and KIN (concentration in mg/1)
NAA KIN NC sc RC
0.01 0.5 20 0 15
0.05 0.5 20 2 (6,4)1 1 1
0.1 0.5 22 0 13
0.05 2.0 20 1 (25) 8
1Numbers in the bracket are the number of shoots for each callus.
NC=Number of Calli; SC=Shoot Calli; RC=Root Calli 71
Figure 5. Shoot formation from 'Altaswede' callus on L2(N0.05K2) medium. Callus had been cultured on SCP medium. Shoots originated from the upper part of the callus, with some having cotyledons and roots. 72
Table 14. Response of 'Norseman' calli to combinations of NAA and KIN (concentration in mg/1).
NAA (mg/1) KIN (mg/1) NC RC (%) GSC (%)
0.01 0.5 48 8 (16.7) 6 (12.5)
0.05 0.5 44 8 (18.2) 8 (18.2)
0.1 0.5 46 11 (23.9) 10 (21.7)
0.5 0.5 46 6 (13.0) 7 (15.2)
0.2 2.0 50 11 (22.0) 20 (40.0)
1 .0 2.0 48 10 (20.8) 25 (52.1)
NC=Number of Calli; RC =Root Calli; GSC=Green-Spotted Calli
1 73 were transferred so that shoots only come from calli which were previously cultured on SCP medium and (2) concentration of NAA is critical because shoots were induced only on media containing 0.05 mg/1 NAA.
In Test 1 more roots were produced when NAA was at
0.001 mg/1, but in Test 3 more roots were resulted at
0.1-0.2 mg/1 NAA at both KIN concentrations. Although light conditions could contribute to the observed difference, the most important factor is callus source. Calli used for Test
3 had been cultured on medium with high level of 2,4-D compared with those used in Test 1. Thus, during the subsequent subculture more rooted calli were produced at higher levels of auxin in Test 3 than in Test 1. It was also apparent that NAA was more critical to root formation than KIN.
4.2.6 NAA and BA in Presence of ADE
After one month of culture no shoots were induced on the four media tested (Table 15). Two rooted calli were produced on medium without NAA but containing 0.2 mg/1 BA.
This result contrasts with the accepted view that root
formation is facilitated by high auxin to cytokinin ratios.
Slightly more green-spotted calli were observed at high levels of NAA. 74
Table 15. Response of 'Norseman' calli on media containing various levels of NAA in combination with BA and ADE1 (concentration in mg/1).
NAA BA NC RC GSC
0 0.2 22 2 3
0.001 0.2 22 0 5
0.01 0.2 22 0 5
0. 1 0.2 22 0 8
1 All the media containing 2 mg/1 ADE.
NC=Number of Calli; RC=Root Calli ; GSC=Green -Spotted Calli 75
4.2.7 Callus Differentiation on Four Media
As shown in Table 16, none of these four media was able to induce shoots from calli. Although only ten calli were cultured for each treatment, the trend was obvious that root formation was inhibited by the presence of auxins, the extent of inhibition was related to auxin concentration and root formation was enhanced by BA and ADE. This observation coincides with the result obtained in the tests on NAA and
BA (Section 4.2.6). Medium without any added auxin but with
BA and ADE present had more rooted calli and the maximum average root number per rooted callus, while the medium
(medium 4) which was similar to that used for root formation from shoots was less efficient for root induction from callus.
4.2.8 Callus Growth and Auxin to Cytokinin Ratio
Results of this test are presented in Table 17. Calli on L2(D11K1) medium were grey to dark-grey in color, friable and showed no differentiation (Figure 6). On L2(D1K10) medium there were basically two callus forms. One form of callus was fast growing and green. Another form of callus was brown-yellow in color. Among 190 calli cultured, 134 were green calli. These green calli, unlike normal green-spotted calli which had isolated green regions, were uniformly green because of the high KIN content in the medium. The contrasting auxin to cytokinin ratios of the two media produced a different callus morphology. 76
Table 16. Differentiation of 'Norseman' calli from L2(D2B2A5) medium on four media (concentration mg/1)
Medium 1 2 3 41 composition
L2 SALTS full full full half
2,4-D 0.01
NAA 0.1
BA 0.2 0.2 0.2
ADE 2 2 2
IAA 0.2
Nicotinic 1.0 ac id
Result
NC 1 0 10 10 10
RC 7 3 3 2
Average 4 4 3 1 Root No.2
1 L2 medium with half strength of salts and vitamins.
2 Based on the number of root calli.
NC=Number of Calli; RC=Root Calli. 77
Table 17. Response of 'Norseman' calli grown on media of high (11) or low (0.1) auxin to cytokinin ratio.
L2(D11K1) L2(D1K10)
NC 190 190
Color white to grey (1)brown-yellow (2 )green-yellow
RC none less than 10
NC=Number of Calli; RC=Root Calli 78
Figure 6. Response of 'Norseman' calli on media with different auxin to cytokinin ratios after five weeks of culture, (a) Calli on L2(D11K1) medium; very friable and grey in color (upper), (b) Calli on L2(D1K10) medium (lower). Two forms of calli resulted. The majority of calli were the green-type (left petri plate). 79
Roots were only formed on the medium with low auxin to cytokinin ratio. This phenomenon, observed in tests with
NAA and BA (Section 4.2.6) and the comparison of four media
(Section 4.2.7), was once again confirmed here.
4.2.9 Callus Growth on SCP Medium
Recovery of Senescing Callus
Recovery frequency of senescing calli to large green-spotted calli is shown in Table 18. These regenerated calli were fast-growing and had isolated green regions.
They were bigger than one centimeter in diameter (Figure 7) were most commonly seen on SCP medium (so-called SCP callus). It is clear from these results that SCP medium was efficient in restoring senescing calli and that the recovery frequency decreased with the length of culture period on LSE medium. Recovery was also efficient when a different callus source was used.
Maintenance of Callus
Most calli on SCP medium grew very fast. Many became large calli with diameter greater than one centimeter within one culture period (3-4 weeks) (Figure 8). They were green-spotted and many of them had nodular structures. In a test of three media, L2(D2N2K2), L2(D2B0.5A2) and
L2(P0.002B2) were compared. On L2(D2B0.5A2) medium, which was similar to SCP, calli grew faster than calli on the other two media and more green-spotted calli were produced. 80
Table 18. Recovery frequency of senescing calli after transfer to SCP medium.
Callus source
1 2 3 4 5
NC 40 48 120 72 144
No. of SCP 5 12 42 48 61 calli
Frequency of 12.5 25.0 35.0 66.7 43.3 SCP calli (%)
NC=Number of Calli 81
Figure 7. Recovery of senescing calli on SCP medium. Before transfer, these calli were darkened and brown in color and had stopped growing. The calli illustrated are after five weeks culture. 82
Figure 8. Callus maintenance on SCP medium. Rapid growth on this medium produced calli with diameters greater than one centimeter within three to four weeks, (a) 'Altaswede' calli (upper); (b) 'Norseman' calli (lower). 83
Calli were easily maintained on SCP medium by
subculturing. Some calli which were originally cultured on
LSE medium were transferred onto SCP on October 7, 1984 and maintained on SCP to July 10, 1985 by subculturing them only
twice (December 9, 1984 and February 9, 1985)
Calli could be maintained on SCP without subculture for
many months. Calli from each of two media: SCP and
L2(P0.001B0.1) were transferred onto SCP on February 9, 1985
and incubated in a growth chamber. When checked on July 10,
they were very large. Although parts of the callus were
less vigorous and not fresh in color, the peripheral regions
were green-yellow and actively growing.
Embryogenesis
Embryogenesis can occur from calli cultured on SCP
medium. This observation was confirmed by microscopic
examination. Callus on this medium contained cell clusters
and in some cases embryos at the globe and heart stages.
Although these embryos did not develop further, they
increased in size, forming large disorganized embryo-like
structures. This fact suggests that the hormone
concentration in this medium was too high for embryos to
complete development. Calli with embryos induced on SCP
medium could be transferred to another medium, eventually
leading to plantlet regeneration. 84
4.2.10 Light Effect on Calli Growing on SCP Medium
The results of this test are shown in Table 19.
Because calli had been cultured on LSE medium prior to transfer and were not vigorous, the frequency of the typical
SCP callus on SCP medium was low, but this frequency
increased with light intensity. It was also obvious from the results that light promoted root differentiation from these calli. However, light quality which was not measured
in this experiment could also have some effect on the
results.
4.2.11 Observations on Embryogenesis
Embryogenesis in culture likely occurs via the
sequence: single cell(?) > cell cluster > globe
stage > heart stage > torpedo stage. Under a light microscope many cell clusters could be seen. These cells
were small and dense in cytoplasm (Figure 9). They were
easily distinguishable from the larger cells usually
encountered in cultures. At the globe stage, the cell
cluster was well shaped and became smooth at periphery.
On SCP medium some calli were found to have embryos
developed, mainly at the globe and early heart stages.
These embryos were not well-shaped and did not develop
further although they could grow larger in size to become
nodular structures.
Upon transfer of these embryogenic calli onto other
media some of the embryos developed normally to the torpedo 85
Table 19. Light effect on growth and differentiation of 'Altaswede' calli on SCP medium.
Intensity No. of Typical Rooted of light calli SCP calli calli (/uEirr 2sec"1)
70 37 2 0
80 49 1 1 0
90 53 18 0
1 00 46 7 0
1 20 51 1 2 0
150 52 15 1
190 51 1 5 1
230 48 8 1
280 50 22 5
450 48 23 8 86
Figure 9. Embryogenic callus of 'Altaswede'. Callus was cultured on L2(P0.001B0.1) medium. Cell clusters contain cells which are small and dense in cytoplasm. Callus shown was unstained (mag. X38). 87 stage and eventully to plantlets (Figure 10). Plantlets formed via embryogenesis developed morphologically like seedlings derived from seeds. Cotyledons were first observable, followed by development of the first leaf consisting of a single leaflet.
4.2.12 Selection and Maintenance of Embryogenic Calli
Results are shown in Table 20 for one subculture of embryogenic calli from LSP medium to LSP medium. Embryos were observed at different developmental stages. Those
embryos which had been developing normally during the culture periods developed into plantlets. Forty to fifty percent of cultures showed plants developed, with 20% of
tubes having more than two plantlets (Figure 11). In some
cultures many embryos at the torpedo stage were observed
growing at the surface of callus. There were a few tubes
where no plantlet or torpedo embryos were observed. However
embryos at earlier stages were observed which could develop
normally after subsequent transfer. In addition, there were
a few tubes (approximately 10%) in which calli were
disorganized or had an unhealthy color.
Embryogenic calli had been maintained in culture for
over one year without lossing the capacity of regenerating
plants.
When shoots originated from such calli were culture on
rooting-medium, plantlets formed and in a few cases
embryogenic callus (with obvious embryos) was produced near 88
Figure 10. Somatic embryo development from embryogenic callus of 'Altaswede'. Calli were cultured on L2(PO.001 BO.1) medium. Embryos were observed by scanning electron mcroscopy. (a) An embryo present in the center. Two cotyledons are observable (upper, mag. x43). (b) Further development of an embryo (lower, mag. X52) 89
Table 20. Embryo development and plantlet formation after subculture of embryogenic calli onto LSP medium.
Culture No1
1 2
No of tubes 84 99
Tubes with plantlet 35 51 development
1-plantlet tube 12 15
2-plantlet tube 5 1 6
3-10 plantlet tube 12 17
>10 plantlet tube 5 3
Tubes with embryos at 41 41 torpedo Stage a few embryos 24 20 many embryos ^5 17 21
Tubes with callus only 8 7 (embryos earlier than torpedo stage)
yellow green callus 2 4
disorganized embryos 6 2
brown or dark callus 0 1
1 These were two batches of cultures from the embryogenic callus. 90
Figure 11. Selection of callus capable of embryogenesis. Embryogenic calli of 'Altaswede' subcultured on L2 medium containing 0.001-0.002 mg/1 PIC and 0.2 mg/1 BA produced embryos continuously and showed plantlet development. 91 the place where roots were intiated (Figure 12).
4.2.13 Growth of Embryogenic and Non-embryogenic Calli
Embryognic calli and non-embryogenic calli were morphologically different. Embryogenic calli had fine grain-like structures (embryos). These structures were very easy to separate from each other. Non-embryogenic calli, although they varied in texture from friable to compact, do not have these structures. Under a light microscope, embryogenic calli had many visible embryos, mostly at the globe and heart stages.
When measured after one month of culture, embryogenic calli, compared with non-embyrogenic calli, grew faster, had fresh yellow-green color and remained alive for a longer time (Figure 13). All treatments showed the same tendency.
Non-embryogenic calli turned brown and showed little or no growth. Embryo-like structures were present in embryogenic cultures, although they became large in size in treatments
(6) and (8). At this time, roots formed on embryogenic calli in treatments (2) and (8). Cotyledons were also observed on the embryogenic calli of treatment (8). After three months without subculture all non-embryogenic calli were dead, whereas most of the embryogenic calli were still alive. 92
Figure 12. Embryogenic callus arising from regenerated plantlet at the point of root origin. 93
Figure 13. Embryogenic and non-embryogenic calli of 'Altaswede'. From left to right, Tubes 1 and 2 contain L2 medium with 0.05 mg/1 NAA and 0.1 mg/1 BA. Tubes 3 and 4 contain L2 medium with 2 g/1 yeast extract and 10 g/1 agar. Tubes 1 and 3 had embryogenic calli while Tubes 2 and 4, non-embryogenic calli. Calli had been cultured on these media for forty days. 94
4.2.14 Summary of Experiments on Callus Growth and
Differentiation
In this series of experiments callus differentiation was evaluated on various media. Some consistent conclusions may be made from these results although the responses varied with individual experiments.
(1) There were differences in callus growth and
differentiation due to cultivar. Usually 'Norseman'
calli grew faster than 'Altaswede' calli and also
produced more green-spotted calli. Shoots were induced
only from 'Altaswede' calli, but more calli with roots
were usually formed from 'Norseman'.
(2) More roots were formed from calli cultured at a low
auxin to cytokinin ratio or in the absence of auxins.
This relationship was apparent in experiments using PIC
and BA (PIC at 0.0005 and 0.001 mg/1) and on NAA and BA
(without NAA) and in the experiment specifically
testing the auxin to cytokinin ratio (more roots at
ratio equal to 0.1). A typical example of this
relationship was observed in the experiment in which
four media were compared. More calli produced roots
and greater numbers of roots per callus on the medium
without auxins than on any one of the other three media
with low or moderate levels of 2,4-D, NAA or IAA.
(3) Callus source is an important facter determining
the result. When non-vigorous calli were used as the
source, less growth and differentiation was observed. 95
In the experiment on PIC and BA, Test 1 showed no root
differentiation and no obvious difference between
different PIC and BA combinations. When other callus
sources were used in Test 3 root formation occurred in
all treatments and differences between treatments were
evident.
(4) Shoots were induced after calli of 'Altaswede'
cultured on SCP medium were transferred onto medium
containing NAA (0.05 mg/1) and KIN with NAA.
(5) NAA concentration was more important than that of
KIN in root or shoot induction, as demonstrated in
the experiment on NAA and KIN. (6)
(6) Additions of amino acids (glutamic acid and
arginine) and casein hydrolysate did not show any
obvious promotive effect on callus growth compared
with calli on media without these additives.
4.2.15 Plant Regeneration from Callus
Plant regeneration from callus was achieved in several cases. In a study of callus culture on the following media:
(1) L2(P0.002B0.2), (2) L2(half strength salts, P0.002B0.2) and (3) L2(NO.05K0.5), one shoot was formed on the
L2(N0.05K0.5) medium. This observation was the first indication of any plant regeneration from callus in this study. Another experiment using NAA and KIN was therefore initiated, leading to regeneration of some plants from calli
(see Section 4.2.5). Calli capable of embryogenesis were 96 subcultured onto LSP medium. Embryos were produced continuously, giving rise to a number of plants (see Section
4.2.13).
4.3 Shoot Tip Culture
4.3.1 Multiple Shoot Induction
Results of multiple shoot induction and statistical analysis are presented in Table 21 and 22. The average number of multiple shoots was based on the number of shoot tips producing multiple shoots. The multiple shoot induction rate of 'Norseman' was higher than that of
'Altaswede'. However, when the shoot tip could be induced to produce any multiple shoots, 'Altaswede' produced more multiple shoots per shoot tip than 'Norseman' (Figure 14).
4.3.2 Multiple Shoot Propagation
As shown in Table 23 'Altaswede' not only had more shoots in the induction culture, but also in the two subcultures. Multiple shoots usually arose from a shoot rosette with callus at its base and green hard tissue at the center. Once the multiple shoot rosette was established additional multiple shoots were easily produced by dividing the rosette into several pieces and subculturing each piece.
Rapid propagation of multiple shoots can be accomplished this way in a short time period. 97
Table 21. Multiple shoot induction from shoot tips of red clover on L2(PO.003B2) medium.
Cultivar Replicate No of shoot No of tips Average Ni tips with MS of MS1 cultured
Altaswede 1 30 9 4.8
2 30 1 2 4.8
3 30 1 1 4.5
4 30 9 4.7
5 30 9 5.0
Total 1 50 50
Norseman 1 30 17 3.9
2 30 18 3.6
3 30 18 3.9
4 30 15 3.9
Total 120 68
1 MS = Multiple Shoots
Table 22. Comparison of two cultivars of red clover for multiple shoot induction.
Cult ivar
Altaswede Norseman
No of tubes
With MS1 10 B2 17 A
Average No of shoots 4.8 A 3.8 B 1 MS = Multiple Shoots
treatments followed by different capital letters are significantly different at P^0.01. 98
e 14. Multiple shoot production in red clover. A single isolated shoot was cultured on L2(P0.003B1) medium and showed multiple shoot proliferation. 'Altaswede' (right) produced more, but smaller shoots than 'Norseman' (left). 99
Table 23. Multiple shoot production by two. cultivars of red clover. Multiple shoot were induced on L2(PO.003B2) medium. They were subcultured on media containing 0.002-0.003 mg/1 PIC and 1 mg/1 BA for propagation at a subculture of one month.
Culture Multiple shoot No / tube
Altaswede Norseman
Induction 4.8 A1 3.8 B culture
First 10.8 a 6.1 b subculture
Second 15.2 A 8.1 B subculture
treatments are significantly different at P^0.01 when followed by different capital letters or at P£0.05 when followed by different small letters. 100
4.4 Growth and Analysis of Regenerated Plants
During this study, plants were regenerated through several different tissue culture pathways. To simplify the discussion, the types of regenerants are described as follows and given an appropriate abbreviated designation:
(1) RG1 plants: those regenerated from multiple shoots induced on multiple shoot induction medium (L2(PO.003B2)) without subculture of the multiple shoots;
(2) RG2 plants: these regenerated from multiple shoots which were derived by subculturing the original multiple shoots twice on L2 medium containing 0.002-0.003 mg/1 PIC and 1 mg/1 BA.
(3) RG3 plants: plants regenerated from multiplication of callus-derived shoots. Shoots were first regenerated from
'Altaswede' calli on L2(N0.05K0.5) and and L2(N0.05K2) media. Calli had been in culture for three months by that time. These shoots were then multiplied twice by shoot tip culture;
(4) RG4 plants: plants regenerated directly from embryogenic calli, which were derived by selecting one 'Altaswede' callus line and subculturing. Before plant regeneration, calli had been maintained in culture for nearly one year.
Plants of all types were derived from 'Altaswede' while regenerants of 'Norseman' were only available as RG1 and RG2 plants. 101
4.4.1 Plant Transfer and Survival
The ultimate objective of the tissue culture cycle must be to produce vigorous regenerated plants. It is important, therefore, to ensure a high rate of survival when plantlets are transferred out of i_n vitro conditions. The transition from the test tube to greenhouse is a significant , environmental change for the plantlets. Appropriate conditions are required to facilitate plant adaptation to the new environment and that conditions be changed gradually.
In the experiment testing the effect of different treatments on plant survival, plants incubated in the
laboratory for the first week were exposed to a relatively
low light intensity (120 iuEm~2sec~1) and high humidity.
Plants were weak and many died in the subsequent transfer to the greenhouse. As a consequence, the survival rate was
low. When plants were transferred to the greenhouse without
Saran wrap protection some plants died only a few days after the transfer. Dessication in the lower humidity environment was a contributing factor in the low survival rate (39%)
(Table 24). The plants which were covered with Saran wrap
for two weeks in the greenhouse had the highest survival
rate (94%).
In the present study five batches of regenerated plants were transferred. During these transfers two other
observations were made regarding the conditions which
supported successful plant transfer. (1) Maintaining shoots 1 02
Table 24. Survival rate of plants transferred to greenhouse after different treatments
Treatment
Laboratory/ Green house Green house green house +Saran wrap -Saran wrap
Plants 112 224 28 transferred
Plants 60 211 11 survived
Survival rate (%) 54 94 39 103 in rooting medium for more than one month prior to transplantation appeared to contribute to better survival.
It might be anticipated that during this extended period, humidity within the culture tubes was somewhat lowered and plants were much bigger. Such plants were more resistant to wilting. (2) In February, natural light intensity is moderate and plants can be transferred to the greenhouse light conditions directly. When light intensity is very high (i.e. during summer days) a period of maintenance at reduced light intensity should precede exposure to the full strength of sunlight. During the transfer of RG4 plants of
'Altaswede' on June 17, both light intensity and temperature in the greenhouse were high. Light intensity was reduced using a screen of black polyethylene film on a frame above the plants. Of the 130 plants transferred more than 95% survived.
4.4.2 Chromosome Number Stability
RG1, RG2, RG3 and RG4 plants of 'Altaswede' were examined for their chromosome number. The normal karyotype of 'Altaswede' is 2n=2X=14. Root tip squashes of seed-derived plants were utilized as controls. The data are
summarized in Table 25. All the control plants showed normal chromosome numbers (Figure 15). Of 45 RG1 plants examined no abnormal chromosome number was observed while
one diploid/tetraploid mosaic plant was detected among 28
RG2 plants. For all the 170 RG3 plants no variation was 104
Table 25. Chromosome numbers of 'Altaswede' plants regenerated via different tissue culture pathways.
Population Number of plants with abnormal tetraploid surveyed chromosome tetraploid /diploid number mosaic
RG1 45 0 0 0
RG2 28 1 0 1
RG3 170 0 0 0
RG4 119 27 23 4
Control 26 0 0 0 Figure 15. Karyotype of normal root tip of red clover cv. 'Altaswede' (2n=2X=14) (mag. X1570). 106 found. In contrast 27 out of 119 RG4 'Altaswede' plants had tetraploid chromosome numbers (28). Most (23) of these 27 plants with altered chromosome number were tetraploids, which were likely produced through chromosome doubling during the callus culture stage. Only four plants were diploid/tetraploid mosaics (Figure 16). These results supported the single cell origin of most embryos, from which these plants were regenerated. The four mosaic plants could come either from embryos which had originated from more than one cell origin or from embryos which had some cell(s) undergo changes in chromosome number during embryogenesis.
It was also demonstrated that plants regenerated from short-term cultures (either multiple shoot cultures or callus cultures) tended to have a normal chromosome number, while chromosome abnormalities could happen to the regenerated plants from cultures with a prolonged culture period. Another interesting aspect of the observation is that no aneuploid chromosome numbers were observed.
4.4.3 Isozyme Analysis
Isozymes are attractive for direct genetic study because they represent, the primary products of structural genes. Changes in coding sequences will in many cases lead to quantitative or qualitative changes in isozyme patterns.
RG3 and RG4 plants of 'Altaswede' were analysed for their isozyme patterns of five enzymes. Comparisons were made to the isozyme patterns from seed-derived plants. 107
Figure 16. Root tip chromosome numbers of two RG4 plants of 'Altaswede' (mag. X1570). (a) 2n=2x=14 (upper); (b) 2n=4x=28 (lower). 108
Results showed that variation existed in the isozyme patterns of all the enzymes but one in seed-derived plants.
This observation is not unusual for an out-crossing species.
In order to determine whether variation occurred in the
regenerated plants it is important to know the original
isozyme patterns of the plants from which calli were induced
and plants were regenerated. Because all the RG3 plants
were from three calli the number of expected isozyme
patterns should not exceed three even if each callus is
different. Four or more patterns for one enzyme will
indicate the occurrence of a non-parental type. Similarly,
the overall isozyme patterns for all the five enzymes should
not exceed three. If two genotypes which differ from each
other in isozyme patterns of two enzymes are used as the
starting materials, the plants regenerated from these two
genotypes should always differ in the two isozyme patterns
provided no variation had happened during the culture
period. In this way the original genotypes and possible
variation could be determined when more than one genotype is
used in culture. For RG4 plants it was easier to determine
the original genotype. Because all the plants were derived
from one genotype, the majority of the regenerated plants
should show the isozyme pattern of the original genotype,
whereas any deviation from the pattern should be in low
frequency.
Two hundred and ten RG3 plants were investigated for
five enzymes. No variation attributable to tissue culture 109 was observed for phosphoglucose isomerase (PGI). Two isozyme patterns were designated for the original genotypes.
These two genotypes differed at one locus with one homozygous and another one heterozygous (Figure 17). The genotype with the heterozygous locus had two more bands than the homozygous one since PGI is a dimeric enzyme. No variation was found in RG3 plants for phosphoglucomutase
(PGM) isozymes. All these plants had three bands, one dense band and two faint bands (Figure 18). The number of bands may vary from three to five among seed-derived plants. For malate dehydrogenase (MDH) all seed-derived and regenerated plants had five bands (Figure 19). It was determined that five loci are likely responsible for the enzyme because it is monomeric. For 6-phosphogluconate dehydrogenase (6-PGDH) four isozyme patterns were obtained. One pattern, represented by only one sample (i.e. plant 170), could be a consequence of the culture process (Figure 20). For shikimate dehydrogenase (SKDH) two isozyme patterns were observed, which were due to two genotypes of the three calli in this enzyme. One pattern had one band and another one had two bands.
Sixty RG4 plants were screened for isozyme patterns of the five enzymes. All these plants had the same pattern for each enzyme as one of the three genotypes which had been observed in RG3 plants, demonstrating that no qualitative variation had been induced from the culture cycle. 110
Figure 17. Zymogram of phosphoglucose isomerase from RG3 plants of red clover cv. 'Altaswede'. Lane 3 from left represents another genotype from which some plants were regenerated. 111
+
Figure 18. Zymogram of phosphoglocomutase from calli of red clover cv. 'Altaswede'. From left to right, lanes 1-19: embryogenic calli and lanes 20-29 non-embryogenic calli. 112
Figure 19. Zymogram of malate dehydrogenase from RG3 plants of red clover cv. 'Altaswede'. All plants had five bands. 1 13
Figure 20. Zymogram of 6-phosphogluconate dehydrogenase from RG3 plants of red clover cv. 'Altaswede'. Lane 3 from left represents another genotype from which some plants were regenerated. Lane 11 from left is an abnormal pattern observed, which had an extra band. 1 14
4.4.4 Nodule Formation and Leghaemoglobin Profile
All plants analyzed for chromosome number and leghaemoglobin profile formed normal symbiotic root nodules.
The normal leghaemoglobin profile of red clover nodules, as shown in Figure 21, has three components, one major and two minor under the CAE conditions used in this study. The major band migrates most slowly. For
'Altaswede' 91 RG1 and RG2 and 33 RG3 plants were analysed.
No qualitative variation (i.e. band missing, new band appearing or altered mobility) was detected. Although quantitative differences (intensity and ratio between bands) were present, these may be a reflection of differences among individuals, sample size and/or infecting Rhizobium strain.
4.4.5 Plant Morphology
Leaflet Number
Among the regenerated plants leaflet number changes from four to seven were observed with high frequency (Table
26). However this variation was also observed on
'Altaswede' control plants. The absence of such variation in 'Norseman' could, in part, be a consequence of the small sample size. All the plants with changed leaflet number were mosaic and had some leaves of normal leaflet number.
It would appear that the tissue culture regime may have a stimulatory effect on the occurrence of altered leaflet 115
+
Figure 21. Cellulose acetate electrophoresis of leghaemoglobin of red clover cv. 'Altaswede'. There are three components, one major and two minor. The major band migrates most slowly. 116
Table 26. Leaflet number in control (seed-derived) and regenerated plants of red clover.
Population Cultivar No of Plants with changed plants leaflet number (%)
RG 1 Altaswede 69 6 (8.7)
RG2 Altaswede 100 8 (8.0)
RG3 Altaswede 263 5 (1.9)
RG4 Altaswede 124 3 (2.4)
RG 1 Norseman 93 23 (24.7)
Control Altaswede 84 5 (6.0)
Control Norseman 20 0 1 17
number, but such a conclusion would require careful comparison and analysis of variation in seed-derived and
regenerated plants.
Length to Width Ratio of Leaflet
The mean, variance, and standard deviation of ratios of
different "populations" are presented in Table 27. RG1,
RG2, RG3 and RG4 plants were analysed for 'Altaswede'.
Because RG4 plants were grown during the summer months an
additional group of control plants (CK2) were grown along
with them. The mean of CK2 plants is a little higher than
that of CK1 probably because at that time both temperature
and light intensity were high and plants were growing more
quickly.
Statistical analysis of these data is shown in Tables
28 and 29. Means of ratios for different populations of
regenerated plants of the two cultivars are not
significantly different from the means of control plants
(P<0.05). That is, the leaflet length to width ratio of
regenerated plants did not become longer or shorter as a
result of iri vitro culture procedures. However, as shown by
the data in Table 29 the variances for RG1, RG2 and RG3
plants of 'Altaswede' are all significantly larger (P^O.01)
than that of control plants. This observation suggests that
in the regenerated plants greater variation existed for the
ratio. Nevertheless the variance of RG4 plants is not
significantly different from that of the control group. 118
Table 27. Means and variances of leaflet length/width ratios for different populations of red clover.
Cultivar Population n1 X s2., n n
Altaswede CK1 51 1 . 127 0.074 5.48X10
RG 1 41 1 . 1 30 0. 149 22.0X10 - 3
RG2 61 1 .162 0.131 17.2X10 - 3
RG3 63 1 .094 0. 125 15.6X10
RG4 1 03 1 .279 0.099 9.80X10 - 3
CK2 49 1 .269 0.092 8.46X10
Norseman CK 35 1 . 156 0.086 7.40X10
RG 1 41 1 .130 0.087 7.57X10
RG2 101 1 . f 7 1 0.077 5.93X10 - 3
1n: sample size 119
Table 28. Comparisons between means of regenerated plants and control plants for leaflet length/width ratio
Cultivar Comparison
Altaswede RG1/CK1 0.118 ns1
RG2/CK1 1 .785 ns
RG3/CK1 1.761 ns
RG4/CK2 0.610 ns
Norseman RG1/CK 1.307 ns
RG2/CK 0.905 ns
1 ns: not significant.
Table 29. Comparisons between variances of regenerated plants and control plants for leaflet length/width ratio
Cultivar Compari son F
Altaswede RG1/CK1 4.019 **1
RG2/CK1 3.139 **
RG3/CK1 2.853 ** .
RG4/CK2 1.158 ns
Norseman RG1/CK 1.023 ns
RG2/CK 0.801 ns
1 **: significant at PS0.01 level; 120
This difference is likely because RG4 plants were regenerated from only one genotype so that less variation might be anticipated. For 'Norseman', however, the result was quite different. The variances of RG1 and RG2 plants were not different from that of control plants. The difference between the two cultivars may be related to ploidy difference, with the tetraploid cultivar 'Norseman' showing more resistance to change.
Morphology of RG4 'Altaswede' Plants
Several albino plants were observed among the RG4 plantlets regenerated from the 'Altaswede' calli. Three of them were isolated and subcultured onto LSP medium; they gradually senesced and died during the first and second subcultures.
In general, regenerated plants grew more quickly and had greener leaves than control plants when recorded ten weeks after the plant transfer. These trends were not obvious when recorded only five weeks after the transfer.
There were some tetraploid plants among RG4 plants, typically reflected in the production of larger leaflets and petioles. 5. DISCUSSION
5.1 Callus Induction
After the explants were inoculated on L2(P0.06B0.2) medium callus production usually commenced within two weeks.
For this study a total of five batches of cultures from two cultivars were initiated, and calli were produced relatively easily. Similarly Phillips and Collins (1979a) observed that one third of 100 genotypes of red clover tested showed excellent response while only one fourth gave poor callus production. Since L2 had been optimized for callus growth by Phillips and Collins (1979a), it is not surprising that
it gave good results for callus induction in this study. L2 medium uses PIC as the sole auxin. For callus induction only 0.06 mg/1 PIC was required. Because PIC is more effective than 2,4-D it is a desirable auxin for use in callus culture. It also has been used as an auxin in experiments in which embryos were formed from leaf-derived callus of pea (Pisum sativum L.) (Jacobsen and Kysely,
1984). PIC may be a promising growth regulator for medium manipulation of cell cultures and plant regeneration from
legumes.
It was observed that calli formed from explants under various light conditions: dark, moderate and high light
intensities. 'Altaswede' appeared to show a higher
frequency of callus induction under low light or dark
conditions than under high intensity light. In 'Norseman'
121 122 such an obvious difference was not observed. Calli formed from both cultivars on either plates or in test tubes.
Calli of different species and even of the same species differ markedly in texture and color (Thorpe, 1982). Callus induced in the dark is white to pale yellow while that initiated under light may be pale yellow or more strongly pigmented. For example red clover callus from hypocotyl explants was white with a slight green coloration when initiated in light.
5.2 Callus Maintenance and Plant Regeneration
5.2.1 Primary Test of Two Media
No shoots (or bud initials) were induced on either one
of the two media (B5 and LSE) on which plant regeneration in red clover has been reported (Beach and Smith, 1979;
Phillips and Collins, 1980). Genotype differences may contribute in part to the difference between the results in this study and those of previous reports because of differences in cultivars. The callus source difference could also account for the different results. Beach and Smith (1979) induced callus on
B5(N2D2K2) medium, while in this work callus induction occurred on L2(PO.06B0.1) medium. The failure of LSE medium to induce bud initials will be discussed in the next section. 1 23
5.2.2 Callus Growth on LSE Medium
In a series of tests designed to induce shoots, calli were transferred onto LSE medium. A poor response was
observed in all these tests. After transfer onto LSE medium, calli showed little growth, began to brown after a
short period in culture and the frequency of green-spotted
calli decreased. After continuous subculture on LSE medium
(approximately two months in culture) calli stopped growing,
turned to brown and black as they died.
Phillips and Collins (1980) reported the induction of
bud formation with LSE medium on 35-40% of plates inoculated
with cell suspension cultures of red clover cultivar
'Arlington'. In their experiment suspension cultures were
established by inoculating hypocotyl and epicotyl sections
into liquid SL-2 medium (Phillips and Collins, 1980). The
suspensions were maintained in liquid culture for six months
using about 5 ml old culture to inoculate 25 ml fresh
medium. Approximately one ml of the suspension was
inoculated onto solid media to induce buds and shoot
induction was verified by subculturing the induced buds onto
medium containing 0.004 mg/1 PIC and 1 mg/1 BA. In
comparing their experimental conditions to those of this
study differences in cultivar and procedure were evident.
Cultivar differences in response have been observed
(Phillips and Collins, 1979a). Differences between the in
vitro behavior of the two cultivars used in the present
study were also obvious. Cultivar differences may be 124 exaggerated or masked by the confounding effect of individual genotypes. From the results of crosses made among regenerated plants, Phillips and Collins (1980) concluded that these plants were all derived from explants of a single seedling. This individual seedling genotype was likely more capable of regeneration than other genotypes.
Therefore, it is possible that one cultivar contains a mixture of genotypes with different capacities for regeneration.
The most likely reason for the discrepancy in observed
response was the procedure used to induce plant
regeneration. Phillips and Collins (1980) used a suspension culture as the inoculum for shoot induction. It is possible
that embryos or pre-embryos had already been initiated
during the cell suspension culture cycle of their
experiment, whereas calli on LSE medium in this study had
not been initiated to undergo embryogenesis. This argument
was supported by the results of another experiment (Section
4.2.4), in which calli from LSE medium did not have embryos
or shoots developed on the media containing 0.0005-0.005
mg/1 PIC and 0.2-1 mg/1 BA. This observation indicated that
calli grown on LSE in these experiments had not passed the
point of determination (Dodds, 1982). Cultures of cells
pre-determined for embryogenesis or embryos may produce
embryogenic calli which can grow and differentiate on a
medium which would not support differentiation or growth of
non-embryogenic calli. As demonstrated in this study, 125 embryogenic and non-embryogenic calli performed differently on all the ten media tested with embryogenic calli more successful than non-embryogenic calli. In addition, embryogenic calli required less exogenous auxin and cytokinin for growth. This observation was reinforced by the fact that calli from one genotype capable of embryogenesis produced embryos on LSP medium.
Non-embryogenic calli were cultured on the same medium, but no response was observed for shoot induction. The failure of shoot induction from calli on LSE medium was probably a consequence of the use of individual genotypes with a low capacity for regeneration or a reflection of the failure to
trigger the process leading to embryogenesis before calli
were transferred onto LSE medium for these genotypes which
were capable of regeneration.
5.2.3 Callus Growth on SCP Medium
SCP medium was shown in these experiments to be able to
revitalize senescing calli. SCP medium may be useful in
cases where calli of poor quality and deteriorating
performance need to be recovered. Calli from different
sources showed a similar response, especially those cultured
on LSE medium. Recovery frequency decreased with the age of
calli cultured on LSE medium, indicating that a threshold
for recovery likely exists.
Calli on SCP medium could be maintained in good
condition for a time period up to four months before 126 subculture. Where callus line maintenance is desirable, SCP can be used to increase the subculture interval from the typical one month to two or three months.
Embryogenesis was also observed on this medium (SCP).
Embryogenesis is usually initiated when callus is transferred from media containing high levels of 2,4-D to media containing little or no 2,4-D (e.g. Reinert et al.,
1977). It is possible to initiate the process on media containing 2,4-D, but obvious embryos can only be observed on media with low or non-existent level of 2,4-D. In the present, study embryos at the globe and heart stages were detected on SCP medium. Progressively longer culture periods for calli eventually lead to disorganization of embryos and embryo-like structures. As a result, many structures which were morphologically similar to embryos at the globe or heart stages were observed, but often much larger in size. Further development of embryos induced on
SCP medium required transfer of the cultures onto other media.
5.2.4 Plant Regeneration: Organogenesis and Embryogenesis
Plants can be regenerated from callus cultures through one of two pathways: organogenesis and embryogenesis (e.g.
Reinert et al., 1977; Narayanswamy, 1977; Bhojwani and
Razdan, 1983). A shoot bud from organogenesis and an embryo from embryogenesis are distinguishable on the basis of recognizable morphological differences. The former is a 1 27 monopolar structure, while the latter is a bipolar one. In some cases, root formation may be incomplete for shoots developed from embryos due to regulators present in the medium. However, it is still possible to determine the origin of regeneration by other criteria. Embryos have a specific morphology and plantlets from these embryos have cotyledons. For example, red clover shoots from embryos are recognizable by three criteria: (1) Embryo structures are visible on callus before shoot development; (2) Shoots have two cotyledons; (3) The first leaf after the cotyledon is unifoliate.
In organogenesis callus cultures are first transferred onto a medium which induces shoots from callus. These shoots are connected anatomically with the callus tissue.
The shoots must be transferred to another medium for root
induction. The most frequent type of differentiation from callus is root formation (Reinert et al., 1977). The primordia of organs are formed, as a rule, from single or
small groups of parenchymatous cells. In tobacco, formation of buds, roots or callus can be controlled at will, depending on the ratio between auxin and cytokinin in the medium (Skoog and Miller, 1957). High concentrations of
kinetin caused bud initiation, whereas high levels of auxin
favored rooting. Other species vary in their requirements
for organogenesis and regeneration may be more complicated.
Plant growth regulators, other medium components and explant
(source, size and physical age) as well as temperature, 128 photoperiod, light intensity, pH and sugar concentration may all play a determining role in organogenesis (Narayanswamy,
1977; Reinert et al., 1977; Street, 1977; Flick et al.,
1983). The early phase of shoot formation via organogenesis is similar to that of root formation. Transfer of tissue to conditions supporting organized growth leads to the appearance of meristemoids, which are located on the surface or embedded in the tissue. Continued division of the surface meristems often leads to formation of small protuberances, giving tissue a nodular appearance (called nodular callus, similar to green-spotted calli here). The meristemoids, initially apolar, are developmentally plastic and capable of giving rise to either root or shoot primordia
(Thorpe, 1982). Generally, roots are endogenously
originated from callus, whereas shoots are formed
exogenously (Bhojwani and Razdan, 1983). The process of
continued primordium development to form organs is
essentially similar to that in the intact plant (Thorpe,
1982) . Often before organogenesis, green-spotted calli (or
nodular calli) have been observed (Reinert et al., 1977;
Ohyama and Oka, 1982; Thorpe, 1982; Bhojwani and Razdan,
1983) . Such callus morphology may be an indicator of
potential shoot formation (Kim and Jang, 1984).
Green-spotted callus showed cell division in the
regions of green color. Areas of high activity in callus
can lead to the formation of meristemoids, which are the
sites of organ formation. Initially, when these structures 129 were observed in this study it was thought that they were indicators of potential shoot formation. However, the results showed that these green spots only formed roots, suggesting that either the environmental factors or endogenous regulation (or both) favored differentiation to roots instead of shoots. As observed in these experiments, when the green spots grew out of the callus, they appeared
initially as green bud-like structures (under illumination).
Further elongation occurred and white hairs emerged from the top region as they became more clearly defined roots. Green
spots on callus in this study were essentially an indication of root organogenesis.
A significant effect on root formation from callus was observed with growth regulators, culture sequence, cultivar and light intensity. More root formation from callus
generally resulted when cultures were grown on media lacking
auxin or with a low auxin to cytokinin ratio. This trend
was observed throughout the study. In tobacco ('Wisconsin
No 38') pith culture Skoog and Miller (1957) have
demonstrated that the relative ratio of auxin (IAA) to
cytokinin (KIN) determine the nature of organogenesis.
Roots formed from the tissue in the absence of kinetin and
in the presence of 0.18-3.0 mg/1 IAA, and shoots formed in
the presence of 1.0 mg/1 kinetin, particularly with IAA
concentrations in the range of 0.005-0.18 mg/1 (Skoog and
Miller, 1957). Since then, quantitative changes have been
found to be decisive in a number of other plant tissue 130 systems (Narayanaswamy, 1977). Root formation in red clover callus cultures was quite different, as demonstrated in this study. Similarly in alfalfa, it was shown that, unlike many conventional systems in which organogenesis has been studied, callus from alfalfa produced roots after induction by four days exposure to relatively high kinetin and relatively low 2,4-D (Walker et al, 1976). Shoots were induced after a four day culture period in relatively high
2,4-D concentrations and low kinetin concentrations.
Nevertheless, it is the ratio of auxin to cytokinin in the
"induction medium" that controlled root or shoot organogenesis.
Shoot formation from callus via organogenesis was not observed. However, shoot organogenesis did occur during propagation of multiple shoot cultures. In this case organogenesis was dependent upon high cytokinin-containing media. Rooting from shoots was relatively easy compared with shoot induction. A rooting frequency of about 85% of the shoots transferred onto the rooting medium was observed and could be improved by elevating IAA concentration slightly.
Plant regeneration may also be achieved via embryogenesis. Although the list of species for which somatic embryogenesis has been reported is long, the number of reports with convicing evidence is smaller (Ammirato,
1983). Somatic embryos should resemble their zygotic counterparts with appropriate root, shoot and cotyledon 131 organs with shoots capable of developing into plants.
Somatic embryogenesis of most species follows a similar pattern: growth initiation on a medium with auxin (2,4-D,
NAA or IAA), appearance of embryos after callus transfer to a medium containing reduced 2,4-D levels, a weaker auxin or no growth regulators (Raghavan, 1976; Reinert et al., 1977;
Evans et al., 1978; Ammirato, 1983). It has been suggested that the successive changes in nutrient media are significant for embryogenesis (Reinert et al., 1977).
Explant source, reduced nitrogen, auxin and other medium components are important factors affecting embryogenesis.
The critical feature of embryo development is the occurrence of bipolarity (Reinert et al., 1977; Brawley et al., 1984).
These treatments favoring embryogenesis of cultured cells should encourage the development of this polarity. Although totipotency is widely accepted in achieving regeneration, the capacity does not in itself ensure that cell differentiation will occur. Callus and cell colonies from various tissues consist essentially of parenchyma cells.
These cells are highly vacuolated and contain inconspicuous nuclei and cytoplasm. A specific type of cell division, leading to the formation of smaller, usually isodiametric cells with prominent nuclei densely stained cytoplasm and microvacuolation, i.e. meristem-like cells, is needed
(Thorpe, 1982). Sometimes the change in one of many factors could benefit the cultured cells to transfer from one state
to another. In anther or pollen cultures of Nicotiana 132 tobaccum embryogenesis and subsequent plant regeneration were studied with exposure during culture to reduced atmospheric pressure, anaerobic environments, water stress as a result of addition of 0.5 M mannitol to the medium and additions of abscisic acid, ascorbic acid, dithiothreitol and each of eight amino acids (Harada and Imamura, 1983).
Results showed that suitable treatments with all these
factors significantly enhanced embryo development in culture.
Plants were regenerated from calli via embryogenesis in
this study. Calli were first cultured on SCP medium containing relatively high 2,4-D and BA concentrations.
Embryos developed on media containing NAA and KIN or
containing PIC and BA. These embryogenic calli can then be
transferred onto LSP medium for embryo development and
eventual plant regeneration. Subculture may be required.
Once the process of embryogenesis has been induced it is
possible to initiate embryo production and have plantlet
development from these embryos on a single medium.
Of several major factors which control iri vitro somatic
embryogenesis the level of reduced nitrogen supplied to
cultures is one of the most important (Kohlenbach, 1978).
In alfalfa embryo formation was stimulated in both number
and quality by amino acid additives (Stuart and Strickland,
1984a; 1984b). Development of embryos to plantlets was also
enhanced by similar additives. In the present study the
addition of amino acids (arginine and glutamic acid) did not 133 enhance callus differentiation. Because these calli were cultured previously on LSE medium and had not been
stimulated to undergo embryogenesis it is perhaps not
surprising that the addition of amino acids did not show any promotional effect on embryo induction while the same
substances have significantly stimulated embryogenesis in
alfalfa (Stuart and Strickland, 1884a). In their experiment
a clone of a highly regenerable genotype (capable of
embryogenesis) was used. Embryogenesis could be easily
triggered by a routine method. Since a callus line which is
highly regenerable was eventually obtained in this study it
should be possible to test whether the addition of amino
acids significantly stimulates embryogenesis in red clover
in comparison with the earlier results reported for alfalfa.
Though plant regeneration from callus cultures
initiated from non-meristem explants of red clover has been
reported (Beach and Smith, 1979; Phillips and Collins,
1979a) it is not always easy to achieve regeneration.
Bhojwani et al. (1984) obtained calli from leaf and
hypocotyl explants which calli did not undergo any
organogenesis except for rare instances of root formation.
Success in regeneration from these cultures mainly depends
on culture procedures and genotype used.
5.2.5 Genotype Effect
Genotypic effects on callus induction, callus
morphology and plant regeneration were observed throughout 134 this study. It is obvious that 'Norseman* and 'Altaswede' calli performed differently on the various media tested for both callus growth and differentiation. 'Norseman' calli grew faster than 'Altaswede' calli. 'Norseman' produced more rooted calli and green-spotted calli, but shoots were
induced only from 'Altaswede' cultures. 'Altaswede' had more shoots per multiple shoot rosette than 'Norseman', while shoots of 'Norseman' were bigger than those of
'Altaswede'.
Differences in the capacity of regeneration has been
reported among cultivars of red clover (Phillips and
Collins, 1979a). Intra-varietal variation for in vitro
plant regeneration was also observed in three Trifolium.
species, including red clover (Bhojwani et al., 1984), while
Phillips and Collins (1979a) reported that cotyledon or stem
petiole explants of red clover yielded calli regenerating
plants from only 1% of the genotypes cultured. Studies
attempting to evaluate variation in callus cultures of red
clover revealed that genetic variance was a significant
source of varibility for most 5^n vitro characters
investigated, which included rapid callus growth, colony
vascularization, root initiation, chlorophyll production and
somatic embryogenesis (Keyes et al., 1980).
Genetic differences seem to be a major factor limiting
both callus induction and plant regeneration (Green et al.,,
1974; Chen et al., 1978; Sharp and Evans, 1982; Subba Rao
and Nitzsche, 1984). Genetic studies on some in vitro 1 35 characters, relating to regeneration, have been carried out by several laboratories (Zamir et al., 1980; Reisch and
Bingham, 1980; Skvirsky et al., 1982; Meins et al., 1983).
In alfalfa, crosses were made between a high frequency of regeneration line and low regenerator (Reisch and Bingham,
1980). The segregation ratio observed among the progeny of these crosses suggested that bud differentiation from callus was controlled by two dominant genes. I_n vitro characters may also be correlated with the expression of other characters such as fertility and yield (Mezentsev, 1980;
Nesticky et al., 1983).
If some rn vitro characters are highly heritable it may be expected that they should respond to breeding and selection in attempts to develop superior populations for use in tissue culture studies. Genetic lines of alfalfa with 67% regeneration were selected from hypocotyl tissue culture regenerated plants with an initial plant regeneration frequency of only 12% (Bingham et al., 1975).
Selection for friability and intensive proliferation of callus from a maize line 'F71' has resulted in three callus lines characterized by uniform structure and improved growth rate (Bartkowiak, 1981). A program aiming to improve maize regeneration was reported using Zea diploperennis as a source of germplasm for its capacity of regeneration
(Sondahl et al., 1984).
In red clover, selection for regenerability may also be possible. Callus lines or plant lines may be obtained by 136 selection techniques for improved ir\ vitro characters, especially plant regeneration. Such lines would greatly facilitate tissue culture studies.
5.3 Shoot Tip Culture
Clonal propagation by multiple shoot production provided a rapid method to propagate valuable materials.
Shoot tip cultures were established from two cultivars, which were significantly different in their response.
Meristem-tip culture of red clover was reported using
L2(P0.004B1) medium (Parrot and Collins, 1983). Shoot tip
cultures were also established using B5 medium and affecting factors were evaluated in another study (Campell and Tomes,
1983). In the present study shoot tips were cultured on L2 media containing different levels of PIC and BA for multiple shoot induction and propagation. Procedures described in these experiments are available for multiplication of plants by in vitro techniques.
5.4 Regenerated Plants
Results from the analysis of leghaemoglobin profiles and isozyme patterns of five enzymes showed that plants regenerated from callus culture were quite stable for these characters. These data indicated either that these characters are very stable genetically so that variation is not easily induced by a single culture cycle or that
somaclonal variation is not common in these genotypes of red 137 clover. The analysis of proteins offers a direct way to evaluate gene products for genetic variation. Somaclonal varition for such qualitative traits may be demonstrable for
species in which this variation has been freqently reported
for quantitative and morphological characters.
When 45 RG1 'Altaswede' plants, which came from the multiple shoots initially induced, were analysed for
chromosome number they were all found to be normal. After
the multiple shoots underwent subculture, one of 28 plants
examined was a diploid/tetraploid mosaic. Plants (170)
regenerated via embryogenesis from short-term callus
cultures (callus had been cultured for three months) did not
show any variation, whereas 23% of 119 plants regenerated
via embryogenesis from long term cultures ( nearly one year)
had changed chromosome numbers, indicating that variation is
related to length of culture period. Chromosome doubling is
a very commen change for cultured cells. It takes place in
some cells by the process of endomitosis, which consists of
a separation of sister-chromatids within the nuclear
membrance but without the formation of a spindle and the
nucleus subsequently undergoes a further DNA replication
(Sunderland, 1977). In some cases, cells with doubled
chromosome numbers increased after a prolonged culture
period (D'Amato, 1977). Accordingly plants regenerated from
long-term cultures tend to have more polyploids. In red
clover previous reports showed plants regenerated from
short-term callus cultures were normal in chromosome number 138
(Beach and Smith, 1979; Phillips and Collins, 1979a).
However, when plants were regenerated from calli of a diploid cultivar which had been cultured in suspension culture for six months before the initiation of regeneration, two out of twenty two plants were tetraploids
(Phillips and Collins, 1980).
In cereals and grasses plant regeneration from
embryogenic calli via embryogenesis has been achieved in
Pennisetum americanum L., Pennisetum purpureum, Pennisetum
americanum L. X P_j_ purpureum hybrid, Panicum maximum and
Triticum aestivum L., high stability of chromosome number in
regenerated plants was observed (Vasil and Vasil, 1981;
Vasil et al., 1982; Vasil, 1983). When 101 plants
regenerated from embryogenic calli of Pennisetum americanum
L. were investigated, one was tetraploid and all the others
were diploids, although a higher percentage of tetraploid
and aneuploid cells were observed in the cultures (Swedlund
and Vasil, 1985). In contrast, when Karp and Maddock (1984)
regenerated plants from cultured immature embryos of four
wheat cultivars (Triticum aestivum L.), 29% of 192 plants
examined were aneuploids and chromosome abnormalities
happened to plants both from embryogenic calli and from the
cultures in which origin of shoots was not identified. The
present study demonstrated that chromosome number change
occurred plants regenerated from embrogenic calli of red
clover. The discrepancy among the reports concerning
chromosome abnormalities could result from differences in 139 length of culture period, culture procedure and materials used.
Somatic embryos are believed to arise from single cells
(e.g. Vasil et al., 1982). The fact that some regenerated plants from one callus were mosaic in the present study suggests that either the somatic embryo originated from more than one cell or the chromosome number changed in some cell(s) at an early stage of embryogenesis.
Leaflet number changes were frequently observed among the plants regenerated either from multiple shoots or from callus cultures. This phenomenon was, however, also observed among seed-derived plants although at lower frequency. It has been reported that the expression of five leaflets in red clover is controlled by two genes with complementary effect (Simon, 1962). When a plant shows only a few leaves with altered leaflet number and the remainder normal, the changes may be a consequence of environmental factors (Dr. U. Simon, Technical university of Munich, personal communication). In this study all the plants with changed leaflet number were mosaic, indicating that they probably arose as a result of phenotypic plasticity rather than from genetic changes. Therefore, the fact that more regenerated plants had varied leaflet numbers than seed-derived plants does not necessarily mean that the tissue culture cycle had induced heritable genetic changes
in plants for this trait. 140
Interestingly, regenerated plants of 'Altaswede' showed more variation in the length to width ratio of leaves than
those of 'Norseman'. Plants which were regenerated from callus cultures of one genotype showed a reduced variation.
Regenerated plants of 'Norseman' did not differ from the
seed-derived controls with respect to this variability. The
tetraploid nature of 'Norseman' may increase its stability
to such changes.
These results demonstrated that variation among
regenerated plants was more likely to occur for quantitative
characters than for qualitative characters. Thus somaclonal
variation is more likely to be reported in work on
quantitative characters such as leaflet length, plant
height, dry weight or grain weight. In sugar cane,
somaclonal variation has been reported frequently but most
of the reports relate to quantitative characters (Section
2.2.1.1.). Usually, relatively high frequencies of variants
were reported. Liu and Chen (1976) reported that 34% of
regenerated plants from one clone F156 differed
significantly from the donor in morphological characters.
However, in another study where five subclones with
distinctive stable and visible markers were selected for
tissue culture, the rate of change of the specific
qualitative characters, leaf freckle and leaf blotch, was
two or three plants per thousand. For two other characters,
missing internode and zig-zag stalk, although changes were
detected, they were not transmissable through vegetative 141 propagation (Irvine, 1984).
Very few reports are available on the analysis of
regenerated plants from cell and callus cultures of legumes
(Groose and Bingham, 1983; Johnson et al., 1983; Damiani et
al., 1985), in part because of the continuing difficulty in
efficient plant regeneration from non-meristem derived
cultures. This problem may be approached by two ways.
First, additional efforts are required to define the proper
culture conditions including medium composition and
environmental regimes. The second approach requires the
development or use of genotypes showing high regenerative
potential from jjn vitro culture. Since a genotypic effect
has been reported in many cases, such lines are possible to
facilitate further research. Nevertheless, good regenerator
genotypes can only be obtained by screening sufficiently
large plant populations to ensure that the range of
variability for regeneration is appropriately sampled.
To provide solid evidence for and to estimate the
frequency of somaclonal variation proper experimental
materials are required. Most of previous reports have dealt
with quantitative traits. These traits are inherently quite
variable and susceptible to existing environmental
conditions at the time of of the experiment. Qualitative
traits should be looked at for the stated purposes. If
plants have one to several single gene-controlled traits and
these traits are available in both heterozygous and
homozygous states, then it will be possible to estimate the 142 frequencies of dominant or recessive mutants after one or more culture cycles. 6. Summary
When hypocotyl explants of red clover cvs 'Altaswede' and 'Norseman' were cultured on L2 medium, 60-85% of
the explants produced calli within two weeks. 'Altaswede'
showed a higher induction frequency than 'Norseman'
(P40.05) in the combined data of the two batches tested.
LSE medium, previously described in culture studies of
red clover, was not successfully used to induce shoots
under the experimental conditions of this study, nor
was it a good medium for callus maintenance.
On SCP medium, calli had a high growth rate. They
could be successfully grown on this medium for up to
five months without subculturing. Embryogenesis was
initiated in some calli on SCP medium, but embryos at
the globe and early heart stages were not able to
develop further normally.
More roots were usually formed from calli cultured at
a low auxin to cytokinin ratio or in the absence of
auxins.
Shoots were formed after calli of 'Altaswede' cultured
on SCP medium were transferred onto media containing
NAA and KIN. NAA concentration was more critical than
KIN concentration.
Additions of amino acids (arginine and glutamic acid)
and casein hydrolysate did not show any promotive
effect on callus growth or differentiation.
143 144
By selecting and subculturing embryogenic calli, a number of plants have been regenerated from culture.
Callus capability for embryogenesis was retained in selected lines for more than one year.
Embryogenic calli grew faster than non-embryogenic calli on ten media used.
Multiple shoots were induced from shoot tips cultured on L2(PO.003B2) medium. These multiple shoots were easily propagated by subculturing them on L2 media containing 0.002-0.003 mg/1 PIC and 1 mg/1 BA.
Usually 'Norseman' calli grew faster and produced more roots than 'Altaswede' calli, while shoots were induced only from 'Altaswede' calli. In multiple shoots culture, 'Norseman' had more shoot tips induced to produce multiple shoots, but the average multiple shoot number for 'Altaswede' was higher than that for
'Norseman'.
Plants were regenerated from multiple shoots, either initial multiple shoots (RG1) or multiple shoots after twice subcultures (RG2), or from callus cultures, either short-term (three months) callus (RG3) or long-term (one year) callus (RG4). 145
12. Regenerated plants of 'Altaswede' were analysed for
chromosome number. All RG1 and RG3 plants were
normal. One of 28 RG2 plants and 27 out of 119 RG4
plants had altered chromosome numbers. All these
plants were tetraploids or diploid/tetraploid mosaics.
No aneuploids were recovered.
13. Regenerated plants showed considerable stability for
leghaemoglobin profiles and isozyme patterns of MDH,
6-PGDH, PGI, PGM and SKDH.
14. For leaflet length/width ratio, different populations
of regenerated plants were compared with those of
controls. RG1, RG2 and RG3 plants of 'Altaswede'
showed more variation for this ratio than control
plants, while RG4 plants of 'Altaswede' and RG1 and
RG2 plants of 'Norseman' were not significantly
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Composition of B5 medium: mineral salts and organic compounds except growth regulators
Macronutrients mg/1
KNO3 2500 CaCl^^O 150
MgSCv7HzO 250
(NH4)2S04 134
NaH2P04.H20 150
Micronutrients mg/1
KI 0.75
H3B03 * 3.0
MnS0;H20 10
ZnS04*7HzO 2.0
Na2Mo04-2H20 0.25
CuS04-5H20 0.025
CoCl2-6H20 0.025
NaaEDTA 37.3 FeSO^I^O 27.8
Organic compounds mg/1
Inositol 100 Nicotinic acid 1.0 Pyridoxine*HCl 1.0 Thiamine-HCl 10.0
Sucrose 20000