Introduction
Callus is defined as an unorganized tissue mass growing on solid
substrate. Callus forms naturally on plants in response to wounding,
infestations, or at graft unions (Bottino, 1981). Callus formation is central to
many investigative and applied tissue culture procedures. Callus can be
multiplied and later used to clone numerous whole plants. Since extensive
callus formation can be induced by elevated hormone levels, tissue culture
media designed to produce callus contain pharmacological additions of
cytokinins and auxins. During callus formation, there is some degree of
dedifferentiation (i.e. the changes that occur during development and
specialization are, to some extent, reversed), both in morphology (a callus is
usually composed of unspecialized parenchyma cells) and metabolism. One
major consequence of this dedifferentiation is that most plant cultures lose the
ability to photosynthesize. This has important consequences for the culture of
callus tissue, as the metabolic profile will probably not match that of the donor
plant. This necessitates the addition of other components such as vitamins and,
most importantly, a carbon source to the culture medium, in addition to the
usual mineral nutrients. Callus culture is often performed in the dark (the lack
of photosynthetic capability being no drawback) as light can encourage
differentiation of the callus. During long term culture, the culture may lose the
requirement for auxin and/or cytokinin. This process, known as habituation, is
common in callus cultures from some plant species (such as sugar beet).Callus
cultures are extremely important in plant biotechnology.
Plant growth regulators are the critical media components in
determining the developmental pathway of the plant cells. The plant growth
53
regulators used most commonly are plant hormones or their synthetic
analogues. There are five main classes of plant growth regulator used in plant
cell culture, Namely:(1) auxins; (2) cytokinins; (3) gibberellins; (4) abscisic
acid; (5) ethylene. Auxins promote both cell division and cell growth. The
most important naturally occurring auxin is indole-3-acetic acid (IAA), but its
use in plant cell culture media is limited because it is unstable to both heat and
light. Occasionally, amino acid conjugates of IAA (such as indole-acetyl-lalanine and indole-acetyl-l-glycine), which are more stable, are used to
partially alleviate the problems associated with the use of IAA. It is more
common, though, to use stable chemical analogues of IAA as a source of auxin
in plant cell culture media. 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most
commonly used auxin and is extremely effective in most circumstances. Other
auxins are available (see Table 2.3), and some may be more effective or
‘potent’ than 2, 4-D in some instances. Cytokinins promote cell division.
Naturally occurring cytokinins are a large group of structurally related purine
derivatives. Of the naturally occurring cytokinins, two have some use in plant
tissue culture media zeatin and42 Plant tissue culture. Auxins and cytokinins
are the most important hormones for transgenic work. Commonly used auxins,
their abbreviations and chemical names
Abbreviation/name Chemical name
2, 4-D 2, 4-Dichloro phenoxy acetic acid
2, 4, 5-T 2, 4, 5-Trichloro phenoxy acetic acid
Dicamba 2-Methoxy-3, 6-dichlorobenzoic acid
IAA Indole-3-acetic acid
IBA Indole-3-butyric acid
MCPA 2-Methyl-4-chloro phenoxy acetic acid
NAA 1-Naphthyl acetic acid
NOA 2-Naphthyloxy acetic acid
54
There are two kinds of cytokinenins one is the naturally occurring
cytokinenis which includes zeatin, 6-ϒ, ϒ-dimethyl allyl amino purine (2iP) and
adenine. There is another type which is synthetic cytokinines. This group
consists of substituted purines i.e. 6-benzyl amino purine (BAP) and 6- furfural
amino purine (kinetin) and phenyl ureas such as thiodiazuron. The synthetic
analogues kinetin and 6-benzylaminopurine (BAP) are therefore used more
frequently. Non-purine-based chemicals, such as substituted phenylureas, are
also used as cytokinins in plant cell culture media. These substituted
phenylureas can also substitute for auxin in some culture systems Picloram 4Amino-3, 5, 6-trichloro- 2 pyridine carboxylic acid Their use is not widespread
as they are expensive (particularly zeatin) and relatively unstable.
There are numerous, naturally occurring, structurally related compounds
termed gibberellins. They are involved in regulating cell elongation, and are
agronomically important in determining plant height and fruit-set. Only a few
of the gibberellins are used in plant tissue culture media, GA3 being the most
common. Abscisic acid (ABA) inhibits cell division. It is most commonly used
in plant tissue culture to promote distinct developmental pathways such as
somatic embryogenesis. Ethylene is a gaseous, naturally occurring, plant
growth regulator most commonly associated with controlling fruit ripening in
climacteric fruits, and its use in plant tissue culture is not widespread. It does,
though, present a particular problem for plant tissue culture. Some plant cell
cultures produce ethylene, which, if it builds up sufficiently, can inhibit the
growth and development of the culture. The type of culture vessel used and its
means of closure affect the gaseous exchange between the culture vessel and
the outside atmosphere and thus the levels of ethylene present in the culture.
Explants, when cultured on the appropriate medium, usually with both
an auxin and a cytokinines, can give rise to an unorganized, growing, and
dividing mass of cells. It is thought that any plant tissue can be used as an
explant, if the correct conditions are found. In culture, this proliferation can be
55
maintained more or less indefinitely, provided that the callus is subculture on to
fresh medium periodically. Manipulation of the auxin to cytokinin ratio in the
medium can lead to the development of shoots, roots, or somatic embryos from
which whole plants can subsequently be produced. Callus cultures can also be
used to initiate cell suspensions, which are used in a variety of ways in plant
transformation studies. Additionally, various genetic engineering protocols
employ callus initiation procedures after DNA has been inserted into cells;
transgenic plants are then regenerated from transformed callus. In other
protocols callus is generated for use in biotechnological procedures such as the
formation of suspension cultures from which valuable plant products can be
harvested. Explant tissues generally show distinct planes of cell division,
various specializations of cells, and organization in to specialized structures
such as the vascular system. Callus formation from explant tissue involved the
development of progressively more random planes of cell division, less
frequent specialization of cells, and loss of organized structures. (Wagley et
al.1987). When sub-cultured regularly on agar media, callus culture will exhibit
an S-shaped or sigmoid pattern of growth. Hence it is importance to study the
callogenesis with different growth regulators like auxins and cytikinins in
different mulberry varieties.
RESULTS
Time Taken for callus initiation (days) from the auxiliary buds at different
concentrations of 2,4 D were significantly variable. In M5 variety (Table 4.1
and Fig 4.1) time taken for callus initiation significantly decreased with the
concentrations from 0.5 to 2.0. The order of decrease at different
concentrations was 0.5 mg-l 2, 4 D > 1.0 mg- 2, 4 D, > 1.5 mg-l 2, 4 D > 2.0
mg-l . A similar trend was also observed in V1 (Table 4.2 and Figure 4.2), S36
(Table 4.3 and Figure 4.3) and Anantha (Table 4.4 and Figure 4.4).
56
From the data presented table 4.5 and figure 4.5, it is observed that
the percent frequency of callus initiation after 20 days at different
concentrations of 2, 4-D (MS medium) in M5. Percent frequency of callus
initiation (days) from the auxiliary buds at different concentrations of 2,4 D
were significantly increased with increased concentrations (0.5mg-l to 2.0 mg-l).
The order of increase Percent frequency of callus initiation (days) at different
concentrations was 0.5 mg-l 2, 4 D < 1.0 mg- 2, 4 D, < 1.5 mg-l 2, 4 D < 2.0
mg-l . A similar trend was also observed in V1 (Table 4.6 and Figure 4.6), S36
(Table 4.7 and Figure 4.7) and Anantha (Table 4.8 and Figure 4.8).
DISCUSSION
In the present investigation auxiliary buds were used as the source
material. Shoot culture (shoot tips or buds) is often sufficiently reliable with
herbaceous plants to be used commercially (Holdgate, 1977). Oka and Ohyama
(1975) performed material for invitro propagation of mulberry. They used three
kinds of explants and various aged auxiliary buds for their study. Young
greenish buds with long or short stem grew into leafy shoots on MS + NAA or
MS + NAA + BA medium respectively. Excised greenish buds did not grow in
var. Ichinose, but they grow in var. Kenmochi when supplemented with BAP.
Excised brownish buds that were more aged than greenish buds did not show
any growth in both varieties.
The physiological status of the explant has been of prime importance
with adult trees. Extensive studies at the Association Forest - Cellulose
(AFOCEL) research institute in France indicated that explants from adult forest
trees were frequently slow to commence growth invitro, or failed completely
unless selected from rejuvenated tissues. When natural rejuvenation is absent, it
has been induced by various treatments such as grafting, shoot pruning,
maintenance of high fertilizer levels, vegetative propagation or spraying with
cytokinines (Franclet, 1979). Hence Two months after pruning, young
57
rejuvenated twigs up to 5th node were collected in the present study. The
response of explants varied depending upon the variety.
The choice of a particular medium depends mainly on the species of the
plant, the tissue or organ to be cultured. In the present investigation, MS media
was tested for callus initiation in M5, V1, S36 and Anantha, days required for
callus initiation and percent frequency of callus in M5, V1, S36 and Anantha
differed considerably in different varieties of Morus Spp. M5, V1, S36 and
Anantha (Plate 1to 4). All mulberry variety responded well on MS medium
fortified with 2mg/l 2, 4-D. This may be due to high nitrate requirement of M5,
V1, S36 and Anantha and nitrate is relatively high in. The present study clearly
demonstrates that there was 100% response in M5 and V1.Callus was yellow
and smooth in M5, V1, S36 and Anantha. During subsequent cultures callus
ceased to grow and appeared brown in colour due to accumulation of phenolic
compounds. This was reported by Tewary et al., (1989) in mulberry and also in
other angiosperm taxa by Shah and Mehta (1976) and Singh et al., (1982). By
using 0.1% activated charcoal and this was prevented in the present
investigation. Activated charcoal adsorbs many organic and inorganic
molecules from the culture medium (James, 1971)
By using 10M 2, 4-D Oka and Ohyama (1973) obtained two types of
calli from stem segments of Mulberry varieties. As for the effect of auxins
better callus formation was observed on medium with 2, 4-D. Oka and Ohyama
(1976) reported that the addition of 2,4-D remarkably enhanced the callus
formation at the lower part of the explants, but the shoots developed rarely.
The effect of different concentrations of 2,4-D (0.5, 1, 1.5, 2mg/l)
sprouting, rooting and callus proliferation was
investigation.
studied in
on
the present
High concentration of 2, 4-D (2mg/l) showed remarkable
suppression of both sprouting and rooting. Gamborg et al. (1976) stated that 2,
4-D is a powerful suppressant of organogenesis and it would not be used in
experiments involving root and shoot initiation.
58
At 2 mg/l there was no sprouting at all and it was represented in Tables
4.1 to 4.8 and figures 4.1 to 4.8. Sekih et al., (1974) reported highest growth
rate of callus obtained with 2, 4-D followed by IAA and NAA in the light
condition. In our present study highest callus initiation was observed at 2mg/l
2, 4-D concentration. The explants cultured on medium without any hormones,
sprouted very little and did not show any further growth or callus proliferation.
Patel et al., (1983) cultured stem, petiole segments, leaf discs on MS medium
alone which has not showed any response, but when supplemented with IAA,
IBA, NAA, 2, 4-D at 0.5, 1, 2 mg-l, they proliferated into callus tissue within 4
weeks. Callus tissue was green and friable on 2, 4-D medium, whereas on NAA
and IAA medium brownish, compact callus was reported. Though the nature of
callus in our present study was smooth and yellow in M5, V1, S36, Anantha,
nodular and yellow colour was observed in M5 in the beginning, it has become
compact and greenish patches appeared when sub-cultured on to the medium
fortified with kinetin and IAA. Tewary et al., (1989) reported that cultured leaf
explants of mulberry on BAP and Kinetin does not provided any result.
However, they found that BAP (2 mg/l) + 2,4-D (0.5mg/1) was most suitable
for callus proliferation and maintenance of callus for long term culture. Many
investigators amply stressed the importance of 2, 4-D on both callus initiation
and proliferation. There is every need to provide considerable interest on the
type of media which also proved the significant effect on callus initiation and
proliferation. Thus the present study provides a scope of rapid callusing of M5,
V1, S36 and Anantha on MS + 2, 4-D (2mg/l). This in vitro technique for rapid
callusing can be exploited for plant regeneration, in M5, V1, S36 and Anantha.
59
Table 4.1: Effect of different concentrations of 2, 4-D 1 (MS medium) time
taken for callus initiation in M5.
Sl.No
Medium (MS) + 2,4-D
(mg/l)
Time Taken for callus
initiation (days)
1
0.5
2
1
3
1.5
4
2
Data represents an average of 10 replicates
15
14
13
11
Fig 4.1: Effect of different concentrations of 2, 4-D 1 (MS medium) time taken
for callus initiation in M5.
60
Table 4.2: Effect of different concentrations of 2, 4-D 1 (MS medium) time
taken for callus initiation in V1..
Sl.No
Medium (MS) + 2,4-D
(mg/l)
Time Taken for callus
initiation (days)
1
0.5
2
1
3
1.5
4
2
Data represents an average of 10 replicates
13
13
10
6
Fig 4.2: Effect of different concentrations of 2, 4-D 1 (MS medium) time
taken for callus initiation in V1.
61
Table 4.3: Effect of different concentrations of 2, 4-D 1 (MS medium) time
taken for callus initiation in S36.
Sl.No
Medium (MS) + 2,4-D
(mg/l)
Time Taken for callus
initiation (days)
1
0.5
2
1
3
1.5
4
2
Data represents an average of 10 replicates
15
12
11
10
Fig 4.3: Effect of different concentrations of 2, 4-D 1 (MS medium) time
taken for callus initiation in S36.
62
Table 4.4: Effect of different concentrations of 2, 4-D 1 (MS medium) time
taken for callus initiation in Anantha.
Sl.No
Medium (MS) + 2,4-D
(mg/l)
Time Taken for callus
initiation (days)
1
0.5
2
1
3
1.5
4
2
Data represents an average of 10 replicates
14
13
12
10
Fig 4.4: Effect of different concentrations of 2, 4-D 1 (MS medium) time
taken for callus initiation in Anantha.
63
Table 4.5: Effect of different concentrations of 2, 4-D 1 (MS medium)
% frequency of callus initiation after 20 days in M5.
Sl.No
Medium (MS) + 2,4-D
(mg/l)
% frequency of callus
initiation
1
0.5
10
2
1
15
3
1.5
33
4
2
100
Data represents an average of 10 replicates and % frequency was recorded at
the end of 20 days. Photo period of 16 hours light and 8 hours dark was
maintained.
Fig 4.5: Effect of different concentrations of 2, 4-D 1 (MS medium)
% frequency of callus initiation after 20 days in M5.
64
Table 4.6: Effect of different concentrations of 2, 4-D 1 (MS medium) %
frequency of callus initiation after 20 days in V1.
Sl.No
Medium (MS) + 2,4-D
(mg/l)
% frequency of callus
initiation
1
0.5
20
2
1
31
3
1.5
50
4
2
100
Data represents an average of 10 replicates and % frequency was recorded at
the end of 20 days. Photo period of 16 hours light and 8 hours dark was
maintained.
Fig 4.6: Effect of different concentrations of 2, 4-D 1 (MS medium)
% frequency of callus initiation, after 20 days in V1.
65
Table 4.7: Effect of different concentrations of 2, 4-D 1 (MS medium)
% frequency of callus initiation after 20 days in S36.
Sl.No
Medium (MS) + 2,4-D
(mg/l)
% frequency of callus
initiation
1
0.5
10
2
1
14
3
1.5
29
4
2
50
Data represents an average of 10 replicates and % frequency was recorded at
the end of 20 days. Photo period of 16 hours light and 8 hours dark was
maintained.
Fig 4.7: Effect of different concentrations of 2, 4-D 1 (MS medium)
% frequency of callus initiation, after 20 days in S36.
66
Table 4.8: Effect of different concentrations of 2, 4-D 1 (MS medium) %
frequency of callus initiation after 20 days in Anantha.
Sl.No
Medium (MS) + 2,4-D
(mg/l)
% frequency of callus
initiation
1
0.5
10
2
1
12
3
1.5
15
4
2
20
Data represents an average of 10 replicates and % frequency was recorded at
the end of 20 days. Photo period of 16 hours light and 8 hours dark was
maintained.
Fig 4.8: Effect of different concentrations of 2, 4-D 1 (MS medium)
% frequency of callus initiation after 20 days in Anantha.
67