Chapter 2 Indirect Organogenesis and histological...
Transcript of Chapter 2 Indirect Organogenesis and histological...
Chapter 2
Indirect Organogenesis and histological analysis of organogenic and non-organogenic calli obtained from
in vitro cultures of Justicia adhatoda L.
2.1. ABSTRACT
Leaf, axillary bud and root tip explants of J. adhatoda were employed
for calli production and indirect organogenesis on Murashige-Skoog media
supplemented with different combinations of indole 3- acetic acid (IAA), α-
naphthyl acetic acid (NAA), indole 3- butyric acid (IBA), 6-
benzylaminopurine (BA) and kinetin (Kn). Combination of 6 mg L-1 IAA
and 6 mg L-1 Kn gave best callusing of leaf explants. Combinations of 3 mg
L-1 IBA, 3 mg L-1 BA and 3 mg L-1 IBA, 6 mg L-1 BA gave maximum callus
response with axillary bud and root tip explants respectively. Multiple shoot
induction occurred per callus within six weeks on medium containing 6 mg
L-1 BA and 4 mg L-1 Kn. High frequency rooting was recorded on
Murashige-Skoog medium with 6 mg L-1 of IBA and NAA. A histological
study of calli of in vitro propagation was carried out. The phenotypic
differences of callus cultures derived from J. adhatoda L. were evaluated
based on their morphology and ultrastructure. The organogenic and non-
organogenic calli are the result of hormonal variation in the medium. In non-
organogenic callus, cells redifferentiated into xylem elements forming
clusters of nest like structures. In organogenic callus, the undifferentiated
cells of callus were found to differentiate into vascular nodules called
meristemoids, which then develop into xylem elements, especially tracheids.
On culturing on the shooting medium, these nodules differentiated into shoot
apical meristem. This adventitious origin provided chances for variability.
Key words: indirect organogenesis, histological study, J. adhatoda L.,
multiple shoot, tracheids, meristemoids, xylem elements.
34 Chapter 2
2.2. INTRODUCTION
With an ever-increasing global inclination towards herbal medicine,
there is not only an obligatory demand for a huge raw material of medicinal
plants, but also of right stage when the active principles are available in
optimum quantities at the requisite time for standardization of herbal
preparations. Ideally, the herbal plants should be grown under uniform
environmental conditions and the planting material must have the same
genetic make- up as of the selected high-yielding clones, which is possible
when they are cloned through an in vitro strategy, i.e. micropropagation, at
least in cases where conventional vegetative propagation methods are
insufficient or wanting to achieve the goal (Chaturvedi et al., 2007).
A number of medicinally important plant species have been
successfully propagated on a mass scale with the use of in vitro techniques.
In vitro propagation helps in production of a very large number of plants
from a tiny explant (Majumder et al., 2011). According to Shanmugapriya
and Sivakumar (2011) tissue culture has been successfully used for the
commercial production of pathogen-free plants to conserve the germplasm of
rare and endangered species.
Morphological and histological studies of callus induction of plant
are important for increasing the incidence of callus production (Feng et al.,
2007; Tan et al., 2009; Yan et al., 2012). In the present study, the
morphology and histology of the callus produced from the leaf, axillary bud
and root tip were systematically studied. The differences in morphological
and histological characteristics between the green compact and white or
brown soft, friable calli were also studied.
Indirect Organogenesis and histological analysis of… 35
2.3. REVIEW OF LITERATURE
Unlimited exploitation of the natural resource for medicine is causing
dwindling of the existing plant population. Large scale cultivation of the
plants is the only remedy for ensuring future availability of medicinal plants.
A major problem faced when we go for large scale cultivation is the scarcity
of the planting materials. Hence there is a necessity for developing an in
vitro culture technique for regeneration of the plants, which yields large
number planting materials at all seasons (Viji and Parvatham, 2011). Large-
scale plant tissue culture offers a controlled supply of biochemicals
independent of plant availability and more consistent product quality
(Nalawade and Tsay, 2004).
Plant regeneration from in vitro culture has been possible via
organogenesis and somatic embryogenesis. Plant tissue culture offers many
unconventional techniques for crop improvement. Callus induction and plant
regeneration is one method of plant propagation useful for experimental
work. Callus is a disorganized mass of undifferentiated tissue comprised of
actively dividing cells. The cells of callus dedifferentiate and thus regain
their meristematic properties, including rapid proliferation (Alatzas et al.,
2008). Due to these meristematic properties, callus cells are totipotent, or
capable of undergoing organogenesis, where they may potentially
differentiate into any plant part, including roots, shoots, flowers or stems
(Razdan, 2003).
Callus is generally induced by a wound response with auxins and
cytokinins which are plant growth regulators or PGRs. Auxins are involved
in cellular division, cell elongation and callus induction. Cytokinins are also
associated with cellular division and cell expansion and are widely used in
36 Chapter 2
conjunction with auxins to induce callus. Cytokinins have been found to
promote shoot regeneration in higher concentrations presumably through the
change in the auxin-to-cytokinin ratio. A wide array of factors can influence
callus growth and morphology, including plant genotype, nutrient medium
composition, the explant material used and abiotic factors such as light and
temperature. Callus may be globular or friable and both may exhibit variable
coloration. The PGRs auxins and cytokinins are required for both callus
induction and organogenesis. Furthermore, the ratio of auxin to cytokinin
concentrations determines if callus, shoots or roots will be induced.
Successful callus induction in other plant systems has been achieved with
mature explants materials such as cotyledons, internodes and petiole.
However, it is believed that the best results are derived from immature
tissues showing meristematic properties, due to their increased culture
survival rates, growth and totipotency in vitro. These tissues include
meristems, leaves, roots, shoots and inflorescences (Carter et al., 2011).
Callus culture and root culture protocols offer the possibility to use
cell/root culture techniques for vegetative propagation and secondary
metabolism studies (Catapan et al., 2002). Somatic embryogenesis generally
occurs through two different pathways, i.e. direct and indirect organogenesis.
Direct organogenesis occurs directly from the explants and indirect
organogenesis, indirectly following callus formation from explants
(Baskaran and Jayabalan, 2010).
Indirect plant regeneration can be employed as an alternative means
for genetic upgrading, and its application largely depends on the reliable
plant regeneration system. A good regenerating system may be suited for
transformation where the production of transformants by direct
organogenesis is desired (Thomas and Sreejesh, 2004).
Indirect Organogenesis and histological analysis of… 37
Production of regenerated plant through indirect organogenesis is one
possible way to contribute to genetic improvement, because there are some
advantages of shoot regeneration from callus over direct shoot regeneration.
A callus phase is commonly included in tissue culture protocols with the
objectives of generating variability to introduce new desirable traits and
generating transgenic plants to introduce traits such as pest resistance in
crops. Moreover, callus production is also a necessary step for obtaining
protoplasts used in protoplast fusion, a useful tool in genetic improvement of
vegetatively propagated plants for introducing useful genes or producing new
crops (Yan et al., 2009).
Genetic transformation is an important and effective technique to
improve the yield and quality of crops. It is a prerequisite for transgenic
studies to establish a highly effective callus induction and regeneration
system (Ruan et al., 2009).
Formation of vascular nodules in callus cultures may represent or be
associated with an early stage of the development of shoot meristems
(Sujatha et al., 2003). They reported that the nodules containing xylem
elements in callus of Pelargonium developed into shoots when moved to an
auxin free medium. Such nodules that protrude out from the callus and leaf
buttresses, later on developed into distinct shoot or leaf primordium. It is also
reported that callus differentiation begins when peripheral meristematic
activity is replaced or supplemented by the formation of centres for cell
division deeper in the tissue.
2.4. SPECIFIC OBJECTIVES:
The present investigation aimed at the development of an indirect
micropropagation for large scale regeneration of plantlets from explants of
38 Chapter 2
mature plants with a view to cloning high alkaloid containing genotypes. A
histological study of different types of calli was also done. The main specific
objectives of this study are,
1. To develop a protocol for indirect micropropagation from the various
explants of J. adhatoda L.
1a) To study the effects of hormones, auxins and cytokinins in
callogenesis.
2. Histological analysis of organogenic and non-organogenic calli
obtained from in vitro cultures of J. adhatoda L.
2.5. MATERIALS AND METHODS
2.5.1. INDIRECT ORGANOGENESIS
2.5.1.1. Plant Material
As mentioned in chapter one (Refer 1.6.1).
2.5.1.2. Inoculation and Incubation
As mentioned in chapter one (Refer 1.6.4.)
2.5.1.3. Subculture
Sub culturing of the cultures were done after every 30 days, using
fresh medium and same culture conditions. The frequency of callus
formation was recorded as percent of the explants forming callus.
After four weeks, the callus formed was sub cultured on MS medium
containing varying concentrations of BA and Kn (Central Drug House (P)
LTD, India) for multiple shoot induction. Excised multiple shoots were
separated after six weeks and transferred to MS medium containing different
concentrations of IAA, IBA and NAA (Central Drug House (P) LTD, India)
Indirect Organogenesis and histological analysis of… 39
for root induction. The rooted plantlets were removed from culture tubes and
transferred to conical flask containing MS medium for two weeks. Then the
rooted plantlets were hardened.
2.5.1.3. Statistical Analysis
Fifty tubes each were inoculated for each hormone concentration and
each explants and this was repeated three times and average callus response
was calculated as a mean of three replicates. Data were expressed as
mean±SE for three replicates each for each hormone combination. Statistical
analysis was done by ANOVA using the statistical package INSTAT and
means were compared by Tukey-Kramer Multiple Comparisons Test.
2.5.2. HISTOLOGICAL EXAMINATION
Samples of calli were prepared for histological examination after 15
days in culture. The calli at different stages of growth after initiation (15, 30,
45, 60, 90 and 120 days) were selected. The samples were fixed in FAA 50
[formalin-acetic acid-70% ethanol (5:5:90)] (E. Merck (India) Limited),
dehydrated in a graded ethanol series (70%, 90% and 100%, three changes in
each concentrations for 30 minutes), xylene (E. Merck (India) Limited),
(three changes, each for 30 minutes) and embedded in paraffin wax (Central
Drug House (P) LTD, India) (melting point: 518–5380C). Serial sections of
10µm thickness were obtained with a rotary microtome (Reichert Jung, 2050
Super cut, Heidelberg, Germany). Sections were stretched on glass slides
previously treated with 100 mg mL-1 poly-L-Lysine (Sigma Aldrich, India),
exposed to xylene-ethanol series to remove paraffin and stained with 0.1%
safranin (Central Drug House (P) LTD, India). They were then mounted in
DPX (dibutyl phthalate xylene/ distrene polystrene xylene) (Sigma Aldrich,
India) and observed under light microscope (Olympus).
40 Chapter 2
2.6. RESULT
2.6.1. INDIRECT ORGANOGENESIS
In the present study micropropagation of the plant was attempted by
using different explants.
2.6.1.1. Callogenesis
In the present work the explants started callusing within two weeks
and callus induction was obtained from leaf, axillary bud and root tip with
different levels of IBA, NAA, IAA, BA and Kn. Different explants
responded differently to various plant growth regulator combinations (Table
2.1, 2.2 and 2.3). In J. adhatoda, leaf explants responded well to callusing.
The nature of callus developed was different when auxins in combination
with cytokinins, were added to the medium. The texture and colour of the
callus depend on the source of origin of cells and the growth regulators in the
medium. In the present study the growth regulator combination in the
medium as well as the type of the explants influenced the mass, the colour
and the texture of callus. The leaf explants showed creamish white callus in
the medium containing IBA or IAA alone (Plate 2.1). With BA and Kn alone
callus produced was loose creamish brown (Plate 2.2). But the same explants
showed green colored compact callus when added with a combination of
IAA, NAA and IBA with BA and Kn (Plate 2.3). The callus developed from
axillary bud was brown in colour, irrespective of the hormone concentration.
Friable white callus was formed from root tip explants with IBA, NAA, BA
and Kn.
Indirect Organogenesis and histological analysis of… 41
Plate 2.1 Friable creamish white callus from leaf explant
Plate 2.2 Loose creamish brown callus from axillary bud
42 Chapter 2
Plate 2.3 Green compact callus from leaf explants
Callusing of leaf explants was at its best when the medium had been
supplemented with 6 mg L-1 IAA and 6 mg L-1 Kn. Significantly higher
percentage callusing was also observed at this concentration (Table 2.1).
Axillary bud explants showed significantly higher callus response at the
hormone concentration of 3 mg L-1 IBA and 3 mg L-1 BA (Table 2.2).
Indirect Organogenesis and histological analysis of… 43
When root tip explants were used for callusing significantly higher
callus response with respect to average days for callusing and average weight
of callus was found when 3 mg L-1 IBA and 6 mg L-1 BA were added to
basal MS medium (Table 2.3).
Table 2.1. Effect of auxins and cytokinins on 50 leaf explants on MS medium after 30 days
Sl.No.
MS medium+ Phytohormones % of callus
induction
Average callus response
(50 explants in three
replicates)
Average wt. of callus in
mg/ 30 days
Average days required for
callusing Nature of callus
IBA IAA KN BA
1 5.5 - - - 57.32 28.6±1.15 195.6±12.5 24.6±0.57 Creamish white
2 6 - - - 76.00 38±2.6 204.3±8.5 23.3±0.57 ,, ,,
3 - 5.5 - - 56.00 28±2.0 170±52.8 26±1.00 ,, ,,
4 - 6 - - 76.00 38±2.64 218±9.4 23.3±1.1 ,, ,,
5 - - - 5.5 58.00 29±1.0 195.6±10.21 27±1.00 Loose
Creamish white
6 - - - 6 72.66 36.3±3.78 201.6±8.08 27±1.00 ,, ,,
7 - - 5.5 - 47.32 23.6±2.08 211±8.0 24±1.00 ,, ,,
8 - - 6 - 60.00 30.0±3.4 214±18.7 22.3±1.52 ,, ,,
9 5.5 - - 5.5 62.66 31.3±3.05 187.6±19.5 23.6±1.53 Green compact
10 5.5 - - 6 64.66 32.3±2.08 172±13.0 22.6±1.52 ,, ,,
11 6 - - 5.5 66.00 33±1.0 173±22.9 22.3±1.15 ,, ,,
12 6 - - 6 78.00 39±2.64 159.6±33.5 20.6±1.15 ,, ,,
13 5.5 - 5.5 - 41.33 20.6±1.15 172.6±20.0 25±1.00 ,, ,,
14 5.5 - 6 - 47.33 23.6±0.57 193.6±8.6 24.3±1.52 ,, ,,
15 6 - 5.5 - 69.33 34.6±4.16 189.3±17.6 23.3±0.57 ,, ,,
16 6 - 6 - 68.66 34.3±5.85 182±17.3 22.6±1.52 ,, ,,
17 5.5 5.5 48.66 24.3±4.72 203±14.0 27±2.00 ,, ,,
18 5.5 6 52.66 26.3±2.08 165±27.5 27±1.00 ,, ,,
19 6 5.5 55.32 27.6±1.52 178±27.3 17.3±1.52 ,, ,,
20 6 6 86.66 43.3±1.52 195±15.39 15.3±1.52 ,, ,,
21 5.5 5.5 47.32 23.6±0.57 195±7.3 25.6±0.57 ,, ,,
22 5.5 6 52.00 26±0.0 219.6±20.3 13.6±1.53 ,, ,,
23 6 5.5 52.00 26±2.0 232±19.46 12.3±1.52 ,, ,,
24 6 6 96.00 48±1.0 291±21.28 10.3±1.53 ,, ,,
(*P <0.0001) CD value 3.76 CD value 16.5 CD value 2.45
44 Chapter 2
Table2.2. Effect of auxins and cytokinins on 50 axillary bud explants on MS medium after 30 days
Sl.No.
MS medium + Phytohormones % of callus
induction
Average callus response
(50 explants in three replicates)
Average wt. of callus in mg/
30 days
Average days required for
callusing
Nature of callus
IBA NAA BA
1 3 - - 63 31.6±2.08 192.6±11.9 23.3±.0.57 creamish brown
2 - 3 - 40 20.3±1.5 162±6.2 24±1.0 ,, ,,
3 - - 3 80 39.6±1.5 230±30.1 23±1.0 ,, ,,
4 2 - 2 19 9.6±1.5 204.3±6.1 20±1.0 brown,soft,
friable
5 2 - 3 30 15±2.8 219±10.8 18.6±0.57 ,, ,,
6 3 - 2 41 24.6±3.0 251.3±30.2 16±1.0 ,, ,,
7 3 - 3 85 42.6±3.0 310.3±19.7 12.3±1.52 ,, ,,
8 - 2 2 31 15.6±1.5 165.3±12.2 25±1.0 ,, ,,
9 - 2 3 34 17.3±2.0 163.6±22.0 23±1.0 ,, ,,
10 - 3 2 48 24±4.3 191±11.1 22±1.0 ,, ,,
11 - 3 3 66 33.3±3.0 194.6±10.5 19±1.0 ,, ,,
(*P <0.0001) CD value 4.42 CD value 16.77 CD value 3.63
Indirect Organogenesis and histological analysis of… 45
Table 2.3 Effect of auxins and cytokinins on 50 root tip explants on MS medium after 30 days
Sl.No.
MS medium + Phytohormones % of callus
induction
Average callus response (50
explants in three replicates)
Average wt. of callus in mg/ 30
days
Average days required for
callusing
Nature of callus
IBA NAA BA KN
1 3 - - - 68.66 34.3±3.5 210±18.2 28.6±1.15 Friable white
2 - 3 - - 57.32 28.3±2.5 170±26.6 30.3±2.08 ,, ,,
3 - - 6 - 73.32 36.6±1.5 216.6±23.4 26±2.0 ,, ,,
4 - - - 6 58.0 29±1.0 173.3±12.6 28±1.0 ,, ,,
5 2 - 5.5 - 13.32 6.6±1.15 186.6±18.3 27.6±0.57 ,, ,,
6 2 - 6 - 8.66 4.3±1.15 173.6±19.5 26.6±1.15 ,, ,,
7 2.5 - 5.5 - 27.32 13.6±1.5 172.3±16.5 23±1.0 ,, ,,
8 2.5 -- 6 - 30.0 15±1.73 245.6±30.2 21±1.15 ,, ,,
9 3 - 5.5 - 63.32 31±2.08 226±13.74 17±1.52 ,, ,,
10 3 - 6 - 91.32 45±2.08 307±10.0 15±1.0 ,, ,,
11 2 - - 5.5 8.66 4.3±2.5 153±21.9 27.6±0.57 ,, ,,
12 2 - - 6 12.0 6±1.0 136±26.0 26.6±1.15 ,, ,,
13 2.5 - - 5.5 15.0 5±2.6 161±10.1 28±1.0 ,, ,,
14 2.5 - - 6 14.66 7.3±0.57 174.3±20.7 26.3±0.57 ,, ,,
15 3 - - 5.5 16.0 8±0.0 151.6±15.3 22±2.0 ,, ,,
16 3 - - 6 16.66 7.6±1.52 24.3±14.5 22.3±1.52 ,, ,,
(*P<0.0001) CD value 5.6 CD value 45.07 CD value 3.87
46 Chapter 2
2.6.1.2. Multiple Shoot Induction
When the callus obtained from leaf, axillary bud and root tip explants
were transferred to shoot inducing medium significantly higher percentage
shoot proliferation and number of total shoots per culture were observed
when the medium was supplied with 6 mg L-1 BA and 4 mg L-1 Kn (Table
2.4), (Plate 2.4). Multiple shoots with green curly leaves appeared at this
concentration. Irrespective of the source the callus obtained from leaf,
axillary bud and root tip showed the same response.
Plate 2.4-Multiple shoot induction
Indirect Organogenesis and histological analysis of… 47
Table. 2.4 Influence of BA and Kn on multiple shoot induction from 25 callus cultures of J adhatoda L. after 30 days.
Sl.No.
Growth regulators(mg/L) %Of callus showing
shoot proliferation No.of total shoots
/culture Average No. of leaves /shoot
BA Kn
1 1 5 22 2.1±0.51 2.8±0.55
2 1 6 23 2.6±0.40 3.0±0.64
3 2 5 20 3.3±0.89 3.4±0.84
4 2 6 24 4.8±1.10 3.4±0.84
5 3 5 25 2.6±0.51 3.8±0.55
6 3 6 35 4.3±0.51 4.6±0.55
7 4 5 42 4.5±1.60 4.6±0.84
8 4 6 53 6.1±0.98 4.2±0.94
9 5 1 22 5.3±0.81 2.6±0.55
10 6 1 30 6.0±1.20 2.6±0.55
11 5 2 21 5.3±0.81 3.0±0.55
12 6 2 22 5.5±0.83 3.8±0.99
13 5 3 62 7.6±0.51 4.4±0.55
14 6 3 83 8.6±1.03 5.2±0.84
15 4 4 51 3.5±0.83 3.8±0.84
16 5 4 72 6.8±0.98 4.0±0.55
17 6 4 89 11.5±1.37 5.5±0.94
(*P<0.0001) CD value 1.82 CD value 1.43
48 Chapter 2
2.6.1.3. Root induction from callus
Calli were rooted on MS medium supplemented with different
concentrations of IBA and NAA. Statistically significant rooting was
reported on MS medium with 6 mg L-1 IBA and 6 mg L-1 NAA (Figure.2.1).
Roots formed in the medium were longer and white in colour (Plate 2.5).
Plate 2.5 In vitro rooting on MS medium
0
10
20
30
40
50
60
70
80
90
100
6IBA-5IAA
6IBA-6IAA
6IBA-5NAA
6IBA-5.5NAA
6IBA-6NAA
6IAA-6NAA
% of in vitro root induction
concentration of growth regulators
Figure2.1 Influence of IAA,IBA & NAA on in vitro root induction from 25 callus cultures of J.adhatoda L after 30 days
%
Indirect Organogenesis and histological analysis of… 49
Table.2.5 Influence of IAA, IBA and NAA on in vitro root induction from 25 callus cultures of J. adhatoda L. after 30 days.
Sl.No.
Growth regulators(mg/L)
% of callus showing root
induction
No. of roots/ culture
Average length of
roots
Nature of root
IBA IAA NAA
1 4 5.5 - 26 2.2±1.30 2.3±.54 Pale,
semifine, tuberous
2 4 6 - 28 2.1±0.70 2.1±0.83 ,, ,,
3 4.5 5.5 - 36 2.3±0.83 3.1±0.70 ,, ,,
4 4.5 6 - 40 3.1±0.70 2.1±0.70 ,, ,,
5 5 5.5 - 24 2.3±0.54 2.6±1.30 ,, ,,
6 5 6 - 44 3.2±0.83 3.1±0.89 ,, ,,
7 5.5 5.5 - 36 3.2±0.54 2.8±1.30 ,, ,,
8 5.5 6 - 46 3.4±0.89 3.7±0.89 ,, ,,
9 6 5 - 54 3.0±0.83 3.2±0.84 ,, ,,
10 6 5.5 - 48 4.2±0.70 2.4±1.10 ,, ,,
11 6 6 - 56 4.6±0.54 2.3±0.83 ,, ,,
12 4 - 6 33 2.6±0.54 3.7±0.89 ,, ,,
13 4.5 - 6 26 2.3±0.83 2.8±1.30 ,, ,,
14 5 - 6 40 4.4±0.83 4.1±1.00 ,, ,,
15 5.5 - 6 30 5.4±0.70 4.0±0.71 ,, ,,
16 6 - 5 56 5.8±0.84 4.3±0.54 ,, ,,
17 6 - 5.5 78 6.0±1.20 4.4±0.84 ,, ,,
18 6 - 6 96 7.4±1.10 5.6±0.89 ,, ,,
19 - 5.5 5 24 2.9±0.70 2.2±1.10 ,, ,,
20 - 6 5 29 3.8±0.55 2.6±0.83 ,, ,,
21 - 5.5 5.5 44 4.2±0.84 2.1±1.00 ,, ,,
22 - 6 5.5 46 4.4±0.70 2.5±0.54 ,, ,,
23 - 5 6 42 3.8±0.83 2.2±0.71 ,, ,,
24 - 5.5 6 44 4.2±0.84 3.8±0.89 ,, ,,
25 - 6 6 48 4.8±0.89 3.2±0.83 ,, ,,
(*P<0.0001) CD value 1.21 CD value 1.34
50 Chapter 2
2.6.1.4. Hardening and Establishment in Pots
The plantlets were planted in poly cups containing sterilized mixture
of sand and soil, irrigated and kept under fluorescent lights, covered with
polythene bags (for maintaining humidity). 16/8h photoperiod and 25±20C
was maintained, for a week, and then transferred to field conditions. The
hardened plants when transferred to field shown 90% survival. So this
process can be adopted as an alternative to propagation through cutting.
Plate. 2.6. In vitro propagated plantlets
2.6.2 Histology
Histological examinations of calli revealed indirect development of few
shoots and no evidence of somatic embryogenesis was found. During the early
stages of callus formation, the parenchyma cells of the mesophyll tissue near the
vascular bundles produced an undifferentiated mass of cells which is called
primary callus (Plate 2.7a). Primary callus underwent division and produced
large parenchymatous cells as derivatives while the initials appeared as darkly
stained clumps (Plate 2.7b). Narrow elongated cells formed procambium and it
developed into distinct vascular elements, especially tracheids (Plate 2.7c).
Vascular nodules were formed (Plate 2.7d). Meristemoid regions were seen
which is characterized with densely stained small cells. On culturing on the
shooting medium from vascular nodules small buds with the tunica corpus
organization of a shoot apical meristem was developed (Plate 2.7e).
Indirect Organogenesis and histological analysis of… 51
Plate 2.7a.Callus with undifferentiated cells Plate 2.7b. Callus with initials
(Darkly stained)
Plate 2.7c Procambium develops into Plate 2.7d Callus with vascular
distinct vascular elements nodules
Plate 2.7e Callus with shoot primordia
52 Chapter 2
2.7. DISCUSSION
It is well known that auxins and cytokinins are effective for callus
and organ formation in tissue culture of many plants (Yakauwa and Harada,
1982). It was clear from the study that the leaf explants showed maximum
callus response (Table 2.1). On increasing the concentrations of growth
regulators gradual increase in percentage of cultures forming callus was
noticed in all cases upto the optimum concentration (Faisal and Anis, 2003).
The nature of callus developed was different when auxins in
combination with cytokinins, were added to the medium. The texture and
colour of the callus depend on the source of origin of cells and the growth
regulators in the medium. In the present study the growth regulator
combination in the medium as well as the type of the explants influenced the
mass, the colour and the texture of callus (Plate 2.1, 2.2 and 2.3).
The histological analysis of the regenerating calli clearly showed that
the shoot buds had emerged from the peripheral nodular structures, which
consisted of closely arranged and highly cytoplasmic cells (Plate2.7a-e). In
some shoots the vascular supply was found to be continuous with the
vasculature of the callus (Thomas and Puthur, 2004).
The division and growth of callus cells continued for some time
resulting in the enlargement of primary callus. According to Sujatha
et al.,(2003) in a root or shoot apex, certain cells of the meristems undergo
divisions in such a way that, one product of a division becomes a new body
cell, called derivative and the other remains in the meristem, called initials. A
similar pattern of meristematic activity was observed in J. adhatoda callus.
Callus cultures contain vascular nodules which comprises vascular elements
and parenchymatous cells. The organogenic and non-organogenic calli are
Indirect Organogenesis and histological analysis of… 53
the result of hormonal variation in the medium. In non-organogenic callus,
cells redifferentiated into xylem elements forming clusters of nest like
structures. In organogenic callus, the undifferentiated cells of callus were
found to differentiate into vascular nodules called meristemoids, which then
develop into xylem elements, especially tracheids. On culturing in the
shooting medium, these nodules differentiated into shoot apical meristem.
The detailed histological analysis shows that the shoots regenerated
from the leaf derived callus of J. adhatoda have no organized cellular
connection with the original explant tissue, indicating an adventitious origin
and hence, chances of genetic variability among the regenerants.
White-friable calli and green-compact calli had similar histological
structures. Shape and sizes of cells, which was composed of these tissues
varied greatly. Compared with the loose morphology outside of calli, cells
inside calli were compact relatively and developed some intercellular spaces.
These two types of calli also showed similar features in ultra structure
(Plate2.7a-e). Result of the histological study showed that cells composed of
overgrown calli had similar morphology. Moreover a structure of a series of
cell clusters could be observed.