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).


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)


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.


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).


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


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


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


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


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

%


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).


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


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.

Chapter 2 Indirect Organogenesis and histological...

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