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NSave Nature to Survive

ISSN: 0973 - 7049

: Special issue, Vol. 2;

Paper presented in International Conference onEnvironment, Energy and Development (from

Stockholm to Copenhagen and beyond)December 10 - 12, 2010, Sambalpur University

IN-VITRO PROPAGATION AND CALLUS FORMATION FROM

SEEDLING OF TERMINALIA ARJUNA ROXB

Chandan Kumar et al.

Terminalia arjuna Callus

Explant

Phytohormones

IBA, NAA, IAA

423-429; 2010

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NSave Nature to Survive

ABSTRACT

The explant of Terminalia arjuna Roxb., a food plant of tasar silkworm (Anteraea mylitta D.)

was cultured on Murashige and Skoog (MS) media supplemented with different concentrations of

6-Benzyalaminopurine (BAP), Indole 3- butyric acid (IBA), Nepthlene acetic acid (NAA), 2,4-

dichlorophenoxy acetic acid (2,4-D) ,Gibberellic acid (GA3), Thidiazuron, Kinetin, TDZ

in

solution and also in combination. The shoot explant in MS media supplemented with Kin., 2,4-

D and GA3 using the concentration from 1-5mg/L developed along with green calli. The nodal

explants was also cultured on MS media supplemented with Thidiazuron (0.5, 1.0 and 2.0mg/

L), BAP (2.0, 3.0 and 4.0mg/L), Kinetin (0.5 and 1.0mg/L), Riboflavin (0.5-1.0mg/L) and

Bio. (0.1 and 0.5mg/L) resulted early bud breakage (6-8days) and shoot proliferation. In T.

arjuna the regeneration process is very slow and it depends upon the explants. The percentage of

responded explant was significant in callus and shoot proliferation.

CHANDAN KUMAR*, AJIT KUMAR SINGH1 AND SHAMSHUN NEHAR

Department of Zoology, Ranchi University, Ranchi - 834 0081Central Tasar Research and Training Institute, Nagri, Ranchi

E-mail: chandanctri@gmail.com

*Corresponding author

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INTRODUCTION

Sericulture is an art of culture or production of silk (queen of textile) which involves host plants. Tasar silk

is one of the natural silk produced by Tasar silkworm (Antheraea mylitta D.) which is reared on three major

food plants viz. Terminalia arjuna, T. tomentosa and Shorea robusta. T. arjuna belongs to Combretaceae

family and is commonly known as Arjun plant. It is a large size deciduous evergreen plant with yellow

flower, conical leaves, dark brown bark and large fruit with hard wings striated with numerous curved

veins. The plant is found in plenty throughout Indo sub Himalayan tracts, Uttar Pradesh, Jharkhand, South

Bihar, Madhya Pradesh, Delhi, Deccan region mainly along riverside, riverlets and ponds. In addition to

timber and other important products such as tannin and oil, it has also therapeutic values. It is an important

cardiotonic plant described in the Ayurveda (Tripathi et al., 1996). The bark extract contains acids (Arjunic

acid, Terminic acid), glycosides (argentine arjunosides I-IV), strong antioxidants (flavones, tannins, oligomeric

proanthocyanidins) and minerals etc. The pharmacological studies have shown that bark of T. arjuna has

antiviral (Kusumoto et al., 1995) anti mutagenic (Kaur et al., 2001) antiplague (Shaila et al., 1997a and b),

anticancer (Avinash et al., 2000) and hypotensive (Takahashi et al., 1997) properties.

Terminalia arjuna is propagated either through seeds or through cuttings. Propagation through cuttings has

additional advantage of by passing the dormancy and juvenile stage besides maintaining genetically stability.

On the other hand, propagation through seeds results into variation in the seedlings because the primary food

plants are cross-pollinated. To overcome these problems large-scale plantation is the only alternative to

thrive by tissue culture method. However, through traditional technique some breeding programmes have

been undertaken by large number of scientists with a little result. Since traditional techniques have its own

limitation, the biotechnological methods specially tissue culture technique could be utilized as an alternate

effective tool to increase biomass and improves the quality of host plants. Very little literature of Terminalia

arjuna micropropagation is available though some attempts are made on micropropagation of Terminalia

arjuna by Ramesh et al., 2001; Tirkey et al., 2000; Nishi et al., 1998; Priyaranjan et al., 1994. Some of the

scientists (Nishi et al., 1998; Ramesh et al., 2000) have been worked on separated tissue of Terminalia

arjuna but there was no response on insect reared tasar food plants. However, Tirkey et al. (1999, 2000)

reported for somatic embryogenesis of some selected genotypes, effect of antioxidant and absorbent in

Terminalia arjuna. In-vitro genotypic response and effect of BAP and Kinetin for callus culture of T. arjuna

palatable to silkworm has been reported by Tirkey et al., 2003; Priyaranjan et al., 1994. The aim of the

present study was to optimize conditions for morphogenesis from upper parts of Terminalia arjuna for

subsequent usage in various in-vitro development progress and to possibilities for reproducible results for

further study.

MATERIALS AND METHODS

Culture material

For the present study the healthy and uniform seeds were collected from CTR and TI, Ranchi during the

month of March –May. Seeds were socked in plane water for 96 hr and kept in the mixture of sterile soil and

sand (1:1) in the month of Aug. – Sept. for seedling. After that shoot, apical bud and leaf were collected

from 10-12 days old seedling. The selected explants were cut into 2-3 cm long segments after washing with

tap water for half an hour and were treated with 2-3 drops of Cetrimide solution for 10-12 minutes. Afterwards

these were soaked in 0.1% Bavistine (fungicide solution) along with few drops of Cetrimide solution for 30

minutes. Explants were again washed in sterilized distilled water (SDW) and were soaked in 70% ethanol

for 30 seconds and were agitated with few drops of Cetrimide solution for 30 min successively. Explants

were then again washed in SDW (at least 3-4 times) and agitated in 25 % bleach solution containing 2-3

drops of Cetrimide for 20 minutes. Finally, explants were washed in SDW (again, at least 3-4 times or until

all traces of detergent was removed). After the necessary treatment the basal part of the explant was

IN-VITRO PROPAGATION OF TERMINALIA ARJUNA

426

remove with the help of sharp scalpel and then implanted vertically in MS media with various concentrations

of phytohormones.

Culture media

The culture medium consisted Murashige and Skoog’s media with 3% sucrose and was supplemented with

1-5 mg/L of 6-Benzyalaminopurine (BAP), 0.5 – 1.0 mg/L, Kinetine, 0.5 – 2.0 mg/L, Nepthlene acetic acid

(NAA), 1-1.5 mg/L, Indole 3- butyric acid (IBA) and 1-5 mg/L Gibberellic acid (GA3)

either individually or

combinations. Chemicals used for media were of analytical grade (Merck and Sigma). Series of concentrated

stock solutions of inorganic salts, iron source, vitamins and amino acids were prepared in advance and kept

under refrigeration and used during media preparation. Media contain macro elements like Ca, Mg, N, P,

K, S and micro elements like Fe, B, Cu, Co, Mn, Mo, Zn, I, Ni, Al and Si. After homogenization of media

the pH was adjusted to 5.7 prior to adding agar and was autoclaved at 121ºC. The cultures were maintained

as 26 ± 2ºC under 16 hr/ day photoperiod.

The media was made semi solid with the addition of 0.8% Difco – Bacto – Agar. The pH of the medium was

adjusted to 5.8 using 0.1 N NaOH or 0.1 N HCl before autoclaving. About 20 mL of hot medium was

dispensed in to each culture test tube (15.0x2.5cm). The test tube or flasks were plugged with non-absorbent

cotton wrapped in cheesecloth. The culture vessel was autoclaved at 1.4Kg/cm2 for 20 minutes.

Culture conditions

The culture was maintained in diffuse light (1000-2000 Lux) at 28± 2ºC and 50-60% relative humidity.

Culture was incubated at 26+2ºC under 16hr photoperiod and 2000 lux with florescent tubes. All experiments

were carried out under sterile condition using laminar air flow chamber. Petridishes used during surface

sterilization of explants and other inoculation tools like forceps, needle, scissor, blade and scalpel were

thoroughly autoclaved. Culture area, glass wares and all tools used were properly sterilized with UV light

under and floor surface was swabbed with 95% ethyl alcohol. Inoculation tools were flamed time to time to

avoid contamination. The cultures were incubated in darkness and the explants were frequently transferred

on a fresh medium whenever browning of the medium was observed (Broom and Zimmermann 1978, 1984;

Litz, 1984).

RESULTS AND DISCUSSION

After 40 days of culture in MS media supplemented with Kin., 2,4-D and GA3 (1-5mg/L) shoot was developed

along with green calli and this green calli produced from the smaller segments of hypocotyls (Fig. 1). The

ability of callus to differentiate into reproductive and vegetative buds depends upon the origin of explant/

culture condition. The higher concentrations of auxin accelerate vegetative bud differentiation. The callus,

which were sub cultured for prolonged period, the regeneration capacity of the callus greatly reduced, but

the addition of IAA, GA3 (1-3mg/L) and Coconut water (20%) increased the capacity of callus development.

The effect of c.water (20%) in combination with 2, 4-D or NAA enhance growth of the callus. Reducing the

concentration of 2, 4-D and omitting cytokinin induced the somatic embryogenesis. Embryos so developed

were isolated from the culture in a medium containing IBA and GA3 for further regeneration to achieve the

plantlets. Callus formation and subsequent organogenesis is affected by the concentration of 2, 4-D ranging

from 0.5-10mg/L. Although, callus formation was observed with concentration as low as 0.5mg/L but the

most effective concentration was found to be 5mg/L (Fig. 2). The higher concentration of 2, 4-D (10mg/L)

and above had toxic effect. The observation indicate that the high concentration of cytokinines (BAP 0.5-

2mg/L) and low auxin (NAA and IBA 0.1-0.5mg/L) results in shoot elongation to about 3-4cm long after 3

weeks. In 3-4mg/L of IAA, the development was very good, with poor or scanty growth of embryo in MS

medium. High dose of NAA, IAA, 2, 4-D (8-10mg/L) applied to T. arjuna stem explants had lethal effect.

Apical bud explants of T. arjuna were cultured on MS media supplemented with different concentration of

BAP and Kin by Tirkey et al., (1999) and the shoot proliferation were achieved. Ramesh et al., (2000)

reported shoot induction and proliferation from mature tree of T. arjuna on TDZ (0.5mg/L) fortified MS

medium. The performance of TDZ as compared to BAP and Kin was found much better. The significant

CHANDAN KUMAR et al.,

427

IN-VITRO PROPAGATION OF TERMINALIA ARJUNA

Figure 3: In-vitro regenerated plantlets of T. arjuna in MS

media with BAP and NAA

Figure 4: Shoot bud break in MS media with Kin 0.5mg/

L) and BAP (3mg/L) in T. arjuna

Figure 1: Callus development from explant of T. arjunaFigure 2: Callus development from the cut ends of T. arjuna

in MS media

result was also observed in shoot proliferation of T. arjuna produced from seedling nodal and apical bud

explant on a medium contain NAA (0.5mg/L), BAP (3mg/L) with C. water (Fig. 3). Rooting was observed

on a medium containing NAA, IBA and Kin (0.1-3mg/L). In the Terminalia species BAP and Adenine

sulphate is most important for enhancing the frequency of embryogenesis as well as shoot bud differentiation.

The nodal explants of T. arjuna was also cultured on MS media supplemented with Thidiazuron (0.5, 1.0 and

2.0mg/L) BAP (2.0, 3.0 and 4.0mg/L), Kinetin (0.5 and 1.0mg/L), Riboflavin (0.5-1.0mg/L) and Bio (0.1 and

0.5mg/L). In all the concentration, early bud breakage (6-8days) and shoot proliferation were observed (Fig.

4). TDZ (0.5mg/L) and BAP (3.0mg/L) were found to be effective in inducing shoot formation. However,

single shoot emerged in case when nodal segment with only one axillary bud was cultured. High concentration

of TDZ (1.0mg/L) resulted in both axillary and adventitious shoot induction. The different concentrations of

IBA and NAA in MS media were effective in inducing root formation from the shoots. Tirkey et al., (1999)

studied the effect of anti-oxidant and absorbent on tissue browning metabolism in T. arjuna nodal explant.

Phenolic exudation in the explants/ cultures was controlled by adding citric acid (10mg/L), Pvp (50-150mg/

L) and ascorbic acid (50-100mg/L) to the media. The significant result was also observed in shoot proliferation

in T. arjuna produced from seedling nodal and apical bud explant on a medium contain NAA (0.5mg/L), BAP

(3mg/L) with c.water. Rooting was observed in the medium containing NAA, IBA and Kin (0.1-3mg/L). In

the present study, upper hypocotyl with shoot apices was observed to be the best explant for plant regeneration.

The exogenous growth regulators required for callus and morphogenic response depends upon the genotype,

the physiological age of the explant and endogenous hormone contents of the tissue (Skoog and Miller, 1957).

The slight decrease and increase in strength of growth regulators gave varied results during work. For

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callus formation and organogenesis 2, 4-D is widely used as an auxin but can not stimulated by IAA for the

growth of a de-differentiated tissue, even in the appropriate ratio of the cytokinine. 2, 4-D usually can not

be replaced by IAA for rooting of explant(Fig.5). In case of T. arjuna, IBA and NAA (1-2.5mg/L) are much

more effective than IAA. By increasing the cytokinine (kin.) and lowring the auxin (NAA) concentration,

the ability to differentiate reproductive and vegetative bud was decreased during several passage of culture.

Auxillary buds and nodal explants of T. arjuna from 25 yr old plant could be induced to form shoots when

cultured in MS media supplemented with BAP in addition to Adenine sulphate in presence of antioxidant and

absorbent. The rhizogenesis in Terminalia species was found to be difficult and require as much more period

of longer in-vitro incubation. Terminalia species is biologically significant system as it forms callus but its

regeneration potential is very low even after two to three years of repeat sub-culturing.

ACKNOWLEDGEMENT

Authors are thankful to Tissue culture division, CTR and TI, Ranchi for providing laboratory facilities.

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