Leaf Callus Induction of Dregea Volubilis

7/27/2019 Leaf Callus Induction of Dregea Volubilis

1/5

Yogananth et al.

JOURNAL OF BIOSCIENCES RESEARCH 3(3):198-202 198

J. B io s c i. Re s .,2012.Vol. 3(3):198-202

Effect of different plant hormones on callus induction in Leaf explant ofDre g e a v o lub i li s Benth. (Asclepiadaceae)

YOGANANTH, N1*, PALANIVEL, S2, PARVATHY, S3, CHANTHURU, A4.

AND BHAKYARAJ, R,51Mohamed Sathak Arts and Science College, Chennai - 600034, Tamil Nadu, India.

2 , 3, Dept of Botany, Govt Arts College, Karur, Tamil Nadu, India4JJ College of Arts and Science, Pudukkottai, Tamil Nadu, India

5 Indian Institute Crop Processing Technology, Thanjavur, Tamil Nadu, India

Abstract

Dregea volubilis is a common shrub plant belonging to the familyAsclepiadaceae. Theleaf juice was anti-inflammatory medicine to treat several diseases including eye ailments,

tracheitis and stomachache. The present work is based on developing a protocol for the

callus induction in Dregea volubilisfrom leaf explants. The sterilized explants were inoculated

in MS media containing various combination of auxins such as Indole acetic acid (IAA),

naphthalene acetic acid (NAA) and 2,4- dichlorophenoxy acetic acid (2, 4-D) and cytokinins

such as kinetin and 6 benzyl amino purine (BAP). The highest efficiency of callus

formation was observed in the medium containing different concentration of 2, 4-D and

kinetin. In vitro generated callus can be used as a source for the isolation of secondary

metabolites from D. volubilis.

Key words: Dregea volubilis, Perukurunchan, callus induction, auxin, cytokinin

For correspondence:[email protected]

Introduction

Dregea volubiliswhich is commonly known

as Perukurunchan in Tamil, is an important

medicinal woody climber belonging to the

family Asclepiadaceae. It is widely used in

Indian traditional medicines and the leaf

paste to treat rheumatic pain, cough, fever

and severe cold (Muthu et al., 2006 and

Rajadurai et al., 2009) leaf paste is takenalong with pepper to treat dyspepsia

(Pandikumar et al., 2007); bark paste, mixed

with hot milk is used internally for treating

urinary troubles (Silija et al., 2008) and leaf

powder is taken orally along with cows milk

have antidiabetic activity (Ayyanar et al.,

2008). The stems and leaves contain a

pigment taraxerol, a triterpenoid,

kaempferol, a glycoside of kaempferol and

saponins (Sauer et al., 1965).

Due to overexploitation and misuse

of medicinal plants, we are faced with the

problem of losing our precious plant

resource in the future. This situation calls

for effective and in time conservation

measures to enrich our lives with theservices of plants. In this regard, Vinod et

al., (2003) stressed the need of conservation

and sustainable utilization of biodiversity.

Different techniques for conservation of

plants have been practiced worldwide, the

most important being tissue culture (Parabia

et al., 2007). Tissue culture is advantageous

ISSN 0976-2272


2/5

Yogananth et al.


in producing multiple copies of a plant

species within minimum time and space.

Various studies have been carried

out on the biochemical (Jeyachandran et al.,

2010 and Maruthupandian and Mohan,

2011), Pharmacological (Thomas et al, 1996;

Biswas et al., 2010, Maruthupandian et al.,2010) and in vitro propagation (Arulanandam

et al., 2011, Vinothkumar et al., 2011,

Yogananth et al., 2011) aspects of Dregea

volubilis, but no such in vitro callus culture

from leaf explants studies have been carried

out in this valuable medicinal plant. The

objectives of this study was to investigate

the influence of plant growth regulators on

induction and growth of D. volubilis callus

culture as a starting point to producebioactive compounds in plant cell culture.

Materials and methods

Young leaves were collected from

mature field grown healthy plant of Dregea

volubilis maintained in green house at J.J.

College of Arts and Science, Pudukkottai,

Tamil Nadu, India and washed thoroughly

under running tap water and then treated

with a few drops of Tween-80 and 1%

Savlon for 10 minutes with constant

shaking. This is followed by successivethree washing with distilled water to make

the material free from savlon. Again the

explants were washed with 70% ethyl

alcohol for few seconds and washed with

distilled water for 3-4 times. After that, the

explants were transferred to laminar air flow

chamber and disinfected with 0.1% HgCl2

for 2 minutes and washed with sterile

distilled water for 5-7 times. Then, the

explants were placed in sterile Petri platesbefore inoculation. The sterilized explants

were injured all over the surface and used

for callus induction.

The excised explants were cultured

on MS (Murashige and Skoog, 1962)

medium augmented with different

concentrations of auxins like IAA, NAA

and 2, 4-D (1.0, 1.5, 2.0, 2.5 and 3.0 mg/l)

along with cytokinins like KIN or BAP (0.5

mg/l) , 3% sucrose and 0.8 to 1% agar with

pH adjusted to 5.8 before the addition of

agar. Culture tubes containing medium were

autoclaved at 121C for 15 lbs/inch2 for 15

min. All the inoculated cultures wereincubated in growth room in controlled

conditions at a temperature of 25 2C, 16

h light/8 h dark photoperiod and

continuous illumination was provided by

cool white fluorescent tubes at 2000 lux.

Each experiment was repeated thrice.

Analysis of variance was carried out and the

differences between the treatments were

determined by DMRT at 5% level of

significance using SPSS (SPSS ver. 16.0).Results and Discussion

Leaf pieces were used as a primary

explants for callus induction. Callus

initiation was observed from cut surface of

leaves after 3 to 4 week of culture initiation.

The leaf explants responded differently

based on the concentrations of auxins and

cytokinin present in the medium. In general,

media containing high auxin and low

cytokinin concentrations promote cell

proliferation resulting in callus formation(Slater et al., 2003).

Plate 1: In v itro callus induction from

leaf explant ofD. vo lub i li s Stage wise

development in 2, 4-D + KIN

concentration

A B C

Callus induction in earlier stage


3/5

Yogananth et al.


D E

Callus in matured stage

Maximum amount of callusing

(81.58 1.36 %) was observed on the

medium supplemented with combination of

2, 4-D and Kin (2.0 mg/l 2, 4-D, and 0.5

mg/l KIN) after 3 weeks of culture

initiation (Fig 3 and Plate E). Minimum

response(54.40 + 0.99 %) was obtained in

NAA 1.0 mg/l and BAP 0.50 mg/l

combination (Fig 2). All the calli derived

from leaf explants were pale green and

friable in nature (Plate 1, 2, and 3). The

results obtained were agreement with the

previous reports by other investigators,

Gymnema sylvestre (Gopi and Vatsala, 2006;

Royet al., 2008) and Ceropegia juncea(Nikam

and Savant, 2009).FIG. 1: EFFECT OF IAA ON CALLUS INDUCTION, CALLUS GROWTH OF YOUNG STEM

EXPLANTS OF Dregea volubilis (L.f.) IN COMBINATION WITH KIN/BAP.

0

0.5

1

1.5

2

2.5

3

3.5

4

1 2 3 4 5 6 7 8 9 10

Conc of IAAwith KIN and BAP

FW/DW

FRESH WEIGHT

DRY WEIGHT

The maximum growth rate in terms

of fresh weight (3.56 0.44 g) and dry

weight (0.33 0.03 g) was observed in the

combination of 2, 4-D 2.5 mg/l and KIN

0.5 mg/l. Minimum growth rate 1.19 + 0.34

g fresh weight and 0.10 + 0.02 g dry weight

was obtained in 1.0 mg/l NAA and 0.5 mg/l

KIN combination.

FIG. 2: INFLUENCE OF NAA ON CALLUS INDUCTION, CALLUS GROWTH OF YOUNG STEM

EXPLANTS IN COMBINATION WITH KIN AND BAP

0

0.5

1

1.5

2

2.5

3

3.5

1 2 3 4 5 6 7 8 9 10

Conc of NAA with KIN and BAP

FW/DW

FRESH WEIGHT

DRY WEIGHT

Among the various concentrations

of auxins tested, 2, 4-D with KIN was more

effective for callus induction than IAA (Fig1) and NAA as a source of auxin in leaf

explants tested. George, (1996) reported

that the 2,4-D shows effect on the RNA

metabolism by inducing the transcription of

messenger RNA capable of coding proteins

required for the growth and hence,

promoting a chaotic cell proliferation, i.e.,

callus formation. In vitro generation of callus

can encourage in vitro mass production of

bioactive compounds of health benefitsfrom Dregea volubilisplant.

FIG. 6: ROLE OF 2,4-D ON CALLUS INDUCTION, CALLUS GROWTH OF YOUNG LEAF

EXPLANTS IN COMBINATION WITH KIN AND BAP

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

1 2 3 4 5 6 7 8 9 10

Conc of 2,4-D with KIN and BAP

FW/DW

FRESH WEIGHT

DRY WEIGHT

References

Arulanandam, L.J.P., Kumar, S.G. and

Sovimini, M. 2011. Micropropagation and

conservation of Medicinal plant Wattakaka


4/5

Yogananth et al.


volubilis Linn. (Stapf). Indian J Biotechnol.

10: 238-241.

Ayyanar, M., Sankara Sivaraman, K. and

Ignacimuthu, S. 2008. Traditional Herbal

Medicines used for the treatment of

Diabetes among two major tribal groups

in South Tamil Nadu, India. EthnoBotanical Leaflet. 47: 389-394.

Biswas, M., Mandal, N.B., Palit, P., Ghosh

A.K., Bannerjee, S. and Haldar Pallab.,K.

2009. In vitro Anti-Leishmanial and Anti-

Tumour Activities of a Pentacyclic

Triterpenoid Compound Isolated from

the Fruits ofDregea volubilisBenth. Tropical

Journal of Pharmaceutical Research, 8 (2): 127-

131.

George, E.F. 1996. Plant propagation bytissue culture: part 1: the technology.

Edington: Exegetics, 574 p.

Gopi, C. and Vatsala, T.M. 2006. In vitro

studies on effects of plant growth

regulators on callus and suspension

culture biomass yield from Gymnema

sylvestreR.Br. African Journal of Biotechnology.

5(12): 1215-1219.

Jeyachandran, R., Baskaran, X. and

Cindrella, L. 2010. Screening of

Phytochemical and Antibacterial Potentialof Four Indian Medicinal Plants. Libyan

Agriculture Research Center Journal Internation.

1 (5): 301-306.

Maruthupandian, A., Mohan, V.R. and

Sampathraj, R. 2010. Antidiabetic,

antihyperlipidaemic and antioxidant

activity of Wattakaka volubilis (l. F) stapf

leaves in alloxan induced diabetic rats.

International Journal of Pharmaceutical Sciences

and Research. 1 (11): 83-90.Maruthupandian, A. and Mohan, V.R. 2011.

GC-MS analysis of ethanol extract of

Wattakaka volubilis (l.f.) stapf. Leaf.

International Journal of Phytomedicine. 3: 59-

62.

Muthu, C., Ayyanar, M., Raja, N. and

Ignacimuthu, S. 2006. Medicinal plants

used by traditional healers in

Kancheepuram District of Tamil Nadu,

India. Journal of Ethnobiology and

Ethnomedicine. 2: 43-52.

Nikam, T.D. and Savant, R.S. 2009. Multiple

shoot regeneration and alkaloid cerpegin

accumulation in callus culture ofCeropegiajuncea Roxb. Physiology and Molecular

BiologyofPlants. 15(1): 71-77.

Pandikumar, P., Ayyanar, M. and

Ignacimuthu, S. 2007. Medicinal plants

used by Malasar tribes of Coimbatore

district, Tamil Nadu. Indian Journal of

Traditional Knowledge. 6: 579-582.

Parabia, F.M., Gami, B., Kothari, I.L.,

Mohan, J.S.S. and Parabia, M.H. 2007.

Effect of plant growth regulators on invitro morphogenesis ofLeptadenia reticulata

(Retz.) W. and A. from nodal explants.

Curr. Sci., 92(9): 1290-1293.

Rajadurai, M., Vidhya, V., Ramya, M. and

Bhaskar, A. 2009. Ethno-medicinal plants

used by the traditional healers of

Pachamalai hills, Tamilnadu, India.

Ethnomedicine. 3: 39-41.

Roy, A., Ghosh, S., Chaudhuri, M. and Saha,

P.K. 2008. Effect of different plant

hormones on callus induction in Gymnemasylvestris R.Br. (Asclepiadaceae). African

Journal of Biotechnology. 7 (13): 2209-2211.

Sauer, H.H., Weiss, E. and Reichstein, T.

1965. Structure of drevogenins Engl.

summ. Helvetica Chimica Acta. 48: 857-878.

Silija, V.P., Samitha Varma, K. and

Mohanan, K.V. 2008. Ethnomedicinal

plant knowledge of the Mullukuruma tribe

of Wayanad district, Kerala. Indian Journal

of Traditional Knowledge. 7: 604-612.Slater, A., Scotee, N. and Wand-Fowler,

M.R. 2003. Plant Biotechnology (The

Genetic Manipulation of Plants). Oxford

University Press.

Thomas, M., Hepsibah, P.T.A., Prasad,

N.B.R. and Sanjeev Kumar, P. 1996.

Pharmacognostical and clinical studies on


5/5

Yogananth et al.


Wattakaka volubilis (Linn.f.). Ancient

Science of Life 15, 4, 277281.

Vinod, A., Shekhawat, N.S. and Singh, R.P.

2003. Micropropagation of Leptadenia

reticulata: A medicinal plant. In vitro cell Dev.

Biol., 39(2): 180-185.

Vinothkumar, D., Murugavelu, S. andSenthikumar, M. 2011.Clonal propagation

of Wattakaka volubilis through nodal

explant culture. Cey. J. Sci. (Bio. Sci).40 (1):

53-58.

Yogananth, N.,Bhakyaraj, R., Chanthuru, A.

and Palanivel, S. 2011. Invitro regeneration

and conservation of rare medicinal plant

Dregea volubilis Benth. Journal of PlantSciences. 6 (6): 225-228.

Documents

Leaf Callus Induction of Dregea Volubilis