Indian Journal of Biotechnology

Vol 15, April 2016, pp 269-271

Somaclonal variation studies in Orthosiphon

stamineus (Benth.) using SDS-PAGE

M Narayani, M Johnson*, A Babu,

T Renisheya Joy Jeba Malar and N Janakiraman

Centre for Plant Biotechnology, Department of Botany,

St. Xavier’s College (Autonomous), Palayamkottai 627002, India

Received 25 September 2015; revised 9 April 2015;

accepted 17 May 2015

The present investigation was intended to reveal somaclonal

variation in Orthosiphon stamineus Benth. using SDS-PAGE as a

tool. For electrophoretic studies, the proteins were isolated from

mother plants, in vitro plantlets raised through nodal segments and

plantlets regenerated from the leaf-derived calli of Orthosiphon

stamineus, and separated using SDS-PAGE. Mother plants and

nodal-segment derived plantlets were confirmed for their genetic

uniformity by expressing similar banding pattern in the gel system.

The proteins PP12 (0.08), PP22 (0.15), PP32 (0.25) and PP51 (0.41)

showed their unique presence in plantlets regenerated from leaf

derived calli and confirmed the occurrence of somoclonal variants.

These protein profiles would be used as a biochemical marker for

plant breeding or genetic improvement programme in future to

identify mother plants and somaclonal variants of O. stamineus

Key words: Biochemical marker, in vitro plantlet, plant breeding,

SDS-PAGE, somaclonal variation

Orthosiphon stamineus (Benth.) is a multipotent herb

(Lamiaceae) and used commonly in Southeast Asia

and European countries for herbal tea, well known as

“Java tea”1. Leaves of O. stamineus have been used in

various ailments and disorders as antioxidant,

hepatoprotective2, antibacterial

3, anti-inflammatory

4,

diuretic and hypoglycemic5. Due to its medicinal

importance, the scientist made attempts to multiply

O. stamineus plants and few reports are available on

the direct and indirect mode of micropropagation6-9

.

However, no information is available on the

somaclonal variation studies on this medicinally

important plant. SDS-PAGE protein profiles and

isoperoxidase profiles are used as tools to identify

somoclonal variants from true to type mother plants

and to classify inter and intra specific variation of

plants10,11

. The present investigation was, therefore,

intended to identify the somaclonal variations of

O. stamineus (Benth.) using SDS-PAGE.

For the electrophoretic studies, the proteins were

isolated from the young leaves of mother plant,

in vitro plantlet raised through the nodal segments and

plantlets regenerated from the leaf-derived calli of

O. stamineus, and separated using SDS-PAGE12

.

After electrophoresis, the gels were stained and the

banding patterns were observed using a Vilber

Loubermat gel documentation system. Then

zymogram was constructed based on the banding

profiles in the gel system. For identification of

somaclonal variants, genetic similarities (GS) were

estimated according to Nei and Li13

.

A total of 19 proteins were expressed (mother

plant, 6; in vitro plantlet raised through nodal

segments, 6; and plantlets regenerated from leaves

derived calli, 7) in the gel system (Table 1). The

proteins PP12 (0.08), PP2

2 (0.15), PP3

2 (0.25) and

PP51 (0.41) showed their unique presence in the

plantlets regenerated from leaf derived calli of

O. stamineus and confirmed the occurrence of

*Author for correspondence:

Tel: +91-9786 924334; Fax: +91-462-2561765

ptcjohnson@gmail.com

Table 1—SDS-PAGE Protein profile of O. stamineus

O. stamineus* MW-Rf Region Position

M N C

0.05 PP11 + +

0.08

1

PP12 +

0.11 PP21 + +

0.15 PP22 +

0.17

2

PP23 + + +

0.22 PP31 + + +

0.25 PP32 +

0.26

3

PP33 + +

0.3 PP41 +

0.37

4

PP42 + +

0.41 PP51 +

0.42 PP52 + +

0.47

5

PP53 + + +

0.65 7 PP71 + + +

0.85 9 PP91 + + +

*M, Leaf of mother plant; N, Leaf of nodal segment derived

plantlet; C, Leaf callus derived plantlets

INDIAN J BIOTECHNOL, APRIL 2016

270

somoclonal variants. Based on the banding pattern of

mother plant, in vitro plantlet raised from the nodal

explants and the calli mediated plantlets, the

zymogram was constructed using MS-Excel (Fig. 1).

The cladogram of O. stamineus displayed two major

clusters (C1 & C2). The cluster C1 showed the genetic

uniformity between the mother plant and nodal

segment derived plantlets of O. stamineus. However,

the cluster C2 showed the genetic variation of in vitro

plantlet regenerated from leaves derived calli and thus

confirms the occurrence of somaclonal variation in

the plantlet regenerated from the leaves derived calli

of O. stamineus (Fig. 2).

In vitro regeneration habitually end results with high

genetic and phenotypic variability in individuals

derived from plant tissue cultures or adventitious

shoots, which is called somatic variation or somaclonal

variation14

. Somaclonal variability frequently occurs in

tissue culture due to epigenetic or modifications in the

genome of differentiating vegetative cells induced by

tissue culture conditions15

. Previous studies on

somaclonal variation revealed that cell or tissue

cultures go through emblematic genetic alterations,

modifications in chromosome number (polyploidy &

aneuploidy), chromosomal structure (deletion,

translocations & duplication) and DNA sequence (base

mutations)16

. The genomic wavering takes place due to

chromosomal reorganization, chromatid interactions

and gene amplification. For proper identification of

genuine herbal plant material and for checking

adulterants, biochemical and molecular markers are of

great value. These can go a long way for

standardization of herbal formulation.

SDS-PAGE is the most economical, simple and

extensively used biochemical technique for analysis of

genetic structure of germplasm17

. The expression of

some proteins and enzymes is affected by growth

stage17-18

. The results of the present study also confirm

the proteomic variations in the cultured plantlets. These

protein profile could be used as a biochemical marker

for the future plant breeding or genetic improvement

programme to identify the mother plant and somaclonal

variant of O. stamineus.

Acknowledgement The authors sincerely thank the St. Xavier’s College

Management for providing infrastructure facilities,

constant support and encouragement. The author TRJJM

sincerely acknowledges the financial assistance provided

by the Department of Science and Technology,

Government of India, New Delhi, through the DST-

INSPIRE Fellowship (Ref. No. DST/INSPIRE

Fellowship/2011/IF 110640 dated 9th December, 2011).

References 1 Jaganath I B & Ng L T, The green pharmacy of Malaysia,

(Vinpress Sdn. Bhd., Kuala Lumpur, Malaysia) 2000, 76-77.

2 Yam M F, Basir R, Asmawi M Z & Ismail Z, Antioxidant and

hepatoprotective effects of Orthosiphon stamineus Benth.

standardized extract, Am J Chin Med, 35 (2007) 115-126.

3 Ho C-H, Noryati I, Sulaiman S-F & Rosma A, In vitro anti

bacterial and antioxidant activities of Orthosiphon stamineus

Benth. extracts against food-borne bacteria, Food Chem, 122

(2010) 1168-1172.

4 Yam M F, Asmawi M Z & Basir R, An investigation of the

anti-inflammatory and analgesic effects of Orthosiphon

stamineus leaf extract, J Med Food, 11 (2008) 362-368.

5 Adam Y, Somchit M N, Sulaiman M R, Nasaruddin A A,

Zuraini A et al, Diuretic properties of Orthosiphon stamineus

Benth., J Ethnopharmacol, 124 (2009) 154-158.

6 Lee W L & Chan L K, Micropropagation of Orthosiphon

stamineus, in Towards bridging science and herbal industry,

Fig. 1—Zymogram of O. stamineus. [M, Leaf of mother plant; N,

Leaf of nodal segment derived plantlet; & C, Leaf callus derived

plantlets]

Fig. 2—Cladogram of O. stamineus based on protein profiles in

SDS-PAGE

SHORT COMMUNICATIONS

271

edited by Y S Chang, M Mastura, S Vimala and Zainon S,

Proc Seminar on Medicinal and Aromatic Plants, held at

Forest Research Institute, Kuala Lumpur, Malaysia, September

12-13, 2000, 170-175.

7 Lee W L & Chang L-K, Plant regeneration from stem nodal

segments of Orthosiphon stamineus Benth., a medicinal plant

with diuretic activity, In Vitro Cell Dev Biol-Plant, 40 (2004)

115-118.

8 Rashid K, Nezhadahmadi A, Mohsin R, Azhar S & Efzueni S,

In vitro propagation of medicinal plant Orthosiphun stamineus

(Misai Kucing) through axillary branching and callus culture,

Life Sci J, 9 (2012) 5283-5294.

9 Sheena E V & Jothi G J, In vitro propagation of Orthosiphon

stamineus Benth. (Lamiaceae) an important medicinal plant

using nodal and leaf explants, Pharm Innov J, 4 (2015) 6-10.

10 Johnson M, Irudayaraj V & Rajkumar S D, Isoperoxidase analysis

on Thelypteris ciliata (Wall. ex Benth.) Holttum

(Thelypteridaceae), Asian Pac J Trop Biomed, 24 (2012) S27-S29.

11 Johnson M & Antonisamy A, Somaclonal variation studies

on Phyllanthus amarus Schum & Thonn, Iran J Biotechnol,

5 (2007) 240-245.

12 Anbalagan K, An introduction to electrophoresis

(Electrophoresis Institute, Yercaud, Tamil Nadu) 1999.

13 Nei M & Li W H, Mathematical model for studying genetic

variation in terms of restriction endonucleases, Proc Nat

Acad Sci USA, 76 (1979) 5269-5273.

14 Kaeppler S M, Kaepller H F & Rhee Y, Epigenetic aspects

of somaclonal variation in plants, Plant Mol Biol, 43 (2000)

179-188.

15 Brown D C & Thorpe T A, Crop improvement through

tissue culture, World J Microbiol Biotechnol, 11 (1995)

409-434.

16 Gonzalo S, Garcia-Cao M, Fraga M F, Schotta G, Peters A

H et al, Role of the RB1 family in stabilizing histone

methylation at constitutive heterochromatin, Nat Cell Biol,

7 (2005) 420-428.

17 Iqbal S M, Ghafoor A & Ayub N, Relationship between

SDS-PAGE markers and Ascochyta blight in chickpea, Pak

J Bot, 37 (2005) 87-96.

18 Platt T Jr, Alfalfa’s potential in dryland crop production

(Washington State University Area Extension Educator,

Davenport, USA) 2003.