Isolation and Characterization of Disease Resistant Cell Lines of Cleome viscosa Using Fungal Elicitor

Amutha K., Rajagopal K., Senthil Kumaran B. and Karthikeyan S.

Department of Biotechnology, School of Life Sciences, VELS University, Velan Nagar, Pallavaram, Chennai- 600 0117, Tamil Nadu, India

ABSTRACT:

Two independent experiments were performed to isolate diseases cell lines of Cleome viscosa from leaf segments. In the first experiment, callus induction was obtained on Murashige and Skoog medium with different concentrations of Thidiazuron (TDZ). Best dedifferentiation rates of 2.08 g fresh weight occurred in the presence of 2.0 mg/l. In the second experiment, Aspergillus nigar and Aspergillus flavus were prominently isolated from garden soil and infected leaves respectively. Biochemical studies of anthocyanin accumulation at the different elicitor treated callus tissue revealed isolation and characterization of diseases resistant cell lines. Among different concentration, 0.5 gm/l mycelia of both fungal strains elucitate the maximam accumulation of anthocyanin. The best accumulation of anthocyanin was achieved with elicitors of A. flavus. Hence, pathogenic fungi are more effective for elicitation and to develop diseases resistant traits.

KEYWORDS: callus, Cleome viscosa, anthocyanin, Aspergillus, medicinal plant

INTRODUCTION:

Cleome is a large genus included in the Capparaceae family, which comprises 427 species occurring in tropical and subtropical regions of the world¹. Many species of the genus were investigated for medicinal properties and showed several important activities. Cleome viscosa L. is a widely distributed sticky herb with yellow flowers and long slender pods containing seeds, which resemble those of mustard. Nayikkadugu (Tamil) found throughout the greater part of India². In Ayurvedic system of medicine, this plant is used in fever, inflammations, liver diseases, bronchitis, diarrhea and infantile convulsions. Cleome viscosa possess various biological activities such as anthelmintic, analgesic, antiinflammatory, immunomodulatory, antiscorbutic, sudorific, febrifuge, cardiac stimulant, antipyretic, psychopharmacological, anti diarrheal, and hepatoprotective activities. Cleome viscosa is a good source of vitamin-C and iron^3,4. Methanolic extract of the whole plant showed the presence of steroids, triterpenoids, flavonoids and tannins². The main prerequisite for the development of high-quality medicinal products is a consistent source of high-quality plant material⁵. In this context, research in the area of plant tissue culture technology has contributed for the production of some important plant pharmaceuticals¹. Anthocyanin pigments and derivatives are flavonoid and their significant role is in plant resistance kingdom. Anthocyanin is also known for anticancer properties⁶. Fungal cell walls and fragments thereof (biotic elicitors) trigger this defense response⁷. It is the first report on in vitro cell culture of Capparaceae species when a cytokinin was used alone. Hence, the main objective of the work was to improve the disease resistance of cell lines of Cleome viscosa through the application of different fungal elicitors.

MATERIALS AND METHODS:

Standardization of Hormone concentration for callus induction:

Fresh healthy leaf explants of C. viscosa plants collected from the herbal garden of the VELS University, Chennai, India. The explants were first washed with detergent several times and rinsed under running tap water, then immersed in 70% (v/v) ethanol for ten seconds before surface-disinfection in 0.1 % HgCl₂ solution under continuous agitation for 3 min and were subsequently rinsed three times with sterile distilled water. After surface sterilization, the leaf explants were cultured in Murashige and Skoog basal medium (MS) supplemented with 30 gl^-1Analytical grade sucrose (Hi-Media, India) and 8.0 gl^-1agar (Hi-Media, India) supplemented with different concentrations (0.5, 1.0, 1.5, 2.0, 2.5, 3.0) of TDZ to obtain callus through dedifferentiation. The cultures were incubated at 24°C to 27°c and 60% relative humidity under light 18 hour/day photoperiod.

Isolation of fungi:

Soil fungi:

Soil sample were collected from the herbal garden of the VELS University. Potato Dextrose Agar (PDA) medium (ref) was prepared amended with chloramphenicol (25mg/100 ml). About 0.1 ml of each aliquot from 10^-3, 10^-4and 10^-5 soil dilutions was spread evenly on surface of PDA medium. Each dilution was plated in duplicates. The plates were sealed and incubated at room temperature.

Plant pathogenic fungi:

Young leaves with lesions were selected, because the fungus will be at its most active. The selected segments should have both infected and healthy part, 80 segments were collected. Surface sterilization is usually needed for leaf material with 0.1% mercuric chloride for one minute, followed by a rinse in sterile tap water to remove surface soil, dust and other contaminants. Eight segments were placed on PDA medium⁸ and incubated at 27 ± 1°C. The fungi that grew out from the leaf segments were periodically observed through a microscope.

Identification of fungi:

Pure cultures of soil and pathogenic fungus can be obtained from the primary isolation plates by colonies initiated from single spores culture. The fungal species were identified using standard characters used for fungal identification⁹.The sterile mycelia that grew out from the tissue were subcultured and exposed to light to induce sporulation⁸.

Fungal elicitor preparation:

The fungal cultures were established in liquid potato dextrose medium and kept at room temperature. The mycelia were separated for the cultures by filtration and washed four times in sterile distilled water. The mycelia were homogenized and the homogenate was used as elicitor.

Effect of Elicitors in Callus induction:

The fungal extracts at different concentration (0, 0.1, 0.25, 0.5, 0.75, 1.0) were added individually to the standardized medium for callus induction i.e. MS medium amended with 2 mg/l TDZ and a piece of callus was subcultured. The growth index was calculated as:-

Growth index = (Final weight-Initial weight) ÷ Initial weight.

Estimation of Anthocyanin:

Acidic methanol (Methanol - 80ml, Distilled water - 20ml and Hydrochloric acid - 1ml) is used as solvent for anthocyanin extraction from wild, elicitor treated callus and control i.e. callus without elicitor treatment. 1 g of sample was taken in 1 ml of acetic methanol and kept 20 hours incubation, followed by centrifugation at 10000 rpm for 5 minutes. The optical density values were absorbed at 525 nm and calculated as described in Teha et al⁶.

RESULTS:

The leaf tissue of C. viscosa was inoculated in MS medium containing different concentrations of TDZ. The callus tissues developed from the explants were studied and tabulated (Table 1). Growth of callus was found to be more (2.08 g) in 2.0 mg/l TDZ amended medium from leaf explants.

Isolated soil and pathogenic fungal strains were identified based on macroscopic and microscopic characteristics. Aspergillus niger was prominently isolated from soil whereas Aspergillus flavus from infected leaves. Hence these two strains were taken from elicitor preparation.

In elicitor-treated cells, changes in anthocyanin accumulation have been observed. Different concentrations of mycelia used from cultures of A. niger and A. flavus, show different values of growth index and anthocyanin accumulation (Table 3) (Plate 7, 8). Among these 0.5 gm/l mycelia of both fungal strains elucitate the maximam accumulation of anthocyanin. Least accumulation of anthocyanin was observed in 0.1 gm/l of A. niger and 0.75 gm/l of A. flavus. Although elicitors of A. niger and A. flavus show more or less similar results A. flavus show significantly higher accumulation of anthocyanin (Table 2).

DISCUSSION:

This study has demonstrated that best callus induction rate could be achieved on the MS medium containing 2.0 mg/l TDZ. Promoting role of TDZ in callus induction has been observed in Leucaena leucocephala, Vitex negundo and Achras sapota¹⁰. In elicitor-treated cells, changes in the phosphorylation status of proteins have been observed¹¹. Exposure of cell suspension cultures of Rauvolfia canescens to a yeast cell wall elicitor leads to the rapid transient induction of endogenous jasmonic acid and methyl jasmonate¹². The effect of different concentrations of L-phenylalanine and calcium chloride in MS medium to increase the anthocyanin content (μg/gm callus fresh weight) was investigated⁶.

Table: 1. Effect of TDZ in the growth of callus from the leaf tissue of C. viscosa on MS medium. This data was obtained after 25 days of inoculation.

S. No	Growth regulator (TDZ) mg/ml	Survival Rate %	Fresh weight (g)	Dry weight (g)
1.	0.5	60	0.98	0.20
2.	1.0	65	1.11	0.25
3.	1.5	68	1.31	0.29
4.	2.0	86	2.08	0.49
5.	2.5	72	1.49	0.32
6.	3.0	74	1.56	0.37

Table 2. Effect of elicitors in the resistant cell line from the callus of C. viscosa on MS medium with 2 mg/l TDZ. This data was obtained after 25 days of inoculation.

S. No

Fungus

Concentration of elicitor (gm/l)

Survival rate %

Fresh weight (gms)

Dry weight (gms)

Growth index

Anthocyanin

Content (µg/gm)

Aspergillus niger

0.1

0.25

0.5

0.75

1.0

2.08

1.04

1.26

1.68

1.32

1.24

0.49

0.23

0.28

0.39

0.31

0.27

5.30

2.15

2.81

4.09

3.00

2.75

56.2

74.5

80.7

105.4

92.5

75.7

Aspergillus flavus

0.1

0.25

0.5

0.75

1.0

2.08

1.29

1.32

1.54

1.19

1.34

0.49

0.28

0.29

0.37

0.24

0.31

5.30

2.90

3.00

3.66

2.60

3.06

56.2

84.6

95.3

108.3

76.2

90.0

In the present investigation anthocyanin content is high when biotic elicitors i.e. fungal mycelia were implemented in MS medium. Cheaper alternatives of gelling agents, carbon source are being used by various workers¹³. In this way this work is a novel economical and also an effective method of using fungal strains from the plant’s surrounding environment such as an infected plant and garden soil. Dietrich et al⁷ have observed elicitor from pathogenic fungus is influenced higher anthocyanin accumulation in callus tissue of M. charantia. Similar results were observed in this experiment with the pathogenic fungus, A. flavus.

ACKNOWLEDGEMENT

Authors are thankful to the Management of Vael’s Educational Trust, Chennai, Tamilnadu, India, for providing the infrastructure for the present study.

REFERENCE:

1. Albarello N, C Simoes, PFG Rosas, TCD Castro, MG Gianfaldoni, CH Callado and Mansur E. In vitro propagation of cleome spinosa (capparaceae) using explants from nursery-grown seedlings and axenic plants. In Vitro Cell. Dev. Biol.—Plant. 2006; 42:601–606.

2. Parimala Devi B, R Boominathan and Subhash CM. Evaluation of antipyretic potential of Cleome viscosa Linn. (Capparidaceae) extract in rats. Journal of Ethnopharmacology. 2003; 87:11–13.

3. Asolkar LV, KK Kakkar and Chakre OJ. Second supplement to glossary of Indian medicinal plants with active principles. Part I. (A–K). Publication and Information Directorate, New Delhi. 1965–1981; p. 217.

4. Chatterjee A and Pakrashi SC. The Treatise on Indian Medicinal Plants PID, vol. 1. CSIR, New Delhi. 1991; p. 155.

5. Murch SJ, CZ Liu, RM Romero and Saxena PK. In vitro culture and temporary immersion bioreactor production of Crescentia cujete. Plant Cell Tiss. Organ Cult. 2004; 78(1): 63–68.

6. Taha, H. S., Abd El-Rahman, R.A., Fathalla, 1 2 2 M. Abd-El-Kareem and 1Aly, U.E. Successful Application for Enhancement and Production of Anthocyantn Pigment from Calli Cultures of Some Ornamental Plants. Australian Journal of Basic and Applied Sciences. 2008; 2(4): 1148-1156.

7. Dietrich A, JE Mayer and Hahlbrock K. Fungal elicitor triggers rapid, transient, and specific protein phosphorylation in parsley cell suspension cultures. J. Biol. Chem. 1990; 265: 6360-6368.

8. Suryanarayanan TS. Light incubation: A neglected procedure in mycology. Mycologist. 1992; 6: 144.

9. Onions AHS, D Allsopp and Eggins HOW. Smith´s Introduction to Industrial Mycology.7^thedition. Edward Arnold,London. 1981; p-118-147.

10. Gill R and Ozias-akins P. Thidiazuron-induced highly morphogenic callus and high frequency regeneration of fertile peanut (Arachis hypogaea L.) plants. In Vitro Cellular and Developmental Biology - Plant. 1999; 35: 445-450.

11. Felix G, DG Gmsskopt, M Regenass and Boller T. Rapid changes of protein phosphorylation are involved in transduction of the elicitor signal in plant cells. Proc. Nati. Acad. Sci. 1991; 88: 8831-8834.

12. Gundlach H, Muller J, Kutchan M and Zenk H. (1992). Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proc. Natl. Acad. Sci. USA. 89: 2389-2393.

13. Joshi P, Trivedi R and Purohit SD. Micropropagation of Wrightia tomentosa: Effect of gelling agents, carbon source and vessel type. Indian J. Biotechnol. 2009; 8:115-120.

Received on 21.07.2010

Accepted on 16.08.2010

Research J. Science and Tech. 2(4): July –August. 2010: 75-77