R.S. Upendra et al.

/ International Journal of Engineering Science and Technology (IJEST)

Turmeric powder (Curcuma longa Linn.) as

an antifungal agent in plant tissue culture
studies
R.S. Upendra1* Pratima Khandelwal2 and A.H. Manjunatha Reddy3
1 & 2:
Asst. professor 2Professor & Head respectively, Department of Biotechnology, New Horizon College of
Engineering, Bangalore 560 103, Karnataka, India
3
Asst. Professor, Department of Biotechnology, RV College of Engineering, Bangalore 560 059, Karnataka,
India
*Corresponding author Email: rsupendra.nhce@gmail.com, +91 9611139872

Abstract:

Culturing the individual plant cells, tissues (explants) and organs in laboratory or in vitro on synthetic media
(MS media) under aseptic conditions is a usual process in plant tissue culture studies. The medium is rich in
nutrients, also supports the growth of variety of microorganisms especially bacteria and fungi, which cause
contamination of the medium, though the media is sterilized by autoclaving. During the process of cooling and
transferring the media, the chances of fungal contamination remain high. This is avoided to the maximum extent
following the good laboratory practices. A novel means could be incorporating turmeric, a well -known anti-
fungal agent, into the media. In the present study, attempts were made to avoid fungal contamination using the
media with various concentration of turmeric powder. Results of the investigation revealed that turmeric powder
used at the concentrations of 0.8 g/L and 1.0 g/L in the media resulted in appreciable control of fungal
contamination.

Key words: Turmeric, Sesamum orientale, Sesamum indicum, Curcumin.

Introduction

Prevention or control of contamination in in-vitro plant culture has been strictly based on the use of sterile

techniques. Any subsequent manipulation of the plant tissue must typically be carried out in a filtered-air

environment, e.g., in a laminar-flow hood (Bottino, 1981). Endophytic or latent type contamination by bacteria

and fungi (Kneifel and Leonhardt, 1992; Ryu and Holt, 1993) is an insidious process that continually threatens

plant tissue culture techniques throughout the duration of the culture period. Chemical anti-microbial agents

(Gilbert, 1991) such as benzyl pencillin, phosphomycin, chloremphenicol, rifamphicin (Haldeman et al., 1987;

Phillips et al., 1981), nalidixic acid, etc., have been used in different crops (Chapman, 1994; Dodds, J. H., and L.

W. Roberts. 1985) to avoid the contamination. These chemical antimicrobial agents generally trigger the normal

physiological and chemical functions of the plant.

Using the environmental friendly materials to check the microbial activity is a beneficiary process,

which reduces the cost as well as the inefficiency. Turmeric (Curcuma longa) is one such compound, which was

ISSN : 0975-5462 Vol. 3 No. 11 November 2011 7899

 R.S. Upendra et al. / International Journal of Engineering Science and Technology (IJEST)

used in traditional medicines as an anti-inflammatory (N. Niamsa and C. Sittiwet; 2009). It was also used in

wide range of conditions including respiratory diseases, liver disorders, sprains and sinusitis. It also has healing

property and ability to help the body to fight off microbes. The turmeric has shown its effect on some of the

diseases viz., antifungal, antimicrobial, antispasmodic, anti-inflammatory, Anti-venom, appetite stimulant,

asthma, bleeding, bloating, boils, bruises, cataracts, colic, contraception, cough, cystic fibrosis, diabetes,

diarrhea, dizziness, epilepsy, gas, gonorrhea, heart damage from Doxorubicin, helicobacter pylori infection,

hepatitis, hepatoprotection, high blood pressure, human papillomavirus (HPV), infections (methycillin-resistant

Staphylococcus aureus) (Kulkarni RR, Patki PS, Jog VP, et al. (1991), insect bites, insect repellent, jaundice,

kidney disease, lactation stimulant, leprosy, liver protection, male fertility, menstrual pain, menstrual period

problems/lack of menstrual period, liver damage from toxins/drugs, multidrug resistance, neurodegenerative

disorders, pain, parasites, ringworm, scarring, scleroderma. Mainly these wide variety of applications were

concentrated on the animal related problems/diseases, while on plant related aspects the applications of turmeric

was meager.

For the first time an attempt has been made to use turmeric in culture media to prevent and control

endophytic and latent contamination (Gilbert et al., 1991; Green, 1993; Haack and Warwick, 1993). The main

aim of the investigation is to test the turmeric as an antimicrobial agent in preventing and controlling the

endophytic and latent contamination in the culture media of sesame. To know at which concentration turmeric

functions effectively as an antibiotic in the plant tissue culture media, and also to know the residual effects of

turmeric on plant tissue of sesame.

Materials and methods

The materials used and methods followed during the study are as represented in the form of the following

Tables:

ISSN : 0975-5462 Vol. 3 No. 11 November 2011 7900

 R.S. Upendra et al. / International Journal of Engineering Science and Technology (IJEST)

Sl.No. Media constituents Formulae Quantity used (mg/L)

1. Ammonium Nitrate NH4NO3 1650

2. Potassium Nitrate KNO3 1900

3. Magnesium Sulphate MgSO4.7H20 370

4. Potassium di-Hydrogen KH2PO4 170
Phosphate
5 Calcium Chloride CaCl2.2H2O 440
6 Potassium Iodide KI 0.83
7 Boric Acid H3BO3 6.2
8 Manganese Sulphate MnSO4.4H2O 22.3
9 Zinc Sulphate ZnSO4.7H2O 8.6
10 Sodium molybdate Na2MoO4.2H2O 0.25
11 Copper Sulphate CuSO4.5H2O 0.025
12 Cobalt Chloride CoCl2.6H2O 0.025
13 Fe-Versenatte (EDTA) EDTA-Na Ferric salt 40
Vitamins
1. Nicotinic Acid C6H5NO2 0.5
2. Thiamine-HCl C8H11NO3HCl 0.5
3. Pyridoxine-HCl C12H18C12N4OS.HCl 0.1
Others
1 Glycine NH2CH2COOH 2
2 Sucrose C12H22O11 30,000
3 Myo-ionositol C6H12O6 100
Table 1: Composition of MS Media

The media was dispensed into test tubes and closed tightly with autoclavable caps and kept for autoclaving at

15lb pressure, 121˚C for 15-20min. After autoclaving these tubes were shifted to the control room for cooling.

Sl.No. Contents Volume taken for preparation of 1 lit of MS-

media

1 Stock solution-I 25ml/L

2 Stock solution-II 10ml/L
3 Stock solution-III 10ml/L
4 Stock solution-IV 10 ml/L
5 Stock solution-V 5ml/L
6 Sucrose 30g/L
7 Myo-ionositol 0.1g/L
8 Agar 0.8%

Table 2: Details of quantity of stocks taken from prepared stock solutions.

ISSN : 0975-5462 Vol. 3 No. 11 November 2011 7901

 R.S. Upendra et al. / International Journal of Engineering Science and Technology (IJEST)

.No Treatment
1 MS+0.2(g/l)Turmeric
2 MS+0.4(g/l)Turmeric
3 MS+0.6(g/l)Turmeric
4 MS+0.8(g/l)Turmeric
5 MS+1.0(g/l)Turmeric
6 MS+2.0(g/l)Turmeric
7 MS+1.0(g/l) Carbendazium
8 MS+2.0(g/l) Carbendazium
9 MS+0.5(g/l)K.Cyclin
10 MS+1.0(g/l)K.Cyclin
11 MS+2.0(g/l)Carbendazium +1.0(g/l)K.Cyclin
12 MS(control)

Table 3: Details of various treatments considered for the study

1. Surface sterilization of explant (Sesamum seed) by using 70% ethanol, 0.01% HgCl2 under laminar air flow

chamber.

2. Preparation of aseptic cotton bottles:

3. Creation of aseptic condition:

4. Inoculation of sesame seed in to cotton bottles

5. Inoculation of germinated sesame seed parts on to the media.

Results and discussion

Turmeric was tested at different concentrations to understand the antifungal activity during the tissue culture
studies of Sesamum.
Table 4: Effect of different antimicrobial agents on media contamination

S.No Treatment (g/L) No. of tubes used for No. of tubes Percentage of
sesame inoculation contaminated contamination
1 MS+ 0.2 Turmeric 20 13 65%
2 MS+ 0.4Turmeric 20 13 65%
3 MS+ 0.6Turmeric 20 7 35%
4 MS+ 0.8Turmeric 20 - 0%
5 MS+ 1.0Turmeric 20 - 0%
6 MS+ 2.0Turmeric 20 7 35%
7 MS+ 1.0 Carbendazium 20 7 35%
8 MS+ 2.0 Carbendazium 20 - 0%
9 MS+ 0.5 K.Cyclin 20 20 100%
10 MS+ 1.0 K.Cyclin 20 20 100%
11 MS+ 2.0 Carbendazium + 1.0 20 - 0%
K.Cyclin
12 MS (control) 20 20 100%

ISSN : 0975-5462 Vol. 3 No. 11 November 2011 7902

 R.S. Upendra et al. / International Journal of Engineering Science and Technology (IJEST)

100% Percent contamination observed with different media

80%
Percentage contamination

60%

40%

20%

0%
MS+ 0.2 MS+ MS+ MS+ MS+ MS+ MS+ 1.0 MS+ 2.0 MS+ 0.5 MS+ 1.0 MS+ 2.0 MS (Control)
Turmeric 0.4Turmeric 0.6Turmeric 0.8Turmeric 1.0Turmeric 2.0Turmeric Carbendazium Carbendazium K.Cyclin K.Cyclin Carbendazium
+ 1.0 K.Cyclin
Types of media used

Figure 1. Percentage of contamination observed in various types of media

Various concentrations of turmeric were used along with MS media under similar environmental
conditions. The percentage of contamination observed is represented in table 4 and figure 1. MS media with 0.8
g/L and 1.0 g/L of turmeric and 2.0 g/L of carbendazium has recorded nil contamination, while highest
contamination was recorded using MS media with K. cyclin (0.5 and 1.0 g/L concentration) and MS media
alone (Figure 2). The contamination was observed even with the turmeric at minimum concentration of 0.6g/L
and at high concentration 2.0 g/L (Figure 3). Hence, it can be depicted that a moderate amount of turmeric
between 0.8 – 1.0 g/L of turmeric can nullify the contamination in tissue culture studies (Figure 4). MS media
with carbendazium has also showed the null contamination at 2.0 g/L. Further the carbendazium is a chemical
compound, which can be hazardous, while the turmeric plays an important role as an effective
antifungal/microbial agent.

Figure 2. Treated with MS media with 0.5 g/L of K.Cyclin Figure 3. Treated with MS media with Turmeric concentration of
showing contamination 2.0 g/L showing contamination

Figure 4. Treated with MS media with Turmeric concentration of Figure 5. Treated with MS media with Turmeric concentration of
1.0 g/L showing no contamination 0.4 g/L showing contamination

ISSN : 0975-5462 Vol. 3 No. 11 November 2011 7903

 R.S. Upendra et al. / International Journal of Engineering Science and Technology (IJEST)

References:

1. Bottino, P. J. 1981. Methods in plant tissue culture. Kemtec Educational Corp., Kensington, Maryland, pp. 72.
2. Kneifel and Leonhardt, 1992, "Testing of different antibiotics against Gram-positive and Gram-Negative bacteria from plant tissue
culture", Plant Cell, Tissue and Organ Culture 29:139-155.
3. Ryu and Holt, 1993, "Growth inhibition of Penicillium expansum by several commonly used food Ingredients"J Food Protection
56(10)862-867.
4. Gilbert et al., 1991, "The use of antibiotics to eliminate latent bacterial contamination in potato tissue Cultures", Ann Appl Biol
119:113-120.
5. Haldeman et al., 1987, "Use of benomyl and rifampicin for invitro shoot tip culture of Camelliasinensis and C. japonica", Hortscience
22(2):306-307.
6. Phillips et al., 1981, "Antibiotics in plant tissue culture: Rifampicin effectively controls Bacterialcontaminants without affecting the
growth of short-term explant cultures of Helianthus Tuberosus",Plant Science Letters 21:235-240.
7. Chapman, 1994, "An effective biocide for in vitro diagnostic reagents and other products", Am ClinLab Aug. 1994, pp.13-14.
8. Dodds, J. H., and L. W. Roberts. 1985. Experiments in plant tissue culture. Second edition.
9. N. Niamsa and C. Sittiwet; 2009. Antimicrobial Activity of Curcuma Longa Aqueous Extract. Journal of Pharmacology and
Toxicology.
10. Kulkarni RR, Patki PS, Jog VP, et al. (1991) Treatment ofosteoarthritis with a herbomineral Formulation: a double-blind, placebo-
controlled, cross-over study. J Ethnopharmacol;33(1-2):91-95.
11. Gilbert et al., 1991, "The use of antibiotics to eliminate latent bacterial contamination in potato tissue Cultures", Ann Appl Biol
119:113-120.
12. Green, 1993, "Efficacy of biocides on laboratory-generated Legionella biofilms", Lett Appl Microbiol 17:158-161.
13. Haack and Warwick, 1993, "Controlling microbial growth inaqueous-based pesticide formulations", In Pesticides Formulations and
Application Systems, Devisetty et al. (eds.), pp. 105-115.

ISSN : 0975-5462 Vol. 3 No. 11 November 2011 7904