EFFECT OF MICROWAVE ON IN-VITRO CULTURED CELLS OF Mentha piperita, A MEDICINAL ...

16 Pages·2015·0.65 MB·English

ISSN: 2394 – 1669 (Online)
International Journal Effect of Microwave on Mentha piperita
for Exchange of Knowledge; 2 (1) : 1-16, 2015 Raghubar Singh & P. C. Thakur
=================================================================================
EFFECT OF MICROWAVE ON IN-VITRO CULTURED CELLS OF
Mentha piperita, A MEDICINAL PLANT
RAGHUBAR SINGH# & P. C. THAKUR*
#Dept. of Physics, Chas College, Chas, Bokaro
* Dept of Botany, Chas College, Chas, Bokaro
E-Mail: [email protected]
ABSTRACT
KEY WORDS
The growing use of microwave in wireless communication especially in mobile
Mobile phone
phones has necessitated the study of its effects on the biological systems.
Therefore, effect of microwave irradiation on in-vitro cultured tissues/cells of
radiation,
Mentha piperita was studied. Mentha piperita, an economically important plant is

Mentha piperita, cultivated mainly for menthol-the peppermint used in confectioneries and
medicine industries. Shoot apex of the plant was cultured on Murashige &
Shoot apex,
Skoog (1962) basal medium supplemented with Benzyle Amino Purine (BAP) 1.0
Differentiation, – 2.5 mg per liter. The cultures were placed inside closed iron sheet chamber
working as Faraday cage and irradiated for 0.5, 1, 2, 3 and 4 hours duration
in vitro
with microwave transmitted from mobile phones.
As compared to controls, the rate of contamination decreased gradually as the
duration of irradiation of shoot apex culture was increased from 0.5 to 4 hours.
Received on:
But the percentage of cultures showing callus initiation decreased gradually.
5.04.2015 New callus formation also decreased with increase in duration of exposure of
culture to the microwave. Increased hour of exposure changed texture of callus

from compact to friable. Similarly, colour of callus changed from green to

Accepted on: yellow at 3 hour duration of exposure and finally at 4 hour of exposure, colour
of callus became brown. Growth rate of callus also decreased with increase in
02.08.2015 hour of exposure. Microwave produced negative effect on the dry weight of the
callus which was decreased from 2.3 to 1.4 grams at four hour duration of
exposure. Microwave negatively affected number of differentiated roots, shoots

and plantlets per culture. At 4 hour duration of exposure, number of

*Corresponding differentiated roots decreased to 10.5 per culture as compared to 15.2 of
controls. Similarly, number of shoots decreased to 9.2 in cultures exposed for
Author four hours. Number of regenerated plantlets per culture also decreased.
Similarly, differentiation of roots per plantlet was decreased to 2.8 as compared

to controls where it was 4.9. It was found that mean length of differentiated

shoots and roots gradually decreased with increase in hour of exposure to
microwave.
INTRODUCTION it does not contain adequate energy to change
Microwaves are electromagnetic radiations chemical nature of substances. Its wave length
having wavelength range from 300 MHz to is 1mm – 1 meter and photon energy is
300GHz. It is a non-ionizing radiation because 1.24meV – 1.24 µeV. Microwaves consist of

1
ISSN: 2394 – 1669 (Online)
International Journal Effect of Microwave on Mentha piperita
for Exchange of Knowledge; 2 (1) : 1-16, 2015 Raghubar Singh & P. C. Thakur
=================================================================================
electric and magnetic field perpendicular to radar technology. Microwaves have also
each other, propagating together in the same attracted the attention of agricultural scientists
direction. The electric field causes heating via for research and development in this field.
two simultaneous mechanisms, namely, Application of microwave in every corner of
dipolar rotation and ionic conduction (Thuery life is increasing, therefore, it opens a vast
1992). Dipolar rotation is due to the alignment area for research and development in physical,
on the electric field of the molecule possessing chemical and biological sciences especially
a dipole movement in both the solvent and the human health, food production and
solid sample. This oscillation produces communication.
collision with surrounding molecule and thus Before the 1990’s radio frequencies were
the liberation of thermal energy into the mainly from a few radio and television
medium. With a frequency of 2.45 GHz (on transmitters located in remote areas. Since the
which domestic and industrial microwaves introduction of wireless telecommunication in
generally work), this phenomenon occurs the 1990’s; the rollout of phone networks has
4.9x109 times per second (Ganzler et al. 1990, caused a massive increase in electromagnetic
Barnabas et al. 1995, Onuska and Terry 1995) pollution (Balmori 2005, Galeev 2000).
due to which heating is very fast. Indeed, the Multiple sources of mobile communication
larger the dielectric constant of the solvent the result in chronic exposure of animals, plants
more optimal the heating (Jassie et al. 1997). and humans. Exposure to microwave from
Unlike classical conductive heating methods, various sources are from GSM and 3G cell
microwave heat the whole sample phones and base stations, all transmitting
simultaneously. towers (TV, Cell phone, etc), wireless local
Application of microwave: Microwave area network (LAN), wireless personal area
radiation is becoming common in the network (WPAN such as blue tooth and Wi-Fi
environment because of exponential use of broad band) and cordless telephones. All
mobile phone technology and wireless animals, plants and humans are subjected to
devices. Most common applications of microwave radiation without studies of its
microwaves are within the 1 to 40 GHz range. environmental impact measuring long-term
Due to high frequency and short wave length, effects. The greater portion of this exposure is
microwaves are used in communication and from mobile telecommunications.
2
ISSN: 2394 – 1669 (Online)
International Journal Effect of Microwave on Mentha piperita
for Exchange of Knowledge; 2 (1) : 1-16, 2015 Raghubar Singh & P. C. Thakur
=================================================================================
Cell phones operate within the frequency band are exposed directly to the radiation 24 hours
of 800 MHz, 900MHz and 1800 MHz and the throughout the year.
latest 3G technology works between 1900- Plants consist of complex and well organized
2200 MHz. A cell phone that is on but not in conductive, insulating, assimilatory and
talk mode also radiates microwave. reproductive systems. Therefore, it is
Computers and laptops operate within the necessary to examine their interaction with
frequency range of 1000-3600 MHz and most increased exposure to radio and microwave
Wi-Fi systems and some cordless phones frequencies. Effect of microwave on different
operate around 2450 MHz which is same species of plants may vary because of
frequency as that of microwave oven. The variation in organization and nature of
growing use of wireless communication in the biochemical molecules present in their body.
last decade has introduced concern about Plant tissue culture: Tissue culture is the
health risks from the so called man made culture and maintenance of plant cells or
electro smog (Kumar Girish 2010). Various organs on sterile growth medium under
epidemiological and experimental studies controlled and supportive environmental
have been carried out and the results have conditions. The cultured tissues of plant
shown to have a close relation between produce callus, shoot & root buds and somatic
biological effects and electromagnetic embryos. Micropropagation through plantlet
radiation. regeneration may also be achieved through
Effect of microwave on Plants: The plants this technique.
play a significant role for living beings, as Mentha piperita: The genus Mentha
they are the primary producers of food and (Lamiacae) includes about 25 species that are
oxygen. They are the most important biotic mostly aromatic perennial herbs with creeping
factor that maintains ecological balance for rhizomes and scented foliage. Several species
the survival of the living beings including of Mentha are cultivated in different parts of
man. And therefore ensure existence of life on the world for their medicinal and commercial
the planet earth. Plants, wild animals and importance. The essential oils of Mentha
microorganisms are more vulnerable to the piperita (peppermint) are widely used in
microwave radiation pollution because they perfumery, food and pharmaceutical industries
3
ISSN: 2394 – 1669 (Online)
International Journal Effect of Microwave on Mentha piperita
for Exchange of Knowledge; 2 (1) : 1-16, 2015 Raghubar Singh & P. C. Thakur
=================================================================================
(Cellarova 1992). M. piperita is cultivated We studied effects of microwave transmitted
mainly for menthol, viridiflorol, menthofuran, from mobile phone tower of “Airtel” which
menthone, etc (Anonymous 1962, Rastogi and transmits microwave frequency of 935 - 960
Malhotra 1993). It has wide range of MHz for GSM900 technology from cell tower
applications in medicine, soap, mouth wash, antennas and Nokia cell phone which
dental cream, alcoholic drinks, candies, transmits 890 - 915 MHz (GSM 900)
chewing gums, confectioneries, etc. As frequency of microwave.
medicine it is used in stomachache, nausea, Mentha piperita: Mentha piperita is an
flatulence, headache, vomiting etc. aromatic, perennial, herbaceous plant native to
MATERIALS AND METHODS Europe but cultivated all over the world
Microwave including India. Experimental mother plant
Microwave radiation is becoming common in (Fig. 1) of M. piperita was six months old
the environment because of exponential use of raised from cuttings growing in the pot.
mobile phone technology and wireless
devices. Cell phones operate within the
frequency band of 800 MHz, 900MHz and
1800 MHz and the latest 3G technology works
between 1900-2200 MHz. A cell phone that is
on but not in talk mode also radiates
microwave. A cell phone transmits 1 to 2 Watt
of power in the frequency range of 824 - 849
MHz (CDMA), 890 - 915 MHz (GSM900)

and 1710 – 1780 MHz (GSM1800). Cell
Fig. 1: Mentha piperita mother plant grown
tower antennas transmit in the frequency
from cuttings
range of 869 - 894 MHz (CDMA), 935 - 960
MHz (GSM900) and 1810 – 1880 MHz METHODS
(GSM1800). Also, 3G has been deployed in a Tissue culture methods
few cities, in which base station antenna Culture Medium Selection
transmits in the frequency range of 2110 –
Based upon previous research reports of
2170 MHz.
4
ISSN: 2394 – 1669 (Online)
International Journal Effect of Microwave on Mentha piperita
for Exchange of Knowledge; 2 (1) : 1-16, 2015 Raghubar Singh & P. C. Thakur
=================================================================================
different scientists and experiments done at Preparation of explants
our laboratory (Apurva et al. 2011), Shoot apices with 2-3 nodes of mature
Murashige and Skoog 1962 (MS 1962) culture experimental mother plant were taken as
medium was selected for experiments of the explants. Explants were surface sterilized
present work. according to the following steps –
Preparation of culture medium (a) Tap water wash – 20 minutes
MS (1962) medium procured from HiMedia (b) Teepol (3-5% v/v) wash – 10 minutes
Laboratories Pvt. Limited, Mumbai was used (c) Again wash in running tap water to
to make basal medium solution. 30g / L remove detergent (teepol)
sucrose as source of carbon and 8g /L agar (d) 70% alcohol treatment for one minute.
were added to this medium. In this mixture, (e) HgCl (1% w/v) treatment – 2.5
2
growth regulators (hormones) were also minutes
added. After that, pH of the solution was (f) Finally, sterile distilled water wash –
adjusted to 5.5 with dil NaOH or dil HCL. 3-4 times
Now the medium is boiled to homogenize the Inoculation of explants to culture medium:
agar and poured into culture tubes and conical The surface sterilized explants were
flasks and finally autoclaved to make the transferred to the culture medium present in
medium sterile. the culture tubes (15cm × 2.5cm) or 125 ml
Selection of Growth regulator Erlenmeyer flasks over flame of the spirit
Following combination of media were lamp inside the laminar flow (Fig. 2).
selected for different experiments as per report Maintenance of cultures: The cultures were
of Apurva et al. (2011) – maintained at 16 hour photoperiod,
1. Callus development medium temperature 23-28ºC, relative humidity 70%
MS + BAP (2.5 mg/l) and illuminance 2500 Lx (diffuse white
2. Callus maintenance medium florescent light) for growth and development
MS + BAP (1.0 mg/l) of cultured explants inside the growth racks
3. Differentiation medium (Fig. 3). The calli were subcultured at 4-5
MS + BAP (2.0 mg/l) weeks interval. Observations were recorded
every week.

5
ISSN: 2394 – 1669 (Online)
International Journal Effect of Microwave on Mentha piperita
for Exchange of Knowledge; 2 (1) : 1-16, 2015 Raghubar Singh & P. C. Thakur
=================================================================================

Fig. 2: Laminar air flow chamber for inoculation of explants to the culture medium

Fig. 3: Tissue culture growth racks for cultured explants
Microwave irradiation methods: Microwave chamber was made with thermocol having all
exposure was carried out in a closed shielded its walls completely layered with metal sheets
chamber designed to act as a Faraday cage to (Galvanized iron; thickness 2 mm) (Fig. 4).
make the experimental environment free from Two commercially available GSM cell phones
outside EMF interferences. The closed made by Nokia Corporation, model no. 2600
6
ISSN: 2394 – 1669 (Online)
International Journal Effect of Microwave on Mentha piperita
for Exchange of Knowledge; 2 (1) : 1-16, 2015 Raghubar Singh & P. C. Thakur
=================================================================================
and model no. 1108 (900 MHz) were used in The phone battery was permanently connected
the present study. They were placed with 12 V DC, 220 V AC adaptor placed 2
horizontally inside the box shaped chamber meter way from the cell phone. Cultured
(Fig. 4). The cell phones were kept at distance tissues and callus were irradiated with mobile
of 16 cm from each other. The cultured tissues phone radiation for ½, 1, 2, 3 or 4 hour. After
and calli to be irradiated were placed in irradiation they were transferred to tissue
between the two mobile phones in the centre culture growth racks (Fig 3). Cultures without
about 8 cm apart from each phones. The irradiation served as controls. All other EMFs
cultured material and mobile phones were (Electro Magnetic Force) from sources inside
kept at the similar height. During exposure, and outside the laboratory were eliminated
cell phones were used in talk mode attached during exposure treatment.
with voice recorders and the box was closed.

Fig. 4: Irradiation chamber with mobile phones for microwave
exposure to cultured tissues/callus of Mentha piperita

Statistical analysis methods: Results were five times ± Standard Error and expressed as
calculated as the mean value of ten replicates % of respective control. Un-irradiated cultures
and each experimental treatment was done were taken as controls for recording the data.
7
ISSN: 2394 – 1669 (Online)
International Journal Effect of Microwave on Mentha piperita
for Exchange of Knowledge; 2 (1) : 1-16, 2015 Raghubar Singh & P. C. Thakur
=================================================================================
RESULTS AND DISCUSSION decreased percentage of callus initiation from
79.5 to 74.1%. However, microwave did not
Effect on establishment of culture
affect texture of callus produced in the culture
Shoot apex explants of M. piperita were
in comparison to the controls. Colour of callus
cultured on MS + BAP (2.5 mg/l) to see effect
was slightly affected which turned to pale
of microwave on establishment of culture. As
green at 3-4 hour exposure. Growth of callus
compared to controls, rate of contamination
was fast but due to effect of microwave the
decreased gradually as the duration of
growth of callus gradually decreased (Table
irradiation of shoot apex culture was increased
1). Therefore, it can be concluded that the
from 0.5 to 4 hours. At 4 hour irradiation
effect of microwave irradiation on
contamination decreased to 5 percent which
establishment of culture was negative except
was 20 percent for the cultures without
one positive effect of reduction in the rate of
irradiation (Table 1). But the negative effect
contamination. The general problem of release
of microwave irradiation was that the
of phenolics from the explants to culture
percentage of cultures showing callus
which cause difficulty in establishment; was
initiation also decreased gradually (Table 1).
not observed in microwave irradiated cultures.
Microwave irradiation for four hours
Table 1: Effect of microwave irradiation on establishment of Shoot apex explants of M.
piperita cultured on MS + BAP (2.5 mg/l)

Duration of Response of culture
irradiation in % of cultures % of culture Texture of Colour of Growth rate
hour (h) contaminated showing callus callus callus of callus
initiation
Control (no 20 79.5 Compact Green Fast
irradiation)
0.5 h 18 78.2 Compact Green Fast
1 h 16 77.6 Compact Green Fast
2 h 12 75.3 Compact Green Moderate
3 h 9 74.8 Compact Pale Green Slow
4 h 5 74.1 Compact Pale Green Slow
*Response recorded after 2 weeks of culture

Many studies have shown that microwave known that weak quantum energy of
cause different types of biological effects. It is electromagnetic force could not break
8
ISSN: 2394 – 1669 (Online)
International Journal Effect of Microwave on Mentha piperita
for Exchange of Knowledge; 2 (1) : 1-16, 2015 Raghubar Singh & P. C. Thakur
=================================================================================
chemical bonds because mobile phone (Table 2). Increased hour of exposure changed
radiation at 0.9 GHz emits 4µeV, which is texture of callus from compact to friable.
much lower energy as compared to the energy Similarly, colour of callus changed from green
of about 1 eV needed to break the weakest to pale yellow at 2 hour duration of exposure
chemical bonds in the nucleic acid or protein (Fig. 5) and finally at 4 hour of exposure,
molecules. When electromagnetic microwave colour of callus became brown. Growth rate of
radiation is absorbed in tissues it causes ionic callus also decreased with increase in hour of
movement and rotation of dipoles due to exposure. Microwave produced negative
which temperature is raised (Krishnan and effect on the dry weight of the callus which
Murugan 2011). It was assumed that this was decreased from 2.3 to 1.4 grams at four
change might have caused different types of hour duration of exposure. In this experiment
variations in the cultured cells and microbes also we found all negative effects of
that cause contamination. This may be a microwave on the callus culture except in the
reason for reduction in contamination and rate of contamination.
callus initiation. Increase in temperature may Time dependant negative impact of
also be responsible for change in the colour of microwave on different responses of the
the callus from green to pale- green and slow culture may be due to rise in temperature with
growth rate of callus. increased duration of time. Similar to our
Effect on callus formation finding of decreased rate of callus formation,
Callus was subcultured on basal medium decrease in growth rate and reduction in dry
supplemented with BAP (1.0 mg/l) to study weight; Sandu et al. (2005) observed a
irradiation effect on the culture. Microwave decrease in chlorophyll content in leaves of
reduced contamination of subcultured callus Robinia pseudoacacia with the treatment of
similar to that of explants culture as described 400 MHz EMF. Lebedev et al. (1977) reported
earlier. New callus formation gradually that the fresh and dry weight of barley root
decreased with increase in duration of and shoot was decreased by 12–48% upon
exposure of culture to the microwave. At 4 exposure to magnetic fields. Maffei (2014)
hour duration of exposure, new callus published general impact of magnetic field on
formation decreased from 88.3 to 75.2 percent plant growth and development.

9
ISSN: 2394 – 1669 (Online)
International Journal Effect of Microwave on Mentha piperita
for Exchange of Knowledge; 2 (1) : 1-16, 2015 Raghubar Singh & P. C. Thakur
=================================================================================
Table 2: Effect of microwave irradiation on callus of M. piperita subcultured
on MS + BAP (1.0 mg/l)

Duration of Response of culture Dry weight
irradiation % of cultures % of culture Texture Colour Growth of callus in
in hour (h) contaminated showing of new of new rate of new gm
formation of callus callus callus
new callus
Control (no 7 88.3 Compact Green Fast 2.3
irradiation)
0.5 h 6 87.5 Compact Green Fast 2.1
1 h 6 87.2 Compact Pale Moderate 2.0
Green
2 h 4.5 84.1 Friable Pale Slow 1.8
Yellow
3 h 3 79.2 Friable Yellow Slow 1.6
4 h 3 75.2 Friable Brown Slow 1.4
*Response recorded after 4 weeks of culture

Fig. 5: Pale yellow callus after 2 hours of irradiation with microwave (x1.4)

10