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 2nd International Conference on Ecological, Environmental and Biological Sciences (EEBS'2012) Oct. 13-14, 2012 Bali (Indonesia)
Evaluation of Using Nanomaterial in Tissue
Culture Media and Biological activity
Kamran Safavi
Abstract— The field of nanotechnology is one of the most
active areas of research in modern materials science. New
applications of nanoparticles and nanomaterials are emerging
rapidly. Nanocrystalline silver particles have found tremendous
applications in the field of high sensitivity biomolecular detection
and diagnostics, antimicrobials and therapeutics, Catalysis and
micro-electronics. The use of nano-sized silver particles as
antimicrobial agents has become more common as technological
advances make their production more economical. TiO 2 is the most
commonly used semiconductor photocatalyst. Among the different
nanomaterials, it is the most studied. The concentration of TiO 2
usually required to kill bacteria varies and depending on the size of
the particles and the intensity and wavelength of the light used.
Plant tissue culture techniques involve the growing and
multiplication of totipotent cells, tissues and organs of plants on
defined solid or liquid media comprising nutrients under an aseptic
and controlled environment. Plant tissue culture has an important
role in the production of agricultural or ornamental plants and in the
manipulation of plants for improved agronomic performance. This
research was planned to evaluate the potential of nano silver and
TiO 2 to remove bacterial contaminants that exist in plant tissue
culture media. The modified MS medium evaluated after four
weeks. The results showed that nano silver and TiO 2 had a good
potential for removing the bacterial contaminants in plant tissue
culture procedures.
Keywords—Nano silver, dioxide titanium, nanotechnology,
Antimicrobial activity.
I. INTRODUCTION
T HE field of nanotechnology is one of the most active
areas of research in modern materials science.
Nanoparticles exhibit completely new or improved
properties based on specific
characteristics such as size, distribution and morphology.
New applications of nanoparticles and nanomaterials are
emerging rapidly. Nanocrystalline silver particles have found
tremendous applications in the field of high sensitivity
biomolecular detection and diagnostics, antimicrobials and
therapeutics, Catalysis and micro-electronics [2].
Kamran Safavi is with the Technology Incubator Center, Khorasgan
(Isfahan) Branch, Islamic Azad University, Isfahan, Iran (phone:
00989131115503; fax:00983115354069; e-mail: k_safavi@ag.iut.ac.ir).
5
The amount of nanoscale silver is increasing rapidly in
consumer products the ability to synthesize these particles on
a large scale improves. Silver nanoparticles (NPs) are toxic
to bacteria, and are currently used in everything from
medical devices to sport socks and washing machines to
deter microbial growth [5].
The use of silver for medicine or local antibacterial agents
was not recognized until the nineteenth century. Since then,
the antimicrobial property of silver has been investigated and
employed more extensively than any other inorganic
antibacterial agent. Silver is known to attack a broad range
of biological processes in microorganisms including the
alteration of cell membrane structure and functions. Silver
also inhibits the expression of proteins associated with ATP
production, although its specific antimicrobial mechanisms
are still not completely understood. Micromolar doses (1 to
10 μM) of silver ions are sufficient to kill bacteria in water,
while silver can be toxic at high doses to mammals and
freshwater and marine organisms, probably compromising
the growth and shape of animal cells by disrupting a variety
of biological functions. Such micromolar concentrations of
silver have no harmful effects on humans. Therefore, silver
has been widely used for the development of many
biological and pharmaceutical processes, products, and
appliances such as coating materials for medical devices,
orthopedic or dental graft materials, topical aids for wound
repair, water sanitization, textile products, and even washing
machines [7].
Toxicity of silver ion and its compounds towards
microbes is well established and this property was used in
wound dressings, silver loaded zeolites etc. because of its
higher stability, stronger antibacterial activity and broad
antibacterial spectrum [1].
The use of nano-sized silver particles as antimicrobial
agents has become more common as technological advances
make their production more economical. One of the potential
applications in which silver can be utilized is in management
of plant diseases. Since silver displays multiple modes of
inhibitory action to microorganisms, it may be used for
controlling various plant pathogens in a relatively safer way
compared to synthetic fungicides [7].
To eliminate bacterial contamination during in vitro
propagation, different methods have been developed in the
last few years. Antibiotics are commonly used in the medium
to eliminate unwanted contaminants from plant systems.
Theoretically, it might seem that all contamination problems
 2nd International Conference on Ecological, Environmental and Biological Sciences (EEBS'2012) Oct. 13-14, 2012 Bali (Indonesia)
could be overcome by the incorporation of one or more
antibiotics into the culture medium. However, antibiotics are
frequently phytotoxic otherwise may retard or inhibit plant
tissue growth [3].
Most antibiotics have been shown inhibitory effects in the
plants. Streptomycin and chloramphenicol are inhibitors of
protein synthesis; rifampicin inhibits nucleic acid synthesis
and penicillin inhibits cell-wall membrane synthesis.
Therefore, using antibiotics without application in the
medium may reduce mutation risks and inhibitory effects on
them. Nano silver has antimicrobial effects at low
concentrations. However, in this research we study the
several article about antimicrobial activities of silver
nanoparticles and then we using NS and non-NS media for
regeneration some plants and evaluate the elimination of
microorganisms in tissue culture procedures.
TiO 2 is the most commonly used semiconductor
photocatalyst. Among the different nanomaterials, it is the
most studied. Activated by UV-A irradiation, its
photocatalytic properties have been utilized in various
environmental applications to remove contaminants from
both water and air. A wealth of information on TiO 2
photocatalytic inactivation of bacteria has been acquired
over the last 20 years. The concentration of TiO 2 usually
required to kill bacteria varies and depending on the size of
the particles and the intensity and wavelength of the light
used.
To eliminate bacterial contamination during in vitro
propagation, different methods have been developed in the
last few years. Antibiotics are commonly used in the medium
to eliminate unwanted contaminants from plant systems.
Theoretically, it might seem that all contamination problems
could be overcome by the incorporation of one or more
antibiotics into the culture medium. However, antibiotics are
frequently phytotoxic otherwise may retard or inhibit plant
tissue growth [3]. Most antibiotics have been shown
inhibitory effects in the plants. Streptomycin and
chloramphenicol are inhibitors of protein synthesis;
rifampicin inhibits nucleic acid synthesis and penicillin
inhibits cell-wall membrane synthesis. Therefore, using
antibiotics without application in the medium may reduce
mutation risks and inhibitory effects on them. Nano silver
has antimicrobial effects at low concentrations. However, in
this research we study the several article about antimicrobial
activities of silver nanoparticles and then we using NS and
non-NS media for regeneration some plants and evaluate the
elimination of microorganisms in tissue culture procedures.
II. MATERIALS AND METHODS
1B
A. Plant materials
3B
In this research we electing tobacco (Nicotinia tabacum
cv. Xanthi) from Biotechnology department of Isfahan
University of Technology and propagated in MS medium
through bud cultivating. Plant grew in the temperature of
6
22°C, under a 16/8 hours (light/darkness) photoperiod and
re-cultivated in MS medium every three weeks. When we
want to re-cultivated their explants in modified media they
were sterilized with 70% ethanol for 1 min and 10% Clorox
(containing 5.25% sodium hypochlorite) for 1 min and then
rinsed four times with sterilized distilled water.
B. Nano materials preparation
4B
The size of nano silver particles used in this research was
35 nm. The base working fluid was pure water. Ag nano-
fluids were prepared using a two-step method. Ag nano-
particles were prepared first. They were produced using a
catalytic chemical vapour deposition method (Nanocid
Company Method). The Ag nano-particles were then added
to pure water. The APS of nano dioxide titanium that used in
this research was 10 nm. The TiO 2 were added in tissue
R R
culture media base on weight percent of the media.
C. Plant Materials
5B
In this research, we elected potato (Solanum tuberosum
L.) and propagated in Murashige and Skoog (MS) medium
through bud cultivating. Plant grew in the temperature of
22°C, under a 16/8 h (light/darkness) photoperiod and re-
cultivated in MS medium every three weeks. When we
wanted to re-cultivate their explants in modified media they
were sterilized with 70% ethanol for 1 min and 10% Clorox
(containing 5.25% sodium hypochlorite) for 1 min and then
rinsed four times with sterilized distilled water.
D.Nano Materials Preparation
6B
The size of nano silver particles used in this research was
35 nm. The base working fluid was pure water. Ag nano-
fluids were prepared using a two-step method. Ag nano-
particles were prepared first. They were produced using a
catalytic chemical vapour deposition method (Nanocid
Company Method). The Ag nano-particles were then added
to pure water.
E. Modification of Plant Tissue Culture Media by
7B
Nanomaterial
In this stage, we added different amounts of nano silver to
tissue culture media. Experiment involved NS with five rates
(5, 25, 50, 75 and 100 mg/l) in MS medium. Explants were
cultured on MS medium and evaluate at four times (1, 2, 3
and 4 weeks). We added different amounts of TiO 2 in two R R R
rates to tissue culture media.
R
F. Data Collection
The percentages of nano materials antimicrobial activity
used in tissue culture media were recorded four weeks after
culture. The experiment was conducted as a completely
randomized design in a factorial arrangement with three
replications.
 2nd International Conference on Ecological, Environmental and Biological Sciences (EEBS'2012) Oct. 13-14, 2012 Bali (Indonesia)
III. RESULT AND DISCUSSION
Several nanomaterials, such as nAg (NS) have found
applications in diverse consumer products and industrial
processes, including water treatment, as antimicrobial agents.
Among all antimicrobial nanomaterial, nAg is probably the
most widely used. The use of silver for medicine or local
antibacterial agents was not recognized until the nineteenth
century. Since then, the antimicrobial property of silver has
been investigated and employed more extensively than any
other inorganic antibacterial agent. Nano silver is a new and
non-toxic which shows high capabilities in eliminating
micro-organisms, fungus, bacteria and viruses. Silver is
known to attack a broad range of biological processes in
micro-organisms including the alteration of cell membrane
structure and functions. The cell wall of the Gram-negative
consists of lipids, proteins and lipopolysaccharides (LPS)
that provide effective protection against biocides, whereas
that of the Gram-positive does not consists of LPS. Silver
nanoparticles exhibit a broad size distribution and
morphologies with highly reactive facets. The major
mechanism through which silver nanoparticles manifested
antibacterial properties is by anchoring to and penetrating
the bacterial cell wall, and modulating cellular signalling by
dephosphorylating putative key peptide substrates on
tyrosine residues. Silver nanoparticles act primarily in three
ways against Gram-negative bacteria : (1) Nanoparticles
mainly in the range of 1-10 nm attach to the surface of the
cell membrane and drastically disturb its proper function,
like permeability and respiration; (2) They are able to
penetrate inside the bacteria and cause further damage by
possibly interacting with sulfur- and phosphorus-containing
compounds such as DNA; and (3) Nanoparticles release
silver ions, which have an additional contribution to the
bactericidal effect of the silver nanoparticles. Although
bacterial cell lysis could be one of the reasons for the
observed antibacterial property, nanoparticles also modulate
the phosphotyrosine profile of putative bacterial peptides,
which could thus affect bacterial signal transduction and
inhibit the growth of the organisms Several nanomaterials,
such as nAg (NS), and TiO2, have found applications in
diverse consumer products and industrial processes,
including water treatment, as antimicrobial agents. Among
all antimicrobial nanomaterials, nAg is probably the most
widely used. The use of silver for medicine or local
antibacterial agents was not recognized until the nineteenth
century. Since then, the antimicrobial property of silver has
been investigated and employed more extensively than any
other inorganic antibacterial agent. Nano silver is a new and
non-toxic which shows high capabilities in eliminating
microorganisms, fungus, bacteria and viruses. Silver is
known to attack a broad range of biological processes in
microorganisms including the alteration of cell membrane
structure and functions. The cell wall of the Gram-negative
consists of lipids, proteins and lipopolysaccharides (LPS)
that provide effective protection against biocides whereas
7
that of the Gram-positive does not consists of LPS [7]. Silver
also inhibits the expression of proteins associated with ATP
production, although its specific antimicrobial mechanisms
are still not completely understood. The low concentrations
of silver have no harmful effects on humans. Therefore,
silver has been widely used for the development of many
biological and pharmaceutical processes [8].
Because of its whiteness and high refractive index, TiO 2
nanoparticles, particularly in the anatase form, are already
used in a large number of consumer products. TiO 2 is
suitable for applications in water treatment because it is
stable in water, non-toxic by ingestion and low-cost. These
properties make the material a candidate for applications
such as medical devices, food preparation surfaces, air
conditioning filters, and etcetera. TiO 2 can kill both Gram-
negative and Gram-positive bacteria, although Gram-positive
bacteria are less sensitive due to their ability to form spores.
More recently, nano-sized TiO2 was also reported to kill
viruses including poliovirus, hepatitis B virus, Herpes
simplex virus, and MS 2 bacteriophage [5].
Up to now, there have been low reports of using
nanomaterial in plant tissue culture for remove of bacteria.
One of the novel reports was concluded that using 100 mg l-
1
NS had a good potential for removing of the bacterial
contaminants in plant tissue culture procedures [3]. On the
other hand, another reports show that using very low
concentration of nano silver in plant tissue culture was
recommended. Until now we have not any reports that used
TiO 2 in plant tissue culture media.
In this research we study the antimicrobial activity of nano
silver and nano dioxide titanium. After confirm the ability of
nano silver and nano dioxide titanium to reduce the
microorganism, we decide to using and adding NS and TiO 2
to MS tissue culture media. The results show that NS and
TiO 2 can reduce and remove microorganisms in MS media
and then the explants can growth very well.
REFERENCES
[1]- A. J. Kora, J. Arunachalam. Assessment of antibacterial activity of
silver nanoparticles on Pseudomonas aeruginosa and its
mechanism of action World J Microbiol Biotechnol DOI
10.1007/s11274-010-0569-2. 2010.
[2]- D. Jain, H. Kumar Daima, S. Kachhwaha, S. L. Kothari. Synthesis of
Plant-mediated silver nanoparticles using papaya fruit extract and
evaluation of their antimicrobial activities. A. Digest Journal of
Nanomaterials and Biostructures: 4, p. 557 – 563. 2009.
[3]- G. Abdi, H. Salehi, M. Khosh-KhuiNano. Silver: a novel nanomaterial
for removal of bacterial contaminants in valerian (Valeriana officinalis
L.) tissue culture. Acta Physiol Plant: 30:709–714. 2008.
[4]- L. Mineo. Plant tissue culture techniques. in Tested studies for
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[5]- Q. Li, sh. Mahendra, D. Y. Lyon, L. Brunet, M. V. Liga, D. Li, P. J.J.
Alvarez. Antimicrobial nanomaterials for water disinfection and
microbial control: Potential applications and implications. A journals of
the international water research. 42, 2008,
 2nd International Conference on Ecological, Environmental and Biological Sciences (EEBS'2012) Oct. 13-14, 2012 Bali (Indonesia)

[6]- T. L. Kirschling. Nanoparticle Interactions with Bacteria: Toxicity and

Chemotaxis. Carnegie Mellon Department of Chemical Engineering.
2009.
[7]- T. Maneerung, S. Tokura, R. Rujiravanit. Impregnation of silver
nanoparticles into bacterial cellulose for antimicrobial wound dressing.
Carbohydrate Polymers. In press.
[8]- Y. K. Jo, B. H. Kim, G. Jung . Antifungal Activity of Silver Ions and
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