Native of the Mediterranean Sea basin, olive (Olea europaea L.) belongs
to the Oleaceae family and its cultivation has been started from ancient times
(Liphschitz et al., 1991). Now, olive is one
of the most important commercial fruits tree grown in regions with Mediterranean
Olive can be propagated by seed, cutting and sucker. The commonest method is
the rooting of stem cuttings under mist system; however the efficiency of this
method is very low. In vitro propagation techniques present a suitable
high efficiency alternative for olive propagation (Rugini
and Pesce, 2006). In vitro produced plants are true to type, clean
(pest and disease free) and suitable for commercial production of a large number
of plants. Successful micropropagation of olive cultivars has been reported
by many researchers during the past three decades (Rugini,
1984; Bartolini et al., 1990; Rama
and Pontikis, 1990; Dimassi-Theriou, 1999; Cozza
et al., 1997; Troncoso et al., 1999).
Too many factors may limit micropropagation of higher plants, especially woody
species (Houng et al., 2001). The most important
limiting factor is in vitro contaminations (e.g., fungal and bacterial
infections). There are some methods and chemicals available to control in
vitro contaminations. However, the efficiency of some of these methods is
low, and/or some of theme are too toxic. Antibiotics are used in controlling
internal bacterial contaminations (Dabai et al.,
2007). Using antibiotics also may adversely affect the growth and response
of explants and may induce resistance in bacteria and generally they are not
suggested for using in plants tissue culture laboratory (Dabai
et al., 2007). Mercury chloride (AgCl) is widely used for controlling
internal contaminations of woody plants. AgCl is very toxic and should be used
with high cautions (Leifert and Woodward, 1997). Such
chemicals not only are too toxic for the explants and the operator, but they
may infect the environment. In addition, Martino et al.
(1999) showed that olive explants are too sensitive to mercury chloride
and it may kill the explants. Under such circumstance, finding an effective
and safe substance for decontamination of the explants (especially for woody
plants) is very important.
Nano-silver is a new and non-toxic material which shows high capabilities in
eliminating microorganisms, e.g., fungus, bacteria and viruses. The detrimental
effects of nano-silver have been shown on more than six-hundred microorganisms
(Abdi et al., 2008). This capability of nano-silver
is due to release of tiny particles of silver and so it is able to destroy not
only the bacteria and fungus, but also the viruses (Sondi
and Salopek-Sondi, 2004).
The aim of the current study was to evaluate the potential of nano-silver particles on decontamination of nodal segments of olive Mission for in vitro propagation.
MATERIALS AND METHODS
Plant materials: The experiment was conducted at tissue culture laboratory of the Department of Horticultural Science of Shiraz University (Iran) from spring to autumn 2008.
Branches of 9 years old olives cv. Mission brought from Kazerun Research Station,
Kazerun, Iran. Explants included the single nodes and shoot apices. Leaves were
removed and 2 cm uniform explants were washed thoroughly in running tap water
for 20 min after pre-washing in solution contained 0.2-0.3% commercial detergent
(Rika-Iran). Explants were submerged in a solution of 100 mg L-1
ascorbic acid plus citric acid 100 mg L-1 for 30 min to control the
phenolic compounds (Brhadda et al., 2003).
Disinfestation by ethanol and clorox: Explants were submerged in 70% ethylic ethanol for 0, 0.5 and 1 min and after 3 times rinsing in sterile distilled water, they were submerged in 10% Clorox (Golrang-Iran) for 0, 5 and 10 min. The Clorox solutions contained 0.1% of a commercial detergent (Rika-Iran) as wetting agent. After Clorox treatments, explants were washed thoroughly for 3 times in sterile distilled water.
Disinfection treatments by nano-silver: Nano-silver (NanoCid L2000-Iran) particles were used as disinfecting treatments after surface sterilization by 70% ethanol and 10% Clorox. Nano-silver treatments included: (1) submersion of explants in nano-silver solutions; (2) adding nano-silver to media.
||Explants were submerged in 0, 100, 200, 300 and 400 mg L-1
nano-silver solutions for 1 h (Abdi et al., 2008).
The nano-silver solutions prepared in sterile distilled water
||Different amount of nano-silver (0, 4, 8 and 16 mg L-1)
were added to the media as disinfection agent and then media were autoclaved.
Thirty days after culture the percentages of infected and developed explants
Medium and culture condition: The explants were placed on Murashige
and Skoog half strength (MS/2) media supplemented with 3.0% sucrose (Brhadda
et al., 2003). The medium was supplemented with 2.1 mg L-1
Benzyl Adenine (BA), 1.26 mg L-1 Gibberellic Acid (GA3)
and 0.6 mg L-1 Naphthalene Acetic Acid (NAA). The pH of medium adjusted
to 5.7 by HCl or NaOH prior to adding 0.8% agar and then autoclaved at 121°C
for 15 min (Bartolini et al., 1990). Culture room conditions were 25±3°C,
16 h photoperiod at 40 W m-2 irradiation.
Statistical analysis: The experimental design was a Complete Randomized Design (CRD) with four replications and 10 vessels per replication and 2 explants per vessel. Data were subjected to ANOVA. Means were compared with Tukeys HSD test at p≤0.01.
Results of disinfestations of olive single nodes by 70% ethanol and 10% Clorox
are shown in Table 1. Results showed that ethanol may not
be used singularly for controlling in vitro contaminations of olive explants.
Clorox treatments reduced the number of infected cultures, significantly. The
best results after using 10% Clorox obtained in 10 min treatment (up to 18%).
However, ethanol treatments increased the efficiency of the Clorox treatments
significantly. Increasing the period of treatments led to better control of
in vitro contaminations and the best results were obtained in 70% ethanol
for 1 min and 10% Clorox for 10 min (up to 51%). The explants were depressed
for 24 to 48 h after long periods of ethanol and Clorox treatments, but no signs
of chlorosis and/or necrosis were observed after these treatments.
Submerging the explants in nano-silver solutions after 70% ethanol and 10% Clorox treatment, wholly prevented the fungus and bacteria contaminations (100%) (Fig. 1). However, nano-silver submersion also affected the olive explants and very few explants developed these treatments (Fig. 1).
Adding nano-silver to the media following the elected 70% ethanol and 10% Clorox
as surface sterilizing treatment decreased the incident of fungus and internal
contaminations significantly (Fig. 2).
||The effects of different time applications of 70% ethanol
and 10% Clorox on controlling the in vitro contaminations
|The means with the same capital or small
case letters were not significantly different according to Tukeys
test at p≤0.01
||The effects of submersion of explants in nano-silver solutions
on decontamination and development of explants. Values are Mean±SE
||Explants grew normally after adding nano-silver particles
to the media. The color of medium also changed from what
to dark yellow
after adding silver particles, though it was still clear
Adding nano-silver particles to the media resulted in reduction of in vitro
contaminations incident less than 5%. The addition of different concentrations
of nano-silver to the media were not significantly different on controlling
the contaminations (Fig. 3). Presence of 6 mg L-1
nano-silver particles in the media seemed to reduce, compared with the other
two concentrations, the growth of olive explants; however the differences were
||The effects of using nano-silver in media on decontamination
and development of olive explants. Values are Mean±SE
Establishing a sterile culture is the most challenging step in micropropagation of woody plants. In addition, this step is so laborious and costly. In the current study, potential of using nano-silver as a cheap and environmental friendly decontamination agent in micropropagation of olive Mission were investigated.
Results of this study showed that 70% ethanol and/or Clorox treatments are
not highly effective in controlling in vitro contamination of olive explants.
Ethanol pretreatments for 1 min before 10% Clorox treatments increase the efficiency
of surface sterilization significantly. Mencuccini et
al. (1997) and Briccoli et al. (2002)
also suggested using of ethanol in decontamination procedure of olive explants.
It has been shown that ethanol by eliminating the air bubbles on explants may
increase the efficiency of Clorox and/or other decontamination treatments. Ethanol
also may dissolve the waxes surrounding the young tissues and improves the penetration
of decontamination factor into the tissues. On the other hand, ethanol also
exerts antimicrobial properties by itself. However, the results of this study
showed that ethanol may not decrease in vitro contaminations significantly.
Based on the results of ethanol and Clorox experiment, the supplementary decontamination
treatments are essential for disinfecting olive explants.
No study investigating the effects of nano-silver on decontamination explants
of woody plants are reported in literature. Present results showed that nano-silver
significantly prevent the incident of in vitro contaminations of olive
explants. Abdi et al. (2008) also showed the
effects of nano-silver particles on controlling the internal contamination of
valerian (Valeriana officinalis L.): these researchers obtained the best
results of decontamination after submersing the surface sterilized valerian
explants in 100 mg L-1 nano-silver solution for 60 min. Submerging
the olive explants in nano-silver solutions after surface sterilization also
showed that nano-silver is very effective in controlling fungus and bacterial
contaminations. However, this method is not applicable for olive explants because
of severe injuries and browning of the explants. On the other hand, adding nano-silver
particles to the media controlled the in vitro contaminations significantly
and did not affect the growth of explants.
The mechanism of action of nano-silver particles in terminating the microorganisms
is not known clearly. However it has been shown that nano-silver particles release
silver ions (Ag+) slowly and the Ag+ can destroy the cell
structure of microorganisms (Lubick, 2008). Dibrov
et al. (2002) stated that the effects of Ag++ on microorganisms
may be due to their chemosmotic activity: these researchers showed that Ag+
affects the phospholipids and destroy the cell membrane of microorganisms; Ag+
also may substitute the sulphur in the -SH groups of cell membrane of
microorganisms and destroy them. Tang et al. (2007)
showed that the destructive effects of Ag+ are related to production
of active silver containing organic compounds, these compounds can attract the
microorganisms and destroy their structure.
In the current study the harmful effects of nano-silver reported on higher
plants for the first time. Such effects also may due to the adverse effects
of high concentrations of Ag+ on cell membrane of explants. These
results are in contrast with those reported by Abdi et
al. (2008) and show that differences in plants tissue and/or prolonged
decontamination treatments may adversely affect the results of using nano-silver.
Martino et al. (1999) also showed the sensitivity
of olive explants to AgCl. However, the results of current study showed that
low concentrations of Ag+ in media are tolerable for olive Mission
explants and may control the incident of contaminations.
The results of this study showed the effects of nano-silver particles on eliminating the fungal and bacterial contaminations of olive explants. Nano-silver eliminates the internal infections of in vitro explants and it is not toxic for the operator and for the environment. As the results showed, submerging of explants in high concentrations of nano-silver may kill them. At the end, it is suggested to add low concentrations of nano-silver particles in media of in vitro cultures of olive. The applicability of using nano-silver in plants tissue culture media as disinfecting agent to explants of other cultivars and/or species should be tested.