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Effect of some environmental stress in tissue culture media on in vitro propagation and antioxidant activity of medicinal plant Ziziphora canescens Benth.


Plant tissue and cells culture technique was used to determine the extent of plant tolerance to environmental stresses such as drought and salinity (Luttus et al, 1999). (Shibli et al, 1992) showed that plant tissue culture provides us with important information about the response of plants to stress of salinity, and the researcher can monitor small changes to the response of shoots and roots with presence of stresses imposed on the plants, because we can control conditions of growth which surrounding with plants in vitro more than ex vivo. so it Possible studying the effect of some of the biotic and abiotic factors that determine spread and growth of plant, and impact of these stresses on in vitro propagation and content the plant of active substances that play a role as antioxidant, Among the abiotic stresses: drought, pH, concentration of nutrient elements and salinity that cause great damage to plants and affect on crops productivity. There are many studies that including effect of environmental stresses on the micropropagation of species, such as: effect of salinity on the in vitro propagation of potato (Khenifi et al, 2011), also effect of the concentration of nutrient elements on the in vitro propagation of Ruscus hypoglossum (Abou Dahab et al., 2005) where used MS, 1/2 MS and 1/4 MS media, and effect of pH on in vitro propagation of Pinus sylvestris (Andersone and Ievinsh, 2008).

Most environmental stresses are affecting on the production of reactive oxygen species (ROS) in plants, causing oxidative stress (Smirnoff, 1993; Hendry, 1994; Bartosz, 1997) and these ROS adversely affect crops yield and quality (Rahimizadeh et al., 2007; Baby and Jini, 2011).

Plants can produce antioxidants for protection against the cytotoxic species of activated oxygen such as superoxide (O2 x -), hydrogen peroxide ([H.sub.2][O.sub.2]), and the hydroxyl radical (OH), The constitutive levels of antioxidant enzymes in plants are usually sufficient to scavenge the free radicals and convert them to harmless metabolites (Rajguru et al., 1999). Antioxidant enzymes activity increases in plant cells as a response to environmental stresses (Mohammadi et al., 2011). Also Many studies showed a correlation between the resistance to environmental stresses and the efficiency of the antioxidant system (Demirel and Turkan, 2005). when increasing the amount of free radicals in the body we have to provide the body with out sourcing of antioxidants, especially natural resources that found in medicinal plants (Devasagayam et al., 2004).

Ziziphora canescens Benth (Ziziphora clinopodioides Lam) Belongs to Lamiaceae, dried aerial parts use for coughs and colds (Verdian-rizi, 2008), also many studies showed the impact of plant as an anti-bacterial (Salehi et al., 2005; Sonboli et al., 2006), antifungal (Behravan et al., 2007), antioxidant (Meral et al., 2002; Salehi et al., 2005) and anti-inflammatory (Ghafari et al., 2006). Where many studies indicated that the most effective components in Z. clinopodioides are: Pulegone, Iso menthone, menthol, menthone 1,8-cineole, thymol, pcymene, carvacrol, terpinen-4-01 and Linalool (Kivanc and Akguel, 1986; Baser et al., 1991).

Salinity is one of the most environmental stresses that affect on growth and productivity of agricultural crops (Alkaraki, 2000; Lopez et al., 2002). Where the salinity in soil influence on the plant by decreasing its ability to absorb water (osmotic stress) and accumulation of absorbed salt in the tissues to toxic levels (ionic stress) (Munns et al., 1995). increasing concentration of salt causes stunting of growth or could be lead to the death of plants (Cavagnaro et al., 2006).

As for pH values, increasing pH of medium above the optimal level leads to the emergence of abnormalities in evolution of plant parts such as the reduction in the growth of stem and roots, as well as changes in color of the leaves (Gurel and Gulsen 1998; Laukkanen et al., 2000). pH of medium affect on absorption of nutrients by plant tissues (Quak, 1977; Hussey, 1986) also pH of medium is one of important environmental chemical and physical factors during the evolution of plant tissue in the conditions of in vitro culture (Williams et al., 1990).

Regarding the mineral nutrient (inorganic) are one of the basic components in tissue culture media that always must be added, in addition to a source of carbon, vitamins, amino acids and plant growth regulators which not necessary to add it to tissue culture media (George and de klerk, 2008). A great time and effort have been make to determine optimal concentrations for each of the basic fourteen mineral nutrients which is added to tissue culture medium (Marschner, 2003). The main difference between the used nutrients media in tissue culture is the macro elements, specifically, concentration of ammonium, nitrate and total concentration of nitrogen (Bell et al., 2009). Also there is inverse relationship between medium strength and secondary metabolite accumulation in spearmint tissue cultures could be possibly explained by the fact that full nutrient media predominantly promote primary metabolism and cellular growth, in some cases hampering morphological and biochemical tissue differentiation (Fadel et al., 2010).

Assessment of plant tolerance to environmental stresses in the field is restricted with agriculture season also influenced by the environment and changed climate surrounding of plants so the credibility of the results could be lost because impact more of one stress on the plants. Therefore, plant tissue culture technique provides the time and effort and gives more realistic evaluation of results. The current research aims to impose artificial stresses on plants in vitro, so we studied effect of different concentrations of NaCl, reduce the concentration of macro elements and different levels of pH on in vitro propagation of Z. canescens to assessment the extent of tolerance this medicinal plant for some prevailing environmental conditions, also we tested effect of these stresses on activity of plant extract as antioxidant where plants of Lamiaceae contain a wide variety of chemicals including phenolic compounds, Flavonoids and phenolic acids, these compounds have antioxidant properties and anti bacteria. On the other hand no previous study about effect of environmental stresses on the in vitro propagation and antioxidant activity of Z. canescens.


This research carried out in the laboratory of plant tissue culture and molecular biology at department of plant biology--Faculty of Sciences--University of Damascus.

1--Plant material and surface sterilization:

Plant samples were collected from mountains of Kalamoon (Assal-Alward) in June 2012. apical and lateral buds were isolated and sterilized using mercuric chloride Mg[Cl.sub.2] 0.1% for two minutes, after that washed with sterilized distilled water and planted on Woody Plant Medium (Lloyd and McCown, 1981), polluted and dead buds were excluded after a month of culture.

2--In vitro propagation of Z. canescens:

Shoots were propagated on medium WPM without plant growth regulators, the results (number and length of formed shoots) were taken after month of culture.

3--Plant extract preparation:

Aerial parts of wild and in vitro plants were collected and dried at room temperature and ground by pistil and mortar to a soft powder. 5 g of powder were immersed in 100 ml of distilled water and was heated in a water bath for 30 min at 95[degrees]C. Then the samples were centrifuged and the supernatants were transferred to new tubes and stored at -20[degrees]C.

4--Free radical scavenging activity test:

The free radical scavenging activity of samples was measured using 2, 2-diphenyl-1-picrylhydrazyl (DPPff), following the method described by Blois (1958) with some modifications (Laouini, et al., 2012). DPPH is reduced to hydrazine when it reacts with hydrogen donors. Briefly, serial dilutions (10, 1, 0.1, 0.01, 0.001 mg/ml) of samples aqueous extracts were tested for radical scavenging activity. 0.2 mM DPPH was prepared in methanol and 500 [micro]l of this solution was added to 1000 [micro]l of a sample at different concentrations. The samples were incubated in the dark at room temperature for 30 min. After that, the absorbance was measured at 517 nm using spectrophotometer. The percentage of radical scavenging activity (RSA%) was calculated using the following equation:

RSA% = [([A.sub.0] - [A.sub.1]/[A.sub.0]] x 100

In which [A.sub.0] is the absorbance of the control reaction, and [A.sub.1] is the absorbance in the presence of sample. In addition, the inhibitory concentration that reduces of 50% of free radicals ([IC.sub.50]) was determined. The experiments were repeated three times.

5--Study effect of some environmental stress on in vitro propagation and the antioxidant activity:

The results (number and length of formed shoots) were taken after month of culture and plant extracts were obtained to determine antioxidant activity.

5-1 Effect of salinity:

Six treatments were prepared to test the effect of salinity, where NaCl was added to media with different concentration (1-2-3-4-5-6 g / l), pH adjusted to 5.7.

5-2 Effect of pH media:

WPM media with different pH levels from 3 to 9 were tested, pH adjusted by HCl and NaOH 1N.

5-3 Effect of macro elements concentration:

Macro elements concentration in WPM medium was reduced to half and quarter, While maintaining the concentration of micro elements and vitamins.

Conditions of Culture:

The plantlets were grown in growth room at 23 [+ or -] 1[degrees]C and 16 h photoperiod with 2000 lux light intensity. pH values were adjusted before adding agar and autoclaved of medium at 121[degrees]C.

Statistical analysis:

For all experiments, the obtained data were analyzed using Analysis of variance (ANOVA). Significant differences between means were determined using the Duncan's test at the 0.05 probability level. each experiment was repeated three times.


1--In vitro shoot formation of Z. canescens:

The results showed that the maximum of shoot production on a medium WPM was 7.5 [+ or -] 0.202 with length 8.7 [+ or -] 0.065 cm after 4 weeks of culture. (Fig.1A)

2--Free radical scavenging activity:

The results of DPPH assay (Table 1) showed increasing antioxidant activity with increasing of aqueous extracts concentration of wild and in vitro plants, it was observed a clear increase of RSA% at concentration 1 mg/ml and the highest value of RSA% was 85.1% of in vitro plants extracts. Also [IC.sub.50] value (0.213) of in vitro plants was better than [IC.sub.50] (0.279) of wild plant extracts.

3--Effect of environmental stress on in vitro propagation and on antioxidant activity of Z. canescens:

3-1: Effect of salinity:

As shown in Table 2, the different applied concentrations of NaCl effected on shoot number and length with significant differences, where the low level of salinity (1, 2 g\l) NaCl in culture medium significantly enhanced and increased shoot length but no significantly of shoot number. High salinity levels (3 to 6 g\l) NaCl caused a decrease in number and length of shoots. (Fig. 1B, 1C, 1D)

There were differences in antioxidant activities with increasing NaCl concentration (Table. 2). at 6 g/l NaCl the [IC.sub.50] was better (0.01 mg/ml) than lower concentrations of NaCl but with significant differences except at 5 g/l NaCl, also increased concentration of NaCl enhanced (decreaced) [IC.sub.50] but no significantly between 3,4,5 g/l.

3-2: Effect of medium pH levels:

The results showed significant differences between pH levels. It was observed that pH level 6 is better than all other pH levels for shoot number (7.6) and length (8.9 cm). and these values decreased at lower and higher levels of pH 6. (Table 1). (Fig.1E, 1F, 1G, 1H)

There were no significant difference between pH levels of 4 and 9 for shoot number, and pH levels of 5 and 7 for shoot length. Also the results of the present study showed that lower pH levels (<6) decreased shoot number and length more than higher pH levels (>6). Also we noticed the beginning of the death and decline in growth tops of shoots that cultured on medium pH = 3 and 4 after 15 days after culture, so we can say Z. canescens tolerance higher pH more than lower pH.

As shown in Table 3, the best value of [IC.sub.50] was 0.08 at pH=9 with significant difference with all of studied values. but no significant difference between [IC.sub.50] at pH (5,6,7) and (3,8) and (3,4). also we noticed that [IC.sub.50] in the case pH>6 better than pH<6.

3-3 Effect of macro elements concentration:

The data in Table (4) clearly showed that significant different between full and half and quarter salt strengths that effected on the growth and development of cultured Z. canescens in vitro. The mean number of formed shoots per explant was the higher in the case of full salt strength (7.5) as compared with 1/2 salt strength (5.2) and 1/4 (4). The longest shoots were found when using full strength (8.7 cm) followed by 1/2 strength (6.3 cm) and 1/4 (4.5 cm). (Fig.1I). whereas [IC.sub.50] decreased in the case of 1/2 WPM (0.2 mg/ml) and 1/2 WPM (0.201 mg/ml) with significantly different compared with WPM (0.213 mg/ml).



1--DPPH radical scavenging activity:

Antioxidants are component which inhibits oxidation or free radicals induced oxidative damage and therefore the importance of the search and exploitation of natural antioxidants, especially of plant origin, has greatly increased in recent years (Gulcin, 2010; Sarikurkcu, 2011). In the current study, DPPH scavenging activity was found in both in vitro propagated and wild plants extract. also in vitro propagated plants extract exhibited a higher antioxidant activity. This result supported by (Nagesh and Shanthamma, 2011) showed that In vitro derived leaf extracts showing more antioxidant activity than natural leaves extracts and also decided that there is correlation between the levels of phenolic content and antioxidant activity.

2--Effect of salinity:

Plantlets grown in vitro presented tolerance but their growth was negatively affected at high salt concentrations. At 2 g/l NaCl the shoot length was increased significantly as compared with the control but shoot number didn't effect. The plantlets in vitro toleranced given range levels of NaCl but shoot length and number decreased. Our results supported by (Bahmani et al, 2012) and also by (Khenifi et al, 2011) showed that the addition of NaCl to the culture media decreased the osmotic potential of the media inducing salinity stress that adversely affected the plants growth of potato cultivars (Solanum tuberosum L). Other parameters were studied by (Shatnawi et al., 2010), Proline and sodium contents increased with elevated salinity, whereas potassium, nitrogen, protein, Chlorophyll and carotenoid decreased of Chrysanthemum morifolium.

Results also showed significant correlations between salinity stress and some antioxidant properties ([IC.sub.50]). (Demirel and Turkan, 2005) showed that Salt stress-induced changes in antioxidant enzymes, such as catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR), CAT and APX activities increased with increasing salt stress. Also plant phenols and antioxidants are inducible by environmental stress (Dixon and Paiva, 1995), and the appropriate plant stress responses may be associated with changes in the level of all these components. Also (Wegener and Jansen, 2013) showed that Drought stress caused a significant decrease in tuber yield but had no significant effect on anthocyanins (Ac), peroxidase enzyme (POD), ascorbic acid equivalent (ACE) and trolox equivalent (TXE), wounding stress significantly induced the POD activity in control and drought stressed tubers of all genotypes. (Zhou, 2004) showed that Positive correlations were detected between the DPPH * scavenging activity and either total solar radiation or average daily solar radiation and suggested that growing conditions may influence the antioxidant properties of wheat.

3--Effect of medium pH levels:

The best medium pH on tissue culture media difference according to stages of micropropagation and roots formation in woody species (von Arnold and Eriksson 1982; Williams et al., 1985; Saborio et al., 1997; Ostrolucka et al., 2004). And (Quak, 1977; Hussey, 1986) reported that pH of the medium has a regulatory effect on nutrient uptake by the explant tissue.

Our experiments showed that the species Z. canescens tolerance different levels of pH but with decrease number and length of shoots. It is clear that pH between 5-8 supported growth, where low values (lower than 5) and high values (higher than 8) stop growth and development plant. May be Plant tissues can reserve on stability pH in cytoplasm relatively, if external medium pH is between 4 to 9 (Caponetti et al., 1971). (Modarres Sanavy and Jami Moeini, 2003) showed that pH 5.5 was the best for all the traits and The reduction growth and rooting of Solanum tuberosum L. was more pronounced at low levels (4.5, 5) than high levels of PH (6, 6.5). also (GUrel and Gulsen, 1998) showed that pH 5.5 was significantly more effective than the other pH levels for both shoot production and shoot growth of Almond (Amygdalus communis L.) and the lowest (4.5) and the highest (6.5) pH levels effected in the normality development of shoots and leaves.

As we notice responses and tolerance of plant to pH difference between species and depend on genetic variation which effect in tissue culture results. Also the best medium pH might be difference between tissue culture media and soil (Sen et al., 1994) and media for acclimatization plant on ex vivo (Ingestad 1979; Bozhkov and von Arnold 1998).

Also our experiments have shown that effect of pH levels on the antioxidant activity as an increase in pH value led to better value of [IC.sub.50]. In plants, the antioxidant activity can mitigate effects of abiotic and biotic stress by scavenging reactive oxygen species (ROS), protecting antioxidant enzymes and interacting with other molecules within the signal transduction pathway (Grace, 2005; Hatier and Gould, 2009). (Moore et al., 2006) showed that each antioxidant property of hard winter wheat bran is influenced differently by genotype and growing conditions. Also (Rajguru et al., 1999) showed that the leaves and callus tissue of some cultivars of cotton exhibit an increase in antioxidant enzyme activity in response to an increase in reactive oxygen species concentrations and, thus, demonstrate some stress resistance.

4--Effect of salt strength:

The other factor we examined was salt strength, the data clearly showed that different WPM salt strengths (full, half and quarter) had a significant effect on the growth and development of Z. canescens cultured in vitro. The number and length of shootlets produced per explant was the greatest in the case of full salt strength as compared with 1/2 and 1/4 salt strength. And disagrees with (Fadel et al., 2010) showed that the highest number and length of shoots of Mentha spicata were observed on half strength medium. But (Abou Dahab et al., 2005) showed that the shoot number of Ruscua hypoglossum cultured in vitro was the greatest in the case of full salt strength as compared with 1/2 and 1/4 salt strength but the longest shoot was found when using 1/2 strength of MS salts. Also (Groll et al, 2002) showed that the maximum number of somatic embryos from manihot esculenta differentiated from a proliferative nodular embryogenic callus on either half or full strength MS medium, and the greatest number of cotyledonary stage embryos were formed on full strength MS medium.

Also our experiments showed that the strength of the culture medium was inversely correlated with the antioxidant activity of plant extracts and Consistent with (Fadel et al., 2010) where showed that the highest average phenolic content was observed on shoot and root extracts cultured onto quarter and half strength medium.


Our present study showed that Z. canescens toleranced different levels of salt NaCl, pH and decrease strength salt and these stresses that imposed on the plant led to increase the antioxidant activity and the best result was when 6 g/l NaCl was added to tissue culture medium. Also we can propagate Z. canescens on WPM medium without add plant growth regulator.


Article history:

Received 25 January 2014

Received in revised form 12 March 2014

Accepted 14 April 2014

Available online 5 May 2014


The authors would like to thank Faculty Members for scientific support in Department of Plant Biology, Damascus University, Damascus.


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Abdulkarim Dakah, Mohamad Suleiman and Salim Zaid

Department of Plant Biology, Faculty of Sciences, University of Damascus, Damascus, Syria

Corresponding Author: Abdulkarim Dakah, Department of Plant Biology, Faculty of Sciences, University of Damascus, Damascus, Syria.

Tel: +963 988074093. E-mail:
Table 1: Radical scavenging activity and [IC.sub.50] [+ or -] SE
of in vitro propagated plants on WPM media using aqueous

Concentration of aqueous
extracts (mg/ml)                  0.001                 0.01

Radical scavenging         27.3 [+ or -] 0.621   35.4 [+ or -] 0.212
activity RSA%
wild plant)

Radical scavenging         29.2 [+ or -] 0.57    38.5 [+ or -] 0.115
activity RSA%
(in vitro plant)

Concentration of aqueous
extracts (mg/ml)                   0.1                    1

Radical scavenging         45.5 [+ or -] 0.012   68.1 [+ or -] 0.076
activity RSA%
wild plant)

Radical scavenging         47.1 [+ or -] 0.057   70.2 [+ or -] 0.088
activity RSA%
(in vitro plant)

Concentration of aqueous
extracts (mg/ml)                   10

Radical scavenging         80.2 [+ or -] 0.065
activity RSA%
wild plant)

Radical scavenging         85.1 [+ or -] 0.057
activity RSA%
(in vitro plant)

Concentration of aqueous
extracts (mg/ml)            [IC.sub.50] (mg/ml)

Radical scavenging           0.279 [+ or -] b
activity RSA%
wild plant)

Radical scavenging         0.213 [+ or -] 0.001a
activity RSA%
(in vitro plant)

Means followed by similar letters in each column are not
significantly different at 5% level according to Duncan's Test.

Table 2: Effect of Concentration of NaCl on in vitro propagation
and antioxidant activity of Z. canescens.

of NaCl (g/l)           Mean shoot           Mean shoot length
on WPM medium    numbers (no [+ or -] SE)     (cm [+ or -] SE)

0                7.5 [+ or -] 0.202a         8.7 [+ or -] 0.065c
1                7.7 [+ or -] 0.209a         9.3 [+ or -] 0.094b
2                7.6 [+ or -] 0.201a        10.1 [+ or -] 0.108a
3                6.1 [+ or -] 0.161b         8.2 [+ or -] 0.117d
4                5.2 [+ or -] 0.074c         7.5 [+ or -] 0.12e
5                  4 [+ or -] 0.159d         6.3 [+ or -] 0.152f
6                2.5 [+ or -] 0.092e         4.5 [+ or -] 0.152g

of NaCl (g/l)
on WPM medium      [IC.sub.50] mg\ml

0                0.213 [+ or -] 0.001e
1                 0.17 [+ or -] 0.017d
2                 0.13 [+ or -] 0.017c
3                 0.08 [+ or -] 0.011b
4                 0.06 [+ or -] 0.015b
5                 0.04 [+ or -] 0.011ab
6                 0.01 [+ or -] 0.004a

Means followed by similar letters in each column are not
significantly different at 5% level according to Duncan's Test.

Table 3: Effect of medium pH on in vitro propagation and
antioxidant activity of Z. canescens.

value of pH on         Mean shoot                 Mean shoot
WPM medium       numbers (no [+ or -] SE)   length (cm [+ or -] SE)

3                0.5 [+ or -] 0.092f          1 [+ or -] 0.207f
4                  2 [+ or -] 0.159e          4 [+ or -] 0.15e
5                4.5 [+ or -] 0.133c        6.8 [+ or -] 0.092b
6                7.6 [+ or -] 0.14a         8.9 [+ or -] 0.067a
7                  5 [+ or -] 0.126b        7.1 [+ or -] 0.097b
8                3.5 [+ or -] 0.092d        6.3 [+ or -] 0.141c
9                  2 [+ or -] 0.159e          5 [+ or -] 0.086d

value of pH on        [IC.sub.50 ]
WPM medium               mg\ml

3                 0.15 [+ or -] 0.023bc
4                 0.17 [+ or -] 0.011c
5                0.213 [+ or -] 0.001d
6                0.213 [+ or -] 0.003d
7                0.212 [+ or -] 0.001d
8                 0.12 [+ or -] 0.017b
9                 0.08 [+ or -] 0.01a

Means followed by similar letters in each column are not
significantly different at 5% level according to Duncan's Test.

Table 4: Effect of % WPM medium on in vitro propagation and
antioxidant activity of Z. canescens.

                  Mean shoot                Mean shoot
Medium     numbers (no [+ or -] SE)   length (cm [+ or -] SE)

WPM        7.5 [+ or -] 0.202a        8.7 [+ or -] 0.065a
1/2 WPM    5.2 [+ or -] 0.101b        6.3 [+ or -] 0.091b
1/4 WPM      4 [+ or -] 0.11c         4.5 [+ or -] 0.06c

Medium             mg\ml

WPM        0.213 [+ or -] 0.001b
1/2 WPM    0.201 [+ or -] 0.000a
1/4 WPM      0.2 [+ or -] 0.002a

Means followed by similar letters in each column are not
significantly different at 5% level according to Duncan's Test.
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Author:Dakah, Abdulkarim; Suleiman, Mohamad; Zaid, Salim
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Date:Apr 1, 2014
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