- Email: [email protected]
Morphogenic and biochemical variations under different spectral lights in callus cultures of Artemisia absinthium L.
Journal of Photochemistry and Photobiology B: Biology 130 (2014) 264–271
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Morphogenic and biochemical variations under different spectral lights in callus cultures of Artemisia absinthium L. Umayya Tariq, Mohammad Ali, Bilal Haider Abbasi ⇑ Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan
a r t i c l e
i n f o
Article history: Received 16 September 2013 Received in revised form 26 November 2013 Accepted 27 November 2013 Available online 4 December 2013 Keywords: Thidiazuron 6-Benzyladenine (BA) Phenolics Flavonoids Protease Peroxidase
a b s t r a c t Through its impact on morphogenesis, light is the key environmental factor that alters plant architectural development; however, the understanding that how light controls plant growth and developmental processes is still poor and needs further research. In this study, we monitored the effect of various monochromatic lights and plant growth regulators (PGRs) combinations on morphogenic and biochemical variation in wild grown-leaf derived callus cultures of Artemisia absinthium L. Combination of a-naphthalene acetic acid (NAA 1.0 mg/l) and Thidiazuron (TDZ 2.0 mg/l) resulted in optimum callogenic frequency (90%) when kept under fluorescent light for 4 weeks (16/8 h). In contrast to the control (white spectrum), red spectrum enhanced peroxidase activity, protease activity, total protein content and chlorophyll a/b ratio. Green spectrum was found to be more supportive for total phenolics, total flavonoids and antioxidant activity. Yellow light enhanced MDA content while white and green light improved total chlorophyll content and carotenoid content. A positive correlation among callogenic response, antioxidant activities and set of antioxidative enzyme activities was also observed in the current report. This study will help in understanding the influence of light on production of commercially important secondary metabolites and their optimization in the in vitro cultures of A. absinthium L. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Artemisia absinthium L., commonly known as ‘‘Wormwood’’ (family; Asteraceae), has been used as herbal medicine throughout Europe, Middle East, North Africa, and Asia ([47]). In the middle Ages, this plant was referred as a ‘‘general remedy for all diseases’’ due to its curative medical powers ([5]). It has been used in China as an antiplasmodial herbal medicine for quinine resistant and quinine analogues resistant patients ([43,56]). Traditionally this species has been used due to its insecticidal, bitter ([3,52]), vermifuge, trematocidal ([15]), diuretic and antispasmodic properties ([33]) and against diarrhoea, cough and common cold ([19]). Phytochemical studies of A. absinthium L. showed the presence of important chemical compounds such as carotenoids [46], flavonoids [8], and many polyphenolic compounds [50]. Chemically major constituents in A. absinthium L. are myrcene, trans-thujones and trans-sabinyl acetate ([29]). The dry leaves and stems of this plant contain 0.25–1.32% essential oil, absinthin, artemisinin, artabsin and matricin ([24]). In vitro regeneration system is very useful for production of phytomedicine because conventional cultivation of plants is subjected to varieties of pests, weather and land availability which adversely affect medicinal qualities of harvested plant [42]. ⇑ Corresponding author. Tel./fax: +92 51 90644121. E-mail address: [email protected] (B.H. Abbasi). 1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2013.11.026
Moreover, callus formation studies are useful to understand metabolic pathways of secondary metabolites (SMs) and somatic embryogenesis [32]. Specific PGRs at appropriate concentrations can perform important function during callogenesis [2]. Among various culture conditions, light illumination influences the accumulation of useful SM [14]. By optimizing in vitro conditions like light sources, SM production can be stimulated [44]. SM from plants have important biological and pharmacological activities, such as anti-oxidative and anti-carcinogenic [6,21, 22,34]. The biological activities of phenolic compounds and flavonoids are associated to their antioxidant potential [16]. The protective action of antioxidant system comes into play under extreme conditions like light, chilling treatment and in defence mechanism against injury. Antioxidant activity has direct relationship with species resistance toward the stressor [51]. Enzymatic and non-enzymatic defence mechanisms have been evolved in plants for the scavenging of reactive oxygen species (ROS). Enzymes involved including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) [26,58]; while the non-enzymatic antioxidants are low molecular weight quenchers such as ascorbic acid (AsA) [23,38,39,55], function together to scavenge reactive oxygen species (ROS). Specific metabolic changes take place when plants are adapted to specific climatic conditions. These changes involve accumulation of protective proteins and regulation of antioxidative enzymes [9].
U. Tariq et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 264–271
Unlike most studies, published to date, on the impact of light on photoregulation during plant development under various illuminations [11,28], the present investigation was undertaken to establish the callus cultures from selected clones of A. absinthium L. under different wavelengths of light, evaluation of hormones required to maintain in vitro leaf derived calli, suitability of specific spectrum for particular biochemical response and correlation between total phenolics, total flavonoids and antioxidant enzyme activities. 2. Materials and methods 2.1. Explant source and surface sterilization Leaf explants were collected from wild grown plants of A. absinthium L. and cultured on MS medium ([35]; Phytotechnology Labs, USA) incorporated with several PGRs. For sterilization purposes, these uncut leaf explants were surface sterilized with 70% ethanol for 60 s followed by immersion in 0.1% (w/v) mercuric chloride solution for 1 min and finally rinsed with sterile distilled water three times. Aseptic transfer was accomplished in Laminar flow hood fitted with a HEPA filter. 2.2. Callus cultures establishment To study the effects of different wavelengths of light and PGRs on callus induction, approximately 1.5 cm of the leaf sections were incubated on Murashige and Skoog (MS) (1962) media supplemented with TDZ, Kin and BA (1.0, 2.0, 3.0, 4.0, and 5.0 mg/l) alone and in combination with NAA (1.0 mg/l). Before autoclaving (121 °C, 20 min, Systec VX 100, Germany), pH of all media was adjusted to 5.8 (Eutech Instruments pH 510, Singapore). All cultures were maintained in a growth room at 25 ± 1 °C under a 16/8-h light/dark photoperiod. Different illumination sources used were sole; cool-white fluorescent tubes (20 W, Toshiba FL20T9D/19) as control 380–780 nm, blue tubes (220 V; 50 Hz, Keliang Ltd.) 380– 560 nm, green tubes (40 W Litex) 480–670 nm, yellow tubes (36 W, Philips Ltd.) 530–780 nm at an intensity ranging from approximately 40–50 lmol m2 s1 or 10,240 lux and red fluorescent tubes (25 W, BINXIANG) 610–715 nm or 640 lux and some cultures were maintained under dark conditions. Light intensity was measured by using Lux meter (SU10, Jeiotech) just under the light source. Changes in callus morphology were recorded on the basis of visual observation. After 3 weeks of culture, the number of responding explants was recorded. Callus subculturing was performed on media with same PGRs composition. 2.3. Analytical methods Extraction of calli was performed according to the protocol described by Giri et al. [17] with minor modifications. Briefly, the dried plant materials were pulverized in chilled pestle and mortar with liquid nitrogen. 0.2 g of the powdered plant tissue was added to 1 ml of 100% pure methanol, mixed and kept for 5 min. Then the samples were sonicated (5 min; Toshiba, Japan), vortexed for 20 min and centrifuged (13,000 rpm, 5 min). The supernatants were collected and either immediately used for analysis or stored at 4 °C. A regression curve of the standard solutions of various concentrations was worked out against their respective absorbance. For antioxidant activity determination, DPPH free radical scavenging assay (FRSA) was used as described by the method of Lee et al. [27] with some modifications. Absorbance was measured at 515 nm using micro-plate reader. The samples which showed more than 70% scavenging at 200 lg/ml concentration were tested
265
at lower concentration by using three fold serial dilutions methodology to find IC50. IC50 values indicate the sample concentration required to scavenge 50% DPPH free radicals ([1]), was calculated by table curve software. The RSA was determined as percentage of DPPH° discoloration using the equation;
% scavenging DPPH free radical ¼ 100 ðAbc Abs =Abc Þ where Abc is absorbance of the negative control and Abs is the absorbance of the DPPH° solution with sample (Methanolic extract) added. Total phenolic content (TPC) was determined by the method of Velioglu et al. [53] by using Folin–Ciocalteu reagent. Absorbance was measured at 630 nm by using UV/VIS–DAD spectrophotometer (Halo DR-20, UV–VIS spectrophotometer, Dynamica Ltd., Victoria, Australia). The calibration curve (0–50 lg/ml, R2 = 0.968) was plotted by using gallic acid as standard. All determinations were performed in triplicate and the TPC was expressed as mg/g gallic acid equivalents (GAE) of dry weight. For total flavonoid content (TFC) determination, aluminium chloride colorimetric method was used as described by Chang et al. [9]. Absorbance of the reaction mixtures was measured at 405 nm by using UV/VIS-DAD spectrophotometer. The calibration curve (0–40 lg/ml, R2 = 0.998) was plotted by using quercetin as standard. The TFC was expressed as mg/g quercetin equivalents (QE) of dry weight. Malondialdehyde content was determined by thiobarbituric acid (TBA) test as described by Bailly et al. [4] with some modifications. Absorbance of the supernatants was recorded at 532 nm and 600 nm. The values of non-specific absorbance at 600 nm were then subtracted from that of 532 nm. Lipid per-oxidation (MDA content) was expressed as lM/g fresh weight by using extinction coefficient of 155 mM1 cm1. Total protein content was determined using method of Lowry et al. [30] with some modifications. Enzyme extract was prepared in the same way as that for antioxidant enzyme assay. The absorbance was then measured at 650 nm using spectrophotometer (Shimadzu, UV-120-01) Standard curve of BSA was then prepared by absorbance versus micrograms protein or vice versa and the unknown protein from the sample was determined from the curve. Chlorophyll ‘‘a’’, ‘‘b’’ and total chlorophyll content were investigated following the method of Hiscox and Israelsham [20] with some modifications. The resulted extract color intensity was measured using spectrophotometer (Shimadzu, UV-120-01) at 645 nm and 663 nm to estimate chlorophyll ‘‘a’’ and chlorophyll ‘‘b’’. The chlorophyll content (mg/g fresh weight) was calculated using the following formulas [13]. Chl a ðmg=g FW of leafÞ ¼ ½ð0:0127ðOD663 Þ 0:00269ðOD645 Þ 100 Chl b ðmg=g FW of leafÞ ¼ ½ð0:0229ðOD645 Þ 0:00468ðOD663 Þ 100 Total chlorophyll content ¼ ½0:0202ðOD645 ÞÞ þ 0:00802ðOD663 Þ 100 Chl a ratio b ¼ Chl a=Chl b
Here, O.D. = Optical Density. The absorbance of the extract was also checked at 480 nm to check total carotenoids. Total carotenoids were then calculated by formula given below.
Carotenoids ðmg=g FW of leafÞ ¼ OD480 0:5 The enzymes extracts for antioxidant enzyme activities were prepared by following the method of Nayyar and Gupta [36] with some modifications. Briefly, 1 g of sample tissue was homogenized with 10 ml of extraction buffer (50 mM potassium phosphate buffer with 1% PVPP at pH 7). The homogenate was then centrifuged at 15,000g at 4 sC for 30 min. and the supernatant was used for enzyme assay. Peroxidase (POD) activity was determined by the
266
U. Tariq et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 264–271
Table 1 PGRs concentrations and combinations, callus formation % and morphology under white light (16-h light and 8-h dark) kept for 4 weeks. Data show mean of three replicates ± SE. Sr. No.
Treatments (mg/l)
Callus initiation (day)
Callus induction frequency (%)
Callus color
Callus texture
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
MS + TDZ 1.0 MS + TDZ 2.0 MS + TDZ 3.0 MS + TDZ 4.0 MS + TDZ 5.0 MS + TDZ 1.0 + NAA 1.0 MS + TDZ 2.0 + NAA 1.0 MS + TDZ 3.0 + NAA 1.0 MS + TDZ 4.0 + NAA 1.0 MS + TDZ 5.0 + NAA 1.0 MS + Kn 1.0 MS + Kn 2.0 MS + Kn 3.0 MS + Kn 4.0 MS + Kn 5.0 MS + Kn 1.0 + NAA 1.0 MS + Kn 2.0 + NAA 1.0 MS + Kn 3.0 + NAA 1.0 MS + Kn 4.0 + NAA 1.0 MS + Kn 5.0 + NAA 1.0 MS + BAP 1.0 + NAA 1.0 MS + BAP 2.0 + NAA 1.0 MS + BAP 3.0 + NAA 1.0 MS + BAP 4.0 + NAA 1.0 MS + BAP 5.0 + NAA 1.0
20 20 20 20 20 20 20 20 20 20 30 30 30 30 30 30 30 30 30 30 20 20 20 20 20
43 56 62 74 67 83 90 70 66 51 70 63 56 41 33 62 77 53 46 39 57 84 77 65 53
DG DG DG DG DG DG DG DG DG DG YG YG YG YG YG YG YG YG YG YG LG LG LG LG LG
F F F F F F F F F F C C C C C C C C C C F F F F F
F = Friable; C = Compact; DG = Dark green; and YG = Yellow green.
Fig. 1. (A) Callus initiation from leaf explants. (B and C) Callus proliferation. (D) Callus after 4 weeks of culture.
method of Lagrimini [25] with some modifications. Protease activity was assayed following the method of McDonald and Chen [31] with minor modifications.
2.4. Data analysis All experiments were repeated twice and each treatment was consisted of three replicates. Mean values of various treatments were subjected to analysis of variance (ANOVA) and significant difference was separated using Duncan’s Multiple Range Test (DMRT). SPSS (Windows version 7.5.1, SPSS Inc., Chicago) was used to determine the significance at P < 0.05 ([12]).
3. Results and discussion 3.1. Callogenesis The efficacy of callogenesis from leaf explants was screened for optimum levels at different wavelengths of light and in response to different plant growth regulators either alone or in combinations (Table 1). Callus was initiated in response to almost all of the hormones tested. However, a combination of TDZ (2.0 mg/l) + NAA (1.0 mg/l) produced optimum response under in vitro conditions (16 h white light/8 h dark; control). Different concentrations (1.0–5.0 mg/l) of BA resulted in direct shooting response (data not shown).
267
U. Tariq et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 264–271 100
Table 2 The application of different wavelengths of light in conjunction with combination of PGRs from T1 to T6 for in vitro callogenesis of Artemisia absinthium L.
Callus induction %
80
60
Treatment
MS + PGRS (mg/l)
Light regimes
Incubation period
T1
MS + TDZ 1.0 MS + TDZ 1.0 MS + TDZ 1.0 MS + TDZ 1.0 MS + TDZ 1.0 MS + TDZ 1.0
2.0 + NAA
Darkness
24-h dark (4 weeks)
2.0 + NAA
White
2.0 + NAA
Red
24-h light 8-h dark (4 weeks) 24-h light (4 weeks)
2.0 + NAA
Blue
24-h light (4 weeks)
2.0 + NAA
Green
24-h light (4 weeks)
2.0 + NAA
Yellow
24-h light (4 weeks)
T2 T3
40
T4 T5
20
T6
MS + TDZ 1.0 MS + TDZ 2.0 MS + TDZ 3.0 MS + TDZ 4.0 MS + TDZ 5.0 MS + TDZ 1.0 + NAA 1.0 *MS + TDZ 2.0 + NAA 1.0 MS + TDZ 3.0 + NAA 1.0 MS + TDZ 4.0 + NAA 1.0 MS + TDZ 5.0 + NAA 1.0 MS + Kn 1.0 MS + Kn 2.0 MS + Kn 3.0 MS + Kn 4.0 MS + Kn 5.0 MS + Kn 1.0 + NAA 1.0 MS + Kn 2.0 + NAA 1.0 MS + Kn 3.0 + NAA 1.0 MS + Kn 4.0 + NAA 1.0 MS + Kn 5.0 + NAA 1.0 MS + BAP 1.0+ NAA 1.0 MS + BAP 2.0+ NAA 1.0 MS + BAP 3.0+ NAA 1.0 MS + BAP 4.0+ NAA 1.0 MS + BAP 5.0+ NAA 1.0
0 100
Treatments (mg/l)
Callogenesis started at cut end of leaf explants. TDZ as callus inducing hormone produced dark green and friable calli (Fig. 1). 74% callogenic frequency was found at 4.0 mg/l TDZ as compared to much improved callogenic response (90%) when TDZ (2.0 mg/ l) was experimented in combination with NAA (1.0 mg/l), followed by 84% callus response at BAP (2.0 mg/l) + NAA (1.0 mg/l) under white spectrum. Similarly, when Kn (1.0 mg/l) was used alone, callogenic response remained less effective (70%) as compared to more pronounced effect (77%) in response to Kin (2.0 mg/l) with NAA (1.0 mg/l) (Fig. 2). These results are in harmony with that reported by Nin et al. [37]; who concluded from their study that Artemisia spp. indicated good responses in media augmented with NAA. The calli were developed by the applications of monochromatic lights with 24 h exposure except the white light (16 h light/8 h dark) which was used as control (Table 2). When investigated under different monochromatic lights, callus formation frequency was highest (90%) under white light, followed by 82% under green light and 70% in dark (Fig. 3). In the hairy root cultures of A. annua L., it was found that the biomass of hairy roots and artemisinin content under red light were 17% and 67% higher than those obtained under white light [54]. Conflicting results from different investigators may be due to species differences and differences in light sources and intensities. In the present study, morphological variations have also been observed in callus cultured under different lights, which is supposed to indicate differences in biochemistry and physiology (Fig. 4).
3.2. Total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity In the current study, 12.215 mg/g DW and 2.015 mg/g DW TPC and TFC respectively were quantified in callus tissues of A. absinthium obtained in response to TDZ and NAA under white light (control) (Fig. 5) followed by 11.259 mg/g DW and 0.87 mg/g DW TPC and TFC under red light, respectively (Fig. 6). In contrast to our findings Singh et al. [49] stated that ethanolic extract of aerial parts of A. absinthium has significantly high concentration of flavonoids
Callus Induction (%)
Fig. 2. Percent callus formation in response to different plant growth regulators. Data were collected after 4 weeks of culture. Values are the mean ± standard error from three replicates.
80
60
40
20
0
Yellow
Green
Blue
Red
White
Dark
Light Treatments Fig. 3. Callus formation % under different light treatments at optimized hormonal concentration (2.0 mg/l TDZ and 1.0 mg/l NAA). Data were collected after 4 weeks of culture. Values are the mean ± standard error from three replicates.
(1108.15 ± 48.78 mg of QUE/g of extract) and phenolics (43.04 ± 0.57 mg of GAE/g of extract). In the present study, a positive correlation has been confirmed between high TPC and antioxidant activities as reported by [8,45] (Fig. 7). In the DPPH assay, the range of IC50 values recorded in calli of A. absinthium was 67.4– 3798 lg/ml DW. IC50 (3222 lg/ml DW) was shown by calli obtained on the medium containing TDZ 2.0 mg/l and NAA 1.0 mg/l while maximum IC50 (3798 lg/ml DW) was recorded in the calli raised on TDZ (5.0 mg/l) + NAA (1.0 mg/l). Among different light treatments given to calli, highest value of IC50 (1070 lg/ml DW) under green light was noticed followed by 882 lg/ml DW DPPH activity under blue light source. 3.3. Peroxidase activity and Malondialdehyde (MDA) content Maximum MDA content of 11.09 lM/g FW was found at Kin (4.0 mg/l) and NAA (1.0 mg/l) with callogenic response of 46% and 10.08 lM/g FW at TDZ (5.0 mg/l) with 67% callogenesis as compared to other hormonal concentrations under white light (Fig. 8). The reason might be that stress exerted on explants by applied light sources and PGRs caused an increase in lipid peroxidation takes place leading to lower rates of physiological processes. Similar increase in MDA content was observed in Toyonoka strawberry during callogenesis under different colored plastic films and gradually decreases with less callogenic percentage [57].
268
U. Tariq et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 264–271
Red
Yellow
White (control)
Dark
Green
Blue
Fig. 4. Effect of different lights on callus growth and morphology under different light sources at same hormonal combination (2.0 mg/l TDZ + 1.0 mg/l NAA).
14
10
2.0
8
1.5
6
1.0
4
0.5
2
0.0
0
-0.5 MS + TDZ 1.0 MS + TDZ 2.0 MS + TDZ 3.0 MS + TDZ 4.0 MS + TDZ 5.0 MS + TDZ 1.0 + NAA 1.0 MS + TDZ 2.0 + NAA 1.0 MS + TDZ 3.0 + NAA 1.0 MS + TDZ 4.0 + NAA 1.0 MS + TDZ 5.0 + NAA 1.0 MS + Kn 1.0 MS + Kn 2.0 MS + Kn 3.0 MS + Kn 4.0 MS + Kn 5.0 MS + Kn 1.0 + NAA 1.0 MS + Kn 2.0 + NAA 1.0 MS + Kn 3.0 + NAA 1.0 MS + Kn 4.0 + NAA 1.0 MS + Kn 5.0 + NAA 1.0 MS + BAP 1.0+ NAA 1.0 MS + BAP 2.0+ NAA 1.0 MS + BAP 3.0+ NAA 1.0 MS + BAP 4.0+ NAA 1.0 MS + BAP 5.0+ NAA 1.0
2.5
Treatments (mg/l)
Fig. 5. Total phenolic content (mg/g DW) and total flavonoid content (mg/g DW) formation in calli induced in response to different plant growth regulators. Values are the mean ± standard error from three replicates.
Interestingly, highest peroxidase activity (POD) 0.3157 nM/ min/mg was obtained at TDZ (5.0 mg/l) along with highest MDA content 10.06 lM/g FW (Fig. 8). Similarly, Cai et al. [7] found a parallel correlation between elevated MDA content and increased activities of peroxidase (POD) supports this notion. The reason might be that under oxidative stress, peroxidase activity is greater in order to remove toxic hydrogen peroxide along with high Malondialdehyde content formation due to lipid peroxidation. In
12
1400 TPC DPPH FRSA
1200
10
1000
8
800 600
6
400
4
200
2
Antioxidant activity (IC 50 µg/ml DW)
12
Total Phenolic Content (mg/g DW)
3.0
TPC TFC
Total Flavonoid Content (mg/g DW)
Total Phenolic Content (mg/g DW)
14
0 Red
Blue
Yellow
Green
White
Dark
Light Treatment Fig. 6. Total phenolics content (mg/g DW) and DPPH (IC50 lg/ml DW) activity in calli under different light treatments at optimized hormonal combination TDZ 2.0 mg/l and NAA 1.0 mg/l. Values are the mean ± standard error from three replicates.
the present study, maximum POD activity was recorded under red followed by blue and green spectra (Fig. 9). Similarly, in Toyonoka strawberry, G-POD activities were highest under the red films followed by green and yellow films [57]. This shows that higher POD activities are involved in protection against stress conditions. 3.4. Protease activity and total protein content Highest protease activity 2.56 U/g FW was observed when Kin (1.0 mg/l) was used singly as compared to protease activity value (1.7 U/g FW) obtained at Kin (2.0 mg/l) + NAA (1.0 mg/l) whereas
269
U. Tariq et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 264–271
3.5
10
3.0 8
2.5 2.0
6
1.5 4
1.0 0.5
2
Malondialdehyde Content (µM/g FW)
4.0
Antioxidant activity IC50 (µg/ml DW)
4.5
12
0.40
8
5.0
TPC DPPH FRSA
7
MDA POD
0.35 0.30
6
0.25 5
0.20
4
0.15
3
0.10
2
0.05 0.00
1
-0.05
0.0
0
MS + TDZ 1.0 MS + TDZ 2.0 MS + TDZ 3.0 MS + TDZ 4.0 MS + TDZ 5.0 MS + TDZ 1.0 + NAA 1.0 MS + TDZ 2.0 + NAA 1.0 MS + TDZ 3.0 + NAA 1.0 MS + TDZ 4.0 + NAA 1.0 MS + TDZ 5.0 + NAA 1.0 MS + Kn 1.0 MS + Kn 2.0 MS + Kn 3.0 MS + Kn 4.0 MS + Kn 5.0 MS + Kn 1.0 + NAA 1.0 MS + Kn 2.0 + NAA 1.0 MS + Kn 3.0 + NAA 1.0 MS + Kn 4.0 + NAA 1.0 MS + Kn 5.0 + NAA 1.0 MS + BAP 1.0+ NAA 1.0 MS + BAP 2.0+ NAA 1.0 MS + BAP 3.0+ NAA 1.0 MS + BAP 4.0+ NAA 1.0 MS + BAP 5.0+ NAA 1.0
0
Red
Blue
Yellow
Green
White
Dark
Light Treatment Fig. 9. Peroxidase activity (nM/min/mg FW) and malondialdehyde content (lM/g FW) formed under different light treatments. Values are the mean ± standard error from three replicates.
Treatments (mg/l)
3.0
800
Protease TPC
700
11
0.35
10
0.30
9
0.25
8
0.20
7
0.15
6
0.10
5
0.05
4
0.00
3
Treatments mg/l
Fig. 8. Peroxidase activity (nM/min/mg FW) and malondialdehyde content (lM/g FW) in response to different PGRs. Values are the mean ± standard error from three replicates.
at TDZ (1.0 mg/l) maximum protein content observed was 577.14 lg BSAE/20 mg FW with protease activity of 2.4 U/g FW (Fig. 10). In strawberry plantlets which were exposed to red light, POD activity increases with other antioxidant enzymes [41]. The protective adaptive abilities of plants under stress conditions may be due to activation of antioxidant mechanism along with protein synthesis and such activation may be the result of shifts in metabolism. Under different colored illuminations tested, highest total protein content 592.14 lg BSAE/20 mg FW was observed with 2.09 U/g FW protease activity under red light as compared to other light sources (Fig. 11). This shows a positive correlation between peroxidase activities with the activation of
600 2.0
500
1.5
400 300
1.0
200 0.5
100
0.0
0 MS + TDZ 1.0 MS + TDZ 2.0 MS + TDZ 3.0 MS + TDZ 4.0 MS + TDZ 5.0 MS + TDZ 1.0 + NAA 1.0 MS + TDZ 2.0 + NAA 1.0 MS + TDZ 3.0 + NAA 1.0 MS + TDZ 4.0 + NAA 1.0 MS + TDZ 5.0 + NAA 1.0 MS + Kn 1.0 MS + Kn 2.0 MS + Kn 3.0 MS + Kn 4.0 MS + Kn 5.0 MS + Kn 1.0 + NAA 1.0 MS + Kn 2.0 + NAA 1.0 MS + Kn 3.0 + NAA 1.0 MS + Kn 4.0 + NAA 1.0 MS + Kn 5.0 + NAA 1.0 MS + BAP 1.0+ NAA 1.0 MS + BAP 2.0+ NAA 1.0 MS + BAP 3.0+ NAA 1.0 MS + BAP 4.0+ NAA 1.0 MS + BAP 5.0+ NAA 1.0
12
MS + TDZ 1.0 MS + TDZ 2.0 MS + TDZ 3.0 MS + TDZ 4.0 MS + TDZ 5.0 MS + TDZ 1.0 + NAA 1.0 MS + TDZ 2.0 + NAA 1.0 MS + TDZ 3.0 + NAA 1.0 MS + TDZ 4.0 + NAA 1.0 MS + TDZ 5.0 + NAA 1.0 MS + Kn 1.0 MS + Kn 2.0 MS + Kn 3.0 MS + Kn 4.0 MS + Kn 5.0 MS + Kn 1.0 + NAA 1.0 MS + Kn 2.0 + NAA 1.0 MS + Kn 3.0 + NAA 1.0 MS + Kn 4.0 + NAA 1.0 MS + Kn 5.0 + NAA 1.0 MS + BAP 1.0+ NAA 1.0 MS + BAP 2.0+ NAA 1.0 MS + BAP 3.0+ NAA 1.0 MS + BAP 4.0+ NAA 1.0 MS + BAP 5.0+ NAA 1.0
Peroxidase Activity (nM/min/mg FW)
POD MDA
Malondialdehyde Content (µM/g FW)
0.40
Protease activity (U/g FW)
2.5
Total Protein Content ( µg BSAE/20 mg FW)
Fig. 7. Total phenolics content (mg/g DW) and DPPH (IC50 lg/ml DW) activity in calli induced in response to different plant growth regulators. Values are the mean ± standard error from three replicates.
0.45
Peroxidase Activity (nM/min/mg FW)
Total Phenolic Content (TPC mg/g DW)
14
Treatments (mg/l)
Fig. 10. Protease activity (U/g FW) and total protein content (lg BSAE/20 mg FW) produced in response to different PGRs. Values are the mean ± standard error from three replicates.
protein synthesis. As Grudkowska and Zagdanska [18] stated that proteases may be involved in turnover of proteins while responding to different abiotic stresses. It suggests that pathway of SM changes in a way that high protease activity may involve the degradation of damaged proteins in case of abiotic stress and finally the biosynthetic pathway provides amino acids for synthesis of proteins required under stress conditions. However protein contents, peroxidase activity, protease activity and lipid peroxidation have an important role in oxidative stress as indicators of cellular damage. 3.5. Chlorophyll ‘‘a’’, chlorophyll ‘‘b’’ and carotenoid content determination It was found that under green and white light sources, total chlorophyll mass was highest (3.742 mg/g FW; 3.532 mg/g FW,
270
U. Tariq et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 264–271 3.0
700
Protease activity (U/g FW)
400 1.5 300 1.0 200 0.5
100
2.5
1.4
Chlorophyll b Chlorophyll a a/b ratio 2.0
1.2 1.1 1.0 0.8
Red
Blue
Yellow
Green
White
0.6 0.5 0.3 0.1
0.85 0.80 0.75
3.5
0.70
3.0
0.65 0.60
2.5
0.55
2.0
0.50 0.45
1.5
0.40
1.0
0.35
Carotenoid Content (mg/g FW)
Total Chlorophyll Content (mg/g FW)
0.90
0.30
0.5
0.25
0.0 White
Yellow
Green
Red
White
Yellow
Green
Blue
Dark
-0.5
Light Treatment
TCC CC
Red
0.0
0.2
Dark
Fig. 11. Protease activity (U/g FW) and total protein content (lg BSAE/20 mg FW) formed under different light treatments. Values are the mean ± standard error from three replicates.
4.0
0.5
0.4
Light Treatment
4.5
1.0
0.7
0.0
0.0
1.5
0.9
Chlorophyll 'b' (mg/g FW)
500
2.0
1.5 1.3
Chlorophyll 'a' (mg/g FW) a/b ratio
600
2.5
Total Protein Content (µg BSAE/20mg FW)
Protease TPC
Blue
Dark
Light Treatment Fig. 12. Total chlorophyll content (mg/g FW) and carotenoid content (mg/g FW) produced in response to different illuminations. Values are the mean ± standard error from three replicates.
respectively), with maximum callogenic frequencies followed by lower callusing under red light with lower total chlorophyll mass (Fig. 12) while chlorophyll a/b ratio was higher (1.304) under red light conditions (Fig. 13). These results are in consonance with the studies of Shin and co-workers [48] who reported significant reduction in chlorophyll content under red light treatment but no significant variation in a/b ratio. It is worthwhile to state that there is direct relationship between chlorophyll content of the callus and its capacity to regenerate. It means that exposure of callus cultures to various light sources influenced total chlorophyll content and SM production. It was reported that chlorophyll may carry precursors of SM which indicates its role in SM biosynthesis [40]. Highest carotenoid content 0.63 mg/g FW was recorded under red light treatment, followed by 0.585 mg/g FW under white light while lowest carotenoid content 0.287 mg/g FW was observed under dark conditions. The present paper describes that light as a physical factor had a positive influence on callus induction and proliferation in A. absinthium L. It can be concluded that different light regimes may help to optimize growth and developmental changes and this makes the practical application of the photo-technology with respect to design of controlled environment for in vitro safe SM production. To
Fig. 13. Chlorophyll ‘‘a’’ (mg/g FW), chlorophyll ‘‘b’’ (mg/g FW) and chlorophyll a/b ratio produced in response to different illuminations. Values are the mean ± standard error from three replicates.
some extent antioxidative enzymes contribute to callus growth in A. absinthium L., but their involvement has yet to be determined at the molecular level which provides bases for scientists to understand their chemistry for future health challenges. In order to determine and characterize effective phytochemicals extracts should be subjected to HPLC analyses in A. absinthium. References [1] N.S. Ahmad, M. Farman, M.H. Najmi, K.B. Mian, A. Hasan, Activity of polyphenolic plant extracts as scavengers of free radicals and inhibitors of xanthine oxidase, J. Basic Appl. Sci. 2 (2006) 1–6. [2] P. Ananthi, B.D. Ranjitha, Kumari and A. Ramachandran, In vitro propagation of Rorippa indica L. from nodal and shoot tip explantsInt. J. Biotechnol. Mol, Biol. Res. 2 (3) (2011) 51–55. [3] F.J. Anderson, An illustrated history of the herbals, Columbia University Press, 1977. [4] C. Bailly, A. Benamar, F. Corbineau, D. Come, Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seed as related to deterioration during accelerated aging, J. Plant Physiol. 97 (1996) 104–110. [5] P. Baker, The book of absinthe: a cultural history, USA, Grove Press, New York, 2001. [6] R. Borneo, E.A. Leo9 n, A. Aguirre, P. Ribotta, J.J. Cantero, Antioxidant capacity of medicinal plants from the Province of Cordoba (Argentina) and their in vitro testing in model food system, Food Chem. 112 (2008) 664–670. [7] F. Cai, L.J. Mei, X.L. An, S.H.U.N. Gao, L. Tang, F.A.N.G. Chen, Lipid peroxidation and antioxidant responses during seed germination of Jatropha curcas, J. Agric. Biol. 13 (2011) 25–30. [8] J.M. Canadanovic-Brunet, S.M. Djilas, G.S. Cetkovic, V.T. Tumbas, Free-radical scavenging activity of wormwood (Artemisia absinthium L.) extracts, J. Sci. Food Agric. 85 (2) (2005) 265–272. [9] C.C. Chang, M.H. Yang, H.M. Wen, J.C. Chern, Estimation of total flavonoids content in propolis by two complementary colorimetric methods, J. Food Drug Anal. 10 (3) (2002) 178–182. [10] E. Chkhubianishvili, N. Kacharava, G. Badridze, S. Chanishvili, T. Kurdadze, Activity of peroxidase, catalase and content of total proteins in leaves of some herbaceous plants of high mountains of the Caucasus, Bull. Georg., Proc. Nat. Acad. Sci. 5 (2) (2011) 96–100. [11] A. Christophe, B. Moulia, C. Varlet-Grancher, Quantitative contributions of blue light and PAR to the photocontrol of plant morphogenesis in Trifolium repens (L.), J. Exp. Bot. 57 (10) (2006) 2379–2390. [12] D.B. Duncan, Multiple range and multiple F-tests, Biometrics 11 (1955) 1–42. [13] R.H. Ellis, E.H. Roberts, in: P.D. Hebblethwaite (Ed.), Towards Rational Basis for Testing Seed Quality, Butterworths, London, 1980, pp. 605–635. [14] R. Endress, in: Plant Cell Biotechnology, Springer-Verlag, Berlin, Germany, 1994, pp. 121–142. [15] F.S. Ferreira, P. Jorge, J. Peaden, Keiser, In vitro trematocidal effects of crude alcoholic extracts of Artemisia annua, A. absinthium, Asimina triloba, and Fumaria officinalis, Parasitol. Res. 109 (2011) 1585–1592. [16] A. Ghasemzadeh, H.Z. Jaafar, A. Rahmat, Antioxidant activities, total phenolics and flavonoids content in two varieties of Malaysia young ginger (Zingiber officinale Roscoe), Molecules 15 (6) (2010) 4324–4333. [17] L. Giri, P. Dhyani, S. Rawat, I.D. Bhatt, S.K. Nandi, R.S. Rawal, V. Pande, In vitro production of phenolic compounds and antioxidant activity in callus
U. Tariq et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 264–271
[18] [19] [20] [21]
[22] [23]
[24]
[25] [26]
[27]
[28]
[29]
[30] [31] [32] [33]
[34]
[35] [36]
[37]
[38]
suspension cultures of Habenaria edgeworthii: a rare Himalayan medicinal orchid, Ind. Crops Prod. 39 (2012) 1–6. M. Grudkowska, B. Zagdanska, Multifunctional role of plant cysteine proteinases, Acta Biochim. Pol. 51 (2004) 609–624 (English edition). M.Q. Hayat, M.A. Khan, M. Ashraf, S. Jabeen, Ethnobotany of the genus Artemisia L. (Asteraceae) in Pakistan, Ethnobot. Res. Applications 7 (2009). J.D. Hiscox, G.F. Israelsham, A method for the extraction of chlorophyll from leaf tissue without maceration, Can. J. Bot. 57 (12) (1979) 1332–1334. V. Katalinic´, M. Miloš, T. Kulišic´, M. Jukic´, Screening of 70 medicinal plant extracts for antioxidant capacity and total phenols, Food Chem. 94 (2004) 550–557. V.I. Kefeli, M.V. Kalevitch, B. Borsari, Phenolic cycle in plants and environment, J. Mol. Cell Biol. 2 (2003) 13–18. G. Kocsy, M. Brunner, A. Ruegsegger, P. Stamp, C. Brunold, Glutathione synthesis in maize genotypes with different sensitivities to chilling, Planta 198 (3) (1996) 365–370. S. Kordali, A. Cakir, A, Mavi, H. Kilic, A. Yildirim, Screening of chemical composition and antifungal and antioxidant activities of the essential oils from three Turkish Artemisia species, J. Agric. Food Chem 53 (2005) 1408–1416. L.M. Lagrimini, Wound-induced deposition of polyphenols in transgenic plants overexpressing peroxidise, Plant Physiol. 96 (2) (1991) 577–583. D.H. Lee, C.B. Lee, Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber: in gel enzyme activity assays, Plant Sci. 159 (1) (2000) 75–85. S.K. Lee, Z.H. Mbwambo, H.S. Chung, L. Luyengi, E.J. Gamez, R.G. Mehta, A.D. Kinghom, J.M. Pezzuto, Evaluation of the antioxidant potential of natural products, Comb. Chem. High T. Scr. 1 (1998) 35–46. S.A. Leicht, J.A. Silander, Differential responses of invasive Celastrus orbiculatus (Clastraceae) and native C. scandens to changes in light quality, Am. J. Bot. 93 (7) (2006) 972–977. D. Lopes-Lutz, D.S. Alviano, C.S. Alviano, P.P. Kolodziejczyk, Screening of chemical composition, antimicrobial and antioxidant activities of Artemisia essential oils, Phytochem. 69 (2008) 1732–1738. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1) (1951) 265–275. C.E. McDonald, L.L. Chen, The Lowry modification of the Folin reagent for determination of proteinase activity, Anal. Biochem. 10 (1965) 175–177. A.A. Meratan, S.M. Ghaffari, V. Niknam, In vitro organogenesis and antioxidant enzymes activity in Acanthophyllum sordidum, Biol. Plant. 53 (1) (2009) 5–10. A.H. Mohamed, M.A. El-Sayed, M.E. Hegazy, S.E. Helaly, A.M. Esmail, N.S. Mohamed, Chemical Constituents and Biological Activities of Artemisia herbaalba, Rec. Nat. Prod. 4 (2010) 1–25. V. Mulabagal, H. Tsay, Plant cell cultures – an alternative and efficient source for the production of biologically important secondary metabolites, Int. J. Appl. Sci., Eng. Technol. 2 (2004) 29–48. T. Murashige, F. Skoog, A revised medium for rapid growth and bio assays with tobacco tissue cultures, Physiol. Plant. 15 (1962) 473–497. H. Nayyar, D. Gupta, Differential sensitivity of C3 and C4 plants to water deficit stress: association with oxidative stress and antioxidants, Environ. Exp. Bot. 58 (1) (2006) 106–113. S. Nin, E. Morosi, S. Schiff, A. Bennici, Callus culture of Atremisia absinthium L.: initiation, growth optimization and organogenesis, Plant Cell, Tissue Organ Cult. 45 (1) (1996) 67–72. G. Noctor, A.C.M. Arisi, L. Jouanin, K.J. Kunert, H. Rennenberg, C.H. Foyer, Glutathione: biosynthesis, metabolism and relationship to stress tolerance explored in transformed plants, J. Exp. Bot. 49 (321) (1998) 623–647.
271
[39] G. Noctor, C.H. Foyer, Ascorbate and glutathione: keeping active oxygen under control, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49 (1998) 249–279. [40] Y.G. Park, M.H. Kim, J.K. Yang, Y.G. Chung, M.S. Choi, Light-susceptibility of camptothecin production from in vitro cultures of Camptotheca acuminata Decne, Biotechnol. Bioprocess Eng. 8 (1) (2003) 32–36. [41] Y. Qin, S. Zhang, A. Syed, L. Zhang, Q. Qin, K. Chen, C. Xu, Regeneration mechanism of Toyonoka strawberry under different color plastic films, Plant Sci. 168 (2005) 1425–1431. [42] G. Sa, M. Mi, Y. He-chun, L. Ben-ye, L. Guo-feng, C. Kang, Effects of ipt gene expression on the physiological and chemical characteristics of Artemisia annua L., Plant Sci. 160 (4) (2001) 691–698. [43] G. Schmid, W. Hofheinz, Total synthesis of qinghaosu, J. Am. Chem. Soc. 3 (1983) 624–625. [44] H. Senger, in: Blue Light Responses: Phenomenona and Occurrence in Plant and Microorganisms, CRC Press, Boca Raton, USA, 1987, pp. 135–138. [45] M. Sengul, H. Yildiz, N. Gungor, B. Cetin, Z. Eser, S. Ercisli, Total phenolic content, antioxidant and antimicrobial activities of some medicinal plants, Pak. J. Pharm. Sci. 22 (1) (2009) 102–106. [46] N.V. Sergeeva, N.D. Zakharova, Qualitative and quantitative study of carotenoids in some galenical preparations, Farmatsiya (Moscow) 26 (1) (1977) 34–38. [47] F.S. Sharopov, V.A. Sulaimonova, W.N. Setzer, Composition of the essentialoil of Artemisia absinthium from Tajikistan, Rec. Nat. Prod. 6 (2012) 127–134. [48] K.S. Shin, H.N. Murthy, J.W. Heo, E.J. Hahn, K.Y. Paek, The effect of light quality on the growth and development of in vitro cultured Doritaenopsis plants, Acta Physiol. Plant. 30 (3) (2008) 339–343. [49] R. Singh, P.K. Verma, G. Singh, Total phenolic, flavonoids and tannin contents in different extracts of Artemisia absinthium, J. Intercult. Ethnopharmacol. 1 (2) (2012) 101–104. [50] J. Slepetys, Biology and biochemistry of wormwood, accumulation dynamics of tanins, ascorbic acid and carotene, Liet. TSR. Mokslu. Akad. Darb. Ser. C 1 (1975) 43–148. [51] N. Smirnoff, Plant resistance to environmental stress, Curr. Opin. Biotechnol. 9 (1998) 214–219. [52] A.E. Smith, D.M. Secoy, Plants used for agriculture pest control in Western Europe before 1850, Chem. Ind. 3 (1981) (1850) 12–15. [53] Y.S. Velioglu, G. Mazza, L. Gao, B.D. Oomah, Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products, J. Agricult. Food Chem. 46 (10) (1998) 4113–4117. [54] Y.C. Wang, H.X. Zhang, B. Zhao, X.F. Yuan, Improved growth of Artemisia annua L. hairy roots and artemisinin production under red light conditions, Biotechnol. Lett. 23 (2001) 1971–1973. [55] G. Wingsle, J.E. Hallgren, Influence of SO2 and NO2 exposure on glutathione, superoxide dismutase and glutathione reductase activities in Scots pine needles, J. Exp. Bot. 44 (2) (1993) 463–470. [56] Xing-Xiang. Xu, J. Zho, D.Z. Huang, W.S. Zhou, Total synthesis of arteannuin and deoxyarteannuin. Tetrahedron, 42, 1986, 819–828. [57] Q. Yonghua, Z. Shanglong, S. Asghar, Z. Lingxiao, Q. Qiaoping, C. Kunsong, X. Changjie, Regeneration mechanism of Toyonoka strawberry under different color plastic films, Plant Sci. 168 (6) (2005) 1425–1431. [58] J. Zhang, S. Cui, J. Li, J. Wei, M.B. Kirkham, Protoplasmic factors, antioxidants responses, and chilling resistance in maize, Plant Physiol. Biochem. 33 (1995) 567–575.
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Morphogenic and biochemical variations under different spectral lights in callus cultures of Artemisia absinthium L. Umayya Tariq, Mohammad Ali, Bilal Haider Abbasi ⇑ Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan
a r t i c l e
i n f o
Article history: Received 16 September 2013 Received in revised form 26 November 2013 Accepted 27 November 2013 Available online 4 December 2013 Keywords: Thidiazuron 6-Benzyladenine (BA) Phenolics Flavonoids Protease Peroxidase
a b s t r a c t Through its impact on morphogenesis, light is the key environmental factor that alters plant architectural development; however, the understanding that how light controls plant growth and developmental processes is still poor and needs further research. In this study, we monitored the effect of various monochromatic lights and plant growth regulators (PGRs) combinations on morphogenic and biochemical variation in wild grown-leaf derived callus cultures of Artemisia absinthium L. Combination of a-naphthalene acetic acid (NAA 1.0 mg/l) and Thidiazuron (TDZ 2.0 mg/l) resulted in optimum callogenic frequency (90%) when kept under fluorescent light for 4 weeks (16/8 h). In contrast to the control (white spectrum), red spectrum enhanced peroxidase activity, protease activity, total protein content and chlorophyll a/b ratio. Green spectrum was found to be more supportive for total phenolics, total flavonoids and antioxidant activity. Yellow light enhanced MDA content while white and green light improved total chlorophyll content and carotenoid content. A positive correlation among callogenic response, antioxidant activities and set of antioxidative enzyme activities was also observed in the current report. This study will help in understanding the influence of light on production of commercially important secondary metabolites and their optimization in the in vitro cultures of A. absinthium L. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Artemisia absinthium L., commonly known as ‘‘Wormwood’’ (family; Asteraceae), has been used as herbal medicine throughout Europe, Middle East, North Africa, and Asia ([47]). In the middle Ages, this plant was referred as a ‘‘general remedy for all diseases’’ due to its curative medical powers ([5]). It has been used in China as an antiplasmodial herbal medicine for quinine resistant and quinine analogues resistant patients ([43,56]). Traditionally this species has been used due to its insecticidal, bitter ([3,52]), vermifuge, trematocidal ([15]), diuretic and antispasmodic properties ([33]) and against diarrhoea, cough and common cold ([19]). Phytochemical studies of A. absinthium L. showed the presence of important chemical compounds such as carotenoids [46], flavonoids [8], and many polyphenolic compounds [50]. Chemically major constituents in A. absinthium L. are myrcene, trans-thujones and trans-sabinyl acetate ([29]). The dry leaves and stems of this plant contain 0.25–1.32% essential oil, absinthin, artemisinin, artabsin and matricin ([24]). In vitro regeneration system is very useful for production of phytomedicine because conventional cultivation of plants is subjected to varieties of pests, weather and land availability which adversely affect medicinal qualities of harvested plant [42]. ⇑ Corresponding author. Tel./fax: +92 51 90644121. E-mail address: [email protected] (B.H. Abbasi). 1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2013.11.026
Moreover, callus formation studies are useful to understand metabolic pathways of secondary metabolites (SMs) and somatic embryogenesis [32]. Specific PGRs at appropriate concentrations can perform important function during callogenesis [2]. Among various culture conditions, light illumination influences the accumulation of useful SM [14]. By optimizing in vitro conditions like light sources, SM production can be stimulated [44]. SM from plants have important biological and pharmacological activities, such as anti-oxidative and anti-carcinogenic [6,21, 22,34]. The biological activities of phenolic compounds and flavonoids are associated to their antioxidant potential [16]. The protective action of antioxidant system comes into play under extreme conditions like light, chilling treatment and in defence mechanism against injury. Antioxidant activity has direct relationship with species resistance toward the stressor [51]. Enzymatic and non-enzymatic defence mechanisms have been evolved in plants for the scavenging of reactive oxygen species (ROS). Enzymes involved including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) [26,58]; while the non-enzymatic antioxidants are low molecular weight quenchers such as ascorbic acid (AsA) [23,38,39,55], function together to scavenge reactive oxygen species (ROS). Specific metabolic changes take place when plants are adapted to specific climatic conditions. These changes involve accumulation of protective proteins and regulation of antioxidative enzymes [9].
U. Tariq et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 264–271
Unlike most studies, published to date, on the impact of light on photoregulation during plant development under various illuminations [11,28], the present investigation was undertaken to establish the callus cultures from selected clones of A. absinthium L. under different wavelengths of light, evaluation of hormones required to maintain in vitro leaf derived calli, suitability of specific spectrum for particular biochemical response and correlation between total phenolics, total flavonoids and antioxidant enzyme activities. 2. Materials and methods 2.1. Explant source and surface sterilization Leaf explants were collected from wild grown plants of A. absinthium L. and cultured on MS medium ([35]; Phytotechnology Labs, USA) incorporated with several PGRs. For sterilization purposes, these uncut leaf explants were surface sterilized with 70% ethanol for 60 s followed by immersion in 0.1% (w/v) mercuric chloride solution for 1 min and finally rinsed with sterile distilled water three times. Aseptic transfer was accomplished in Laminar flow hood fitted with a HEPA filter. 2.2. Callus cultures establishment To study the effects of different wavelengths of light and PGRs on callus induction, approximately 1.5 cm of the leaf sections were incubated on Murashige and Skoog (MS) (1962) media supplemented with TDZ, Kin and BA (1.0, 2.0, 3.0, 4.0, and 5.0 mg/l) alone and in combination with NAA (1.0 mg/l). Before autoclaving (121 °C, 20 min, Systec VX 100, Germany), pH of all media was adjusted to 5.8 (Eutech Instruments pH 510, Singapore). All cultures were maintained in a growth room at 25 ± 1 °C under a 16/8-h light/dark photoperiod. Different illumination sources used were sole; cool-white fluorescent tubes (20 W, Toshiba FL20T9D/19) as control 380–780 nm, blue tubes (220 V; 50 Hz, Keliang Ltd.) 380– 560 nm, green tubes (40 W Litex) 480–670 nm, yellow tubes (36 W, Philips Ltd.) 530–780 nm at an intensity ranging from approximately 40–50 lmol m2 s1 or 10,240 lux and red fluorescent tubes (25 W, BINXIANG) 610–715 nm or 640 lux and some cultures were maintained under dark conditions. Light intensity was measured by using Lux meter (SU10, Jeiotech) just under the light source. Changes in callus morphology were recorded on the basis of visual observation. After 3 weeks of culture, the number of responding explants was recorded. Callus subculturing was performed on media with same PGRs composition. 2.3. Analytical methods Extraction of calli was performed according to the protocol described by Giri et al. [17] with minor modifications. Briefly, the dried plant materials were pulverized in chilled pestle and mortar with liquid nitrogen. 0.2 g of the powdered plant tissue was added to 1 ml of 100% pure methanol, mixed and kept for 5 min. Then the samples were sonicated (5 min; Toshiba, Japan), vortexed for 20 min and centrifuged (13,000 rpm, 5 min). The supernatants were collected and either immediately used for analysis or stored at 4 °C. A regression curve of the standard solutions of various concentrations was worked out against their respective absorbance. For antioxidant activity determination, DPPH free radical scavenging assay (FRSA) was used as described by the method of Lee et al. [27] with some modifications. Absorbance was measured at 515 nm using micro-plate reader. The samples which showed more than 70% scavenging at 200 lg/ml concentration were tested
265
at lower concentration by using three fold serial dilutions methodology to find IC50. IC50 values indicate the sample concentration required to scavenge 50% DPPH free radicals ([1]), was calculated by table curve software. The RSA was determined as percentage of DPPH° discoloration using the equation;
% scavenging DPPH free radical ¼ 100 ðAbc Abs =Abc Þ where Abc is absorbance of the negative control and Abs is the absorbance of the DPPH° solution with sample (Methanolic extract) added. Total phenolic content (TPC) was determined by the method of Velioglu et al. [53] by using Folin–Ciocalteu reagent. Absorbance was measured at 630 nm by using UV/VIS–DAD spectrophotometer (Halo DR-20, UV–VIS spectrophotometer, Dynamica Ltd., Victoria, Australia). The calibration curve (0–50 lg/ml, R2 = 0.968) was plotted by using gallic acid as standard. All determinations were performed in triplicate and the TPC was expressed as mg/g gallic acid equivalents (GAE) of dry weight. For total flavonoid content (TFC) determination, aluminium chloride colorimetric method was used as described by Chang et al. [9]. Absorbance of the reaction mixtures was measured at 405 nm by using UV/VIS-DAD spectrophotometer. The calibration curve (0–40 lg/ml, R2 = 0.998) was plotted by using quercetin as standard. The TFC was expressed as mg/g quercetin equivalents (QE) of dry weight. Malondialdehyde content was determined by thiobarbituric acid (TBA) test as described by Bailly et al. [4] with some modifications. Absorbance of the supernatants was recorded at 532 nm and 600 nm. The values of non-specific absorbance at 600 nm were then subtracted from that of 532 nm. Lipid per-oxidation (MDA content) was expressed as lM/g fresh weight by using extinction coefficient of 155 mM1 cm1. Total protein content was determined using method of Lowry et al. [30] with some modifications. Enzyme extract was prepared in the same way as that for antioxidant enzyme assay. The absorbance was then measured at 650 nm using spectrophotometer (Shimadzu, UV-120-01) Standard curve of BSA was then prepared by absorbance versus micrograms protein or vice versa and the unknown protein from the sample was determined from the curve. Chlorophyll ‘‘a’’, ‘‘b’’ and total chlorophyll content were investigated following the method of Hiscox and Israelsham [20] with some modifications. The resulted extract color intensity was measured using spectrophotometer (Shimadzu, UV-120-01) at 645 nm and 663 nm to estimate chlorophyll ‘‘a’’ and chlorophyll ‘‘b’’. The chlorophyll content (mg/g fresh weight) was calculated using the following formulas [13]. Chl a ðmg=g FW of leafÞ ¼ ½ð0:0127ðOD663 Þ 0:00269ðOD645 Þ 100 Chl b ðmg=g FW of leafÞ ¼ ½ð0:0229ðOD645 Þ 0:00468ðOD663 Þ 100 Total chlorophyll content ¼ ½0:0202ðOD645 ÞÞ þ 0:00802ðOD663 Þ 100 Chl a ratio b ¼ Chl a=Chl b
Here, O.D. = Optical Density. The absorbance of the extract was also checked at 480 nm to check total carotenoids. Total carotenoids were then calculated by formula given below.
Carotenoids ðmg=g FW of leafÞ ¼ OD480 0:5 The enzymes extracts for antioxidant enzyme activities were prepared by following the method of Nayyar and Gupta [36] with some modifications. Briefly, 1 g of sample tissue was homogenized with 10 ml of extraction buffer (50 mM potassium phosphate buffer with 1% PVPP at pH 7). The homogenate was then centrifuged at 15,000g at 4 sC for 30 min. and the supernatant was used for enzyme assay. Peroxidase (POD) activity was determined by the
266
U. Tariq et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 264–271
Table 1 PGRs concentrations and combinations, callus formation % and morphology under white light (16-h light and 8-h dark) kept for 4 weeks. Data show mean of three replicates ± SE. Sr. No.
Treatments (mg/l)
Callus initiation (day)
Callus induction frequency (%)
Callus color
Callus texture
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
MS + TDZ 1.0 MS + TDZ 2.0 MS + TDZ 3.0 MS + TDZ 4.0 MS + TDZ 5.0 MS + TDZ 1.0 + NAA 1.0 MS + TDZ 2.0 + NAA 1.0 MS + TDZ 3.0 + NAA 1.0 MS + TDZ 4.0 + NAA 1.0 MS + TDZ 5.0 + NAA 1.0 MS + Kn 1.0 MS + Kn 2.0 MS + Kn 3.0 MS + Kn 4.0 MS + Kn 5.0 MS + Kn 1.0 + NAA 1.0 MS + Kn 2.0 + NAA 1.0 MS + Kn 3.0 + NAA 1.0 MS + Kn 4.0 + NAA 1.0 MS + Kn 5.0 + NAA 1.0 MS + BAP 1.0 + NAA 1.0 MS + BAP 2.0 + NAA 1.0 MS + BAP 3.0 + NAA 1.0 MS + BAP 4.0 + NAA 1.0 MS + BAP 5.0 + NAA 1.0
20 20 20 20 20 20 20 20 20 20 30 30 30 30 30 30 30 30 30 30 20 20 20 20 20
43 56 62 74 67 83 90 70 66 51 70 63 56 41 33 62 77 53 46 39 57 84 77 65 53
DG DG DG DG DG DG DG DG DG DG YG YG YG YG YG YG YG YG YG YG LG LG LG LG LG
F F F F F F F F F F C C C C C C C C C C F F F F F
F = Friable; C = Compact; DG = Dark green; and YG = Yellow green.
Fig. 1. (A) Callus initiation from leaf explants. (B and C) Callus proliferation. (D) Callus after 4 weeks of culture.
method of Lagrimini [25] with some modifications. Protease activity was assayed following the method of McDonald and Chen [31] with minor modifications.
2.4. Data analysis All experiments were repeated twice and each treatment was consisted of three replicates. Mean values of various treatments were subjected to analysis of variance (ANOVA) and significant difference was separated using Duncan’s Multiple Range Test (DMRT). SPSS (Windows version 7.5.1, SPSS Inc., Chicago) was used to determine the significance at P < 0.05 ([12]).
3. Results and discussion 3.1. Callogenesis The efficacy of callogenesis from leaf explants was screened for optimum levels at different wavelengths of light and in response to different plant growth regulators either alone or in combinations (Table 1). Callus was initiated in response to almost all of the hormones tested. However, a combination of TDZ (2.0 mg/l) + NAA (1.0 mg/l) produced optimum response under in vitro conditions (16 h white light/8 h dark; control). Different concentrations (1.0–5.0 mg/l) of BA resulted in direct shooting response (data not shown).
267
U. Tariq et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 264–271 100
Table 2 The application of different wavelengths of light in conjunction with combination of PGRs from T1 to T6 for in vitro callogenesis of Artemisia absinthium L.
Callus induction %
80
60
Treatment
MS + PGRS (mg/l)
Light regimes
Incubation period
T1
MS + TDZ 1.0 MS + TDZ 1.0 MS + TDZ 1.0 MS + TDZ 1.0 MS + TDZ 1.0 MS + TDZ 1.0
2.0 + NAA
Darkness
24-h dark (4 weeks)
2.0 + NAA
White
2.0 + NAA
Red
24-h light 8-h dark (4 weeks) 24-h light (4 weeks)
2.0 + NAA
Blue
24-h light (4 weeks)
2.0 + NAA
Green
24-h light (4 weeks)
2.0 + NAA
Yellow
24-h light (4 weeks)
T2 T3
40
T4 T5
20
T6
MS + TDZ 1.0 MS + TDZ 2.0 MS + TDZ 3.0 MS + TDZ 4.0 MS + TDZ 5.0 MS + TDZ 1.0 + NAA 1.0 *MS + TDZ 2.0 + NAA 1.0 MS + TDZ 3.0 + NAA 1.0 MS + TDZ 4.0 + NAA 1.0 MS + TDZ 5.0 + NAA 1.0 MS + Kn 1.0 MS + Kn 2.0 MS + Kn 3.0 MS + Kn 4.0 MS + Kn 5.0 MS + Kn 1.0 + NAA 1.0 MS + Kn 2.0 + NAA 1.0 MS + Kn 3.0 + NAA 1.0 MS + Kn 4.0 + NAA 1.0 MS + Kn 5.0 + NAA 1.0 MS + BAP 1.0+ NAA 1.0 MS + BAP 2.0+ NAA 1.0 MS + BAP 3.0+ NAA 1.0 MS + BAP 4.0+ NAA 1.0 MS + BAP 5.0+ NAA 1.0
0 100
Treatments (mg/l)
Callogenesis started at cut end of leaf explants. TDZ as callus inducing hormone produced dark green and friable calli (Fig. 1). 74% callogenic frequency was found at 4.0 mg/l TDZ as compared to much improved callogenic response (90%) when TDZ (2.0 mg/ l) was experimented in combination with NAA (1.0 mg/l), followed by 84% callus response at BAP (2.0 mg/l) + NAA (1.0 mg/l) under white spectrum. Similarly, when Kn (1.0 mg/l) was used alone, callogenic response remained less effective (70%) as compared to more pronounced effect (77%) in response to Kin (2.0 mg/l) with NAA (1.0 mg/l) (Fig. 2). These results are in harmony with that reported by Nin et al. [37]; who concluded from their study that Artemisia spp. indicated good responses in media augmented with NAA. The calli were developed by the applications of monochromatic lights with 24 h exposure except the white light (16 h light/8 h dark) which was used as control (Table 2). When investigated under different monochromatic lights, callus formation frequency was highest (90%) under white light, followed by 82% under green light and 70% in dark (Fig. 3). In the hairy root cultures of A. annua L., it was found that the biomass of hairy roots and artemisinin content under red light were 17% and 67% higher than those obtained under white light [54]. Conflicting results from different investigators may be due to species differences and differences in light sources and intensities. In the present study, morphological variations have also been observed in callus cultured under different lights, which is supposed to indicate differences in biochemistry and physiology (Fig. 4).
3.2. Total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity In the current study, 12.215 mg/g DW and 2.015 mg/g DW TPC and TFC respectively were quantified in callus tissues of A. absinthium obtained in response to TDZ and NAA under white light (control) (Fig. 5) followed by 11.259 mg/g DW and 0.87 mg/g DW TPC and TFC under red light, respectively (Fig. 6). In contrast to our findings Singh et al. [49] stated that ethanolic extract of aerial parts of A. absinthium has significantly high concentration of flavonoids
Callus Induction (%)
Fig. 2. Percent callus formation in response to different plant growth regulators. Data were collected after 4 weeks of culture. Values are the mean ± standard error from three replicates.
80
60
40
20
0
Yellow
Green
Blue
Red
White
Dark
Light Treatments Fig. 3. Callus formation % under different light treatments at optimized hormonal concentration (2.0 mg/l TDZ and 1.0 mg/l NAA). Data were collected after 4 weeks of culture. Values are the mean ± standard error from three replicates.
(1108.15 ± 48.78 mg of QUE/g of extract) and phenolics (43.04 ± 0.57 mg of GAE/g of extract). In the present study, a positive correlation has been confirmed between high TPC and antioxidant activities as reported by [8,45] (Fig. 7). In the DPPH assay, the range of IC50 values recorded in calli of A. absinthium was 67.4– 3798 lg/ml DW. IC50 (3222 lg/ml DW) was shown by calli obtained on the medium containing TDZ 2.0 mg/l and NAA 1.0 mg/l while maximum IC50 (3798 lg/ml DW) was recorded in the calli raised on TDZ (5.0 mg/l) + NAA (1.0 mg/l). Among different light treatments given to calli, highest value of IC50 (1070 lg/ml DW) under green light was noticed followed by 882 lg/ml DW DPPH activity under blue light source. 3.3. Peroxidase activity and Malondialdehyde (MDA) content Maximum MDA content of 11.09 lM/g FW was found at Kin (4.0 mg/l) and NAA (1.0 mg/l) with callogenic response of 46% and 10.08 lM/g FW at TDZ (5.0 mg/l) with 67% callogenesis as compared to other hormonal concentrations under white light (Fig. 8). The reason might be that stress exerted on explants by applied light sources and PGRs caused an increase in lipid peroxidation takes place leading to lower rates of physiological processes. Similar increase in MDA content was observed in Toyonoka strawberry during callogenesis under different colored plastic films and gradually decreases with less callogenic percentage [57].
268
U. Tariq et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 264–271
Red
Yellow
White (control)
Dark
Green
Blue
Fig. 4. Effect of different lights on callus growth and morphology under different light sources at same hormonal combination (2.0 mg/l TDZ + 1.0 mg/l NAA).
14
10
2.0
8
1.5
6
1.0
4
0.5
2
0.0
0
-0.5 MS + TDZ 1.0 MS + TDZ 2.0 MS + TDZ 3.0 MS + TDZ 4.0 MS + TDZ 5.0 MS + TDZ 1.0 + NAA 1.0 MS + TDZ 2.0 + NAA 1.0 MS + TDZ 3.0 + NAA 1.0 MS + TDZ 4.0 + NAA 1.0 MS + TDZ 5.0 + NAA 1.0 MS + Kn 1.0 MS + Kn 2.0 MS + Kn 3.0 MS + Kn 4.0 MS + Kn 5.0 MS + Kn 1.0 + NAA 1.0 MS + Kn 2.0 + NAA 1.0 MS + Kn 3.0 + NAA 1.0 MS + Kn 4.0 + NAA 1.0 MS + Kn 5.0 + NAA 1.0 MS + BAP 1.0+ NAA 1.0 MS + BAP 2.0+ NAA 1.0 MS + BAP 3.0+ NAA 1.0 MS + BAP 4.0+ NAA 1.0 MS + BAP 5.0+ NAA 1.0
2.5
Treatments (mg/l)
Fig. 5. Total phenolic content (mg/g DW) and total flavonoid content (mg/g DW) formation in calli induced in response to different plant growth regulators. Values are the mean ± standard error from three replicates.
Interestingly, highest peroxidase activity (POD) 0.3157 nM/ min/mg was obtained at TDZ (5.0 mg/l) along with highest MDA content 10.06 lM/g FW (Fig. 8). Similarly, Cai et al. [7] found a parallel correlation between elevated MDA content and increased activities of peroxidase (POD) supports this notion. The reason might be that under oxidative stress, peroxidase activity is greater in order to remove toxic hydrogen peroxide along with high Malondialdehyde content formation due to lipid peroxidation. In
12
1400 TPC DPPH FRSA
1200
10
1000
8
800 600
6
400
4
200
2
Antioxidant activity (IC 50 µg/ml DW)
12
Total Phenolic Content (mg/g DW)
3.0
TPC TFC
Total Flavonoid Content (mg/g DW)
Total Phenolic Content (mg/g DW)
14
0 Red
Blue
Yellow
Green
White
Dark
Light Treatment Fig. 6. Total phenolics content (mg/g DW) and DPPH (IC50 lg/ml DW) activity in calli under different light treatments at optimized hormonal combination TDZ 2.0 mg/l and NAA 1.0 mg/l. Values are the mean ± standard error from three replicates.
the present study, maximum POD activity was recorded under red followed by blue and green spectra (Fig. 9). Similarly, in Toyonoka strawberry, G-POD activities were highest under the red films followed by green and yellow films [57]. This shows that higher POD activities are involved in protection against stress conditions. 3.4. Protease activity and total protein content Highest protease activity 2.56 U/g FW was observed when Kin (1.0 mg/l) was used singly as compared to protease activity value (1.7 U/g FW) obtained at Kin (2.0 mg/l) + NAA (1.0 mg/l) whereas
269
U. Tariq et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 264–271
3.5
10
3.0 8
2.5 2.0
6
1.5 4
1.0 0.5
2
Malondialdehyde Content (µM/g FW)
4.0
Antioxidant activity IC50 (µg/ml DW)
4.5
12
0.40
8
5.0
TPC DPPH FRSA
7
MDA POD
0.35 0.30
6
0.25 5
0.20
4
0.15
3
0.10
2
0.05 0.00
1
-0.05
0.0
0
MS + TDZ 1.0 MS + TDZ 2.0 MS + TDZ 3.0 MS + TDZ 4.0 MS + TDZ 5.0 MS + TDZ 1.0 + NAA 1.0 MS + TDZ 2.0 + NAA 1.0 MS + TDZ 3.0 + NAA 1.0 MS + TDZ 4.0 + NAA 1.0 MS + TDZ 5.0 + NAA 1.0 MS + Kn 1.0 MS + Kn 2.0 MS + Kn 3.0 MS + Kn 4.0 MS + Kn 5.0 MS + Kn 1.0 + NAA 1.0 MS + Kn 2.0 + NAA 1.0 MS + Kn 3.0 + NAA 1.0 MS + Kn 4.0 + NAA 1.0 MS + Kn 5.0 + NAA 1.0 MS + BAP 1.0+ NAA 1.0 MS + BAP 2.0+ NAA 1.0 MS + BAP 3.0+ NAA 1.0 MS + BAP 4.0+ NAA 1.0 MS + BAP 5.0+ NAA 1.0
0
Red
Blue
Yellow
Green
White
Dark
Light Treatment Fig. 9. Peroxidase activity (nM/min/mg FW) and malondialdehyde content (lM/g FW) formed under different light treatments. Values are the mean ± standard error from three replicates.
Treatments (mg/l)
3.0
800
Protease TPC
700
11
0.35
10
0.30
9
0.25
8
0.20
7
0.15
6
0.10
5
0.05
4
0.00
3
Treatments mg/l
Fig. 8. Peroxidase activity (nM/min/mg FW) and malondialdehyde content (lM/g FW) in response to different PGRs. Values are the mean ± standard error from three replicates.
at TDZ (1.0 mg/l) maximum protein content observed was 577.14 lg BSAE/20 mg FW with protease activity of 2.4 U/g FW (Fig. 10). In strawberry plantlets which were exposed to red light, POD activity increases with other antioxidant enzymes [41]. The protective adaptive abilities of plants under stress conditions may be due to activation of antioxidant mechanism along with protein synthesis and such activation may be the result of shifts in metabolism. Under different colored illuminations tested, highest total protein content 592.14 lg BSAE/20 mg FW was observed with 2.09 U/g FW protease activity under red light as compared to other light sources (Fig. 11). This shows a positive correlation between peroxidase activities with the activation of
600 2.0
500
1.5
400 300
1.0
200 0.5
100
0.0
0 MS + TDZ 1.0 MS + TDZ 2.0 MS + TDZ 3.0 MS + TDZ 4.0 MS + TDZ 5.0 MS + TDZ 1.0 + NAA 1.0 MS + TDZ 2.0 + NAA 1.0 MS + TDZ 3.0 + NAA 1.0 MS + TDZ 4.0 + NAA 1.0 MS + TDZ 5.0 + NAA 1.0 MS + Kn 1.0 MS + Kn 2.0 MS + Kn 3.0 MS + Kn 4.0 MS + Kn 5.0 MS + Kn 1.0 + NAA 1.0 MS + Kn 2.0 + NAA 1.0 MS + Kn 3.0 + NAA 1.0 MS + Kn 4.0 + NAA 1.0 MS + Kn 5.0 + NAA 1.0 MS + BAP 1.0+ NAA 1.0 MS + BAP 2.0+ NAA 1.0 MS + BAP 3.0+ NAA 1.0 MS + BAP 4.0+ NAA 1.0 MS + BAP 5.0+ NAA 1.0
12
MS + TDZ 1.0 MS + TDZ 2.0 MS + TDZ 3.0 MS + TDZ 4.0 MS + TDZ 5.0 MS + TDZ 1.0 + NAA 1.0 MS + TDZ 2.0 + NAA 1.0 MS + TDZ 3.0 + NAA 1.0 MS + TDZ 4.0 + NAA 1.0 MS + TDZ 5.0 + NAA 1.0 MS + Kn 1.0 MS + Kn 2.0 MS + Kn 3.0 MS + Kn 4.0 MS + Kn 5.0 MS + Kn 1.0 + NAA 1.0 MS + Kn 2.0 + NAA 1.0 MS + Kn 3.0 + NAA 1.0 MS + Kn 4.0 + NAA 1.0 MS + Kn 5.0 + NAA 1.0 MS + BAP 1.0+ NAA 1.0 MS + BAP 2.0+ NAA 1.0 MS + BAP 3.0+ NAA 1.0 MS + BAP 4.0+ NAA 1.0 MS + BAP 5.0+ NAA 1.0
Peroxidase Activity (nM/min/mg FW)
POD MDA
Malondialdehyde Content (µM/g FW)
0.40
Protease activity (U/g FW)
2.5
Total Protein Content ( µg BSAE/20 mg FW)
Fig. 7. Total phenolics content (mg/g DW) and DPPH (IC50 lg/ml DW) activity in calli induced in response to different plant growth regulators. Values are the mean ± standard error from three replicates.
0.45
Peroxidase Activity (nM/min/mg FW)
Total Phenolic Content (TPC mg/g DW)
14
Treatments (mg/l)
Fig. 10. Protease activity (U/g FW) and total protein content (lg BSAE/20 mg FW) produced in response to different PGRs. Values are the mean ± standard error from three replicates.
protein synthesis. As Grudkowska and Zagdanska [18] stated that proteases may be involved in turnover of proteins while responding to different abiotic stresses. It suggests that pathway of SM changes in a way that high protease activity may involve the degradation of damaged proteins in case of abiotic stress and finally the biosynthetic pathway provides amino acids for synthesis of proteins required under stress conditions. However protein contents, peroxidase activity, protease activity and lipid peroxidation have an important role in oxidative stress as indicators of cellular damage. 3.5. Chlorophyll ‘‘a’’, chlorophyll ‘‘b’’ and carotenoid content determination It was found that under green and white light sources, total chlorophyll mass was highest (3.742 mg/g FW; 3.532 mg/g FW,
270
U. Tariq et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 264–271 3.0
700
Protease activity (U/g FW)
400 1.5 300 1.0 200 0.5
100
2.5
1.4
Chlorophyll b Chlorophyll a a/b ratio 2.0
1.2 1.1 1.0 0.8
Red
Blue
Yellow
Green
White
0.6 0.5 0.3 0.1
0.85 0.80 0.75
3.5
0.70
3.0
0.65 0.60
2.5
0.55
2.0
0.50 0.45
1.5
0.40
1.0
0.35
Carotenoid Content (mg/g FW)
Total Chlorophyll Content (mg/g FW)
0.90
0.30
0.5
0.25
0.0 White
Yellow
Green
Red
White
Yellow
Green
Blue
Dark
-0.5
Light Treatment
TCC CC
Red
0.0
0.2
Dark
Fig. 11. Protease activity (U/g FW) and total protein content (lg BSAE/20 mg FW) formed under different light treatments. Values are the mean ± standard error from three replicates.
4.0
0.5
0.4
Light Treatment
4.5
1.0
0.7
0.0
0.0
1.5
0.9
Chlorophyll 'b' (mg/g FW)
500
2.0
1.5 1.3
Chlorophyll 'a' (mg/g FW) a/b ratio
600
2.5
Total Protein Content (µg BSAE/20mg FW)
Protease TPC
Blue
Dark
Light Treatment Fig. 12. Total chlorophyll content (mg/g FW) and carotenoid content (mg/g FW) produced in response to different illuminations. Values are the mean ± standard error from three replicates.
respectively), with maximum callogenic frequencies followed by lower callusing under red light with lower total chlorophyll mass (Fig. 12) while chlorophyll a/b ratio was higher (1.304) under red light conditions (Fig. 13). These results are in consonance with the studies of Shin and co-workers [48] who reported significant reduction in chlorophyll content under red light treatment but no significant variation in a/b ratio. It is worthwhile to state that there is direct relationship between chlorophyll content of the callus and its capacity to regenerate. It means that exposure of callus cultures to various light sources influenced total chlorophyll content and SM production. It was reported that chlorophyll may carry precursors of SM which indicates its role in SM biosynthesis [40]. Highest carotenoid content 0.63 mg/g FW was recorded under red light treatment, followed by 0.585 mg/g FW under white light while lowest carotenoid content 0.287 mg/g FW was observed under dark conditions. The present paper describes that light as a physical factor had a positive influence on callus induction and proliferation in A. absinthium L. It can be concluded that different light regimes may help to optimize growth and developmental changes and this makes the practical application of the photo-technology with respect to design of controlled environment for in vitro safe SM production. To
Fig. 13. Chlorophyll ‘‘a’’ (mg/g FW), chlorophyll ‘‘b’’ (mg/g FW) and chlorophyll a/b ratio produced in response to different illuminations. Values are the mean ± standard error from three replicates.
some extent antioxidative enzymes contribute to callus growth in A. absinthium L., but their involvement has yet to be determined at the molecular level which provides bases for scientists to understand their chemistry for future health challenges. In order to determine and characterize effective phytochemicals extracts should be subjected to HPLC analyses in A. absinthium. References [1] N.S. Ahmad, M. Farman, M.H. Najmi, K.B. Mian, A. Hasan, Activity of polyphenolic plant extracts as scavengers of free radicals and inhibitors of xanthine oxidase, J. Basic Appl. Sci. 2 (2006) 1–6. [2] P. Ananthi, B.D. Ranjitha, Kumari and A. Ramachandran, In vitro propagation of Rorippa indica L. from nodal and shoot tip explantsInt. J. Biotechnol. Mol, Biol. Res. 2 (3) (2011) 51–55. [3] F.J. Anderson, An illustrated history of the herbals, Columbia University Press, 1977. [4] C. Bailly, A. Benamar, F. Corbineau, D. Come, Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seed as related to deterioration during accelerated aging, J. Plant Physiol. 97 (1996) 104–110. [5] P. Baker, The book of absinthe: a cultural history, USA, Grove Press, New York, 2001. [6] R. Borneo, E.A. Leo9 n, A. Aguirre, P. Ribotta, J.J. Cantero, Antioxidant capacity of medicinal plants from the Province of Cordoba (Argentina) and their in vitro testing in model food system, Food Chem. 112 (2008) 664–670. [7] F. Cai, L.J. Mei, X.L. An, S.H.U.N. Gao, L. Tang, F.A.N.G. Chen, Lipid peroxidation and antioxidant responses during seed germination of Jatropha curcas, J. Agric. Biol. 13 (2011) 25–30. [8] J.M. Canadanovic-Brunet, S.M. Djilas, G.S. Cetkovic, V.T. Tumbas, Free-radical scavenging activity of wormwood (Artemisia absinthium L.) extracts, J. Sci. Food Agric. 85 (2) (2005) 265–272. [9] C.C. Chang, M.H. Yang, H.M. Wen, J.C. Chern, Estimation of total flavonoids content in propolis by two complementary colorimetric methods, J. Food Drug Anal. 10 (3) (2002) 178–182. [10] E. Chkhubianishvili, N. Kacharava, G. Badridze, S. Chanishvili, T. Kurdadze, Activity of peroxidase, catalase and content of total proteins in leaves of some herbaceous plants of high mountains of the Caucasus, Bull. Georg., Proc. Nat. Acad. Sci. 5 (2) (2011) 96–100. [11] A. Christophe, B. Moulia, C. Varlet-Grancher, Quantitative contributions of blue light and PAR to the photocontrol of plant morphogenesis in Trifolium repens (L.), J. Exp. Bot. 57 (10) (2006) 2379–2390. [12] D.B. Duncan, Multiple range and multiple F-tests, Biometrics 11 (1955) 1–42. [13] R.H. Ellis, E.H. Roberts, in: P.D. Hebblethwaite (Ed.), Towards Rational Basis for Testing Seed Quality, Butterworths, London, 1980, pp. 605–635. [14] R. Endress, in: Plant Cell Biotechnology, Springer-Verlag, Berlin, Germany, 1994, pp. 121–142. [15] F.S. Ferreira, P. Jorge, J. Peaden, Keiser, In vitro trematocidal effects of crude alcoholic extracts of Artemisia annua, A. absinthium, Asimina triloba, and Fumaria officinalis, Parasitol. Res. 109 (2011) 1585–1592. [16] A. Ghasemzadeh, H.Z. Jaafar, A. Rahmat, Antioxidant activities, total phenolics and flavonoids content in two varieties of Malaysia young ginger (Zingiber officinale Roscoe), Molecules 15 (6) (2010) 4324–4333. [17] L. Giri, P. Dhyani, S. Rawat, I.D. Bhatt, S.K. Nandi, R.S. Rawal, V. Pande, In vitro production of phenolic compounds and antioxidant activity in callus
U. Tariq et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 264–271
[18] [19] [20] [21]
[22] [23]
[24]
[25] [26]
[27]
[28]
[29]
[30] [31] [32] [33]
[34]
[35] [36]
[37]
[38]
suspension cultures of Habenaria edgeworthii: a rare Himalayan medicinal orchid, Ind. Crops Prod. 39 (2012) 1–6. M. Grudkowska, B. Zagdanska, Multifunctional role of plant cysteine proteinases, Acta Biochim. Pol. 51 (2004) 609–624 (English edition). M.Q. Hayat, M.A. Khan, M. Ashraf, S. Jabeen, Ethnobotany of the genus Artemisia L. (Asteraceae) in Pakistan, Ethnobot. Res. Applications 7 (2009). J.D. Hiscox, G.F. Israelsham, A method for the extraction of chlorophyll from leaf tissue without maceration, Can. J. Bot. 57 (12) (1979) 1332–1334. V. Katalinic´, M. Miloš, T. Kulišic´, M. Jukic´, Screening of 70 medicinal plant extracts for antioxidant capacity and total phenols, Food Chem. 94 (2004) 550–557. V.I. Kefeli, M.V. Kalevitch, B. Borsari, Phenolic cycle in plants and environment, J. Mol. Cell Biol. 2 (2003) 13–18. G. Kocsy, M. Brunner, A. Ruegsegger, P. Stamp, C. Brunold, Glutathione synthesis in maize genotypes with different sensitivities to chilling, Planta 198 (3) (1996) 365–370. S. Kordali, A. Cakir, A, Mavi, H. Kilic, A. Yildirim, Screening of chemical composition and antifungal and antioxidant activities of the essential oils from three Turkish Artemisia species, J. Agric. Food Chem 53 (2005) 1408–1416. L.M. Lagrimini, Wound-induced deposition of polyphenols in transgenic plants overexpressing peroxidise, Plant Physiol. 96 (2) (1991) 577–583. D.H. Lee, C.B. Lee, Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber: in gel enzyme activity assays, Plant Sci. 159 (1) (2000) 75–85. S.K. Lee, Z.H. Mbwambo, H.S. Chung, L. Luyengi, E.J. Gamez, R.G. Mehta, A.D. Kinghom, J.M. Pezzuto, Evaluation of the antioxidant potential of natural products, Comb. Chem. High T. Scr. 1 (1998) 35–46. S.A. Leicht, J.A. Silander, Differential responses of invasive Celastrus orbiculatus (Clastraceae) and native C. scandens to changes in light quality, Am. J. Bot. 93 (7) (2006) 972–977. D. Lopes-Lutz, D.S. Alviano, C.S. Alviano, P.P. Kolodziejczyk, Screening of chemical composition, antimicrobial and antioxidant activities of Artemisia essential oils, Phytochem. 69 (2008) 1732–1738. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1) (1951) 265–275. C.E. McDonald, L.L. Chen, The Lowry modification of the Folin reagent for determination of proteinase activity, Anal. Biochem. 10 (1965) 175–177. A.A. Meratan, S.M. Ghaffari, V. Niknam, In vitro organogenesis and antioxidant enzymes activity in Acanthophyllum sordidum, Biol. Plant. 53 (1) (2009) 5–10. A.H. Mohamed, M.A. El-Sayed, M.E. Hegazy, S.E. Helaly, A.M. Esmail, N.S. Mohamed, Chemical Constituents and Biological Activities of Artemisia herbaalba, Rec. Nat. Prod. 4 (2010) 1–25. V. Mulabagal, H. Tsay, Plant cell cultures – an alternative and efficient source for the production of biologically important secondary metabolites, Int. J. Appl. Sci., Eng. Technol. 2 (2004) 29–48. T. Murashige, F. Skoog, A revised medium for rapid growth and bio assays with tobacco tissue cultures, Physiol. Plant. 15 (1962) 473–497. H. Nayyar, D. Gupta, Differential sensitivity of C3 and C4 plants to water deficit stress: association with oxidative stress and antioxidants, Environ. Exp. Bot. 58 (1) (2006) 106–113. S. Nin, E. Morosi, S. Schiff, A. Bennici, Callus culture of Atremisia absinthium L.: initiation, growth optimization and organogenesis, Plant Cell, Tissue Organ Cult. 45 (1) (1996) 67–72. G. Noctor, A.C.M. Arisi, L. Jouanin, K.J. Kunert, H. Rennenberg, C.H. Foyer, Glutathione: biosynthesis, metabolism and relationship to stress tolerance explored in transformed plants, J. Exp. Bot. 49 (321) (1998) 623–647.
271
[39] G. Noctor, C.H. Foyer, Ascorbate and glutathione: keeping active oxygen under control, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49 (1998) 249–279. [40] Y.G. Park, M.H. Kim, J.K. Yang, Y.G. Chung, M.S. Choi, Light-susceptibility of camptothecin production from in vitro cultures of Camptotheca acuminata Decne, Biotechnol. Bioprocess Eng. 8 (1) (2003) 32–36. [41] Y. Qin, S. Zhang, A. Syed, L. Zhang, Q. Qin, K. Chen, C. Xu, Regeneration mechanism of Toyonoka strawberry under different color plastic films, Plant Sci. 168 (2005) 1425–1431. [42] G. Sa, M. Mi, Y. He-chun, L. Ben-ye, L. Guo-feng, C. Kang, Effects of ipt gene expression on the physiological and chemical characteristics of Artemisia annua L., Plant Sci. 160 (4) (2001) 691–698. [43] G. Schmid, W. Hofheinz, Total synthesis of qinghaosu, J. Am. Chem. Soc. 3 (1983) 624–625. [44] H. Senger, in: Blue Light Responses: Phenomenona and Occurrence in Plant and Microorganisms, CRC Press, Boca Raton, USA, 1987, pp. 135–138. [45] M. Sengul, H. Yildiz, N. Gungor, B. Cetin, Z. Eser, S. Ercisli, Total phenolic content, antioxidant and antimicrobial activities of some medicinal plants, Pak. J. Pharm. Sci. 22 (1) (2009) 102–106. [46] N.V. Sergeeva, N.D. Zakharova, Qualitative and quantitative study of carotenoids in some galenical preparations, Farmatsiya (Moscow) 26 (1) (1977) 34–38. [47] F.S. Sharopov, V.A. Sulaimonova, W.N. Setzer, Composition of the essentialoil of Artemisia absinthium from Tajikistan, Rec. Nat. Prod. 6 (2012) 127–134. [48] K.S. Shin, H.N. Murthy, J.W. Heo, E.J. Hahn, K.Y. Paek, The effect of light quality on the growth and development of in vitro cultured Doritaenopsis plants, Acta Physiol. Plant. 30 (3) (2008) 339–343. [49] R. Singh, P.K. Verma, G. Singh, Total phenolic, flavonoids and tannin contents in different extracts of Artemisia absinthium, J. Intercult. Ethnopharmacol. 1 (2) (2012) 101–104. [50] J. Slepetys, Biology and biochemistry of wormwood, accumulation dynamics of tanins, ascorbic acid and carotene, Liet. TSR. Mokslu. Akad. Darb. Ser. C 1 (1975) 43–148. [51] N. Smirnoff, Plant resistance to environmental stress, Curr. Opin. Biotechnol. 9 (1998) 214–219. [52] A.E. Smith, D.M. Secoy, Plants used for agriculture pest control in Western Europe before 1850, Chem. Ind. 3 (1981) (1850) 12–15. [53] Y.S. Velioglu, G. Mazza, L. Gao, B.D. Oomah, Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products, J. Agricult. Food Chem. 46 (10) (1998) 4113–4117. [54] Y.C. Wang, H.X. Zhang, B. Zhao, X.F. Yuan, Improved growth of Artemisia annua L. hairy roots and artemisinin production under red light conditions, Biotechnol. Lett. 23 (2001) 1971–1973. [55] G. Wingsle, J.E. Hallgren, Influence of SO2 and NO2 exposure on glutathione, superoxide dismutase and glutathione reductase activities in Scots pine needles, J. Exp. Bot. 44 (2) (1993) 463–470. [56] Xing-Xiang. Xu, J. Zho, D.Z. Huang, W.S. Zhou, Total synthesis of arteannuin and deoxyarteannuin. Tetrahedron, 42, 1986, 819–828. [57] Q. Yonghua, Z. Shanglong, S. Asghar, Z. Lingxiao, Q. Qiaoping, C. Kunsong, X. Changjie, Regeneration mechanism of Toyonoka strawberry under different color plastic films, Plant Sci. 168 (6) (2005) 1425–1431. [58] J. Zhang, S. Cui, J. Li, J. Wei, M.B. Kirkham, Protoplasmic factors, antioxidants responses, and chilling resistance in maize, Plant Physiol. Biochem. 33 (1995) 567–575.