1.0 INTRODUCTION:

Phenolic compounds are most commonly distributed in plant kingdom. No tissue lacks phenolic compounds and high concentrations can be found in actively growing cells^[1]. The potential antioxidant activity of phenolic compounds from callus culture of medicinal plants such as Psoralea corylifolia and Rosmarinus officinalis was studied^[2,3].

Biotic and abiotic elicitors can enhance secondary metabolites production. The stimuli are perceived by receptors activating secondary messengers. These transmit signals into the cell through signal transduction pathways leading to gene expression and biochemical changes ^[4] resulting in compounds formation.

Plants interact with their environment by producing a diverse array of secondary metabolites ^[5]. Many of these compounds are valued for their pharmacological activities and industrial or agricultural properties which increase the commercial value of crops ^{[6, 7]}.

There is an emerging body of evidence, based on biological activities in vitro and on the correlation between rates of accumulation and expression of resistance in vivo, which indicates that isoprenoid and phenylpropanoid compounds play a role in the induced and constitutive response of plants to biotic and abiotic agents ^[8].

Plant tissue cultures are exposed to stresses and stress combinations that they may not have encountered in nature in their long evolution. It is a remarkable reflection on the plasticity of the plant genome that it can decipher and respond to novel in vitro stresses. Today various tissue culture techniques are used to enhance yield of secondary metabolites by triggering stress response like using elicitors, precursors and biotransformation, change in environmental conditions, change in medium constituents etc., ^[9]. Summart et al. [10] found that the significant different in growth and callus morphology under the influences of incubation temperatures (25±2°C and 30±2°C) on growth of rice callus. Apart from this the culture temperature played an important role in the growth and isoflavone production from callus cultures of Pueraria candollei ^[11]. The present study was focused to investigate the callus induction, biomass, total phenolic, flavonoids content and free radical scavenging activity of callus from internode explants of Heliotropium indicum L.

2.0 MATERIALS AND METHODS:

2.1 Explants and Sterilisation:

The wildly grown plant Heliotropium indicum L. near Siksha-Bhavana, Visva-Bharati, Santiniketan was located for the collection of explants. Healthy, young and disease free plant twigs of Heliotropium indicum were washed thoroughly with tap water. The internode explants were excised from the plant and they were surface cleaned with 5% teepol solution for 5min. and the explants were kept it in running tap water for 10 min. The explants were disinfected with 70% ethanol for 30 sec. and followed by 0.1%HgCl₂ for 5min., the explants were rinsed with autoclaved distilled water at least three time followed by each disinfection treatment.

2.2 Callus induction, culture medium and culture condition:

The internode explants were inoculated on MS ^[12] medium supplemented with pre-standardised plant growth regulators NAA 2.0 mg/L with BA 0.5 mg/L for callus induction with three percentage (w/v) of sucrose as a carbon source, 0.8 percentage of agar as a gelling agent and the media pH 5.8 adjusted with 1 N NaOH before autoclaving at 121 °C for 15 min. The cultures tubes were incubated at 25±2 °C with a photoperiod of 16 h with 60 µmol m⁻² s⁻¹of light intensity. The callus was proliferated followed by sub-culturing the callus in a regular interval. The callus were harvested and dried after 30 days of inoculation.

2.3 Incubation temperatures on callus culture:

The callus were subcultured on MS medium supplemented with NAA 2.0 mg/L and BA 0.5 mg/L and they were kept under various temperatures such as 20⁰C, 25⁰C, 30⁰C and 32⁰C. The callus tissues from various temperatures were used to analyse the various parameters.

2.3.1 Callus biomass:

Two hundred milligrams (200 ± 2 mg) of callus were subcultured on MS medium supplemented with NAA 2.0 mg/L and BA 0.5 mg/L and they were kept under various temperatures. After 30 days of subculture the fresh weight of callus from each treatment was measured. The fresh weight of callus and callus morphology such as colour and texture were noted after 30 days of subculture. Based on the initial (FW1) and final (FW2) fresh weight of callus the relative growth rate (RGR) was calculated according to the following formula: RGR = (FW2 – FW1)/Number of days. The photographs of the callus were taken by Nikon D60 camera and the three dimensional views of photos were taken by stereomicroscope.

2.3.2 Determination of total phenolic content:

The callus from various incubation treatments were used to estimate the total phenolic content according to the method described by Makkar et al. ^[13]. Aliquots (200µL) of the extracts were taken from each sample and the volume made up to 1mL with methanol. To the extracts, 0.5 mL of Folin-Ciocalteu reagent (1:1 with water) and 2.5 ml of sodium carbonate solution (20%) was added sequentially in each tube. The tubes were vortexed and were incubated at room temperature in dark for 40 min. Absorbance was measured at 725 nm against the reagent blank. The results are means of three repetitions expressed in the form of gallic acid (GA) equivalents per gram of extract.

2.3.3 Determination of flavonoids content:

Aluminium chloride colorimetric technique ^[14] was used to estimate the flavonoids from the callus grown under various incubation treatments. Aliquots (200µl) of each extract were taken and the volume made up to 1.8 mL with methanol, 0.1 mL of 10% aluminium chloride, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water were also added to make up the final volume of 5ml. It was left at room temperature for 30 min, after which the absorbance of the reaction mixture was measured at 415 nm. Total flavonoids contents were expressed as mg Quercetin equivalents (QE) per gram dry mass.

2.3.4 DPPH (2,2-diphenyl-1-picryhydrazyl) radical scavenging activity:

The DPPH assay for the determination of free radical scavenging activity of callus from various temperature treatments was done ^{[15, 16]}. 0.5 ml of methanolic extract of samples or 0.005 ml of standards such as gallic acid and ascorbic acid (mg/ml) were taken in separate test tubes. The volume was adjusted to 1ml by adding MeOH and 5 ml of freshly prepared DPPH solution (0.004% w/v) was added in each of these test tubes. After 30 min. the absorbance was taken at 517 nm. The DPPH solution without sample solution was used as control and methanol was used as blank. Percent scavenging of the DPPH free radical was measured using the following equation:

% DPPH radical scavenging (%) = [1-(As/Ac)] ×100.

Where, Ac=absorbance of control, As =absorbance of sample solution.

2.4 Statistical analysis

All the experiments were done in triplicate and the data were subjected to a one-way analysis of variance (ANOVA) and the significance of the difference between means was determined by Duncan’s multiple range test (P < 0.05) using the SPSS statistics. Values expressed are mean of three replicate determinations ± standard error.

3.0 RESULT AND DISCUSSION:

3.1. Incubation temperatures on callus culture

Plants often meet the critical growth conditions. Temperature is one of the climatic factors which act as major abiotic environmental stressor that limit plant growth and development. With respect to this phenomenon, the culture temperature also plays vital role in cell culture techniques for cell growth, morphology and secondary metabolites production.

The callus culture of Heliotropium indicum L. maintained on MS medium containing optimal concentrations of growth regulators under the exposure of various incubation temperatures such as 20, 25, 30 and 32°C for 30 days to study the morphogenetic response, total phenolic, flavonoids content and free radical scavenging activity of the callus. The callus grown under various temperatures show different in callus biomass and slight variation in callus morphology.

The influences of incubation temperatures (25±2°C and 30±2°C) on growth of rice callus were investigated. Significant different in growth and callus morphology was observed when they were cultured in these temperature ^[10]. Similarly in our study, while comparing the callus biomass of Heliotropium indicum L. which were produced under various temperatures with the callus grown under control temperature i.e 25° were significantly different. The callus biomass in all the concentrations was reduced than the control. The callus grown under 20 and 30°C were produced 0.43 ± 0.02 and 0.44 ± 0.02 g /tube respectively with the relative growth rate of 0.008 g/day. Moderate amount of callus was proliferated (0.63 ± 0.01 g/tube) under the temperature 30°C with the relative growth rate of 0.014 g/tube.

Table 1: Influence of incubation temperatures on callus biomass and callus morphology

Temperature	Callus biomass	Relative growth	Callus morphology
Temperature	g/tube	rate of callus (g/day)	(colour and texture)
20°C	0.43 ± 0.02^c	0.008	Brown/ Compact
25°C	1.92 ± 0.01^a	0.057	Dark brown/ Compact
(Control)
30°C	0.63 ± 0.01^b	0.014	Blackish brown/ Compact
32°C	0.44 ± 0.02^c	0.008	Blackish brown/ Compact

Fresh weight of callus and callus morphology were noted after 30 days of subculture.

Values for callus biomass represent mean ± standard error of three replicates.

Mean values followed by different letters are significantly different from each other at

P < 0.05 level comparison by Duncan’s multiple range test (DMRT)

Under all the temperature stress the texture of the callus was compact but there was insignificant variation in the colour. The temperature 20 and 25°C influenced brown colour callus formation where as 30 and 32°C alter the colour in to blackish brown (Table 1 and Figure 1 and2).

3.2. Influence of various incubation temperatures on total phenolics, flavonoids content and free radical scavenging activity of callus from Heliotropium indicum L.

The total phenolics and flavonoids content were considerably altered in the callus tissue by the various temperatures. The remarkable content of total phenolic 10.29 ± 0.09 mg/g were estimated in the callus tissues grown under the temperature of 30°C than the other callus tissue produced by various temperature. The temperature at 32°C also influenced the synthesis of total phenolics (9.08 ± 0.07 mg/g) which is equal to the total phenolic content of callus tissue from the control temperature at 25°C. The lowest temperature at 20°C was highly reduced the total phenolics synthesis (6.72 ± 0.07 mg/g) of callus tissue. Like this the culture temperature played an important role in the growth and isoflavone production from callus cultures of Pueraria candollei. Over twofold of growth and threefold of isoflavone production were demonstrated at 32±2ºC ^[11].

While estimating the flavonoids content from the callus tissue, the temperature at 32°C influenced to increase the flavonoids content (1.07 ± 0.03 mg/g) however it was less than the flavonoids (1.67 ± 0.04 mg/g) produced under the control temperature. Temperature at 20°C and 30°C were not favoured in synthesis of flavonoids like other temperatures, when we compare these both temperatures there was no significant difference in the flavonoids content (Table 2).

Table 2: Influence of various incubation temperatures on total phenolics and flavonoids content from callus culture of Heliotropium indicum Linn.

after 30 days of inoculation.

Temperature (mg/L)	Total phenolics	Flavonoids (mg/L)
20°C	6.72 ± 0.07^d	0.88 ± 0.03^c
25°C	9.97 ± 0.06^b	1.67 ± 0.04^a
(control)
30°C	10.29 ± 0.09^a	0.82 ± 0.03^c
32°C	9.08 ± 0.07^c	1.07 ± 0.03^b

Each values represents mean triplicate determination ± standard error of three replicates.

Mean values followed by different letters in each coloum are significantly different from each other at P < 0.05 level comparison by Duncan’s multiple range test (DMRT).

Figure 3: Percentage of free radical scavenging activity from methanolic extract of callus (Heliotropium indicum L.) under the influence of various incubation temperatures. Values are mean of triplicate determinations ± SE. Bars having different letters are significantly different (P < 0.05).

The antioxidant activity of callus tissues from various temperature treatments were studied by DPPH free radical scavenging method. Among the various temperature treatment the temperature at 30°C produced callus shows highest percentage of free radical scavenging activity (53.17 ± 1.43) than the other treatments. Callus produced under the temperature at 32°C also moderate percentage of free radical scavenging activity (50.79 ± 1.43). The lowest percentage of free radical scavenging activity was assayed from the callus tissue which was grown under 20°C it was comparatively less than the control temperature (25°C) produced callus (Figure 3).

4.0 CONCLUSION:

The callus grown under various incubation temperatures was studied for their biomass, total phenolic, flavonoids content and free radical scavenging activity. The callus biomass was not increased in the treatments than the control. In callus morphology there was no difference in the texture but the callus colour among the treatments show slight variations. There was no positive correlation between the callus growth and total phenolic content among the various treatments. However the callus produced under 30°C shows both maximum amount of phenolic content and highest percentage of free radical scavenging activity.

5.0 REFERENCES:

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Received on 08.09.2012 Accepted on 14.10.2012

Asian J. Pharm. Res. 2(4): Oct. - Dec. 2012; Page 148-152