THE EFFECT OF NICKEL CHLORIDE (NICL2) ON VARIOUS CALLUS GROWTH DYNAMICS OF COLEUS BLUMEI (BENTH.) CULTURES

Amandeep Chabhal and Gurdeep Singh Virk * . Department of Botanical and Environmental Sciences, Guru Nanak Dev University, 143005, Amritsar. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


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Nickel as a heavy metal present in soil and water, in trace amounts is considered to be an essential micronutrient improving yield and quality of plants.It becomes toxic when present in higher concentrations (Kumar et al., 2012).The significance of Ni as a pollutant was recognized two decades back (Iljin, 1991).At higher concentrations, Nickel is highly phytotoxic at morphological, physiological and biochemical levels probably due to direct toxicity of metal or its tendency to compete with Ca 2+ , Mg 2+ , Fe 2+ and Zn 2+ cations causing their deficiency in plants (Seregin and Kozhevnikova, 2006;Sengar et al., 2008).The symptoms of Ni toxicity in plants are reported as inhibition of seed germination, growth, photosynthesis, sugar transport (Dubey and Pandey, 2011).Ni toxicity leads to induction of chlorosis, necrosis and wilting due to deficiency of essential micronutrients Zinc or Iron in the plant tissues (Anderson et al;1973;Madhava Rao andSresty, 2000, Pandey andSharma, 2002).
As an essential tool of biotechnological research, plant tissue culture techniques are of important use in plant propagation, disease free production, plant improvement and production of secondary metabolites.It is an efficient technology which provides the conductive environmental conditions for their growth and multiplication.The cultures are grown under stress conditions to study various physiological and biochemical parameters.These conditions are provided under proper supply of nutrients, pH medium, adequate temperature and humidity, proper gaseous and liquid environment.These micropropagation techniques have several advantages over the traditional methods of propagation such as seeds, cuttings, grafting, etc. (Nowak, 1998;Hussain et al., 2012).

Source of Plant Material:-
The aseptically grown in vitro shoots were transferred for callus induction into standardized 2, 4dichlorophenoxyacetic acid (2, 4-D) concentrations fortified to MS solid medium (Murashige and Skoog, 1962).The induced callus was then aseptically transferred to the test tubes (150 mm×25 mm) containing standardized MS Medium for further callus multiplication.

Supplementation of MS medium with Nickel Chloride Hexahydrate (NiCl 2 .6H 2 O):-
The mother stock solution of 1M NiCl 2 .6H 2 O was prepared and Seitz filtered through 0.22µm Millipore filter membranes under sterile conditions in the laminar flow cabinet and stored in an amber colored reagent bottle at 4˚C for further use.The different range of heavy metal NiCl 2 .6H 2 O concentrations were prepared from the mother stock solution by employing dilution equation (C 1 V 1 = C 2 V 2 ).The autoclaved MS medium was immediately transferred to the laminar flow cabinet where the different Ni metal concentrations calculated in the range of 0mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0mM were added from the mother stock solution of NiCl 2 .6H 2 O to the different culture flasks containing culture medium under aseptic conditions.The Ni supplemented MS medium was poured into culture tubes and flasks, sealed and marked with the respective concentrations of heavy metal and transferred to the culture room maintained at 25±1ºC and 70% relative humidity under 40 µmole/m 2 /sec cool white fluorescent lights for 16 h photoperiod/8h dark period daily for solidification and further use in experiments.

Determination of Callus Growth Dynamics:-
The growth rate of callus was monitored after 30 days of culture by measuring the percent callus survival, percent callus multiplication, average fresh and dry weights, percent growth index and percent dry matter content of callus from the data recorded on in vitro cultures.Approximately, 500 mg of 30 days old callus tissues were transferred to each culture tube containing the fortified MS medium supplemented with different Ni concentrations.Each experimental set up was recorded after 30 days of inoculation and repeated in 3 replicas.The rate of callus growth dynamics are calculated as follows:-

The rate of callus survival was expressed as:
The percent callus multiplication is calculated as: The fresh weight was calculated as an average of three fresh weights of callus cultures grown in three different culture flasks.The fresh callus measured in grams (g) was then oven dried at 80˚C for 48 hours prior to the dry weight determination.The dry weight of callus was calculated in grams (g) by subtracting the initial weight of 865 empty petriplate from the final weight of the petriplate containing dried callus.The experiment was performed in three replicas.
The growth index of callus was measured in terms of initial and final fresh weights at an interval of 21 days.The measured weight of callus (500 mg) was inoculated on the fortified MS medium at different NiCl 2 concentrations.After 7 days, the callus from three culture tubes was removed and their average initial fresh weight was recorded at each concentration.After 28 days of inoculation, the final average fresh weight of callus was measured at each concentration.

The percent callus dry matter content determined according to Khater et al. (2013) was calculated as:-
In the present investigation, the IC 50 value of the heavy metal nickel was decided on the basis of 50% decrease in the percent callus survival, percent callus multiplication, percent callus growth index, average fresh and dry weight of callus with respect to the control on the basis of regression equation.

Statistical Analysis:-
The data were analyzed for mean, standard error (SE) and linear regressions using self coded computer software in MS Excel and the values were expressed as Mean ± SE.

Results and Discussion:-Effect of Nickel (Ni) on callus growth parameters:-
The data tabulated in Table 1 represents the effect of NiCl 2 concentrations on the various growth parameters of in vitro callus cultures.It is evident that with increasing metal treatments there was a decline in percent callus survival, percent callus multiplication, fresh weight, dry weight and percent growth index of callus.As per regression equation, there is 50% decrease in values at 0.4 mM Ni treatment with respect to control.Further there was significant decrease in the growth parameters from 0.4 mM to 0.8 mM treatments as compared to control treatments (Figure 1).
In case of percentage callus dry matter content, an increase in trend was observed with increasing metal concentrations.The percentages of callus dry matter content ranging from 0.1 mM to 0.8 mM concentrations were higher as compared to the control treatments (Figure 1).Similarly, Fathalla et al. (2011) reported in Roseus (L.) G. Don.Callus Cultures that with increasing concentrations of mercury (Hg) metal there was significant decrease in the different growth parameters of shoot and root derived callus.However, the percentage of callus dry matter content significantly increased with increasing Hg metal concentrations than the control treatment.Table 1:-Effect of different concentrations of nickel chloride (NiCl 2 ) on percent callus survival, percent callus multiplication, fresh weight (g), dry weight (g), callus growth index (%) and callus dry matter content (%) of in vitro cultures of C. blumei (Benth.)inoculated on MS medium (observation recorded after 30 days of inoculation).

Figure 1 :
-Response of callus growth dynamics represented as (a) percent callus survival, (b) percent callus multiplication, (c) average fresh weight (g), (d) average dry weight (g), (e) callus growth index (%) and (f) callus dry matter content (%) of in vitro C. blumei (Benth.)callus cultures inoculated on MS medium supplemented with various nickel concentrations (observations recorded after 30 days of inoculation in triplicates).