Rapid industrialization , urbanization processes and poor management of industrial effluent has led to the incorporation of pollutants such as pesticides, petroleum products, acids and heavy metals in the natural resources like soil, water and air thus degrading not only the quality of the environment, but also affecting both plants and animals. Seed is a developmental stage that is highly protective against external stresses in the plant life cycle. Seed germination, seedling development and its growth rate play a key role in phytoremediation and Plants have evolved strategies to combat heavy metal stress. A few studies have reported the genetic and biochemical elements in plants helping them overcome heavy metal stress so that the present study was conducted to determine the effect of Cu,Ni,Pb and Cr on seed germination and seedling growth of marigold plant species Tagetes erecta and Calendula officinalis. Both the plant species were germinated in soil containing metal concentration ranges from 50-400 mg/kg of soil. Our results showed that when increased the concentration of Cu,Ni,Pb and Cr specifically above 100 mg/kg decreased in the percentage of seed germination, seedling growth and increased in the time of seed germination in both species of marigold as compare to control pot. This study showed that the both species of marigold very well tolerate to high concentration of metal upto 100 -300mg/kg in soil. The highest germination percentage as well as seedling growth of both species was estimated in the pot containing lead, followed by nickel, copper and chromium.
Environmental pollution caused by natural processes or anthropogenic activities is a major global problem. Environmental contamination has become a concerning issue worldwide due to number of risks it poses to human health and ecosystem functioning (Malik, 2017). Heavy metals can enter a water supply by industrial and consumer waste, or even from acidic rain breaking down soils and releasing heavy metals into streams, lakes, rivers, and groundwater. Evaluated heavy metals contaminated soils are widely spread and concerns have been raised over the potential risks to humans, animals and agriculture crops (Moraghebi, 2011). Heavy metals are great interest for research purpose with respect to toxicological importance to human health, plants and animals (Tchounwou PB, 2012). Excessive concentration of heavy metals viz., Cr, Cd, As, Ni, Se and Pb have been found in soils of agricultural land nearby cities, mines and industrial areas around the world (Dotaniya, 2016). The bioavailability of metals in soil is a dynamic process that depends on specific combinations of chemical, biological, and environmental parameters (Wati Ibnu Hajar, 2014). In heavy metal polluted soils, plant growth can be inhibited by metal absorption. However, some plant species are able to accumulate fairly large amounts of heavy metals without showing stress, which represents a potential risk to animals and humans (Oliver, 1997). The level of plant tolerance to heavy metals is related to the balance between the rate at which metal ions are taken up and the efficiency with which they are detoxified within the plant. Thus the same amount of a metal present in plant tissues may be detrimental for one species while not at all for others (Wati Ibnu Hajar, 2014).
In this study two species of Marigolds were selected as they can grow in polluted areas and they can tolerate contaminant toxicity. Marigold plant species recently reported by various researcher for their heavy metal accumulation potential (Choudhury, 2015). So that present investigation taken to evaluate the effect of selected heavy metals like Cu,Ni, Pb,and Cr on seed germination, and seedling growh of margold plant species Tagetes erecta and Calendula officinalis
Material and Methods
Seed samples and chemicals: Seed samples of tagetes erecta and calendula officencilis were purchased from Suman farms and garden pvt. Ltd.Nagpur ,India . All metal salt were used in this study purchased from High media pvt.ltd.
Soil collection and characterization: Soil was collected from garden of Dr. Ambedkar College, Deekshabhoomi. Nagpur (Maharashtra), India; at 10 cm to 20 cm deep surface by using soil sampler. Soil was dried, sieved through 2 mm screens and made ready to determine physical and chemical properties (Rahul Anand, 2015).
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Determination of moisture content: 100 grams of soil was weighed with water contents and after that the soil sample was dried in the hot air oven and after proper drying the soil sample was weighed again and finally the moisture content of soil was calculated as mentioned below. Moisture content was determined by the ratio of the weight of water in the soil to the weight of dry soil and expressed in % (Estefan G, 2012)
Determination of pH: The pH of the soil is an important factor of determining the growth rate of plant because pH regulates nutrients availability in plant. 20 grams weighed soil was placed into 100 ml polyethylene beakers and 40 ml water was added to make a saturated paste. Then it was shaken for 2 hrs or was stirred well with glass rod and electrode was immersed in the suspension. pH was reading when reading was stabilized on pH meter (Harold, 1934).
Determination of Particle size: 40 g air-dry soil (2-mm) was weighed and 60-Ml dissolving solution [(NaPO3)13 + (Na2CO3)] was added to this soil. A beaker was taken and covered with a watch glass, and left for overnight. Then Stir the suspension at high speed for 3 minutes using the stirrer. Stirring paddles was washed into a cup, and allow standing for 1 minute. Quantitatively suspension was move to a 1-L calibrated a cylinder (hydrometer jar), and bring to volume with water. Then suspension was poured through a 50 μl sieves to determine the sand clay and slit (Rahul Anand, 2015).
Determination of Electrical Conductivity: The same soil suspension prepared for Determination of pH was used for conductivity determination. After soil pH was recorded allow the soil suspension in the beaker to settle for maximum of ½ hours. The conductivity and 20 ml distilled water and 4 ml of reagent B was added. OD was taken on spectrophotometer after 10 minutes and value was calculated against the standard curve (Olsen S.R. and Sommers L.E.Phosphorous, 1982).
Determination of Potassium: 5 gm soil was placed in a 150 ml flask and 25 ml of neutral N ammonium acetate was added. After shaking for 5 minutes filter the solution to Whatman no. 1 filter paper. First few ml. of the filtrate was rejected. A solution was mixed and concentration of K was estimated by the flame photometer (Manohar, 2012) .
Determination of Nitrogen content in soil : 5g of soil was taken in a digestion tube and little water was added into it. Then 20 ml. of 0.32% KMnO4 solution was taken and added to sample and fit the tube in the distillation unit. 20 ml of 2.5% NaOH solution was added through the distyl-em-dosing pump. Then 20 ml. of 2.5% of boric acid was pipette out in a conical flask and clip the receiving end of the distyl-em in it. From the tube distil ammonia gas was collected in the received acid. 5 drops of mixed indicators were added and was titrated with 0.02N H2SO4. Blank correction (without soil) was to be made for final calculations (Rahul Anand, 2015).
Experimental Pots Preparation, Sowing and Cultivation of Plants: Equal quantity of soil (1kg) was taken in a rectangular container aluminum foil (15/20 cm) and various Concentration of different metal salt of copper (Copper Sulphate), Nickel (Nickel Chloride), lead(lead II acetate trihydride), chromium(chromium trioxide) was added in the experimental aluminum container separately (table no. 1). A triplicate of each respective metal salt concentration was prepared. These metals were properly mixed with soil. 10 seeds of each species of tagetes plant were sown with each metal treated and control soil pots separately .These pots were kept in green houses properly, regularly irrigated and observed daily to determine seed germination percentage and length of seedling .
Result and Discussion
Physiochemical analysis of soil: Soil analysis: Soil was collected from garden of Dr. Ambedkar College, Deekshabhoomi. Nagpur (Maharashtra), India. The various physical properties of soil were examined and mentioned in Table-2. In this study pH of the soil was 7.2. Therefore, this pH value was suitable for metal ions uptake by plants.
In this investigation carried out the effect of copper, nickel, lead, chromium metals on seed germination and seedlings growth of marigold species Tagetes erecta and Calendula officinalis and result obtained in this study presented in table no.3-6. Results of pot experiments showed that when increased the concentration of Cu,Ni,Pb and Cr decreased in the percentage of seed germination , seedling growth and increased in the time of seed germination in both species of marigold as compare to control pot.
Copper showed similar effect on seed germination of both species of marigold but seedling growth of Calendula officinalis less affected as campare to Tagetes erecta (table-3).Copper (Cu) has been reported to be toxic to sunflower seedlings inducing oxidative stress via generation of reactive oxygen species (ROS) and by decreased catalase (CAT) activity via oxidation of protein structure. Cu stress leads to reduced germination rate (Pena LB, 2011) (Sfaxi-Bousbih A, 2010)
Nickel (Ni) is reported to be toxic to most plant species affecting amylase, protease and ribonuclease enzyme activity thus retarding seed germination and growth of many crops. It has been reported to affect the digestion and mobilization of food reserves like proteins and carbohydrates in germinating seeds (Ahmad MS, 2011 ) in our study also observed similar effect of Nickel on both Tagetes erecta and Calendula officinalis (table-4). In this study lead also shows negative effect on seed germination, seedling growth and delay in germination on both species Tagetes erecta and Calendula officinalis (table-5). Chromium showed highest negative effect on Tagetes erecta and Calendula officinalis against seed germination as well as seedling growth and significantly changes observed in germination period as compare to control(table-6). The highest germination percentage as well as seedling growth of both species was estimated in the pot containing lead, followed by nickel, copper and chromium.
The study was carried out to evaluate the effect of the Cu , Ni, Pb, Cr on seed germination and seedling growth of Tagetes erecta and Calendula officinalis at all concentrations i.e. 50, 100, 200, 300 and 400 mg/Kg badly affected the germination period and partial decreased in germination percentage as well as growth of seedling. But both the species of marigold able to tolerate Cu,Ni,Pb, and Cr. in high concentration upto 300 mg/kg and able to grow in soil containing such a high concentration of toxic metals only period of germination badaly affected . Hence it is suggested that the use of both Tagetes erecta and Calendula officinalis to understand the unknown molecular mechanisms by which germinating seeds combat heavy metal stress. The future scope of this study remains in understanding the biochemistry of heavy metal toxicity in germinating seeds.
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