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INTRODUCTION INTRODUCTION Field view of pea Field view of maize Environmental stresses such as salinity, drought, heat, cold, flooding and heavy metal toxicity are major threat to the agricultural productivity worldwide (Gaafar et al., 2012). Heavy metals (HMs) are among the major environmental contaminants and pose a severe threat to human and animal health by their long-term persistence in the environment (Gisbert et al., 2003; Halim et al., 2003; Jabeen et al., 2009), if they are excess in the food, water and in the air they may cause lot of problems (Salama and Radwan, 2005; Itumoh et al., 2011; Omaka, 2012; Omaka et al., 2012; Itumoh et al., 2013). They are given special attention throughout the world due to their toxic effects even at very low concentrations (Salama and Radwan, 2005) or at high concentrations (Stevovic et al., 2010). Today, several cases of human diseases, disorders, malfunction and malformation of organs due to metal toxicity have been reported (Angelin-Brown et al., 1995; Stoica, 1999; Salama and Radwan, 2005; Omaka, 2008; Itumoh et al., 2011; Itumoh et al., 2013). Concentration of these toxic metals has accelerated dramatically since the beginning of the industrial revolution (Ana et al., 2009) thus, posing problems to health and environment (Nriagu, 1979). Once the heavy metals contaminate the ecosystem, they remain a potential threat for many years. Heavy metal contaminants causing ecological problems are of global concern. Heavy metal refers to metals and metalloids with atomic densities greater than 4 g cm-3 or 5 times or more greater than water and is usually associated with pollution and toxicity although; some of these 1 INTRODUCTION elements (essential metals) are required by organisms at low concentrations (Hawkes, 1997; Adriano, 2001). However, chemical properties of the heavy metals are the most influencing factors compared to their density. Heavy metals include lead (Pb), cadmium (Cd), nickel (Ni), cobalt (Co), iron (Fe), zinc (Zn), chromium (Cr), iron (Fe),arsenic (As), silver (Ag) and the platinum group elements (Nagajyoti et al., 2010). Heavy metals are significant environment pollutants (Berry, 1986). Plants are susceptible to heavy metal toxicity, and their toxicity is a problem of increasing significance for ecological, evolutionary, nutritional and environmental reasons. There are different sources of heavy metals in the environment such as: (i) natural sources (ii) agricultural sources (iii) industrial sources (iv) domestic effluents (v) atmospheric sources Heavy metal pollution can originate from both natural and anthropogenic sources. Activities such as mining and smelting operations and agriculture have contaminated extensive areas of world such as Japan, Indonesia and China mostly by heavy metals, i.e. Cd, Cu and Zn (Herawati et al., 2000), Cu, Cd and Pb in North Greece (Zanthopolous et al., 1999) in Albania (Shallari et al., 1998) and Cr, Pb, Cu, Ni, Zn and Cd in Australia (Smith, 1996). The most important natural source of heavy metals is geologic parent material or rock outcroppings. The geologic plant materials generally have high concentrations of Cr, Mn, Co, Ni, Cu, Zn, Cd, Sn, Hg and Pb (Pacyna, 1986; Nagajyoti et al., 2010). During the last two decades there has been an increasing awareness of the potential adverse effects of soil pollution by trace metals (Adriano, 2001; Remon et al., 2013). Their availability in soils depends on natural procedure, especially lithogenic and pedogenic soils and anthropogenic factors such as mining, combustion of fossils fuels, urban waste disposal, soil runoff, metal working industries, boating activity, and phosphate fertilizer application. An increase in heavy metals in the soils could also be attributed to factors such as soil properties or different agricultural practices eg., application of sludge to agricultural land (Foy et al., 1978). The household municipal and industrial wastes are also sources of heavy metals to soils (Alloway, 1995). Soil contaminated with heavy metals above the permissible limit 2 INTRODUCTION lead to decreased agricultural yields (Nellessen and Fletcher, 1993; Akinola and Ekiyoyo, 2006). There is a two way relationships between the high concentration of heavy metals in the soil and the expression of toxicity. On the one hand, heavy metals compete with essential mineral nutrients for uptake thereby disturbing the mineral nutrition of plants (Clarkson and Luttge, 1989) and on the other hand after uptake by the plant, it accumulates in plant tissue and cell compartments and hampers the general metabolism of the plant (Taylor, 1988, Turner, 1997; Hasan et al., 2009). The time of exposure, the degree of toxicity influenced by biological availability of metals and interactions with other metals in the soil, nutritional status, age and mycorrhizal infection of the plant (Anna-Maj, 1989) are some of the factors with contribute to the phytotoxicity of the metals. Heavy metal accumulation in soils is of concern in agricultural production due to the adverse effects on food safety and marketability, crop growth due to phytotoxicity, and environmental health of soil organisms (Nagajyoti et al., 2010).The heavy metal accumulation by crop species decreases in the following order: leaf vegetables>root vegetables>grain crops (Puschenrieter et al., 2005; Korkmaz et al., 2010). The vegetables absorb and adsorb these metals from the ground as well as from the parts of vegetable exposed to air from polluted environment (Vausta et al., 1996; Oti Wilberforce and Nwabue, 2013). There differences can occur between different parts of the crops and the edible parts are the most relevant as heavy metals can be easily transferred from them to human food chain. Except for roots, the highest concentrations are found in leaves, whereas the lowest are typically observed in seeds (Ivanova et al., 2003; Korkmaz et al., 2010). Heavy metals are accumulated in the environment and lead to reduced root and shoot growth, low yield production, low nutrient uptake and impaired homeostasis. Growth inhibition is a general phenomenon associated with most of heavy metals (Peralta et al., 2001), although the tolerance limits for heavy metals toxicity are specific not only for species but also for each variety of crop plants (Metwally et al., 2008). The acidification of the rhizosphere and exudation of carboxylase are considered to be potential targets of enhanced metal accumulation (Clemens et al., 2002; Yang et al., 2005). Heavy metals taken up by plants from contaminated soil and water are toxic to growth performance of plants and posses a hidden threat to consumers. The toxic levels of heavy metals change the pattern of biomass productivity, plant growth, photosynthetic pigments, protein, amino acids, starch, soluble sugars, 3 INTRODUCTION and essential nutrients uptake. Plants have multiple direct and indirect effects on plant growth and alter many physiological functions due to heavy metals toxicity (Wool House, 1983) by forming complexes with O, N and S ligands (Van Assche and Clijsters, 1990). Thus, heavy metals interfere with mineral uptake, protein metabolism, membrane functioning (Azevedo et al., 2005) seed germination and water relations (Kastori et al., 1992). Many heavy metals like Fe, Cu, Cd, Cr, Zn. etc have been shown to cause oxidative damage in higher plants (Prasad et al., 1999; Panda and Patra, 2000). Highly reactive free radicals are produced with the exposure of plants to a range of abiotic stresses which include water, salt and heavy metal toxicity etc. These reactive oxygen species (ROS) have been implicated directly with molecular damage in plant cells. The heavy metal toxicity is also attributed to the generation of ROS leading to oxidative injury (Hendry et al., 1992; Gallego et al., 1996; Chaoui et al., 1997; Goel, 2012). As, certain metals are known to act as catalysts for the production of Free radicals in radicals in biological system. Reactive oxygen species (ROS) can damage biological molecules including DNA, RNA, protein and lipid by inducing peroxidation (Shah et al., 2001). Lipid peroxidation occurs in plants as a consequence of high ROS level when excessive ROS cannot be scavanged immediately and effectively and finally result in the disruption of plant growth and development. Malondialdehyde (MDA) is one of the ultimate products as a result of lipid peroxidation (Bailly et al., 1996). Therefore, antioxidant enzymes activities and MDA content often serve as important physiological indicators for the resistant abilities of plants under stress conditions (Yin et al., 2009). The over production of ROS is a common response of plants to different stress factors. An oxidative stress is defined as a shift of the balance between prooxidative and antioxidative reactions. The sources of reactive oxygen includes various environmental and biological factors such as hyperoxia, light, draught, high salinity, cold, metal ions, pollutants, xenobiotics, toxins, reoxygenation after anoxia, experimental manipulations, pathogen, infection and aging of plant organs. The production of ROS during environmental stress is one of the main cause of decreases in productivity, injury and death that accompany these stresses in plants. Generation of reactive oxygen species (ROS) has been identified as an inevitable process of normal aerobic metabolism in plants and the four major types of ROS are singlet oxygen (1O2), superoxide (.O2-), hydrogen peroxide (H2O2) and hydroxyl radicals 4 INTRODUCTION (OH-) (Dinakar et al., 2010). The reactive oxygen species are also generated in plant cells during normal metabolic processes. When plants are exposed to toxic metals, apoplastic transport followed by cytosolar uptake and distribution of metals to organelles which causes ROS generation which affects sub-cellular metabolism (Sharma and Dietz, 2009). Fig. 1. Occurrence of heavy metals in India (www.google.com) 5 INTRODUCTION 1.1 Cadmium The regulatory limit of Cadmium (Cd) in agricultural soil is 100 mg/kg soil (Salt et al. 1995). Cd is a heavy metal that normally occurs in low concentrations in soils and up to 100-120 mg/kg dry weight was reported by Lombi et al. (2002) and Nwaichi et al. (2010). In plants, the accumulation of Cd can cause numerous morphological and physiological changes. Plants grown in soil containing high levels of Cd show visible symptoms of injury reflected in terms of chlorosis, necrosis, leaf roll, growth inhibition, browning of root tips and finally death (Sanita di Toppi and Gabbrielli, 1999; Benavides et al., 2005; Guo et al., 2008). Cadmium structure At the physio-logical level, excess Cd results in an inhibition of photosynthesis and transpiration (Mobin and Khan, 2007; Shi and Cai, 2008; Shi et al., 2010), imbalance of mineral nutrients, induction of oxidative stress (Sandalio et al., 2009), changes in enzyme activity (Hasan et al., 2009), and modifications to gene expression (Herbette et al., 2006). Photosynthesis, one of the major determinants to biomass production, has been shown to be very sensitive to Cd in higher plants (Ekmekci et al., 2008; Liu et al., 2011). The inhibition of root Fe (III) reductase induced by Cd led to Fe (II) deficiency, and it seriously affected photosynthesis (Alkantara et al., 1994). In general, Cd has been shown to interfere with the uptake, transport and use of several elements (Ca, Mg, P and K) and water by plants (Das et al., 1997). Cd also reduced the absorption of nitrate and its transport from roots to shoots, by inhibiting the nitrate 6 INTRODUCTION reductase activity in the shoots (Hernandez et al., 1996). Cadmium treatments have been shown to reduce ATPase activity of the plasma membrane fraction of wheat and sunflower roots (Fodor et al., 1995). Cadmium produces alterations in the functionality of membranes by inducing lipid peroxidation (Fodor et al., 1995) and disturbances in chloroplast metabolism by inhibiting chlorophyll biosynthesis and reducing the activity of enzymes involved in CO2 fixation (De Filippis and Ziegler, 1993; Nagajyoti et al., 2010). 1.2 Chromium Chromium (Cr) is one of the most toxic heavy metals found abundantly in the earth’s crust (Panda and Choudhory, 2005; Nematshahi et al., 2012), which attenuates the environment. Although toxic, it has nutritive importance too (Kabata-Pendias and Pendias, 2001). Chromite structure Chromium can exist in several chemical forms, displaying oxidation numbers from 0 - VI. Cr compounds are highly toxic to plants and are detrimental to their growth and development. Although some crops are not affected by low Cr (3.8 x 104 μM) concentrations (Huffman and Allaway, 1973a,b), Cr is toxic to higher plants at 100 kg-1 dry weight (Davries et al., 2002). Since seed germination is the first physiological process affected by Cr, the ability of a seed to germinate in a medium containing Cr would be indicative of its level of tolerance to this metal (Peralta et al., 2001). High levels (500 ppm) of hexavalent Cr in soil reduced germination up to 48% in the bush bean Phaseolus vulgaris (Parr and Taylor, 1982). Peralta et al. 7 INTRODUCTION (2001) found that 40 ppm of Cr (VI) reduced by 23% the ability of seeds of Lucerne (Medicago sativa cv. Malone) to germinate and grow in the contaminated medium. Reductions of 32–57% in sugarcane bud germination were observed with 20 and 80 ppm Cr, respectively (Jain et al., 2000). The reduced germination of seeds under Cr stress could be a depressive effect of Cr on the activity of amylases and on the subsequent transport of sugars to the embryo axes (Zeid, 2001). Protease activity, on the other hand, increases with the Cr treatment, which could also contribute to the reduction in germination of Cr-treated seeds (Zeid, 2001). Decrease in root growth is a well-documented effect due to heavy metals in trees and crops (Tang et al., 2001). Chromium stress is one of the important factors that affect photosynthesis in terms of CO2 fixation, electron transport, photophosphorylation and enzyme activities (Clijsters and Van Assche, 1985). In higher plants and trees, the effect of Cr on photosynthesis is well documented (Van Assche and Clijsters, 1983). However, it is not well understood to what extent Cr-induced inhibition of photosynthesis is due to disorganization of chloroplasts ultra structure (Vazques et al., 1987), inhibition of electron transport or the influence of Cr on the enzymes of the Calvin cycle. Chromate is used as a Hill reagent by isolated chloroplast (Desmet et al., 1975). The more pronounced effect of Cr(VI) on PS I than on PS II activity in isolated chloroplasts has been reported by Bishnoi et al. (1993a, b) in peas. Nevertheless, in whole plants, both the photosystems were affected. Chromium stress can induce three possible types of metabolic modification in plants as follows: (i) alteration in the production of pigments, which are involved in the life sustenance of plants (e.g., chlorophyll, anthocyanin) (Boonyapookana et al., 2002). (ii) increased production of metabolites (e.g., glutathione, ascorbic acid) as a direct response to Cr stress, which may cause damage to the plants (Shanker et al., 2003b). (iii) alterations in the metabolic pool to channelize the production of new biochemically related metabolites, which may confer resistance or tolerance to Cr stress e.g., phytochelatins, histidine (Schmoger, 2001). Induction and activation of superoxide dismutase (SOD) and of antioxidant catalase are some of the major metal detoxification mechanisms in plants (Shanker et al., 2003a). Gwozdz et al. (1997) found that at lower heavy metal concentrations, activity of antioxidant enzymes increased, whereas at higher concentrations, the SOD 8 INTRODUCTION activity did not increase further and catalase activity decreased (Nagajyoti et al., 2010). 1.3 C3 and C4 Plant System In this study two plants such as pea (a C3 plant) and maize (a C4 plant) were taken as test plants. Their physiological and morphological details are discussed below: In nature three different plant systems exist viz., C3, C4 and CAM, characterized on the basis of CO2 trapping mechanisms, however; C4 and CAM plants essentially follow C3 pathway to trap CO2 as an initial step. C4 is a characteristic photosynthesis syndrome of angiosperms. Generally, phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) enzyme is widespread among all plants, including C3 (e.g., Pisum sativum, Gossipium hirsutum, Oryza sativa, Brassica campestris, Triticum aestivum, Avena sativa) C4 (e.g., Zea mays, Saccharum officinarum, Sorghum spp., Vetiveria zizanioides, Cyanadon dactylon) and CAM (e.g., members of Orchidaceae, Polypodiaceae (ferns)) species and is responsible for the initial carbon fixation in C4 and CAM plants. C3 and C4 plants have unique carbon trapping mechanisms. The general enzymatic system involves in CO2 fixation in C3 and C4 are Ribulose-1-5-bisphosphate carboxylase oxygenase (Rubisco EC 4.1.1.39) and phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), Pyruvate, phosphate dikinase (PPDK) respectively. The leaves of C4 plants display Kranz anatomy whereby an outer layer of mesophyll cells containing chloroplast surrounds vascular bundles with an inner layer of bundle sheath cells (Dengler and Nelson, 1999). In C3 plants, mesophyll cells are devoid of chloroplast and CO2 is fixed in bundle sheath cells by Rubisco. The chloroplasts of C3 plant contain a complete Calvin cycle and are able to assimilate CO2 to convert it to the principle 3 carbon compound (triose phosphate), on the other hand CO2 is distributed in two cells viz., mesophyll and bundle sheath in C4 plants and converted primarily in 4 carbon compound (acid oxaloacetate) by the action of PEPC in mesophyll cells which is then transported in bundle sheath cells where by the acids from mesophyll cells provide carbon dioxide. Extensive research literature is available on the comparative account of C3 and C4 plant systems (Du and Fang, 1982; Derner et al., 2003; Sage, 2004; Edwards et al., 2005; Caird et al., 2007; Brautigam et al., 2008; Doubnerova and Ryslava, 2011). However, very few and 9 INTRODUCTION scattered information is available on the comparative account of C3 and C4 plants and their growth performances under extreme environmental conditions. Photosynthesis in C 4 plants does not saturate but increases at high light intensities and can continue at very low CO 2 concentrations (Sage, 2004; Brautigam et al., 2008). Subsequently, these plants have rapid growth rates and higher biomass and economic yields as compared to the C 3 plants. There are evidences from researches that C 4 plant such as vetiver grass (Vetiveria zizanioides L. Nash) can withstand harsh environmental conditions (Chen et al., 2004, Chiu et al., 2006). A comparative study performed on two separate species belonging to C 3 and C4 systems respectively show that the environmental tolerance depends on the high biomass production which is higher in case of C 4 plants (Ali et al., 2002). However; there is lack of information regarding the biochemical differences among C 3 and C4 plant systems exposed to toxic environment for e.g., the extent of detoxification mechanism, mycorrhization, proteomes (expression of genes). Toxic response of particular plant variety belonging to C 3 and C4 type indicate that there is a high tolerance in C4 plants as compared to the C 3 plants which may or may not be true for the entire group of plants belonging to these systems (Chapin, 1991; Ali et al., 2002). C4 photosynthesis allows fast biomass accumulation with high nitrogen and water use efficiency (Leegood and Edwards, 1996; Sage, 2004) which is desired set of traits to increase the productivity of crop plants (Matsuoka et al., 1998) and a required character for successful phytoremediation (Srivastava et al., 2012). Pea (Pisum sativum L.) The pea is a cool-season annual vine that is smooth and has a bluish-green waxy appearance. Vines can be up to 9 ft long, however modern cultivars have shorter vines, about 2 ft long. The stem is hollow, and the taller cultivars cannot climb without support (Elzebroek and Wind, 2008). Leaves are alternate, pinnately compound, and consist of two large leaf like stipules, one to several pairs of oval leaflets, and terminal tendrils (McGee, 2012).Pea is an important vegetable in India; the crop is generally cultivated for its green pods. It is highly nutritive and is rich in protein. It is used as a vegetable or in soup, canned frozen or dehydrate. It is cooked as a vegetable along or with potatoes. Split grains of pea are widely used for dal. Pea straw is a nutritious fodder. 10 INTRODUCTION Fig. 2. The global cultivation of Pisum sativum (http//en.wikipedia.org/wiki/file:peayield.png) with seed yields Peas are adapted to many soil types, but grow best on fertile, light-textured, well-drained soils (Hartmann et al., 1988; Elzebroek and Wind, 2008). Peas are sensitive to soil salinity and extreme acidity. Pea is a cool season crop and performs best at 10°C to 18°C. The flower and young pods are badly affected by frost. The germination of seeds takes place at 3.3°C soil temperature. Boswell (1920) reported that as the temperature increases during the growing season the yield decline sharply. The optimum mean monthly temperature for pea is 12.8°C to 18°C. Pea is currently grown in temperate regions, at high elevations, or during cool seasons in warm regions throughout the world (Elzebroek and Wind, 2008). Major pea producers are China, India, Canada, Russia, France and the United States (FAO, 2012) as shown in Fig. 2. 1.4 Harvesting Peas are harvested for table use when the pods are filled and the young tender peas changing in colour from dark to light green. Peas may be picked in 45 to 60 days, 75 days and 100 days according to early, mid and late season. Airtimes respectively, 3 to 4 pickling are done within the interval of 2 to 10 days. Fresh unshielded peas may be kept two at °C and 90-95 percent relative humidity. 11 INTRODUCTION 1.5 Commercial Crop Peas are a cool-season crop grown for their edible seed or seed pods. Garden or green peas are harvested before the seeds are mature for the fresh or fresh-pack market (Elzebroek and Wind, 2008). Sugar snap peas and snow peas lack the inner pod fiber and are also harvested early for the fresh or fresh-pack market (McGee, 2012). Field peas, including fall-sown Austrian winter peas, are harvested when seeds are mature and dry, and are primarily blended with grains to fortify the protein content of livestock feed. Dried peas are also sold for human consumption as whole, split or ground peas. Peas are a nutritious legume, containing 15 to 35% protein, and high concentrations of the essential amino acids lysine and tryptophan (Elzebroek and Wind, 2008). 1.6 Forage Crop Peas are grown alone or with cereals for silage and green fodder (Elzebroek and Wind, 2008). Peas can also be grazed while in the field. Young Austrian winter pea plants will regrow after being grazed multiple times (Clark, 2007). 1.7 Rotational Crop Peas and other legumes are desirable in crop rotations because they break up disease and pest cycles, provide nitrogen, improve soil microbe diversity and activity, improve soil aggregation, conserve soil water, and provide economic diversity. 1.8 Green Manure and Cover Crop Peas are grown as green manures and cover crops because they grow quickly and contribute nitrogen to the soil (Clark, 2007). Pea roots have nodules, formed by the bacteria Rhizobium leguminosarum, which convert atmospheric nitrogen to ammonia. Peas also produce an abundance of succulent vines that breakdown quickly and provide nitrogen (Clark, 2007). Austrian winter peas are the most common type of pea used as a green manure or cover crop because they are adapted to cold temperatures and fit in many rotations. Therefore, Pea plants were chosen to reveal consequences of Cadmium toxicity in-vivo and in vitro. Pea is a self pollinated crop plant, commonly known as seed-pod of the legume. Botanically it is a fruit, treated as a vegetable in cooking, grown once in a year in cool climatic surrounding depending on location altitude. The 12 INTRODUCTION seeds are used as a vegetable, fresh, frozen or canned, and are also dry peas in the form split pea as pulses. They do not thrive in the summer heat of warmer temperate and tropical climates but do grow well in cooler and high altitude tropical areas. Generally, many cultivars reach maturity in about 60 can also be grown outdoors during the winter. The protein concentration of pea seeds ranges from 15.5-39.7% (Bressani and Elias, 1988) along with other nutritional characteristics as shown in Table 1. Table 1. Nutritional values of raw green pea Raw Green Pea (Nutritional value 100g seeds) Energy 339 kJ (81 kcal) beta-carotene 449 μg (4%) Carbohydrates 14.5 g Thiamine (Vit. B1) 0.3 mg (23%) Sugars 5.7 g Riboflavin (Vit. B2) 0.1 mg (7%) Dietary fiber 5.1 g Niacin (Vit. B3) 2.1 mg (14%) Fat 0.4 g Pantothenic acid (B5) 0.1 mg (2%) Protein 5.4 g Vitamin B6 0.2 mg (15%) Vitamin A equiv. 38 μg (4%) Folate (Vit. B9) 65 μg (16%) Vitamin C 40.0 mg (67%) Iron 1.5 mg (12%) Calcium 25.0 mg (3%) Phosphorus 108 mg (15%) Magnesium 33.0 mg (9%) Potassium 244 mg (5%) Zinc 1.2 mg (12%) lutein and zeaxanthin 2593 μg (Source: USDA Nutrient database; www.google.com) Maize (Zea may L.) Maize (Zea mays L.) is the most important grain crop. Maize is widely cultivated throughout the world, and a greater weight of maize is produced each year than any other grain. Maize (Zea mays L.), is world’s one of the three most popular cereal crops for both livestock feed and human nutrition. With its high content of carbohydrates, fats, proteins, some of the important vitamins and minerals, maize is the main food of the lower-socio-economic populations and in 13 INTRODUCTION some regions it represents more than 80% of daily food consumption. The United States produces 40% of the world's harvest; other top producing countries include China, Brazil, Mexico, Indonesia, India, France and Argentina (Fig. 3). Maize is the most widely grown grain crop throughout the Americas, with 332 million metric tons grown annually in the United States alone. Approximately 40% of the crop 130 million tons is used for corn ethanol. Maize kernels are used in cooking as a starch. The most suitable soil for maize is one with a good effective depth, favourable morphological properties, good internal drainage, an optimal moisture regime, sufficient and balanced quantities of plant nutrients and chemical properties that are favourable specifically for maize production. Although largescale maize production takes place on soils with a clay content of less than 10 % (sandy soils) or in excess of 30 % (clay and clay-loam soils), the texture classes between 10 and 30 % have air and moisture regimes that are optimal for healthy maize production. Therefore, maize plants were chosen to reveal consequences of Chromium toxicity in-vivo and in vitro. Maize (Zea mays L.) is one of the most important cereal crops and comprises some heavy metal tolerant genotypes (Clark, 1977; Liu et al., 2001). Some maize cultivars with capability of absorbing and accumulating extraordinarily high amounts of heavy metals from soil (Liu et al., 2006). Zea mays L. is one of the most important agricultural crops. Being rich source of nutrition (72% starch, 10% protein, 8.5% fiber & 4.8% edible oil), maize is one of the major sources of food, sugar, cooking oil and animal feed all over the world (Dowswell et al., 1996; Hussain et al., 2012). The nutritional values are given to Table 2. 14 INTRODUCTION Fig.3. The global cultivation of Zea mays (http//en.wikipedia.org/wiki/file:Zeayield.png) L. with seed yields Table 2. Nutritional values of raw yellow corn Raw Yellow corn (Nutritional value 100g seeds) Energy 360 kJ (86 kcal) Thiamine (Vit. B1) 0.200 mg (17%) Carbohydrates 19.02 g Niacin (Vit. B3) 1.700 mg (11%) Sugars 3.22 g Vitamin B6 0.093 mg (7%) Dietary fiber 2.7 g Folate (Vit. B9) 0.093 mg (7%) Fat 1.18 g lutein and zeaxanthin 644 μg Protein 3.22 g Potassium 270 mg (6%) Vitamin A equiv. 9 μg (1%) Iron 0.52 mg (4%) Vitamin C 6.8 mg (8%) (Source: USDA Nutrient database; www.google.com) 1.9 Harvesting and Uses It is very important to harvest sweet corn at the proper stage of maturity. The critical time is the milk stage, a stage when the juice in the kernel appears milky when you puncture the kernel with your thumbnail. Sweet corn remains in the milk stage for 15 INTRODUCTION a relatively short period, so check the ears frequently. Corn that is too young will ooze a watery material, while ears that are too old will have a tough, doughy kernel. During the milk stage, the unhusked ear should feel firm, have full kernels at the tip of the ear, and have brown, dry silks. Generally, ears should be ready about three weeks from silking time. Field corn in the U.S. is used mainly to feed livestock, but in other countries is used for human consumption as well. Indian corn was originally the term applied to what we now know as maize or corn, to differentiate it from the generic term of “corn” Europeans used for all grains at that time. Now, it usually refers to any corn that has different colored kernels. Usually it is dried and used for ornamental purposes. Popcorn, the ability of maize kernels to “pop” and expand upon heating, was also discovered by the Native Americans. Maize is able to pop because, unlike other grains, its kernels have a hard moisture-sealing hull and a dense starchy filling. When heated, pressure builds inside the kernel until an explosive "pop” results, and the starch expands and then hardens in the cooler air. Many maize varieties will pop, but some varieties have been specifically cultivated for this purpose. Maize flour, or meal, is made into a thick porridge in many cultures (polenta, Italy; angu, Brazil; mãmãligã, Romania; sadza, nshima, ugali, and mealie pap, Africa). Maize meal is also used as a replacement for wheat flour, to make cornbread and other baked products. Popcorn Maize flour Fig. 4. The various maize products used by the society 16 Corn syrup INTRODUCTION Corn syrup is used as a sweetener instead of sugar in thousands of products, including soda, candy, cookies and bread as shown in Fig. 4. The effects of chromium on some metabolic activities of maize (Zea mays L.) have caused visible lesions of interval chlorosis. The young leaves showed vein clearing margins of leaves with curling and appearance of pale colour due to loss in chlorophyll (Sharma et al., 2003; Labra et al., 2006; Zou et al., 2009). The growth of pea (Pisum sativum L.) plants treated with CdCl2 for 15 days produced a reduction in the number and length of lateral roots and changes in structure of the principal roots affecting the xylem vessels. Cadmium induces reduction in glutathione and ascorbate content, and also reduces activities of catalase, glutathione reductase and guaiacol peroxidase (Serrano et al., 2006; Popova et al., 2008; Smiri et al., 2010; Bavi et al., 2011; Januskaitiene, 2012). Keeping above details in view, our study is aimed to investigate to the following: 1. The effect of heavy metals on seed germination, growth and biomass characteristics of pea and maize plants. 2. The effect of heavy metals (Cadmium and Chromium) on physiological and biochemical characteristics on pea and maize crop plants. 17