Study of heavy metals effect in response to linum seed germination

The aim of the present study is to develop phenotypically stable varieties for those soils that are contaminated with mercury or cobalt. This is a novel report about their ability to grow in the contaminated soil. The benefit of this technology is the potential for low cost remediation. Highly significant differences have been observed among the varieties of Linum usitatissimum for all the characters in all the treatment or environment. There has been considerable amount of variability for all the characters in all the treatments. The 10 -5 M HgCl2 treatment shows the inhibitory effect in all the varieties and there is no further seedling establishment after seed germination. The highest toxic effect has been observed for the seedling vigour and seed vigour index. The 10 -5 M HgCl2 concentration is the last limit of tolerance in plants while 10 -4 M CoCl2 could be the last tolerance limit and there may be a specific gene in plants which monitor the toxicity level or tolerance capacity. The maximum magnitude of positive correlation coefficient has been found between seedling fresh and dry weight and negative for 1000 seed weight and germination rate index. The seedling length exerts maximum positive direct effect on seedling vigour followed by absolute seedling water content.


INTRODUCTION
Among all the oil seed crops, linseed (Linum usitatissimum L.) ranks fourth in importance in term of area as well as production.It is cultivated in almost all the countries of the world.It is chiefly grown as a fibre crop in European and other temperate countries while in India it is exclusively cultivated as oil seed crop.Heavy metals contamination has been recognized as a major environmental concern due to their pervasiveness and persistence.These heavy metals are not bio-degradable, hence there is a need to develop such a remediation technique, which should be efficient, economical and rapidly deployable in a wide range of physical settings.It is also necessary to integrate different areas, such as biology, phytogeography, soil sciences and even anthropology to get a more dynamic view of the problem.Such changes might add significant new information to our knowledge on the subject.Elevated levels of heavy metals in contaminated soils are widely spread and concerns have been raised over the potential risks to humans, animals and agricultural crops.As a rule, heavy metal has a negative effect on the growth of organisms as it can greatly depress their numbers.Contamination of wastewater with high rates of heavy metals caused a significant decrease in biodiversity.
Heavy metal contamination affects the biosphere in many places worldwide (Cunningham, et al., 1997;Raskin and Ensley, 2000;Meagher, 2000).Metal concentrations in soil range from less than 1 mg/kg (ppm) to high as 100,000 mg/kg, whether due to the geological origin of the soil or as a result of human activity (Blaylock and Huang, 2000).Excess concentrations of some heavy metals in soils have caused the disruption of natural aquatic and terrestrial ecosystems (Gardea et al., 1996;Meagher, 2000).There is a great lack of knowledge defining the precise quantitative limits of tolerance that is the actual dosage level at which a chemical is toxic and the point beyond which no further adaptation can be achieved by a species (Dickinson et al., 1991).There seems to be no work on heavy metal toxicity in Linum except the one of Tkachuk and Kuzina (1972), who reported the mercury levels in flax seeds.
Heavy metals are included in the main category of environmental pollutants as they can remain in the environment for long periods; their accumulation is potentially hazardous to humans, animals and plants (Benavides et al., 2005;Gratão et al., 2005a).In the last few decades a dramatic, worrisome increase in contamination of the environment, including soil, air and water.It would appear that humans are the only ones to blame, because anthropogenic activities are the main source of the pollution that is causing the contamination (Gratão et al., 2005a).It is quite obvious from the studies carried out along the years that heavy metals have adverse effects on plants as well as animals and their productivity, although some metals are essential for plant growth in small quantities (Gomes et al., 2006).The accumulation of metals in plants is particularly important because some species have been characterized as hyper accumulators.They may be used, as along with specifically designed transgenic plants, in phytoremediation (Cherian and Oliveira, 2005;Eapen and D'Souza, 2005;Gratão et al., 2005b).Moreover, the use of bio-remediation techniques (Lynch and Moffat, 2005) has been replacing whenever possible the traditional engineering approaches.Such a situation has led to investigations on a wide range of aspects related to heavy metals.For instance, there has been intensive research on metals in soil related to plant nutrition, general effects on plant metabolism, tolerancesusceptibility, and environmental effects as well as on how contaminated areas can be reclaimed (Gratão et al., 2005a;Pilon, 2005;Taulavuori et al., 2005).In first few years of the 21st century a boom in the research in defense system in response to heavy metal-induced stress in plant tissues.It is quite difficult to say exactly which specific aspects have received most attention.
There is still plenty for such analyses; however, feel that along with such re-search, other approaches should now be included to consolidate the work on stress induced by metals, or even other stresses.For instance, few has been published on research related to proteomics, metabolomics, metallomic and metalloproteins (Garcia et al., 2006).Molecular techniques, such as northern blots and PCR, which have been used to detect the level of gene expression, have been available for quite some time; however, they still do not predominate in this area; their importance in elucidating biochemical problems is undeniable.Such approaches are much expensive and can not be adopted rapidly by researchers working on heavy-metal effects on plants.
Currently, cleanup processes of heavy metal pollution are expensive and environmentally destructive (Nanda et al., 1995;Moffat, 1995;Meagher, 2000).Recently, scientists and engineers have started to generate costeffective technologies that include the use of microorganisms, biomass, and live plants in the cleaning process of polluted areas (Miller, 1996;Boyajian and Carreira, 1997;Dushenkov et al., 1997;Ebbs and Kochian, 1998;Wasay et al.,1998;Gardea et al., 1996).Some heavy metals at low doses are essential micronutrients for plants, but in higher doses they may cause metabolic disorders and growth inhibition for most of the plants species (Fernandes and Henriques, 1991;Claire et al., 1991).Researchers have observed that some plants species are endemic to metalliferous soils and can tolerate greater than usual amounts of heavy metals or other toxic compounds.Evolved tolerance to toxic concentrations of heavy metals in plants inhabiting spoil heaps of mines is a well known phenomenon that has been the subject of much research in the last two decades.These plants are useful models for studying processes involved in the early stages of the speciation of edaphic endemics.Consequently, the information on variance, genetic variability, correlation coefficient and path coefficient is not available.Recent work has revealed the importance of several phenomena in the differentiation of tolerant populations, including natural selection, founder effects and 'hitch-hiking' .The present study is an endeavour in this direction.

MATERIALS AND METHODS
The experiments were carried out in the laboratory at room temperature.The effect of two heavy metals that is cobalt and mercury was recorded on the ten varieties of L. usitatissimum namely TLP-1, RLC-29, LC-54, LC-185, T-397, Kiran, Nagarkot, Neelum, Shubra and Gaurav with the help of unsoaked and soaked control experimental sets.For the control set, the tap water was taken.For heavy metal sets, 10 -5 , 10 -6 and 10 -7 molar concentration solution of both the heavy metals were prepared in tap water.The experiments were conducted in glass Petri dishes having four replications.Each replication has 10 seeds.The seeds were imbibed in different concentrations of heavy metals along with control one for 24 h before sowing.After 24 h these seeds were continuously washed with running tap water and transferred to Petri dishes having wetted pads of same concentrations.The solution was applied in such a manner that the pads and seeds always remain wet.The visual emergence or protrusion of radicle was taken as the criteria for germination.The seed germination test was carried out according to rules laid down by International Seed Testing Association (1976).The percentage of seed germination was recorded till the fifth day after sowing.The germination rate index has been calculated with the help of modified method of Srivastava and Sareen (1972) and Heydecker (1973) using the following formula: Germination rate index = 4 (5g+4g+3g+2g+g) g = The number of germinated seed after each 24 h.
The radicle and hypocotyl length has been measured from 5th to 7th day after sowing.The organ elongation rate has been calculated by Heydecker (1973) modified method.
(Mean length of the organ / Days to first count +++ Mean increase in length /Days to last count) The seedling length has been measured on seventh day after sowing.The cotyledonary area that is, length and breadth of cotyledons has been measured on seventh day of sowing.At the time of termination of experiments, ten seedlings were weighed and the same were dried in a hot air oven at 60°C for 24 h for obtaining the fresh and dry weight of seedlings.The absolute and specific seedling water content was calculated with the help of following formulas.
Absolute seedling water content = seedling fresh weight -seedling dry weight.Specific seedling water content = Absolute seedling water content / Seedling dry weight.
The seedling vigour and seed vigour index has been calculated with the help of the formula given by Abdulbaki and Anderson(1973).
Seedling vigour = Percentage germination x Seedling length Seed vigour index = Percentage germination x Mean dry weight The tolerance index (T.I.) was estimated by Wilkins (1978)  The data thus collected were subjected to the following statistical and biometrical analysis.The analysis of variance was calculated by Panse and Sukhatme (1961) formulae.Phenotypic coefficient of variance (P.C.V.), genotypic coefficient of variance (G.C.V.), and heritability in broad sense were calculated using the formulae as suggested by Burten (1952).The expected genetic advance at 5% intensity of selection differential was calculated by the Johanson et al. (1955) formulae and genetic advance as in percentage of mean according to Allard (1960) formula.The genotypic and phenotypic correlation coefficient among all characters under/study was estimated as given by Searle (1961).The direct and indirect effects were estimated in path coefficient analysis as suggested by Dewey and Lu (1959).Simmonds (1962) states that the range of variability depends upon the selection pressure under domestication that is, upon the system of agriculture.He further states that the imposition of new norms of selection, allowing the survival of only favored genotypes and variability is destroyed at a considerable rate.The rate and magnitude of variability depends upon factors such as system, growth habit and population size.We classified the G.C.V., P.C.V., heritability and genetic advance into high, moderate and low magnitudes for our convenience for the presentation and analysis of results.
However, there is no such particular criteria for the classification of G.C.V., P.C.V. and G.A. except in case of heritability where Robinson (1966) has given such range.In the present communication, we have considered the following range of G.C.V., P.C.V., heritability and genetic advance.The magnitude of G.C.V. and P.C.V. up to 10% as low, 10 to 20% moderate and above the 20% considered as high.The magnitude of heritability in broad sense has been considered as low below the 0.3, between 0.3 to 0.6 as moderate and above 0.6 as high.The magnitude of G.A in percentage of mean up to 7% as low, between 7 to 15% as moderate and above 15% as high.

RESULTS AND DISCUSSION
Seed germination and seedling establishment are the two most important events in the life cycle of plants.Heavy metal pollution has created a great ecological crisis.Modern civilization introduces a wide range of pollutants to the atmosphere through various anthropogenic activities such as industry, mining, transportation, etc.
Despite the fact that, it is almost impossible to visualize a soil without trace levels of heavy metals and most of the heavy metals are essential elements for living organisms, but their excess amounts are generally Jain 95 harmful to plants, animals and human health (Azevedo and Lea, 2005;Jarup , 2003).Currently, contamination of soil in cultivated fields with toxic heavy metals has emerged as a new threat to agriculture (Singh et al., 2007).The present study reveals that there was no further growth after germination in 10 -5 M HgCl 2 concentration.All characters in all the varieties of L. usitatissimum showed significant differences in different environments except character like absolute seedling water content in unsoaked control environment (Table 1).The error variance is higher than their G.C.V. and P.C.V.
for the characters like germination rate index, seedling vigour and vigour index in all the concentrations or environments.The percentage germination also shows similar results in unsoaked control, soaked control, 10 -5 M, 10 -6 M and 10 -7 M CoCl 2 .These findings indicate that these characters are mostly influenced by environment.
The percentage germination in 10 -6 M, 10 -7 M CoCl 2 and 10 -6 M HgCl 2 shows lower error variance value then their respective G.C.V. and P.C.V.The characters like radicle length, radicle elongation rate, hypocotyl elongation rate, seedling length, cotyledonary area, seedling fresh weight and specific seedling water content have lesser value of error variance then their respective G.C.V. and P.C.V. in all the environments.These results indicate that the characters in reference though controlled genetically but are greatly affected by environment.(Tables 1 and 2) The decrease in seed germination and seedling growth due to heavy metal treatment is in conformity with the findings of other researchers (Ayaz and Kadioglu, 1997;Morzek and Funiceli, 1982;Iqbal and Mehmood, 1991;Jamal et al., 2006).For example, Rahman et al. (2010) observed a reduction in seed germination and seedling growth in chickpea treated with 50, 100, 200 and 400 ppm of nickel and cobalt.Singh et al. (2007) observed a reduction in germination percentage and early growth stage of wheat treated with copper at 5, 25, 50, and 100 ppm.Treatment of Leucaena leucocephala with 25, 50, 75 and 100 ppm of lead and cadmium showed a gradual reduction in seed germination and seedling growth (Shafiq et al., 2008).However, germination test showed a non significant effect on germination percentage of corn treated with low levels of zinc and copper (6 to 12 ppm) (Mahmood et al., 2005).According to Shafiq et al. (2008), decrease in seed germination of plant can be attributed to the accelerated breakdown of stored food materials in seed, by the application of heavy metal mixture.Reduction in seed germination can also be attributed to alterations of selection permeability properties of cell membrane.The G.C.V. is lower than P.C.V. for all the characters in all the concentrations (environment).This indicates that these characters are sensitive to environmental influence and the direct selection for these traits on phenotypic basis could be done reliably.The influence of environment of each trait could be determined by the difference in P.C.V. and G.C.V.When the difference between P.C.V. and G.C.V. is less, it suggests that these traits are least affected by environment.This is also supported by very high value of heritability (Table 2).
High heritability with high/moderate genetic advance has been recorded for all the characters in all the treatment in all the studied varieties.It indicates that expression of these attributes is governed by additive gene effects.High heritability with high genetic advance provides good scope of further improvement by selection.These characters can be subjected to mass progeny, family or any other modified selection scheme for exploiting the additive genetic variance.High heritability estimates have been found to be helpful in making selection of superior genotypes on the basis of phenotypic performance of characters.(Table 2).Plant species shows a wide genetic diversity that offer great sensitivity and selectivity.They have a number of general and specific mechanisms in gene expression that they use to response to unfavorable conditions.These genes linked with a variety of toxic compound response.
The seedling vigour is an important attribute in seed technology.The capacity of seed germination and seedling establishment of variety in a range of environment (climatic condition and heavy metal pollution) has an importance equal to that of its growth and yield potential.Eberhart and Russell (1966) have pointed out that the validity of character's stability increase as the number of environment increases.Hutchinson (1984) and Dueck et al. (1987) are of the opinion that the difference in toxicity level or tolerance is either due to genetic variability or the interactive effect of combination of contamination.Kulkarni and Nayeem (1986) found that in wheat varieties there is genotypic difference in emergence.Gartside and Mc Neily (1974), Bradshaw (1984), Baker and Proctor (1990) and Bradshaw (1991) accepted that only those plant species possessing the required genetic variation can develop tolerance in their population.Bates (1940), suggested that albinism in citrus seedling treated with mercury may be due to primarily to heterozygosis, and perhaps to the presence of unstable genes.In the present study we have used two heavy metals with three concentrations of each and compare it with the control, to obtain the true nature and interaction of heavy metals on seed germination and seedling vigour.The above view is in conformity with those reported by Wilkins (1978) and Coughtrey et al. (1979).Wilkins (1978) stated that ideally a concentration should be used which slightly reduce the growth of tolerant plant.Coughtrey et al. (1979) reported that the use of single metal in tolerance test may not give the reflection of the true nature of interaction between plants and metals.Baker (1987) and Mcnair (1990) found that if a single metal solution is employed over a fixed time period, the data can be highly misleading.Monzuroglu and Geckil (2002) reported the complete inhibition on germination in wheat and cucumber at concentration > 1.5 mM in cucumber and 1.7 mM in wheat.
A perusal of data reveals that the maximum toxic effect and minimum tolerance capacity in 10 -5 M CoCl 2 has been recorded for the characteristics like seed vigour index, seedling vigour and absolute seedling water content.The maximum toxicity and minimum tolerance capacity in 10 - 6 M CoCl 2 has been recorded for the characteristics or variables like seed vigour index, absolute seedling water content and seedling vigour.The 10 -7 M CoCl 2 showed the maximum toxic effect and minimum tolerance for the characters like absolute seedling water content; seed vigour index and seedling dry weight.The minimum toxic effect and maximum tolerance capacity has been observed for the characters like specific seedling water content in all the concentration except in 10 -7 M CoCl 2 where it is observed for character like cotyledonary  particular region or character.The difference in toxicity level or tolerance capacity is either due to genetic variability or the interaction of contamination.The present findings on cobalt toxicity effect are in agreement to those reported by Haselhoff (1985).Brenchley (1938) and Vergano and Hunter (1952).Haselhoff (1895) reported that 1 ppm cobalt in culture solution was toxic to beans and corns, while Vergano and Hunter (1952) reported that solution culture containing as low as 0.1 ppm cobalt produce adverse or toxic effect on many crop plant.Blaylock et al. (1986) observed that cobalt generally decreased the photosynthesis and chlorophyll in soybean and tomatoes.Recently, Xu et al. (1993) reported that Co at low concentration allowed continued growth cycle of groundnut cells (>0.5 mM).At high concentration (1 mM) these ions inhibit the growth cycle of cell.We considered that 1x10 -4 M CoCl 2 concentration is the maximum toxicity and there after no germination is possible.Mitchell (1945) considered the 0.7 ppm ratio of cobalt content in plant is the minimum tolerance limit.Chatterjee and Chatterjee (2000) also found similar result in cauliflower.Palit et al. (2008) reported that cobalt act as pre-prophase poison and thus retarded the process of karyokinesis and cytokinesis.The distribution of cobalt in plant is entirely species dependent.
When the seeds of Linum allowed to germinate in 10 -

5
M HgCl 2 , it showed highest toxic effect.Here only seed germinated and no further seedling establishment was noticed.In this concentration the seeds have loose their tolerance capacity.The 10 -6 HgCl 2 Concentration shows less toxic effect in comparison to 10 -5 M. In this concentration, the maximum toxic effect and minimum tolerance capacity has been observed for the followings variables: seedling vigour, absolute seedling water content, seedling dry weight and seed vigour index.However, in 10 -7 M concentration of HgCl 2 , the maximum toxic effect has been observed for the seedling vigour, seed vigour index and absolute seedling water content.
The minimum toxic effect and maximum tolerance capacity has been observed for the specific seedling water content in both the concentration of HgCl 2 in different varieties.These findings also suggest the last limit that is with maximum toxic effect of mercury for these plant species is 10 -5 M HgCl 2 concentration.The 10 -

7
M HgCl 2 concentration has least toxic effect (Table 3).The above findings are more or less similar to those reported by Mukherji and Ganguly (1974).They stated that root growth inhibition was stronger than shoot growth inhibition by Hg treatment.Sharma (1983) soaked the seeds of Pisum sativum cultivars namely T-163, Bonneville and Arkel for 24 h in 4.98x10 -5 and 2.49x10 -4 M concentration of mercuric acetate.He observed that in 2.49x10 -4 concentration the germination and growth was inhibited.Sharma (1984) reported that in Cucumis utilissimus and Luffa aegyptiaca on allocation of lower concentration of mercury showed that hypocotyl growth was more affected that of radicle.Jamal et al. (2006) observed the highest reduction was in root length rather than shoot and seedling length due to mercury concentration < 0.05 in two wheat varieties.
Generally, it makes negative effect on their metabolisms by influencing the activity of cellular enzymes (Yang et al., 1986).Many studies have been carried out on the effects of heavy metals on plants.They showed that, cadmium in certain amounts inhibited the germination and development of the plants ( Aydinalp and Marinova, 2009).Cadmium caused chlorophyll aberrations at very high concentrations (Reddy and Vaidyanath, 1978), reduction of mitotic index in root cells (Zhang and Yang, 1994), chromosomal abnormalities and micronucleus formation (Li and Zheng, 1992), disorder in nucleus structure (Jiang et al., 1994) and abnormalities in (deoxyribonucleic acid) DNA and Ribonucleic acid (RNA) synthesis (Enger et al., 1997).
Several authors reported that, the inhibition of root elongation caused by heavy metals may be due to metal interference with cell division, including inducement of chromosomal aberrations and abnormal mitosis (Jiang et al., 2001;Huillier et al., 1996;Radha et al., 2010;Liu et al., 2003), which can be effected on seedling growth and explain the inhabitation of seedling growth in this investigation.It was also observed that mercury toxicity was more than cobalt and the higher concentration showed more toxic effect than the lower.The present study further indicates that there are limits to the degree of tolerance that plants can achieve.The quantitative limits of tolerance and toxicity have great importance for determining the actual dosage at which a chemical becomes toxic as also the point beyond which no further adaptation can be achieved by a plant species or variety.It is inferred that the evaluation of germination and seedling growth may be helpful in assessing the relative resistance or susceptibility to heavy metal.So the choice of resistant cultivars for cultivation would be meaningful in minimizing the pollution hazards.The possible reason of inhibition of seedling growth due to mercury and cobalt may be to its association with cell wall or cell membrane inhibiting water absorption and interference with mobilization of reserve food from residual cotyledons to the developing seedling.The reason for low tolerance against heavy metal mixtures might be due to changes in the physiological mechanism in seed germination and seedling growth of safflower.Shafiq and Iqbal (2005) reported similar results for low tolerance in Cassia siamea seedlings at 100 ppm of lead and cadmium treatments as compared to control.General observation in this study can conclude that, heavy metal mixture treatment produced toxic impact on germination and seedling growth .Increase in the concentrations of heavy metals mixture in the soil, brought up changes in most of the growth parameters of crop.
Contamination of wastewater with high concentration of heavy metals caused a significant decrease in the number of survivals .The toxicity of heavy metals on organism depends mainly upon two factors, namely, metal species and concentration.Other factors such as pH, influent strength are also reported to affect the toxicity of metals, though to a lesser degree.Heavy metals change the structure of the flora and fauna by modifying both cell density and species richness.Due to the toxic behavior of metal ions resulting in a lower demand of dissolved oxygen.Metal ion complexion with heavy metal effects the growth of living being and it's the cause of death.When a critical amount of heavy metal is applied to the seeds or seedlings either the cell's metabolic activity decreases or the cells die.It could be possible that there is a specific gene which monitors the toxic level.

Correlation coefficient
As revealed from Table 4, the highly significant positive   The significant positive correlation coefficient has been observed among the 1000 seed weight with seedling dry weight, the seed size with cotyledonary area, seedling fresh weight, absolute seedling water content and specific seedling water content.The seedling vigour with seed germination and germination rate index, the absolute water content with radicle length, radical elongation rate and hypocotyl elongation rate, the seedling dry weight with radicle length, radicle elongation rate and hypocotyl elongation rate, the seedling fresh weight with radicle length, radicle elongation rate and hypocotyl elongation rate, the cotyledonary area with hypocotyl length and hypocotyl elongation rate, the seedling length with radicle length and radicle elongation rate.
The significant negative correlation coefficient has observed between 1000 seed weight with germination rate index.
These findings are similar to those of reported by Carleton and Cooper (1972).They found the positive correlation between seed size and seedling vigour in three forage legume and same was observed by Gelmond (1972) in cotton.Mulett and Wilkinson (1979) reported that hypocotyl length had significant positive correlation with fresh and dry weight of seedling in Pisum sativum.Hussaini et al. (1984) stated that seed size positively affect the seed germination and vigour in maize.Nayeem and Deshpande (1987) reported that in wheat, the seed vigour index had significantly positive correlation with radicle length, seedling fresh and dry weight.Eduardo et al. (2007) observed the positive correlation in growth, water content and chlorophyll concentration in Rumex induratus and Marrubium vulgare.
The above discussed results clearly indicate that the genotypic correlation coefficient has found to be greater, than phenotypic and environmental correlation in almost all the characters.This indicates that though these have a high degree of association between two traits at genotypic level, its phenotypic expression has lessened due to the influence of the environment.The negative correlation might arise in character is favoured over the other in the developing stage, when the nutrient supply is limited.In other words this is a case of physiological incompatibility which leads to conclusion that intensification of one such character will be at the expense of other.Possible genetic reason for negative correlation among these components could be pleiotropy.

Path coefficient analysis
The results of Table 5 indicated that the seedling length exerted maximum positive direct effect on seedling vigour followed by characters like absolute seedling water content, percentage germination, seed vigour index and specific seedling water content at phenotypic level.However, seedling fresh weight showed maximum direct effect followed by seedling length, seed vigour index, radicle elongation rate, specific seedling water content, germination rate index and cotyledonary area at genotypic level.
The hypocotyl elongation rate has maximum positive indirect effect on seedling vigour via characteristics seedling length followed by hypocotyl length, seed vigour index, seedling fresh weight, absolute seedling water content, seedling dry weight, radicle elongation rate, radicle length, cotyledonary area, specific seedling water content and 1000 seed weight.The seedling dry weight exert maximum positive indirect effect on seedling vigour via seed vigour index followed by seedling fresh weight, absolute seedling water content, radicle length, radicle elongation rate, seedling length, hypocotyl elongation rate and hypocotyl length.These findings suggest that seedling vigour is a complex character which is governed by many parameters and is an important attribute in seed technology.The direct and indirect effects of characters help in designing appropriate selection strategies and pinpoint the actual parameters to be manipulated.The high positive direct effect which was reflected in its positive and significant correlation coefficient may be regarded as the prime character for selection.This seems to be effective in obtaining superior seedling vigour.However, the characters which have positive and significant correlation coefficient with seedling vigour, but the direct effect was negligible or negative, the indirect effect seem to be cause of positive correlation, therefore, for improving the seedling vigour, the indirect causal factors are to be considered simultaneously for selection.This correlation coefficient has been found to be almost equal to its indirect effect.
The hypocotyl length has maximum negative direct effect on seedling vigour followed by radicle length.These negative direct effects indicate the difficulty to improve the seedling vigour through selection, but compensated through the positive indirect effect via other characters which ultimately resulted in the significant positive correlation.Thus these characters in a balanced proportion and are mainly responsible for the improvement in seedling vigour.The maximum positive direct effect of seedling length on the seedling vigour has been nullified by the negative indirect effect via hypocotyl and radicle length.To overcome this problem a special technique should be adopted to break this linkage.It is interesting to note that the direct and indirect effect have positive value at phenotypic level and negative value at genotypic level or vice versa.It shows that the involvement of these characters in seedling vigour are variable, and the time of improvement if the due weight age would be given to these characters, thus their direction must be considered separately at genotypic and phenotypic level.The residual effect has negative value at genotypic level and positive value at phenotypic level with considerably low magnitude which indicates that most of the important attributed characters enhancing seedling vigour ratio have been taken into account in the study.Basak et al. (2001) observed that heavy metal stressed the chlorophyll content along with Hill activity.Although, the effects of the individual heavy metals on plants have been evaluated by many studies (Brown and Wilkins, 1986;Shafig et al., 2008;Dharam et al., 2007;Shafiq and Iqbal, 2005;Kabir et al., 2008), limited information is available on the effects of heavy metal mixture on plant species.There is the need to study the combined effects of heavy metals on plants because most of them are present in an environment at the same time or on the same environment at different times.Linseed is economically an important annual oilseed crop.It has been traditionally grown for its oil, food, fabrics animal meal, bird feed, medicinal uses, as a potential candidate crop for production of plant made pharmaceuticals .To our knowledge, limited information is available on the effects of heavy metals on seed germination and seedling growth of this plant.Hence, the objective of this study was to evaluate the various effects of mixed heavy metals at different levels on Linum in early growth stages.

Conclusion
India as well as world's environment is becoming fragile and environmental pollution is one of the undesirable side effects of industrialization, urbanization, population growth and unconscious attitude towards the environment.At present, environmental protection is the main need of the society.Though industrialization and development in agriculture are necessary to meet the basic requirement of people, at the same time it is necessary to preserve the environment.In India, too, the environmental pollution has become a cause of concern at various levels.In India, due to lack of sewage treatment plants, generally untreated sewage effluents are released either on agricultural land for irrigation or disposed of in nearby water bodies.In general, sewage effluents from industries and municipal origin contain appreciable amounts of plants nutrients and variable amount of metallic cations like Hg, Co, Zn, Cu,Fe, Mn, Pb, Ni, Cd, etc.Long-term irrigation with such effluents increases heavy metals accumulation in soils and the chances of their entrance in food chain and this ultimately causes significantly health concern.Thus, it becomes necessary to study the composition of sewage waters and heavy metals accumulation, with the help of advance techniques.The effects of toxic substances on plants are dependent on the amount of toxic substance taken up from the given environment.Germination and seedling  establishment are vulnerable stages in the plant life cycle.Seedling growth is considered as an indicator of metal stress on plant ability to survive.The seed germination and seedling establishment is notoriously sensitive to heavy metals often a difference i.e., a noticeable change in growth rate.There may be a specific gene in plants which monitor the toxicity level.This gene is responsible for the different strength of tolerance to a variety of toxic compound in different plant species or varieties.The present study emphases the importance of several parameters for assessment of the comparative resistance of cultivars of a crop species.The evaluation of germination and seedling growth may be helpful in assessing the relative resistance or susceptibility of cultivars to heavy metals for cultivation would be meaningful in minimizing the heavy metals effect.This study is crucial to be delivery of the potential of possible loss in yield beyond the trials and research farms to agriculture in general, because crop performance is entirely determined by a complex genotype and environment interaction.
formula.T.I. (%) = Organ growth in solution with metal / Organ growth in solution without metal.The toxicity level was determined with the help of following formula Toxicity level (%) = 100 -Tolerance index

Table 1 .
Analysis of variance in different concentrations in Linum usitatissimum L. seed germination.

Table 2 .
Analysis of variability in different concentrations in Linum usitatissimum L. seed germination.
area.These results indicate that in cobalt the 10 - 5 M concentration is much toxic than 10 -6 M concentration and 10 -7 M CoCl 2 concentration seem to be normal, but it shows toxicity to some extent.The same toxicity level shows difference for the characters in different concentration.It could be due to the disrupting capacity of the applied concentration of metal to disrupt the integrity of biomolecules of the

Table 3 .
Tolerance index and toxicity level in linum usitatissimum seed germination.

Table 4 .
Estimation of correlation coefficient Among various characters of seed germination in Linum usitatissimum.

Table 5 .
Path coefficient analysis showing direct and indirect effect on seedling vigour in linum usitatissimum L.