11 Int.J.Curr.Biotechnol. Volume 3; Issue 4; Apr, 2015 Ganesan Vijaiyan Siva and Vidhya Udayakumar, Effect of Heavy Metal Chromium on Germination, Root length and Cytology of germinated Allium cepa L. seeds, Int.J.Curr.Biotechnol., 2015, 3(4):11-16. Effect of Heavy Metal Chromium on Germination, Root length and Cytology of germinated Allium cepa L. seeds Ganesan Vijaiyan Siva* and Vidhya Udayakumar Department of Biotechnology, University of Madras, Guindy Campus, Chennai – 600 025. ARTICLE INFO ABSTRACT Article History: Received 16 April 2015 Received in revised form 21 April 2015 Accepted 26 April 2015 Available online 30 April 2015 Key words: A.cepa, Chromium, Tolerance in- dex, Mitotic index. Heavy metals are currently of much environmental concern and are harmful to humans, animals and tend to bio accumulate in the food chain. The present study was designed to examine the effect of Chromium (Cr) on germination, root length and cell divisions in the root meristems of Germinated Allium cepa seeds (GAS) to reveal the cytogenic effect and chromosonal abnormalities induced by Cr. The A.cepa seeds were germinated with Chromium (Cr) solution at three different concentrations. The accumulation, root length and tolerance index of GAS on treatment with different concentrations Cr were estimated. The mitotic index and chromosomal abnormalities are used to evaluate genotoxicity and micronucleus analysis used to verify effect of heavy metal Cr. The results showed that GAS has the ability to accumulate Cr in their tissue. Heavy metal concentrations increased in roots and results are compared with those of control. The root length and tolerance index of GAS on treatment with different concentrations Cr solution significantly decreased. The cytological study revealed a concentration dependent mito depressive effect of Cr. Introduction Heavy metal contamination is a cause of major environmental hazards worldwide, leading to losses in agricultural yields and harmfully affecting human health when contaminants enter the food chain. Chromium (Cr) is the seventh most abundant element on earth (Panda and Choudhory, 2005). Chromium has different oxidation states that in nature. It exists in both trivalent (Cr III) and hexavalent (Cr VI) forms, of which the latter is more toxic (Panda and Patra, 1997). Uptake and toxic effects of Cr depend on its oxidation state. Chromium enters the food chain through consumption of plant material. Cr compounds cause environmental pollution as a result of a large number of industrial operations, including mining, pigment manufacturing, petroleum refining, leather tanning, wood preserving, textile manufacturing, pulp processing and fungicide development (Wang and Xiao, 1995). Cr disrupts several physiological and cytological processes in cells. According to Shanker et al., 2005 effect of Cr leads to reduced root growth and seed germination. Previous studies have shown that Cr induced various chromosomal abnormalities in plant cells thereby severely reducing mitotic index and root growth (Olorunfemi et al., 2010). The aim of the present study was to evaluate the cytological effects of Cr on GAS and to investigate the effect of Cr on germination percentage and root length. Materials and Methods Preparation of onion seeds The healthy, homogenous certified seeds of onion (Allium cepa) were obtained from DOGR, Nasik. The seeds were subjected to surface sterilization with 0.3% sodium hypochlorite solution for 10 min and then rinsed with double distilled water. After washing of seeds in water, seedlings were raised in petriplates. Chromium Treatment Four replicates each with 10 seeds were incubated in a clean pertidishes lined with three layers of Whatman filter paper (No. 1), soaked in distilled water (control), 1, 10 and 100 ppm Potassium dichromate solutions at room temperature. Petridishes were sealed by Para film to prevent evaporation. An emerged radicle of 1-mm from the seed was the criterion for germination. Seed Germination percentage Percentage of seeds germination is the indicator of Cr uptake. An emerged radicle was the criterion for International Journal of Current Biotechnology Journal Homepage : http://ijcb.mainspringer.com *Corresponding author. Email address: [email protected]ISSN: 2321 - 8371
Ganesan Vijaiyan Siva and Vidhya Udayakumar, Effect of Heavy Metal Chromium on Germination, Root length andCytology of germinated Allium cepa L. seeds, Int.J.Curr.Biotechnol., 2015, 3(4):11-16.
Effect of Heavy Metal Chromium on Germination, Root length and Cytology of germinatedAllium cepa L. seeds
Ganesan Vijaiyan Siva* and Vidhya Udayakumar
Department of Biotechnology, University of Madras, Guindy Campus, Chennai – 600 025.
A R T I C L E I N F O A B S T R A C T
Article History:Received 16 April 2015Received in revised form 21 April 2015Accepted 26 April 2015Available online 30 April 2015
Heavy metals are currently of much environmental concern and are harmful to humans,animals and tend to bio accumulate in the food chain. The present study was designedto examine the effect of Chromium (Cr) on germination, root length and cell divisionsin the root meristems of Germinated Allium cepa seeds (GAS) to reveal the cytogeniceffect and chromosonal abnormalities induced by Cr. The A.cepa seeds weregerminated with Chromium (Cr) solution at three different concentrations. Theaccumulation, root length and tolerance index of GAS on treatment with differentconcentrations Cr were estimated. The mitotic index and chromosomal abnormalitiesare used to evaluate genotoxicity and micronucleus analysis used to verify effect ofheavy metal Cr. The results showed that GAS has the ability to accumulate Cr in theirtissue. Heavy metal concentrations increased in roots and results are compared withthose of control. The root length and tolerance index of GAS on treatment withdifferent concentrations Cr solution significantly decreased. The cytological studyrevealed a concentration dependent mito depressive effect of Cr.
IntroductionHeavy metal contamination is a cause of majorenvironmental hazards worldwide, leading to losses inagricultural yields and harmfully affecting human healthwhen contaminants enter the food chain. Chromium (Cr)is the seventh most abundant element on earth (Pandaand Choudhory, 2005). Chromium has different oxidationstates that in nature. It exists in both trivalent (Cr III) andhexavalent (Cr VI) forms, of which the latter is more toxic(Panda and Patra, 1997). Uptake and toxic effects of Crdepend on its oxidation state. Chromium enters the foodchain through consumption of plant material. Crcompounds cause environmental pollution as a result ofa large number of industrial operations, including mining,pigment manufacturing, petroleum refining, leathertanning, wood preserving, textile manufacturing, pulpprocessing and fungicide development (Wang and Xiao,1995).
Cr disrupts several physiological and cytologicalprocesses in cells. According to Shanker et al., 2005 effectof Cr leads to reduced root growth and seed germination.Previous studies have shown that Cr induced variouschromosomal abnormalities in plant cells thereby severely
reducing mitotic index and root growth (Olorunfemi etal., 2010). The aim of the present study was to evaluatethe cytological effects of Cr on GAS and to investigatethe effect of Cr on germination percentage and rootlength.
Materials and MethodsPreparation of onion seedsThe healthy, homogenous certified seeds of onion(Allium cepa) were obtained from DOGR, Nasik. Theseeds were subjected to surface sterilization with 0.3%sodium hypochlorite solution for 10 min and then rinsedwith double distilled water. After washing of seeds inwater, seedlings were raised in petriplates.
Chromium TreatmentFour replicates each with 10 seeds were incubated in aclean pertidishes lined with three layers of Whatman filterpaper (No. 1), soaked in distilled water (control), 1, 10and 100 ppm Potassium dichromate solutions at roomtemperature. Petridishes were sealed by Para film toprevent evaporation. An emerged radicle of 1-mm fromthe seed was the criterion for germination.
Seed Germination percentagePercentage of seeds germination is the indicator of Cruptake. An emerged radicle was the criterion for
germination. Germination was observed every 48 hoursup to 8 days. Germinated A.cepa seeds were countedand removed from the dishes of C and 1, 10 and 100 ppmCr treatment. Cumulative germination percentage wascalculated using the formula,
% of germination = No. of seeds germinated x 100
Total No. of seeds sownRoot lengthRoot length was taken from the plates after 8 days fromthe date of plating in randomly selected 4 seedlings fromeach replicate. The weight of the seedlings from thereplicates was also measured. Root length of the treatedGAS was measured and growth inhibitory rate of rootwere calculated.
Tolerance indexTolerance index (TI) indicates the efficiency of the plantto tolerate heavy metals. It is a ratio of the size of theheavy metal treated roots to that in the control roots.The phytoremediation of the GAS with the Cr treatmentof 1, 10 and 100 are estimated by Tolerance indexparameter (Adesodun et al., 2010) and it is calculatedusing the formula,
Tolerance index (TI) = Mean root size with Cr treat ment x 100
Mean root size in Control
Cytological studies Cytological studies of Cr treated GAS were done by roottip squash method (Girija et al., 2013). The mitotic index,Micronucleus assay and chromosomal aberrations inGAS roots were observed (Mahamuni et al., 2012). 1-2cm of root tips of growing GAS roots are cut off and fixedin fixative (Acetic acid: Ethanol) by washing. The roottips are then put into test tube containing 1 NHydrochloric acid (HCl) for exactly 40 minutes. The roottips are washed and transferred into mordant (Iron Alum)and left for 45 minutes. Again the root tips are washedand stained with Hematoxylin for 40 minutes. Then theyare washed with distilled water and transferred onto aclean microscope slide. 4-5 mm of the root tips are cutand gently broken up with a mounted needle by a processcalled maceration. The squashed root tip is used forrecording mitotic index and the nuclei of the cells arelocated. The regularly shaped, actively dividing cells arefocused and counted for every phases of mitosis. Theobservations included analysis of the MI and scoring ofcytological abnormalities like fragments, bridges,micronuclei (MN), multipolarity and vagrants indicatingvarious clastogenic and physiological disturbances. Thedata obtained are recorded and tabulated.
Mitotic index = Cells observed with visible chromosome
Total no. of cells visible
Results and DiscussionGermination percentageThe effect of different concentrations of Cr affectsgermination of A.cepa seeds. The metal concentrationand duration of exposure at which germination of A.cepaseeds inhibited are summarized in graph depicted in Fig.1. Germination percentage was taken with reference to
control from initial germination to 100% germination. Theimprovement in germination percentage of in control was50 – 100% from the 2nd to the 8th day.
The germination percentage decreased to 10% in 100 ppmwhen compared to 50% in the control seeds after 2 daysexposure. The germination percentage of 1, 10 and 100ppm of Cr treatment after 4 days exposure produced 70,60 and 30% respectively. Similarly, these percentagesincreased to 90, 80 and 60 after 6 days of exposure. Thegermination percentages on the final day of treatmentwere 100, 90 and 70% in 1, 10 and 100 ppm Cr treatment.Similar results of reduced germination of seeds under Crstress would be due to the depressive effect of Cr on thesubsequent transport of sugars to the embryo axis (Zeid2001). A report (Parr and Taylor, 1982) showed that athigh levels of Cr (VI) affect the germination and growthof bush bean.
Root lengthRoot length of germinated seeds showed significantlygreater response for seeds exposed to different Crconcentrations. However, there was a reduction in rootlength compared to unexposed controls for exposures,including 1, 10 and 100 ppm (Fig.2). The maximumreduction of 80% in root length was observed in 100 ppmfollowed by 10 and 1 ppm of Cr concentration. Seed rootlength was greatest in control which is 14 cm and reducedto 9.5, 6 and 3 in 1, 10 and 100 ppm of Cr exposure. Similarreport that the effect of Cr on root growth is due to thefact that heavy metals (Cr) accumulated on root due tobinding of metals (Cr) on the cell wall of root and retardcell division and cell elongation (Woolhouse 1983).
Tolerance index (TI)The GAS was tested for tolerance to heavy metals, usingdifferent concentrations of Cr. There is a gradual decreasein tolerance with the increasing concentrations of Cr ascompared to control (Fig.3). Chromium treatments at 1,10 and 100 ppm produced 67.85, 42.85 and 21.42 % oftolerance in GAS, respectively. According to toleranceindices, Cr treatment at 100 ppm concentration showedlowest percentage of tolerance in GAS as compared tocontrol. Khan (2003) correlated varying metal toleranceresponse with physiological variations during growthactivities in wheat cultivars.
Cytological studiesTo study the effect of genetic damage, the meristematiccells of GAS roots were analysed to record the mitoticindex, incidence of micronuclei and mitotic anomalies.Data presented in Table- 1 reveals a concentrationdependent mito depressive effect of Cr. Germinated roots,exposed to low concentration of 1 ppm concentrationdid not show significant mitotic inhibition. The higherconcentrations of 10, 100 ppm Cr treatment reduced themitotic activity of GAS roots. A significant increase inthe incidence of micronuclei was observed in GAS rootsexposed to different concentrations of Cr though thehighest concentration did not induce micronuclei.Sporadic instances of mitotic anomalies (Fig. 5) forexample, anaphase bridges, laggards were recorded inGAS roots exposed to Cr, though a concentrationdependent effect was not observed. Mitodepressiveeffect of heavy metal was also studied by Deysson, 1968.
ConclusionThis study concluded that root length of all theconcentrations were sensitive to Cr. Root growth of the
Figure -5: Microphotographs of squash preparation of GAS roots with mitotic anomalies
Cells showing mitotic anomalies such as micronucleus, anaphase bridges and laggard after treatment with differentconcentration of chromium.Micronucleus B,C,D. Anaphase bridges and laggard
Treatment concentration
Control 1 ppm 10 ppm 100 ppm
Cells analysed 8000 8000 8000 8000
Number of dividing 419 246 61 22
Cells at interphase 7581 7880 7939 7978
Mitotic index (%) 5.23 1.37 0.76 0.27
Micronuclei
n - 44 21 -
% - 0.551 0.264 -
Mitotic anomalies%
n - 1 4 -
% - 0.41 6.55 -
Table - 1: Cytogenetic effects of chromium in meristematic cells of the GAS roots
GAS were more accurate to measure the toxicity of Cr.There is an inverse relationship between concentrationof Cr and root growth. The tolerance index limitationshowed GAS viability against Cr metal. The growthresponses are less harmed at lower Cr treatment whencompared to higher Cr treatments and differentconcentrations of Cr treatment showed different levelsof tolerance. Different concentrations of Cr showedlagging chromosomes, anaphase bridge andmicronucleus. Cr showed mitodepressive effect indifferent concentrations. The present study revealedcytotoxic and clastogenic properties of Cr.
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