Clastogenic and Aneugenic Responses of Supplemental UV-B Radiation on Root Meristems of Lentil

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1 Vol. 4, Issue 1, pp: (1-8), Month: Jnury - Mrh 2016, Aville t: Clstogeni nd Aneugeni Responses of Supplementl UV-B Rdition on Root Meristems of Lentil * 1 Ash Pndey, 2 G. Kumr 1,2 Plnt Genetis Lortory, Deprtment of Botny, University of Allhd, Indi Astrt: All living orgnisms re inseprly interdependent with nturl environment nd intert with eh other. UV-B rdition is prt of sun s energy whih due to depletion of ozone veil penetrtes the erth s surfe in n elevted level nd disrupts the vitl metoli proesses of plnts nd indues geneti instility, whih will ultimtely e trnsloted to vrious trophi levels. Considering the ove forementioned risk of UV-B on plnts, the present experimentl work hs een designed to study the effet of UV-B exposure on root meristems of Lentil (Lens ulinris Medik). Experiment ws rried out in four sets viz. A, B, C nd D. A,B,C sets were treted with UV-B rditions for 20min, 40min,nd 60min, suessively for 1 dy, 2dy nd 3dy. Set D remin unrdited eing kept s ontrol. After eh irrdition, roots were ut off from seeds nd fixed in Crnoy s fixtive (GAA: AA, 1:3) for 24 hours then trnsferred in 90% lohol. With the help of sqush tehnique ytologil slides were prepred. Vrious types of hromosoml nomlies were reported however the more prevlent were unorienttion, preoious hromosomes, stikiness, sttering, -mitosis, non-synhronistion, lggrds, ridges et. It ws oserved tht the hromotoxi effet of UV-B ws exggerted s exposure durtion elevted s evident in 60 minute treted sets. Hene, it ws onluded tht the higher intensity of UV-B exposure durtion is ftl nd genotoxi for plnt's geneti mhinery nd indues genome instility. Keywords: UV-B, irrdition, hromosoml nomlies, genotoxi, Lens ulinris Medik. 1. INTRODUCTION All living orgnisms require energy to enle them to mintin their vitl proesses. Energy rdited y sun lled solr energy is the primry soure of energy for ll living retures. From the 20 th entury, understnding of the reltionship etween plnts nd inident solr rys is mjor onern s it is the resoure se nd plys n importnt role s regultor nd ontroller of growth nd development. Solr rdition is the set of eletromgneti rdition emitted y sun. However, not ll the rditions pss through the erth s surfe s ultrviolet whih re too short, re sored y the gses present in erth s tmosphere silly CO 2, ozone nd wter vpour. Due to destrutive nthropogeni tivities nd extreme pollution, the ozone veil is eing dmged nd s onsequene more UV rys would reh the erth s surfe. Of ll the living orgnisms, plnts re more prone nd sensitive to elevted UV rditions s they re fully dependent on solr rdition for their survivl. Aording to the wvelength, UV rys hs een tegorised into UV-C ( nm) whih is extremely hrmful to orgnisms, ut not relevnt under nturl onditions, UV-B ( nm) is of prtiulr interest euse this wvelength represents ner out the 1.5% of the totl spetrum ut n indue vriety of dmging effets in plnts, UV-A ( nm) represents 6.3% of inoming solr rdition nd is the less hzrdous prt of UV rdition [1]. A smll derese in ozone level my use lrge reltive inrese in iologilly effetive UV rdition [2], [3]. UV-B n use severe deleterious effets in iologil orgnisms, despite representing only smll mount of totl solr rditions [4], [5], [6]. UV-B n dversely ret with mny iologil moleules inluding mino ids, nulei ids, proteins, lipids nd eliits stress responses t moleulr, ellulr nd whole orgnism levels [7], [8]. Grinis et. l., 2005 stted tht UV-B n dmge DNA y reting yloutne pyrimidine dimers nd pyrimidine (6-4) pyrimidine dimer, whih n led to point nd rek muttions if not orretly repired. Certin responses were eliited y plnts inluding inhiition of hypootyls elongtion nd root growth [10], otyledon opening Reserh Pulish Journls Pge 1

2 Vol. 4, Issue 1, pp: (1-8), Month: Jnury - Mrh 2016, Aville t: [11] stomtl losure [12] nd ntomil hnges ssoited with UV-B protetion. In reent yers, extensive reserhes were done y sientists to work out the plnt responses ginst UV-B rdition. During seed germintion root is the first to emerge, hene hromosoml errtions ourred in root meristems, will led to vrious impirments nd norml growth nd development. Although numerous studies hve een done with physil mutgens in diverse plnt speies ut knowledge out tivity of meristemti ells of erly roots will e of supreme importne to study proper growth nd developmentl pttern nd muttions in genotypi nd phenotypi hrters of plnts s onsequene of UV-B rditions. As the plnts re the est model system for iologil studies nd heper, esily ville, hene present experimentl work hs een designed in iming to determine the effets of UV-B on rte of mitoti division in root meristems of Lentil whih is n importnt leguminous rop. Lentil (Lens ulinris Medik) is onsidered s siling of en nd is rih soure of protein, rohydrte, nd minerls. It serves s poor people s mel in undeveloped ountries nd n lterntive of niml proteins in developed ountries due to its low holesterol ontent. As result of detiled literture study, this work represents first study on this sujet eing n implition of UV-B on Lentil tht will prove to e light for future studies. A. Seed prourement: 2. MATERIAL AND METHODOLOGY Seeds of Lens ulinris Medik were olleted from Indin Institute of Pulse Reserh (IIPR), Knpur, Uttr Prdesh. Seeds were onsistently seleted nd thorough wshing ws done upto 10 minutes with distilled wter nd surfe sterilized with 0.1% HgCl 2. B. Experimentl Design: Fresh seeds of Lentil were pre-soked in distilled wter for 12 hours nd then kept in sterilized petripltes with wet whtmnn filter pper s se for germintion in seed germintor t 25±2 o C with humidity 60-80%. Regulrly whtmnn filter ppers were llowed to hnge nd sprinkle with distilled wter. Four sets were prepred i.e. Set A for 20 minutes, Set B for 40 minute, Set C for 60 minute tretment nd Set D s ontrol. Nine replites were mintined for eh tretment. C. UV-B Tretment: Seeds with erly roots of length etween 5mm to 30mm of eh sets A, B, C were irrdited with fluoresent UV-B ( nm) lmps longwith white light. Firstly 9 replites of eh sets were irrdited for1 dy of time durtion 20, 40, 60 minute. Out of whih three replites from eh set were removed off from UV-B hmer for fixtion nd mitoti study. Then next 6 replites of eh set were gin irrdited with UV-B of respetive time durtion on seond dy nd three replites were removed off. Remining three replite out of 9 in eh sets were irrdited on third dy nd sme proess ws followed for eh set. Fourth set D ws without UV B tretment. The tretment ws strted in morning, 9m to 10 m every dy. D. Fixtion: Irrdited germinted seeds of eh sets were fixed in Crnoy s fixtive (glil eti id : solute lohol, (1:3) fter llowing for one hour reovery in their respetive ottles long with ontrol set. After 24 hours ll of them were removed from fixtive nd trnsferred into ottles ontining only 90% lohol. E. Mitoti preprtion: The roots of ontrol nd irrdited seedlings were exised, proessed nd hydrolysed in 1N HCL y djusting wter th t 60 0 for 5-10 minutes so tht tissues of root tips get soften. Then wshed under running wter to remove exess HCL nd llowed to keep on lotting pper for dehydrtion. Dried root tips were stined with 2% etormine (30 minute). By using sqush tehnique mitoti slides were prepred nd oserved ells were snpped under Nikon Reserh Eletron Mirosope using Olympus PCTV Vision Softwre. Approx. 10 mirosopi field views were reorded from eh slide. Soring ws done from 3 roots of eh replite. D. Formul used for soring of dt: The rry of mitoti indies nd vrious normlities were sreened out y pplying elow mentioned formul: Ative mitoti index (AMI) % = (Totl numer of dividing ells/totl numer of oserved ells)*100 Totl normlity perentge (TAB) % = (Totl numer of norml ells/totl numer of oserved ells)*100 Reserh Pulish Journls Pge 2

3 Vol. 4, Issue 1, pp: (1-8), Month: Jnury - Mrh 2016, Aville t: E. Sttistil nlysis: The dt otined ws nlysed using sttistil softwre, SPSS 16 nd mens were ompred using DUNCANS multiple rnge test (DMRT) (P 0.05). All the results were expressed in form of Men ± Stndrd Error. The grph ws plotted y using Sigmplot softwre. 3. RESULT The dt sored fter UV B tretment ws doumented in form of tle 1 nd tle 2 nd most frequent hromosoml normlities oserved were shown through photo mirogrphs in form of ytologil pltes in figure 1. Tle 1: Showing the ount of AMI (%) nd TAB (%) indued y UV-B in root meristems of Lens ulinris Medik TREATMENT (UV-B)* DOSES (MINUTES) AMI (%) ** (MEAN ± S.E.) TAB (%) *** (MEAN ± S.E.) CONTROL ± DAY 20 MIN ± ± MIN ± ± MIN 9.87 ± ± 0.21 CONTROL ± DAY 20 MIN ± ± MIN ± ± MIN 8.52 ± 0.28 d 6.21 ± 0.15 CONTROL ± DAY 20 MIN ± ± MIN 9.53 ± ± MIN 7.81 ± 0.30 d 7.45 ± 0.33 *Arevitions: UV-B*- Ultrviolet B rdition, AMI (%) **- Ative mitoti index, TAB (%) ***- Totl normlity perentge. Mens re followed y lowerse letter is sttistilly signifint t p<0.05. Tle 2: Showing vrious normlities indued y UV-B rditions in root meristems of Lens ulinris Medik. TREATMENT (UV-B) DOSES (MINUTES) CONTROL METAPHASIC ABNORMALITY (MEAN ± S.E.) ANAPHASIC ABNORMALITY (MEAN ± S.E.) SC CM PR ST UN BRDG ST UN LG OTH DAY 20 MIN 0.34± ± ± ± ± ± ± ± ± ± MIN 0.54± ± ± ± ± ± ± ± ± ± MIN 0.44± ± ± ± ± ± ± ± ± ±0.11 CONTROL DAY 20MIN 0.41± ± ± ± ± ± ± ± ± ± MIN 0.31± ± ± ± ± ± ± ± ± ± MIN 0.47± ± ± ± ± ± ± ± ± ±0.11 CONTROL DAY 20MIN 0. 24± ± ± ± ± ± ± ± ± ± MIN 0.56± ± ± ± ± ± ± ± ± ± MIN 0.60± ± ± ± ± ± ± ± ± ±0.15 *Arevitions: SC- Sttering, CM-C-mitosis, PR-Preoious movement, ST-Stikiness, UN-Unorienttion, BRDG-Bridge, LG-Lggrds, OTH-Other normlities. Mens followed y lowerse letter is sttislly signifint t p<0.05 Reserh Pulish Journls Pge 3

4 Vol. 4, Issue 1, pp: (1-8), Month: Jnury - Mrh 2016, Aville t: Figure 1: Showing UV-B indued hromosoml errtions: A: Prophse; B: Metphse (2n = 14); C: Anphse (14: 14); D: Unoriented metphse; E: Preoious movement of hromosomes with stiky metphse; F: Sttering t metphse; G: C-mitosis t metphse; H: Stiky metphse; I: Non-synhronistion t nphse; J: Forwrd movement of hromosomes t nphse; K: Unorienttion t nphse; L: Segmentl seprtion t nphse; M: Loop forming lggrd hromosome t nphse; N: Unequl seprtion t nphse; O: Single ridge t nphse; P: Multiple ridges t nphse. [Sle Br: Length = 2.16 µm, Width= 2.09 µm]. Reserh Pulish Journls Pge 4

5 Vol. 4, Issue 1, pp: (1-8), Month: Jnury - Mrh 2016, Aville t: A. Cytology of ontrol sets: Mitosis ws studied in meristemti ells of root tips. The present study reveled norml diploid hromosome omplement of Lens ulinris Medik (2n=14) whih were ligned in form of equtoril plte t metphse (2n =14) figure 1(B) nd these were eqully seprted (14:14) towrds eh pole t nphse figure 1 (C).The MI ws found to e highest (13.10%) nd no osure hromosoml nomlies were oserved. B. Cytology of UV B irrdited sets: The UV-B irrdited root tips showed devition ginst norml mitoti stges of ell division. Effet on tive mitoti index (AMI): AMI is mjor of ell ount undergoing ellulr division nd governing the rte of meristemti tivity. The genotoxiity level of UV-B rdition n e governed y the inrese or derese in the rte of tive mitoti index. Tle 1 shows the trend of progressive deline in AMI s the exposure dys were inresed longwith elevted durtion of UV- B. The AMI of 20 minute treted sets ws reorded to e highest 2.54 ±0.19 in 1 dy exposure nd it seems to e mildly delined to 11.39±0.15 on 3 dy UV-B exposure. However in 40 minute treted set severe deline ws noted from 11.73±0.15 to 9.53±0.59. Aprt from this drsti deline in AMI ws reorded in 60 minute treted sets from 9.87±0.34 to 7.81±0.30 d s ompred to ontrol set. Figure 2 eluidtes the pttern of grdul derese in mitoti index on inresing the durtion of supplementl UV-B irrditions. Hene it ws lerly envisged from present study tht the elevted level of UV-B exposure is mitolsti in its tion nd inhiits the norml mitoti yle y reduing the rte of tive mitoti index. ACTIVE MITOTIC INDICES (%) UV-B 1 DAY UV-B 2 DAY UV-B 3 DAY d d 0 CONTROL 20 MIN 40 MIN 60 MIN UV-B EXPOSURE (MINUTES) Figure 2: Supplementl effet of UV-B exposure on Ative Mitoti Indies (AMI) in root meristems of Lens ulinris Medik exposed for 20 min., 40 min. nd 60 min. Eh rshown in vertil lines represents the men of three smples ssyed in triplite. Brs showing different letters differ signifintly t P<0.05. Reserh Pulish Journls Pge 5

6 Vol. 4, Issue 1, pp: (1-8), Month: Jnury - Mrh 2016, Aville t: Effet of Chromosoml Orgnistion: The root mitosis shows vrious distortions in pttern of hromosoml orgnistion in UV-B treted sets tht leds to the indution of vrious types of hromosoml errtions oth lstogeni nd neugeni type. The ount of totl norml ells hs een doumented in form of totl normlity perentge (TAB) in tle 1. The rte of TAB (%) inreses from 0.44±0.08 (20 minutes) to 7.45±0.33 (60 minutes) s the exposure durtion ws inresed. The wide rnges of hromosoml errtions were oserved suh s unoriented metphse (figure 1.D), preoious movement of hromosomes with stiky metphse (Figure 1.E), sttered metphse (figure 1.F), C-mitosis (figure 1.G), stiky nphse (Figure 1.H), non-synhronistion (figure 1.I), loop formtion (figure 1.M) nd ridge formtion (Figure1.O) et. The most pronouned metphsi nd nphsi normlities indued on1 dy tretment ws preoious hromosomes (0.63±0.05 ) nd on 2 nd dy stikiness (1.02±0.13 ) nd unorienttion (1.46±0.17 ) t 60 minutes. However, on 3 rd dy stikiness ws found to e most dominnt (1.28±0.26 ) nd (1.36±0.28 ) t 60 minutes. Figure 3 shows the omprtive trend of indued hromosoml nomlies long with inresed level of UV-B exposure durtion. TOTAL ABNORMALITY PERCENTAGE (TAB %) MIN 40MIN 60MIN UVB 1 DAY UVB 2 DAY UVB 3 DAY UV-B EXPOSURE (MINUTES) Figure 3: Comprtive ount of hromosoml errtions indued y UV-B exposure in root meristemsof Lens ulinris Medik exposed for 20 min, 40 min, nd 60 min. Eh r represents the men of three smples ssyed in triplite nd rs re shown in vertil lines signifintly different t P<0.05. Hene, on the ount of ove forementioned oservtions, it ws lerly mnifestted tht the elevted UV-B exposure will led to indution of vrious hromosoml errtions in plnt ells. 4. DISCUSSION The result of present study eluidtes signifint derese in mitoti index long with dose dependent inrese in perentge of hromosoml errtions due to elevted exposure of supplementl UV-B irrditions. The studies onduted y Csill I. Br, (2009) nd Hopkins et l., (2002), were in fvour of present study showing redued mitoti indies. The redued AMI my our due to rekdown of plnt self protetion system nd further inhiition of ell DNA replition, trnsription nd protein synthesis [15]. Proly, more ells were rrested t interphse stge tht leds to deline in ell division. The deresed ATP level nd pressure exerted y energy produing entre my e the prole use for inhiition of DNA synthesis nd redued ATP [16]. Joze Bvoon nd Nd Gl (1996) were lso reported Reserh Pulish Journls Pge 6

7 Vol. 4, Issue 1, pp: (1-8), Month: Jnury - Mrh 2016, Aville t: deresed mitoti tivity nd lesser vitlity due to influene of UV-B irrdition in Pie ies. Wellmen (1983) stted tht slowing of mitosis is protetive plnt retion, s DNA is most sensitive to UV-B during replition. The enhned UV-B rdition doses indue different types of hromosoml errtions hrterized y hnge in totl hromosoml numer or struture. If the ells re irrdited during nd fter DNA synthesis (S nd G2) errtions re of hromtid type. However, if efore G 1, errtions re of hromosoml types [19]. Stikiness (Figure 1.H) ws found to e most dominnt hromosoml errtions reorded in the metphse nd nphse of mitosis. Chromosome stikiness leds to intivtion of DNA replition, inresed hromosoml ontrtion nd ondenstion or nuleoproteins proly leding to ell deth [20]. Preoious movement of hromosomes (Figure 1.E) oserved t metphse might e formed due to erly hism terminlistion or univlent hromosome formtion t end of prophse. Unorienttion (Figure1D, K) my our due to disruption of spindle struture or funtion s per UV-B rdition, leding to the spindle fire imlne on oth sides of entromere trtion power or hromosome entri frture nnot use hromosome norml movement [21]. C- mitosis t metphse ws deteted (Figure 1.G) firstly disovered y Levn (1938) in root tips of Allium ep used y intivtion of the spindle fire followed y rndom sttering of the hromosomes over the ell. The loop forming lggrds t nphse (Figure 1.H) might hve originted due to filure of kinetohores to tth with spindles nd leding to the joining of ends forming loops. Suh disorders my led to muttions. Formtion of Chromosome Bridge ws nother dominnt nomly reorded t nphse (Figure 1.O nd P). Aording to [15], the hromosome ridge might hve resulted due to enhned tivity of UV-B rditions, mking hromosome reks, then the two hromosome sides re respetively heled, produing with doule entromere hromosomes i.e. hromosome ridges. Hliem et l., (2013) re of the opinion tht the DNA dmges indued y UV-B rditions might hve influened the expression of numer of genes leding to ltertions in proteins tht ontrol mny metoli proesses like plnt development, ell yle, fertilistion nd seed formtion. 5. CONCLUSION As Lentil is n importnt pulse ommodity, hene seleted to study the UV-B effet in root meristemti ells. From present study it ws inferred tht if plnts re exposed for longer durtion to diret UV-B rditions, it will ffet root growth due to inhiition of ell division. Hene, this type of reserhes will further e reiproted to understnd the genotoxi effets of UV-B on whole plnts nd enhning wreness to onsumers out ill effets of UV-B poisoned plnts nd to devise protetive mesures ginst them. ACKNOWLEDGEMENT The uthors re grteful to Prof. Anupm Dixit, Hed of Botny Deprtment, University of Allhd, Allhd for providing essentil reserh filities. Thnks re lso due to Indin Institute of Pulse Reserh (IIPR), Knpur, Uttr Prdesh for providing Lentil seeds to ondut the experiment suessfully. REFERENCES [1] Hollosoy F., (2002) Effet of ultrviolet rdition on plnt ells.miron 33, [2] Mndronih, S., (1992). Implitions of reent totl tmospheri ozone mesurements for iologilly tive ultrviolet rdition rehing the erth s surfe. Geophys.Res. Lett. 19, [3] Mndronih, S., (1993). UV rdition in the nturl nd pertured tmosphere. In: Tevini, M. (Ed.) Effets of UV-B Rdition on Humns, Animls,Plnts, Miroorgnisms nd Mterils. Lewis Pulishers, Bo Rton, FL, pp [4] Cldwell M M., Bjorn L O., Bornmn J F., Flint S D., Kulndivelu G., Termur A H., nd Tevini M., (1998). Effets of inresed solr ultrviolet rdition on terrestril eosystems. J Photohem Photoiol B 46: Reserh Pulish Journls Pge 7

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