EFFECT OF SEED TREATMENT WITH CHITOSAN ON THE GROWTH OF RICE (Oryza sativa L.) SEEDLINGS cv. INCA LP-5 IN SALINE MEDIUM

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1 Cultivos Tropiles, 15, vol. 36, no. 1, pp Jnury-Mrh IS print: IS online: Ministry of Higher Eution. Cu Ntionl Institute of Agriulturl Sienes EFFECT OF SEED TREATMENT WITH CHITOSAN ON THE GROWTH OF RICE (Oryz stiv L.) SEEDLINGS v. INCA LP-5 IN SALINE MEDIUM Efeto el trtmiento ls semills on quitosn en el reimiento e plántuls e rroz (Oryz stiv L.) ultivr INCA LP-5 en meio slino Lisel Mrtínez González ), Ynelis Reyes Guerrero, Alejnro Flón Roríguez n Mirim Núñez Vázquez ABSTRACT. The hitosn is wiely use in griulture for hving strong fungistti effet, lso it hs other properties suh s extening the post hrvest life of fruits n vegetles n stimulting the plnt growth, mong others. However, the use of this iopolymer for inuing slinity tolerne hs een few stuie, so the ojetive of this work ws to hek if hitosn-trete sees re le to reverse the effets use y slinity in the rie seeling growth n some iohemil initors ssoite to this response. To o this, rie (Oryz stiv L.) sees vriety INCA LP-5 were trete for hours with ifferent onentrtions of hitosn (, 1 n 5 mg L -1 ). The germinte sees were trnsferre to pots ontining ilute Hogln nutritive solution supplemente or not with NCl 1 mmol L -1 n they were ple in growth hmer with ontrolle onitions. The growth n iohemil initors were evlute eleven ys fter stress tretment. Sees trete with hitosn 1 mg L -1 stimulte shoot length n ry mtter in sline meium grown seelings n lowere mlonilehye n inrese proline levels. Both hitosn onentrtions enhne the tivities of tlse n peroxise enzymes, lthough higher effet ws otine with hitosn 5 mg L -1. RESUMEN. L quitosn es us mplimente en l griultur por su efeto fungistátio, emás lrg l vi postoseh e frutos y vegetles y estimul el reimiento e ls plnts. Sin emrgo, h sio poo estuio el uso e este iopolímero en l inuión e tolerni l slini, por lo que el ojetivo e este trjo fue ompror si el trtmiento ls semills on quitosn revertí los efetos inhiitorios el estrés slino en el reimiento e plántuls e rroz, sí omo en lgunos iniores ioquímios soios est respuest. Pr ello se trtron semills e rroz (Oryz stiv L.) vrie INCA LP-5, urnte hors, on iferentes onentriones e quitosn (, 1 y 5 mg L -1 ). Ls semills germins se trnsfirieron potes, los que se les iionó soluión nutritiv Hogln ilui, suplement o no on NCl 1 mmol L -1 y se oloron en un urto e reimiento on oniiones ontrols. Ls evluiones e reimiento y los iniores ioquímios se relizron los one ís espués e estleio el estrés. El trtmiento ls semills on l onentrión e 1 mgl -1 e quitosn estimuló l longitu y l ms se e l prte ére e ls plántuls reis en meio slino, sí omo isminuyó los niveles e mlonilehío e inrementó los e prolin. En unto l tivi enzimáti ms onentriones e quitosn estimulron ls enzims tlss y peroxiss, sieno el efeto más notle on l onentrión e 5 mg L -1. Key wors: rie, growth, hitosn, slt stress Plrs lve: rroz, reimiento, quitosn, estrés slino INTRODUCTION Instituto Nionl e Cienis Agríols (INCA), gvet postl 1, Sn José e ls Ljs, Myeque, Cu. CP 37. ) lisel@in.eu.u; ynelisrg@in.eu.u; lflon@in.eu.u; mnunez@in.eu.u Chitosn is sfe n ioegrle ompoun minly otine from the exoskeleton of r, shrimp n loster (1, ). It is use for its potentilities in ifferent fiels inluing iotehnology, griulture, foo n phrmeutil inustries (3,, 5). 136

2 Cultivos Tropiles, 15, vol. 36, no. 1, pp Jnury-Mrh Chitosn hs een wiely use in griulture minly euse of its ntifungl n ntimiroil tivity (6, 7). It is le to stimulte the enzyme proution relte to efensive responses in plnts, suh s hitinses, glunses n phenyllnine mmoni lyse (PAL) (8). Besies, it hs een foun tht these ompouns, epening on their onentrtion n egree of etyltion, re le to tivte PAL enzyme n other resistne omponents inue in plnts (9). Slinity is n ioti stress whih uses low proutivity in most rops roun the worl (1). Suh stress ereses germintion n provokes n uneven emergene of seelings, therey reuing popultion ensity, n spet tht ffets rop estlishment. On the other hn, slinity is known to inhiit plnt growth use, t first phse, y wter uptke reution in roots, whih is lle osmoti stress or wter stress phse (11). There is little informtion out the use of hitosn to relieve the verse effets of slinity on plnt growth n there is not ny kgroun knowlege in rie rop. Therefore, the im of this stuy ws to evlute whether sees trete with hitosn were le to reverse the verse effets of slinity on rie seeling growth of vriety INCA LP-5, s well s to evlute some iohemil initors ssoite to this nswer. MATERIALS AND METHODS Rie (Oryz stiv L.) sees from vriety INCA LP-5 were trete for hours y ifferent onentrtions (, 1 n 5 mg L -1 ) of hitosn polymer with the following hrteristis: moleulr mss 93,3 kd n 31,69 % egree of etyltion. Susequently, the trete sees were ple in Petri ishes with istille wter for its germintion in the rk t 5± o C. After 8 hours, germinte sees were trnsferre to pots, to whih 5 ml of ilute Hogln nutrient solution ws e (1:), supplemente or not with 1 mmol NCl L -1. Then, sees per pot n six pots per tretment were use, tht is, 1 seelings. Pots were ple in growth hmer t 1-hour photoperio n temperture of ±, o C. Eleven ys lter, the lengths of roots n eril prts were mesure in 5 plnts per tretment, n five smples of five plnts eh were forme to evlute the ry mss of eril prts n roots. At this very moment, some iohemil etermintions were performe. Thus,.5 g fresh tissue were tken of eh tretment n merte in liqui nitrogen, ing.5 ml phosphte uffer 1 mmol L -1, ph 7 n then entrifuge t 1, rpm for minutes t C. The superntnt otine llowe to estimte lipi peroxition y mesuring mlonilehye (MDA) (1), the enzyme tivity of totl peroxises (13) n tlses (1), n totl solule proteins y MiroLowry metho (15). Moreover, for etermining proline mino i,,5 g fresh tissue were tken of eh tretment n merte in liqui nitrogen, ing 1 ml wter t 1 ºC. Proline ontent ws quntifie y Btes metho (16), using L-proline (Sigm) s stnr. The experiment ws repete twie with similr performne. Results from one replite re presente. Dt were sttistilly proesse y using Single Clssifition Vrine Anlysis n mens were ompre y Tukey Multiple Rnge test t p<,5. RESULTS AND DISCUSSION Figure 1 shows the results of vrile performne in rie seeling growth of v. INCA LP-5, ultivte oth in nutrient solution lone n in nother one supplemente with NCl. As it n e seen, the presene of 1 mmol L -1 NCl erese signifintly the length n ry mss of the eril prt; however, it h no effets on roots. Chitosn onentrtion of 1 mg L -1 h signifint stimultion on the length n ry mss of the eril prt of those seelings whose sees were trete with hitosn (Figure 1A n C). The tretment of 1 mg L -1 erese root ry mss in stresse seelings (Figure 1D). It is interesting to remrk tht sees trete with hitosn h no effet on seeling growth in nutrient solution. This result oul e ue to the onentrtions use, s it hs een oserve tht growth is inue y lower onentrtions. Chitosn not only hs effets ginst pthogeni fungi, ut lso it generlly hs growth-promoting effets, lthough it hs een so fr hrly stuie. Some uthors point out tht rop yiels re higher when sees re trete with hitosn. Rie yiel inrese to 3 % fter folir sprying n see tretment with hitosn, respetively (17, 18). When nlyzing hitosn tretments pplie to sees, positive effet of this polymer ws oserve on rie seeling growth of vriety INCA LP-5 in sline meium. The tretment of 1 mg L -1 ws the est, sine it fully n prtilly reverse the negtive impt of slt on the ry mss n length of the eril prt, respetively (Figure 1). Previous stuies foun similr performne in Isgol (Plntgo ovt Forsk) plnts, where hitosn (, %) inue shoot n root lengths, s well s root ry mss uner slt stress onitions (19). With hitosn pplitions to soyen seelings uner slt stress onitions, higher ry mss ws lso oserve (). Similrly, sfflower (Crthmus tintorius L.) sees trete with hitosn inrese seeling growth uner wter efiit onitions (). 137

3 Cultivos Tropiles, 15, vol. 36, no. 1, pp Jnury-Mrh A B Length (m) mg L -1 Chitosn 5 mg L -1 Chitosn =,53*** Length (m) =,7** 1 mg L -1 Chitosn 5 mg L -1 Chitosn +NCl +NCl C D Dry mss (mg plnt -1 ) mg L -1 Chitosn 5 mg L -1 Chitosn =,8*** Dry mss (mg plnt -1 ) 8 6 =,6** 1 mg L -1 Chitosn 5 mg L -1 Chitosn +NCl +NCl Culture meium Culture meium Common letters o not iffer oring to Tukey test (p.5) n= 6 A. Length of the eril prt. B. Root length. C. Dry mss of the eril prt. D. Root ry mss Figure 1. Influene of hitosn tretment on some vriles of rie seelings of v. INCA LP-5 grown in nutrient solution supplemente (+NCl) or not () with NCl 1 mmol L -1 Zeng n Luo reporte something similr, s they foun tht sees ote with hitosn enhne germintion rte n plnt growth of whet rop uner rought onitions (1). Other uthors hve reporte tht orn sees trete with hitosn t,5 % improve germintion rte n the length n ry mss of seeling roots n shoots uner low temperture stress onitions (). Mlonilehye (MDA) is one of the finl lowmoleulr-mss prouts from lipi hyro peroxie eomposition, whih is the most ommonly use ompoun to mesure lipi peroxition in ell memrne. As shown in Figure A, seelings grown in nutrient solution with NCl showe higher MDA levels. This inrese MDA onentrtion to vrious stresses is wiely esrie in literture (3, 5) n it is iret onsequene of the tion of retive oxygen speies (EAO). Sees trete with oth hitosn onentrtions erese MDA levels in stresse seelings. Similr results hve een reporte in sunflower plnts (6). Besies, it hs een oserve tht MDA ontent erese in tomto seelings folirly sprye with 15 mg L -1 hitosn n sujete to slt stress onitions (7). Also, oligohitosn pretretment with,65 % reue MDA levels in whet plnts grown uner sline onitions (8). Likewise, hitosn onentrtions (up to,5 %) erese MDA levels of sfflower seelings sujete to osmoti stress (). At low temperture onitions, hitosn tretment lso erese MDA ontent in orn plnts (). Higher plnts my umulte L-proline (Pro) in response to vrious environmentl stresses. In this experiment, slt stress inrese this mino i onentrtion in rie seelings over the ontrol. 138

4 Cultivos Tropiles, 15, vol. 36, no. 1, pp Jnury-Mrh mmol TBARS g -1 MF µg Pro g -1 MF 3,5 3,5 1,5 1,5,5,,3,,1 Es x =,57*** Es x =,31*** A 1 mg L -1 Chitosn 5 mg L -1 Chitosn +NCl Culture meium B 1 mg L -1 Chitosn 5 mg L -1 Chitosn Culture meium +NCl Common letters o not iffer oring to Tukey test (p.5) n= 6 Figure. Influene of hitosn tretment t mlonilehye (A) n proline (B) levels on rie seelings of v. INCA LP-5 grown in nutrient solution supplemente (+NCl) or not () with 1 mmol L -1 NCl Chitosn tretment t onentrtion of 1 mg L -1 inrese these levels even more. This sme onentrtion signifintly erese this initor in unstresse seelings. Although the ext role of proline on slt stress is ontroversil, sine not lwys inreses of this mino i hve een orrelte with higher stress tolerne (9), in reent yers it hs een shown tht the exogenous pplition of proline provies protetive tion ginst oxitive mge inue y slt stress (3, 3). Furthermore, it hs een shown tht proline umultion stilizes memrnes n suellulr omponents inluing omplex II of mitohonril eletron trnsport hin. Stuies hve suggeste proline s finl eptor of free rils (33) n reox potentil stilizer y resupplying NADP + supplies (3). Results of this stuy showe tht hitosn tretment inrese proline levels uner sline onitions; however, only the onentrtion of 1 mg L -1 signifintly inrese this initor in reltion to the ontrol tretment. Inrese proline hs een foun in whet n tomto plnts sujete to sline stress (7, 8), wheres sees trete with hitosn h n opposite effet in sunflower plnts (6). Totl protein onentrtion erese with slt stress n hitosn tretment worsene this performne (Figure 3A). Contrry to this result, it ws foun tht tretment with low hitosn onentrtions in sfflower seelings sujete to wter stress inrese solule protein onentrtion (). It is interesting to oserve how the polymer tretment t the onentrtion of 5 mg L -1 in unstresse seelings lso erese protein ontent. Similr results were reore in sfflower seelings erive from hitosn-trete sees (). Among the key enzymes removing toxi oxints re peroxises n tlses. Figure 3 (B n C) shows tht, in the se of the ontrol tretment, slt stress erese peroxise enzyme (POX) tivity n h no effet on tlses (CAT). Chitosn tretment inrese the tivity of oth enzymes in stresse seelings n its effet epene on the onentrtion. It is interesting to point out tht the onentrtion of 1 mg L -1 erese oth enzymti tivities in unstresse seelings; however, the onentrtion of 5 mg L -1 signifintly inrese enzyme tivity. Perhps, it is euse higher onentrtion oul put the ell on the lert, strengthening efenses. There re numerous stuies out slt stress effets on plnt ntioxint enzymes, ut they re sometimes ontritory. Regring peroxises, slt stress inreses this enzyme tivity in rie plnts (3, 35, 36). Peroxise tivity of potto lluses inrese uner slt stress onitions, ut t higher onentrtions (15 mmol L -1 NCl) this tivity erese (37). Similrly, in otton, it hs een reporte tht POX inreses in slt-tolernt rops n ereses or remins onstnt in sltsensitive rops (38, ). However, slinity erese POX enzyme in whet, wheres inrese MDA levels n root superoxie proution (1), similr to this stuy. Conerning tlse enzyme, something similr ours euse some uthors hve reore stimultion of this enzyme tivity through tivting Ct, n Ct3 () genes. Nevertheless, others foun CAT erese in rie leves y NCl tretment (3, ). On the other hn, it hs lso een oserve tht this enzyme oes not vry in rie seelings uner slt stress (5). 139

5 Cultivos Tropiles, 15, vol. 36, no. 1, pp Jnury-Mrh Protein onentrtion (mg prot g-1 PF) POX (umol H O min -1 mg -1 prot) CAT (umol H O min -1 mg -1 prot),6,5,,3,,1,5,,3,,1,1,9,6,3 e A 1 mg L -1 Chitosn B C =,56*** +NCl =,86*** +NCl =,15*** +NCl Men of ultivtion 5 mg L -1 Chitosn Common letters o not iffer oring to Tukey test (p.5) n= 6 Figure 3. Influene of hitosn tretment on totl solule protein onentrtion (A), peroxise (B) n tlse (C) enzyme tivities of rie seelings of v. INCA LP-5 grown in nutrient solution supplemente (+NCl) or not () with 1 mmol L -1 NCl So fr s the stimultion of CAT enzymti tivity y hitosn tretment is onerne, similr results were otine in tomto seelings sprye with hitosn n sujete to slt stress (7). Besies, inreses in POX, CAT n superoxie ismutse (SOD) enzymti tivities were foun in whet seelings trete with oligohitosn n sujete to stress (8). However, uner norml onitions, POX n CAT tivities were stimulte in sunflower seelings fter hitosn tretment, ut these sme onentrtions reue oth enzyme tivities uner sline onitions (6). Furthermore, it ws foun tht hitosn tretment stimultes ntioxint enzyme tivity in plnts sujete to other kins of stress, suh s low tempertures in orn plnts () n wter stress in whet plnts (1). Reently, hitosn hs reeive speil ttention for its ility s n ntioxint, whih epens on the moleulr mss n egree of etyltion (6). Severl stuies hve onfirme tht hitosn my hve potentil s free ril trpper (6, 7). This ompoun n th the ril hyroxyl n superoxie n it hs een reporte for its protetive DNA properties (8). The mehnism y whih hitosn ts oul e ttriute to its struture with lrge numer of ville hyroxyl n mino groups reting with tive EAO oxygen speies (9). In generl, hitosn tretment tivte POX n CAT ntioxint enzymes in seelings grown in sline meium, therey lipi peroxition erese in the memrne. Moreover, lthough hitosn lso inrese proline levels in seelings grown in sline meium, only the onentrtion of 1 mg L -1 inue n inrese whih iffere sttistilly from the other tretments, oiniing with the onentrtion tht reverse growth inhiition use y slinity in the eril prt of seelings, suggesting the importne of this metolite uner these onitions, s it is known tht proline n t s n ntioxint n s osmotilly tive solute. Tking into ount tht these re the first results from hitosn effet on rie seeling growth in sline meium, prtiulrly in v. INCA LP-5, it is suggeste to keep on eepening into these stuies, espeilly y onsiering the importne of this vriety in our rie proution n the onstnt enlrgement of slinityffete res. 1

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