The effect of Zinc oxide nano particles and Humic acid on morphological characters and secondary metabolite production in Lilium ledebourii Bioss

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1 IRANIAN JOURNAL of GENETICS n PLANT BREEDING, Vol. 4, No. 2, Ot 2 The effet of Zin oxie nno prtiles n Humi i on morphologil hrters n seonry metolite proution in Lilium leeourii Bioss Esmeil Chmni *, Skineh Krimi Ghlehtki 2, Mehi Moheoini 3, Alirez Ghnri 4 Professor, Deprtment of Hortiulturl Sienes, Fulty of Agriulture n Nturl Resoures, University of Mohghegh Arili, Aril, Irn. 2 M.S. stuent, Deprtment of Hortiulturl Sienes, Fulty of Agriulture n Nturl Resoures, University of Mohghegh Arili, Aril, Irn. 3 Assoite Professor, Deprtment of Hortiulturl Sienes, Fulty of Agriulture n Nturl Resoures, University of Mohghegh Arili, Aril, Irn. 4 Asistnt Professor, Deprtment of Hortiulturl Sienes, Fulty of Agriulture n Nturl Resoures, University of Mohghegh Arili, Aril, Irn. * Corresponing uthor, E-mil: ehmni@um..ir. Tel: Reeive: 2 Jul 2; Aepte: 9 Jun 26. Astrt The effets of ifferent onentrtions of Zin oxie nno prtiles (ZnONP;,, 2,, 7 n mg L - ) n Humi i (HA;,,, n mg L - ) on root length n numer, lef length n numer, ul imeter, n hlorophyll, totl phenol, nthoynin n flvonoi ontents were exmine uner in vitro onitions. Totl phenol, flvonoi n nthoynin ontents were signifintly (P.) ffete. The mximum phenoli ontent of plntlets ws otine in the meium ontining 7 mg L - ZnONP. The highest nthoynin ontent ws oserve in plntlets trete with HA t mg L -. The highest flvonoi ontent mesure t 27, 3 n 33 nm wvelengths were otine with ZnONP t 2 mg L -, ZnONP t 2 mg L - n HA t mg L -, respetively. Explnts trete with HA proue the highest root length, lef length n numer, ul numer n hlorophyll ontent in the mei ontining,,, n mg L - HA, respetively. However, the mei ontining,,, 7 n mg L - ZnONP proue the highest root length, lef length n numer, ul numer n hlorophyll, respetively. Key wors: Humi i, Lilium leeourii Bioss, Seonry metolites, Zin oxie nno prtiles. INTRODUCTION Seonry metolites in plnts re iverse iohemil sustnes with numerous iologil funtions. Mjor roles of plnt seonry metolites inlue plnt protetion from ioti n ioti stresses. Plnts n e unique soures of seonry metolites for phrmeutils, foo itives, flvors n other inustril sustnes. Aumultion of these seonry metolites often ours in plnts sujete to stresses meite y vrious hemil eliitors n signl moleules. Plnt ell n tissue ulture hs een use s mens of prouing seonry metolites whose hemil synthesis is iffiult (Zho et l., 2). Moreover, the evolving ommeril importne of seonry metolites hs resulte in n interest in the possiility of enhning the proution of iotive plnt metolites y tissue ulture tehnologies (Vnisree et l., 24). Lilium leeourii Boiss. (Fmily Liliee) is lle Susn-e-Chelhergh in Persin. It is the only enngere Lilium speies in Irn. Wil popultions re urrently t risk of erition euse of irregulr grzing n pohing (Azi n Khosh-Khui, 27).

2 Chmni et l. L. leeourii grows on the estern slopes of the Alurz Mountins, where it is now protete in smll re of egre eeh forest roun Dmsh villge (Bkhshie et l., 2; Azi n Khosh-Khui, 27). It is perennil plnt with lneolte yellowish sles, thik stem n eret leves. Its flowers re white n lrge (Frsm et l., 23). Little or no investigtion hs een rrie out on the phytohemistry of this speies. However, there re reports on the phytohemistry of other Liliums. The fresh n rie uls of L. nium hve een suggeste useful in the tretment of gyneologil isorers, uler, urns, injuries (Frsm et l., 23). Primry qulittive phytohemil tests showe steroil sponins, phenoli glyosies, flvonoi lklois, n pyrroline-pyrroliine lklois (Frsm et l., 23). Humi i (HA) hs een reognize to hve morphologil n physiologil effets on higher plnts. However, reltionships etween HA struture n iologil tivity re not ler (Mor et l., 2). Positive influenes of HA on plnt growth n evelopment hve een reporte. Yilirim (27) reporte tht HA pplie in the soil n sprye on tomto plnts, h inrese prout qulity n quntity. In this ontext potentil effets on ntioxint tivity hve reeive little ttention (Aminifr et l., 22). Stuies on lettue n tomto revelenutrient soures i not ffet the totl phenoli ontent. In ontrst, flvonois in tomtoes n ntioxints in strwerries were inrese in orgni rop mngement prties. As reporte in onestuy, orgni strwerries h higher levels of totl phenolis, Ellgi i, n flvonols ompre to onventionlly ulture strwerries (Aminifr et l., 22). Flvonois enompss lrge su-group of phenoli plnt seonry metolites tht hve in vitro ntioxint free ril svenging tivity (Mithell et l., 27). They re proue through the phenyl propnoi pthwy. The key enzyme tht tlyzes their iosynthesis is phenyllnine mmoni-lyse. Stresses inluing nutrient efiieny, wouning, pthogens, n UV rition re known to tivte the iosynthesis of phenyl propnoi ompouns (Mithell et l., 27). Environmentl ftors use hnges in the growth of meiinl plnts, inluing the quntity n qulity of seonry metolites. These ftors inlue ulture meium mironutrients, ie. Zn. Nnoprtiles (smll size n lrge surfe re) re onsiere iel for elivering Zn fertilizer to plnts (Prs et l., 22). Most stuies hve een rrie out on the effet of Zn nnoprtiles on the growth ftors of plnts, ut there re no stuies on the effet of Zn nnoprtiles on the proution of seonry metolites. Zin is n essentil mironutrient n limite stuies hve shown its enefiil effet on essentil oils s well s on plnt growth. Zn is importnt s metl omponent of ertin enzymes n/or s funtionl struturl regultory oftor. It is essentil for protein synthesis, photosynthesis, uxin synthesis, ell ivision, memrne struture n funtion, n sexul fertiliztion (Si et l., 2). Previous stuies hve shown tht hevy metls n inrese the tivity of the ntioxint enzymes tlse (CAT) n sorte peroxise (APX). As yet, there is no informtion on ZnO nnoprtile (ZnONP) effets on seonry metolites (Hernnez-Viezs et l., 2). The present reserh investigte the effets of some eliitors t vrious onentrtions on in vitro growth n seonry metolites proution in L. leeourii. Using these eliitors n proly stimulte seonry metolite synthesis, genetilly n physiologilly. MATERIALS AND METHODS Plnt Mteril Bul sles otine y in vitro miro-propgtion in MS meium (Murshing n skoog, 962) were use s soure for plntlet proution. Sles of L. leeourii were seprte n ulture in ifferent onentrtions of HA solutions (,,, n mg L - ) n ZnONP (,, 2,, 7 n mg L - ) in the MS meium. After six suultures, plntlets were use for the evlution of root length n numer, lef length n numer, ul imeter, n hlorophyll n seonry metolites proution. The experiment ws ompletely rnomize esign with six replites. All ultures were mintine t 23 ± 2 C n 6-hour photoperio with light intensity of 2 lux t plnt level provie y ool white fluoresent lmps. Chlorophyll ontent ws mesure y SPAD hlorophyll meter. Lef, root n ul numer ounting ws one y oservtion. Length ws mesure y igitl ruler. Totl phenoli ontent Totl phenoli ontent ws etermine using the Folin Ciolteu ssy (Singleton n Rossi, 96) with minor moifition. Dilute extrts were iretly ssye t 6 nm with glli i s the stnr. Results were expresse s totl phenols in miromoles of Glli i equivlents (GAE) per gr of fresh weight. 2

3 IRANIAN JOURNAL of GENETICS n PLANT BREEDING, Vol. 4, No. 2, Ot 2 Tle. Anlysis of vrine for effets of HA on root n lef length, lef n ul numer, hlorophyll, phenol, flvonoi (27, 3 n 33 nm) n nthoynin ontents. Soure of vritions f Men of squre Root length Lef length Lef numer Bul numer Chlorophyll HA 3 27.* * 9.39* 3.793* 9.47* Error Phenol Flvonoi 27 Flvonoi 3 Flvonoi 33 Anthoynin HA **.294** 9.3**.3**.826** Error *,**: Signifint t % n % proility level, respetively. Totl nthoynin Anthoynin ontent ws exmine oring to Msuksu et l. (23). Frozen tissue (.2 gr) ws homogenize with ii MeOH (MeOH n HCl t 99:) n entrifuge t 2 g for min. The superntnt ws kept in rk overnight. Anthoynin ontent ws etermine s sorne t nm using the extintion oeffiient of 33, m - M - n ws expresse s µm/g FW. Totl flvonois Totl flvonoi ontent ws etermine oring to Krizek et l. (993). Frozen tissue (.2 g) ws homogenize with ii EtOH (EtOH n HOAC t 99:) n entrifuge t 2 g for min. The superntnt ws hel in n 8 C wter th for min. After ooling, sorne of flvonois ws t 27, 3 n 33 nm. Flvonois ontent ws etermine y using extintion oeffiient of 33 m - M - n ws expresse s µm/g FW. RESULTS AND DISCUSSION Effets of HA Anlysis of vrine inite tht ifferent onentrtions of HA signifintly (P.) ffete ll hrteristis (Tle ). Men omprison y Dunn multiple rnge tests showe tht the mximum root n lef length, lef numer, ul numer n hlorophyll ontent ws hieve in the mei ontining,,, n mg L - HA, respetively (Figure 2 A, B, C, D n E). The highest phenol ontent of plntlet ws otine in the meium ontining mg L - HA (Figure 2 F). However, ll HA onentrtions signifintly (P.) improve totl phenoli ontent in plntlets ompre to ontrol n no signifint ifferenes (P.) were foun in, n mg L - HA tretments (Figure 2 F). All HA onentrtions signifintly (P.) improve nthoynin ontent in the trete plntlets ompre to ontrol (Tle ). The highest ontent of nthoynin ws otine from trete plntlets y HA t mg L - (Figure 2 G). It is onlue tht inresing HA onentrtions higher thn mg L - erese nthoynin ontent. However, no signifint ifferenes (P.) were foun in n n lso n mg L - HA tretments (Figure 2 G). All HA onentrtions signifintly (P.) improve flvonoi ontent in 27, 3 n 33 nm wvelengths ompre to ontrol (Tle ). The results lso revele tht the highest onentrtion of flvonoi in 27, 3 n 33 nm wvelengths were otine in the meium supplemente y mg L - HA (Figure 2 H, I n J). The results of men omprison showe tht inresing HA onentrtions higher thn mg L - use erese in flvonoi ontent t ll mesure wvelengths (Figure 2 H, I n J). Among vrious onentrtions of HA, only mg L - tretments proue the lowest flvonoi ontent in plntlets t ll mesure wvelengths (Figure 2 H, I n J). Effets of Zin oxie nno prtiles Anlysis of vrine inite tht the effets of vrious onentrtions of Zin oxie nno prtiles on root length n numer, length of lef, numer of uls, hlorophyll, totl phenol ontent, flvonoi n nthoynin ontent signifintly iffere ompre to the ontrol (Tle 2). Men omprison y Dunn multiple rnge tests showe tht the mximum root length, lef numer n length, ul numer n hlorophyll ontent ws otine in the mei ontining,,, 7 n mg L - Zin oxie nno prtiles, respetively (Figure D n E n Figure 3 A, B, C, D n E). Mximum 3

4 Chmni et l. Figure. Effet of ifferent onentrtions of HA n ZnONP on growth ftors of Lilium leeourii (A: mg L - HA, B: mg L - HA n C: mg L - HA, D: mg L - Zin oxie n E: 7 mg L - Zin oxie). Tle 2. Anlysis of vrine for the effet of ZnO on root n lef length, lef n ul numer, hlorophyll, phenol, flvonoi (27, 3 n 33 nm) n nthoynin ontents. Soure of vritions f Men of squre Root length Lef length Lef numer Bul numer Chlorophyll ZnO 342.6* 79.6*.944* 49.2** 33.7* Error Phenol Flvonoi 27 Flvonoi 3 Flvonoi 33 Anthoynin ZnO 8.94**.28** 28.32**.86**.3** Error *,**: Signifint t n % proility level, respetively. mount of phenoli ontent of plntlet ws otine in the meium ontining 7 mg L - Zin oxie nno prtiles (Figure 3 F). However, ll Zin oxie nno prtile onentrtions signifintly (P.) improve totl phenoli ontent in plntlets ompre to ontrol. In se of totl phenoli ontent in plntlets, no signifint ifferenes (P.) were foun mong, 2, n mg L - Zin oxie nno prtiles tretments (Figure 3 F). The results lso revele tht the highest onentrtions of flvonois mesure t 27, 3 n 33 nm wvelengths were otine with Zin oxie nno prtiles t 2 mg L - (Figure 3 G, H n I). It is onlue tht inresing Zin oxie nno prtiles onentrtions higher thn 2 mg L - erese flvonois ontent t ll mesure wvelengths (Figure 3 G, H n I). The results lso revele tht the highest onentrtions of nthoynin ontent of plntlet ws otine in the mei ontining 7 mg L - Zin oxie nno prtiles (Figure 3 J). However, ll Zin oxie nno prtile onentrtions signifintly (P.) improve nthoynin ontent in plntlets ompre to ontrol. In se of nthoynin ontent in plntlets, 4

5 Flvonoi Content 33 nm Anthoynin Content Flvonoi Content 27 nm Flvonoi Content 3 nm Bul Numer Chlorophyl (Sp Unit) Phenol Content Root Lenght (mm) Lef Lenght (mm) Lef Numer IRANIAN JOURNAL of GENETICS n PLANT BREEDING, Vol. 4, No. 2, Ot 2 A B C Humi Ai Conentrtion Humi Ai Conentrtion Humi Ai Conentrtion D E F Humi Ai Conentrtion Humi Ai Conentrtion Humi Ai Conentrtion G H.. I Humi Ai Conentrtion Ai Humi Conentrtion Humi Ai Conentrtion J 2.. Humi Ai Conentrton Figure 2. Effets of HA on root n lef length (A, B), lef n ul numer (C, D), hlorophyll (E), phenol (F), nthoynin (G) n flvonoi (27, 3 n 33 nm) (H, I, J) ontents.

6 Anthoynin Flvonoi Content 27 nm Flvonoi Content 3 nm Flvonoi Content 33 nm Bul Numer Chlorophyl (Sp Unit) Phenol Content Root Lengh (mm) Lef Lenght (mm) Lef Numer Chmni et l. A B C Zin Oxie Conentrtion Zin Oxie Conentrtion Zin Oxie Conentrtion D 3 2 E F Zin Oxie Conentrtion Zin Oxie Conentrtion Zin Oxie Conentrtion G 3 2 H I Zin Oxie Conentrtion Zin Oxie Conentrtion Zin Oxie Conentrtion J Zin Oxie Conentrtion Figure 3. Effets of Zin oxie nno prtiles on root n lef length (A, B), lef n ul numer (C,D), Chlorophyll (E), phenol (F), flvonoi (27, 3 n 33 nm) (G, H, I) n nthoynin (J) ontents. 6

7 IRANIAN JOURNAL of GENETICS n PLANT BREEDING, Vol. 4, No. 2, Ot 2 no signifint ifferenes (P.) were foun mong 2,, 7 n mg L - Zin oxie nno prtiles tretments (Figure 3 J). The result of this stuy showe tht the mximum root length ws otine in the meium ontining mg L - HA. Bnier et l. (29) reporte humi sustnes le to reution in root imeter n n inrese in speifi root length in rish (Rphnus stivus). HAs hve lso een shown to e le to stimulte the prolifertion of lterl roots long with the tivtion of plsmlemm n vuolr H + -ATPses n tonoplst H + -PPse. In greement, it hs reently een shown tht HAs oul intert with root orgni i exution n re le to hnge root re, primry root length, numer of lterl roots n lterl root ensity (Znni et l., 29). The results of our experiment revele tht the highest lef numer n length ws otine from trete plntlets y HA t mg L -. However, plnt growth signifintly erese when the HA onentrtions ws inrese. The sme result ws foun in the stuy rrie out y Atiyeh et l. (22) whih revele tht the generl pttern of plnt growth ws inrese in response to low onentrtions of HA ut erese signifintly when the exeeing onentrtions of HA were pplie. It is suggeste tht its mehnism might e hormone-like tivities of HA, suh s n uxin tivity, sine plnt hormones suh s uxin when pplie t high onentrtions (eyon n optimum level) oul reue the growth n evelopment of plnts. In stuy rrie out y Osuwn et l. (2) it ws shown tht humi sustnes enhne the growth y inresing the uptke of mironutrients.so fr, few stuies hve investigte the influene of orgni mngement on ul proution in plnts. Ael- Rzzk et l. (23) reporte tht the pplition of HA on grli inrese ul numer. However, it seems tht these growth responses were most proly ue to the hormone-like tivity of HAs. In our stuy the highest hlorophyll ontent ws otine in the mei ontining mg L - HA. The effet of HA on the vilility of mironutrients hs een given prtiulr ttention euse of oserve inreses in the uptke rtes of these nutrients following pplition of HA on Frgri nnssvr: Cmros (Ameri n Tehrnifr, 22). Humi sustnes hve positive effets on plnt physiology, so tht HA t 2 ppm ws effetive in inresing hlorophyll ontent. It uses reution of Fe 3+ to Fe 2+ n me iron heltes, whih re reily ville to the plnts (Ameri n Tehrnifr, 22). It is onlue tht inresing of HA n improve phenol, nthoynin n flvonoi ontents. Chssy et l. (26) reporte tht HA signifintly inrese levels of solule solis, flvonois, totl phenolis, n sori i in orgni tomtoes ompre to their onventionl ounterprts grown in moel plots over 3-yer perio. Strwerries grown orgnilly h higher levels of ntioxints inluing totl phenoli, Ellgi i, n flvonols ompre to onventionlly grown strwerries (Amimifr et l., 22). Prnin n Smvt (22) reporte tht HA signifintly inrese the mount of nthoynin in Lilium flowers. Mithell et l. (27) reporte tht the levels of flvonois inrese over time in orgnilly trete smples, wheres the levels of flvonois i not vry signifintly in onventionl tretments. The result of our experiment showe tht the mximum root length ws hieve in the meium ontining mg L - Zin oxie nno prtiles. In ontrst, root length erese t Zn onentrtions higher thn mg L - (Figure 3 A). Luo et l. (2) reporte tht the first visile mge, ue to exessive Zin, ws on root growth use y reution in ell ivision. Ghoke et l. (2) reporte tht with inresing onentrtions of the nno prtiles, the elongtion of the roots ws severely inhiite y Zin oxie NPs toxiity in the ulture Allium ep (onion uls). It ws reently reporte tht the NPs my inue the formtion of new n lrge-size pores n routes for the internliztion of lrge NPs through ell wlls (Ghoke et l., 2). Although, the moleulr mehnism of the toxiity ue to Zin oxie NPs in the plnt roots is not ler n requires further investigtion. However, it shoul e losely relte to the hemil omposition, hemil struture, prtile size, n surfe re of the NPs (Ghoke et l., 2). Our results showe tht the highest lef length n numer ws otine in the trete plntlets y ZnO t mg L - (Figure 3 B n C). Mhjn et l. (2) reporte tht with n inrese in nno-zno onentrtion, the root n shoot growth of mung (Vign rit) n grm (Cier rietinum) h inrese. However, fter ertin onentrtions, the growth of root n shoot ws foun to eline. Luo et l. (2) reporte tht the iomss of plntlets otine from otyleons, hypootyls of Jtroph urs seeling, inrese grully up to.2,. n. mm Zin, respetively n then erese. 7

8 Chmni et l. Therefore, in vrious plnt speies there re eferent growth retions. Sugr eet shoot, fresh root n ry mss erese progressively when Zn onentrtion in the nutrient solution inrese (Sgroy et l., 28). Mximum numer of uls ws otine in the meium ontining 7 mg L - Zin oxie nno prtiles (Figure 3 D). Khlif et l. (2) showe tht the folir sprying of Zin sulphte (4.g/l) t ll onentrtions signifintly inrese growth prmeters suh s ullets numer of Iris plnts n yiel/plnt ompre to the ontrol. In our stuy, the mximum n minimum hlorophyll ontents in plntlets were otine in the mei ontining n mg L - ZnO, respetively (Figure 3 E). In ft, y inresing ZnO onentrtion, hlorophyll ontent erese n lef toxiity symptoms were oserve. It hs een reporte tht Zin inhiits Fe trnslotion n in the young lef inues hloroti leves, showing Zn 2+ toxiity (Rout n Ds, 23). Zin toxiity symptoms inlue reue yiels n stunte growth, Fe-efiieny inues hlorosis through reutions in hlorophyll synthesis n hloroplst egrtion (Mrtin, 26). Leves of sugr eet (Bet vulgris L.) trete with n µm Zn evelope symptoms of Fe efiieny, inluing erese in Fe, hlorophyll n rotenoi onentrtions, inrese in rotenoi hlorophyll rtios. Exess Zin lso erese the numer of leves n lef re, lef mrgins were istorte or wrinkle n leves were rolle inwrs n showe hlorosis symptoms (Sgroy et l., 28). The result of the present stuy revele phenoli ontent n nthoynin in Lilium leeourii Boiss. Plntlets expose to exessive Zin, showe very slight n non signifint growth y inresing Zin onentrtions to 7 mg L -. But flvonoi ontent in three wvelengths (27, 3 n 33 nm) inrese y inresing Zin onentrtions from to 2 mg L -. Nevertheless, eyon this onentrtion, the flvonoi ontent grully erese y inresing Zin onentrtions. There hve een numerous reports on the inue umultion of phenoli ompouns n peroxise tivity in plnts uner stress. The roots of mny plnts expose to hevy metls show high levels of phenolis. In plnts, phenoli ompouns suh s flvonols n phenyl propnois t s potentil ntioxint ompouns y onting eletrons to guiol peroxises (GPX) for etoxifition of high mounts of H 2 O 2 proue uner stress onitions (Brtwl et l., 23). Vrious stuies hve een rrie out to explore the effet of nnoprtiles on the growth of plnts ut few stuies were foun to onentrte on the effet of nnoprtiles on plnts seonry metolites. Zin is one of the mironutrient of plnts, n limite stuies hve een one on the enefiil effet of Zin n other essentil mironutrients on plnt growth. However, it is well known to e neessry for plnt growth n evelopment. However, exessive Zn in plnts n profounly ffet norml ioni homeostti systems y interfering with the uptke, trnsport, osmoti n regultion of essentil ions n results in the isruption of metoli proesses suh s trnspirtion, photosynthesis n enzyme tivities relte to metolism (Mhjn et l., 2). Zin is metl omponent of vrious enzymes or s funtionl struturl or regultory oftor n for protein synthesis, photosynthesis, the synthesis of uxin, ell ivision, the mintenne of memrne struture n funtion, n sexul fertiliztion (Si et l., 2). In ontrst, Zn phytotoxiity lso inues oxitive stress y generting free rils n retive oxygen speies (ROS). CONCLUSION The present stuy showe the signifint effets of HA n ZnO nnoprtiles on in vitro multiplition n some seonry metolite proution in L. leeourii. It ws foun tht HA n ZnO might e use s eliitor. For this purpose, estlishment of suspension ultures from this plnt in future stuies woul e useful. Thus, our next investigtion will fous on suh omponents to evlute seonry metolite proution. REFERENCES: Ael-Rzzk H. S., n El- Shkwy G. A. (23). Effet of Biofertilizer n humi i pplition on growth, yiel, qulity n storilityof two Grli (Allium stivum L.) ultivr. Asin Journl of Crop Siene, : AmeriA., n Tehrnifr A. (22). Effet of humi i on nutrient uptke n physiologil hrteristi Frgri nnss vr: Cmros. Journl Biology Environmentl Siene, 6: Aminifr M. H., Aroee H., Azizi H., Nemti M., n Jfr Z. E. (22). Effet of humi i on ntioxint tivities n fruit qulity of hot pepper (Cpsium nnuum L.). Journl of Hers, Spies & Meiinl Plnts, 8: Atiyeh R. M., Lee S., n Ewrs C. A. (22). The influene of humi is erive from erthwormproesse orgni wstes on plnt growth. Bioresoure Tehnology, 84: 7 4. Azi P., n Khosh-Khui M. (27). Miropropgtion of Lilium leeourii Bioss.s ffete y plnt growth regultor, surose onentrtion, hrvesting seson n ol tretment. Eletroni Journl of Biotehnology, : 8

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