THE SALINITY EFFECT ON MORPHOLOGY AND PIGMENTS CONTENT IN THREE PAU- LOWNIA CLONES GROWN EX VITRO

5 Bulgrin Journl of Agriulturl Siene, 9 (), 5 56 Agriulturl Ademy THE SALINITY EFFECT ON MORPHOLOGY AND PIGMENTS CONTENT IN THREE PAU- LOWNIA CLONES GROWN EX VITRO K. MILADINOVA, K. IVANOVA, T. GEORGIEVA, M. GENEVA nd Y. MARKOVSKA * Biotree, 8 Iliensko shoes str., BG Sofi, Bulgri Sofi University, Fulty of Biology, BG 64 Sofi, Bulgri Bulgrin Ademy of Sienes, Institute of Plnt Physiology nd Genetis, BG Sofi, Bulgri Astrt MILADINOVA, K., K. IVANOVA, T. GEORGIEVA, M. GENEVA nd Y. MARKOVSKA,. The slinity effet on morphology nd pigments ontent in three Pulowni lones grown ex vitro. Bulg. J. Agri. Si., Supplement, 9: 5 56 We evluted the effet of slt stress on growth prmeters, hlorophyll, hlorophyll nd rotenoids ontent in Pulowni lones (P. elongt x fortunei x elongt T, P. elongt x elongt T4, P. elongt x kwkrnii EK) grew s hydroponi ulture t three levels of slinity, 5 mmol.l, mmol.l, mmol.l sodium hloride (NCl) solution. The root nd stem length, s well s lef numer nd totl lef re of T lone were redued insignifintly in omprison with these of T4 nd EK lones during NCl tretment. Control of T lone ws hrterized with pproximtely twie higher totl dry mss per plnt thn EK nd T4 lones. The root dry mss to shoot dry mss rtios of three lones hnged in different mnner with inresing slinity levels nd ws highest in EK lone t mmol.l NCl, followed y T4 nd T. The totl lef re showed the pility of plnt in forming of photosyntheti surfe nd ws redued more in T4 nd EK lones under slt stress. Our results suggest tht T lone ws more tolernt to slt stress thn EK nd T4 lones. Key words: hydroponi, morphology, Pulowni, pigments, slinity Arevitions: Chlorophyll hl ; hlorophyll hl, rotenoids r Introdution The environmentl degrdtion, minly soil pollution nd erosion use ioti nd ioti stress in plnts nd limited their growth nd development (Sho et l., 5). Among ioti stresses, drought nd slinity re mjor stresses tht hrm rop losses worldwide (Vinour et l., 5). High slinity uses severe dmge to plnts, inluding growth inhiition, impired metolism, nerosis, nd loss of prodution nd qulity (Sivritepe et l., 999). The reserh to improve slt tolerne in plnts is minly foused on iohemil nd physiologil spets nd the genes responsile for slt tolerne in some speies hve een identified (Shleiff, 8). Stress might our s omplex mehnism of severl interting environmentl ftors tht use vritions in plnt phenotypes, s plnts respond to omplex growth onditions (Sho et l., 7). The plnt ell nd tissue ulture methods ould e useful in studying the slinity tolerne mehnisms in plnts nd their effets on rop prodution when re not evidently known (Akini et l., 4). Pulowni is ntive from Chin. Pulowni tomentos hs een introdued into USA nd Europe s n ornmentl plnt nd is still widely used for this purpose. Trees introdued in Bulgri reh m verge height nd.4 m verge dimeter during 7 yers (Klmukov, 995). This high-yielding tree n e used for the prodution of energy, pper pulp nd wooden uilding mterils. The genetilly tissue ultured Pulowni seedlings produed y The World Pulowni Institute (WPI) llow prodution of iofuels fter introduing of ultivrs without detrimentl impts on food supply or the environment. Reserh on in vitro propgtion of P. elongt nd P. fortunei hs een reported (Bergmnn et *E-mil: pmmster@yhoo.om

The Slinity Effet on Morphology nd Pigments Content in Three Pulowni Clones Grown Ex Vitro 5 l., 997). Applition of this tehnology for miropropgtion of tree speies offers rpid mens of produing lonl plnting stok for fforesttion, woody iomss prodution nd it is effetive wy to mintin the geneti gin (Prk et l., 99). Plnts used in the urrent pper re propgted nd rooted ording tehnology registered y BioTree Ltd., Bulgri. This lortory is lrgest produer nd supplier of genetilly superior Pulowni tissue ultures in vitro plnts. In this reserh, the effet of NCl on the growth nd plstid pigments ontent in leves of three Pulowni lones (P. elongt x fortunei x elongt T, P. elongt x elongt T4, P. elongt x kwkrnii EK), grown s hydroponi ulture fter trnsplnttion the plnts were ompred to provide fundmentl se for vegettion restortion in slinized soils. Mterils nd Methods Plnt mteril. In vivo explnts from the speies P. elongt nd their hyrids with P. fortunei nd P. kwkrnii were used for developing of in vitro multiplition protool. For indution of shoots, explnts were ultured on Murshige nd Skoog (MS) nutrient medium inluded.5% (w/v) surose,.8% (w/v) gr nd vitmins. For shoots multiplition MS medium supplemented with 4.49 μm 6-enzylminopurine (ВАР) nd.57 μm indolileti id (IAA) ws used. After multiplition the shoots were trnsferred to rooting medium sed on hlf strength sl slts MS medium, % surose, 6 % gr nd vitmins supplemented with 4.9 μm indole--utyri id (IBA) nd.75 μm IAA. The ph of ll medi ws djusted to 5.7 using. N HCl nd. N NOH efore utolving. All ultures were inuted under ontrolled onditions 6 h photoperiod, light intensity of 5 μmol m s nd 4/8±ºC dy/night temperture. After three weeks of rooting, the shoots were rinsed with.5 ml/l Proplnt solution. Hydroponi experiment. The experiments were set s four tretments inluding ontrol, eh tretment with replitions. The uniform plnts were seleted nd trnsplnted to polyethylene vessels ontining. l of /4 Hellriegel solution (Hellriegel, 898) with n ddition of A-Z miroelements fter Hoglnd (ph 5.9) in growth hmer with 6-h photoperiod (PAR μmol m s on the upper lef surfe, 5/±ºC dy/night temperture, reltive humidity 6/7%). Eh vessel ontined two plnts whih represented one replition. After dys of ultivtion the plnts were trnsferred to / Hellriegel solution with the ddition of A-Z miroelements (ph 5.9). The slt tretment ws pplied on the 48 th dy fter trnsplnting of plnts when the plnts hd dpted to the onditions of / Hellriegel nutrient solution nd (ontrol), 5,, nd mmol.l NCl ws dded. The solutions were erted every dy nd were hnged every d to prevent depletion of nutrients nd NCl. Plnts were hrvested fter d of tretment. Toxiity symptoms (e.g. disolortion, pigmenttion, yellowing nd stunting) were ssessed y eye throughout the experiment. Mesurement of plnt growth. At the end of the experiment the plnt smples were olleted, wshed with tp wter nd rinsed with distilled wter efore eing seprted into lef, petiole, stem nd root nd fresh mss of eh plnt smple were mesured grvimetrilly. Dry mss of shoots nd roots were determined fter oven drying (6 o C) for dys until onstnt weight ws otined. Lef re ws lulted using softwre progrm SigmSn Pro 5. Pigment determintion. Smples were olleted from the fully developed leves of three plnts grown in vrying onentrtions of NCl. Smples of the ontrol plnts of three Pulowni lones were lso olleted t these times. For pigment extrtion, mg of fresh mteril from the middle prt of fully developed leves of eh plnt ws extrted with 5 ml 8% etone nd filtered through glss filter G4. The pigment ontent ws determined spetrophotometrilly (Lihtenthler, 987). Sttistil nlysis. All vlues reported in this work were men of t lest three independent experiments. The men vlues ±SD nd ext numer of experiments re given in the tles. The signifine of differenes etween ontrol nd eh tretment ws nlyzed y Fisher LSD test (P.5) fter performing ANOVA multiftor nlysis. Results nd Disussion Effet of slt stress on plnts growth. The seedlings growth is normlly limited y inresing onentrtion of NCl (Sreenivsulu et l., ). In our study, with inresing slinity levels, the root nd stem length, lef numer nd totl lef re in the three plnts were redued (Tle ). The root nd stem length of EK lone ws redued more thn tht of T4 nd T lone. The vlues for stem length mesured t mmol.l NCl for T lone ws higher thn ontrol. The lef numer of EK lone rose, ut the totl lef re delined shrply with inresing slinity levels. Mximum redution of the totl lef re ws oserved t mmol.l NCl y 55% for T4, 4% for EK nd 7% for T, respetively, ompred to the ontrol. The totl lef re showed the pility of plnt in forming of photosyntheti surfe. The totl DW per plnt ws inresed for

54 K. Mildinov, K. Ivnov, T. Georgiev, M. Genev nd Y. Mrkovsk T4 y 8% nd 47% under low slinity levels (5 mmol.l nd mmol.l NCl), ut ws deresed y 7% t mmol.l NCl. The totl DW per plnt were redued strongly for T nd EK lones with inresing slinity levels. Under highest slinity level ( mmol.l NCl) the vlues deresed out 45% for EK nd out 66% for T lone, respetively, ompred to the ontrol. The roots to shoot dry mss rtios were inresed in different mnner with inresing slinity levels. The highest vlues ws oserved t mmol.l NCl for T4 lone (~6%), followed y EK lone (~%) nd T lone (~%). Effet of slt stress on pigment ontent. Higher slinity redued the hlorophyll ontent in the leves of three lones. High NCl onentrtions resulted in signifint derese the hlorophyll, nmely in leves of T4 nd T lones (Figure A nd B). Both pigments dereses were reported in spinh (Spini oleree) exposed to slinity stress (Di Mrtino et l., ). The rotenoid ontent signifintly deresed in the leves of EK nd T lones grown in the highest onentrtions of NCl, ut inresed in the leves of T4 plnts (Figure A nd C). In sweet herry rootstok Gisel 5 (Prunus ersus x nesens) derese in hlorophyll ontent ws oserved s result of NCl exposure (, 5, nd 5 mm) (Erturk et l., 7) nd in ens green nd yellow pigments deresed with inresing slinity ( mm NCl) (Stoev et l., 8). During the plnt growth nd development, the form nd funtions of orgns undergo onsiderle hnge, nd the ility of plnts to ret to slinity stress depends on the genes tht re expressed t the stge of development during whih the stress is imposed. The mehnism for slinity tolerne eomes even more omplited when response to slinity stress of plnt speies vries with the growth stge of its life yle. In order to develop prtile strtegies for seleting slt tolernt lones/genotypes of potentil rops, there is need to gin detiled informtion on whether hnges in physiologil nd iohemil prmeters due to slt stress re ttriutle to detrimentl effets of slt stress, or re omponents of the dpttion mehnism (Ashrf, 4). Our results showed tht Pulowni elongt x fortunei x elongt T lone is more tolernt to slt stress t the slinity onditions tested thn P. elongt x elongt T4 nd P. elongt x kwkrnii EK lones. Further investigtions re needed to determine the influene of NCl on Pulowni lones, in order to eluidte the mehnisms utilized y these lones. In other studies (Ayl Astorg et l., 9; Ayl Astorg et l., ) with Pulowni imperilis nd Pulowni fortunei speies grown in vitro, it is reported they survived t 6 mm NCl onentrtion dded to ulture medium, nd higher onentrtions indued nerosis nd deth. Otined results re not ompred euse Pulowni lones re imposed to inresing slinity levels in hydroponi experiment wheres the other plnts re in vitro grown t different NCl onentrtions. Tle. Men vlues ± SD (n = 5-6) of the root nd stem length, lef numer, totl lef re, totl dry weight nd root/shoot rtios of Pulowni lones (T4, T, EK), grown s hydroponi ulture in response to slt stress Tretments Root length [m] Stem length [m] Lef numer Totl lef re [m plnt ] Totl DW [g plnt ] root DW / shoot DW T4 Control. ±. 8. ±.4. ±.9 4.7 ±.8.54 ±.8.68 5 mm.l NCl 6. ±.8 6.6 ±.8. ±.5 94.8 ± 8.4.658 ±.78.5 mm.l NCl 4.5 ± 4.9 7. ±.. ±.7 79. ± 46..76 ±.6.4 mm.l NCl 7.8 ±.9d 5.6 ±.d 9. ±. 88.6 ± 9.8d.4 ±.58d.69 T Control 9. ± 5.5 8.8 ±.. ±. 4. ± 6.4.4 ±.4.6 5 mm.l NCl 7.5 ±.8 8.5 ±.. ±. 45.9 ± 8.8.86 ±.6.75 mm.l NCl 5. ±.9 7.6 ±.9. ±.5 9.6 ±..68 ±.4.9 mm.l NCl 6. ± 4.d 9.6 ±.7 8. ±.5 5.4 ± 6.5.458 ±.8d.68 EK Control 7.8 ±. 7.9 ±. 8. ±.7 8. ± 68.8.66 ±.5. 5 mm.l NCl 4.4 ±.9 7.9 ±.. ±. 7. ±..68 ±.7. mm.l NCl.7 ±. 6.6 ±.5 9. ±. 49.9 ± 7.9.454 ±.5. mm.l NCl.6 ±.5d 6.5 ±. 9. ±. 98.6 ±.7.4 ±.d.7 Note: Vlues with the sme letter re not signifintly different when mens re seprted y Fisher`s LSD test (P <.5).

The Slinity Effet on Morphology nd Pigments Content in Three Pulowni Clones Grown Ex Vitro 55 Pigments [mg.g - FW] A B Pigments [mg.g - FW] C Pigments [mg.g - FW].5.5.5.5.5.5.5 Control 5 NCl [mm.l - ] Chl [mg.g- FW] Chl [mg.g- FW] Crotenoids [mg.g- FW].5.5 d Control 5 NCl [mm.l - ] Chl [mg.g - FW] Chl [mg.g - FW] Crotenoids [mg.g - FW] d d d Control 5 NCl [mm.l - ] Chl [mg.g - FW] Chl [mg.g - FW] Crotenoids [mg.g - FW] Fig.. Chnges in hlorophyll, hlorophyll nd rotenoid ontents in the leves of Pulowni lones, grown s hydroponi ulture in response to slt stress: A/ P. elongt x fortunei x elongt - T; B/ P. elongt x elongt - T4; C/ P. elongt x kwkrnii EK. In generl, there is muh work to do to improve rops euse the prolem is inresing with slinity, drought, nd other worldwide ftors tht ffet rops. Conlusion We exmined the physiologil inditors in order to develop prtile strtegie for seleting slt tolernt lones of Pulowni (P. elongt x fortunei x elongt T, P. elongt x elongt T4, P. elongt x kwkrnii EK), whih were produed y BioTree Ltd., Bulgri. Control of T lone ws hrterized with pproximtely twie higher totl dry mss per plnt thn EK nd T4 lones.the root nd stem length, s well s lef numer nd totl lef re of T lone were redued insignifintly in omprison with these of T4 nd EK lones during tretment with inresing onentrtions of NCl from 5 mmol.l to mmol.l. The hlorophyll nd rotenoid ontents in the leves of three lones remin reltively invrile. Our results suggest tht T lone ws more tolernt to slt stress thn EK nd T4 lones. Referenes Akini, I. nd M. Simsek, 4. Ameliortive effets of potssium nd dmium on the slinity stress in emryo ulture of uumer (Cuumis stivus L.), J. Biol. Si., 4: 6 65. Ashrf, M., 4. Some importnt physiologil seletion riteri for slt tolerne in plnts. Flor, 99: 6-76. Ayl Astorg, G. I., L. Alrz-Melendez, P. F. Ayl nd A. E. Cstellnos, 9. Effet of sodium hloride stress in Pulowni imperilis (Sieold & Zrini) nd Pulowni fortunei (Seemnn & Hemsley) plnt growing in vitro. Agrohimi, 5: 66 78. Ayl Astorg, G.I. nd L. Alrz-Melendez,. Slinity effets on protein ontent, lipid peroxidtion, pigments, nd prline in Pulowni imperilis (Sieold & Zrini) nd Pulowni fortunei (Seemnn & Hemsley) plnt grown in vitro. Eletr. J. Biotehn., : 5. http://dx.doi.org/ 5/vol -issue5-fulltext-. Bergmnn, B. A. nd H.-K. Moon, 997. In vitro dventitious shoot prodution in Pulowni. Plnt Cell Rep., 6: 5 9. Di Mrtino, C., D. Sestino, R. Pizzuto, F. Loreto nd A. Fuggi,. Free mino id nd glyine etine in lef osmoregultion of spinh responding to inresing slt stress. New Phytol., 58: 455 46. Erturk, U., N. Sivritepe, C. Yerliky, M. Bor, F. Ozdemir nd I. Turkn, 7. Response of the herry rootstok to slinity in vitro. Biol. Plnt., 5: 597 6. Hellriegel, H., 898. Die methode der sndkulture. Ar. Lndwirtsh. Ges., 4: 5 8. Klmukov, K., 995. Influene of primry density upon the pro-

56 K. Mildinov, K. Ivnov, T. Georgiev, M. Genev nd Y. Mrkovsk ess of growth nd development of some fst growing tree speies used for iomss. In: Ed. V.N. Brezin 7 Yers Forestry edution in Bulgri. Juilee Sienes Session, 7 9. VI. 995, Sofi, v. I, pp. 9 7. Lihtenthler, N. K., 987. Chlorophylls nd rotenoids: pigments of photosyntheti iomemrnes. In: Methods in Enzymol., 48: 5-8. Prk, Y. S. nd J. M. Bong, 99. Conifer miropropgtion: its funtion in tree improvement progrms. In: Ahuj, M.R. (ed.) Miropropgtion of woody plnts. Ed. M.G. Ahuj, Kluwer Ademi, Dordreht, pp. 457 47. Shleiff, U., 8. Anlysis of wter supply of plnts under sline soil onditions nd onlusions for reserh on rop slt tolerne. J. Ag. Crop Si., 94: 8. Sho, H.-B. nd L.-Y. Chu, 5. Plnt moleulr iology in Chin: opportunities nd hllenges. Plnt Mole. Biol. Re.p., : 45 58. Sho, H.-B., Q.-J. Guo, L.-Y. Chu, X.-N. Zho, Z.-L. Su, Y.- C. Hu nd J.-F. Cheng, 7. Understnding moleulr mehnism of higher plnt plstiity under ioti stress. Colloids nd surfes, 54: 7 45. Sivritepe, N. nd A. Eris, 999. Determintion of slt tolerne in some grpevine ultivrs (Vitis vinifer L.) under in vitro onditions. Turk J. Biol., : 47 485. Sreenivsulu, N., B. Grimm nd U. Wous,. Differentil response of ntioxidnt ompounds to slinity stress in slt tolernt nd slt sensitive seedlings of foxtil millet. Physiol. Plnt., 9: 45 44. Stoev, N., nd M. Kymknov, 8. Effet of slt stress on the growth nd photosynthesis rte of en plnts (Phseolus vulgris L.). J. Centrl Eur. Agr., 9: 85 9. Vinour, B. nd A. Altmn, 5. Reent dvnes in engineering plnt tolerne to ioti stress: hievements nd limittions. Curr. Opinion Biotehn., 6:.