Effect of Growing Factors on Productivity and Quality of Lemon Catmint, Lemon Balm and Sage under Soilless Greenhouse Production: II.

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1 Mediinl nd Aromti Plnt Siene nd Biotehnology 211 Glol Siene Books Effet of Growing Ftors on Produtivity nd Qulity of Lemon Ctmint, Lemon Blm nd Sge under Soilless Greenhouse Prodution: II. Nitrogen Stress Artur Mnukyn Center of Greenhouses nd Lortories Dürnst, Center of Life Sienes Weihenstephn, Tehnil University of Munih, Dürnst 4, 8535 Freising-Weihenstephn, Germny Corresponding uthor: * mnukyn@wzw.tum.de ABSTRACT Vrious environmentl nd growing ftors n ffet not only the primry, ut lso the seondry metolism in hers. Nitrogen stress onsidered to e one of the min environmentl ftors influening the plnt growth nd qulity. This study investigted the impt of different nitrogen onentrtions in nutrient solution (5, 15 nd 3 mg/l) on herl yield, ontent nd omposition of essentil oils nd polyphenols, s well s on ntioxidtive pity of polyphenol-rih extrts of lemon tmint (Nepet tri L. f. itriodor), lemon lm (Meliss offiinlis L.) nd sge (Slvi offiinlis L.) under soilless greenhouse ultivtion. It ws found tht high nitrogen onentrtion of 3 mg/l ws effetive for herl yield of lemon tmint. By ontrst, low nitrogen onentrtion of 5 mg/l ws positive for herl produtivity of sge, while for lemon lm oth 5 nd 15 mg/l onentrtions provided mximum results. Nitrogen stress hd n impt on essentil oil ontent of lemon lm nd sge, ut not of lemon tmint. In se of lemon tmint high nitrogen onentrtion of 3 mg/l ws fvourle for mximum yield of essentil oil, while for sge medium onentrtion of 15 mg/l ws the est nd for lemon lm there ws no signifint differene etween the tretments. The essentil oil omposition of seleted mediinl nd romti plnts ws minly ffeted y tested nitrogen onentrtions. The influene of nitrogen stress ws lso signifint for ontent nd omposition of polyphenols in studied hers, s well s for ntioxidtive pity of polyphenol-rih extrts y the ABTS system. Keywords: iotive ompounds, hydroponis, Meliss offiinlis, Nepet tri f. itriodor, nitrogen onentrtion, Slvi offiinlis INTRODUCTION Environmentl nd growing ftors n influene the yield nd iohemistry of hers. Besides limti onditions t the growing site, others re edphi ftors, suh s nitrogen stress, whih re of prtiulr importne s nturl tools for improvement or limiting produtivity nd qulity of plnts. Some reports lredy desried the impt of nutrition on the herl yield nd ontent of iotive ompounds of mediinl nd romti plnts (Ruminsk 1978; Frnz 1983; Rtnyk et l. 1998; Brievi et l. 1999; Mrtinetti et l. 26; Aziz et l. 28; Sid-Al Ahl et l. 29; Colling et l. 21; Mtos et l. 21). Though, for suh rops, the yield of seondry plnt sustnes is predominnt, little is known yet on seondry metolism s influened y nitrogen onentrtion in nutrient solution under soilless greenhouse onditions. The im of this study is to investigte the effet of nitrogen stress on herl yield, mount nd omposition of essentil oils (EOs) nd flvonoids, s well s on ntioxidtive pity of flvonoidrih extrts of some vlule mediinl plnts under soilless greenhouse onditions. Hving suh informtion on the effets of different nitrogen onentrtions, the herl yield, quntity nd qulity of vlule phrmeutil sustnes in plnt mteril n e mnged under proteted soilless ultivtion. MATERIALS AND METHODS Plnt mteril nd experimentl design The experiments were onduted t the Center of Greenhouses nd Lortories Dürnst of the Center of Life Sienes Weihenstephn, Tehnil University of Munih. Mediinl nd romti plnts to study were lemon tmint (Nepet tri L. f. itriodor), lemon lm (Meliss offiinlis L.) nd sge (Slvi offiinlis L.) - (ll three speies elong to the Lmiee fmily). Cultivtion ws strted y sowing seeds, derived from hydroponilly grown plnts, in ommon pet ( Florton 3 ) in greenhouse. Seeds were strtified y pplition of 2 g/l KNO 3 solution (Merk) nd 1 C for one week. Priking ws done one month lter into ell trys with ommon pet sustrte ( Florton 2 ). The experiment strted nother month lter with trnsplnting of the seedlings into 3 l (lemon tmint nd sge) nd 5 l (lemon lm) pots. The plnts were ultivted in utomtilly ontrolled greenhouse. For ll speies unomposted wood fire Tores speil (Ts) ws used s sustrte, whih hd ph 5.9 nd ontined.8 g/l wter-solule slts (233 mg/l N, 9 mg/l P 2 O 5 nd 9 mg/l K 2 O). For eh speies 3 e- nd flood tles were used with 2. m 2 surfe re for eh with 9 plnts per m 2 (45 plnts per tretment with 4 repetitions). The plnts were utomtilly fed twie dy for the durtion of 6 minutes. The nutrient solution ws mde up from 7g Flory sis 1 (P-43; K-22; Mg-21; B-.1; Cu-.15; Fe-1; Mn-.2; Mo-.3; Zn-.25 mg/l) in ddition to 5, 15 or 3 mg/l nitrogen (omposed of mmonium nitrte (Merk) nd lium nitrte (Merk)) with ph of nd EC of , nd ms/m, respetively. Plnts were hrvested t week 8 fter trnsplnting. The riterion for lemon tmint hrvest ws the time of flower emergene; the other two speies were hrvested lmost t the sme time hving minimum 8 g/plnt fresh iomss. The fresh mediinl mteril ws dried t 35 C for 5-7 dys. Anlyses EO from fresh herl mteril of seleted mediinl nd romti plnts were extrted y stem distilltion nd pentne (Crl Roth) extrtion for 1 h (SDE). The extrts were dried over sodium Reeived: 2 My, 211. Aepted: 3 June, 211. Originl Reserh Pper

2 Mediinl nd Aromti Plnt Siene nd Biotehnology 5 (2), Glol Siene Books sulfte (Merk) nd onentrted under strem of nitrogen. The mount of essentil oil ws determined grvimetrilly. The omposition of the essentil oil ws nlyzed y GC nd GC-MS. The oils were diluted in etone (Crl Roth) (split 1: 4) with seprtion of the ompounds y GC (Fisons Instruments Meg 536, Itly) on Supelo-Wx pillry olumn (6 m, i.d. =.32 mm,.25 m film thikness) with helium s rrier gs (.8 ml/min) nd temperture progrm: 5 C (3 min), 1 C/min, 12 C (2 min), 2 C/min, 155 C ( min), 8 C/min, 24 C. Identifition of EO min ompounds ws performed with GC-MS system (HP 589 Series II/HP 5971 A, HEWLETT PACKARD) on the sme olumn nd with the sme temperture progrm y eletron impt ioniztion t 7 ev. Mss spetr were evluted y omprison of retention times nd mss spetr (Wiley 199) nd with n own terpenoid mss spetr dtse. Polyphenols were extrted from.4 g dry mteril (powder) with 8% MeOH (Crl Roth) nd seprted y HPLC. Extrted polyphenols were resolved in 5 μl CH 3 CN (Merk) nd 5 μl EtOH (Merk), injetion volume ws 1 μl. The nlytil HPLC ws rried out on dionex system (pump P58, utosmpler Gin 5) using 25 mm 4,6 mm RP-18 olumn (phenomenex Hydro-RP) with gurd olumn. A grdient sequene using (A) wter, etonitrile nd eti id (Merk) (97: 2: 1) nd (B) etonitrile in the following proportions: % B ( min), 5% B (5 min), 95% B (1 min), % B (14 min) t flow rte of 1 ml/min. Detetion nd quntifition were performed using Diode Arry detetor (Dionex UVD 34S) nd hromeleon (Dionex) softwre. Folin-Ciolteu test (Merk) pplied to determinte the totl ontent of polyphenolis in plnt mteril (Singleton et l. 1998). The ABTS-system ws used to evlute the ntioxidtive pity of polyphenol-rih extrts (Arno et l. 1999; Re et l. 1999). The ility to redue the peroxidse (myelogloin/h 2 O 2 )-generted ABTS + [2,2'-zino-is(3-ethylenzthizoline-6-sulfoni id) rdil tion] hs een used to rnk the ntioxidnt tivity of vrious gents inluding dietry flvonoids nd hlones (Chn et l. 23). In this system, myogloin (Sigm) nd H 2 O 2 (Merk) oxidise ABTS (Merk) to the green ABTS + rdil tion. 2,2'-zino-is(3-ethylenzthizoline-6-sulfoni id) rdil tion (ABTS +) is stle hromophore whih sors strongly t 734 nm. This retion n e followed photometrilly. The lower rte of ABTS + formtion indites reduing or Fe-helting properties of the extrt. The nlyses were performed with 3-4 replitions. The sttistil nlyses were performed ording to Dospekhov (1985). RESULTS The produtivity of hers ws minly ffeted y the level of nitrogen in nutrient solution under soilless growing onditions. It ws found ht high nitrogen onentrtion of 3 mg/l ws fvorle for herl yield of lemon tmint, while low nd medium onentrtions (5 nd 15 mg/l) were Tle 1 Influene of nitrogen onentrtion on herl yield. Speies Nitrogen onentrtion, Fresh mediinl Rw mediinl mteril mg/l mteril, g/plnt g/plnt % (dry weight) Lemon tmint LSD 5* Lemon lm LSD 5* Sge LSD 5* * Within the sme speies optiml for high produtivity of lemon lm. In the se of sge omprtively low nitrogen onentrtion of 5 mg/l provided mximum results. In the se of lemon tmint nd sge 5 mg/l nitrogen onentrtion resulted lower perent of dry weight (Tle 1). Different nitrogen onentrtions hve onsiderle influene on EO ontent of lemon lm nd sge, ut not of lemon tmint. The tretments with omprtively high nitrogen vilility in nutrient solution (15 nd 3 mg/l) provided mximum ontent of EO in lemon lm, while for sge 15 mg/l ws optiml (Fig. 1). Clultions for EO yield showed tht high nitrogen stress (3 mg/l) provided mximum yield in the se of lemon tmint, while in lemon lm there were no signifint differenes etween the tretments. In the se of sge, 15 mg/l nitrogen onentrtion provided mximum yield of EO (Fig. 2). EO omposition of lemon tmint depended on tested nitrogen levels (Tle 2). The identified min sustnes of lemon tmint EO were gerniol ( %), gernil ( %), nerol ( %), nerl ( %) nd itronellol ( %). Twenty-two identified ompounds represented pproximtely 97% of the totl oil. Different nitrogen onentrtions hd onsiderle influene on the ontent of numer of ompounds in lemon tmint EO. The inrese of nitrogen onentrtion in nutrient solution from 5 up to 3 mg/l deresed the ontent of some min ompounds, suh s nerl nd gernil, while in se of itronellol nd nerol it ws on the ontrry. The ontent of gerniol ws not essentilly ffeted y nitrogen onentrtion in nutrient solution. It ws lso notle tht the ontent of phytol ws more thn 1 times,22,2,18,16 oil ontent [%],14,12,1,8,6,4,2 5 mg/l N 15 mg/l N 3 mg/l N 5 mg/l N 15 mg/l N 3 mg/l N 5 mg/l N 15 mg/l N 3 mg/l N Lemon tmint Lemon lm Sge Fig. 1 Influene of nitrogen onentrtion on essentil oil ontent of hers. Vlues represent men ± stndrd devition (SD). Sttistis re mde within the sme speies. 127

3 Nitrogen stress nd soilless ulture of hers. Artur Mnukyn,4,35,3 oil yield [ml/plnt],25,2,15,1,5 5 mg/l N 15 mg/l N 3 mg/l N 5 mg/l N 15 mg/l N 3 mg/l N 5 mg/l N 15 mg/l N 3 mg/l N Lemon tmint Lemon lm Sge Fig. 2 Influene of nitrogen onentrtion on essentil oil yield of hers. Vlues represent men ± stndrd devition (SD). Sttistis re mde within the sme speies. Tle 2 Influene of nitrogen onentrtion on lemon tmint essentil oil omposition. Compound* Nitrogen onentrtion, mg/l Content in essentil oil, % is-oimene.12 ** is-3-hexenl trns-oimene is-3-hexenyl ette methyl-5-hepten-2-on trns-3-hexen-1-ol is-3-hexen-1-ol trns-2-hexen-1-ol itronelll linlool ryophyllene itronellyl ette humulene nerl neryl ette gernil gernyl ette itronellol nerol gerniol ionone phytol * Only identified ompounds re presented ** Sttistil nlyses re mde within the sme ompound Tle 3 Influene of nitrogen onentrtion on lemon lm essentil oil omposition. Compound* Nitrogen onentrtion, mg/l Content in essentil oil, % -myrene.6 **.2.8 is-oimene is-3-hexenl trns-oimene methyl-5-hepten-2-on trns-3-hexen-1-ol trns-2-hexen-1-ol oten-3-ol itronelll linlool not identified not identified ryophyllene humulene nerl neryl ette gernil gernyl ette itronellol nerol gerniol is-9-otdeenoi id methyl ester * Only identified nd not identified min sustnes of out or > 1% re presented ** Sttistil nlyses re mde within the sme ompound higher t high nitrogen stress (3 mg/l). EO of lemon lm omposed mostly of gernil ( %), nerl ( %) nd gerniol ( %) under soilless greenhouse onditions (Tle 3). Twenty identified ompounds represented out 97, 97 nd 96% of the totl oil t 5, 15 nd 3 mg/l nitrogen onentrtions, respetively. Exept of three mjor onstitutions, the ontents of ll other identified ompounds were ffeted y nitrogen onentrtions in nutrient solution. Among the twenty-five identified ompounds in sge EO, -thujone ( %), mphor ( %), -thujone ( %) nd 1,8-ineole ( %) were the mjor ones (Tle 4). Identified ompounds represented out 97, 97 nd 98% of the totl oil t 5, 15 nd 3 mg/l nitrogen, respetively. Different nitrogen onentrtions hd onsiderle influene on ontent of ll identified ompounds in sge EO. The inrese of nitrogen onentrtion from 5 up to 15 mg/l deresed the ontent of -thujone, ut further inrese up to 3 mg/l produed mximum mount of this ompound (48.8%). In se of mphor, the inrese of nitrogen onentrtion influened negtively the ontent of this sustne in sge oil. The ontents of -thujone nd 1,8-ineole peked up t 15 mg/l. The influene of tested nitrogen onentrtions on the ontent of polyphenols in studied hers ws lso signifint (Tle 5). The inrese of nitrogen onentrtions in nutrient solution mximized the ontent of flvonoids in ll three speies. At the sme time the hnges in the polyphenoli ontent were more essentil for lemon lm nd the differene etween the minimum nd mximum results ws more thn 1%. HPLC nlysis of polyphenols showed differenes in sensitivity of min polyphenols to nitrogen stress s well. In lemon tmint, three mjor identified polyphenoli ompounds (ffei, rosmrini nd p-oumri ids) enrihed with highest nitrogen onentrtion (3 mg/l) (Fig. 3). In lemon lm, rosmrini id (pek 2) rehed its mximum t 5 mg/l, while ffei id (pek 1) nd melitri id (pek 3) showed no essentil differenes (Fig. 4). In sge, rosmrini id (pek 2) ws mximl t high nitrogen stress tretment, while the high onentrtion of ffei id (pek 1) ws reorded t 15 mg/l. Melitri id (pek 3) 128

4 Mediinl nd Aromti Plnt Siene nd Biotehnology 5 (2), Glol Siene Books Tle 4 Influene of nitrogen onentrtion on sge essentil oil omposition. Compound* Nitrogen onentrtion, mg/l Content in essentil oil, % -pinene 2.46 ** mphene pinene sinene myrene terpinene limonene ,8-ineole is-oimene is-3-hexenl terpinene p-ymole terpinolene trns-3-hexen-1-ol trns-2-hexen-1-ol thujone thujone mphor ornyl ette ryophyllene humulene orneol myrtenol viridiflorol mnool * Only identified ompounds re presented ** Sttistil nlyses re mde within the sme ompound Tle 5 Influene of nitrogen onentrtion on polyphenol ontent of hers. Speies Nitrogen onentrtion, mg/l Glli id equivlents- GAE [mm] Lemon tmint 5.65 ± ± ±.3 LSD 5* Lemon lm 5.83 ± ± ±.5 LSD 5* Sge ± ± ±.5 LSD 5* * Within the sme speies showed no essentil differenes in this ontext nd ws detetle only in very smll mounts (Fig. 5). A higher ntioxidtive pity of polyphenol-rih extrts of lemon lm nd sge ws noted even in 1 fold lower onentrtion (1:2) (Fig. 7, 8) ompred to lemon tmint t 1: 2 (Fig. 6). In se of lemon tmint, the polyphenoli extrts otined from 5 mg/l nitrogen tretment showed little it higher ntioxidtive pity y the ABTS-system (Fig. 6). Polyphenoli extrts of lemon lm nd sge grown under different nitrogen levels showed orreltions etween their ntioxidtive pity nd ontent of rosmrini id. The mximum onentrtion of rosmrini id t 5 mg/l in lemon lm (Fig. 4) nd t 3 mg/l in sge (Fig. 5) provided ordingly high ntioxidtive pity y the ABTS-system. Additionlly, the shpes of the urves differ onsiderly with omprtively longer lg phse for 5 mg/l tretment in lemon lm (Fig. 7) nd for 3 mg/l tretment in sge (Fig. 8). DISCUSSION This study onfirms tht nitrogen stress is n importnt externl ftor tht influenes the produtivity nd qulity of seleted mediinl nd romti plnts under soilless growing onditions. Herl yield, ontent nd omposition of EO were mostly ffeted y different nitrogen onentrtion in nutrient solution. The nlysis showed some diret orreltions etween the nitrogen level in fertilizer nd the mount of some voltile sustnes in hers. In prtiulr, the inrese of nitrogen onentrtion in nutrient solution from 5 up to 3 mg/l inresed the ontent of 6-methyl-5- hepten-2-on (in lemon lm) nd deresed the ontent of trns-oimene, itronellol (in lemon lm) nd mphor, ornyl ette (in sge). The tested nitrogen onentrtions hd signifint effet on ontent nd omposition of polyphenols, s well s on ntioxidtive pity of flvonoidrih extrts y the ABTS-system. In spite of growing interest during reent yers for nturl wys to produe high qulity herl mteril with vlule phrmeutil ompounds without ny geneti mnipultion, the studies on the effets of nitrogen stress on herl yield nd prodution of iotive ompounds under ontrolled soilless onditions re still omprtively seldom. Severl uthors hve pulished dt on the effet on nitrogen onentrtion in nutrient solution nd nitrogen stress on herl yield, ontent nd omposition of iotive sustnes of mediinl nd romti plnts under soil nd soilless onditions. In one study it ws reported tht there ws no remrkle influene of levels, forms nd time of dding nitrogen fertilizer on the totl lkloid ontent of Dtur innoxi Mill. (Ruminsk et l. 1978). In other field experiment, it ws found tht the EO ontent of the peppermint (Menth piperit L.) nd sweet sil (Oimum silium L.) inresed proportionlly with N-supply, ut it hnged ording to the optiml urve in the green dill (Anethum grveolens L.) (Hornok 1983). Nitrogen dressing inresed iomss nd EO yield of peppermint (Menth x piperit L.),16 pek / internl stndrd,14,12,1,8,6,4,2 5 mg/l N 15 mg/l N 3 mg/l N 5 mg/l N 15 mg/l N 3 mg/l N 5 mg/l N 15 mg/l N 3 mg/l N Pek 1 (ffei id) Pek 2 (rosmrini id) Pek 3 (p-oumri id) Fig. 3 Influene of nitrogen onentrtion on quntity of mjor polyphenols in lemon tmint. Vlues represent men ± stndrd devition (SD). Sttistis re mde within the sme ompound. 129

5 Nitrogen stress nd soilless ulture of hers. Artur Mnukyn pek / internl stndrd mg/l N 15 mg/l N 3 mg/l N 5 mg/l N 15 mg/l N 3 mg/l N 5 mg/l N 15 mg/l N 3 mg/l N Pek 1 (ffei id) Pek 2 (rosmrini id) Pek 3 (melitri id) Fig. 4 Influene of nitrogen onentrtion on quntity of mjor polyphenols in lemon lm. Vlues represent men ± stndrd devition (SD). Sttistis re mde within the sme ompound pek / internl stndrd mg/l N 15 mg/l N 3 mg/l N 5 mg/l N 15 mg/l N 3 mg/l N 5 mg/l N 15 mg/l N 3 mg/l N Pek 1 (ffei id) Pek 2 (rosmrini id) Pek 3 (melitri id) Fig. 5 Influene of nitrogen onentrtion on quntity of mjor polyphenols in sge. Vlues represent men ± stndrd devition (SD). Sttistis re mde within the sme ompound. sorption 734 nm 1,9,8,7,6,5,4,3,2,1 Control 5 mg/l N 15 mg/l N 3 mg/l N retion time [min] Fig. 6 Influene of nitrogen onentrtion on ntioxidtive pity of polyphenol-rih extrts of lemon tmint. sorption 734 nm 1,9,8,7,6,5,4,3,2,1 Control 5 mg/l N 15 mg/l N 3 mg/l N retion time [min] Fig. 8 Influene of nitrogen onentrtion on ntioxidtive pity of polyphenol-rih extrts of sge. sorption 734 nm 1,9,8,7,6,5,4,3,2,1 Control 5 mg/l N 15 mg/l N 3 mg/l N retion time [min] Fig. 7 Influene of nitrogen onentrtion on ntioxidtive pity of polyphenol-rih extrts of lemon lm. ultivr "Itlo-Mithm", ut deresed the perentge of leves over iomss (Pigli et l. 1993). Another study found tht N rtes inrese thyme (Thymus vulgris L.) rop, ut differenes in the yield of EO were not remrkle (Brnuskienè et l. 23). Other reserh reported on the effet of N fertiliztion on growth nd EO of Eulyptus torqut nd Eulyptus ngulos. It ws found tht the two levels of N (2 nd 4 kg/fed) gretly inresed the perentge of EO of oth speies. N t 2 kg/fed gve the highest perentge of ineole. Wheres, N t 4 kg/fed, inresed the perentge of itronellol of Eulyptus torqut nd Eulyptus ngulos oils (Mhdi et l. 1987). High levels of N promoted inrese in leves weight, ut less oil ontent with low menthol ontent in Menth rvensis L. (Nilson et l. 21). Other study reported on the effet of different nitrogen onentrtion (1, 15 nd 2 mg/l) in the nutrient solution on the voltile onstituents of leves of Slvi frutios Mill. in NFT. It ws found tht the onstituents of the EO showed qulittive nd quntittive differenes due to the smpling t different periods s well s different onentrtions of nitrogen in the nutrient solutions. The effet of nitrogen onentrtion ws minor with remrkle derese to 2 mg of N/l t the end of the seed 13

6 Mediinl nd Aromti Plnt Siene nd Biotehnology 5 (2), Glol Siene Books formtion stge (Krioti et l. 23). The derese in EO ontent t 2 mg/l grees with the results on Menth rvensis L. snd ulture. It ws shown tht the EO ontent inresed with n inrese in nitrte-n (NO 3 -N) up to 16 mequiv/l, eyond whih it deresed (Singh et l. 1989). Other study found no effet of NO 3 -N level t similr onentrtions (1, 15, nd 2 mg/l) on the EO ontent in leves nd rts of Orignum ditmnus L. grown with NFT (Eonomkis et l. 1999). The herl yield of hydroponilly grown sil (Oimum silium L.) ws improved y inresing the nutrient solution nitrogen onentrtion up to 3 mg/l. EO yield did not show ny signifint hnges relted to tretment vriles nd ws etween.2-.5% (v/w). Nitrogen supply did not hve mrked effet on oil omposition (Smith et l. 1997). In other experiment, the EO ontent of fennel (Foeniulum vulgre Mill.) ws multiplied y inresing N-portion. The highest EO ontent (7.25 g/1 g) ws t 2 kg/h N-portion. The inrese of ws 81.25% ompred to the ontrol (Omidigi et l. 1992). The yield ontriuting hrters, drug nd strw yields, ontent of the EO nd flvonoids of different momile genotypes signifintly ffeted y the sowing sesons nd fvoured y inresed nitrogen level (Lethmo 1992). Another study investigted the produtivity of sprgus (Asprgus offiinlis L.) in eroponis nd wter ulture. It ws found out tht 1 mg/l nitrogen level in nutrient solution (Flory 1) is more eptle for getting mximum yield with high ontent of ville rohydrtes in plnt roots (Mnukyn et l. 25). The effet of N supply on the prodution of hyperiins (hyperiin nd pseudohyperiin) in leves of St. John's wort (Hyperium perfortum L.) ws exmined using plnts grown in snd ulture nd soil. In snd ulture, 56 dys with deresed N levels resulted in inresed prodution of hyperiins in leves. A short-term low N stress in snd ulture lso resulted in inresed prodution of lef hyperiins. Growth in low N- ontining soil resulted in elevted levels of hyperiins; suh prodution ws deresed y supplementtion of the soil with dditionl N. Inresed prodution of hyperiins in leves did not require the N supply to e deresed to levels tht resulted in N defiieny symptoms. Moreover, ltertion in the prodution of lef hyperiins ourring with hnges in N supply did not lter the onentrtion rtio of pseudohyperiin nd hyperiin (Briskin et l. 2). Other reserh studied the fesiility of mptothein (CPT) prodution from Cmptothe umint in hydroponi ulture. High onentrtions of CPT were found in leves of plnts in hydroponi ulture, ut the N onentrtion in the ulture medium did not ffet tissue CPT onentrtion, plnt weight, plnt height, lef numer, lmin dry weight per re, totl stem weight, totl lef weight, or totl lef CPT yield (Li et l. 25). In following study the effet of nitrogen nd phosphorus supply ws investigted on 3-yer-old grden thyme (Thymus vulgris L.) popultion. After the pplition of six different dosges of nitrogen (, 1, 15, 2, 25, nd 3 kg/h) nd phosphorus (, 5, 1, 15, 2, nd 25 kg/h), it ws determined tht N nd P fertiliztion hd signifint effet on her yield nd EO ontent ut did not hnge the thymol ontent (Omidigi et l. 22). The tivities of ntioxidnt enzymes viz. glutthione redutse, GR; superoxide dismutse, SOD; peroxidse, POD; tlse, CAT nd glutthione-s-trnsferse, GST nd lkloid umultion were investigted in lef pirs (pil, middle, sl) nd in roots of Cthrnthus roseus (Mdgsr periwinkle) seedlings under the onditions of different nitrogen soures (2 mm KNO 3 nd 2 mm NH 4 Cl) nd slinity. Chnges in ntioxidnt enzyme tivity used y different nitrogen soures differed in ll lef pirs, s well s in roots of C. roseus. Ammonium-fed plnts showed higher CAT, GR nd GST tivity in lef pirs s well s in roots, while POD nd SOD tivity were higher in nitrtefed plnts. Higher peroxidse tivity onomitnt with the inresed umultion of lkloid ws found in ll lef pirs, s well s in roots of C. roseus of NO 3 fed plnts s ompred to NH 4 + fed ontent (Misr et l. 26). In other experiment with periwinkle, the effet of N fertiliztion on the ontent of nti-ner nd nti-hypertension lkloids of two high lkloid ontent mutnts of periwinkle ws studied, in omprison with their prentl vriety, to determine the possiility of further inresing their lkloid ontents. The three genotypes were evluted t three levels of N (, 1 nd 15 kg/h) fertiliztion in split plot experiment. N fertiliztion signifintly inresed the ontent of lkloids oth in leves nd roots of ll genotypes. Over genotypes, pplition of 15 kg/h of N resulted in n inrese of 42 nd 32% in the ontent of lef nd root lkloids, respetively. However, the inrese ws highest in genotypes with the lowest ontent of lef or root lkloids. The high lkloid mutnts grown t 15 kg h 1 of N fertiliztion exhiited n inrese of 87 nd 56% in the ontent of lef nd root lkloids, respetively, when ompred with their prentl vriety grown without nitrogen fertiliztion (Sreevlli et l. 24). In order to investigte the effet of different levels of nitrogen nd potssium in Johnson solution on EO ontent of peppermint (Menth piperit) in hydroponis, n experiment ws rried out were peppermint plnts grown in snd medi under open-ir soilless ulture ondition nd the system ws g ulture method. The tretments inluded N in five levels (15, 135, 165, 195 nd 225 ppm) nd K in three levels (138, 178 nd 218 ppm). The results showed tht the highest EO prodution ws in N t 195 ppm nd K t 218 ppm onentrtion (Mollfili et l. 21). In nother study, the effets of long-term nitrogen defiieny (N.1 mm for 4 months) on growth, phenoli ontent nd tivity of phenyllnine mmoni lyse (PAL; EC ) were investigted in the leves, infloresenes nd roots of yrrow (Ahille ollin Beker ex Rh.) grown in hydroponis. Nitrogen strvtion deresed plnt growth nd the leves totl nitrogen, mino ids, proteins, hlorophylls nd rotenoids ontents inditing tht the primry metolism ws severely limited y low nitrogen vilility. The mount of totl phenolis nd the ntioxidnt pity were higher in leves nd roots of nitrogenstrved ompred to ontrol plnts. Nitrogen strvtion signifintly inresed the ontents of phenoli ids (5-Offeoylquini id (hlorogeni id) nd 3,5 nd 4,5-di-Offeoylquini ids) nd the PAL tivity in lef nd root tissues (Giorgi et l. 29). The following study imed to exmine the effets of nitrogen fertiliztion on the growth performne, etine nd polyshride onentrtions in different plnt prts of Lyium rrum. L. rrum seedlings were sujeted to orgni (Org 1: 15 g N/pot; Org 2: 3 g N/pot) nd hemil (Chem 1: 2.5 g N/pot; Chem 2: 5 g N/pot) N fertilizer tretments. Results showed tht the yield nd uptke of N in plnts of Org 2 (high orgni fertilizer) tretment were the highest. Although plnts of Chem 2 (high hemil fertilizer) tretment demonstrted to ontin the highest N onentrtion, its yield in iomss ws the lowest. A derese in solule sugr, polyshride nd etine onentrtions ws noted with inresing pplition of hemil N fertilizer. However, n inrese in the orgni N fertilizer fvors the etine synthesis (Chung et l. 21). The next study investigted the intertive effets of light intensity nd ontrolled-relese nitrogen fertilizer (CRNF) supply on growth, gs exhnge, nd hlorophyll (Chl) fluoresene prmeters of two speies of potted Host seedlings (Host lus vr. enst nd Host ventrios). N 4 (4 g of CRNF per pot), N 8 (8 g of CRNF per pot) nd sometimes N 12 (12 g of CRNF per pot) signifintly inresed totl dry weights, net photosyntheti rte (P N ), stomtl ondutne (g s ), trnspirtion rte (E), the mximum quntum yield of PSII photohemistry (F v /F m ), the mximum rtio of quntum yields of photohemil nd onurrent non-photohemil proesses in PSII (F v /F ), tul effiieny of photohemil energy onversion in PSII under light ( PSII ), nd photohemil quenhing oeffiient (qp), ut signifintly deresed internl CO 2 onentrtion (C i ) nd nonphotohemil Chl fluoresene quenhing (NPQ) ompred to ontrol plnts t different growth stges of the two Host speies in two levels of light 131

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