ISSN Vol. 8, No. 2, July-December, Appl. Hort. Journal of THE SOCIETY FOR ADVANCEMENT OF HORTICULTURE

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1 ISSN Vol 8, No 2, July-Decemer, 26 Appl Hort Journl of THE SOCIETY FOR ADVANCEMENT OF HORTICULTURE

2 JOURNAL OF APPLIED HORTICULTURE Vol 8, No 2, July-Decemer, 26 CONTENTS Trnsgenic tomto (Lycopersicon esculentum) overexpressing capx exhiits enhnced tolernce to UV-B nd het stress -Yueju Wng, Michel Wisniewski, Richrd Meiln, Minggng Cui nd Leslie Fuchigmi (USA) 87 The effect of nti-hil nets on fruit protection, rdition, temperture, qulity nd profitility of Mondil Gl pples -Ignsi Iglesis nd Simó Alegre (Spin) 91 Induction of phenolic compounds iosynthesis with light irrdition in the flesh of red nd yellow pples -D Bkhshi nd O Arkw (Jpn) 11 * vlues to follow lycopene concentrtion during ripening nd storge of tomto (cv Cruso) -Axelle Schuwers, Ad MCN Roch nd Alcin MMB Moris (Portugl) 15 Effects of usive tempertures on the posthrvest qulity of lettuce leves: scoric cid loss nd microil growth -Mrí del R Moreir, Alejndr G Ponce, Crlos E del Vlle, R Ansoren nd SI Rour (Argentin) 19 Shelf-life nd qulity of pple fruits in response to posthrvest ppliction of UV-C rdition -Syvsh Hemmty, Noorollh Mollemi nd Lotfli Nseri (Irn) 114 Effects of sugrs nd minooxycetic cid on the longevity of pollinted Dendroium (Heng Beuty) flowers -S Chndrn, CL Toh, R Zulin, YK Yip, H Nir nd AN Boyce (Mlysi) 117 Agronomic ttriutes of sffron yield t groecosystems scle in Irn -A Koocheki, MA Behdni nd M Nssiri (Irn) 121 Effect of ud scle removl nd AOA on ud rek nd ACC content of Musct Biley A grpevines -Ali R El-Shereif, Fuso Mizutni, Justus M Onguso nd ABM Shrif Hossin (Jpn) 125 Effect of exogenous ppliction of nti-stress sustnces nd elementl sulphur on growth nd stress tolernce of tissue culture derived plntlets of dte plm (Phoenix dctylifer L) cv Khls during cclimtiztion -Mohmed A Awd, A A Soud nd S M El-Konissi (UAE) 129 Impct of Anthurium spp genotype on cllus induction derived from lef explnts, nd shoot nd root regenertion cpcity from cllus -Duong Tn Nhut, Nguyen Duy, Nguyen Nhu H Vy, Chu Diem Khue, Dinh Vn Khiem nd Do Nng Vinh (Vietnm) 135 A one step in vitro cloning procedure for Red Gloe grpe: The influence of sl medi nd plnt growth regultors -MS Brreto, A Nookrju, NVM Hrini nd DC Agrwl (Indi) 138 Anthocynin ccumultion in the hypocotyl nd petl of Red Agti (Sesni grndiflor), n ornmentl legume -Kitti Bodhipdm, Sompoch Noichind, Ssikn Udomrti, Gorvis Nthlng, Boonyuen Kijwijn nd Dvid WM Leung (Thilnd) 143 Effect of different stlk lengths nd certin chemicl sustnces on vse life of gerer (Gerer jmesonii Hook) cv Svn Red -MA Memn nd KM Dhi (Indi) 147 Micropropgtion of Prthenocissus quinquefoli (L) from seedling explnts -Yun Li, Dling Feng, Zhenyun Sun nd Chen Niu (Chin) 151 Effects of orgnic mnure on okr (Aelmoschus esculentus (L) Moench) production -J Ofosu-Anim, ET Bly, nd ME Frempong (Ghn) 155 Economic rtionle of commercil orgnic fertilizer technology in vegetle production in Osun Stte of Nigeri -T Alimi, OC Ajewole, OO Oluode-Awosol nd EO Idowu (Nigeri) 159 Technicl nd economic spects of utilizing firous wool composts in horticulture -KC Ds, PA Annis, E WTollner nd S Dudk (USA) 165 Effect of pollen source on productivity, mturity nd fruit qulity of 'Hyyni' dte plm -J Muhtse nd H Ghnim (Jordn) 17

3 Forthcoming Ppers Ectopic expression of Mn-SOD in Lycopersicon esculentum leds to enhnced tolernce to slt nd oxidtive stress-yueju Wng, Richrd Meiln, Michel Wisniewski, Sndr L Urtsu, Minggng Cui, Ahy Dndekr nd Leslie Fuchigmi (USA) Posthrvest ppliction of exogenous sucrose in roccoli: Some physiologicl responses nd chnges in the ctivities of glutmine synthetse- Dewoowoogen P Bclyon, Toshiyuki Mtsui, Hruo Suzuki nd Yusuke Kosugi (Jpn) Developmentl influence of in vitro light qulity nd cron dioxide on photochemicl efficiency of PS II of strwerry leves (Frgri x nnss)- John H Mirnd nd Richrd Willims (Austrli) Effect of forcing t different times on ud urst, flowering, nd fruit development of low-chill pech cultivr Premier -Sutsinee Mneethon, Kenji Beppu, Noko Kozi, Ryosuke Mochiok nd Ikuo Ktok (Jpn) Chrcteriztion of environmentl stress-regulted nthocynin production nd growth of crnerry cllus -Yu Zhou nd Bl Rm Singh (USA) Fctors ffecting fruit ortion in gynoecious cucumer cultivr-a Tzuke, P Boonkorkew, S Hikosk nd N Sugiym (Jpn) Supplement of 2,4-D nd NAA mitigtes utotoxicity of strwerry in hydroponics- H Kitzw, T Aso, T Bn, Y Hshimoto, T Hosoki (Jpn) Sucrose synthse nd cid invertse ctivities in reltion to the florl structures ortion in pepper (Cpsicum nnuum L) grown under low night temperture-néji Trchoun, Slh Rezgui nd Adelziz Mougou (Tunisi) Tolernce of lilyturf (Liriope muscri) nd four perennil ornmentl grsses to preemergent hericides- Jmes T Cole nd Jnet C Cole (USA) Effect of onion cultivrs on storge losses under hot conditions-nser Alemzdehnsri Ansri (Irn) N-NO 3 from cellulr extrct s indictor of nutritionl sttus of cntloupe muskmelon in fertigtion-mrí Remedios Cigles Rivero nd Octvio Pérez Zmor(Mexico) Thinning response of Aé Fetel per to lime sulphur-pi Grriz, HL Alvrez, GM Colvit nd MS Gjdos (Argentin) Trnsgenic crntions with reduced ethylene production trnsformed with ACC oxidse cdna in sense orienttion-tkyuki Inokum, Tomoyuki Kinouchi nd Shigeru Stoh (Jpn) Growth, fruit yield nd qulity of Golden Delicious pple trees under fixed prtil root zone drying-g Tlluto, V Frin nd R Lo Binco (Itly) Soil, plnt nd cnopy resistnce to wter flow in ell pepper (Cpsicum nnuum L) s ffected y fertigtion regimes- SO Agele (Nigeri) Chlorine disinfection: Effects on hydroponics lettuce- Zdenk Premuzic, Hemilse E Plmucci, Jun Tmorene, Mrtin Nkm (Argentin) Identity nd efficiency of pollintors of wtermelon (Citrullus lntus (Thun) Mnsf) t Ytt, Keny-GN Njoroge, B Gemmill, R Bussmnn, LENewton nd VW Ngumi (Keny) Tulip cultivr response to Flurprimidol preplnt ul soks-brin A Krug 1, Brin E Whipker, Ingrm McCll, nd John M Dole (USA) Low-temperture threshold nd Growth Degree Dy (GDD) for two pistchio cultivrs-yousef Gholipour (Irn)

4 Journl of Applied Horticulture, 8(2): 87-9, July-Decemer, 26 Trnsgenic tomto (Lycopersicon esculentum) overexpressing capx exhiits enhnced tolernce to UV-B nd het stress Appl Journl Yueju Wng A *, Michel Wisniewski B *, Richrd Meiln C, Minggng Cui A nd Leslie Fuchigmi A A Deprtment of Horticulture, Oregon Stte University, Corvllis, OR, , USA, B USDA-ARS, 45 Wiltshire Rod, Kerneysville, WV, , USA, CForestry & Nturl Resource Deprtment, Purdue University, West Lfyette, IN, , USA, *Current ddress: Children s Blood Foundtion Lortories, Weill Medicl College of Cornell University, New York, NY 121, USA *E-mil: MichelWisniewski@rsusdgov Astrct Rective oxygen species (ROS), such s hydrogen peroxide, superoxide nd hydroxyl rdicls, re y-products of iologicl redox rections ROS cn denture enzymes nd dmge importnt cellulr components Plnts develop ntioxidnt enzymes, such s superoxide dismutse (SOD) nd scorte peroxidse (APX) to scvenge ROS nd detoxify them The effect of incresed cytosolic scorte peroxidse (capx) on het nd UV-B stress tolernce ws studied using trnsformed tomto (Lycopersicon esculentum cv Zhongshu No 5) plnts This reserch demonstrtes, in either lortory or field tests, the potentil to enhnce tolernce to het, UV-B, nd sunscld stress y gene trnsfer Overexpression of capx in trnsgenic tomto enhnced resistnce to het (4 C) nd UV-B stress compred to wild-type plnts When lef disks were plced t 4 C for 13 hours, the electrolyte lekge of disks from wild-type were 93%, wheres two tested trnsgenic lines (A9, A16) exhiited 24% nd 52% lekge respectively When fruits of wild-type nd trnsgenic plnts were exposed to UV-B (25mW cm -2 ) for five dys, the extent of rowning ws 95%, nd 33%, nd 37%, respectively In field tests, the detched fruits from field-grown trnsgenic plnts showed more resistnce to exposure to direct sunlight thn fruits from wild-type plnts APX ctivity in leves of capx trnsgenic plnts ws severl folds higher thn in leves of wild-type plnts when exposed to het, UV-B, nd drought stresses Key words: Lycopersicon esculentum, overexpression, scorte peroxidse (APX), het, UV-B, oxidtive stress, sunscld Introduction Rective oxygen species (ROS) including superoxide (O 2- ), hydrogen peroxide (H 2 O 2 ), nd hydroxyl rdicl (OH - ), cn hve deleterious effects such s lipid peroxidtion, DNA muttion nd protein denturtion in cells (Bowler et l, 1992; Scndlios, 1993; Apel nd Hirt, 24) ROS my e produced in response to iotic nd iotic stresses, nd re ssocited with numer of physiologicl disorders in plnts (Allen, 1995) These stresses, to vrying degrees, ffect crop growth, resulting in huge losses of plnts nd plnt products nnully throughout the world (Scndlios, 1993) Plnts hve evolved efficient non-enzymtic nd enzymtic systems to cope with ROS Nonenzymtic systems, such s scorte, glutthione, α-tocopherol, nd crotenoids, cn rect directly with ROS (Allen, 1995) Enzymtic systems like SOD scvenge the superoxide nion APX removes H 2 O 2 Glutthione reductse (GR) lso cn remove H 2 O 2 vi the scorte-glutthione cycle (Noctor nd Foyer, 1998; Scndlios, 1993) Scvenging of ROS is importnt for mintennce of norml plnt growth (Allen, 1995) Het nd ultrviolet B (UV-B, nm) stimulte the production nd ccumultion of toxic ROS, which results in lipid peroxidtion nd memrne injury (A-H-Mckerness, 2; Dvidson et l, 1996; He et l, 22; Jing nd Hung, 21; Sirm et l, 2) An interction of high tempertures nd light intensity (UV-B) cn induce sunscld in fruits of mny horticulture crops (Rinowitch et l, 1983; Renquist et l, 1989) Sunscld of fruits is mnifested s either tissue rowning or desicction (Renquist et l, 1989) Tolernce of tomto fruits to sunscld dmge y controlled het tretment ws ccompnied y n increse in superoxide dismutse (SOD) ctivity (Rinowitch et l, 1982; Rinowitch nd Skln, 198) Rinowitch nd Skln (198) reported tht SOD ctivity levels were high in immture green fruits nd declined to minimum in the mture-green nd reker (erly ripening) stges, which re known to e most susceptile to sunscld During tomto ripening, oxidtive processes such s lipid peroxidtion, protein oxidtion, nd hydrogen peroxide content, increse t the reker stge In contrst, ntioxidnt enzyme ctivities of SOD nd APX decresed t the reker stge of ripening (Jimenez et l, 22) Tolernce to het nd UV-B correltes with n incresed cpcity of the plnts to scvenge or detoxify ctivted oxygen species (Chitny et l, 22; Dvidson et l, 1996; Mzz et l, 1999; Sirm et l, 2), suggesting tht incresed ntioxidnt enzyme ctivity might protect plnt tissues ginst sunscld Wisniewski et l (22) reported tht trnsgenic pple plnts tht overexpressed capx hd improved resistnce to het stress Lef disks from wild-type (WT) pple plnts exhiited 1% electrolyte lekge, wheres trnsgenic lines exhiited 4% to 75% lekge Trnsgenic pple plnts lso showed tolernce to UV-B nd freezing stress Chen nd Pn (1998) reported tht

5 88 Trnsgenic tomto overexpressing capx exhiits enhnced tolernce to UV-B nd het stress overexpression of Cu/Zn-SOD cn enhnce the tolernce to het stress or UV-B rdition in Aridopsis thlin The overexpression of enzymes involved in scvenging ROS in plnts y gene trnsfer technology my e le to increse tolernce of plnts to oxidtive stresses nd improve plnt performnce under these conditions (Sen Gupt et l, 1993, ; Kuo et l, 1995; McKersie et l, 1999; Wng et l, 1999; Wng et l, 25; Yoshimur et l, 2) The ojective of this study ws to determine the tolernce of tomto plnts nd fruits to het nd UV-B stresses when capx gene is introduced into the plnts Mterils nd methods Plnt mteril nd growth conditions: Tomto (Lycopersicon esculentum cv Zhongshu No 5) ws previously trnsformed y Agrocterium tumefciens with inry vector contining pe capx cdna (Mittler nd Zilinsks, 1991) Independentlytrnsformed capx lines of T 2 genertion nd wild-type (WT) plnts were used in the experiments Cuttings from regenerted trnsgenic T 2 plnts were rooted in rooting medium complimented with ntiiotic (MS + 5 mg L -1 knmycin + 2 mg L -1 NAA + 4 mg L -1 cefotxime + 7 g L -1 gr) nd trnsplnted in pet moss soil (Lkelnd Pet Moss, Inc, Edmonton, Alert, Cnd) They were grown in greenhouse with nturl lighting supplemented with sodium vpor lmps (1W, Philips, Inc, Eindhoven, Netherlnds) to provide 16-h photoperiod t pproximtely 23/21 C (±2 C, dy/night temperture) Het, UV-B, sunscld, nd drought stress tests Het stress tests: Lef discs from WT nd T 2 plnts were het stressed nd the disruption of memrne integrity ws estimted y electrolyte lekge Two lef discs (62 cm in dimeter) were punched out with cork orer from the youngest fully expnded leves of T 2 trnsgenic nd WT plnts of the sme ge The lef discs were immerged in test tue contining 1 ml of deionized, distilled wter The se of the tue ws sumerged in wter th t 4 C nd removed fter 5, 2, 3, 5, 7, 9, 11 nd 13 h for testing Following het tretment, electrolyte lekge ws mesured using conductivity meter (ElectroMrk Anlyzer, Mrkson Science, Inc, Del Mr, CA) Determinintion of percent electrolyte lekge ws done sed on the method of Wisniewski et l (1997) Mens for ll vlues re n verge of two susmples in ech plnt with three replictions The significnce of differences etween mens were determined using Duncn s multiple rnge test t P < 5 level (NCSS-PASS softwre, NCSS Inc, Willimsport, PA) Arcsine squre root trnsformtion ws performed efore dt nlysis Nontrnsformed mens re presented Discs (62 cm) from fourth or fifth leves of WT nd T 2 trnsgenic capx lines were plced in n eppendorf tue (15 ml) nd were incuted in wter th (42 C) for 4 h The smples were then frozen in liquid nitrogen nd stored t 8 C for further enzyme ctivity gel nlysis UV-B stress tests: Uniform mture green tomto fruits, rndomly detched from WT nd T 2 plnts grown in the field (Lewis-Brown Frm t Corvllis, Oregon), were exposed to UV-B rdition provided y UV-B fluorescent lmps (Blk-Ry lmp, UVP, Inc, Sn Griel, CA, USA) in room with no other source of light The fruits were plced 2-cm wy from the light source (25 mw cm -2 ) nd exposed 1 h per dy for 3, 4, or 5 dys The extent of injury ws sed on colors from white to yellow color of the epidermis, followed y rowning when the injury ws more severe UV-B fruit injury ws ssessed y estimting the percent of exposure re tht exhiited rowning The mens of injury vlues re n verge of three replictes The significnce of differences were estimted y Duncn s multiple rnge test t P < 5 level For UV-B stress enzyme ctivity gel nlysis, shoots with three to four leves from greenhouse-grown WT nd T 2 plnts were plced in tues contining 5 ml of distilled wter The shoots were plced 2 cm elow the UV light source for 4 h After tretment, lef discs (62 cm) were excised nd frozen in liquid nitrogen efore eing stored t 8 C Sunscld tests: In field test, mture green tomto fruits, rndomly detched from WT nd T 2 plnts were exposed to field conditions for 15 d (from Septemer 8 to 23, 22, Lewis-Brown Frm, Corvllis, Oregon) Fruit injury ws visully recorded s descried y Rinowitch et l (1986) Sunscld injury ws chrcterized y the leching (with rown or yellow hlo round the leched re) nd necrosis of the pericrp The injured re eventully ecme sunken nd dry The percent of injured fruit re ws estimted visully Men vlues re n verge of five replictes The significnce of differences etween mens were determined y Duncn s multiple rnge test t P < 5 level Drought stress test: For drought stress test, 15 d-old rooted WT nd trnsgenic plnts were grown in plstic pots (15 cm dimeter; 145 cm height) on pet moss (Lkelnd Pet Moss, Inc, Edmonton, Alert, Cnd) After four weeks in greenhouse, the plnts were sujected to drought stress y withholding wter for 7 d Lef discs (12 cm in dimeter) were excised from the fourth or fifth lef of the treted plnts, frozen in liquid nitrogen, nd stored t 8 C for lter use in enzyme gel ctivity experiments APX enzyme gel ctivity ssy: Aout 1 mg stored lef tissue ws ground to fine powder in the liquid nitrogen nd homogenized in 2 µl of grinding uffer (1 mm NPO 4, ph7; 5mM scorte; 1 mm EDTA, ph 8; 1% glycerol; nd 1% romophenol lue), nd centrifuged t 13,g for 6 min t 4 C The superntnt ws collected nd protein concentrtion ws determined using Protein Assy System (Bio- Rd; Hercules, CA) Approximtely 7 µg of totl protein ws loded into ech lne of non-denturing, 1% polycrylmide gel nd electrophoreticlly (PAGE) seprted for 5 h t 4ºC in 1X Tris-glycine uffer (24 mm Tris, 192 mm glycine), with susequent stining for APX ctivity s descried y Mittler nd Zilinsk (1993) Results Het stress tests: The electrolyte lekge of the lef discs re presented in Fig 1 The mount of lekge incresed with time of exposure The differences in het stress resistnce etween the WT nd capx- expressing plnts (A9 nd A16) were sttisticlly significnt fter 2 h tretment (P <5) After 13 h tretment, the

6 Trnsgenic tomto overexpressing capx exhiits enhnced tolernce to UV-B nd het stress 89 Electrolyte lekge (%) WT A16 A Durtion (h) Fig 1 Electrolyte lekge of lef discs of wild-type (WT), nd trnsgenic capx (Lines A9 nd A16) tomto plnts following het stress (4 C) of vrying durtion (5, 3, 5, 7, 9, 11, nd 13 h) Vlues re mens ±SE (n=3) Different letters indicte significnt differences (P<5) etween mens within ech smpling time (Duncn s multiple rnge test) electrolyte lekge in WT plnts ws 93%, wheres tht in trnsgenic tomto lines A9 nd A16 ws 24 nd 52%, respectively UV-B stress tests: The first visile sign of dmge ws seen on dy 3, with trnsgenic A9, A16 nd WT fruits showing out 2, 34 nd 61% injury, respectively After 5 d, WT nd trnsgenic A9, A16 fruits showed 95%, 33% nd 37% injury, respectively, s evidenced y rown discolortion of the exocrp tissue (Fig 2) Compred to WT fruits, trnsgenic fruits suffered significntly less UV-B rowning injury (P <5) Sunscld tests: Exposure of detched fruits to nturl sunlight under field conditions resulted in sunscld injury to ll fruits fter 15 d However, the fruits of the trnsgenic tomtoes were less injured (Fig 3) Sunscld injury in WT fruit verged out 21% In contrst the capx- trnsgenic tomtoes (A3, A9, A13, A16, A24 nd A3) exhiited 1% to 1% sunscld injury Significnt differences (P <5) in fruit sunscld injury etween trnsgenic APX lines nd WT fruit were oserved (Fig 3) APX enzyme ctivity: The APX enzyme ctivity gel densitometric ssy reveled higher APX ctivity in trnsgenic plnt lines (A9, A16) thn in WT plnts fter UV-B (9- to 1- fold), het (3- to 32-fold), nd drought stress (5- to 6-fold) tretment (Fig 4) UV-B injury (%) WT A9 A16 c c c c Sunscld injury (%) WT A3 A9 A13 A16 A24 A3 Fig 3 Percent sunscld injury to detched fruit from wild-type (WT) nd trnsgenic capx (A3-A3) tomto plnts under field conditions for 15 d Vlues re mens ± SE (n=5) Different letters indicte significnt differences (P<5) y Duncn s multiple rnge tests Discussion The overexpression of the capx enzymes in tomto plnts incresed the resistnce of their lef nd fruit tissues to het nd UV-B stresses, supporting the results reported y previous work in pple (Wisniewski et l, 22) Electrolyte lekge in stressed trnsgenic plnts ws much less thn in WT plnts (Fig 1) providing strong evidence tht overexpression of capx resulted in enhnced protection of memrne lipid peroxidtion cused y ROS during het stress (Hung et l, 21) After UV-B stress, WT fruits showed more rowning dmge thn trnsgenic fruits (Fig 2), indicting tht overexpression of ntioxidnts might ply n importnt role in detoxifying hevy lods of ROS during UV-B stress to provide protection (Blkumr et l, 1997; Mzz et l, 1999) Severl trnsgenic fruit lines showed significntly less sunscld dmge thn WT fruits (P <5) (Fig 3), suggesting tht overexpression of ntioxidnt genes might increse tolernce to sunscld APX enzyme ctivity ws higher in capx-expressing plnts fter het, UV-B, nd drought stress (Fig 4), which suggests tht the overexpression of APX my ply role in protecting plnt ginst ROS during vrious physiologicl stress (Allen, 1995) We lso tested SOD enzyme ctivity y gel ssy nd found tht fter het nd UV-B stresses, the SOD nds were very fint, indicting tht SOD enzyme ctivity ws very low (dt not shown) The reson for this low enzyme ctivity is not known ut my e relted to the loss of SOD ctivity following long exposure to the stress tretment It is lso possile tht the effect of SOD is indirect, ecuse the product of SOD ctivity WT A9 A16 WT A9 A16 WT A9 A16 3-d 4-d 5-d Durtion (dy) Fig 2 Percent injury (exposed re exhiiting rowning) in fruit of wild-type (WT) nd trnsgenic capx (A9 nd A16) tomto plnts fter UV-B (25 mw/cm2) tretment for 3, 4, nd 5 dys Vlues re mens ± SE (n=3) Different letters indicte significnt differences (P<5) etween mens within smple time (Duncn s multiple rnge tests) UV Het Drought Fig 4 APX enzyme ctivity gel ssy UV-B (25 mw cm -2 ) for 4 h; het (42 C) for 4 h; nd drought stress (withholding wter for 7 d) Aout 7 µg protein ws loded in ech lne of the ntive gel

7 9 Trnsgenic tomto overexpressing capx exhiits enhnced tolernce to UV-B nd het stress is hydrogen peroxide, which hs een implicted s n elicitor of genes relted to stress tolernce (Sen Gupt et l, 1993; McKersie et l, 1996; Prsd et l, 1994) Overexpression of APX in trnsgenic tomto plnts provides etter protection ginst het, UV-B nd sunscld This elevted enzyme ctivity might ply n importnt role in incresing stress tolernce ginst fruit sunscld Given these results, long with those of other studies showing het nd UV-B effects on SOD or APX in trnsgenic plnts (Chen nd Pn, 1998; Wisniewski et l, 22), we elieve tht similr pproch might e pplicle to other importnt fruits tht re sensitive to sunscld, such s pple, to improve their tolernce ginst this stress Acknowledgments We re grteful to the Wshington Tree Fruit Reserch Commission nd Oregon Stte University for their finncil support of this work We lso thnk Dr Liling Cheng (Cornell University; Ithc, NY), Dr Jmes Myers for technicl dvice, nd Dr Steve Struss (Oregon Stte University; Corvllis, OR) for providing fcilities for much of the moleculr studies References A-H-Mckerness, S 2 Plnt responses to ultrviolet-b (UV-B: nm) stress: Wht re the key regultors? 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Ntl Acd Sci, USA, 93: He, YY, M Klisch nd DP Hder, 22 Adption of cynocteri to UV-B stress correlted with oxidtive stress nd oxidtive dmge Photochem Photoiol, 76(2): Hung, B, X Liu nd Q Xu, 21 Suproptiml soil tempertures induced oxidtive stress in leves of creeping entgrss cultivrs differing in het tolernce Crop Sci, 41: Jing, Y nd R Hung, 21 Drought nd het stress injury to two cool-seson turfgrsses in reltion to ntioxidnt metolism nd lipid peroxidtion Crop Sci, 41: Jimenez, A, G Creissen, B Kulr, J Firmin, S Roinson, M Verhoeyen nd P Mullineux, 22 Chnges in oxidtive processes nd components of the ntioxidnt system during tomto fruit ripening Plnt, 214: Kuo, A, H Sji, K Tnk nd N Kondo, 1995 Expression of Aridopsis cytosolic scorte peroxidse gene in response to ozone or sulfur dioxide Plnt Mol Biol, 29: Mzz, CA, D Bttist, AM Zim, M Szwrcerg-Brcchitt, CV Giordno, A Acevedo, AL Scopel nd CL Bllre, 1999 The effects of solr ultrviolet-b rdition on the growth nd yield of rley re ccompnied y incresed DNA dmge nd ntioxidnt responses Plnt Cell Environ, 22(1): 61-7 McKersie, BD, SR Bowley, E Hrjnto nd O Leprince, 1996 Wter-deficit tolernce nd field performnce of trnsgenic lflf overexpressing superoxide dismutse Plnt Physiol, 111: McKersie, BD, SR Bowley nd KS Jones, 1999 Winter survivl of trnsgenic lflf overexpressing superoxide dismutse Plnt Physiol, 119: Mittler, R nd BA Zilinsk, 1991 Moleculr cloning nd nucleotide sequence nlysis of cdna encoding pe cytosolic scorte peroxidse FEBS Lett, 2: Mittler, R nd BA Zilinsks, 1993 Detection of scorte peroxidse ctivity in ntive gels y inhiition of the scorte-dependent reduction of nitrolue tetrzolium Anl Biochem, 212: Noctor, G nd CH Foyer, 1998 Ascorte nd glutthione: Keeping ctive oxygen under control Annu Rev Plnt Physiol Plnt Mol Biol, 49: Prsd, TK, MD Anderson, BA Mrtin nd CR Stewrt, 1994 Evidence for chilling-induced oxidtive stress in mize seedlings nd regultory role for hydrogen peroxide Plnt Cell, 6: Rinowitch, HD nd D Skln, 198 Superoxide dismutse: A possile protective gent ginst sunscld in tomtoes (Lycopersicon esculentum Mill) Plnt, 148: Rinowitch, HD, D Skln nd P Budowski, 1982 Photo-oxidtive dmge in the ripening tomto fruit: Protective role of superoxide dismutse Plnt Physiol, 54: Rinowitch, HD, B Ben-Dvid nd M Friedmnn, 1986 Light is essentil for sunscld induction in cucumer nd pepper fruits, wheres het conditioning provides protection Sci Hortic, 29: Rinowitch, HD, M Friedmnn nd B Ben-Dvid, 1983 Sunscld dmge in ttched nd detched pepper nd cucumer fruits t vrious stges of mturity Sci Hortic, 19: 9-18 Renquist, RR, HG Hughes nd MK Rogoyski, 1989 Comined high temperture nd ultrviolet rdition injury of red rsperry fruit HortScience, 24(4): Sirm, RK, GC Srivstv nd DC Sxen, 2 Incresed ntioxidnt ctivity under elevted tempertures: mechnism of het stress tolernce in whet genotypes Bio Plnt, 43(2): Scndlios, JG 1993 Oxygen stress nd superoxide dismutse Plnt Physiol, 11: 7-12 Sen Gupt, AS, JL Heinen, AS Holdy, JJ Burke nd RD Allen, 1993 Incresed resistnce to oxidtive stress in trnsgenic plnts tht overexpress chloroplstic Cu/Zn superoxide dismutse Proc Ntl Acd Sci USA, 9: Sen Gupt, AS, RB We, AS Holdy nd RD Allen, 1993 Overexpression of superoxide dismutse protects plnts from oxidtive stress (induction of scorte peroxidse in superoxide dismutse-overexpressing plnts) Plnt Physiol, 13: Wng, J, H Zhng nd RD Allen, 1999 Overexpression of n Aridopsis peroxisoml scorte peroxidse gene in tocco increses protection ginst oxidtive stress Plnt Cell Physiol, 4(7): Wng, Y, M Wisniewski, R Meiln, M Cui, R We nd L Fuchigmi, 25 Overexpression of cytosolic scorte peroxidse in tomto (Lycopersicon esculentum L) confers tolernce to chilling nd slt stress J ASHS, 13(2): Wisniewski, M, J Suter, L Fuchigmi nd V Stepien, 1997 Effects of ner-lethl het stress on ud rek, het-shock proteins nd uiquitin in dormnt poplr (Populus nigr chrkowiensis x P nigr incrsst) Tree Physiol, 17(7): Wisniewski, M, L Fuchigmi, Y Wng, C Srinivsn nd J Norilli, 22 Overexpression of cytosolic scorte peroxidse gene in pple improves resistnce to het stress XXXVI Interntionl Horticulturl Congress & Exhiition p 147 (Astr) Yoshimur, K, Y Yut, T Ishikw nd S Shigeok, 2 Expression of spinch scorte peroxidse isoenzymes in response to oxidtive stresses Plnt Physiol, 123:

8 Journl of Applied Horticulture, 8(2): 91-1, July-Decemer, 26 The effect of nti-hil nets on fruit protection, rdition, temperture, qulity nd profitility of Mondil Gl pples Appl Journl Ignsi Iglesis* nd Simó Alegre Institut de Recerc i Tecnologi Agrolimentàries (IRTA) - Estció Experimentl de Lleid, Avd Rovir Roure, Lleid, Spin *Corresponding uthor E-mil: ignsiiglesis@irtes Astrct The effects of crystl (trnsprent) nd lck nets on the protection of fruits from hil, the interception of light, temperture, humidity nd fruit qulity were evluted over four sesons (from 2 to 23) t the IRTA-Experimentl Sttion, Lleid (NE-Spin) on Mondil Gl pples (Mlus x domestic Borkh) Nets demonstrted their efficiency for fruit protection ginst hil; decresed mximum orchrd tempertures nd incresed minimum tempertures nd reltive humidity Bsed on PAR vlues, on sunny dys, the lck net intercepted 25% more incident rdition thn the control nd the crystl net intercepted 12% more The use of lck net resulted in significnt reduction of colour intensity nd dys tken for mturity, nd provided lower verge yields for fruit hrvested t the first picking The crystl net ws ssocited with intermedite vlues etween lck net nd control, or similr vlues to those of the control Both nets reduced fruit temperture nd the incidence of sunurn improving glol skin qulity The lck net incresed the vigour of the trees Fruit firmness ws not ffected y the use of nets Solule solid content decresed when lck net ws used, while mturity ws delyed in some sesons There were no consistent effect with respect to titrtle cidity nd fruit crcking The nnul cost of the nti-hil nets ws 1874 to 1612 h -1, respectively, for crystl nd lck nets, depending minly on the durility of the net The nnul cost of insurnce ws 76 h -1 nd ws determinted y site, cultivr, yield nd price insured, nd ws lower thn tht of covering y nets The gross profit corresponding to the crystl net (8896 h -1 ) ws lower to the control/insurnce (9223 h -1 ) nd greter to the lck net (7842 h -1 ) ecuse of the reduction in fruit colour With Mondil Gl pples, the use of oth colour nets ws not economiclly eneficil compred to the control Key words: Apple, Mlus x domestic Borkh, Mondil Gl, net, hil protection, insurnce, rdition, temperture, humidity, vigour, fruit colour, qulity, cost, enefit Introduction Apple production is importnt in Spin, which hd totl surfce re of h in 22 The Ero Vlley is the min re of production, nd Ctloni - with h - is the most importnt region (MAPA, 23) The min cultivrs re Golden nd Gl, which together ccount for round 6% of the totl re dedicted to pple production An increse in the dmge to crops cused y hil storms hs een oserved over the lst 15 yers, prticulrly in the months of April nd My This hs encourged fruit growers in the Lleid re to estlish network of silver iodure urners covering lmost the whole re Even so, in some yers nd under certin conditions, this remedy does not produce completely stisfctory results In ddition to using silver iodure urners, spnish fruitgrowers hve lso insured their crops with the pulic compny Agroseguro, which receives susidies from oth ntionl nd regionl governments One of the most common options is to insure crops ginst hil dmge with lower cost thn nti-hil nets, ut in the cse of hil, the loss of production presents prolem for fruit industry compnies tht wish to mintin customers nd lso results in n increse in totl costs (ssocited with unused grding mchines, cold store fcilities, etc) Furthermore, in the cse of some new cultivrs tht hve een developed ccording to the clu formul, insurnce compnies estlish mximum price pyle (31 kg -1 in the cse of Pink Ldy ), wheres their plnting/production costs nd dded vlue re much higher thn those of stndrd cultivrs In the min pple producing countries of the EU (Itly, Frnce, Austri, etc) nd Americ (Argentin, Chile, Mexico, etc), the nets hve een incresingly employed to protect fruit ginst hil dmge in recent decdes minly ecuse the huge increse of the insurnce cost This technique now constitutes the only effective wy to protect fruit ginst this thret The efficiency of nets in fruit protection nd their effects on the interception of light, fruit colour nd qulity, sunurn, instlltion systems, costs, nd modifictions of orchrd climte hve ll een widely documented y uthors from Itly, Frnce, Austri, Argentin, Chile, Mexico nd South Afric, etc (Andrews nd Johnson, 1996; Bru, 1996; Coreu et l, 1997; Reigne, 1997; Vercmmen et l, 1998; Vercmmen, 1999; Crété, 2; Yuri et l, 2; Peno et l, 21; Dussi et l, 25; Gind nd Wnd, 25, 26; Vittone et l, 26) However, little informtion is ville relting to the effects nd efficiency of this technique with respect to deciduous fruit crops grown in Spin, lthough some preliminry results hve een reported in NE-Spin y Iglesis nd Alegre (24) This pper is synthesis of dt collected during the 2-23 growing sesons t the IRTA-Experimentl Sttion of Lleid with Mondil Gl pples The ojective of the tril ws to evlute the effects of two different coloured nets on the protection of fruit ginst hil, fruit colour nd qulity nd the effect on orchrd climte (temperture nd humidity) nd light interception Also

9 92 Effect of nti-hil nets on fruit protection, rdition, temperture nd qulity of Mondil Gl pples n economicl nlysis of using the two colours of nets hs een done Mterils nd methods Study site, plnt mteril, climtic conditions nd net chrcteristics: The study ws conducted in the 2, 21, 22 nd 23 sesons in plnttion of Mondil Gl pple trees on M9 Pjm 2 rootstock, plnted t the IRTA-Estció Experimentl de Lleid (Molleruss, NE Spin), in 1994 The rows were oriented from NE to SW Trees were trined with modified centrl-leder system, spced t 4 x 14 m nd grown on Tipic Xerofluvent, corse-silty, mixed (clcreous), mesic soil, with n verge depth of 85 m Irrigtion ws sme for ll the tretments nd pplied sed on evpotrnspirtion (Etc) minus the effective rinfll; ws provided dily y mens of drip system, which consisted of two 4 L h -1 drippers per tree This prticulr re of Spin is sujected to periods of high summer tempertures (>3ºC) nd very low rinfll (413, 296, 43 nd 528 mm, respectively, for the 2, 21, 22 nd 23 sesons) In the pre-hrvest nd hrvest period (from 1 st July to 15 th August), importnt differences etween sesons were recorded with respect to temperture nd rinfll Lower tempertures were registered in 2 nd 22, tempertures were norml in 21 nd extremely dry nd typiclly hot conditions occurred in 23 (Fig 1) Nets were instlled in My 1998 The orchrd ws covered with nets from the end of April (fter looming) to Octoer The net chrcteristics were: Blck net: Blck polyethylene net, dimeter 28 mm, Benigro GVM 25 x 3 (cell size 3 x 74 mm) Non-overlpping system with slight inclintion towrds the centre of the interrows Estimted lifespn: 15 yers Crystl net: Trnsprent polyethylene net, dimeter 28 mm, Benigro GVM 25 x 3 (cell size 3 x 74 mm), with the sme system s the lck net Estimted lifespn: 8 yers Five-metre high poles, with 14/16 cm dimeter, locted t 125 m intervls, were used to support the nets Estimted lifespn: 24 yers Experimentl design: A completely rndomised lock design ws used, with four locks ssigned to ech of three tretments: lck net, crystl net nd control This design ws used to control the effect due to the possile vritions in tree vigour due to the effect of nets Ech tretment nd lock consisted of five rows of 15 trees Two trees per tretment were selected from the centrl row of ech lock sed on uniform crop lod nd vigour Light interception y nets: The effect of nets on the interception of light ws mesured nnully s percentge of totl ovecnopy Photosyntheticlly Active Rdition (PAR), using Ceptometer mod Sun Scn SS1-UM-15 (Delt-T Devices Ltd Cmridge, UK) with 64 sensor photodiode linerly sorted in 1 cm length sword Sun Scn response ws lmost entirely within the PAR wvelength nd of 4-7 nm The sword ceptometer ws plced in the middle of two rows nd lso prllel to them for ech tretment, positioned 11 m ove ground level Five redings per tretment were tken t 2 hour intervls, etween 9 nd 21 (7 nd 19 solr time) severl times in ech seson (July-August), fter shoot growth hd stopped, on oth sunny nd cloudy dys Light distriution in reltion to tree height ws lso mesured t 7 heights etween 5 nd 35 m ove the ground on 1 th August 2 t 14 nd 18 (12 nd 16 solr time) nd compred with incident rdition recorded t height of 45 m for ech tretment Orchrd temperture, fruit temperture nd reltive humidity: The effect of nets on orchrd temperture nd reltive humidity ws recorded using Hoo Pro RH/Temp/2x Externl mod H8-7-2 utomtic sensors (Equipos de Instrumentción y Control SL, Mdrid, Spin), instlled within the cnopy, nd locted 12 m ove the ground level t the centre of the lock of ech tretment Fruit temperture ws mesured three times in sunny dys Fig 1 Dily mximum nd minimum tempertures over the period 1st July 15th August for the 2, 21, 22 nd 23 sesons

10 Effect of nti-hil nets on fruit protection, rdition, temperture nd qulity of Mondil Gl pples 93 nd three more times in cloudy dys of July nd August 2 These mesurements were mde using Crison Model 637 (Crison Instruments, Brcelon, Spin) digitl thermometer The temperture sensor ws introduced 3 mm under the pple skin tngentilly to the surfce of the exposed side of the fruit, s proposed y Ryll nd Pentzer (1982) At ech mesurement dte, fruit temperture ws recorded for 1 fruits per tretment (5 fruits/tree x 2 trees) picked t round 16-2 m ove ground level on the side directly exposed to sunlight during the period of mximum dily temperture, from 15: to 15:3 (13: to 13:3 solr time) Fruit colour nd size mesurements: Apple colour ws mesured with Minolt Chrom Meter CR-2 portle tristimulus colorimeter (Minolt Corp, Osk, Jpn) nd fruit chromticity ws recorded ccording to Commission Interntionle d Eclirge L*, * nd * colour spce coordintes (Hunter, 1975) Hue ngle ws clculted s descried y McGuire (1992) nd expressed in degrees In ech seson, from 2 to 23, 2 fruits were selected nd mrked in the inner (round the centrl xis) nd outer (periphery re) cnopies on ech of two trees per tretment-lock on 25 th June; 1 fruits were selected from ech of two tree sides; five from the top (12-2m ove ground level) nd five from the ottom (6-12 cm) Fruit colour mesurements were recorded in situ on ech one of the four smpling dtes (five smpling dtes in 22 nd 23), from 5 th July through to commercil hrvest on 1 th August (Fig 4) Colour vlues were mesured t oth the reddest (exposed side) nd greenest (shded side) points on the fruit equtor Hrvest dte ws 138 dys fter full loom, when flesh firmness ws <175 l nd solule solid content ws >13% Commercil vlue of the crop depends on oth, fruit size nd fruit colour At commercil hrvest, fruit size distriution sed on 5 mm-intervl fruit dimeter ctegories nd verge of red colour fruit-surfce coverge were determined for the whole crop for two trees per tretment nd lock on which fruit colour ws recorded y using n electronic grding mchine (SAMMO srl, Model S21, Itly) Four complementry trees per repliction were mrked nd their fruits were picked in two hrvests The criteri estlished for the first hrvest were: fruit colour >4% of fruit surfce nd fruit size dimeter >7 mm Professionl pickers were used nd instructed to hrvest only those fruits which hd sufficiently red colour to comply with the EU extr colour grde (1/2 of their surfce with good red colour) Fruit qulity prmeters: The sme 2 fruits mrked per tree (4 fruits per lock-tretment) were lso used t commercil hrvest for fruit qulity determintions Flesh firmness ws determined with tle penetrometer (Penefel, Cop technologies, Frnce) with n 11 mm dimeter tip nd expressed in l Two redings were tken from opposite peeled sides of 4 fruits per lock-tretment on which colour ws recorded Solule solid concentrtion (SSC) were determined y mesuring the refrctive index of lended composite of wedges tken from 4 unpeeled pples per lock/tretment, using n Atgo-Plette 1 digitl refrctometer (Atgo Co, Tokyo, Jpn) nd expressed s % Titrtle cidity (TA) ws determined for the sme composite y titrting to finl ph vlue of 82 with 1 N NOH nd expressed in g of mlic cid L -1 The effect of nets on fruit mturity ws sed on strch conversion nd determinted using the Ctifl-Eurofru code with vlues rnging from 1 (immture) to 1 (mture) Complementry determintions: The verge numer of fruits dmged y hil ws visully recorded for the whole production when fruits were grded t commercil hrvest The effect of nets on the incidence of sunurn ws evluted with respect to the sme smple used to determine fruit qulity prmeters Tree vigour ws determinted on the sis of mesurements of trunk-cross-sectionl-res (TCSA) tken 2 cm ove the grft union, mesured t the strt of the experiment nd nnully in winter time Dt nlysis: Anlysis of vrince ws preformed seprtely ech yer for PAR vlues, orchrd nd fruit temperture, chromticity vlues, yields, fruit size, fruit colour nd qulity, ccording to complete rndomized lock model with ech lock eing repliction unit, using the Sttisticl Anlysis System softwre (SAS Institute Cry, NC, 1997): sttisticl significnce ws tested t P = 5 In the cse of chromticity vlues, dt from ech lock represented the men for two fruit sides (E: exposed side, S: shded side) for 2 fruits per tree nd two trees per lock nd tretment Plots were rrnged in rndomized complete lock design When the nlysis ws sttisticlly significnt (F-test), men seprtion ws crried out y Tukey s test t P=5, using the men squre error for ech smpling dte nd prmeter evluted Differences etween tretments nd sesons were evluted y nlysis of vrince nd were tested using the Generl Liner Model procedure (PROC GLM of SAS) s rndomized complete lock design to determine the sttisticl significnce of single nd doule-wy interctions Seson nd tretment were considered s fixed effects, while locks, trees, nd fruits were designted s rndom effects Results nd discussion Effects of nets on the interception of rdition, orchrd temperture nd humidity: The vlues for rdition nd light interception were similr for ll four sesons (2-23) PAR vlues recorded under the nets on sunny dy (19 th July) nd on cloudy dy (17 th July) in 2, re presented in Fig 2 Greter verge vlues for interception were recorded etween 13: nd 15: (11: nd 13: solr time), the period when the incidence of sunlight is most verticl On sunny dys, oth nets intercepted more light rdition thn the control without net: verge light interception vlues were 25 nd 12% for lck nd crystl nets, respectively Light interception y lck nets ws twice s gret s for crystl nets on sunny dys, wheres the difference ws smller on cloudy dys Clouds cused reduction in totl incident rdition of round 5% (Fig 2) In the period from the 1 st July to 17 th August of the four sesons studied (2-23) only men of 5 dys were cloudy, the rest were sunny s usul in wrm res Asolute vlues of PAR nd the effects of nets on light interception were similr to those reported y Peno et l (21), Vercmmen (1999) nd Crété et l (21) Dussi et l (25) recorded respective decreses in PAR of 28 nd 45%, using 15 nd 55% density shde nets in Argentin (Alto Vlle)

11 94 Effect of nti-hil nets on fruit protection, rdition, temperture nd qulity of Mondil Gl pples The solr rdition registered on sunny dys resulted in high PAR vlues (mximum 16-2 µmol m -2 s -1 ), which re common in southern Europe rid conditions On cloudy dys (complete clouds cover), the mximum vlues rnged from 8-9 µmol m -2 s -1 In oth cses, the registered vlues were twice s gret s those for northern Europe (Belgium, Germny, etc) nd for this reson rdition disponiility is not limittion even when lck nets is used (mximum PAR vlues round 12 µmol m -2 s -1 ) Mesurements of light intercepted t different tree heights (from 5 to 35 m) in the middle row of ech tretment were tken, efore hrvest, t two different times on 28 th July 2 t 14: nd 18: (12: nd 16: solr time) provided similr vlues The recorded differences etween tretments were significnt nd very similr t oth mesurement times At 14:, the verge of light intercepted, did not depend on tree height ecuse the incidence of sunlight ws verticl At 18:, the interception of sunlight incresed with incresed tree height due to the shding of the lower prts of the trees (dt not shown) This explins the incresed exposure to light of the upper prts of the trees nd their consequent etter fruit colourtion, ecuse sunlight is the most importnt fctor regulting nthocynin synthesis nd colour s reported y Sure (199) nd Lncster (1992) in pple skin nd Dussi nd Huysmer (1995) in per skin Mximum, men nd minimum dily tempertures for the period 1 st July- 17 th July 21 (period representtive of summer conditions in wrm regions) showed tht the use of nets exerted limited influence on orchrd temperture (Fig 3) Mximum tempertures tended to e lower under the nets (3 ºC), due to the interception of rdition or shde effect which is greter thn the gin of temperture cused y the use of nets due to their role in the interception of ir circultion or greenhouse effect Bigger differences were recorded on right nd sunny dys (1 st, 2 nd July,) nd lesser ones on cloudy dys (8 th July, 18 th July) Minimum tempertures tended to e lower in the control PAR PAR July 2 (cloudy) 19 July 2 (sunny) Blck Crystl Control 9h 11h 13h 15h 17h 19h 21h Blck Crystl Control 9 h 11 h 13 h 15 h 17 h 19 h 21 h Time Fig 2 Diurnl curve of photosyntheticlly ctive rdition (PAR±SE) (μmol m -2 s -1 ) s ffected y different coloured nets or no net on sunny dy (19th July) nd cloudy dy (17th July) 2 y 1 ºC (Fig 3) thn in the nets ecuse of the greenhouse effect nd the low rdition t this time of the dy Similr results were reported y Vercmmen (1999) nd Crété et l (21), indicting tht the influence of nets upon mximum orchrd tempertures nd their role in incresing minimum tempertures ws not clerly demonstrted Reigne (1997) found moderte increse in mximum tempertures ssocited with the use of nets nd Vysse (1997) reported moderte decrese (<1 ºC) Peno et l (21) did not find ny cler temperture effect ssocited with the use of nets nd only noted tht mximum Mx Temp (Control) Men Temp (Control) M in Temp (Control) Mx Temp (Crystl) Men Temp (Crystl) Min Temp (Crystl) Mx Temp (Blck) Men Temp (Blck) Min Temp (Blck) 3 5 Temperture ( o C) Dtes (July) Fig 3 Mximum (upper), men (centre), minimum (lower) dily nd men tempertures per tretment (right) ffected y the use of two different coloured nets during the period 3rd -19th July 21

12 Effect of nti-hil nets on fruit protection, rdition, temperture nd qulity of Mondil Gl pples 95 were slightly incresed It would e interesting to quntify the interception of ir circultion y the nets in order to know the greenhouse effect Orchrd humidity incresed y the use of oth nets ut no significnt differences were oserved with respect to mximum reltive humidity (dt not shown) These results were in line with those reported y Crété et l (21), indicting 2-6% increse in humidity ssocited with the use of nets These uthors lso reported decrese in evportion y11% in July ssocited with the use of nets nd significnt reduction in wind speed, which lso resulted in decrese in skin ruising Fruit temperture: Temperture mesurements registered during the mximum dily orchrd tempertures, in summer 2, showed the positive effects of nets on oth, reductions in fruit temperture (Tle 1) nd in the incidence of sunurn (Tle 3) This reduction in temperture ws relted to rdition intercepted y the nets (Fig 2) The lck net ws ssocited with greter temperture reduction (round 4 ºC) thn the crystl net (25 ºC) when compred to the control On cloudy dys, without direct sun rdition over the cnopy, fruit tempertures were lower nd differences mong tretments were minor (1-2 ºC) Also the differences etween ir nd fruit tempertures were smller compred to sunny dys It is lso interesting to note tht the higher fruit tempertures ssocited with sunny dys in summer (471 ºC), were round 12 ºC higher thn ir tempertures due to direct exposure to sunlight (Tle 1) For this reson the use of shde nets, evportive cooling nd kolin-sed prticle film technology were the three techniques commonly used to reduce rdition nd fruit temperture in pple nd per, s reported y Dussi et l (1997, 25), Gind nd Wnd (26) nd Iglesis et l (22, 25) Tle 1 Internl fruit temperture y nd orchrd ir temperture ( C) of `Mondil Gl' pples ffected y the use of two different coloured nets under different conditions t 15: (13 solr time) on six different dtes in 2 Tretment/ conditions Sunny Dte Men Nets vs control 25 th July 6 th Aug 1 th Aug Blck 416* c -41 Crystl Control Air temperture Cloudy 21 th July 3 th Aug 14 th Aug Blck Crystl Control Air temperture (*):Ech vlue is the men of 1 mesurements on the exposed side of the fruit Y Different letters in the sme column represent significnt difference t P < 5 y Tukey rnge test if the F-test ws significnt in the ANOVA Fruit colour: The evolution of fruit colour, sed on Hue vlues (with higher vlues indicting less colour), over the 4 weeks efore commercil hrvest nd Hue vlues t commercil hrvest, reveled significnt differences etween tretments oth t hrvest nd lso during the 2-3 weeks efore hrvest, for ll sesons except 2 (Fig 4), s result of optiml tempertures for colour development This ws ecuse this is the period of mximum nthocynin iosynthesis in Gl pples (Iglesis, 1996; Iglesis et l, 25) When four sesons were compred, Hue vlues prior to hrvest nd t commercil hrvest showed significnt differences from seson to seson, nd the interction seson x tretment ws significnt The crystl net produced either similr vlues to the control (21 nd 22) or vlues etween the lck net nd the control (23) The higher vlues ssocited with use of the lck net prlleled reduction in fruit colour with respect to the control nd lso evidenced dely nd reduction in fruit colour development This could e explined y lower vilility of crohydrtes necessry for nthocynin iosynthesis due to the reduction of light disponiility It is well known tht nthocynin iosynthesis in pple is directly ffected y light (Lncster, 1992; Arkw, 1988) nd temperture (Frgher, 1983; Arkw, 1991) The reduction of rdition is responsile for down-regultion of photosynthetic cpcity of leves nd consequently lower light sturted photosynthetic rte compred to the control (Gind nd Wnd, 26) Tkos et l (26), more recently, reported tht in red skin pple cultivrs severl flvonoid genes required for nthocynin synthesis were coordintely trnscried in reponse to light exposure When fruit colour on oth sides of the tree (est-west) nd for different heights were compred, lower vlues were recorded on the west sides nd in the upper prts of trees (dt not shown) This ws relted to the greter exposure to light on the higher prts of the cnopy nd into the sunset side of the tree, s reported y Jckson et l (1977) nd Dussi et l (25) The interctions tretment x tree side nd tretment x cnopy height were not significnt, nd the lck net reduced fruit colour for the whole cnopy In 23, tempertures were unusully high during the summer period nd this resulted in poor colour development compred with 2 nd 22, even in the cse of optimum exposure to light on the exposed sides of fruits without nets Under these conditions the effect of oth high tempertures nd significnt reductions in exposure to light ssocited with the use of nets explined the drmtic reduction in fruit colour with respect to other sesons Under these criticl conditions, even the slight reduction in exposure to light occsioned y the crystl net resulted in reduction in fruit colour with respect to the control in 23, ut this did not occur in 2, 21 nd 22 (Fig 4), when tempertures were more fvourle for colour development Averge of fruit colour re importnt in order to determine its commercil vlue, nd minimum of 5% of fruit surfce should e coloured to comply with the EU Extr colour grde requirements nd more thn 8% for Extr Fncy requirements Averge vlues for fruit colour distriution for the totl yield of ech tretment t commercil hrvest re presented in Fig 5 Significnt differences were oserved etween sesons nd the interction seson x tretment ws significnt with respect to fruit colour nd Hue vlues The gretest vlues for fruits with <4% of fruit colour were recorded in 23 Intermedite vlues were otined in 21 nd the lowest vlues in 2 nd 22 When the verges of fruits >8% of fruit colour were compred, the gretest vlues were otined for 2 nd 22 sesons These results were similr to those discussed ove for Hue vlues (Fig 4), nd showed the negtive effect of the lck net in reducing verge vlues for fruits with >8% nd 4-8% of fruit colour

13 96 Effect of nti-hil nets on fruit protection, rdition, temperture nd qulity of Mondil Gl pples Hue( o ) Hue( o ) July Blck Crystl Control 6 Blc k Cr ys tl Contr ol July 29 - July 1 - August 5 - July 17 - July July 1 - August c July July July 1 - August 1 - August 5 - July July July 1 -August 1 -August Dte Dte Fig 4 The effect of two different coloured nets on fruit colour evolution (Hue) for Mondil Gl pples in the prehrvest period of the 2, 21, 22 nd 23 sesons Tretments with the sme letter for the sme seson nd dt re not sttisticlly different ccording to the Tukey Test (P<5) (Seson x tretment interction for hue vlues t hrvest ws significnt t P<1) Ech point represents the men of two fruit sides of 2 fruits per tree nd 8 trees per tretment (2 treeslock-1 x 4 locks) Fruit (%) Blck 21 8 Crystl Control 6 Fruit (%) c Fruit (%) < 4% 4-8% >8% < 4% 4-8% >8% Percent of fruit colour Percent of fruit colour Fig 5 The effect of two different coloured nets on the verge of fruit colour for ll the yield of Mondil Gl pples t commercil hrvest in the 2 ferent ccording to the Tukey Test (P<5) (Seson x tretment interction for % of fruits for ech different degree of colour ws significnt t P<1) Fruit (%)

14 Crystl net provided similr vlues to the control, except in 23 when reduced the verges of 4-8% nd incresed those of <4%, indicting reduction of fruit colour (Fig 5) The totl yield hrvested t the first picking dte (>7 mm size nd >5% of coloured surfce) reveled lower vlues ssocited with the use of the lck net nd similr vlues etween the crystl net nd the control in 2, 21 nd 22, nd intermedite vlues for the control nd use of the lck net in 23 Even in sesons fvourle to fruit development, such s 2, the lck net reduced fruit colourtion Men vlues for ll sesons only indicted significnt differences etween the control nd lck net (Fig 6) Importnt differences were oserved etween sesons, with higher verge vlues eing otined in 2 nd lower vlues in 23 For this reson, the interction seson x tretment ws lso significnt with regrd to verge vlues ssocited with fruit hrvested t the first picking All previously presented results relting to fruit colour confirm those reported y other uthors working with icoloured cultivrs such s Royl Gl, Jongold nd Elstr (Reigne, 1997; Vercmmen, 1999; Crété, 2; Peno et l, 21) They lso illustrte the negtive effect of the lck net on fruit colour; in the cse of the crystl net, similr vlues were otined to the control for sesons with norml or optimum tempertures, nd vlues etween those otined using the lck net nd the control were otined in wrm sesons, such s 23 Dussi et l (25) working with the cultivr Fuji, in Argentin, nd Gind nd Wnd (26) working with Royl Gl in South Afric, lso reported significnt reduction in fruit colour under nti-hil nets Red colour development ws influenced y the seson due to the direct effect of tempertures on nthocynin iosynthesis in Mondil Gl pples s reported y Iglesis et l (22) Tree vigour nd yield: No difference etween tretments were oserved t the end of the first seson (2) fter the instlltion of the nets on tree vigour The nnul increse in TCSA showed greter vlues for trees under the lck net in 22 nd 23 nd intermedite vlues for crystl net in 21 (Tle 2) Furthermore, nnul shoot growth ws significntly higher in ll sesons when using the lck net in comprison to the crystl net nd the control, s reported y Peno et l (22) Shding y nets (specilly lck net) hs effects similr to over-tree evportive cooling; decresing plnt temperture nd wter stress, nd reducing the incidence of sunurn This hs Yield hrvested (%) Effect of nti-hil nets on fruit protection, rdition, temperture nd qulity of Mondil Gl pples 97 Blck Crystl Control First pick B A AB 1 een widely documented y severl uthors (Prchomchuk nd Meheriuk, 1996; Recsens et l, 1998; Gind nd Wnd, 25, 26; Iglesis et l, 25) As discussed ove, fruit colour ws significntly reduced y net shding ecuse of decresed direct sunlight on the fruits nd consequently reduction of nthocynin ioshynthesis rte Tle 2 The effect of two different coloured nets on tree vigor z, mesured s the trunk-cross-sectionl-re (TCSA), of Mondil Gl pples over 1999 to 23 sesons Tretment TCSA (cm 2 )** 1999* Blck Crystl Control Significnce (P) z Different letters in the sme column represent significnt difference t P 5 y Tukey rnge test if the F-test ws significnt in the ANOVA (*): Initil TCSA, efore instlltion of nets **: TCSA mesured in Novemer The tril ws locted in wrm region with sunny dys nd high rdition levels (Fig 2) over the most prt of summer In these conditions, light disponiility compred to cold regions of the North Europen countries, is not limittion, even under lck nets For this reson the increse of vigour could e explined y the positive effect of shding (lck net) which resulted in significnt reduction of oth, the rdition reching trees nd consequently the level of evpotrnspirtion This resulted in reduction in plnt wter stress, n increse in photosynthesis nd incresed vilility of crohydrtes: ll of these circumstnces re conducive to n increse in vigour, s oserved in commercil orchrds The reduction in stress is lso fvoured y reduction in mximum tempertures nd increse in orchrd humidity ssocited with the use of nets In these conditions we suggest tht this reduction is more importnt thn the reduction in photosynthesis cused y the interception of rdition y the nets Gind nd Wnd (25) reported down-regultion of photosynthetic cpcity due to nti-hil net which resulted in reduced fruit size on Royl Gl nd Cripps Pink pples Furthermore, the greter humidity found under the nets leds to decrese in plnt wter stress This confirms the dt presented y Crété et l (21), which indicted tht it is possile to reduce irrigtion needs y out 15% with respect to the control ecuse of n 1% evportion decrese c Men Men Fig 6 Percentge of yield hrvested y picking for Mondil Gl pples for the period 2-23, s ffected y the use of different coloured nets Columns with the sme letter for the sme seson nd picking were not sttisticlly different ccording to the Tukey Test (P<5) (Seson x tretment interction for % of yield hrvested y picking ws significnt t P<1) Blck Crystl Control Second pick c c A AB B

15 98 Effect of nti-hil nets on fruit protection, rdition, temperture nd qulity of Mondil Gl pples Tle 3 Effect of two different coloured nets on men yields, fruit size, qulity prmeters, strch index, incidence of crcking nd sunurn nd hil dmge, for Mondil Gl pples t commercil hrvest in the 2, 21, 22 nd 23 sesons Seson Tretment Yield Weight Size mm (% yield) Firmness SSCs TA Strch Crck Sunurn Hil (kg tree -1 ) (g fruit -1 ) <7 7-8 >8 (l) (ºBrix) (g L -1 ) index (%) (%) ffe (%) Blck Crystl Control Blck Crystl Control Blck Crystl Control Blck c 23 Crystl Control Columns with the sme letter for the sme seson were not stticlly different ccording to the Tukey Test (P<5) Men yields (kg tree -1 ) recorded during the period 2-23 were not significntly ffected y the use of nets (Tle 3) These results support those presented y Reigne (1997), ut Peno et l (22) reported decrese y the effect of nti-hil nets due to modifictions in the plnt physilogy The effect of nets on fruit size, fruit mturity nd qulity prmeters: Fruit size distriution, fruit weight nd fruit firmness were not significntly ffected y the use of nets (Tle 3) Similr oservtion hve een reported y Borin nd Soncell (2) nd Crété (2) Solule solid content decresed when the lck net ws used, ut vlues were similr for the crystl net nd the control These results gree with those reported y Coreu et l (1997), Crété et l (21), Peno et l (21) on Gl nd Fuji pples, nd confirm tht shding reduces the solule solid content of fruits, delying their ripening In opposite, etter exposition of the leves to light hs een relted with etter fruit sugr content (Jckson et l, 1977; Dussi et l, 25) Nets hd no effect either on titrtle cidity or on fruit firmness, ut mturity, expressed y strch conversion, ws delyed y out week in 2 nd 23, ut not in the other sesons Similr results were reported y severl other workers (Borin nd Soncell, 2; Peno et l, 21) However, Ghind nd Wnd (26) did not find ny differences in fruit firmness, solule solid content nd strch conversion when using shde net with Royl Gl pples According to Reigne (1997), the use of nets did not hve ny influence upon the incidence of pests (Cydi, Aphis, etc) or diseses (Venturi, Podospher, etc) Demri et l (26), reported significnt decrese of moth pest popultion nd reduction of fruit-dmges with nti-hil nets The effect of nets on fruit sunurn nd crcking: The use of nets hd positive effect on the reduction in numer of fruits ffected y sunurn, in spite of Mondil Gl is not very sensitive cultivr to this disorder compred with Fuji or Grnny Smith (Cró et l, 24) Crystl net produced intermedite results etween lck net nd the control (Tle 3) The reduction of sunurn ssocited with the use of nets, especilly the lck net, nd the increse in overll skin qulity (ut decrese of fruit colour) hs een widely reported for the cultivrs Golden, Gl, Grnny Smith, Fuji nd Pink Ldy (Andrews nd Johnson, 1996; Yuri et l, 1996; Crété, 2; Peno et l, 21; Dussi et l, 25; Gind nd Wnd, 25, 26) This cn e explined y oth, the reduction in direct incident rdition on the fruit (Fig 2), nd y the reduction in fruit temperture under the net (Tle 3) This finding is in greement with most of the reserch indicting high tempertures nd intense solr rdition s the min cuses of sunurn in pples (Yuri et l, 1998) For this reson, shding y nets hs een used in severl prts of the world with high tempertures nd intense solr rdition to reduce sunurn nd increse skin qulity in pples (Andrews nd Johnson, 1996; Yuri et l, 1996; Dussi et l, 25; Gind nd Wnd, 25; 26), specilly on non coloured pples such s Golden or Grnny Smith As reported in wrm res, the reduction of fruit colour oserved on icoloured or red cultivrs should e considered nd the use of white or grey nets is recommended (Peno et l, 21), in spite the minor protection ginst sunurn Fruit crcking ws not ffected y the nets, except in 23 when greter vlues of fruit crcking were recorded, proly due to the unusully high summer tempertures, which lso reduced fruit colour In this seson nets significntly reduced the verges of fruit crcking compred to the control (Tle 3) Efficiency of nets ginst hil protection: The only hil storm recorded took plce in My 23 The results otined showed the efficiency of the nets in protecting fruit compred to the control (Tle 3) The stility offered y this system in windy storms hs lso een reported y severl reserchers from different countries, working with the sme system (Bru, 1996; Vercmmen, 1999) Economic evlution Instlltion costs: Benigro-type GVM 25 x 3, crystl nd lck coloured nets were used in the tril The totl cost of net instlltion (nets, poles, poles nchorges, rented mchinery, net instlltion, lours, etc) ws h -1 The cost of the support structure for plnted orchrd (poles + support cles) ws 6625 h -1 In the cse of new plnttion, the sme poles used to support the nets cn support the trees nd this mkes it possile to shre the cost of supporting trees, which is estimted t round 2 h -1 (the cost of poles nd their positioning)

16 Effect of nti-hil nets on fruit protection, rdition, temperture nd qulity of Mondil Gl pples 99 Annul cost of nets nd insurnce: These costs hve een clculted for the orchrd where the tril ws crried out in the region of Lleid (Molleruss) Annul costs of using nets (nets, instlltion, structure, rented mchinery, lour, fold nd unfold the nets ech yer, etc) were 1874 nd 1612 h -1, for crystl nd lck nets, respectively (Tle 4) We consider lifespn for the support structure (poles, support cles) of 24 yers, nd lifespns of 8 nd 15 yers, respectively, for the crystl nd lck nets The nnul cost of using nets ws compred with the nnul cost of insurnce (only protection ginst hil) for the cultivr Mondil Gl, considering men yield of 5 tons h -1 t the Molleruss site The finl nnul cost of insurnce ws 76 h -1 (Tle 4) nd ws susidised y ENESA (48%) nd DARP-Generlitt de Ctluny (15%) The cost ( kg -1 ) of insurnce ws lower thn tht of covering the orchrd with nets nd directly depended on the yield insured: the cost incresed s the yield incresed The greter cost of the crystl net ws due to its lower life expectncy (8 yers) thn the lck net (15 yers) In contrst with the cse of insurnce, the cost ssocited with the use of nets ( kg -1 ) ws inversely relted to the yields otined The effect of nets on gross income: As explined erlier, the use of nets influenced some prmeters of fruit qulity s fruit colour, dmge y sunurn nd crcking, nd consequently fruit price Furthermore, s the insurnce (control), the instlltion of nets resulted in nnul recurring cost Both fctors re considered to evlute the effect of the nets on the gross income without considering the other costs of production -lour, inputs, irrigtion, etc-, ecuse these re the sme for the three tretments of 1 h of Mondil Gl pples Considering the sme yield for the three tretments nd sed on the men dt corresponding to the 2-23 period, shown in Fig 5 (fruit colour distriution), Fig 6 (yields hrvest y the two hrvests) nd Tle 3 (fruit size, dmge y crcking nd sunurn), the totl income from fruit sle, the cost of protection ginst hil, nd the enefit re presented in Tle 4 Compred to the control (insurnce without using net), the enefits (totl gross profit) of using crystl net ws lower thn using lck net Crystl net reduced sunurn, without ffecting negtively fruit colour, ut the nnul cost of its instlltion is more thn two times compred to the insurnce nd greter thn lck net The decrese of enefit when using lck net, even fter reduction of crcking nd sunurn, is due to the decresed fruit color: verge of yields on the first picking nd increse of non mrketing yield The results discussed ove re from Mondil Gl, with limited insurnce price In the cse of new developed cultivrs s Pink Ldy, Knzi, etc, the fruitgrower price is greter (5-6 kg -1 ), ut the price to insure is limited (31 kg -1 ) nd cn t cover the vlue of the fruit In this cse the enefit of using crystl net (lck net reduces colour) increse the enefit compred to the control which is nowdys used in the min res of fruit production in Spin On the sis of the results presented, we cn conclude tht the system evluted ws very effective t protecting fruit ginst hil dmge A study of nnul enefits shows similr enefits when the control nd the crystl net re compred nd the disdvntge of using lck nets due to the reduction of fruit colour on Mondil Gl pples Net cost ws higher for the crystl net thn for the lck one due to its shorter lifespn (8 nd 15 yers, respectively) This reltive cost decresed when yields incresed; just the opposite to wht hppened in the cse of insurnce The use of nets offers gretest return in cse of cultivrs with high mrket vlues, due to the limits of mximum insurle vlue Tle 4 Differences on gross profit due to the use of two coloured nets nd control (without nets), over 1 h orchrd surfce of `Mondil Gl' pples t Lleid (Spin) Concept per h Tretment Control Blck net Crystl net Totl yield (kg h -l )* % of totl yield < 7 mm * % of totl yield ffected y sunurn* % of totl yield ffected y crcking* Commercil yield (kg h -1 ) Yield hrvested t 1st picking ** (%) (dimeter > 7mm nd > 5% fruit Yield decrese t 1st picking with respect to the control (%) _ Hrvest t 1st picking* (kg) Price 1st picking ( kg -1 ) *** Income 1st picking ( ) Yield t 2nd picking**(%) Hrvest t 2nd picking (kg) Yield <4% fruit colour *(%) Price yield no mrket ( kg -1 )*** Income yield no mrket (+) Price 2nd picking ( kg -1 )*** Income 2nd picking ( ) Totl fruit income ( h -1 ) Difference to control ( h -1 ) Insurnce cost ( h -1 yer -1 ) Totl nets cost ( h -1 yer -1 ) Difference to control ( h -1 ) Totl gross profit' ( h -1 ) (*): dt from Tle 3; (**): dt from Figure 6; (***): men fruit grower prices from 2 to 23 (y) : without considering production costs (lour, inputs, irrigtion, etc) For ll of the fctors studied, the lck net lwys hd greter impct thn the crystl net, resulting in reduction of fruit colour, solule solid content nd sunurn, nd cusing susequent dely in mturity It would therefore divisile to use crystl nets in wrm res with poorly or medium coloured cultivrs It would lso e interesting to study the effect of using nets on plnt hydric sttus nd wter needs nd their influence upon tree vigour These re considertions of potentil interest due to the effects of nets on rdition, humidity, evpotrnspirtion nd temperture Ackowledgements This work hs een prtilly supported y Project INIA RTA1-85 (21-23) We thnk Mrc Btlle, Nuri Crull nd Jordi Plà, for their collortion in collecting nd prtilly processing the dt Thnks to NOVAFRUT for technicl support in the

17 1 Effect of nti-hil nets on fruit protection, rdition, temperture nd qulity of Mondil Gl pples design for nets instlltion, nd for help with cost dt We lso thnk NUFRI SAT to provide the dt for the study of economics References Andrews, H nd P Johnson, 1996 Physiology of sunurn development in pples Good Fruit Grower, July: Arkw, O 1988 Chrcteristics of colour development in some pple cultivrs: chnges in nthocynin synthesis during mturtion s ffected y gging nd light qulity J Jp Soc Hort Sci, 57: Arkw, O 1991 Effect of temperture on nthocynin ccumultion in pple fruit s ffected y cultivr, stge of fruit ripening nd gging J Hort Sci, 56: Borin, M nd C Soncell, 2 Impinto di rete ntigrndine, spetti tecnici ed economici L informtore Agrrio, LVI (29): Bru, M 1996 Les filets prgrêle u crile Fruits et Legumes, 14: 3-31 Cró, J, M Csls, I Iglesis, JM Pgès nd J Bonny, 24 El golpe de sol en mnzno: cuss y control Acts de ls VI Jornds de Experimentción en Fruticultur Acts de Horticultur, 43: Coreu, C, A Oser nd M Trillot, 1997 Protection contr l grêle Infos-Ctifl, 3-6pp Protection contre l grêle Infos-Ctifl, 128: 3-6 Crété, X 2 Filets prgrêle, des incidences sur l qulité Fruits et Legumes, 191: Crété, X, JL Regnrd, G Ferre nd C Tronel, 21 Effects secondires et conséquences sur l conduite du verger L roriculture fruitière, 553: Demri, D, I Mrtini nd A Alm, 26 Influenze dell reti ntigrndine sulle poluplzioni e sull gestione di Cydi pomonell (L) in Piemonte Rivist di Frutticoltur, 11: Dussi, MC nd M Huysmer, 1995 Severe posthrvest summer pruning of mture Forelle per trees influences cnopy light distriution, nd fruit nd spur chrcteristics in the following sesons JS Afr Soc Hort Sci, 5(2): 57-6 Dussi, MC, D Sugr, AN Azrenko nd T Righetti, 1997 Effect of cooling y overtree sprinkler irrigtion on fruit colour nd firmness in Senstion Red Brtlett Per HortTech, 7(1): Dussi, MC, G Girdin nd P Ree, 25 Shde nets effect on cnopy light distriution nd qulity of fruit nd spur lef on pples cv Fuji Spnish Journl of Agriculturl Reserch, 3(2): Frgher, JD 1983 Temperture regultion of nthocynin ccumultion in pple skin J Exp Bot, 34: Gind, J nd SJE Wnd, 25 Sunurn in pples nd effectiveness of control mesures Comined Congress, 1-13 Jnury 25, Potchefstroom, South Afric, Astrct pp 43 Gind, J nd SJE Wnd, 27 Do fruit sunurn control mesures ffect lef photosynthetic rte nd stomtl conductnce in Royl Gl pple Environmentl nd Experimnetl Botny, 59(2): Hunter, RS 1975 The mesurement of ppernce John Willey & Sons, Inc New York Iglesis, I 1996 Influenci del mteril vegetl y del riego por spersión en l clorción de vrieddes rojs de mnzn (Mlus x domestic Borkh) PhD, Universitt de Lleid Iglesis, I, J Slví, L Torguet nd C Cús, 22 Orchrd cooling with overtree microsprinkler irrigtion to improve fruit colour nd qulity of Topred Delicious pples Scienti Horticulture, 93: Iglesis, I nd S Alegre, 24 Influenci de ls mlls ntigrnizo en l protección de los frutos, l intercepción de l rdición y l clidd de mnzns cv Mondil Gl Acts de ls VI Jornds de experimentción en Fruticultur Acts de Horticultur, 43: Iglesis, I, J Slvi, L Torguet nd M Montserrt, 25 The evportive cooling effects of overtree microsprinkler irrigtion on Mondil Gl pples Scienti Horticulture, 13: Jckson, JE, JW Plmer, MA Perring nd RO Shrples, 1977 Effects of shde on the growth nd cropping of pple trees III Effects on fruit growth, chemicl composition nd qulity t hrvest nd lter storge J Hort Sci, 52: Lncster, JE 1992 Regultion of skin colour in pples Crit Rev in Plnt Sci, 1: MAPA 23 Encuest ncionl de plntciones frutles, 22 Secretri Generl Técnic McGuire, RG 1992 Reporting of ojective colour mesurements HortScience, 27: Prchomchuck, P nd M Meheriuk, 1996 Orchrd cooling with pulsed irrigtion to prevent solr injury nd improve fruit qulity of Jongold pples HortScience, 31: Peno, C, G Gicolone, A Bosio, G Vittone nd G Bounous, 21 Influenz delle reti ntigrndine sull qulit delle mele Rivist di Frutticoltur, 9: Peno, C, G Vittone, G Giclone nd S Aimr, 22 Influenz delle reti ntigrndine sull produzione Informtore Agrrio, 28: Recsens, DI, J Recsesns nd J Brrgn, 1988 Sprinkler irrigtion to otin refreshing microclimte Effect on fruit growth rtes nd qulity of Jonee nd Golden Smoothee pples Act Horticulture, 228: Reigne, M 1997 Des incidences gronomiques mesures Fruits et Legumes, 158: Ryll, AL nd WT Pentzer, 1982 Tretments efore shipment or storge In: Trnsporttion nd storge of fruits nd vegetles, 2 nd Ed, Vol 2 AVI pulictions Comp Inc Westport-USA SAS Institute, 1997 SAS/STAT user s guide Version 612 SAS Inst, Cry, NC Sure, MC 199 Externl control of nthocynin formtion in pple Sci Hort, 42: Tkos, AM, SP Roinson nd AR Wlker, 26 Trnscriptionl regultion of the flvonoid pthwy in the skin of drk-grown Cripps Red pples in reponse to sunlight J Hortic Sci Biotechnol, 81: Vysse, P 1997 Incidences climtique, phytosnitrie et gronomique des filets Infos-Ctifl, 128: 6 Vercmmen, J, P Vn Ler nd D Vn Leeuw, 1998 Les filets nti-grele: premiere experience en Belgique Le fruit Belgue, 473: Vercmmen, J 1999 Les filets nti-grele: premiere experience en Belgique Le fruit Belgue, 482: Vittone G, P Welschen nd S Pellegrino, 26 Reti ntigrndine semplificte, nere o colorte, per l protezione dei meleti piemontesi Rivist di Frutticoltur, 11: Yuri, JA, J Vásquez, C Torres nd JL Vásquez, 1996 Golpe de sol, l experienci chilen Proc Coloquio de Pomáces University of Tlc, Chile, Octoer, pp Yuri, JA, C Torres, R Bstis nd A Neir, 1998 Estudio del golpe de sol en mnznos y lgunos fctores que lo inducen Proc IX Congreso Lt De Hortic And XLIX Congreso Agron de Chile Sntigo de Chile, 3 Novemer Ref 26 Yuri, JA, C Torres nd J Vsquez, 2 Golpe de sol en mnzns I- Evlución del dño y métodos de control Agro-Cienci, 16(1): 13-21

18 Journl of Applied Horticulture, 8(2): 11-14, July-Decemer, 26 Induction of phenolic compounds iosynthesis with light irrdition in the flesh of red nd yellow pples Appl Journl D Bkhshi nd O Arkw Fculty of Agriculture nd Life Science, Hiroski University, 3 Bunkycho, Hiroski, Aomori, , Jpn E-mil: orkw@cchiroski-ucjp Astrct Effect of light irrdition on the ccumultion of phenolic compounds ws investigted in the flesh slices of three pple (Mlus domestic Borkh) cultivrs Fuji nd Jonthn with red skin nd Orin yellow-green one were used in this study The irrdition ws crried out t 1, 17, 24 nd 3 for 96 hours, using mixture of white plus ultrviolet fluorescents Phenolic cids, nthocynin nd flvonols were the phenolics tht incresed rpidly y irrdition wheres flvnols, procynidins nd dihydrochlcones did not chnge in either mture or in ripe fruits of ll the three cultivrs There ws positive correltion etween nthocynins, phenolic cids nd flvonols in exmined cultivrs oth t the mture nd ripe stges Optimum temperture for the synthesis of phenolic cids, nthocynins nd flvonols ws 24 regrdless to the mturity stge nd vriety Totl phenolic content of Fuji incresed through ripening ut it decresed in Jonthn nd Orin Therefore, the irrdition to the flesh might e very useful method for the study of the regultion mechnism of the phenolic compounds ccumultion Key words: Apple, light irrdition on flesh, ultrviolet-b, flvonoids, phenolic cids, high performnce liquid chromtogrphy Introduction Apple fruit is known to contin lrge mounts of phenolics such s chlorogenic cid, flvnols, flvonols, procynidins nd nthocynins (Awd nd de Jger, 22; Awd et l, 2) The studies using nimls nd in vitro work hve een conducted to define mechnisms y which pples my help in preventing chronic disese cused y lipid nd DNA oxidtion (Boyer nd Liu, 24) The ntioxidtive potentil of pple, however, is known to depend on the concentrtion nd composition of phenolics (Kondo et l, 22), which is influenced y mny physiologicl nd environmentl fctors (Lncster, 1992) Moreover, distriution of these metolites differs within fruit (Awd et l, 2) The ility for nthocynin ccumultion in pple skin vries within vrieties Red-skinned pples ccumulte nthocynin, while yellow-green ones do not nd most of the commercil cultivrs with red skin ccumulte nthocynin in the skin ut not in the flesh Although the iosynthesis mechnisms of nthocynin nd other phenolics hve een widely studied in the skin, limited informtion out the flesh is ville Stimultive effect of irrdition on the nthocynin ccumultion, influenced y gging, temperture nd mturity hs een reported, recently These studies show tht UV-B stimultes nthocynin synthesis in the pple skin (Arkw et l, 1985; Arkw, 1988; Arkw, 1991; Wng et l, 2) Becuse of lower content of phenolics, specificlly flvonols, in the flesh compred to the skin, pulp my hve lower ioctivity thn the peel Mny reserches hve een conducted on the regultion of phenolics ccumultion in the skin, nd little is known out the flesh The im of this study ws to investigte the regultion mechnism of nthocynin nd other phenolic compounds ccumultion in the flesh, with specil ttention given to light nd temperture s two key regultory fctors Mture nd ripe fruits of three cultivrs with vried genetic ckground were studied Mterils nd methods Fruits of Jonthn nd Fuji (red cultivrs) nd Orin (yellowgreen ) were hrvested from 15-yer-old trees grfted onto Mlus prunifoli Borkh vr ringo Asmi rootstocks The trees were grown t the experimentl orchrd of the Fculty of Agriculture nd Life Science, Hiroski University Smpling dtes for mture nd ripe stges of ech cultivr were: Jonthn ; 123 nd 145 dys fter full loom (DAFB), Fuji ; 134 nd 165 DAFB nd Orin ; 131 nd 17 DAFB, respectively Three fruits of ech cultivr were equtorilly cut nd immeditely dipped in solution of Polyclr SB 1 (2 g L 1 ), in order to protect the tissues from rowning, nd then sterilized with 5 percent ethnol for 1 minute Three sectors of these smples were put into petri dishes nd covered with cler polyethylene sheet to mintin the moisture of the tissues during irrdition The other three hlves were wrpped in luminum foil, to mintin drk conditions during irrdition, s controls The irrdition ws crried out t 1, 17, 24 nd 3 using mixture of white fluorescent lmp (FL2-S W, Toshi, Tokyo; 4 W m -2 ) nd UV-B fluorescent (FL2-S E, Toshi Tokyo; 13 W m -2 ) with filter eliminting rdition elow 29 nm The irrdition ws crried out for 96 hours, continuously, nd some wter ws dily spryed on the smples during the irrdition period The irrdited surfce (lmost one millimeter in thickness) ws then seprted nd frozen in liquid nitrogen then kept t 8 until nlysis Phenolic extrction ws crried out with 15 percent cetic cid in methnol dded to 1 g of finely powdered tissue nd then kept in 4 for 2 hours These smples were centrifuged t 1, g for 15 minutes t (Himc CR 15, HITACHI, Jpn) The

19 12 Biosynthesis of phenolic compounds in pple flesh superntnt of smples ws filtered through 45μm disposle syringe filter nd used for flvonoids quntifiction y high performnce liquid chromtogrphy (HPLC) coupled to diode rry detector with wvelength set t 28, 35 nd 53 nm The column ws 15 mm ID 25 mm (Grnd C18-UG 12-5 SE, MASIS, Inc, Aomori, Jpn) with 15 mm ID 35 mm gurd column Elution solvents were () 15 percent phosphoric cid in wter of which the flow ws 9 μl min -1 nd () 15 percent phosphoric cid, 2 percent formic cid nd 25 percent cetonitrile in wter with 1 μl min -1 Smples (5 μl) were injected on to the column which ws mintined t 3 According to stndrd nlysis, identified sustnces were ctegorized to six groups; nmely, phenolic cids (chlorogenic cid), nthocynins (cynidin 3-glctoside), procynidins (procynidin B2), flvnols (ctechin nd epictechin), flvonols (quercetin glycosides), nd dihydrochlcones (phloridzin) Mture nd ripe fruit flesh of three cultivrs were used for iochemicl nlysis Results Accumultion of phenolic cids, nthocynin nd flvonols in response to UV-B irrdition: Irrdited flesh of mture nd ripe fruits of ll three cultivrs ccumulted phenolic cids, flvonols nd nthocynin (Figs 1 nd 2) Fuji nd Orin ccumulted the highest flvonols nd phenolic cids t 24 nd 3 ; wheres for Jonthn 24 ws optimum However, the concentrtion of nthocynin ws lower thn phenolic cids nd flvonols in three vrieties Chlorogenic cid of Orin ws the predominnt phenolic compound fter irrdition Totl phenolic content of Fuji incresed from mture stge to ripening ut in Jonthn nd Orin it decresed Interestingly, totl phenolic content of Fuji (efore nd fter irrdition), oth t mture nd ripe stges, ws higher thn tht of Jonthn followed y Orin Jonthn ccumulted the highest nthocynin, resulted in redder color, followed y Fuji nd Orin Anthocynin iosynthesis in the pith of receptcle re ws higher thn the cortex of receptcle Lck of response of flvnols, procynidins nd dihydrochlcones to UV-B irrdition: Irrdition did not stimulte the ccumultion of flvnols, procynidins nd dihydrochlcones oth in mture nd ripe stges in ll three cultivrs (Figs 1 nd 2) In ripe Orin the flvnols incresed slightly t 24, ut it ws not significnt Flvnols incresed with fruit ripening regrdless to irrdition temperture Flvnol content of Jonthn ws higher thn tht of Fuji nd Orin Correltion etween the ccumultion of nthocynin nd other phenolics: There ws not sme pttern of correltion etween nthocynin iosynthesis nd other phenolics in the exmined cultivrs (Tle 1) In Fuji nd Jonthn, phenolic cids nd flvonols showed positive correltion with nthocynin ccumultion, which ws not ffected y ripening In Orin, on the other hnd, the correltion coefficient vlues etween nthocynin versus phenolic cids nd flvonols decresed from mture stge to full ripening Discussion Our results showed tht the flesh of ll three cultivrs used in this study hd the potentil for ccumulting nthocynin s well s mny other polyphenolics, lthough temperture nd mturing stge ffected it The flesh of Jonthn ccumulted the highest -1 Anthocynin ( g g FW) o C Fuji Jonthn Orin 17 o C 24 o C Irrdition temperture 3 o C -1 Phenolics ( g g FW) o C Flvnols Procynidins Phenolic cids Flvonols Dihydrochlcones 17 o C 24 o C Irrdition temperture Fuji 3 o C -1 Phenolics ( g g FW) o C Flvnols Procynidins Phenolic cids Flvonols Dihydrochlcones 17 o C 24 o C Irrdition temperture Jonthn 3 o C -1 Phenolics ( g g FW) o C Flvnols Procynidins Phenolic cids Flvonols Dihydrochlcones 17 o C 24 o C Irrdition temperture 3 o C Orin Fig 1 Effect of UV-B irrdition on the ccumultion of nthocynin nd other phenolic compounds in three pple cultivrs t mture stge Phenolic content of the three cultivrs (µg g -1 FW), irrdited t 1, 17, 24 nd 3 Verticl rs represent SE

20 Biosynthesis of phenolic compounds in pple flesh 13-1 Anthocynin ( g g FW) o C Fuji Jonthn Orin 17 o C 24 o C Irrdition temperture 3 o C -1 Phenolics ( g g FW) o C Flvnols Procynidins Phenolic cids Flvonols Dihydrochlcones 17 o C 24 o C Irrdition temperture 3 o C Fuji -1 Phenolics ( g g FW) o C Flvnols Procynidins Phenolic cids Flvonols Dihydrochlcones 17 o C 24 o C Irrdition temperture Jonthn 3 o C -1 Phenolics ( g g FW) o C Flvnols Procynidins Phenolic cids Flvonols Dihydrochlcones 17 o C 24 o C Irrdition temperture 3 o C Orin Fig 2 Effect of UV-B irrdition on the ccumultion of nthocynin nd other phenolic compounds in three pple cultivrs t ripe stge Phenolic content of the three cultivrs (µg g -1 FW), irrdited t 1, 17, 24 nd 3 Verticl rs represent SE nthocynin followed y Fuji nd Orin, respectively, which is sme s the order of the nthocynin synthesis ility of the skin of these cultivrs suggesting tht the light threshold of the flesh is sme s tht of the skin While pple flesh contins low level of flvonols s reported y Awd et l (2), it incresed with light irrdition showing tht the synthesis of this group of flvonoids is light-dependent process A positive correltion etween nthocynins, phenolic cids nd flvonols, ut not the other phenolics, show tht the expression of the genes controlling the synthesis The quntity nd qulity of phenolics chnged with irrdition, depending on the irrdition tempertures Optimum temperture for the synthesis of phenolic cids, nthocynins nd flvonols ws 24, regrdless to the mturity stge nd vriety The response of flesh to light irrdition is different from tht of skin Our previous study showed tht temperture hd different effects on phenolic ccumultion in skin depending on the mturity stge; mture nd ripe fruits ccumulted the highest phenolics t 17 nd 24, respectively (Bkhshi nd Arkw, in press) Activtion of the expression of the flvonoid iosynthetic genes y enhnced light hs recently een reported in other studies (Lncster, 1992; Jkol et l, 24) All the three cultivrs exmined in this study ccumulted low mounts of phenolics t 1 which coincides with our former results on the skin Although low tempertures hve een suggested to induce Phenyllnine Ammoni-lyse (PAL) ctivity (Lncster, 1992), it seems tht low temperture could reduce the UV-B enhncement of quercetins nd chlorogenic cid Phenolic cids, procynidins, ctechins nd dihydrochlcones were the phenolics identified in the flesh mong which phenolic cids incresed significntly with irrdition This might e relted to the induction of PAL ctivity y UV-B, which medites the synthesis of coumroyl CoA Comroyl CoA then gives rise to phenolic cids nd flvonoids Anthocynin iosynthesis in the irrdited flesh of Orin tht does not nturlly ccumulte red color might e relted to the simultneous effect of UV-B nd wounding on the induction of PAL ctivity (Lncster, 1992) However, pple sectors kept in the drk during irrdition did not show ny chnge in their phenolic content showing tht only wounding is not enough for phenolic ccumultion (dt not Tle 1 Correltion coefficient vlue (r) of nthocynin versus other identified phenolic compounds in the light (white plus UV-B) irrdited flesh of Fuji, Jonthn nd Orin cultivrs Mture Ripe Phenolic clsses Fuji Jonthn Orin Fuji Jonthn Orin Flvnols 39 ns 51 ns 55 ns 47 ns 56 ns 38 ns Procynidins 19 ns 22 ns 28 ns 36 ns 58 ns 21 ns Phenolic cids 61 * 77 ** 9 ** 64 * 68 * 65 * Flvonols 84 ** 92 ** 88 ** 81 ** 93 ** 79 ** Dihydrochlcones 21 ns 34 ns 16 ns 36 ns 28 ns 12 ns *, ** Significnt t P=5% nd 1%, respectively; ns- not significnt

21 14 Biosynthesis of phenolic compounds in pple flesh shown) Other thn nthocynin nd quercetin, phenolic cids incresed with irrdition This coincides with the result of Awd et l (2) who reported tht the level of chlorogenic cid in the shded side of skin (receiving low light) ws lower thn tht of the sun exposed side In the lst two decdes, interest in using nturl ingredients in foods hs incresed drmticlly The emphsis given to nturl ntioxidnts results from concerns over the toxicity of some synthetic ntioxidnts nd from the reserch findings tht point to reltionship etween ctive dietry ingredients, such s nturl ntioxidnts, nd their protection of cells from free rdicl-induced oxidtive stress in the humn ody (Vlenzuel et l, 23) Our results show tht pple fruit is potentilly good source of phenolic compounds s nturl ntioxidnts The present study reveled tht light nd temperture hve strong effect on the phenolics ccumultion in the pple flesh s well s the skin This finding offers the exciting possiility of improving the polyphenolic content y optimizing the environmentl fctors Light irrdition on the flesh of pple lso seems to e good method for the study on the expression of the genes controlling the ccumultion of phenolic compounds References Arkw, O 1988 Photoregultion of nthocynin synthesis in pple fruit under UV-B nd Red light Plnt Cell Physiol, 29(8): Arkw, O 1991 Effect of temperture on nthocynin ccumultion in pple fruits s ffected y cultivr, stge of fruit ripening nd gging J Hortic Sci, 66(6): Arkw, O, Y Hori nd R Ogt, 1985 Reltive effectiveness nd interction of ultrviolet-b, red nd lue light in nthocynin synthesis of pple fruit Physiol Plnt, 64: Awd, M A nd A de Jger, 22 Formtion of flvonoids, especilly nthocynin nd chlorogenic cid in Jonglod pple skin: influences of growth regultors nd fruit mturity Sci Hortic, 93: Awd, MA, A de Jger nd LM Westing, 2 Flvonoid nd chlorogenic cid levels in pple fruit: chrcteristion of vrition Sci Hortic, 83: Bkhshi, D nd O Arkw, 26 Effects of UV-B irrdition on phenolic compound ccumultion nd ntioxidnt ctivity in Jonthn pple: influenced y gging, temperture nd mturtion J Food, Agric Environ, 4(1) (in press) Boyer, J nd RH Liu, 24 Apple phytochemicls nd their helth enefits Jkol, L, K Mtt-Riihinen, S Krenlmpi nd A Hohtol, 24 Acvtivtion of flvonoid iosynthesis y solr rdition in ilerry (Vccinium myrtillus L) leves Plnt, 218: Kondo, S, K Tsud, N Muto nd J Ued, 22 Antioxidnt ctivity of pple skin or flesh extrcts ssocited with fruit development on selected pple cultivrs Sci Hortic, 96: Lncster, J E 1992 Regultion of skin color in pple Crit Rev Plnt Sci, 1(6): Lncster, J E, P F Rey, J N Norris nd RC Butler, 2 Induction of flvonoids nd phenolic cids in pple y UV-B nd temperture J Hortic Sci Biotechnol, 75(2): Vlenzuel, A, J Snchuez nd S Nieto, 23 Cholesterol oxidtion: Helth hzrd nd the role of ntioxidnts in prevention Biol Res, 36(3-4): Wng, H, O Arkw nd Y Motomur, 2 Influence of mturity nd gging on the reltionship etween nthocynin ccumultion nd Phenyllnin mmoni-lyse (PAL) ctivity in Jonthn pple Posthrvest BiolTechnol, 19:

22 Journl of Applied Horticulture, 8(2): 15-18, July-Decemer, 26 * vlues to follow lycopene concentrtion during ripening nd storge of tomto (cv Cruso) Appl Journl Axelle Schuwers*, Ad MCN Roch** nd Alcin MMB Moris*** * KHo Sint-Lieven Cmpus Rot, Deprtement Industrieel Ingenieur, Ger Desmetrt 1, 9 Gent Belgium; **Fculdde de Ciêncis d Nutrição e Alimentção, Universidde do Porto, Ru Dr Roerto Fris, Porto, Portugl; ***Corresponding uthor: Escol Superior de Biotecnologi, Universidde Ctólic Portugues, Ru Dr António Bernrdino de Almeid, Porto, Portugl E-mil: moris@esucppt Astrct The ripening of tomto fruit is highly regulted process during which colour, flvour, rom nd texture chnge in coordinted mnner This reserch work ims to correlte the colour chnges mesured ojectively with the lycopene concentrtion in tomtoes during ripening t room temperture (21 nd 26 C) These results were compred with colour nd lycopene content of pink nd light red tomtoes stored t 14 C, temperture used to prevent ripening nd therefore extend the shelf life of the fruits The durtion of het tretment t 1ºC ws previously optimized in order to relese the mximum lycopene from chromoplsts during extrction An * vlue of 2 for the peel corresponds to n increse of lycopene content of Cruso tomto from 9 to 43 mg/1 g TSS, t room temperture The shelf life of pink nd light red tomtoes cn e extended to two weeks t 14ºC without loss of lycopene content, presenting the sme content s green tomtoes ripened t room temperture for one week Keywords: Tomto, Lycopersicon esculentum Mill, lycopene, colour, ripening, storge Introduction The composition of tomto (Lycopersicon esculentum Mill) in crotenoids differs qulittively nd quntittively with fctors such s cultivr or vrietl differences, stge of mturity, climtic or geogrphic effects, prt of the plnt, conditions during production, posthrvest hndling, processing nd storge (Deli nd Rodriguez-Amy, 23) Crotenoids re responsile for the plesing yellow, ornge or red colour of mny foods The role of some of these compounds s provitmin A precursors hs een known for yers Other eneficil effects to humn helth which hve een more recently ttriuted to crotenoids re of enhncement of the immune response nd reduction of the risk of degenertive diseses such s cncer, crdiovsculr diseses, ctrct nd msculr degenertion, these ctions not eing restricted to the provitmin A (Olson, 1999) Since the humn ody is unle to synthesize crotenoids from endogenously produced iochemicls, the ody is totlly dependent on dietry sourced (exogenous) crotenoids The principl crotenoids in tomto re et-crotene, etcryptoxnthin, lycopene, lutein nd zexnthin Lycopene is inctive vitmin A ut is more efficient ntioxidnt thn et-crotene (Di Mscio et l, 1989) It hs een linked with reduction of the risk of cncer, especilly lung, stomch nd prostte cncer (Giovnnucci, 1999) In nture, crotenoids re protected y the cellulr structure of the plnt tissues On the other hnd, this nturl protection lowers iovilility The intctness of the cellulr mtrix determines the iovilility of different nutrients (Vn het Hof et l, 2) Het tretment cn hve deleterious effect on the micronutrient content of vegetles ut t the sme time the iovilility of some nutrients cn increse There exist lot of different opinions whether processed tomtoes contin higher lycopene concentrtion or not Food processing my improve lycopene iovilility y reking down cell wlls, which wekens the onding forces etween lycopene nd tissue mtrix, thus mking lycopene more ccessile (Deli nd Rodriguez-Amy, 23; Dewnto et l, 22; Gärtner et l, 1997; Sthl nd Sies, 1992; Thompson et l, 2) Other uthors lso estlished heting t 1 C to relese the mximum lycopene from the chromoplsts (Shrm nd Le Mguer, 1996) In other studies, heting of tomto products under different processing conditions my cuse degrdtion of lycopene (Cole nd Krpur, 1957; Klui nd Buernfeind, 1981; Miki nd Aktsu, 197; Nole, 1975) Dt on the effects of processing on the crotenoid content of foods re therefore conflicting The wy of clculting the lycopene concentrtion is most importnt When heting tomto pulp the lycopene content in pulp increses However, this increse is directly relted to the increse in tomto totl solule solids (TSS) concentrtion during heting In fct, lycopene content in pulp my decrese (Shrm nd le Mguer, 1996) when expressed per mss of TSS In severl of pulished ppers (Gärtner et l, 1997; Sthl nd Sies, 1992; Thompson et l, 2; Khchik et l, 1992; Nguyen nd Schwrtz, 1999; Nicoli et l, 1999), the uthors did not tke into ccount the increse of tomto TSS In consequence, it seemed tht the lycopene level incresed Tomto ripening is regulted process during which colour, flvour, rom nd texture chnge in coordinted mnner Tomto fruit colour is n importnt qulity ttriute, nd it is the initil spect evluted y the consumers Fruit colour is

23 16 Lycopene in tomto t different stges of ripening relted to the lycopene nd chlorophyll content chnges during the ripening process (Brmley, 1997) Tomto fruits re chilling sensitive nd the recommended storge temperture vries with the mturity stge (Hrdenurg et l, 1986) Temperture mngement is criticl to mintin qulity Ripe tomto fruits cn e stored t 1 C, without visile symptoms of chilling injury, lthough flvour nd rom my e negtively ffected (Mul et l, 2) This reserch work imed to correlte the colour chnges mesured ojectively with the lycopene concentrtion in tomtoes during ripening t room temperture (21 nd 26 C) An ojective of the work ws lso to compre these results with colour nd lycopene content of pink nd light red tomtoes stored t 14 C, temperture used to extend the shelf life of the fruits (Hrdenurg et l, 1986) Mterils nd methods Plnt mteril: Tomto (cv Cruso) t different stges of ripening (ccording to the experiment) were purchsed t locl hypermrket (Mtosinhos, Portugl) Het tretment: Red tomtoes (stge 5, light red, nd stge 6, red, USDA, 1975) were cut into pieces nd the seeds were removed The tomto ws lended in plstic eker covered with luminium foil, to prevent the destruction of the lycopene y light The lending ws continued till rough, smooth tomto pulp ws formed Afterwrds the rough tomto pulp ws further homogenized for 1 min with homogenizer (Ultr-Turrx T25, Jnke & Kunkel, IKA-Lortechnik, Germny) The homogente ws divided into two frctions One frction ws kept to nlyze the lycopene content in the fresh tomto nd the other frction ws heted to 1 C with mgnetic stirrer t mximum speed After strting to oil, the tomto pulp ws left simmering for different periods of time (5 to 15 min) t 1 C Both heted nd non-heted smples were collected nd 5 g from ech smple were weighed in glss ekers covered with luminium foil Lycopene content ws determined in these smples Ripening experiment: Tomtoes t the stge of rekers (stge 2, USDA, 1975) were exposed to light t room temperture (21 nd 26 C) nd left to ripen during 1 dys This colour stge ws chosen ecuse consumers my purchse tomtoes t this stge nd leve them to ripen t room temperture Tomtoes were nlysed for colour nd lycopene content ech dy Three replictes of three tomtoes were used for ech dy Storge experiment: Pink nd light red tomtoes (stges 4, pink, nd 5, light red, USDA, 1975) were stored t 14 C (this temperture is recommended to stop the ripening process nd store tomtoes of stge 4 nd stge 5) nd reltive humidity 9-95% for two weeks (Hrdenurg et l, 1986) These colour stges were chosen ecuse consumers usully uy tomtoes t these ripening stges The colour nd lycopene content were nlysed during tht time Three replictes of three tomtoes were used for ech colour stge nd for ech dy Colour determintion: The colours of peel nd pulp were mesured y using hnd-held tristimulus reflectnce colorimeter (Minolt CR-3, Minolt Corp, Rmsey, New Jersey, USA) Colour ws expressed s the CIE-L* * * uniform colour spce L* indictes lightness, * chromticity on green (-) to red (+) xis, * the chromticity on lue (-) to yellow (+) xis Numericl vlues of * nd * were converted into Hue ngle (H = tn -1 * /* ) nd chrom ( Chrom = (* 2 + * 2 ) 1/2 ) (Frncis, 198) Ten mesurements were performed on ech tomto Tomto colour ws lso clssified ccording to the USDA colour chrt (USDA, 1975) Lycopene determintion: The method used for the determintion of lycopene concentrtion ws dpted from Goul et l (26) A smple of 5 g of homogenized tomto pulp (fresh or processed t 1 C) ws mixed with cetone nd petroleum ether nd stirred in glss eker The mixture ws trnsferred to Kitzto nd connected to vcuum pump The extrct ws wshed three times with distilled wter The cetone ws then removed helping to prevent the formtion of stle emulsions The sornce ws mesured t 55 nm using Perkin-Elmer Lmd 15 UV/VIS spectrophotometer (Shimdzu Corp, Tokyo, Jpn) fter clirtion with petroleum ether Lycopene determintion ws performed in triplicte The extinction coefficient used in the clcultions ws 282 for 1% (w/v) solution (Person, 1976) nd the lycopene content expressed in mg/ 1 g TSS Totl solule solids determintion: The mount of totl solule solids ws determined t room temperture y using refrctometry (hnd-held Atgo refrctometer model ATC1) Sttisticl nlysis: The descriptive nlysis consisted of the clcultion of men nd stndrd devition for crdinl vriles nd of frequencies for ordinl vriles Kolmogorov-Smirnov test ws used to verify the normlity of the distriution of crdinl vriles The Mnn Whitney test ws used to compre medium orders of two independent smples nd the Kruskl-Wllis to compre medium orders of three or more independent smples (non prmetric tests) The Spermn correltion coefficient ws clculted to quntify the intensity etween pirs of vriles (non prmetric correltion) (Finney, 198) P level of criticl significnce ws used to reject null hypothesis (rejected when P<5) Results nd discussion Het tretment: The effect of different heting times ws investigted In the eginning, it ecme cler tht the heting time optimum ws etween the 5 nd 1 min heting The mximum lycopene content seems to correspond to heting time etween 5 nd 7 min (dt not shown) For further experiments 6 min heting time ws used in order to mximize the relese of lycopene from tomto tissues Ripening experiment: At colour stge 1, tomtoes re still green, mening, there is no red pigment, so there is no lycopene present At colour stge 2 the level of lycopene is still very low Only when tomto chnged from colour stge 4 to stge 5, the lycopene level increses rpidly ecuse during this chnge the tomto turns from pink to light red Differences etween lycopene concentrtion of level 4 nd 5 were sttisticlly significnt (P<2) t oth tempertures Levels of round 4 mg/ 1 g

24 Lycopene in tomto t different stges of ripening 17 TSS were chieved for tomtoes t colour stges 5 nd 6 (Tle 1) Stge 5 ws chieved fter 7 dys of storge while stge 6 ws chieved fter 1 dys Lycopene concentrtion did not increse from stge 5 to stge 6 (Tle 1) Tle 1 Lycopene concentrtion in heted tomto smples during ripening t 21nd t 26ºC Ripening temperture ( C) Colour stge (USDA, 1975) Lycopene (mg/ 1 g TSS) Stndrd devition A strong positive curviliner reltionship ws found etween the * vlues of tomto peel nd pulp nd lycopene concentrtion t oth tempertures (21 nd 26ºC) Low lycopene contents correspond to low * vlues For * vlues round 15 (pulp) nd 2 (peel) the lycopene concentrtion incresed ruptly, corresponding to the chnge from stge colour 4 to stge colour 5 (Figs 1 nd 2) For high vlues of * (colour stge 5 nd 6), the peel presented higher vlues thn the pulp This mens tht the red colour is more intense in the peel, proly ecuse the peel contins higher lycopene concentrtion thn the pulp No correltion ws found for the other colour prmeters (L*, *, hue nd chrom) (dt not presented) Storge experiment: After two weeks of storge t 14 C pink nd light red tomtoes presented no symptoms of chilling injury No pitting or decy (Wills et l, 1998) were oserved which indictes tht this temperture ws dequte for storge of tomtoes t these mturity stges After two weeks, tomtoes t these stges of colour exhiited lycopene contents (Tle 2) similr to the green tomtoes (stge 2) ripened to the sme stges of colour (ripening experiment) fter 6 nd 7 dys of storge, respectively The sme tendency ws oserved for the peel nd pulp (Figs 3 nd 4) Tle 2 Lycopene concentrtion of heted tomto smples fter two week storge t 14ºC Colour stge Lycopene Stndrd (USDA, 1975) (mg/ 1 g TSS) devition Processing tomtoes is vlue-dded step in terms of lycopene iovilility By heting processed tomtoes (lended nd simmered) t 1 C for 6 min, the iovilility of lycopene seems to increse, due to rekdown of the cell wlls of the tomto pulp The * prmeter (CIE-L***) is good prmeter for quntifying the lycopene level in tomtoes The evolution from colour stge 4 to colour stge 5 t room temperture (21 nd 26 C), involves drmtic increse in the lycopene content from 9 mg 1g -1 TSS to 43 mg 1g -1 TSS This hppens for * vlues round 15 for the pulp nd 2 for the peel The ltter is good Lycopene concentrtion (mg 1 g -1 TSS) Peel Pulp * vlue Fig 1 Lycopene concentrtion of heted tomto smples in function of * vlue during ripening t 21ºC Lycopene concentrtion (mg 1 g -1 TSS) Peel Pulp * vlue Fig 2 Lycopene concentrtion of heted tomto smples in function of * vlue during ripening t 26ºC Lycopene concentrtion (mg 1 g -1 TSS) 14º C 21º C 26º C Peel * vlue Fig 3 Lycopene concentrtion of heted tomto smples in function of * vlue of the peel during ripening t 21ºC nd 26ºC nd during storge t 14ºC Lycopene concentrtion (mg 1 g -1 TSS) 14º C 21º C 26º C Pulp * vlue Fig 4 Lycopene concentrtion of heted tomto smples in function of * vlue of the pulp during ripening t 21ºC nd 26ºC nd during storge t 14 o C

25 18 Lycopene in tomto t different stges of ripening indictor for the consumption of tomto (cv Cruso) since it corresponds to high lycopene content nd colour mesurement of the peel y CIE- L*** which is not destructive method The storge temperture (21 or 26ºC) does not influence the lycopene content for specific stge of ripening Pink nd light red tomtoes (colour stges 4 nd 5), usully purchsed y consumers, cn e successfully stored for two weeks t 14 C, presenting lycopene contents similr to green tomtoes (colour stge 2) fter eing exposed to room temperture for one week, turning to light red (colour stge 5) tomtoes References Brmley, PM 1997 The regultion nd genetic mnipultion of crotenoid iosynthesis in tomto fruit Pure & Applicle Chemistry, 69(1): Cole, ER nd NS Krpur, 1957 The stility of lycopene II Oxidtion during heting of tomto pulps Journl Science Food Agriculture, 8: Deli, B nd DB Rodriguez-Amy, 23 Enhncing the crotenoid levels of foods through griculture nd food technology Deprtmento de Ciênci de Alimentos, Fculdde de Engenhri de Alimentos, Universidde Estdul de Cmpins em Brsil Dewnto, V, XZ Wu, KK Adom nd RH Liu, 22 Therml processing enhnces the nutritionl vlue of tomtoes y incresing totl ntioxidnt ctivity Journl Agriculturl Food Chemistry, 5: Di Mscio, P, SP Kiser nd H Sies, 1989 Lycopene s the most efficient iologicl crotenoid singlet oxygen quencher Arch Biochemistry nd Biophysics, 274: Finney, DJ 198 Sttistics for iologists Chpmn nd Hll London Frncis, DM 198 Breeding for color nd lycopene content in dpted tomto germplsm Dept of Horticulture nd Crop Science, Ohio Agriculturl Reserch nd Development Center, 168 Mdison Ave, Wooster, OH Gärtner, C, W Sthl nd H Sies, 1997 Lycopene is more ioville from tomto pste thn from fresh tomtoes Americn Journl Clinics Nutrition, 66(1): Giovnnucci, E 1999 Tomtoes, tomto-sed products, lycopene nd cncer Review of the epidemiologic literture Journl of the Ntionl Cncer Institute, 91(4): Goul, AM, KG Admopoulos, PC Chtzitkis nd VA Niks, 26 Prediction of lycopene degrdtion during drying process of tomto pulp Journl of Food Engineering, 74(1): Hrdenurg, RE, AE Wtd nd CY Wng, 1986 The commercil storge of fruits, vegetles, nd florist nd nursery stocks USDA Agriculturl Hndook 66, 13 pp Khchik, F, MB Goli, GR Beecher, J Holden, WR Lusy, MD Tenorio nd MR Brrer, 1992 Effect of food preprtion on qulittive nd quntittive distriution of mjor crotenoid constituents of tomtoes nd severl green vegetles Journl of Agriculture nd Food Chemistry, 4: Klui, H nd JC Buernfeind, 1981 Crotenoids s colournts nd vitmin A precursors Acdemic Press, London, pp Mul, F, SA Srgent, CA Sims, EA Bldwin, MO Bln nd DJ Huer, 2 Recommended commercil storge tempertures ffect tomto flvor nd rom qulity Journl of Food Science, 65(7): Miki, N nd K Aktsu, 197 Effect of het steriliztion on the colour of tomto juice Nihon Shokuhin Kogyo Gkki, 17: Nguyen, ML nd SJ Schwrtz, 1999 Lycopene: Chemicl nd iologicl properties Food Technology, 53(2): Nicoli, MC, M Anese nd M Prpinel, 1999 Influence of processing on the ntioxidnt properties of fruit nd vegetles Trends in Food Science nd Technology, 1: 94-1 Nole, AC 1975 Investigtion of the colour chnges in het concentrted tomto pulp Journl of Agriculture nd Food Chemistry, 23: Olson, JA 1999 Crotenoids In: Modern Nutrition in Helth nd Disese, eds ME Shils, JA Olson, M Shike, AC Ross, 9th edition, Willims & Wilkins, Bltimore, pp Person, D 1976 Tomto products In: The chemicl nlysis of foods, 7 th edition, Churchill Livingstone, Edinurg, London, New York, pp Shrm, SK nd M Le Mguer, 1996 Kinetics of lycopene degrdtion in tomto pulp solids under different processing nd storge conditions Food Reserch Interntionl, 29(3-4): Sthl, W nd H Sies, 1992 Uptke of lycopene nd its geometricl isomers is greter from het-processed thn from unprocessed tomto juice in humns Journl of Nutrition,122: Thompson, KA, MR Mrshll, CA Sims, CI Wei, SA Srgent nd JW Scott, 2 Cultivr, mturity nd het tretment on lycopene content in tomtoes Journl of Food Science, 65(5): USDA 1975 Visul Aid TM-L-1, Nutrient Dt L, Agriculture Reserch Service, US Deprtment of Agriculture, Beltsville Humn Nutrition Reserch Center, Riverdle, MD Vn het Hof, KH, BCJ De Boer, LBM Tijurg, BHM Lucius, I Zijp, CE West, JGAJ Hutvst nd JA Weststrte, 2 Crotenoid iovilility in humns from tomtoes processed in different wys determined from the crotenoid response in the tri-glyceride-rich lipoprotein frction of plsm fter single consumption nd in plsm fter four dys of consumption Journl of Nutrition, 13: Wills, RBH, B McGlsson, D Grhm nd D Joyce, 1998 Posthrvest: An introduction to the physiology nd hndling of fruits, vegetles nd ornmentls University of New South Wles Press, 262 p

26 Journl of Applied Horticulture, 8(2): , July-Decemer, 26 Effects of usive tempertures on the posthrvest qulity of lettuce leves: scoric cid loss nd microil growth Appl Journl Mrí del R Moreir *, Alejndr G Ponce, Crlos E del Vlle, R Ansoren nd SI Rour Universidd Ncionl de Mr del Plt, Argentin; Consejo Ncionl de Investigciones Científics y Técnics (CONI- CET), Argentin*Corresponding uthor: Moreir Mrí del Rosrio, Grupo de Investigción en Ingenierí en Alimentos, Fcultd de Ingenierí, Universidd Ncionl de Mr del Plt (UNMDP)- Argentin Jun B Justo 432, CP: 76 Mr del Plt Provinci de Buenos Aires Argentin E-mil: moreir_mri@fimdpedur Astrct Chnges in lettuce lef qulity (scoric cid contents nd microil popultions) t two usive tempertures (8 nd 15ºC), simulting the commercil storge conditions for fresh vegetles were nlyzed A storge temperture of 8 C ws chosen to simulte usive refrigerted storge nd storge temperture of 15 C ws chosen to simulte room temperture Qulity indictors evlution in smples, stored t usive tempertures were compred with smple from optiml storge temperture ( C) First order kinetics is ssumed for scoric cid degrdtion Ascoric cid degrdtion rte in lettuce leves stored t usive tempertures ws from 27 to 29 times fster thn t C The growth curve of totl microil counts ws fitted with the Gompertz nd Logistic models These models llowed us to predict the vegetle microiologicl shelf life Temperture is the controlling fctor for lettuce shelf life nd qulity; microil qulity ws retined 16 nd 4 times longer t ºC with respect to 8 nd 15ºC, respectively Key words: Ausive temperture storge, nutritionl qulity, microiologicl qulity, shelf life, growth models, lettuce, Lctuc stiv, scoric cid retention Introduction Vegetles re importnt components in lnced humn diet They re low in ft, low in clorie with high crohydrte nd fier contents They provide significnt levels of some micronutrients Fresh vegetles hve short shelf life when they re exposed to conditions tht destroy their qulity ttriutes Fresh vegetle qulity is ffected y vrious fctors, including post-hrvest hndling nd storge conditions (temperture, reltive humidity, light nd tmosphere composition) Minimum sfe low tempertures nd high reltive humidity control re the most importnt tools for extending the shelf life of most fresh vegetles Temperture is the single most importnt vrile nd its improper mnipultion cuses evident chnges in the sensory chrcteristics of fresh vegetles tht condition consumers cceptility Ausive storge tempertures lso led to reduce levels of some nutrients, minly scoric cid, nd fvor microorgnisms prolifertion to levels tht my exceed tolerle levels (Frncis et l, 1999) Fresh fruits nd vegetles proly receive the gretest temperture use t the retil level Temperture use is function of time nd temperture during holding nd the reltive perishility of prticulr commodity (Pull, 1999) Fresh vegetles re mjor source of scoric cid, nutrient tht esides its vitmin ction, is vlule for its ntioxidnt effects, its stimultion of the immune system nd other helth enefits tht re eing ctively investigted nd reported (Ginnkourou nd Toukis, 23) While the ssessment of scoric cid content does not represent complete nutritionl evlution, this compound is very lile nd therefore it gives sensitive index of reltive nutritionl qulity (Perrin nd Gye, 1986) Another sic phenomenon tht confronts the extension of fresh vegetles shelf life is the microorgnisms prolifertion Disese-cusing microorgnisms growth is food sfety concern, especilly with high ph vegetles Visile growth nd off-odors cused y microorgnisms re lso ojectionle Vrious tretments nd strtegies hve een evluted to id in the mngement of post-processing microil popultions, eg use of snitizing solutions (chlorine wter, hydrogen peroxide, sodium dichloroisocynurte, etc), modified tmosphere control nd irrdition with gmm source (Zgory, 1999) However, most of the reserch evluting the efficiency of snitizer for eliminting microorgnisms to dte hs not een promising (Brckett, 1999) The first few hours fter hrvest cn e crucil for the fresh vegetles shelf life Although tempertures close to C re recommended, fresh vegetles my e prepred, shipped nd stored t 5 C nd, sometimes s high s 1 C (Wtd et l, 1996) Hndling t this elevted level cn hsten deteriortion ecuse Q 1 of iologicl rections rnge from 3 to 4 nd possily s high s 7 within this temperture region (Wiley, 1997) The prmount mrketing tool for fresh vegetles is strict voidnce of temperture use during processing, distriution, nd merchndising to chieve the required or ner mximum shelf life for ech product (Schlimme, 1995) Although reserch re needed to determine the kinetic prmeters tht help to model the chnges in qulity ttriutes of fresh products (Ginnkourou nd Toukis, 23), these studies were done in similr conditions found in commercil prctices The significnce of lortory studies nd

27 11 Effects of usive tempertures on lettuce qulity to lesser extent simulted shipping studies my not e relevnt to commercil prctices in mny cses (Pull, 1999) The purpose of the present work ws to nlyze chnges in lettuce lef qulity kept t two usive tempertures (8 nd 15 ºC) simulting the commercil sles prctice of fresh vegetles A storge temperture of 8 C ws chosen to simulte usive refrigerted storge nd storge temperture of 15 C ws chosen to simulte room temperture Qulity indictors evlution in smple stored t usive tempertures ws compred to the optiml storge temperture ( C) Qulity chnges of lettuce leves were nlyzed through nutritionl indictors (scoric cid retention), microiologicl (totl microil counts) nd other physicochemicl prmeters such s solule solids contents, wter loss nd weight loss In the present work, only tp wter immersion ws used to eliminte superficil microflor; in this wy, only temperture effects on microil evolution ws evluted In ddition, Gompertz nd Logistic models were pplied to predict the vegetle microiologicl shelf life Mterils nd methods Smple preprtion: Heds of Romine lettuce (Lctuc stiv, type Cos, vriety Logifoli) were hrvested t optiml mturity when they reched mrketle size (pproximtely 2-24 leves per hed) They were immeditely trnsported to the lortory To reduce nturl vriility mong smples, lettuce leves were sorted for integrity, color nd size uniformity, nd lck of defects Outer leves were discrded nd only photosynthetic leves (green leves) were included in the smples Lettuce leves were dipped in tp wter for 4 min t room temperture (2 C) in rtio of one prt of lettuce for 1 prts of wter Lettuce leves were then centrifuged for 3 sec t 2 rpm in sld spinner to remove excess wter Leves were piled up in 12 g stcks nd plced in polyethylene gs (25 x 2 cm, useful volume: 18 l) with n O 2 permeility of 52-4 cm 3 m -2 dy -1, CO 2 permeility of 39-1 cm 3 m -2 dy -1 nd wter vpor of 4-1 g m -2 dy -1 Smples were plced in oxes with overll dimensions of 4 x 3 x 3 m, mde of hevy-duty, 6 cm thick, trnsprent crylic, with 97-99% reltive humidity, nd stored t, 8 nd 15 C for pproximtely 2 h In smples stored t 15ºC scoric cid nd microiologicl determintions were stopped erlier ecuse, from sensoril point of view, they were deteriorted eyond ny commercil vlue Physicochemicl determintions: Smples were weighed to determine weight loss during storge Moisture ws determined y the weight loss of 1 g smples fter 24 h t 8 C (Bstrsh et l, 1993) 3 g of lettuce lef juice ws otined with home juice mker nd centrifuged t 2 rpm for 5 min Juice smples were diluted (1:1) with distilled wter Solule solids were determined on the diluted smples with n Ae refrctometer (Kim et l, 1993) At ech smpling period, two smples from ech experimentl condition were tken for the nlysis Anlyses were performed on ech smple in duplicte Determintion of scoric cid: Ascoric cid contents were determined ccording to Moreir et l (23) Lettuce leves were ground with tissue homogenizer (Multiquick, MR 555 CA, Brun) for 1 min Twenty grms of lettuce ws homogenized with 4 ml of oxlic cid solution 5 g / 1 (w/w) The mixture ws filtered through Whtmn # 42 filter pper Aliquots (1 ml) of the filtrte were titrted with 2,6-dichloroindephenol To prepre solutions, copper-free wter ws used Since copper my e present in the lettuce smples, titrtion ws performed rpidly to minimize interfering effects Ascoric cid contents re reported s mg / 1g smple on wet sis Ascoric cid determintion ws performed on three different experimentl runs t ech storge temperture For ech experimentl run two smples were tken nd nlysis ws performed in triplicte Microil popultion: Ground lettuce (1 g) ws mcerted in uffer solution (PO 4 K 3, ph = 72) Totl microil counts were done on plte count gr (PCA) fter incution for 48 h t 35 C (ICMSF, 1983, Mossel nd Moreno Grcí, 1985) Microil counts in fresh lettuce leves were performed on three independent experimentl runs for ech storge condition nlyzed At ech smpling time, two smple lots were nlyzed y duplicte Primry growth models ppliction: Experimentl vlues were fitted with the Gompertz model (Zwietering et l, 199) nd the Logistic model (Ricker, 1979) The Gompertz model is: log N Where, log N is the deciml logrithm of microorgnisms counts t time t; A is the symptotic vlue when time decreses indefinitely (pproximtely equivlent to the deciml logrithm of the initil microorgnisms counts); C represents the increse in the logrithm of microorgnisms counts when time increses indefinitely (numer of log cycles); B is the mximum rte of growth relted to time M nd M is the time required to rech the mximum rte of growth The logistic model is: (Ricker, 1979) log N Where, Log N, A nd C hs the sme mening s in the Gompertz model D is dimensionless prmeter nd F is the rte of growth reltive to the men time of the exponentil phse Sttisticl nlysis: Liner correltions significnce ws tested ccording to Volk (198) Differences mong slopes for the evolution of scoric cid t different storge tempertures were tested s indicted y Volk (198) Whenever differences re reported to e significnt, 95% confidence level ws used Estimtion of the prmeters in oth Gompertz nd the Logistic models ws done with the function lsqcurvefit from Mthl 7 Results nd discussion Physicochemicl prmeters: Lettuce leves weight losses during storge re presented in Fig 1 The weight loss incresed with the storge tempertures nd, fter 17 h of storge, they mounted to 6, 1 nd 16 g / 1 g (w/w) in smples held t, 8 nd 15 C, respectively Weight loss of fresh vegetles cn e ttriuted to: (1) evportion of surfce wter remining on the product fter wshing; (2) dehydrtion, tht is wter loss due to the difference in wter vpor pressure etween the surrounding tmosphere nd the foodstuff; nd (3) respirtion, which consist in the rekdown of crohydrtes to produce cron dioxide nd wter Since the smples were kept in polyethylene gs with very low permeility to wter vpor nd with reltive humidity

28 Effects of usive tempertures on lettuce qulity 111 Weight loss(%) Storge time (hour) Fig 1 Lettuce lef weight loss, s percentge of fresh weight, during storge t ºC ( ) (y= 43x 343, R= 94), 8ºC ( ) (y= 59x , R= 97) nd 15ºC ( ) (y= 94x + 432, R= 98) ove 98%, weight loss would e due to respirtion Initil wter contents in lettuce leves were 9456 ± 53 g / 1 g (w/w) After 17 h of storge the wter contents of smples stored t C were 9484 ± 21 g / 1 g (w/w) In smples stored t 8 C they were 9514 ± 29 g / 1 g nd in smples stored t 15 C they were 9583 ± 6 g / 1 g (w/w) These increses in wter contents would correspond to wter produced y respirtion While cron dioxide is lost to the tmosphere, wter is retined y the food nd weight loss is independent of moisture loss (Sstry nd Buffington, 1982) Polyethylene gs provide high reltive humidity, so dehydrttion typiclly is not prolem However, moisture my condense on the inner surfce of the g, detrcting visul ppernce nd providing conditions for microil growth Initil solule solid contents in lettuce leves were 39 ± 24 Brix After 17 h of storge, the solule solids contents of smples held t nd 8 C were not significntly different from the initil vlue (P<1) However, in smples stored t 15 C the finl solule solids contents were significntly lower (P<1) with vlue of 36 ± 32 Brix This decrese in solule solids could e ttriuted to sugr consumption through respirtion Ascoric cid contents: The initil scoric cid contents of lettuce leves rnged etween 6 nd 166 mg/1 g of smple with men vlue of 19 ± 29 mg / 1 g (n=4) Moreir et l (25) nd Rour et l (23) reported similr initil men vlues for scoric cid contents in lettuce leves (83 ± 1 nd 94 ± 16 mg / 1 g of smple, respectively) The wide rnge of vlues for the initil scoric cid contents in lettuce leves would reflect the influence of vrious fctors such s climte conditions, culturl prctices, mturity t hrvest, hrvesting method nd post-hrvest hndling conditions (Lee nd Kder, 2) Of course, the inherent vriility in iologicl systems would lso contriute to this vriility First order kinetics is ssumed for scoric cid degrdtion Therefore, the nturl logrithm of scoric cid contents rtio to the initil scoric cid contents ginst time should fll on stright line with the slope representing the pprent rection constnt Ascoric cid contents in lettuce leves kept t the different storge temperture re presented in this wy in Fig 2 The slopes of the stright tendency lines for smples stored t 8 nd 15 C were not significntly different etween themselves Ln AA/AA Storge time (hour) Fig 2 Ascoric cid degrdtion (ln AA/AA ) in lettuce leves stored t ºC ( ) (y= -36x 184, R= 76, N= 7), 8ºC ( ) (y= -98x + 538, R= 93, N= 56) nd 15ºC ( ) (y= -16x - 65, R= 92, N=32) AA is the scoric cid content (mg AA/1 g smple) t ny time of storge nd AA (mg AA /1 g smple) t time zero Ascoric cid determintion ws performed on three different experimentl runs t ech storge temperture For ech experimentl run two smples were tken nd nlysis were performed in triplicte ut were significntly different (P<1) from the slope of the tendency line for smples stored t C These results indicte tht scoric cid degrdtion rte in lettuce leves stored t usive tempertures ws from 27 to 29 times fster thn t C After 2 h of storge t C scoric cid retention in lettuce leves ws out 46 g / 1 g In smples kept t 8 C, the retention ws out 14 g / 1 g Smples held t 15 C were only kept for 1 h, time t which they were completely spoiled At tht time, the scoric cid retention ws lredy down to 22 g / 1 g Tulio et l (22) nlyzing the effects of storge temperture on the posthrvest qulity of jute leves reported tht the scoric cid content declined in ll storge temperture conditions s storge period ws incresed However, the reduction in the scoric cid contents ws grdul t low storge tempertures ut rpid t high storge tempertures Lefy vegetles lose scoric cid in posthrvest opertions nd it hs een ttriuted to oth temperture nd wter loss (Kder, 1999) Chnges in wter contents in the smples kept in polyethylene gs were miniml nd would not suffice to explin the losses in scoric cid Ascoric cid losses could e ttriuted to temperture effects nd tissue structurl chnges due to iologicl deteriortion fctors (senescence nd microil ctivity) Microil growth: Initil totl microil counts were in the rnge of 4 to 44 x 1 6 CFU/ g Similr results hd een found in previous work (Rour et l, 23) Totl microil counts on lettuce leves during storge re presented in Fig 3 s log CFU/ g versus time Experimentl results re widely scttered This my e ecuse determintions were done on three different experimentl runs on smples of n ctul foodstuff, with the concurring vriility ssocited to iologicl mteril The growth curve of totl microil counts fitted with the Gompertz nd Logistic models including the r 2 vlues re shown in Fig 3 Prmeter A in oth models ws fixed t the log of the men initil counts on lettuce leves (682 CFU/ g) Both models produced very similr ptterns for totl microorgnisms growth in smples stored t C (Fig 3A), with n incution

29 112 Effects of usive tempertures on lettuce qulity log (CFU/g) log (CFU/g) log (CFU/g) 8 78 A Storge time (hour) B Storge time (hour) C Storge time (hour) Fig 3 Growth curve of totl microil during storge t ºC (A), 8ºC (B) nd 15ºC (C) fitted with the Gompertz (--x--, R= 87) nd Logistic models ( _ _, R= 85) phse lsting out 2-25 h, logrithmic growth phse nd stiliztion phse with logrithmic of microil popultions round 765 CFU/ g For smples stored t 8 C, however, the est fit for the logistic model indictes tht the growth of totl cteri is still in the exponentil phse fter 2 h of storge (Fig 3B) On the other hnd, the est fit for the Gompertz model, indictes tht the stiliztion phse would hve een reched y 175 h of storge The logrithm of microil popultions reched t 8ºC ws round 87 CFU/ g, tht is, out one log cycle ove the stiliztion vlue for smples stored t C Finlly, for smples stored t 15 C, est fits for oth models indicted tht the totl microil growth ws in the exponentil phse fter 1 h of storge (Fig 3C) It should e pointed out tht smples were stored t 15 C for only 1 h ecuse, y tht time, they hd deteriorted eyond ny commercil vlue French regultions imposed 8ºC s mximum storge temperture for minimlly processed vegetles This limit ws lowered to 4ºC, ut vegetles re often stored or distriuted t higher tempertures This legisltion llows mximum of 5 x 1 7 CFU/ g for the sfe consumption of minimlly processed vegetles (Frncis et l, 1999) Accepting this vlue s threshold tht must not e surpssed, from cteriologicl point of view, the keeping time of fresh lettuce would e round 8 dys for lettuce kept t C; 5 dys for lettuce kept t 8 C nd 2 dys for lettuce kept t 15 C The min spects of this study were the estlishment of vitmin C losses nd microil counts for Romine lettuce leves kept t two usive tempertures nd the comprtive estimtion of their shelf life with respect to the idel storge temperture (ºC) For scoric cid degrdtion nd microil evlution, the profiles otined were supported through high numer of experimentl dt ( men of 52 dt per fitted curve) during 1-2 hours fter hrvest, in order to e le to predict the qulity prmeters in relile wy Exposure to undesirle tempertures resulted in fster scoric cid degrdtion rtes detrimentl to nutritionl qulity The rte of degrdtion of scoric cid in lettuce leves stored t usive tempertures would e from 27 to 29 times fster thn t C If fruits or vegetles re hndled t usive tempertures, snitizer gents pplied to reduce initil microil counts will do little to extend shelf life or limit microorgnism growth Mishndling of these products, exposing them to usive tempertures, would mke microil levels go right ck up to pre-wsh levels or higher In the present work, only tp wter immersion ws used to eliminte superficil microflor; in this wy, only temperture effects on microil counts were evluted Model cn e powerful tool for microil nlysis, quickly providing n initil estimte of microorgnism s ehvior In this work, two primry models (Gompertz nd Logistic), were compred for their ility to descrie the totl microil counts s function of storge tempertures pplied to lettuce These models llowed us to predict the vegetle microiologicl shelf life Temperture is the controlling fctor for lettuce shelf life nd qulity; microil qulity ws retined 16 nd 4 times longer t ºC with respect to 8 nd 15ºC, respectively Acknowledgments This work ws supported y Consejo Ncionl de Investigciones Científics y Técnics (CONICET) nd Universidd Ncionl de Mr del Plt (UNMDP) References Bstrsh, S, J Mkhlouf, F Cstigne nd C Willemot, 1993 Optiml controlled tmosphere conditions for storge of roccoli florets J Food Sci, 58: , 36 Brckett, RE 1999 Incidence, contriuting fctors, nd control of cteril pthogens in produce Posthrvest Biol Technol, 15: Frncis, G A, C Thoms nd D O Beirne, 1999 The microiologicl sfety of minimlly processed vegetles Int J Food Sci Technol, 34: 1-22 Ginnkourou, MC nd PS Toukis, 23 Kinetics modeling of Vitmin C loss in frozen green vegetles under vrile storge conditions Food Chem, 83(1): ICMSF, 1983 Métodos recomenddos pr el nálisis microiológicos en limentos In: Microorgnismos de los limentos I Técnics de nálisis microiológicos Acrii SA, Zrgoz, Espñ, 2d Ed Kder, AA 1999 Posthrvest technology of horticulturl crops Division of Agriculture nd Nturl Resource University of Cliforni, Dvis, CA Kim, DM, NL Smith nd CYC Lee, 1993 Qulity of minimlly processed pple slices from selected cultivrs J Food Sci, 58:

30 Effects of usive tempertures on lettuce qulity 113 Lee, S nd A Kder, 2 Prehrvest nd posthrvest fctors influencing vitmin C content of horticulturl crops Posthrvest Biol Technol, 2: Moreir, MR, S Rour, nd C del Vlle, 23 Qulity of Swiss chrd produced y conventionl nd orgnic methods Leens-Wiss Technol, 36: Moreir, M, A Ponce, C del Vlle nd S Rour, 25 Inhiitory prmeters of essentil oils to reduce foodorne pthogen Leens- WissTechnol, 38(5): Mossel, D A nd B Moreno Grcí, 1985 Microiologí de limentos Acrii SA Zrgoz Espñ 2 nd Edn Pull, RE 1999 Effects of temperture nd reltive humidity on fresh commodity qulity Posthrvest Biol Technol, 15: Perrin, PW nd MM Gye, 1986 Effect of simulted retil disply nd overnight storge tretment on qulity mintennce in fresh roccoli J Food Sci, 51: Ricker, WE 1979 Growth rtes nd models Fish Physiol, 8: Rour, SI, M del R Moreir, A Ponce nd del Vlle, 23 Dip tretments for fresh romine lettuce Itl J Food Sci, 3(15): Sstry, SK nd DE Buffington, 1982 Trnspirtion rtes of stored perishle commodities: mthemticl model nd experiments on tomtoes ASHARAE Trnsctions, 88(1): Schlimme, DV 1995 Mrketing lightly processed fruits nd vegetles HortSci, 3(1): Tulio Artemio Z, OSE Kimiko, Chchin Kzuo nd Ued Yoshinori, 22 Effects of storge tempertures on the posthrvest qulity of jute leves (Corchorus olitorius L) Posthrvest Biol Technol, 26(3): Volk, W 198 Applied Sttistics for Engineers, Correltion-Regression Mc Grw-Hill, Inc New York, 1 st Edn Wtd, AE, NP Ko nd D A Minott, 1996 Fctors ffecting qulity of fresh cut horticulturl products Posthrvest Biol Technol, 9: Wiley, CR 1997 Fruts y hortlizs mínimmente procesds y refrigerds En: Mossel, D A nd Moreno Grcí, B Microiologí de limentos Acrii SA Zrgoz Espñ, 1 st Edn Zgory, D 1999 Effects of post-processing hndling nd pckging on microil popultions Posthrvest Biol Technol, 15: Zwietering, M, I Jongenurger, F Romouts nd K Vn t Riet, 199 Modeling of the cteril growth curve Appl Environ Microiol, 56(6):

31 Journl of Applied Horticulture, 8(2): , July-Decemer, 26 Shelf-life nd qulity of pple fruits in response to posthrvest ppliction of UV-C rdition Appl Journl Syvsh Hemmty,, Noorollh Mollemi nd Lotfli Nseri c Jhd-e-Dneshghi, Urmi University, Urmi, Irn; Deprtment of Horticulture, Shhid Chmrn University of Ahvz, Ahvz, Irn; c Deprtment of Horticulture, Urmi University, Urmi, Irn E-mil: syvshhort@gmilcom Astrct In this study, UV-C irrdition ( w cm -2 ) ws used to mintin fruit qulity of Red Delicious nd Golden Delicious pple cultivrs during storge Apple fruits were irrdited in three different tretments (, 5 nd 15 min), nd were stored in cold storge t 1±1ºC with 85-95% RH for 6 months At the end of storge, irrdited fruits for 15 min hd lower ph nd totl solule solids/titrtle cids rtio nd higher titrtle cids nd firmness thn irrdited fruits for 5 min nd control fruits A significnt difference ws oserved mong totl solule solids of irrdited Red Delicious fruits for 15 min, irrdited fruits for 5 min nd control fruits t the end of storge Red Delicious pples hd lower totl solule solids nd totl solule solids/titrtle cids rtio nd higher firmness thn Golden Delicious pples fter 6 months Our results showed tht UV-C irrdition cn e used to reduce loss of fruit qulity during long period storge of pples Key words: UV-C irrdition, pple, Golden Delicious, Red Delicious, fruit qulity Introduction Apple (Mlus domestic Borkh) fruits re commonly stored for long periods t low tempertures nd under controlled tmosphere During this period, usully qulity nd nutritionl vlue of fruit decreses A numer of techniques like prestorge het tretment (Klein nd Lurie, 1992), tretment with chemicl compounds (Leverentz et l, 23) nd modified tmosphere (Hertog et l, 21) hve een used However, some chemicl compounds my pose serious helth hzrd nd environmentl risks Additionlly, consumers prefer griculturl products without chemicls residues nd, hence, lterntive methods to control posthrvest disese nd to extend product shelf-life re required (Mrquenie et l, 23) UV-C is generlly hrmful ut cn produce eneficil effect on horticulturl crops t low doses, phenomenon known s hormesis (the stimultion of plnt response y low levels of inhiitors or stress) (Lukey, 1982) UV-C rdition hs een shown to e promising posthrvest tretment, ecuse it delys the ripening process nd does not ffect qulity during storge (Mhrj et l, 1999) Ripening of fruit involves complex physiologicl chnges The ripening process is generlly ccompnied y incresed pigmenttion, sugr, ph, ethylene nd decresed firmness, strch nd scoric cid (Wills et l, 1981) Purohit et l (23) concluded tht Ber (Zizyphus muritin Lmk) fruits irrdited for 6 hours with UV-C rdition hd lower TSS nd TSS/TA rtio nd higher TA thn other tretments nd control fruits Mhrj et l (1999) nd Vicente et l (24, 25) lso reported increse in fruit firmness due to UV-C irrdition This experiment ws conducted to exmine the effect of UV-C tretment on shelf life nd the physico-chemicl chnges in Golden Delicious nd Red Delicious pples Mterils nd methods Golden Delicious nd Red Delicious pple fruits were hrvested mnully on optiml dte for commercil hrvesting (15 dys fter full loom for Red Delicious nd 17 dys fter full loom for Golden Delicious pples) (Nseri, 24) from 15-yers old stndrd trees from n orchrd in Urmi, Irn, during growing yer Fruits of uniform shpe, size nd free from fungl infection were selected UV-C rdition ws provided y fluorescent germicidl lmp (3 w, 9 cm) with pek emission t 254 nm Irrdition ws crried out under mient condition for, 5 nd 15 min The intensity of rdition ws w cm -2 Fruits were plced t pproximtely 25 cm from the lmp nd rotted so tht their lossom nd stem ends fced the lmp to ensure uniform irrdition After irrdition, fruits were stored t 1±1 C nd 85-95% RH for 6 months After smpling from cold stored fruits, they were stored for 7 dys t 25ºC Fruit physicl nd chemicl prmeters were mesured periodiclly fter tretment nd 45 dys storge t 1±1 C plus 7 dys t 25 C in 12-pples smples per tretment (3 pples per repliction) per cultivr Totl solule solids (TSS) were determined with hnd-held refrctometer Titrtle cids (TA) ws determined y titrtion with 1 N NOH nd expressed s % mlic cid (AOAC, 198) Fruit firmness ws determined on opposite sides of the fruit fter peel removl using penetrometer with 8 mm dimeter tip ph ws mesured with ph meter TSS/TA rtio ws lso clculted Completely rndomized fctoril design with four replictions ws used Every replicte hd 24 fruits An nlysis of vrince ws used to nlyze difference etween mens The Duncn s multiple rnge test ws pplied for men seprtion t P=5

32 Shelf-life nd qulity of pple fruits in response to posthrvest ppliction of UV-C rdition 115 Results nd discussion Qulity mesurements reveled tht during storge, in irrdited fruits, ph, totl solule solids nd TSS/TA rtio incresed nd firmness nd titrtle cids decresed slowly thn control fruits (Fig 1) After 6 months storge t 1±1 C plus 7 dys t 25 C, irrdited fruits for 15 min hd significntly low ph nd TSS/TA rtio nd high titrtle cids nd firmness thn other tretment nd control fruits Totl solule solids were not ffected y UV-C rdition (Tle 1) In Red Delicious pples, irrdited fruits for 5 nd 15 min hd low TSS thn control fruits But in Golden Delicious pples, there weren t significnt differences etween tretments Differences for ph etween irrdited Red Delicious nd Golden Delicious pples were non-significnt (Fig 2) Tle 1 Effect of UV-C rdition on firmness, titrtle cids (TA), ph nd TSS/TA rtio fter 6 months t 1±1 ºC plus 7 dys t 25 ºC Irrdition time (min) Firmness (N) TA (%) ph TSS/ TA rtio (control) 254 c 1 c c c Similr letters in columns hve no significnt differences t P<5 After 6 months storge t 1±1 C plus 7 dys t 25 C, Golden Delicious fruits hd high TSS nd TSS/TA rtio nd low firmness thn Red Delicious fruits ph nd TA were not ffected y cultivr (Tle 2) Tle 2 Effect of cultivr on firmness, totl solule solids (TSS) nd TSS/TA rtio fter 6 months t 1±1 ºC plus 7 dys t 25 ºC Cultivr Firmness (N) TSS (%) ph Red Delicious Golden Delicious Similr letters in columns hve no significnt differences t P<5 Like other plnt development processes, fruit ripening is significntly influenced y growth regultors of which ethylene plys key role, however, polymines hve lso een implicted to control ripening (Pndey et l, 2; Shm nd Alderson, 25) In climcteric fruits, urst of ethylene production is first detectle sign of ripening tht occurs during ripening nd precedes the respirtory climcteric During ripening, chnges in color nd texture re under the control of ethylene wheres flvor development in fruit is not much influenced (Shm nd Alderson, 25) Polymines re group of nitrogen-contining compounds tht ccumulte in plnts in response to environmentl stress (Evns nd Mlmerg, 1989; Brk et l, 2) Polymines nd ethylene my compete for the intermedite S-denosyl methionine (SAM) which produces the propylmine moiety for their iosyntheses (Pndey et l, 2; Shm nd Alderson, 25) Mhrj et l (1999) showed tht optiml doses of UV- C produced higher levels of free nd conjugted polymines prticulrly putrescine, compred with the control in mture green tomto fruits Level of putrescine seems to increse in plnts sujected to stress nd these includes UV irrdition tretments (Shm nd Alderson, 25) The increse in TSS my e relted to the moisture loss nd hydrolysis of polyscchrides UV rdition checks the moisture loss, therey, incresing TSS retrdtion (Lu et l, 1993) ph Titrtle cids (%) Firmness (N) TSS/Titrtle cids rtio Totl solule solids (N) Fig 1 Chnges of ph, totl solule solids, firmness, titrtle cids nd TSS/TA rtio during storge t 1±1 ºC plus 7 dys t 25 ºC ph 49 Red Delicious Golden Delicious min 5 min 15 min min 5 min 15 min min 5 min 15 min 5 15 Irrdition time (min) c c Totl solule solids (%) min 5 min 15 min Red Delicious Golden Delicious min 5 min 15min Dys t 1+1 ºC plus 7 dys t 25 º C 5 15 Irrdition time (min) Fig 2 Interction effect of UV-C rdition nd cultivr on totl solule solids nd ph fter 6 months t 1±1 ºC plus 7 dys t 25 ºC (P 5)

33 116 Shelf-life nd qulity of pple fruits in response to posthrvest ppliction of UV-C rdition Therefore, lower rte of increse in TSS of UV-C treted fruits could e ttriuted to reduced moisture losses nd reduced hydrolysis of polyscchrides The rte of respirtion nd ethylene production reduces fter UV-C tretment (Mhrj et l, 1999; Vicente et l, 24) Mhrj et l (1999) suggested tht since UV rdition cused shift in ethylene production rtes, the climcteric respirtion pttern ws lso shifted The shift in ethylene production pek is ssocited with UV treted fruit suggests tht SAM ws proly limiting fctor in UV treted fruit UV could hve lso cused irreversile memrne deteriortion, which could hve ffected the ctivity of ACC oxidse (loosely ound to memrne), nd thus explin the reduced ethylene production in such fruit (Mhrj et l, 1999) The higher TA in irrdited fruits could e due to lower respirtion rte in the fruits nd lower usge of these compounds in respirtion process The lower ph in irrdited fruits is relted to high TA in these fruits The low TSS/TA rtio resulted in higher retention of TA in irrdited fruits Mhrj et l (1999) proposed tht polymines ply role like conjugtion of clcium to pectic cid nd other polyscchrides nd, therefore, mintin cell wll from hydrolytic enzymes Additionlly, Knee nd Brtley (1981) proposed tht loss in cohesion of cell wlls my result from S-denosyl methionine methyltion of the free croxylic groups in the pectin mteril nd disruption of clcium cross linkge of djcent polyuronides Since SAM is proly routed to polymines synthesis, it wsn t redily ville for disruption of clcium ridges tht could led to incresed firmness of the UV treted fruit (Mhrj et l, 1999) Furthermore, Krmer et l (1989) hve shown tht polymines inhiit softening y reducing the ctivity of cell wll degrding enzymes such s polyglcturonse Brk et l (2) reported tht UV-C tretments could reduce the ctivity of cell wll degrding enzymes, ie polyglcturonse, pectin methyl esterse, xylnse, β-d-glctosidse nd protese, nd dely softening y ffecting the cell wll disssemly rte Reduction in polyglcturonse ctivity in UV-C treted fruits of tomto ws reported y Stevens et l (24) These could explin the higher level of firmness found in UV-C treted pples Present investigtion reveled tht irrdited Red Delicious fruits for 15 min hd higher vlues of totl solule solids t the end of storge Red Delicious pples fter 6 months hd lower totl solule solids nd totl solule solids/titrtle cids rtio nd higher firmness thn Golden Delicious pples The results reveled tht UV-C irrdition cn e used to reduce losses of fruit qulity during long storge period of pple References AOAC 198 Officil methods of nlysis Assocition of Officil Anlyticl Chemists, Wshington, DC, 13 th Edn Brk, EA, S Klntri, J Mkhlouf nd J Arul, 2 Impct of UV-C irrdition on the cell wll-degrding enzymes on ripening of tomto (Lycopersicon esculentum L) fruits Journl of Agriculturl Food Chemistry, 48: Evns, PT nd RL Mlmerg, 1989 Do polymines hve roles in plnt developments? Annul Review of Plnt Physiology nd Plnt Moleculr Biology, 4: Glston, AW nd R Kur-Swhney, 199 Polymines in plnt physiology Plnt Physiology, 94: Hertog, MLATM, SE Nicholson nd NH Bnks, 21 The effect of modified tmospheres on the rte of firmness chnge in Breurn pples Posthrvest Biology nd Technology, 23: Klein, JD nd S Lurie, 1992 Pre-storge heting of pple fruit for enhnced posthrvest qulity: interction of time nd temperture HortScience, 27: Knee, M nd IM Brtley, 1981 Composition nd metolism of cell wll polyscchrides in ripening fruits In: J Friend nd MJC Rhodes (eds) Recent Advnces in the Biochemistry of Fruit nd Vegetles Acdemic Press, New York, p Krmer, GF, CY Wng nd WS Conwy, 1989 Correltion of reduced softening nd incresed polymine levels during low-oxygen storge of McIntosh pples Journl of Americn Society for Horticulturl Science, 114: Leverentz, B, WS Conwy, WJ Jnisiewicz, RA Sftner nd MJ Cmp, 23 Effect of comining MCP tretment, het tretment, nd iocontrol on the reduction of posthrvest decy of Golden Delicious pples Posthrvest Biology nd Technology, 27: Lu, JY, SM Lukomo, C Stevens, VA Khn, CL Wilson, PL Pusey nd E Chlutz, 1993 Low dose UV nd gmm rdition on storge rot nd physico chemicl chnges in peches Journl of Food Qulity, 16: Lukey, TD 1982 Hormesis with ionizing rdition CRC Press, Florid, 222 p Mhrj, R, J Arul nd P Ndeu, 1999 Effect of photochemicl tretment in the preservtion of fresh tomto (Lycopersicon esculentum cv Cpello) y delying senescence Posthrvest Biology nd Technology, 15: Mrquenie, D, AH Geerered, J Lmmertyn, C Soontjens, JFV Impe, CW Michiels nd BM Nicoli, 23 Comintion of pulsed white light nd UV-C or mild het tretment to inctivte conidi of Botrytis cinere nd Monili fructigen Interntionl Journl of Food Microiology, 85: Nseri, L 24 Effect of folir ppliction of ure nd hrvesting dte on fruit qulity of two pple cultivrs Journl of Reserch in Agriculturl Sciences, 3: Pndey, S, SA Rnde, PK Ngr nd N Kumr, 2 Role of polymines nd ethylene s modultors of plnt senescence Journl of Bioscience, 25: Purohit, AK, TS Rwt nd A Kumr, 23 Shelf life nd qulity of Ber (Ziziphus muritin Lmk) fruits cv Umrn in response to posthrvest ppliction of ultrviolet rdition nd pcloutrzol Plnt Foods for Humn Nutrition, 58: 1-7 Shm, G nd P Alderson, 25 UV hormesis in fruits: concept ripe for commercilistion Trends in Food Science nd Technology, 16: Stevens, C, J Liu, VA Khn, JY Lu, MK Kwe, CL Wilson, ECK Igwege, E Chlutz nd S Droy, 24 The effects of low-dose ultrviolet light-c tretment on polyglcturonse ctivity, dely ripening nd Rhizopus soft rot development of tomtoes Crop Protection, 23: Vicente, AR, B Repice, GA Mrtinez, AR Chves, PM Civello nd GO Sozzi, 24 Mintennce of fresh oysenerry fruit qulity with UV-C light nd het tretments comined with low storge temperture Journl of Horticulturl Science nd Biotechnology, 79: Vicente, AR, C Pined, L Lemoine, PM Civello, GA Mrtinez nd AR Chves, 25 UV-C tretments reduce decy, retin qulity nd llevite chilling injury in pepper Posthrvest Biology nd Technology, 35: Wills, R, TH Lee, D Grhm, B Mcglsson nd EG Hll, 1981 Posthrvest: An introduction to the physiology nd hndling of fruit nd vegetles New South Wles University Press, Kensington, New South Wles, Austrli 176 p

34 Journl of Applied Horticulture, 8(2): , July-Decemer, 26 Effects of sugrs nd minooxycetic cid on the longevity of pollinted Dendroium (Heng Beuty) flowers Appl Journl S Chndrn 1, CL Toh 1, R Zulin 1, YK Yip 1, H Nir 2 nd AN Boyce 1 1 Institute of Biologicl Science, University of Mly, 563 Kul Lumpur; 2 Asin Institute of Medicine, Science nd Technology, 2 Persirn Cempk, Amnjy, 8 Sungi Petni, Kedh Drul Amn, Mlysi E-mil: chndrn@umedumy Astrct The vse life of detched pollinted Dendroium (Heng Beuty) orchids re ffected y loss of energy source nd the production of pollintion-induced ethylene The physiologicl chnges tht follow these two events re discolortion, thinning of petls nd hyponsty In order to circumvent this prolem, individul detched pollinted Dendroium (Heng Beuty) flowers were treted with solutions contining different concentrtions of sucrose or glucose, Aminooxycetic cid (AOA) nd comintion of sugrs nd AOA Discolourtion, petl thickness nd hyponsty were oserved nd dt ws recorded dily Weight loss of flowers nd ph of ll solutions were lso mesured dily Results showed tht the est tretment solution in extending the longevity of the flowers were solutions contining 4% sucrose + 5mM AOA Flowers held in this tretment lso showed dely in discolortion, thinning of petls nd hyponsty The inclusion of AOA into solutions resulted in low ph nd contriutes to etter wter uptke nd delyed turgor loss in flowers Key words: Dendroium, pollintion, ethylene, AOA, flowers, discolortion, sucrose, glucose Introduction The cut flower industry is US$ 2 illion industry nd is n importnt revenue for some of the mjor cut flower producers in the world Orchids re mong the highly demnded cut flowers nd re produced y countries like Thilnd, the Netherlnds, Hwii, Singpore nd Itly However, like ny other cut flower, orchids fce vse life-relted prolems such s excessive wter loss, decline in respirle sustrtes nd sensitivity to exogenous or endogenous ethylene tht hstens senescence nd wilting of the flowers (Hew, 1994) Vse-life is yrdstick for the longevity of cut flowers nd is n importnt trget for improving flower chrcteristics, whether y chemicl tretment or plnt reeding (Ymd et l, 22) The influence nd effect of sugrs nd ethylene re two mjor res which hve een vstly studied to tckle the prolem of flower qulity nd vse life of cut flowers Severl studies hve focused on the effect nd mechnism of ethylene inhiitors Compounds such s minooxycetic cid (AOA), minoethoxyvinylglycine (AVG) nd silver thiosulphte (STS) effectively dely senescence of climcteric flowers y inhiiting the synthesis or ction of 1-minocyclopropne-1 croxylte synthse (ACCS) (Yng, 1984) Petl senescence is generlly ccelerted y tretment with exogenous ethylene nd delyed y nionic STS, which prevents ethylene ction (Beyer, 1976) For exmple, gldiolus flowers were reported to lst longer (Hwng et l, 1995) when they were treted with silver STS Furthermore, the use of sugrs in holding solution of cut flowers hs een extensively studied nd hs yielded gret mount of success in vriety of cultivrs Inclusion of sugrs in the vse solution of crntion flowers reduced their sensitivity to ethylene (Myk nd Dilley, 1976) Sucrose, tht extended vse-life of rose (Kuiper et l, 1995) nd sweet pe (Ichimur nd Hiry, 1999), lso extended vse-life of gldiolus (Mrousky, 1971; Brvdo et l, 1974) From these oservtions, it is cler tht sugrs like sucrose hve negtive effect on the process of cell deth leding to petl senescence This study ims to further estlish the effectiveness of AOA nd sugrs in prolonging the vse life of pollinted Dendroium (Heng Beuty) Mterils nd method Plnt mteril: Dendroium (Heng Beuty) orchids were tken from the glsshouse in the University of Mly Flowers were cut t the peduncle, nd were hnd-pollinted y removing the pollini from the nther to the stigm on the sme flower using forceps The cut flowers were held in glss vils contining 22ml of distilled wter (control) or solutions contining chemicls It ws importnt for the peduncle of the flowers to e immersed in the solutions to enle efficient uptke of wter nd nutrient The weight of the flowers were etween g All flowers were held t room temperture (26 ± 4 o C) Ethylene mesurements: Ethylene production of detched unpollinted nd pollinted Dendroium flowers ws mesured using Hewlett-Pckrd Gs Chromtogrphy every 12 hours Flowers were weighed s individul flowers were held in 3 ml glss centrifuge tue throughout the experiment Tues were seled for 2 hours efore redings were tken After ech reding, the sels were removed until the next time point Preprtion of chemicls: AOA (Sigm) ws tested t concentrtion of 25 nd 5mM Glucose (Sigm) nd sucrose (Sigm) were used t 4% All solutions were prepred t the

35 118 Effects of sugrs nd minooxycetic cid on the longevity of pollinted Dendroium eginning of the experiments nd were not renewed ph redings: The ph of the solutions ws determined dily with ph meter (HANNA Instruments) Wter uptke nd fresh weight: The difference etween consecutive weighing of the vil plus solution (without the flower) ws used to clculte the wter uptke Evportive wter loss from the surfce of the solution ws negligile The weight of ech flower ws determined dily y sutrcting the weight of the vil nd solution from weight of vil, solution nd flower Mesurement of thickness: Thickness of ech flower ws mesured dily y using micrometer (Mitutoyo Micrometer) Flower wilting: Vse life of flowers ws considered terminted when the flowers reched full closure The experiment ws run ginst flower treted with distilled wter s control Colour mesurement: The colour of petls ws mesured dily using Minolt CR-2 Chrommeter using the L Munsell Colour System L* indicted vlue, * nd * mesures hue nd chrom Colour chnge ws clculted using L*, * nd * vlues nd the formul: L* x * / * Results In this experiment, ethylene production of detched pollinted Dendroium flowers were mesured ginst unpollinted Dendroium flowers to confirm the climcteric response in the flowers As shown in Fig 1, pollinted flowers produced ethylene t 6 hours (125 μg g -1 h -1 ), 84 hours (14 μg g -1 h -1 ) nd 18 hours (13 μg g -1 h -1 ) fter pollintion Therefter the ethylene production decresed nd remined t μg g -1 h -1 Unpollinted flowers (control) did not produce ny ethylene throughout the experiment Pollinted Dendroium held in distilled wter (control), hd the shortest vse life (3 dys) compred to pollinted flowers in the other tretments Flowers treted with 5mM AOA, 4% glucose with 5 mm AOA nd 4% sucrose with 5 mm AOA proved to e the etter tretments in extending longevity (Tle 1) The comintion of 4% sucrose nd 5mM AOA ws the est tretment ecuse of its ility to extend vse life up to 13 dys Tle 1 Vse life of pollinted Dendroium (Heng Beuty) held in different tretments Tretment Full closure (dy) Distilled wter Glucose 4% Sucrose 4% 25mM AOA 5mM AOA Sucrose 4% + 5mM AOA Glucose 4% + 5mM AOA 6 ±3 8 ±5 7 ±1 7 ±5 11 ±3 13 ±5 11 ± Generlly, ll the tretments showed weight increment in the first 3 dys nd egn to decrese therefter The increment my e due to the initil wter uptke from the solutions to fulfil the nutrient requirement of the cut flowers However, t dy 3, the wter uptke ws no longer effective to increse the fresh weight of the flowers nd the weight egn to drop The fresh weight ecme lower when the flowers strted to close (result not shown) Control showed drop in weight of flowers t dy 2 nd the rpid loss of weight continued throughout the experiment On -1-1 Amount of ethylene ( g g h ) weight (g) Time(hour) Fig 1 Ethylene production of unpollinted nd pollinted detched Dendroium Heng Beuty Time (d) Fig 2 Weight of detched pollinted Dendroium Heng Beuty held in different tretment solutions the other hnd, flowers treted with 5mM AOA, sucrose 4% + 5mM AOA nd glucose 4% + 5mM AOA mnged to dely the onset of weight loss to dy 4 nd dy 5 with more stle weight loss throughout the experiment (Fig 2) Tle 2 shows the totl wter uptke of Dendroium flowers held in different tretments throughout the tretment Flowers held in 4% sucrose +5mM AOA showed the highest mount of wter uptke (446 ml), followed y flowers held in 5mM AOA (394 ml) nd flowers held in 4% glucose+ 5mM AOA (383 ml) Totl wter uptke y the flowers held in the rest of the tretment solutions ws not significntly different from the control Tle 2 Totl wter uptke y pollinted Dendroium (Heng Beuty) flowers held in different tretments Tretments Unpollinted Pollinted Distilled wter Glucose 4% Sucrose 4% 25mMAOA 5mM AOA Sucrose 4%+5mMAOA Glucose 4%+5mMAOA Distilled wter 5 mm AOA 4% glucose+5 mm AOA 4% sucrose+5 mm AOA Totl wter uptke (ml) Fig 3 shows the chnge in thickness of flower petls of pollinted Dendroium throughout the experiment In this oservtion, the positive results coincided with the longevity of flowers This is evident s flowers treted with 5mM AOA, 4% glucose with 5 mm AOA nd 4% sucrose with 5 mm AOA mnged to show dely in decrement of petl thickness which only occurred t dy 6 (4% sucrose + 5mM AOA) nd dy 4 (5mM AOA, 4% glucose) compred to control which showed decrese in petl thickness on dy 1

36 Effects of sugrs nd minooxycetic cid on the longevity of pollinted Dendroium 119 Thickness (mm) Time (d) Fig 3 Thickness of petls of pollinted Dendroium (Heng Beuty) flowers held in different tretment solutions L* x */* The decrese in L vlue is n indiction of discolortion of petl, from white to eventully trnsprent Flowers held in 5mM AOA, 4% glucose+5mm AOA nd 4% sucrose+5mm AOA showed lower rte of discolourtion of petls compred to the control (Fig 4) Tle 3 shows the chnge in ph vlues of the different tretments It ws found tht the ph of solutions contining AOA (25 mm AOA, 5 mm AOA, sucrose 4% +5mM AOA nd glucose + with 5mM AOA) were mintined round 3 throughout the experiment Tretments with sugrs however, showed higher ph vlues (5-6) throughout the experiment The ph vlues of the solutions directly influence the wter reltions of the flowers Tle 3 ph vlue of tretment solutions of t dy nd dy of termintion of the experiment Tretment Distilled Wter 4% Glucose 4% Sucrose 25 mm AOA 5 mm AOA 4% Glucose + 5 mm AOA 4% Sucrose + 5 mm AOA Discussion Distilled wter 5 mm AOA 4% glucose+5 mm AOA 4% sucrose+5 mm AOA Distilled wter 5 mm AOA 4% glucose+5 mm AOA 4% sucrose+5 mm AOA Time (dys) Fig 4 Colour chnge of petls of pollinted Dendroium (Heng Beuty) flowers held in different tretment solutions ph of solution t initil wilting ph of solution t dy of termintion It is well known fct tht the effect of ethylene on wilting cn e inhiited y ethylene inhiitors Woltering nd vn Doorn (1988), clssified petl senescence of 93 species from 22 fmilies into three types s follows: (i) Type I, wilting pprently medited y ethylene; (ii) Type II, wilting pprently not medited y ethylene; (iii) Type III, scission pprently medited y ethylene In the cut flower species showing Types I nd III of petl senescence, ie ethylene-sensitivity, vse-life could e improved y tretment with ethylene inhiitors or y genetic trnsformtion with ethylene relted genes (Reid nd Wu, 1992; Chng et l, 1993) The Dendroium flls under the ctegory of flowers where the senescence nd scission re medited y ethylene Furthermore this prticulr flower lso shows pollintion induced ethylene production which eventully cts s signl for the flower to undergo senescence-like physiologicl chnges Inhiitors of ethylene production nd ction hve shown to slow down florl scission (Sexton et l, 1985) Aminooxycetic cid (AOA) is n inhiitor of pyridoxl phosphte-requiring enzymes including 1-minocyclopropne-1-croxylic cid (ACC) synthse, key enzyme of ethylene synthesis (Aeles et l, 1992) The results from this experiment further estlishes the effectiveness of AOA s n ethylene inhiitor s the tretment with 5 mm AOA resulted in significnt extension of vse life compred to tht of the control The post hrvest life of flowers is strongly dependent on the crohydrte sttus nd the cceptle mounts of metolic sugrs re fctors tht ffect the rte of senescence Keeping flowers in vse solutions contining sucrose hs een shown to extend their vse-life (Ho nd Nichols, 1977) Supplying cut flowers with exogenous sugr mintin the pool of dry mtter nd respirle sustrtes, especilly in petls, thus promoting respirtion nd extending longevity (Coorts, 1973; Rogers, 1973) Arts (1957) suggested tht exogenous sucrose in some wy mintins the structure nd semi-permeility of the plsm memrne It ws demonstrted tht sucrose ntgonized the effect of scisic cid in promoting the senescence of roses (Borochov et l, 1976) Furthermore, tretments of cut flowers with sucrose re found to e eneficil in delying senescence processes (Ykimov et l, 1996) Exogenous supply of sugrs delys wilting in mny flowers nd this effect is due to mintennce in strch nd sugr levels in cut flowers (Rttnwislnon et l, 23) Previous studies hve found tht sucrose interct with other internl plnt hormones in regulting the process of senescence It ws shown tht sucrose enhnces the effect of cytokinins in delying senescence of flowers nd reduces the effect of ethylene in promoting it (Myk nd Dilley, 1976) Sugr is found to e involved in enhncing the function of certin orgnelle such s mitochondri Kltler nd Steponkus (1976) found tht in mitochondri isolted from cut flowers pretreted with sucrose, respirtory control vlues were mintined over longer periods Hence, they concluded tht the min effect of pplied sugr in extending longevity is to mintin mitochondril structure nd functions All of these findings show close reltionship with the results nd effect of sugrs oserved in this experiment However, the min disdvntge of sugrs in the vse solution is the promotion of cteril growth when not ccompnied y

37 12 Effects of sugrs nd minooxycetic cid on the longevity of pollinted Dendroium n dequte ntimicroil gent; nd it my therefore clog the xylem vessels nd inhiit the uptke of oth wter nd dissolved sugrs (vn Doorn, 1997) As is shown from the results, the effectiveness of sugr s continuous energy supply for cut flowers ws retrded due to the sence of n ntimicroil gent cusing stem lockge Hence, the turgidity nd overll qulity of the vse life ws not significntly mintined throughout the experiment Rttnwsilnon et l (23) found tht inclusion of AOA in the vse wter, together with sugr, hd positive effect on the time to flower senescence In pollinted flowers, the effect of AOA on wilting my e due to reduction of endogenous ethylene synthesis; however, such n effect is present only when AOA is comined with sugrs (Rttnwsilnon et l, 23) Tretments with AOA or AOA + sucrose effectively retrded the longevity of cut spry-crntion flowers (Ykimov et l, 1997) Our result is in greement with oth these findings s the comintion of AOA nd sucrose showed the longest vse life of pollinted Dendroium AOA therefore, cn ct s n nticteril gent, which inhiits cteril growth (Rttnwsilnon et l, 23) The ility of AOA s n ntimicroil gent is ttriuted to the mintennce of low ph which results in non-conducive environment for cteril growth Low ph seems to ply the role of n nticteril gent s cteril growth is virtully hlted t ph of three or lower (Kets nd Nrku, 21) Wter uptke ws lso enhnced y cidic (ph 3 to 4) nd wrm wter (43 o C) (Dole nd Schnelle, 22) Therefore, solutions with low ph throughout the experiment (5mM AOA sucrose 4% + 5mM AOA) considerly delyed oth scission nd petl wilting s wter reltions were improved nd mintined In this experiment post pollintion symptoms oserved were discolourtion of petls, chnge in petl thickness, weight loss nd chnges in ph vlues Sucrose 4% with 5mM AOA ws proved to e the est chemicl tretment in delying the petl senescence of Dendroium (Heng Beuty) The tretment ws le to mintin the highest wter uptke y the flowers throughout the experiment The lowest rte of thickness decrement of this tretment indicted their ility to slow down the pectin hydrolysis of petls throughout the experiment It remins to e the centre of future studies to investigte the consequences of different ethylene inhiitors for their contriution in prolonging the vse life of cut flowers References Arts, JFT 1957 On the keepility of cut flowers Meded Lndouwhogesch Wgeningen 57: 1-62 Aeles, FB 1973 Ethylene in plnt iology Acdemic Press, New York pp Beyer, EMJ 1977 Ethylene: its incorportion nd oxidtion to cron dioxide y cut crntions Plnt Physiol, 6: Borochov, A, S Myk nd AH Hlevy, 1976 Comined effects of cid nd sucrose on growth nd senescence of rose flowers Plnt Physiol, 36: Brvdo, B, S Myk nd Y Gvijel, 1974 Sucrose nd wter uptke from concentrted sucrose solutions y gldiolus shoots nd the effect of these tretments on floret life Cn J Bot, 52: Chng, C, SF Kwok, AB Bleecker nd Meyerowitz, 1993 Aridopsis ethylene-response gene ETR1: similrity of product to two-component regultors Science, 262: Coorts, GD 1973 Internl metolic chnges in cut flowers HortScience, 8: 195 Dole, MJ nd MA Schnelle, 22 The cre nd hndling of cut flowers Division of Agriculturl Sciences nd Nturl Resources, Oklhom Stte University Hew, CS 1994 Orchid cut- flower production in ASEAN countries In: Orchid Biology: Reviews nd Perspectives, Vol VI, ed J Arditti (John Wiley nd Son Inc, New York) pp Ho, L nd R Nichols, 1977 Trnsloction of 14 C-sucrose in reltion to chnges in crohydrte content in rose corolls cut t different stges of development Ann Bot, 41: Hwng, MJ nd KS Kim, 1995 Posthrvest physiology nd prolonging vse life of cut gldiolus J Kor Soc Hort Sci, 36(3): Ichimur, K nd T Hiry, 1999 Effect of silver thiosulfte complex (STS) in comintion with sucrose on the vse life of cut sweet pe flowers J Jpn Soc Hort Sci, 68: Kltler, REL nd PL Steponkus, 1976 Fctors ffecting respirtion in cut roses J Amer Soc Hort Sci, 11: Kets, S nd N Nrku, 21 Effect of Aminooxycetic cid nd sucrose on vse life of cut roses Act Hortic, 543: Kuiper, D, SA Riot, HS Vn Reenen nd N Mrissen, 1995 The effect of sucrose on the flower ud opening of Mdelon cut roses Sci Hortic, 6: Mrousky, FJ 1971 Influence of 8-hydroxyquinoline citrte nd sucrose on crohydrte content of leves nd florets of cut gldiolus spikes Act Hortic, 23: Myk S nd DR Dilley, 1976 Effect of sucrose on response of cut crntion to kinetin, ethylene nd scissic id Am Soc Hort Sci, 11: Rttnwislnon, C, S Kets nd WG vn Doorn, 23 Effect of minooxycetic cid nd sugrs on the vse life of Dendroium flowers Posthrvest Biology nd Technology, 29: 93-1 Reid, MS nd MJ Wu, 1992 Ethylene nd flower senescence Plnt Growth Regul, 11: Rogers, M 1973 An historicl nd criticl review of post hrvest physiology reserch on cut flowers HortScience, 8: Sexton, R, LN Lewis, AJ Trewvs nd P Kelly, 1985 Ethylene nd scission In: Ethylene nd Plnt Development JA Roerts nd GATucker (Eds) Butterworths, Boston, MA, USA, pp Vn Doorn, WG 1997 Effect of pollintion on florl ttrction nd longevity Journl of Experimentl Botny, 48: Woltering, EJ nd WG Vn Doorn, 1988 Role of ethylene in senescence of petls-morphologicl nd txonomicl reltionships J Exp Bot, 28: Ykimov, E, BB Atnssov nd V Kpchin-Totev, 1997 Longevity nd some metolic events in post-hrvest Sprycrntion flowers Bulg J Plnt Physiol, 23: Ymd, T, Y Tktsu, T Mne, M Ksumi nd W Mrushi, 23 Suppressive effect of trehlose on poptotic cell deth leding to petl senescence in ethylene-insensitive flowes of gldiolus Plnt Science, 164: Yng SF nd NE Hoffmn, 1984 Ethylene iosynthesis nd its regultion in higher plnts Annu Rev Pl Physiol, 35:

38 Journl of Applied Horticulture, 8(2): , July-Decemer, 26 Agronomic ttriutes of sffron yield t groecosystems scle in Irn Appl Journl A Koocheki 1, MA Behdni 2 * nd M Nssiri 1 1 Center of Excellence for Specil Crops, Ferdowsi University of Mshhd, Mshhd, Irn; 2 Fculty of Agriculture, The University of Birjnd, POBox 97175/ 331, Birjnd, Irn; *E-mil: mehdni@yhoocom Astrct In order to study effective fctors in production of sffron, series of studies ws crried out during 21 nd 22 In these studies, four selected loction, Birjnd, Qen, Gond nd Tort-Hydrieh were spotted s the min sffron producing res in Irn Dt ws collected from 16 sffron frms, ged etween 1 nd 5 yers Results indicted tht ge of sffron frms, corm size, irrigtion intervl, nd summer irrigtion hd positive liner reltionship with yield Age of sffron frms hd the most pronounced effects on yield nd ws the most importnt component in ll liner equtions Age of frms, irrigtion intervls nd corm size were mjor fctors contriuting to yield The longest irrigtion intervl ws oserved for Gond (24 dys) nd the shortest ws for Tort-Hydrieh (12 dys) Highest ctul yield ws for Tort-Hydrieh which is n indiction of etter frm mngement in comprison with other res Mximum yield of 4 kg h -1 ws frequent ut mny frms produced over 7 kg h -1 yield Key words: Sffron, irrigtion intervl, summer irrigtion, corm size Introduction Sffron is n expensive spice nd hs een grown for long time in mny prts of the world including Spin, Itly, Greece nd Irn Tody more thn 95% of sffron in the world is produced in Irn nd most of this production is from centrl nd southern Khorsn (Kfi et l, 22) Sffron is n importnt csh crop for the smll holding in Khorsn province nd more thn 85 frmers re involved in its production (Kfi et l, 22) Sffron is fmily sed crop nd most of frming prctices prticulrly picking flowers re crried out y fmily memers or community coopertion Not only this crop is csh crop for the frmers ut lso it hs strong tie with their socil life Sffron is used loclly nd it hs trditionl medicinl uses (Koocheki, 24) Recent reports on sffron s cncer curing gent hs rought more ttention to this crop (Adullev, 22) Sffron production does not require much wter ut timeliness of irrigtion, prticulrly the first irrigtion is very importnt for flower emergence nd length of flowering period (Kfi et l, 22) Low temperture in utumn is crucil fctor for flower emergence (Molin et l, 24) There re mny fctors contriuting to the yield of sffron The most importnt fctors re environmentl conditions nd frming prctices such s ge of frm, corm size, method of plnting, irrigtion ppliction, irrigtion intervl nd recent prctice of summer irrigtion which is not usul prctice for sffron The purpose of present study ws quntittive evlution of the mgnitude of fctors ffecting crop yield y comprehensive survey t frm level for two yers Mterils nd methods Sffron producing re of southern Khorsn, in which 95% sffron is produced, were investigted in four min counties nmely Birjnd, Qen, Gond nd Tort Hydrieh in two growing sesons 21 nd 22 Dt were collected from 16 frms with very diverse criteri including the size (5 m 2 to 2 h), ge of sffron fields (1 to 5 yer), frming prctice (frmers skill) nd frming ckground A comprehensive survey ws mde during two yers, ssocited with frming prctices such s dte of plnting, time of first irrigtion, time of first flowering period, the mount of mnure used, irrigtion frequency (including summer irrigtion, if ny), size of corm, plnting method nd yield, y personl reference to the frmers nd direct monitoring Climte dt were collected from the nerest climte recording sttion Sttisticl nlyses were mde on the reltionship etween yield nd yield ttriuting fctors nd correltion coefficients were clculted ccordingly Frming prctices nd phenologicl stges of plnt prticulrly first time of flower ppernce nd length of flowering were correlted to the frming prctices such s time of irrigtion nd corm size for the whole re Anlyses were mde y Excel, SigmStt nd SPSS softwre Results nd discussion Dte of plnting: Tle 1 indictes tht the most frequent dte of plnting in the re ws 1 1 th of Septemer, however in Torthydrieh this prctice ws more frequent in 1-2 th of August nd in Gond 11-2 th of Septemer Dte of plnting vried with different geogrphicl loctions In Spin corms re plnted during mid My to erly June (Behni, 1991) nd in Kshmir mid July to lte August (Frooq nd Koul, 1993) Plnting method: In generl, two plnting methods of hill nd row re in prctice (Tle 2) In trditionl systems, more tendency is towrds hill plnting where 1 to 15 corms re locted in ech hill (Mollfili, 24) In generl, 14 % of frms re plnted through this method (Tle 2) In row plnting, corms re plnted in rows which re 2cm prt from ech other Age of frm: In Fig 1, reltionships etween yield nd ge of

39 122 Agronomic ttriutes of sffron yield t groecosystems scle in Irn Tle 1 Distriution frequency of plnting dte in different res (%) Counties 1-2 August August 1-1 Septemer 11-2 Septemer 21-3 Septemer 1-1 Octoer 11-2 Octoer Brijnd Qen Gond Tort- hydrieh Totl re Tle 2 Frequency(%) nd yield (kg h -1 ) of sffron under different plnting methods in different res Counties Brijnd Qen Gond Tort-Hydrieh Totl re Plnting method Row Hill Percentge Yield Percentge Yield Percentge 3 7 Yield Percentge Yield Percentge Yield frms up to 5 yers re presented Yield in the first yer ws low nd the mximum yield ws otined in 5th yers, ut usully sffron frms re kept up to 1 yers (Behni, 1991; Kfi et l, 22; Negi, 1999) With incresing ge of frm from 1 to 5, yield ws incresed (Fig 1) This trend is most pronounced for Tort Hydrieh followed y Birjnd Higher yield in Tort Hydrieh nd Birjnd is ssocited with more suitle frming prctices Age of frms in Irn, which t present re more thn 8 yers, hs een recommended to e reduced to 4 or 5 yers, ecuse yield cn e improved y shortening the verge ge of sffron frms from 8-1 to 4 or 5 yers Size of corm: The correltion etween size of corm nd yield is shown in Fig 2 With incresing size of the corm, yield lso incresed, nd there ws good correltion etween these two vriles Big corms cuse erlier nd vigorous flower emergence nd therefore higher yield is otined This type of corm produces igger dughter corms for next sesonl growth (DeMsstro nd Rut, 1993; McGimpsey et l, 1997) In Tle 3 proportion of corms with different size is shown As it is seen in Tort Hydrieh smll size corm ws not used nd the proportion of smll size corm ws less thn 5 % for other counties Medium size corm 7 to 1 g ws used more thn other types in different counties There re references (Sdeghi, 1998), indicting tht corms with less thn 7 g hve low flowering potentil nd corms with 9 g re the most frequent with optimum flowering potentil nd corms with 15 g (not frequently used), yield more flower nd sffron yield of 7 kg h -1 in first yer (Kfi et l, 22) Tle 3 Distriution (%) of corm size in different counties Counties Corm size (g) Brignd Qen Gond Tort-Hydrieh Totl re Yield (kg h -1 ) Yield (kg h -1 ) Yield (kg h -1 ) Yield (kg h -1 ) Yield (kg h -1 ) c d e y = 13881x R 2 = 9356 y = 5915 x R 2 = 9711 y = 8725 x R 2 = 9665 y = x R 2 = y = 1259x R 2 = Age of frm (yer) Fig 1 Reltionship etween yield of sffron nd ge of frm in Birjnd (), Qen (), Gond (c), Tort-Heydrieh (d) nd men of whole re (e)

40 Agronomic ttriutes of sffron yield t groecosystems scle in Irn 123 Yield (kg h -1 ) Corm size(g) Fig 2 Reltionship etween yield nd corm size y =5868x R 2 = Irrigtion intervl: There ws good correltion etween irrigtion intervls nd yield of sffron (Fig 3) By reducing irrigtion intervl, yield incresed for different ge groups nd lso for the verge of ll ge groups Higher yield otined with lower irrigtion intervl hs lso een confirmed elsewhere (Mosferi, 21) Three different irrigtion intervls tht were used for different counties re shown in Fig 4 It is oserved tht irrigtion with 24- dys intervl ws most frequent in Birjnd, Qen nd Gond nd 12-dys intervl ws most frequent in Tort Hydrieh, reson for higher yield There ws no irrigtion intervl with 12 dys in Gond Summer irrigtion: Bsed on the physiologicl chrcteristics of sffron (growth strt in erly utumn with decresing temperture of the re), first irrigtion is required to stimulte flower emergence After flower emergence nd hrvesting, which normlly lst 3 dys, vegettive growth strts nd the leves emerge During winter dys growth of leves continue nd y the end of My Percent Birjnd Qen Gond Tort Irrigtion intervl Fig 4 Percentge of frms with different irrigtion intervls in different counties Yield (kg h -1 ) Yield (kg h -1 ) Yield (kg h -1 ) y = -113x R 2 = Irrigtion Intervl (dy) c y = -149x R 2 = Irrigtion Intervl (dy) e y = -1725x R 2 = Irrigtion Intervl (dy) Yield (kg h -1 ) Yield (kg h -1 ) Yield (kg h -1 ) y = -1128x + 51 R 2 = Irrigtion Intervl (dy) y = -147x R 2 = Irrigtion Intervl (dy) y = -1586x R 2 = Irrigtion Intervl (dy) Fig 3 Reltionship etween irrigtion intervls nd yield of sffron in different ged frms,, c, d, nd e, represent frms with 1, 2, 3, 4, nd 5 yers old, respectively f illustrte the men of different ged frms in whole surveyed re d f

41 124 Agronomic ttriutes of sffron yield t groecosystems scle in Irn leves re dried nd the plnt goes under dormncy Flower initition strt in erly July (Frooq nd Koul, 1983) Therefore, first irrigtion is normlly pplied t the eginning of the fll without ny summer irrigtion pplied However, in recent yers one summer irrigtion is prcticed etween end of July to mid August It is elieved tht ppliction of one irrigtion t the time of erly flowering helps this process nd results higher rte of flowering (Frooq nd Koul, 1983; Sdeghi, 1993) Investigtion shows tht 45% of the frms, in the re, received one summer irrigtion (Tle 4) except in Gond Men yield for the frms with summer irrigtion ws higher thn those with no summer irrigtion (49 nd 335 kg/h, respectively) nd s whole, nerly 6% of yield ws otined from the frms in which summer irrigtion ws prcticed There re references showing tht one irrigtion in mid Augest led to yield increse while one irrigtion in mid July resulted in 17% reduction weight of flowers (Mosferi, 21) Tle 4 Frequency (%) nd yield of sffron frms (kg h -1 ) with nd without summer irrigtion County Frm/yield chrcters No summer irrigtion Birjnd Qen Gond Tort Hydrieh Totl re Summer irrigtion is pplied Frms (%) Frequency Yield % From totl yield Frms (%) Frequency Yield % From totl yield Frms (%) 4 Frequency 1 Yield % From totl yield Frms (%) Frequency Yield % From totl yield Frms (%) Frequency Yield % From totl yield In conclusion, frm prctices relted to irrigtion intervl, summer irrigtion, ge of frms nd corm size re the min ttriutes of sffron yield Incresing irrigtion intervls decrese yield however, summer irrigtion t flower differentition stge will led to yield increse While sffron frms re used up to 1 yers fter estlishment, the highest yield is otined from 5 yers old frms nd yield will e decresed in the following yers Lrge corms lso hve promotive effect on flower emergence, yield nd lso on producing igger dughter corms References Adullev, FI 22 Cncer chemopreventive nd tumoroicidl properties of sffron (Crocus stivus L) Exp Biol Med, 227: 2-25 Behni, MR 1991 Sffron cultivtion Tehrn University Press, Thern DeMsttro, G nd C Rut, 1993 Reltion etween corm size nd sffron (Crocus stivus L) flowering Act Horticulture, 344: Frooq, S nd K Koul, 1983 Chnges in gierellins like ctivity in corms of sffron plnt (Crocus stivus L) during dormncy nd sprouting J Plnt Biochem, 178: Kfi, M, MH Rshed, A Koocheki nd A Mollfili, 22 Sffron (Crocus stivus L), production nd processing Center of excellence for gronomy, Fculty of Agriculture Ferdowsi University of Mshhd, Irn Koocheki, A 24 Indigenous knowledge in griculture with prticulr reference to sffron production in Irn Act Horticulture, 65: McGimpsey, JA, MH Dougls nd AR Wllce, 1997 Evlution of sffron (Crocus stivus L ) in New Zelnd Nz J Crop Hort Sci, 25: Molin, RV, M Vlero, Y Nvro, A Grci-luis nd JL Grdiol, 24 Temperture effects on flower formtion in sffron (Crocus stivus L) Scienti Horticulture, 13(): Mollfili, A 24 Experimentl finding of production nd ecophysiologicl spects of sffron (Crocus stivus L) Act Horticulture, 65: Mosferi, H 21 Effect of different regimes of irrigtion on sffron yield M Sc Thesis of irrigtion nd dringe, Ferdowsi University of Mshhd, Irn Negi, M, 1999 Sffron cultivtion: pst, present nd future prospects In: Sffron (Crocus stivus L), Negi, M (ed), Hrwood Acdemic Pulishers, Amsterdm, pp 1-18 Sdeghi, B 1993 Effect of corm weight on sffron flowering IROST Mshhd Center, Irn Sdeghi, B 1998 Effect of summer irrigtion on sffron yield IROST Mshhd Center, Irn

42 Journl of Applied Horticulture, 8(2): , July-Decemer, 26 Effect of ud scle removl nd AOA on ud rek nd ACC content of Musct Biley A grpevines Appl Journl Ali R El-Shereif* 1, Fuso Mizutni, Justus M Onguso nd ABM Shrif Hossin Fculty of Agriculture, Ehime University, Httnji 498, Mtsuym City, Ehime , Jpn; 1 Horticulture Deprtment, Fculty of Agriculture, Kfr El-Sheikh University, Kfr El-Sheikh, Egypt*E-mil: elshereif@yhoocom Astrct A study ws crried out to exmine the effect of ud scle removl (BSR) nd minooxy cetic cid (AOA) on ud rek nd 1- minocyclopropne-1-croxylic cid (ACC) content of Musct Biley A grpevines using single-eye cuttings Smples were collected monthly from Octoer to Ferury Single-eye cuttings were sujected to these tretments; BSR, BSR + AOA, control nd AOA The results show tht in Octoer nd Novemer, BSR nd BSR + AOA were more effective on ud rek without ig difference etween the two tretments Wheres, control nd AOA were found to e more effective from Decemer up to Ferury In Octoer, ACC content recorded mrked increse fter one week nd decresed fterwrds under BSR nd BSR + AOA However, it showed continuous increse under control nd reverse trend under AOA In Novemer, it incresed fter one week nd decresed in the fourth week under ll tretments A continuous increse ws recorded in Decemer under ll tretments In Jnury, there ws no significnt chnge under control with time nd AOA tretment exhiited decline with time, while BSR nd BSR + AOA tretments recorded smll increment nd then decresed In Ferury, it decresed under ll tretments with time The results indicte tht ud rek of grpevine seems to e ssocited with the promotion of ethylene iosynthesis cused y wounding stress Key words: Aminooxy cetic cid, AOA, 1-minocyclopropne-1-croxylic cid, ud rek, ud scle removl, dormncy, ethylene iosynthesis, grpe Introduction In temperte zone deciduous fruit trees, the endo-dormncy period is overcome y exposure to low tempertures (Smish, 1954; Sure, 1985; Kwmt, et l, 22; Kurod et l, 22) The mount of chilling required for ud rek is specific for species nd cultivrs Dormnt grpevine uds hve chilling requirements However, these requirements for grpe re generlly thought to e less thn those of most deciduous fruit species Chilling is not n solute requirement for ud rek, ecuse high tempertures (Tohe et l, 1998), ud scle removl (Iwski nd Wever, 1977; Iwski, 198; Mizutni et l, 1995) which implies wounding stress, nd neroic conditions (Erez et l, 198) cn replce the chilling requirements Ethylene production increses following vrious disturnces or stresses in plnts These disturnces cn e induced y iotic or iologicl gents (Aeles et l, 1992) Environmentl stresses tht induce ethylene production include physicl wounding nd cutting, chilling, drought, nd wter flooding (Yng nd Oetiker, 1998) 1-Aminocyclopropne-1-croxylic cid (ACC), precursor for ethylene synthesis, incresed during the trnsition from dormncy to the ctive stte in Prunus vium L nd Prunus serrult Lindl (Wng et l, 1985) Aminooxy cetic cid (AOA) hs minooxy groups tht re effective inhiitors of ethylene production vi inhiiting ACC synthse (Aeles et l, 1992) Tohe et l (1998) reported tht minooxy cetic cid ws found to inhiit ud rek In the present study we exmined the effect of ud scle removl nd AOA on ud rek nd ACC content of grpevine using single-eye cuttings to know whether ud rek of grpevine is ssocited with the promotion of ethylene iosynthesis cused y wounding stress or not Mterils nd methods Cnes were collected monthly from mture Musct Biley A grpevines (Biley x Musct Hmurg) grown t Ehime University Frm from 28 Octoer, 23 until 24 Ferury, 24 On ech smpling dte, single eye-cuttings were prepred from the collected cnes nd mounted through sheet of styrene fom, which ws floted on wter in plstic continer nd plced in growth chmer under continuous white fluorescent light t 24 o C These single eye-cuttings were sujected to the following tretments: 1) Control (uds without removing scles), 2) Aminooxy cetic cid (AOA), 3) ud scle removl (BSR), 4) BSR + AOA Bud scle removl ws done using forceps nd the tretment of AOA ws done using queous solution (1mM) of AOA y employing sorent cotton Ech tretment ws represented y 5 single-eye cutting The percentge of ud rek ws clculted every week Bud rek ws indicted y the presence of green tissues eneth the ud scles ACC content ws determined s follows: Buds (scleless uds) were dissected from cuttings, weighed nd extrcted with 8% ethnol contining 5% (v/v) 2-mercptoethnol The extrct ws filtered nd evported in vcuo to dryness The residue ws tken up in 4 ml distilled wter nd n liquot of the solution ws ssyed for ACC content ccording to the method of Lizd nd Yng (1979) ACC content ws determined t the smpling

43 126 Effect of ud scle removl nd AOA on ud rek nd ACC content of Musct Biley A grpevines uds dte ( week), fter 1 week nd 4 weeks with n exception in Jnury nd Ferury smples It ws determined t, 1 nd 2 weeks ecuse of rpid ud rek in these two months Results nd discussion In Octoer nd Novemer, BSR nd BSR + AOA tretments recorded the highest percentge of ud rek nd ud rek strted erlier thn those of control or AOA From Decemer until Ferury the highest percentge ws recorded under control Bud rek (%) Oct Nov Contol AOA BSR BSR+AOA nd AOA tretments without much differences etween oth of them (Fig 1) The time required to chieve 5% ud rek ws shorter under BSR nd BSR + AOA thn control or AOA tretments in Octoer nd Novemer (Fig 2) However, the reverse ws true from Decemer to Ferury These results re in greement with those of Iwski nd Wever (1977), Iwski (198) nd Mizutni et l (1985) They reported tht ud scle removl ws much more effective in stimulting ud rek of grpe cuttings thn leving them intct Spires (1972) found tht ud scle removl in tung trees gretly reduced ud dormncy t n erly collection period However, this effect decresed with time nd completely disppered two months fter the onset of tretments Also, Iwski (198) found tht ud scle removl ws more effective in ud rek thn control from August to Novemer Wheres, in Decemer they recorded the sme ud rek percentge nd it ws higher under control therefter until Ferury In this study the sme tendency ws oserved s in Spires (1972) nd Iwski (198) results Bud scle provide not only protective covering for the ud, ut lso ct s uffer ginst resumption of growth, where it ws found tht inhiitive sustnces exist in the scles nd its levels incresed grdully, nd then dropped until ud rek (Iwski nd Wever, 1977) Therefore, the effect of ud scle Dec Bud rek (%) Bud rek (%) Jn Weeks fter tretment Fig 1 Effect of ud scle removl (BSR) nd AOA on ud rek percentge of grpevine uds Bud rek (%) Bud rek (%) Fe Weeks fter tretment

44 Effect of ud scle removl nd AOA on ud rek nd ACC content of Musct Biley A grpevines uds 127 W eeks Control AOA BSR BSR+AOA Oct Nov Dec Jn Fe Smpling time Fig 2 The time required for 5% ud rek of grpevine uds s ffected y ud scle removl (BSR) nd AOA tretments removl seemed to e mostly due to mechnicl injury, greter ese of gs exchnge nd removl of inhiitory fctors involved in the scles Fig 3 indicte tht the effect of the tretments on ACC content ws dependent on smpling dte or the intensity of ud dormncy ACC content recorded mrked increse in Octoer fter one week nd decresed fterwrds under BSR nd BSR + AOA However, it showed continuous increse under control nd reverse trend under AOA Wheres, in Novemer it incresed fter one week nd decresed in the forth week under ll tretments A continuous increse ws recorded in Decemer under ll tretments nd there ws no significnt difference etween control nd BSR + AOA nd etween AOA nd BSR especilly fter one week In Jnury, there ws no significnt chnge under control with time nd AOA tretment exhiited decline with time, while BSR nd BSR + AOA tretments recorded smll increment fter one week nd then decresed to e lower thn control in the second week In Ferury, the generl trend ws the decrese under ll tretments with time The effect of ud scle removl on incresing ACC content is supported y Mizutni et l (1995) They found tht shrp increse in the ACC content in the scle removed uds nd the high ACC levels in treted uds were mintined until dy 2 ut the content decresed to the control level t dy 3 Also they stted tht ud scle removl implies wounding stress Wounding, in generl, is known to produce ethylene in plnt tissues, wheres Iwski (198) reported tht it is difficult to ssume tht the termintion of ud rest is hstened y ethylene relesed y ud scle removl My e the ud rek ws enhnced ecuse of HCN which is co-product of the conversion of ACC to ethylene These results indicte tht ud scle removl which implies wounding stress is more effective within the prdormncy nd endodormncy stge nd less effective therefter AOA inhiits ethylene iosynthesis effectively when it is comined with ud scle removl thn with intct uds Also, it indicte tht ud rek of grpevine seems to e ssocited with the promotion of ethylene iosynthesis cused y wounding stress ACC (nmol/g fw) ACC (nmol/g fw) ACC (nmol/g fw) ACC (nmol/g fw) ACC (nmol/g fw) Octoer Novemer week 1 week 2 week Jnury Ferury week 1 week 2 week week 1 week 2 week Decemer week 1 week 2 week week 1 week 2 week Control C AOA BSR BSR+ AOA Fig 3 Effect of ud scle removl (BSR) nd AOA on ACC content of grpevine uds Verticl rs indicte SE

45 128 Effect of ud scle removl nd AOA on ud rek nd ACC content of Musct Biley A grpevines uds Acknowledgements The uthors re grteful to the Ministry of Eduction, Culture, Sports, Science nd Technology, Jpn for supporting this work through scholrship References Aeles, FB, PW Morgn nd ME Sltveit, 1992 Ethylene in Plnt Biology Acdemic Press, Sn Diego Erez, A, GA Couvillon nd SL Kys, 198 The effect of oxygen concentrtion on the relese of pech lef uds from rest HortScience, 15: Iwski, K nd RJ Wever, 1977 Effects of chilling, clcium cynmide, nd ud scle removl on ud rek, rooting, nd inhiitor content of uds of Zinfndel grpe (Vitis vinifer L) J Amer Soc Hort Sci, 12: Iwski, K 198 Effect of ud scle removl, clcium cynmide, GA 3, nd ethephon on ud rek of Musct of Alexndri grpe (Vitis vinifer L) J Jpn Soc Hort Sci, 48: Kwmt, M, E Nishid, H Ohr, K Ohkw nd H Mtsui, 22 Chnges in the intensity of ud dormncy nd internl compositions of current shoot in fig J Jpn Soc Hort Sci, 71: Kurod, H, T Sugiur nd D Ito, 22 Chnges in hydrogen peroxide content in flower uds of Jpnese per (Pyrus pyrifoli Nki) in reltion to reking of endodormncy J Jpn Soc Hort Sci, 71: Lizd, MCC nd SF Yng, 1979 A simple nd sensitive ssy for 1-minocyclopropne-1-croxylic cid Anl Biochem, 1: Mizutni, F, M Isogi nd K Kdoy, 1985 Role of ud-scles in dormncy nd ud rek of grpe vines I Inhiitive sustnces of scles for ud rek Mem Coll Agr, Ehime Univ, 29: Mizutni, F, A Hino, S Amno, K Kdoy, J Wtne nd H Akiyoshi, 1995 Effect of clcium cynmide, GA 3 nd scle removl on ud rek, ethylene production nd ACC content in grpevine uds Mem Coll Agr, Ehime Univ, 4: Smish, RM 1954 Dormncy in woody plnts Annu Rev Plnt Physiol, 5: Sure, MC 1985 Dormncy relese in deciduous fruit trees Hort Rev, 7: Spires, JM 1972 Effects of defolition nd ud-scle removl on ud ctivity in tung J Amer Soc Hort Sci, 97: Tohe, M, R Mochiok, S Horiuchi, T Ogt, S Shiozki nd H Kurook, 1998 Role of ACC nd glutthione during reking of dormncy in grpevine uds y high temperture tretment J Jpn Soc Hort Sci, 67: Tohe, M, R Mochiok, S Horiuchi, T Ogt, S Shiozki nd H Kurook, 1998 The influence of sustnces relted to ethylene iosynthesis on reking ud dormncy in grpevines J Jpn Soc Hort Sci, 67: Wng SY, M Fust nd GL Steffens, 1985 Metolic chnges in cherry flower uds ssocited with reking of dormncy in erly nd lte looming cultivrs Physiol Plnt, 65: Yng, SF nd JH Oetiker, 1998 Moleculr iology of ethylene iosynthesis nd its ppliction in horticulture J Jpn Soc Hort Sci, 67(6):

46 Journl of Applied Horticulture, 8(2): , July-Decemer, 26 Effect of exogenous ppliction of nti-stress sustnces nd elementl sulphur on growth nd stress tolernce of tissue culture derived plntlets of dte plm (Phoenix dctylifer L) cv Khls during cclimtiztion Appl Journl Mohmed A Awd 1 *, AA Soud nd SM El-Konissi Deprtment of Aridlnd Agriculture, College of Food nd Agriculture, UAE University, POBox Al-Ain, United Ar Emirtes *E-mil: Mohmedwd@ueuce; 1 Permnent ddress: Fculty of Agriculture, Pomology Deprtment, Mnsour University, El-Mnsour/Egypt Astrct There is high demnd for dte plm plntlets regenerted vi tissue culture techniques However, such plntlets require long cclimtiztion period extending months efore trnsplnting in the open field The effect of folir nd soil ppliction of ntistress sustnces nd elementl sulphur, respectively, on growth nd survivl percentge of tissue culture-derived Khls dte plm plntlets during cclimtiztion were studied The results showed tht ppliction of slicylic cid, cetyl slicylic cid (spirin), elementl sulphur, plntcur-e ( vitmin E formultion contining 25% -tocopherol) t 1%, nd oleic cid t 1 ppm, significntly incresed plntlet survivl percentges compred to the control In this respect, gmm minoutyric cid (GABA) t 2 mm ws the most effective tretment compred to 1 mm nd the control Slicylic cid, spirin, elementl sulphur nd plntcur-e (t 2%) significntly incresed the concentrtions of Fe, Mn, Zn, nd Cu in leflets compred to the control However, the mcro nutrients showed no cler response to the pplied tretments Appliction of 25 ppm of the ethylene iosynthesis locker, ABG-3168 (ABG), inhiited the growth of plntlets, nd completely suppressed growth t 5 ppm, suggesting the potentil role of ethylene iosynthesis in susequent plntlet development Irrigtion with 1, ppm se wter for two months decresed chlorophyll concentrtion nd incresed electrolyte lekge y 2-3 fold compred to the control nd the other tretments GABA t 2 mm significntly incresed chlorophyll concentrtion nd decresed electrolyte lekge of leflets compred to ll the sline wter tretments In contrst, ABG t 25 ppm significntly decresed chlorophyll concentrtion nd incresed electrolyte lekge of leflets y out 3-fold compred to ll the sline wter tretments These results show potentil role of GABA, slicylic cid, spirin nd oleic cid conducive for improved survivl percentge of plntlets nd stress tolernce during cclimtiztion Key words: Tissue culture, cclimtiztion, elementl sulphur, gmm minoutyric cid, slicylic cid, spirin, vitmin E, oleic cid, ABG-3168, Phoenix dctylifer L Introduction The numer of nturlly produced offshoots y fruiting plms re not sufficient to meet the high demnd for the new plnttions Thus, there is specil interest in dte plm plnts regenerted vi tissue culture since this technique cn provide lrge numer of homogenous plnts tht re true to type nd free of diseses nd cn e produced in lrge scle (Zid nd de Wet, 1999) Generlly, these plntlets require n cclimtiztion period of out months efore trnsplnting in open field conditions The VP1-stge (in vitro plnt in stge-1) is the most sensitive nd criticl stge due to the stress cused y moving the plntlets from the controlled lortory conditions nd trnsplnting in pots under greenhouse conditions for 2 to 4 months During this period, the survivl rte of plntlets is low which might, in the long run, led to economic losses (Zid nd de Wet, 1999; Frg et l, 22) Anti-stress sustnces my enhnce the plntlets tolernce to environmentl stresses thus incresing the survivl rte (Sreenivsulu et l, 2) Slicylic cid hs een found, mong mny other functions, to control ion uptke y roots, stomt conductivity nd to increse the ntioxidnt cpcity of plnts (Rskin, 1992) Alph tocopherol (vitmin E) nd scoric cid (vitmin C) re ntioxidnt sustnces concentrted in the chloroplst nd protect the photosynthetic pprtus when plnt is sujected to stress, y scvenging the excessively rective oxygen species known s free rdicls (Fryer, 1992; Krnner et l, 22) Environmentl stresses eg het, drought, slt nd mechnicl stress increse GABA ccumultion in plnt tissues The mechnicl dmge in soyen leves incresed GABA levels y 1 to 25-folds within 1 to 4 min of the strt of the stimulus (Kthiresn et l, 1997) Also, it hs een reported tht the kinetics of GABA ccumultion in plnts revels stress-specific pttern of ccumultion tht is consistent with physiologicl role for GABA in stress mitigtion (Kinnersley nd Turno, 2) Increse in free unsturted ftty cids, eg oleic cid, due to hydrolysis of memrne phospholipids, often occur during plnt senescence nd under dverse conditions including wounding, freezing, drought, slt, nd pthogen elicittion The olete-stimulted phospholipse-d nd phosphtidic cid decresed H 2 O 2 -induced cell deth in ridopsis (Wng nd Wng, 21; Zhng et l, 23) Ethylene hs significnt effect on plnt development nd influences the stges from

47 13 Stress tolernce of tissue culture derived plntlets of dte plm during cclimtiztion seed germintion to orgn senescence However, ecuse of the diversity of ethylene ction, it ws difficult to ssign definitive role for it in growth responses (Nqvi, 1994) Generlly, slinity nd drought stress increse ethylene level in vegettive plnt tissues (Nilsen nd Orcutt, 1996) Elementl sulphur is strongly involved in improving nutrient ssimiltion nd in stimulting the nti-oxidtive defense system of plnts through its metolite glutthione (Gondent nd Ullmn, 2; Tusz et l, 2) Also, the cidity produced during elementl sulphur oxidtion increses the vilility of nutrients such s P, Mn, C, nd SO 4 in soils which my enhnce growth performnce of plnts (Mrschner, 1995) The im of the present study ws to investigte the effect of exogenous ppliction of nti-stress sustnces nd elementl sulphur on survivl percentge, nutrient uptke nd slinity tolernce of tissue culture derived dte plm plntlets cv Khls during the cclimtiztion Mterils nd methods Plnt mterils nd experimentl procedure: This experiment ws conducted during the period from 24 to 25 on tissue culture-derived plntlets of the commercilly importnt Khls dte plm cultivr t the Dte Plm Reserch nd Development Unit (DPRDU), nd the Horticulture Lortory, Deprtment of Aridlnd Agriculture, College of Food nd Agriculture, UAE University, Al-Ain, UAE Helthy nd uniform tissue culture-derived plntlets were trnsplnted in pper pots (9 x 8 cm), mintined in greenhouse under controlled temperture (28 C nd 8-9% reltive humidity) nd received the norml cclimtiztion progrm (irrigtion nd fertiliztion, pest nd disese control) developed y DPRDU The plntlets were sujected to one of the following tretments with elementl sulphur lone s soil ppliction nd the other tretments s folir spry A complete rndomized design with 3 replictes (15 plntlets ech) per tretment ws dopted (Steel nd Torrie, 198) Elementl sulphur ws pplied s fine prticles in pots t three different levels of 283, 1415, or 283g/pot (these rtes correspond to 1, 5 nd 1 ton/h, respectively) Slicylic cid nd cetyl slicylic cid were pplied t 5 mm or 1 mm Plntcur-E ws pplied t 1 or 2% Oleic cid ws pplied t 1 ppm GABA ws pplied t 1 mm or 2 mm, only in the second experiment (GABA ws not ville t the time of conducting experiment 1) ABG ws pplied t 25 ppm or 5 ppm Elementl sulphur ws pplied once t trnsplnting, wheres, the other chemicls were pplied twice, t 2-dys nd t 2-weeks from trnsplnting All tretments, except elementl sulphur, were comined with 1% Tween-2 (polyoxyethylene soritn monolurte) s wetting gent In control, plntlets were spryed with only wter plus 1% Tween-2 This experiment ws repeted to confirm the otined results During the VP1-stge of cclimtiztion (in vitro plntlets under cclimtiztion procedure extending -4 months t 28 C ccording to the definition provided y the DPRDU), the numer of survived, well rooted plntlets nd suitle to move to VP2- stge were recorded t 2 nd 4 months from trnsplnting The survived plntlets were trnsplnted in plstic pots (25 x 16 cm) At the end of the VP2-stge (in vitro plntlets under cclimtiztion procedure extending 4-12 months t 3 C), ll the plntlets in ll tretments survived These plntlets were trnsplnted in lrger plstic pots (4 x 24 cm) During the VP3-stge (in vitro plntlets under the cclimtiztion procedure extending months t out 35 C), leflets t middle ge were excised from plntlets nd trnsferred to the horticulture lortory for nutrient nlysis During the VP3 stge, the plnts of ech tretment were irrigted with se wter (diluted to 1, ppm) (8ml/pot/week) for two months The control tretment ws sudivided into two equl groups, one group irrigted with diluted se wter nd the other group irrigted with norml wter At the end of the two months, leflets t middle ge were excised from the plntlets nd immeditely trnsferred to the horticulture lortory for chlorophyll nd electrolyte lekge mesurements Nutrient nlysis of the leflets: During the VP3-stge, efore the strt of se wter irrigtion, rndom leflet smples from only experiment 1 were collected from ech replicte of ech tretment for nutrient nlysis The collected leflets were wshed with deionized wter nd oven dried for 48 h t 65 C The smples were crushed nd pssed through 2-mesh stinless steel sieve Smples were digested y the dry shing method s descried y Jones nd Cse (199) Totl content of micronutrients (Fe, Mn nd Zn) were determined y the tomic sorption spectrophotometer Vrin, model SpectrAA 22 FS Sulphur content ws mesured using ICPAES, Vrin model Vist MPX Phosphorus ws determined colorimetriclly ccording to the method descried y Kuo (1996) Totl nitrogen concentrtion in leves ws determined, fter the wet digestion ccording to Jones nd Cse (199), y stem distilltion using the semi utomtic Kjeldhl method Totl chlorophyll mesurement: Totl chlorophyll ws mesured in leflets y the procedure of Hiscox nd Isrelstm (1979) Two hundred milligrms of lef tissue in frctions ws plced in vil contining 14 ml dimethyl sulphoxide (DMSO) nd chlorophyll ws extrcted into the fluid without grinding t 65 o C y incuting for 8 hours The extrct ws then trnsferred to grdute tue nd mde up to totl volume of 2 ml with DMSO The OD vlues t 645 nd 663 nm were red in Beckmn DBG spectrophotometer ginst DMSO lnk Electrolyte lekge mesurement: During the VP3-stge two leflets of middle ge, ech from different plntlet, were collected for ech replicte/tretment The collected leflets were directly wshed with tp wter nd rinsed in deionized wter to remove the dust nd electrolytes dhering to the surfces nd then lightly clened with tissue ppers Lef segments of 3 x 3 cm were cut from the middle of ech leflet (pinn), cut into 2- pieces, plced in ech test tue contining 4 ml of deionized wter then loosely covered with luminum foil Three replictes, ech with two leflets were used for ech tretment The tues were then incuted in the refrigertor t 6 o C over night efore electricl conductivity of ech solution ws determined In the next dy, the tues were tken from the refrigertor, wrmed up to room temperture (22 o C±2), plced in shker (Gesllschft FurLortechnik, GFL, mh-model 315, Germny) for one hour to diffuse electrolytes, vortexed for few seconds Electrolyte lekge efore killing ws mesured with digitl electricl conductivity meter (Orion- model 15- USA) The leflet

48 Stress tolernce of tissue culture derived plntlets of dte plm during cclimtiztion 131 smples were killed y utoclving (JKA-J39 Autoclve, Jpn) t 121 o C for 2 min to relese ll electrolytes, cooled to 22±2 o C fter which they were left on the shker for 1 hour, vortexed for few seconds Totl electrolyte lekge ws mesured y using the sme digitl conductivity meter Percentge of electrolyte lekge ws clculted for ech smple using the rtio of the initil (efore killing) to the finl (fter killing) mesurements (Ingrm nd Buchnn, 1984; Jing nd Hung, 21; Frg et l, 22) Sttisticl nlysis: The dt were sujected to nlysis of vrince (ANOVA) using the sttisticl pckge MSTATC Progrm (Michign Stte University, Est Lnsing, MI) Comprisons etween mens were mde y lest significnt differences (LSD) t 5% level Results Percentge of successful plntlets t VP1-stge: Most of the pplied sustnces positively influenced Khls plntlets growth during the VP1-stge of the cclimtiztion period (estimted s the percentge of successful plntlets tht were redy to move to the next stge of the cclimtiztion progrm or VP2-stge) (Tle 1) In experiment 1, elementl sulphur ppliction, especilly t the low nd the moderte levels, incresed plntlets growth fter 2 months during the VP1-stge Aspirin nd slicylic cid were the most effective tretments, especilly t 5 mm, in incresing growth nd development of plntlets oth fter 2 nd 4 months However there ws no significnt difference etween the high nd the low concentrtion of spirin nd slicylic cid on the survivl percentge of plntlets The ABG tretment t 25 ppm significntly inhiited susequent plntlet growth nd completely Tle 1 Successful plntlet percentge of Khls dte plm during the VP1-stge of the cclimtiztion period s ffected y elementl sulphur nd nti-stress sustnces Tretments Successful plntlet (%) during the VP1-stge Experiment 1 Experiment 2 After 2 months After 4 months After 2 months After 4 months Control Sluphur t 283g sulphur/pot Sulphur t 1415g sulphur/pot Sulphur t 283g sulphur/pot Aspirin t 5 mm Aspirin t 1 mm Slcylic cid t 5 mm Slcylic cid t 1 mm Plntcur-E t 1% Plntcur-E t 2% ABG t 25 ppm ABG t 5 ppm Oleic cid 1 ppm GABA 1 mm GABA 2 mm F-test *** *** *** *** LSD (P=5) *** significnt t level P = 1; - not clculted prevented plntlet development t 5 ppm Similrly, plntcur- E nd oleic cid lso hd no significnt effect on the survivl percentge of plntlets oth fter 2 nd 4 months (Tle 1) In experiment 2, similr trend ws oserved, however, the positive effect of sulphur, spirin nd slicylic cid ws less pronounced on the survivl percentge of plntlets thn in experiment 1 In contrst to the results of experiment 1, plntcur-e (especilly t 1%) nd oleic cid t 1 ppm ppliction hd significnt positive effects on the survivl percentge of plntlets oth fter 2 nd 4 months Interestingly, GABA t oth concentrtions (1 nd 2 mm) showed cler positive effect on the survivl percentge of plntlets In this respect, GABA t the high concentrtion of 2 mm ws more effective (91% survived plntlets) thn t the low concentrtion of 1 mm (73% survived plntlets) (Tle 1) At the end of the VP2-stge ll plntlets in ll the tretments survived Nutrient concentrtion of leflets t the VP3 stge: Slicylic cid nd its derivtive cetyl slicylic cid (spirin) ppliction, especilly t the lower concentrtion, significntly incresed the concentrtions of Fe, Mn, Zn, nd Cu in the leflets compred to the control (Tle 2) Also, elementl sulphur ppliction significntly incresed the concentrtion of Fe, Mn, Zn nd Cu However, the concentrtion of Cu ws significntly decresed t the highest level of sulphur ppliction compred to the control Plntcur-E ppliction, only t the higher concentrtion, significntly incresed the concentrtion of Fe, Mn, Zn, nd Cu in the leflets compred to the control (Tle 2) Oleic cid ppliction significntly incresed the concentrtion of N, Fe nd Mn ut the other nutrients were not ffected The untreted (control) plntlets contined significntly higher concentrtion of sulphur thn ll other tretments However the concentrtions of N, P nd K were only slightly ffected y the pplied tretments (Tle 2) Chlorophyll concentrtion nd electrolyte lekge of leflets fter 2-months of irrigtion with se wter: After two months irrigtion with se wter (diluted to 1, ppm slinity) ll plnts survived in ll tretments including the control However, the irrigtion with se wter significntly decresed the concentrtion of chlorophyll in ll the tretments compred to the control in oth experiments (Tle 3) ABG ppliction t 25 ppm significntly decresed chlorophyll concentrtion compred with the control nd most of the other tretments in oth experiments Plntlets treted with 5 mm spirin contined significntly higher concentrtion of chlorophyll thn those treted with 1 mm in only experiment 1 There ws no significnt difference in chlorophyll concentrtion etween the different sulphur tretments in oth the experiments GABA ppliction t 2 mm significntly incresed the concentrtion of chlorophyll compred to the se wter tretment nd most of the other tretments (Tle 3) Electrolyte lekge of leflets significntly incresed (2-3 fold) y se wter tretment in oth experiments (Tle 3) ABG ppliction t 25 ppm significntly incresed electrolyte lekge (out 3-fold) compred with the control nd the other tretments in oth experiments In experiment 1, elementl sulphur t oth low nd high levels significntly decresed electrolyte lekge compred to the se wter tretment Plntlets treted with spirin nd slicylic cid t oth 5 mm nd 1 mm showed significntly lower percentges of electrolyte lekge thn those

49 132 Stress tolernce of tissue culture derived plntlets of dte plm during cclimtiztion Tle 2 Nutrient concentrtion of Khls dte plm plnt leflets t the VP3-stge of the cclimtiztion period s ffected y elementl sulphur nd nti-stress sustnces Tretments N% P% K% S% Fe Mn Zn Cu (ppm) (ppm) (ppm) (ppm) Control Sluphur t 283g sulphur/pot Sulphur t 1415g sulphur/pot Sulphur t 283g sulphur/pot Aspirin t 5 mm Aspirin t 1 mm Slcylic cid t 5 mm Slcylic cid t 1 mm Plntcur-E t 1% Plntcur-E t 2% Oleic cid 1 ppm F-test *** *** *** *** *** *** *** *** LSD (P=5) (***)-significnt t level P = 1; (-)-not clculted Tle 3 Totl chlorophyll concentrtion nd electrolyte lekge (%) of Khls dte plm plnt leflets treted with se wter t the VP3-stge of the cclimtiztion period s ffected y elementl sulphur nd some nti-stress sustnces Tretments Experiment (1) Experiment (2) treted with only se wter in experiment 1 Among ll the tretments, oleic cid significntly decresed electrolyte lekge In experiment 2, elementl sulphur t the high level significntly decresed electrolyte lekge compred to the se wter tretment Interestingly, GABA ppliction t 2 mm significntly decresed electrolyte lekge thn the se wter nd most of the other tretments (Tle 3) Discussion Chlorophyll (mg/g fw) Electrolyte lekge (%) Chlorophyll (mg/g fw) Electrolyte lekge (%) Control (norml wter) Se wter Sluphur t 283g sulphur/pot Sulphur t 1415g sulphur/pot Sulphur t 283g sulphur/pot Aspirin t 5 mm Aspirin t 1 mm Slcylic cid t 5 mm Slcylic cid t 1 mm Plntcur-E t 1% Plntcur-E t 2% ABG t 25 ppm Oleic cid 1 ppm GABA 1 mm GABA 2 mm F-test *** *** *** *** LSD (P=5) (***)-significnt t level P = 1; (-)not clculted Our results showed tht the ppliction of nti-stress sustnces GABA, plntcur-e, slicylic cid, spirin, oleic cid, nd elementl sulphur incresed survivl percentge of the plntlets during the VP1-stge of the cclimtiztion period which would otherwise led to economic loss (Tle 1) The plntlets during VP1-stge ws most sensitive to stress since during oth VP2 nd VP3 stges ll the plntlets survived in ll the tretments including the control The results showed tht, in oth experiments, irrigtion with se wter for two months significntly decresed the concentrtion of chlorophyll nd incresed electrolyte lekge of the plnt leflets compred to the control (Tle 3) In this context, most of the pplied sustnces especilly GABA, slicylic cid nd spirin incresed chlorophyll concentrtion nd clerly decresed the electrolyte lekge in the plnt leflets compred to those treted only with se wter (Tle 3) Such effects might e due to protecting the endogenous nti-oxidnt systems often correlted with incresed resistnce to oxidtive stress nd/or controlling the level of free rdicls within plnt tissues (Sreenivsulu et l, 2) It is lso possile tht the nti-stress sustnces were effective in mintining the memrne integrity to reduce the lekge of electrolyte through its positive effect on the ntioxidnt enzymes system s hs een demonstrted y Pinhero nd Fletcher (1994) in corn seedlings It is generlly known tht most of environmentl stresses hve in common similr mechnism in ffecting plnt growth nd performnce Under stress conditions, the genertion of free rdicls nd low nutrient uptke re elieved to e the min cuse for dmging nd dis-functioning of plnt cells (Bohnert et l, 1995; Sreenivsulu et l, 2) Elementl sulphur is strongly involved in improving nutrient ssimiltion nd in the ntioxidtive defense systems of plnts through its metolite glutthione Glutthione is lso necessry for the efficient-functioning of other defense systems such s scorte nd -tocopherol regenertion nd crotenoids nd protein thiols conservtion (Gondent nd Ullmn, 2; Tusz et l, 2) Hence, in the present study we propose tht the protection confined y elementl sulphur ws due to similr mechnism

50 Stress tolernce of tissue culture derived plntlets of dte plm during cclimtiztion 133 of enhnced free rdicl scvenging systems in the plnt Our results showed positive effects of most of the pplied ntistress sustnces especilly slicylic cid, spirin nd elementl sulphur on the concentrtions of Fe, Mn, Zn, nd Cu in the leflets compred to the control (Tle 2) These nutrients hve een reported to e connected with the improved tolernce of severl plnts (Kinnersley nd Turno, 2) Elementl sulphur lso incresed the level of these nutrients in the leflets proly y incresing their vilility nd uptke vi modifying soil ph s reported y Mrschner (1995) The results on slicylic cid nd spirin re in ccordnce with those of Rskin (1992) who reported tht slicylic cid, mong mny other functions, controls ion uptke y roots nd stomtl conductivity nd increses the ntioxidnt cpcity of plnts These results suggest slicylic cid nd spirin s meditor of minerl cquisition in stressrelted metolism It hs een reported tht slicylic cid nd its derivtive spirin induced multiple stress tolernce in en nd tomto plnts ginst drought, het, chilling, nd slinity when seeds imied in queous solutions (1-5 mm) or when pplied to the plnts s folir spry or even s soil drenches (Senrtn et l, 2) Moreover, slicylic cid hs een found to protect whet plnts from drought nd slinity stress, diminished the ltertion of phytohormones, nd incresed the level of cell division within the picl meristem of seedling roots which cused n increse in plnt growth (Shkirov et l, 23) Our results on the positive effects of GABA ppliction (Tles 1 nd 3) confirm those of Kinnersley nd Turno (2) who reported tht the kinetics of GABA ccumultion in plnts revels stress-specific pttern of ccumultion tht is consistent with physiologicl role for GABA in stress mitigtion To the est of our knowledge, this is pioneer study investigting the role of GABA s potentil nti-stress sustnce for improving growth nd stress tolernce ility of dte plm plntlets (Tles 1 nd 3) Such positive effects of GABA hve een confirmed on Khdrwy dte plm plntlets, however, the lower concentrtion (1 mm) ws more effective thn the higher one (2 mm), indicting cultivr dependent concentrtion (unpulished dt) The ppliction of the ethylene iosynthesis locker, ABG t 25 ppm gretly inhiited nd completely suppressed susequent plntlet development nd growth t 5 ppm (Tle 1) Also, the ABG-treted plntlets contined lower chlorophyll concentrtion nd showed higher electrolyte lekge, fter se wter irrigtion, thn the control (Tle 3) These results suggested n importnt role of ethylene in susequent plntlet development This study might provide certin recommendtions to enefit oth the tissue culture-sed dte plm industry nd growers Acknowledgements This work ws finncilly supported y the Reserch Affirs t the UAE University under the contrct No /4 We thnk Mr Aou-Mosslm Az, Rshid Adel-Ftth, Sd Khlil nd Azhri Omer, College of Food nd Agriculture, UAE University for their indispensle technicl support We lso thnk the stff of CLU, UAE University for their support in sulphur nlysis The contriution of Dte Plm Reserch nd Development Unit, UAE University is gretly pprecited References Bohnert, H, ED Nelson nd RG Jensen, 1995 Adpttion to environmentl stresses The Plnt Cell, 7: Frg, KM, S Al-Konnissi nd A Zid, 22 Tolernce of Brhi nd Khls dte plm plnts reproduced y tissue culture to het nd slt stresses nd tretments to enhnce their tolernce Proceedings of the Fourth Annul UAE University Reserch Conference, UAE, pp Fryer, MJ 1992 The ntioxidnt effects of thylkoid vitmin E ( - tocopherol) Plnt Cell Environ, 15: Gondent, L nd P Ullmn, 2 Glutthione metolism in plnts in reltion to stress tolernce Plnt Sulfur Reserch in Europe, Cost Action 829, Instit Plnt Nutri Soil Science, Federl Agric Res Centre, Brunschweig, Germny, pp Hiscox, JD nd GF Isrelstm, 1979 A method for the extrction of chlorophyll from lef tissue without mcertion Cn J Bot, 57: Ingrm, DL nd D Buchnn, 1984 Lethl high tempertures for roots of three citrus rootstocks J Amer Soc Hort Sci, 19: Jing, Y nd B Hung, 21 Physiologicl responses to het stress lone or in comintion with drought: A comprison etween tll Fescue nd perennil Ryegrss HortScience, 36: Jones, JB nd VW Cse, 199 Smpling, Hndling, nd Anlyzing Plnt Tissue Smples In: Soil Testing nd Plnt Anlysis,Westermn (ed) Book Series no 3 Soil Science Society Americ, Mdison WI, pp Kthiresn, A, P Tung, CC Chinnpp nd DM Reid, 1997 Gmm minoutyric cid stimultes ethylene iosynthesis in sunflower Plnt Physiol, 115: Kinnersley, AM nd FJ Turno, 2 Gmm minoutyric cid (GABA) nd plnt responses to stress Criti Rev Plnt Sci, 19: Krnner, I, RP Beckett, S Wornik, M Zorn nd HW Pfeifhofer, 22 Revivl of resurrection plnt correltes with its ntioxidnt sttus Plnt J, 31: 13-2 Kuo, S 1996 Phosphorus In: Methods of Soil Anlysis, prt 3, DL Sprks (ed) Chemicl Methods Soil Sci Soc Am, Book Series no 5 Mdison, WI, USA Mrschner, H 1995 Minerl Nutrition of Higher Plnts 2 nd Edition Acdemic Press, London Nqvi, SSM 1994 Plnt hormones nd stress phenomen In: Hndook of Plnt nd Crop Stress, M Pessrkli (ed), Mrcel Dekker, New York - Bsel, Hong Kong, p383-4 Nilsen, E T nd DM Orcutt, 1996 Physiology of plnts under stress; iotic fctors John Wiley & Sons, INC, pp Pinhero, RG nd RA Fletcher, 1994 Pcloutrzol nd ncymidol protect corn seedlings from high nd low temperture stress Plnt Growth Reg, 15: Rskin, I 1992 Role of slicylic cid in plnts Annu Rev Plnt Physiol Plnt Mol Biol, 43: Senrtn, T, D Touchell, E Bunn nd K Dixon, 2 Acetyl slicylic cid (spirin) nd slicylic cid induce multiple stress tolernce in en nd tomto plnts Plnt Growth Regul, 3: Shkirov, FM, AR Skhutdinov, MV Bezrukov, RA Ftkhutdinov nd DR Ftkhutdinov, 23 Chnges in the hormonl sttus of whet seedlings induced y slicylic cid nd slinity Plnt Sci, 164: Sreenivsulu, N, B Grimm, U Wous nd W Weschke, 2 Differentil response of ntioxidnt compounds to slinity stress in slt-tolernt nd slt-sensitive seedlings of foxtil millet (Setri itlic) Physiologi Plntrum, 19: Steel, RGD nd JH Torrie, 198 Principles nd procedures of sttistics, A iometricl pproch 2 nd Edition, McGrw-Hill, New York, USA, 198

51 134 Stress tolernce of tissue culture derived plntlets of dte plm during cclimtiztion Tusz, M, A Wonishch, M Muller nd D Grill, 2 The role of glutthione in the development of stress nd dmge to plnts Plnt Sulfur Reserch in Europe, Cost Action 829, Instit Plnt Nutri Soil Science, Federl Agric Res Centre, Brunschweig, Germny, pp Wng, C nd X Wng, 21 A novel phospholipse D of Aridopsis tht is ctivted y oleic cid nd ssocited with the plsm memrne Plnt Physiol, 127: Zid, A nd PF de Wet, 1999 Dte plm propgtion In: Dte plm cultivtion, A Zid nd EJ Aris (eds), FAO plnt production nd protection pper, No 156 p Zhng, W, C Wng, C Qin, T Wood, G Olfsdottir, R Welti nd X Wng, 23 The olete-stimulted phospholipse D, PLDs, nd phosphtidic cid decrese H 2 O 2 -induced cell deth in Aridopsis The Plnt Cell, 15:

52 Journl of Applied Horticulture, 8(2): , July-Decemer, 26 Impct of Anthurium spp genotype on cllus induction derived from lef explnts, nd shoot nd root regenertion cpcity from cllus Appl Journl Duong Tn Nhut 1, Nguyen Duy 1, Nguyen Nhu H Vy 1, Chu Diem Khue 1, Dinh Vn Khiem 1 nd Do Nng Vinh 1 Deprtment of Plnt Biotechnology, Dlt Institute of Biology, 116 Xo Viet Nghe Tinh, D Lt, Lm Dong, Vietnm; 2 Institute of Agriculturl Genetics, H Noi, Vietnm E-mil duongtnnhut@yhoocom Astrct In this study, the most criticl step in Anthurium micropropgtion ws the induction of primry clli from lef segments Genotype plyed n importnt role during in vitro multipliction of Anthurium Cllus induction from lef segments ws exmined in ten Anthurium cultivrs: Crnvl, Neon, Choco, Sonte, Midori, Pistche, Tropicl, Sfri, Arizon nd Cncn on MS medium supplemented with 1 mg L -1 BA, 8 mg L -1 2,4-D, 3 g L -1 glucose, 8 g L -1 gr nd djusted to ph 6 After 1 dys, lef segments of eight genotypes formed clli, mong them, cultivr Pistche hd the highest cllus induction rtio (651%) nd two genotypes, Crnvl nd Cncn, showed no response After multipliction, clli were sucultured on shoot regenertion medium, 1/2 MS with NH 4 NO 3 level djusted to 26 g L -1, dded with 2 g L -1 glucose, 1 mg L -1 BA, 8 g L -1 gr nd djusted to ph 6 Shoots were otined from ll cultivrs with different potentil of shoot regenertion The verge numer of shoots per explnt in Tropicl (11) ws higher thn tht of Choco (43) nd Pistche (35), nd shoots (t lest 1 mm high) were excised nd cultured on rooting medium, 3 g L -1 glucose, 8 g L -1 gr nd 1 g L -1 ctivted chrcol dded to 1/4 MS medium All shoots consistently formed roots fter 3 dys nd plntlets developed well fter eing trnsferred to the nursery The propgtion process took 1 nd hlf months to complete Key words: Anthurium ndrenum, genotypes, lef explnts, cllus induction, shoot regenertion, root regenertion Introduction Anthurium sp, perennil herceous plnt, is n economiclly importnt genus of Arcee It is ntive to the tropics of Centrl nd South Americ, nd is enjoyed for its colorful ttrctive, luxurious flowers nd exotic folige It is cultivted commercilly s cut flower nd potted plnt throughout the world The necessity to find rpid nd effective propgtion method is required to stisfy the incresing demnd of Anthurium Different explnts, leves, immture petioles, inflorescence stlks, spthes, spdices nd xillry nodes hve een used for micropropgtion Hmidh et l (1997) induced somtic emryogenesis from leves, petioles nd roots on medium to which vrious concentrtions of sugr nd severl types of growth regultors were dded For somtic emryos, lef pieces from micropropgted plnts were found to e est on MS medium (Murshige nd Skoog, 1962) with 1 mg L -1 2,4-dichlorophenoxycetic cid (2,4-D) nd high sucrose concentrtion (6%), ut low glucose concentrtion (3%) ws etter to induce cllus MS with 1 mg L -1 6-enzyldenine (BA) ws the est medium for shoot regenertion nd 1/2 MS plus 1 mg L -1 α-nphthlene cetic cid (NAA) for shooting (Hmidh et l, 1997) Pierik (1976) developed method of Anthurium propgtion using solid nd liquid medi lterntively Geier (1986) used culture medi derived from Nitsch s sl medium (1969), which contined minerls, vitmins, sucrose nd gr Of the vrious medi tested, including NH 4 NO 3 concentrtion nd the presence or sence of different uxins nd cytokinins, NH 4 NO 3 level hd the most significnt effect on cllus nd shoot formtion, low level eing est for regenertion in ll the genotypes investigted Cytokinin ws lso necessry to induce cllus from lef explnts, ut if uxin nd cytokinin were comined, shoot induction nd development were etter in suculturing Kuehnle nd Sugii (1991) exmined the influence of growth regultors on cllus induction derived from lef segments Cllus ws produced most successfully on modified Pierik medium contining 2,4-D nd BA, while sucrose ws etter thn glucose s cron source Another fctor tht plys n importnt role during Anthurium in vitro propgtion is the effect of genotype Pierik (1976) exmined 38 genotypes of Anthurium ndrenum nd oserved moderte to strong cllus formtion from lef segments in 31 genotypes, very poor cllus formtion in four nd no response in the remining three genotypes In nother study, Geier (1986) found tht out of 18 genotypes, three did not show ny regenertion, five produced only cllus nd ten formed culogenic cllus when lef explnts were cultured on Nitsch medium Not only cllus induction ut lso shoot regenertion from cllus ws dependent on the genotype Oserving the verge numer of shoots per culture, Geier (1986) otined <1 in five genotypes, 1-1 in three genotypes, nd >1 in two genotypes This result showed tht different genotypes hd different responses to the sme medium nd the identicl procedure could not e pplied for the in vitro propgtion of ll Anthuriums Although mny reserches hve lredy een conducted, the min ojective of this study ws to investigte the effect of genotype on cllus induction derived from lef explnts nd its influence on shoot nd root regenertion cpcity, which llows estlishing

53 136 Impct of Anthurium spp genotype on cllus induction derived from lef explnts suitle procedure suited to ech Anthurium, which cn e used for commercil production Mterils nd methods Plnt mteril: Lef segments of ten vlule cultivrs of Anthurium ndrenum: Crnvl, Neon, Choco, Sonte, Midori, Pistche, Tropicl, Sfri, Arizon nd Cncn were used s mteril Young leves hving pproximtely 1/2-2/3 of the finl length were surfce-sterilized with HgCl 2 1%, nd then sectioned to out 1 cm 2 pieces Cllus induction nd multipliction: To investigte the effect of genotype on cllus induction derived from lef explnts, 1 lef explnts from ech genotype were trnsplnted onto cllus induction medium: 1/2 MS sl medium supplemented with 3 g L -1 glucose, 1 mg L -1 BA, 8 mg L -1 2,4-D nd 8 g L -1 gr Two lef segments were plced in ech culture vessel, nd cllus ws induced in drkness t 2 ± 2 o C, 75-8% reltive humidity When cllus hd formed nd chieved the required size (out 2 to 25 cm 2 ), it ws divided nd three 5 cm 2 pieces were trnsferred to cllus multipliction medium (1/2 MS medium with 2 g L -1 glucose, 1 mg L -1 BA nd 8 g L -1 gr) to get lrge mount of clli Shoot regenertion: To investigte the effect of genotype on shoot regenertion from cllus, clli otined in four month sucultured Tropicl, Choco nd Pistche were trnsferred to shoot regenertion medium: 1/2 MS with NH 4 NO 3 lowered to 26 g L -1, 2 g L -1 glucose, 1 mg L -1 BA nd 8 g L -1 gr Frile, etiolted clli were chosen to increse the frequency of shoot regenertion nd ech culture vessel contined three 7 cm 2 cllus pieces Rooting: For more rpid nd consistent rooting, 1 mm tll shoots of Tropicl, Choco nd Pistche were isolted nd trnsplnted to rooting medium: 1/4 MS medium supplemented with 3 g L -1 glucose, 8g L -1 gr nd 1 g L -1 ctivted chrcol Culture conditions (in vitro nd ex vitro): ph ws djusted to 6 in ll medi except for the rooting medium (ph 62) nd medi were utoclved for 3 minutes t 121 o C nd 1 tm Cllus multipliction, shoot regenertion nd root formtion were crried out under 1-hour photoperiod, 45 μmol m -2 s -1 illumintion, nd t 25 ± 2 o C nd 7-75% reltive humidity Plntlets of Tropicl, Choco nd Pistche with more thn 2 well-developed roots nd 25-3 cm in height were isolted nd trnsferred to 5-7% of dylight shded nursery Plntlets were cultivted on tree fern fier sustrte t 22 ± 2 o C nd 8-85% reltive humidity, spryed with wter 2 times dy, Komix BFC s fertilizer 3 times week nd 5 g L -1 fungicide solution mixture including Zodic 8 WP nd Mnozep 8 WP 2 times week Dt ws nlyzed for significnce y ANOVA with the men seprtion y Duncn s multiple rnge test (Duncn, 1995) Results nd discussion Effects of genotype on cllus induction from lef segments: Two 1 cm 2 lef pieces were cultured in ech cllus inducing medium vessel After 3 dys of culture in drkness, cllus formtion ws first oserved in Midori nd Sfri with 182% nd 125% explnts inducing cllus, respectively (Tle 1) Lef pieces of other cultivrs were green ut without trces of cllus Tle 1 Effect of genotype on cllus induction of Anthurium fter different periods Cultivr Cllus induction rte (%) 3 dys 6 dys 1 dys Crnvl Neon 2 2 Choco Sonte Midori Pistche Tropicl Sfri Arizon Cncn Explnts were continuously left in the drk After 6 dys of culture, ll cultivrs induced cllus except for Crnvl nd Cncn While the cllus formtion rte of cultivrs Midori nd Sfri ws slow (182 to 2% in Midori nd 125 to 167% in Sfri ), some cultivrs which did not form cllus fter 3 dys of culture, hd high rtes of cllus induction (286% in Sonte, 343% in Tropicl nd 618% in Pistche ), even higher thn those of genotypes inducing clli erly Explnts of other genotypes lso formed cllus ut t lower rtes: 125% in Choco, 98% in Arizon nd 2% in Neon And fter 1 dys, the regenertion rte of the ten cultivrs grdully incresed with corresponding rise in fresh weight of clli In Choco, Pistche nd Sfri, lef explnts strted to turn yellow, yet clli still developed Sfri explnts induced cllus erly ut hd low induction rte (28%) while Pistche hd the highest cllus induction rte (651%) The induction rte of Midori nd Tropicl were similr (out 352 nd 35%, respectively), however the size of Tropicl clli were profusely lrger In ddition, 286% of Sonte lef explnts induced lrge clli, which developed quickly fterwrds Other cultivrs showed little chnge: low induction rte in Choco (125%), Arizon (17%) nd Neon (2%) Crnvl nd Cncn did not induce cllus These results indicte tht ten Anthurium genotypes hd different responses to the sme cllus induction medium nd culture conditions They could e clustered into four groups: group of Pitsche hving high cllus induction rte; group of Midori, Tropicl, Sonte, Sfri, Choco nd Arizon hving verge cllus induction rte; group of Neon hving low cllus induction rte nd group of Crnvl nd Cncn, which could not induce cllus Effect of genotype on shoot regenertion from cllus: Shoot formtion ws investigted in Choco, Pitsche nd Tropicl, which hd different cllus induction rtes After 3 dys of culture, shoot regenertion ws first oserved in Tropicl, while other cultivrs did not form shoots Clli of three cultivrs still survived fter 1 dys of culture Among them, the highest numer of regenerted shoots ws otined in Tropicl (11 shoots per explnt), which were firm with smll leves; moreover, much culogenic clli ws induced Choco hd lower numer of regenerted shoots (43 per explnt), which hd lrge leves nd developed slowly, ut its clli turned drker The lowest numer of shoots formed in Pistche (35 per explnt) (Tle 2)

54 Impct of Anthurium spp genotype on cllus induction derived from lef explnts 137 Fig 1 Micropropgtion y lef explnt culture of Anthurium spp: (), () cllus induced from lef segment; (c) shoots regenerted from cllus; (d), (e) root formtion; (f) plntlets; (g) ex vitro plntlets; (h) Anthurium Choco flower Tle 2 Effect of genotype on shoot regenertion from cllus of Anthurium Cultivrs Cllus survivl rte (%) Numer of shoots per explnt Choco 1 43 x Pistche 1 35c s Tropicl 1 11 s x Different letters within column indicte significnt differences t P < 5 y Duncn s multiple rnge test Effect of genotype on root formtion from shoots: Shoots of Choco, Pistche nd Tropicl were isolted nd trnsferred to shoot formtion medium without plnt growth regultors (1 shoots per vessel), which is necessry for firm nd rpid root formtion Although shoots nd roots could regenerte nturlly when cultured over long period nd under illumintion on MS medium, root formtion depended on the size nd developmentl stge of shoots, nd culture medi Shoots formed roots when plced on medi with low concentrtion of mcroelements nd without cytokinin, BA Results showed tht ll shoots of the three cultivrs formed fully developed root systems fter 3 dys of culture with comprle numer of roots However, sed on the chrcteristics of ech cultivr, formed roots hd different shpes nd sizes Shoots with smll leves of Tropicl formed long nd thin roots, while shoots with lrger leves of Choco nd Pistche formed short ut thick roots Effect of genotype on ex vitro plntlet development: Five hundred homomorphous plntlets ech of Choco, Pistche nd Tropicl were plced in the nursery All of them survived nd developed well fter 3 dys of culture Some plntlets with very short roots or even without roots lso grew nd formed roots post plnting, lthough it took more time Anthurium micropropgtion y lef explnt culture ws chrcterized y the use of different medi t different stges of morphogenesis The most difficult step in this propgtion process ws cllus induction from lef explnt This is due to the restricted dedifferentition of mture tissue nd depends strongly on genotype Anthurium Pistche could est induce clli on 1/2 MS medium supplemented with 3 g L -1 glucose, 1 mg L -1 BA, 8 mg L -1 2,4-D, 8 g L -1 gr nd djusted to ph 6 (651% explnts induced clli fter 1 dys of culture), wheres Crnvl nd Cncn could not Genotype ffected shoot regenertion slightly ut did not ffect root formtion The criticl fctor in shoot regenertion stge ws the effect of NH 4 NO 3 concentrtion Tropicl hd the highest numer of regenerted shoots per explnt (11 shoots per explnt fter 1 dys) when its clli were cultured on 1/2 MS with NH 4 NO 3 lowered to 26 g L -1, 2 g L -1 glucose, 1 mg L -1 BA, 8 g L -1 gr (ph 6), nd ll genotypes formed roots normlly (fter 3 dys) on 1/4 MS medium supplemented with 3 g L -1 glucose, 8 g L -1 gr, 1 g L -1 ctivted chrcol (ph 62) All plntlets developed well when trnsferred to the nursery According to the results of this study, the propgtion process took ten nd hlf months to complete: four months for cllus induction, two nd hlf months for mss multipliction, three months for regenerting shoots nd one month for forming roots This process ws est for Pistche, pproprite for Tropicl, ut hd to e djusted to otin more regenerted shoots of Pistche nd Tropicl clli More reserches re required to define the pproprite requirements for ech of the other cultivrs Acknowledgement We thnk Ntionl Project KC 4 19 for finncil support References Duncn, DB 1995 Multiple rnge nd multiple F test Biomet, 11: 1-42 Geier, T 1986 Fctors ffecting plnt regenertion from lef segments of Anthurium scherzerinum Schott (Arcee) cultured in vitro Plnt Cell Tiss Org Cult, 6: Hmidh, M, AGA Krim nd P Deergh, 1997 Somtic emryogenesis nd plnt regenertion in Anthurium scherzerinum Plnt Cell Tiss Org Cult, 48: Kuehnle, AR nd N Sugii, 1991 Cllus induction nd plntlet regenertion in tissue cultures of Hwiin Anthurium HortSci, 26: Murshige, T nd F Skoog, 1962 A revised medium for rpid growth nd iossys with tocco tissue culture Plnt Physiol, 15: Nitsch, JP 1969 Experimentl ndrogenesis in Nicotin Phytomor, 19: Pierik, RLM 1976 Anthurium ndrenum plntlets produced from cllus tissues cultivted in vitro Plnt Physiol, 37: 8-82

55 Journl of Applied Horticulture, 8(2): , July-Decemer, 26 A one step in vitro cloning procedure for Red Gloe grpe: The influence of sl medi nd plnt growth regultors Appl Journl MS Brreto, A Nookrju, NVM Hrini nd DC Agrwl* Plnt Tissue Culture Division, CSIR Ntionl Chemicl Lortory, Pune 411 8, Indi *E-mil: dcgrwl@nclresin Astrct Erlier studies hve shown tht the degree of success t ech stge of micropropgtion in grpevine is genotype dependent; hence it ecomes impertive to optimize culture conditions for rpid propgtion of vriety Present report descries two pproches of in vitro propgtion of Vitis vinifer cultivr, Red Gloe In one pproch, whole plnts could e developed from single node segments y ud rek nd direct rooting in vitro Eight different sl medi tried showed different morphogenetic responses In second pproch, multiple shoots were induced in nodl segments cultured on MS sl medium supplemented with BA (888 µm) Also, second crop of shoots could e induced in left over nodl segments devoid of shoots Rooting of shoots could e induced in vitro, oth in semi-solid or liquid medi nd lso ex vitro y pulse tretment of IAA (285 µm) + NAA (27 µm) Plnt estlishment in lter cse ws 8% A simple procedure descried here cn complement conventionl methods, currently eing used in propgtion of this importnt grpe vriety Key words: Auxin pulse, enzyldenine, grpe, micropropgtion, Red Gloe, Vitis vinifer Introduction Due to heterozygous nture grpe vrieties re mostly propgted y vegettive mens Appliction of plnt tissue culture techniques in propgtion nd improvement of grpevines hs een reviewed y severl workers (Krul nd Mowry, 1984; Gry nd Meredith, 1992; Torregros et l, 21) The technique hs een used to propgte pthogen free grpevine stock (Durn-Vil et l, 1988) Micropropgtion complements the conventionl technique when lrge numer of propgules of prticulr vriety re required in shorter time Erlier studies on in vitro propgtion of Vitis hve indicted tht the degree of success t ech stge of culture is genotype dependent nd vries under given set of culture conditions (Brlss nd Skene, 198; Monette, 1988; Botti et l, 1993) Hence, it ecomes essentil to optimize culture conditions for prticulr clone / cultivr / rootstock or newly red line tht needs lrge scle plnting ut vilility of sufficient plnting stock is limittion The present communiction descries influence of eight sl medi nd growth regultors on micropropgtion of Red Gloe, Vitis vinifer cultivr The vriety is in gret demnd due to its ttrctive reddish-purple colour; tste ud rousing flvour nd ppeling lrge plum-size erries with uniform unches Mteril nd methods Twigs of field grown, disese free vines of Red Gloe were collected from the vineyrd of Ntionl Reserch Centre for Grpes, Pune These were defolited nd cut into single node segments (2 cm) The explnts were dipped in 1% Lolene solution for 1 min; rinsed with tp wter; sumerged in 1% Bvistin solution; kept on shker (12 rpm) for 2 h nd therefter rinsed three times with sterile wter in lminr flow hood These were then disinfected with 1% mercuric chloride solution for 1 min nd rinsed three times with sterile wter The explnts were finlly lotted dry on sterile filter pper nd inoculted on medium in glss test tues (15 X 25 mm) For udrek, eight different sl medi MS (Murshige nd Skoog, 1962), WPM (Lloyd nd McCown, 198), NN (Nitsch nd Nitsch, 1969), B5 (Gmorg et l, 1968), ER (Eriksson, 1965), LS (Linsmier nd Skoog, 1965), C 2 d (Chee nd Pool, 1987) nd GNMG (Glzy et l, 199) devoid of growth regultors were tested Another experiment with MS medium nd rnge of BA concentrtions (4 to 111 µm) ws undertken to mximize udrek To otin second crop of shoots, primry nodl segments left fter excising the grown xillry shoot (hereinfter referred to s mother explnt) insted of its discrd, were trnsferred to WPM or MS with or without BA (444 nd 888 µm) For induction of multiple shoots, xillry shoots otined from primry nodl segments were inoculted (S) in test tues hving MS medium with BA (222 to 888 µm) After 3 d, explnts showing multiple shoots were trnsferred to fresh medium in glss ottles This ws continued t n intervl of 3 d until five trnsfers (from S1 to S5) To test the effect of inoculum s density per culture vessel, two, three, four or five shoot clumps per culture ottle were inoculted on MS with BA t 444 or 888 µm Two sets of experiments were crried out for elongtion of in vitro shoots In the first set, shoots less thn 3 cm in length were inoculted on WPM supplemented with or without BA ( µm) In the second set, multiple shoot clumps with shoots of <15 cm in length were kept for elongtion These were inoculted in glss ottles contining MS supplemented with BA (222 or 444 µm) nd NAA (54 µm) For in vitro rooting, shoots more thn 3 cm were inoculted in test tues contining hlf or full strength MS or WPM supplemented

56 A one step in vitro cloning procedure for Red Gloe grpe 139 with NAA (54-17 µm) or IAA ( µm) or IBA (49-98 µm) or IPA (53-16 µm) with or without gr Liquid medi hd filter pper ridges In vitro rised shoots, more thn 7 cm in length were given pulse tretment of different uxins, IAA ( µm) or IBA ( µm) or IPA ( µm) or NAA ( µm) either singly or in comintion for 1 min nd then plnted in plstic cups contining mixture of coco-pet + soil + snd (1:1:1) Untreted shoots served s control Shoots rooted were tken out of the culture tues, their roots gently wshed with wter to remove dhering medium nd were trnsferred to plstic cups contining the ove mentioned mixture Plnts were cclimtized y the Schet technique (Rvindr nd Thoms, 1985) Plnts fter trnsfer to cups were kept in continuous light of 244 µmol m -2 s 1 t 25 ± 2 C Therefter, these were shifted to nother growth room t mient temperture (35 ± 2 C) Estlishment of plnts ws recorded fter 3 d All the medi were supplemented with sucrose 2 g L -1 nd gelled with gr 7 g L -1 The ph of the medi ws djusted to 58 efore utoclving t 121 C for 2 min Cultures were incuted under 16 h photoperiod otined with cool light fluorescent tues with light intensity of 244 µmol m -2 s 1 t 25 ± 2 C Experiments were repeted t lest three times Oservtions were recorded t monthly intervl The experiments were conducted in Completely Rndomized Design nd the results were sujected to nlysis of vrince Results nd discussion Bud rek in nodl segments commenced from the fifth dy of inocultion nd continued up to 2 th dy nd therefter shoots put forth rpid growth Among the eight sl medi tested, C 2 d, LS nd WPM without ny growth regultors induced 92, 9 nd 84% udrek, respectively Induction of two or more shoots in mximum explnts (76%) ws oserved in C 2 d medium NN induced the minimum response (Tle 1) In ddition to udrek, nodl explnts oth in WPM nd B5 induced rooting t the se in 7% of explnts In cse of NN, it ws 46% In ll other medi rooting ws very low These rooted nodl segments with primry shoots could e estlished on potting nd were hrdened y the Schet technique Thus, no specil difficulty ws fced with nodl culture producing entire plntlet Eight different nutrient medi induced different morphogenetic responses in nodl segments Shoots in C 2 d were found to e stunted, succulent with light green, thick leves nd glossy in ppernce Bsl medi LS nd Eriksson showed necrosis in shoot tip, which continued to the entire shoot nd cused drying of the whole shoot Shoots in NN lcked vigour, hd thin stems with drk green leves MS resulted into comprtively etter shoots with norml internode nd light green leves Also, shoots on MS were most vigourous s compred to other medi tested The shoots in B5 were similr to those oserved in MS except tht the internode ws slightly thicker The shoots in WPM lcked vigour nd hd thin, lnky stems showing twining hit with thin folige Of the eight medi tested, MS ws found to e the most suitle medium resulting into vigorous shoots Hence, for multiple shoot induction experiment, only MS ws used In similr study on sl medium, Reisch (1986) oserved significnt differences in growth in grpe cultivr White Riesling with MS hlf nd MS full medium However, in contrst to the present study, Gry nd Benton (1991) oserved stunted growth in shoots of Muscdine grpe cultivrs when WPM ws used Genotypic vriility within Vitis vinifer cultivrs cultured in vitro hs erlier een reported (Hrris nd Stevenson, 1982; Chee nd Pool, 1983; Glzy et l, 199) Vrying response of different genotypes to different sl medi could e due to vritions in nutrient compositions For exmple, mount of CCl 2 is higher in MS, LS nd Eriksson s compred to WPM nd NN, while in C 2 d nd GNMG, it is sustituted y C(NO 3 ) 2 Similrly, Potssium Iodide (KI) is sent in WPM, NN, C 2 d nd Eriksson while it is present in GNMG, B5, LS nd MS though in different quntities Also mounts of MnSO 4 vry in the eight sl medi tested Glzy (1969) demonstrted tht minerl requirement vried with the morphogenic process: strong K nd N concentrtions proved fvorle to shoot development ut impeded root growth Chee nd Pool (1987) working with grpe tissues hve reported tht lower concentrtions of KI nd MnSO 4 in the medium were good for mximum shoot production nd incorportion of C(NO 3 ) 2 insted of CCl 2 produced shoots of good qulity Present study corroortes these findings since mximum results of udrek of shoots were otined from explnts inoculted on C 2 d though shoots otined from explnts inoculted on MS medium were comprtively helthy nd vigorous Besides nutrients, differences in in vitro response etween genotypes of different species my e relted to differences in endogenous Tle 1 Effects of eight sl medi on morphogenesis in nodl segments of Vitis vinifer cv Red Gloe Bsl medium Explnts showing ud-rek* (%) Explnts showing single shoots (%) Explnts showing 2 or more shoots(%) Totl numer of shoots otined Soots elongted (%) Averge shoot height (cm) ± SD Explnts showing rooting t the se (%) NN ± 1 46 C 2 d ± 46 2 B ± 23 7 MS ± 35 1 LS ± ER ± 1 4 WPM ± 5 7 GNMG ± 18 LSD (P=1) 53 * Bsed on 5 explnts, ± SD = Stndrd devition

57 14 A one step in vitro cloning procedure for Red Gloe grpe 12 1 Explnts showing ud rek Explnts showing 2 or more shoots Explnts showing single shoot BA ( M) Fig 1 Effects of BA concentrtion on udrek nd numer of shoots in nodl segments of Red Gloe following 3 d of culture, sl medium Murshige nd Skoog, (1962); rs indicte stndrd error Averge numer of shoots/explnt Averge numer of elongted shoots/clump μm 444 μm Numer of pssges Fig 2A Effects of BA concentrtion nd numer of pssges on shoot prolifertion in Red Gloe grpe 222 μm 444 μm Numer of pssges Fig 2B Effect of BA concentrtion nd numer of pssges on elongtion of shoots in Red Gloe grpe levels of phytohormones (Looney et l, 1988; Alvrez et l, 1989; Gronroos et l, 1989) In the second experiment, BA t 666, 888 nd 111 µm resulted into 1, 9666, nd 9666% of explnts showing udrek There ws mrginl difference in the response of BA levels from 4 to 89 µm However, liner increse in numer of two or more shoots per explnt ws oserved on increse in BA concentrtion from 4 to 111 µm Mximum response (9%) of two or more shoots per explnt ws recorded with BA t 111 µm (Fig 1) Addition of BA in MS not only induced ud rek in higher numer of nodl explnts ut shoots were of etter qulity in terms of vigour nd lef colour Positive influence of BA in estlishment of xenic shoots in grpes hs erlier een documented in severl reports (Chee nd Pool, 1983; Reisch, 1986; Lee nd Wetzstein, 199; Rocker nd Chng, 1992; Torregros nd Bouquet, 1995; Mhtre et l, 2) A second crop of shoots could e induced in mother explnts cultured on WPM or MS with BA t 444 or 888 µm The mximum shoot induction ws otined in MS with BA (888 µm) considering oth single nd two or more shoots in explnts (dt not shown) Primry shoot used s explnt, induced mximum multiple shoots (227) per explnt on n verge in MS supplemented with BA (888 µm) fter 3 dys of inocultion (S) Though mrginlly higher, liner increse in numer of shoots ws oserved on increse in BA concentrtion from 222 to 888 µm though reverse ws true for numer of shoots elongted per explnt Medium without BA (served s control) showed the lest numer of shoots s well s lest numer of elongted shoots per explnt On trnsfer of these shoots to fresh medi (S1) in glss ottles, numer of multiple shoots incresed severl fold nd showed liner increse with increse in BA concentrtion (Fig 2A) The sme trend ws oserved with numer of elongted shoots per explnt (Fig 2B) It ws oserved tht BA concentrtions t 666 nd 888 µm showed higher numer of shoots nd elongted shoots per explnt from sucultures S to S1, however, shoots produced were hyperhydric nd showed normlities in lef shpe The leves were drk green with glossy ppernce Also, shoots

58 A one step in vitro cloning procedure for Red Gloe grpe 141 Tle 2 Effect of sl medi nd BA on shoot elongtion in cv Red Gloe Bsl medium + BA (µm) Percent of shoots elongted* Averge shoot length (cm) ± SD WPM + BA (444) ± 45 WPM + BA (888) ± 45 WPM ± 17 MS + BA (444) ± 26 MS + BA (888) ± 3 MS 3 1 ± 6 LSD (P=1) 56 * Bsed on 6 explnts, ± SD = Stndrd devition were short nd compct in the form of clumps Hence for further sucultures, these two BA concentrtions were discontinued On repeted su-cultures of these shoot clumps from S1 to S4 t n intervl of 3 d, drstic nd liner increse in numer of shoots ws oserved (Fig 2A) As oserved erlier this could e due to xillry rnching in shoots clusters or occurrence of dventitious orgnogenesis (Chee nd Pool, 1985) However, in S5, numer of shoots per clump decresed drsticlly indicting toxicity It ws found tht the density of clumps inoculted per culture vessel influenced the rte of shoot prolifertion for ll the BA levels An inverse correltion ws oserved etween BA concentrtion nd density of clumps Higher the BA concentrtion more the numer of shoots per clump while reverse ws true with density ie higher the numer of clumps per vessel lesser the numer of shoots per clump The mximum numer of verge shoots (154) per clump ws otined with two clumps per culture vessel in medium contining BA 888 µm (dt not shown) Since ll the shoots in multiple shoot clumps did not elongte, it ws necessry to crry out seprte experiment for elongtion of shoots In one experiment, shoot clump ws kept s explnt on two medi contining MS s sl medium supplemented with NAA t 54 µm nd BA t two concentrtions 222 nd 444 µm At oth the BA concentrtions, there ws very mrginl difference in verge numer of shoots elongted 1295, 1363 with verge shoot length of 59 nd 533 cm, respectively Inocultion of shoot clumps for elongtion of shoots hs dvntge since severl shoots elongted simultneously insted of one, sving time nd lour In the other experiment, single shoots (2-3 cm) isolted from multiple shoot clumps were kept on WPM nd MS sl medi without BA or with BA t 444 nd 888 µm Mximum percentge of shoots (9333%) elongted on MS with BA (444 µm) giving n verge shoot length of 675 cm followed y WPM (6667 %) giving n verge shoot elongtion of 75 cm BA concentrtions lower thn 444 µm showed olting of shoots while BA t higher thn 888 µm resulted into thick, stunted, succulent nd hyperhydric shoots (dt not shown) This is in conformity with oservtions recorded in three grpevine cultivrs y Mhtre et l (2) Bsl medium without growth regultors showed shoot necrosis nd ws not effective in shoot elongtion Rooting of in vitro shoots is influenced y severl fctors, of which growth regultor requirement is of mjor importnce Though rooting of in vitro rised shoots could e induced in MS hlf or full strength sl medi (gr solidified or liquid) In vitro shoots nd hrdened plnts In vitro shoots Hrdened plnts Months Fig 3 Numer of in vitro shoots nd hrdened plnts otined in spn of seven months (strting with 1 nodl segments) supplemented with or without NAA (54-17 µm) or IAA ( µm) or IBA (49-98 µm) or IPA (53-16 µm) however, qulity of roots ws etter on incorportion of NAA t µm in the medium Numer of dys required for rooting ws less for the shoots inoculted in the liquid medium s compred to the solidified medium In MS hlf or MS full medium devoid of growth regultors, the qulity of roots ws poor nd shoots lcked vigour Addition of NAA in the rooting medium induced longer roots with primry nd secondry rnching This ws reflected in the higher survivl of rooted shoots (83%) when treted with NAA (dt not shown) In erlier reports on grpevine, it ws documented tht uxin stimulted root initition ut inhiited susequent root growth (Glzy, 1969), nd tht its pproprite concentrtion ws of criticl importnce In previous studies, it ws oserved tht effects of uxins on rooting depend on minerl composition of the nutrient medi (Novk nd Juvov, 1983; Zlenko et l, 1995) Root initition ws not influenced y slt concentrtion, ut root growth ws enhnced when slt concentrtion of rooting medi ws reduced (Hrris nd Stevenson, 1979) Ex vitro uxin pulse tretment of in vitro shoots for 1 min induced direct roots Shoots given pulse tretment with uxin mixture of IAA (285 µm) nd NAA (27 µm) showed 8% plnt estlishment Pulse tretment of IAA (57 µm) or NAA (54 µm) lone gve rise 73 nd 7% estlishment, respectively A mixture of IAA (57 µm) nd NAA (54 µm) resulted into lower percent (53%) of estlishment Shoots directly trnsferred to potting mixture without ny uxin pulse did not induce roots nd could not estlish Plnts could e cclimtized y the schet technique which is simple, effective nd does not require ny sophisticted set-up It ws found tht shoots plnted in mixture of coco-pet + soil + snd (1:1:1) showed plnt survivl of 75% Thus, present communiction descries two routes of micropropgtion in grpevine cultivr Red Gloe In one route, whole plnts could e developed from single node segments y ud rek nd direct rooting To our knowledge no such systemtic study on sl medi hs een reported so fr for tissue culture of grpevines In second route, lrger numer of plnts could e otined y multiple shoot induction, shoot prolifertion nd ex vitro rooting y uxin pulse tretment Within seven months period, out 1 single node segments could give rise to out 5442 in vitro shoots nd 4354 estlished plnts compred to conventionl vegettive cutting method where ech three to five node cutting yields only one plnt (Fig 3) Tissue culture plnts produced, hve een supplied to Ntionl

59 142 A one step in vitro cloning procedure for Red Gloe grpe Reserch Center for Grpes (NRCG), for its performnce in the field A simple in vitro propgtion procedure descried here cn complement conventionl methods, currently eing used in propgtion of this importnt grpevine vriety Acknowledgements Authors cknowledge finncil support from Deprtment of Biotechnology (DBT), New Delhi nd Council of Scientific nd Industril Reserch, Govt of Indi nd the supply of plnt mteril of Red Gloe y Dr GS Krisspp, Ntionl Reserch Centre for Grpes, Pune References Alvrez, R, SJ Nissen nd EG Sutter, 1989 Reltionship etween indole-3-cetic cid levels in pple (Mlus pumil) rootstocks cultured in vitro nd dventitious root formtion in the presence of indole-3-utyric cid Plnt Physiol, 89: Brlss, M nd KGM Skene, 198 Studies on the frgmented shoot pex of grpevine II Fctors ffecting growth nd differentition in vitro J Expt Bot, 31: Botti, C, L Gry nd G Reginto, 1993 The influence of culture dtes, genotype nd size nd type of shoot pices on in vitro shoot prolifertion of Vitis vinifer Cvs Thompson seedless, Riier nd Blck seedless Vitis, 32: Chee, R nd RM Pool, 1985 In vitro vegettive propgtion of Vitis: the effects of orgnic sustnces on shoot multipliction Vitis, 24: Chee, R nd RM Pool, 1987 Improved inorgnic medi constituents for in vitro shoot multipliction of Vitis Scienti Hortic, 32: Chee, R nd RM Pool, 1983 In vitro vegettive propgtion of Vitis: Appliction of previously defined culture conditions to selection of genotypes Vitis, 22: Durn-Vil, N, J Jurez nd JM Arregui, 1988 Production of viroidfree grpevines y shoot tip culture Am J Enol Viticult, 39: Eriksson, T 1965 Studies on the growth mesurements of cell cultures of Hplopppus grcilis Physiol Plnt, 18: Glzy, R 1969 Remrques sur l croissnce de Vitis rupestris cultivee in vitro sur differents milieux nutritifs Vitis, 8: Glzy, R, V Hffner nd D Compn, 199 Influence of three fctors on the growth nd nutrition of grpevine micro cuttings J Expt Bot, 41: Gmorg, OL, RA Miller nd K Ojim, 1968 Plnt cell culture I Nutrient requirement of suspension culture of soyen root cells Exp Cell Res, 5: Gry, DJ nd CM Benton, 1991 In vitro micro propgtion nd plnt estlishment of muscdine grpe cultivrs (Vitis rotundifoli) Plnt Cell Tiss Org Cult, 27: 7-14 Gry, DJ nd CP Meredith, 1992 Grpe In: Biotechnology of perennil fruit crops, Hmmerschlg, FA nd Litz, RE (ed) Biotechnology in Agriculture No 8 Wllingford pp Gronroos, L, B Kut, S von Arnold nd L Elissone, 1989 Cytokinin contents in shoot cultures of four Slix clones J Plnt Physiol, 135: Hrris, RE nd JH Stevenson, 1982 In vitro propgtion of Vitis Vitis, 21: Hrris, RE nd JH Stevenson, 1979 Virus elimintion nd rpid propgtion of grpes in vitro Proc Int Plnt Prop Soc, 29: Krul, WR nd GH Mowry, 1984 Grpes In: Hndook of Plnt Cell Culture, Vol 2, WRShrp, DA Evns, PV Ammirto nd Y Ymd (ed), Mcmilln Pulishing Co Inc NY pp Lee, N nd HY Wetzstein, 199 In vitro propgtion of muscdine grpe y xillry shoot prolifertion J Am Soc Hort Sci, 115: Linsmier, EM nd F Skoog, 1965 Orgnic growth fctor requirements of tocco tissue culture Physiol Plnt, 18: Lloyd, G nd B McCown, 198 Commercilly fesile micropropgtion of mountin lurel, Klmi ltifoli, y use of shoot-tip culture Proc Intl Plnt Prop Soc, 3: Looney, WE, JS Tylor nd RP Phris, 1988 Reltionship of endogenous gierellin nd cytokinin levels in shoot tips to picl form in four strins of McIntosh pple J Am Soc Hort Sci, 113: Mhtre, M, CK Slunkhe nd PS Ro, 2 Micropropgtion of Vitis vinifer; towrds n improved protocol Scienti Hortic, 84: Monette, PL 1988 Grpevine (Vitis vinifer L) In: Bjj, YPS (ed), Biotechnology in Agriculture nd Forestry, Vol 6 Berlin, Springer- Verlg pp 3-37 Murshige, T nd F Skoog, 1962 A revised medium for rpid growth nd iossys with tocco tissue cultures Physiol Plnt, 15: Nitsch, JP nd C Nitsch, 1969 Hploid plnts from pollen grins Science, 163: Novk, FJ nd Z Juvov, 1983 Clonl propgtion of grpevine through in vitro xillry ud culture Scienti Hortic, 18: Rvindr, MB nd P Thoms, 1985 Schet technique n efficient method for the cclimtiztion of micro propgted grpes Curr Sci, 68: Reisch, BI 1986 Influence of genotype nd cytokinins on in vitro shoot prolifertion of grpes J Am Soc Hort Sci, 111: Rocker, CD nd CJ Chng, 1992 Shoot tip culture of muscdine grpe to eliminte Pierce s disese cteri HortSci, 27: Torregros, L nd A Bouquet, 1995 In vitro propgtion of Vitis x Muscdini hyrids y micro-cuttings or xillry udding Vitis, 34: Torregros, L, ABouquet, nd P G Goussrd, 21 In vitro culture nd propgtion of grpevine In: Moleculr Biology nd Biotechnology of the Grpevine, KA Rouelkis-Angelkis (ed), Netherlnds, Kluwer Acdemic Pulishers pp Zlenko, VA, LP Troshin nd IV Kotikov, 1995 An optimized medium for clonl micropropgtion of grpevine Vitis, 34(2):

60 Journl of Applied Horticulture, 8(2): , July-Decemer, 26 Anthocynin ccumultion in the hypocotyl nd petl of Red Agti (Sesni grndiflor), n ornmentl legume Appl Journl Kitti Bodhipdm 1, Sompoch Noichind 1, Ssikn Udomrti 1, Gorvis Nthlng 2, Boonyuen Kijwijn 3 nd Dvid WM Leung 4 1 Deprtment of Agro-Industril Technology, Fculty of Applied Science, King Mongkut s Institute of Technology North Bngkok, Bngsue, Bngkok, 18, Thilnd; 2 Deprtment of Biology, Fculty of Science, Mhidol University, Bngkok, 14, Thilnd; 3 Deprtment of Biology, Fculty of Science, Khon Ken University, Khon Ken, 42, Thilnd; 4 School of Biologicl Sciences, University of Cnterury, Privte Bg 48, Christchurch 1, New Zelnd, E-mil: dvid leung@cnterurycnz Astrct Seeds of Red Agti (Sesni grndiflor), n ornmentl leguminous tree, were germinted in vitro under oth light nd drk conditions for 7, 1, 15, 2 nd 25 dys The locliztion of nthocynin-contining cells nd level of totl nthocynin content of hypocotyl from severl developmentl stges were determined In the hypocotyl of light-grown seedlings, nthocynin-contining cells were oserved in epiderml nd su-epiderml lyer nd peripherl cortex while none ws found in tht of drk-grown seedlings On dy 7, the hypocotyl of light-grown seedlings hd the highest nthocynin content (29 μg/g FW) Moreover, Red Agti s petl t vrious developmentl stges ws lso exmined for the totl nthocynin content It ws found tht the petl of 3 cm length hd the highest totl nthocynin level (455 μg/g FW) It is concluded tht the hypocotyl of light-grown Red Agti seedlings is n ttrctive lterntive source of nthocynins to the petl s the seedlings cn e rised nd e mde ville throughout the yer Key words: Red Agti, nthocynin, Sesni grndiflor, histology, development Introduction Red Agti (Sesni grndiflor), perennil nitrogen-fixing ornmentl tree, elongs to the fmily Fcee The origin of this leguminous plnt is not known ut it is considered ntive to Southest Asin countries In Thilnd, it is commonly grown in ckyrd grdens This plnt nd closely relted species, Agti (Sesni grndiflor), re useful for reforesttion s they hve extremely fst growth rte They re lso used for forge, firewood, pulp nd pper, food, green mnure nd lndscpe decortion (Ntionl Reserch Council, 1979; Thiengurnthum, 1993; Bry, 1994; Gutteridge, 1994 nd Jensen, 21) The petl of Red Agti is lrge, distinctively rose pink or red in color while tht of relted species, Agti, is white or yellowish, reflecting the differing nthocynin content in the vcuole (Mrkhm et l, 2) Anthocynins re plnt phenolic compounds in the flvnoid group They generlly re responsile for the red, purple nd lue colors in plnt prts such s fruits, vegetles, flowers, leves, roots nd tuers All nturl nthocynins re glycosides clled nthocynidin Anthocynins re receiving renewed ttention for their potentil helth enefits s ntioxidnts nd nti-inflmmtory gents (Klt nd Dufour, 1997; Wng et l, 1997; Wng et l, 1999) Plnt tissue culture is one of the importnt iotechnologicl tools which hs een pplied in vrious spects, such s, micropropgtion (Bodhipdm nd Leung, 22; Hw nd Keng, 23), in vitro flowering nd fruiting (Bodhipdm nd Leung, 22; Bodhipdm nd Leung, 23) nd secondry metolite, especilly nthocynin production (Ro nd Rvishnkr, 22; Filippini et l, 23) Though there were some pulictions on tissue culture of Sesni, the ojective of most of them ws to propgte this plnt (Shnker nd Rm, 199; Shnker nd Rm, 1993; Sinh nd Mllick, 1993; Detrez et l, 1994) Unlike Agti, Red Agti hs remrkly red petl tht would e good source of nthocynin However, the hypocotyl of Red Agti seedling is lso red This could e more ttrctive nd convenient source for nthocynin production thn petl With this gol in mind, the present study ws undertken to compre the mount of nthocynin extrcted from the hypocotyl nd petl of Red Agti nd investigte the time during their development when mximl ccumultion of nthocynin might occur in the pproprite orgn Mterils nd methods Plnt mterils: Mture dry seeds of Red Agti were otined from locl grden of Nonthuri province To void possile complictions from microil contmintion during rising seedlings from seeds for the present experiments, the Red Agti seeds were germinted under septic conditions: they were immersed in sterile distilled wter overnight efore they were surfce-sterilized y soking in 7% (v/v) ethnol for 3 seconds, 15% (v/v) sodium hypochlorite for 15 minutes nd rinsing them three times with sterile distilled wter After seeds germinted in vitro on the medium (see Medium) under oth light nd drk conditions for 7, 1, 15, 2 nd 25 dys, hypocotyl from these seedlings were cut nd exmined for totl nthocynin content

61 144 Anthocynin ccumultion in the hypocotyl nd petl of Red Agti (Sesni grndiflor), n ornmentl legume nd its locliztion Flowers of Red Agti were collected from locl grden of Nonthuri province Only petl prts 3, 6 nd 9 cm in length were used to nlyze for totl nthocynin content The collection ws mde fter the opening of the petls in 4, 7 nd 12 dys, respectively Medium: MS sl medium (Murshige nd Skoog, 1962) in this study were djusted to ph 57, gelled with 8% (w/v) gr, nd utoclved t 121 C nd 15 psi for 2 minutes All the cultures were kept in growth room t 25 C under 16 hours illumintion from white fluorescent lmps (3,18 Lux) or in the drk Determintion of totl nthocynin content: Totl nthocynin content ws mesured using method modified from tht of Fuleki nd Frncis (1968) 15 g of hypocotyl from seedlings grown in vitro under oth light nd drk conditions fter 7, 1, 15, 2 nd 25 dys, or petl (3, 6 nd 9 cm in length) were lended with mixture of 95% (v/v) ethnol nd 15 N HCl (128: 23 ml) One ml of the extrction mixture ws mixed with 95% ethnol nd 15 N HCl (175: 188 ml) in 2-mL vil wrpped with luminum foil This ws gently shken, kept overnight t 4 C nd then centrifuged t 6,87 x g for 1 min The sornce of the superntnt ws red t 535 nm Totl nthocynin content ws clculted ccording to Fuleki nd Frncis (1968) Anthocynin locliztion: Hypocotyls of Red Agti seedlings grown in vitro under oth light nd drk conditions for 7, 1, 15, 2 nd 25 dys were cut independently with rzor lde The free hnd sections were then plced in drop of wter on glss slide, covered with cover slip nd exmined under light microscope to loclize the nthocynin-contining cells Results Morphology of Red Agti seedlings: Red Agti seeds, germinted in vitro developed normlly into helthy seedlings in the light ut not in the drk (Fig 1) On dy 7 in the light, the cotyledons strted expnding nd emerging from the seed cot while in the drk y dy 25, the cotyledons still remined inside the seed cot On dy 1, true leves could e found on some seedlings under light conditions It ws clerly seen tht plnt height ws greter in the drk thn in the light throughout the experiment (Fig 2) Hypocotyls of drk-grown seedlings were white while those grown in the light were red However, the red color ppered lighter s the hypocotyls grew Locliztion of nthocynin in hypocotyl of Red Agti: When free hnd sections of Red Agti hypocotyls, cultured under oth light nd drk conditions in vitro, were exmined under light microscope, it ws reveled tht nthocynin could not e found in hypocotyls of drk-grown seedlings t ll developmentl stges In contrst, nthocynin ws evidently present in the hypocotyls of light-grown seedlings (Fig 3) The nthocynincontining cells were found in the epiderml nd su-epiderml lyer Besides, some could e seen in the peripherl cortex of the hypocotyl The hypocotyl on dy 7 ppered to hve the highest numer of nthocynin-contining cells Totl nthocynin content in Red Agti hypocotyl: Totl nthocynin content ws mesured in hypocotyls of germinted seeds in vitro under oth light nd drk conditions for 7, 1, 15, 2 nd 25 dys It ws reveled tht hypocotyls of light-grown seedlings hd the highest nthocynin content (29 μg/g FW) on dy 7 (Fig 4) Then totl nthocynin content decresed shrply In contrst, nthocynin were not detectle in hypocotyl of Drk Light Plnt height (cm) Fig 1 Red Agti grown in vitro on 7, 1, 15, 2 nd 25 dys under light (, c, e, g nd i, respectively) nd drk (, d, f, h nd j, respectively) conditions Dys Fig 2 Height of Red Agti grown in vitro t vrious developmentl stges (vlues re mens of 2 replictions ± SD) Vlue in dy 25 dropped ecuse explnts mostly died nd men of this stge re included ll of those 2 replictions

62 Anthocynin ccumultion in the hypocotyl nd petl of Red Agti (Sesni grndiflor), n ornmentl legume 145 drk-grown seedlings (Fig 4) Totl nthocynin content in Red Agti petl: During Red Agti flower development, the petl incresed in size nd length The petl of 3 cm in length hd the highest totl nthocynin 45 4 Anthocynin content (μg /g FW) Fig 3 Cross section of Red Agti s hypocotyl grown in vitro on 7, 1, 15, 2 nd 25 dys under light (, c, e, g nd i, respectively) nd drk, d, f, h nd j, respectively) conditions Scle r = 1 μm Anthocynin content (μg /g FW) Drk Light Dys Fig 4 Totl nthocynin content in hypocotyl of Red Agti t vrious developmentl stges (vlues re mens of 9 replictions ± SD) Florl length (cm) Fig 5 Totl nthocynin content in petl of Red Agti t vrious developmentl stges (vlues re mens of 9 replictions ± SD) content (455 μg gfw) Totl nthocynin content oviously decresed when the petl length incresed further (Fig 5) Discussion The demnd of nturl nthocynins hs een growing due to their eneficil ttriutes for humn helth nd well-eing when compred to synthetic dyes (Myer nd vn Stden, 1995; Stintzing nd Crle, 24) Though leves nd fruits from other plnts re the good source of nthocynins, such s, 188 nd 375 μg/gfw of totl nthocynin content were found in purple sil lef nd cerol fruit, respectively (Simon, et l, 1999; Vendrmini nd Trugo, 24), hypocotyl nd flower of Red Agti could provide high mount of nthocynins s well In this reserch, totl nthocynin content of Red Agti in reproductive (petl) nd nonreproductive (hypocotyl) tissues were investigted for the first time Reddening ws found in hypocotyl of Red Agti seed germinted under light condition This phenomenon, the presence of red color in seedling, ws lso discovered in severl Pinus species nd mny dicotyledonous epigeous seedlings, especilly hypocotyl prt which developed red pigmenttion shortly fter it emerges from the soil into light (Nozzolillo et l, 22) In Red Agti hypocotyl, nthocynin ws oserved in the epiderml nd su-epiderml lyer nd peripherl cortex There hve een mny reports showing tht locliztion of nthocynin vries depending on the species Mostly, nthocynins re found in or just elow the epidermis (Chlker-Scott, 1999; Nozzolillo et l, 22) However, Hr et l (23) found tht the loction of nthocynin-contining cells in rdish hypocotyls chnged during growth On dy 14, these cells were seen in the peripherl cortex ut they were mostly found in the pericycle cells on dy 21 This finding differed from the present result s the distriution of nthocynin-contining cells of Red Agti hypocotyl ws still in the su-epiderml lyer nd peripherl cortex nd remined or unchnged throughout the experiment

63 146 Anthocynin ccumultion in the hypocotyl nd petl of Red Agti (Sesni grndiflor), n ornmentl legume There hve een numer of reports showing tht light is n importnt fctor for nthocynin synthesis (Mtsumoto et l, 1973; Chlker-Scott, 1999) Our results re lso in greement with these studies Nevertheless, cllus culture of Vitis nd strwerry produced lrge mounts of nthocynin in the drk (Ymkw et l, 1983; Nkmur et l, 1999) Totl nthocynin content in Red Agti hypocotyl on dy 7 under light conditions ws comprle to tht of the 3-cm long petl Furthermore, the developmentl chnges in their totl nthocynin content ppered similr Anthocynin ccumulting pttern in Red Agti hypocotyl of seedlings grown under light nd drk condition ws totlly different The locliztion of nthocynin were mostly found in su-epiderml lyer nd peripherl cortex t ll developmentl stges The erly developmentl stge of oth hypocotyl nd petl hd the higher mount of totl nthocynin content thn the lte developmentl stge Thus from the present results, the suitle time to hrvest Red Agti hypocotyls for nthocynin extrction would e on dy 7 fter sowing seeds under septic conditions Similrly, petl of 3 cm long would lso e idel Although totl nthocynin content ws less in hypocotyl thn in petl, the former is more redily ville nd would e more fvorle source or n ttrctive lterntive to petl of Red Agti to use for nthocynin production Further studies would e needed to exmine if the nthocynnin profiles in the hypocotyl nd petl of Red Agti re comprle References Bodhipdm, K nd DWM Leung, 22 Fctors importnt for somtic emryogenesis in zygotic emryo explnts of Cpsicum nnuum L J Plnt Biol, 45: Bodhipdm, K nd DWM Leung, 22 In vitro flowering of plntlets regenerted vi somtic emryogenesis from immture zygotic emryo explnts of Cpsicum nnuum L cv Sweet Bnn Phyton, 42: Bodhipdm, K nd DWM Leung, 23 In vitro fruiting nd seed set of Cpsicum nnuum L cv Sweet Bnn In Vitro Cell Dev Biol Plnt, 39: Bry, RA 1994 Diversity within tropicl tree nd shru legumes In: Forge Tree Legumes in Tropicl Agriculture, Gutteridge, RC & Shelton, HM (eds), CAB Interntionl, Wllingford, UK, pp Chlker-Scott, L 1999 Environmentl significnce of nthocynins in plnt stress responses Photochem Photoiol, 7: 1-9 Detrez, C, S Ndiye nd B Dreyfus, 1994 In vitro regenertion of the tropicl multipurpose leguminous tree Sesni grndiflor from cotyledon explnts Plnt Cell Rep, 14: Filippini, R, R Cnito, A Piovn nd EM Cppelletti, 23 Production of nthocynins y Cthrnthus roseus Fitoterpi, 74: Fuleki, T nd FJ Frncis, 1968 Quntittive methods for nthocynins: 1 Extrction nd determintion of totl nthocynin in crnerries J Food Sci, 33: Gutteridge, RC 1994 The perennil Sesni species In: Forge Tree Legumes in Tropicl Agriculture, Gutteridge, RC & Shelton, HM (eds), CAB Interntionl, Wllingford, UK, pp Hr, M, K Oki, K Hoshino nd T Kuoi, 23 Enhncement of nthocynin iosynthesis y sugr in rdish (Rphnus stivus) hypocotyl Plnt Sci, 16: Hw, AB nd CL Keng, 23 Micropropgtion of Spilnthes cmell L, io-insecticide plnt, through prolifertion of multiple shoots J Appl Hort, 5: Jensen, M 21 Trees nd Fruits of Southest Asi - An Illustrted Field Guide Orchid Press, Bngkok, Thilnd Klt, W nd D Dufour, 1997 Helth functionlity of lueerries Hortic Technol, 7: Mrkhm, KR, KS Gould, CS Winefield, KA Mitchell, SJ Bloor nd MR Bose, 2 Anthocynic vcuolr inclusion their nture nd significnce in flower colourtion Phytochemistry, 55: Mtsumoto, T, K Nishid, M Nogichi nd E Tmki, 1973 Some fctors ffecting the nthocynin formtion y Populus cells in suspension culture Agr Biol Chem, 37: Myer, HJ nd J vn Stden, 1995 The in vitro production of n nthocynin from cllus cultures of Oxlis lineris Plnt Cell Tiss Org Cult, 4: Murshige, T nd F Skoog, 1962 A revised medium for rpid growth nd iossys with tocco tissue cultures Physiol Plnt,15: Nkmur M, Y Tkeuchi, K Miyng, M Seki nd S Furuski, 1999 High nthocynin ccumultion in the drk y strwerry (Frgri nnss) cllus Biotech Lett, 21: Ntionl Reserch Council, 1979 Tropicl Legumes: Resources for the Future Ntionl Acdmy of Sciences, Wshington DC, USA Nozzolillo, C, P Iselle, OM Andersen nd M Aou-Zid, 22 Anthocynins of jck pine (Pinus nksin) seedlings Cn J Bot, 8: Ro, SR nd GA Rvishnkr, 22 Plnt cell cultures: Chemicl fctories of secondry metolites Biotech Adv, 2: Shnker, S nd HYM Rm, 199 Plntlet regenertion from tissue cultures of Sesni grndiflor Curr Sci, 59: Shnker, S nd HYM Rm, 1993 Aerrnt chromosome numers in the cllus nd regenerted shoot uds in Sesni grndiflor (L) Pers Phytomorphology, 43: 75-8 Simon, JE, MR Morles, WB Phippen, RF Vieir nd Z Ho, 1999 Bsil: source of rom compounds nd populr culinry nd ornmetl her, In: Perspectives on new crops nd new uses, Jnick, J (ed), ASHS Press, Alexndri, VA, pp Sinh, RK nd R Mllick, 1993 Effect of gmm-rdition on in vitro cllus growth nd regenertion of Sesni grndiflor (L) Poir Cytoios, 76: Stintzing, FC nd R Crle, 24 Functionl properties of nthocynins nd etlins in plnts, food, nd in humn nutrition Trends Food Sci Tech, 15: Thiengurnthum, W 1993 Dictionry of Flowering nd Decortive Plnts in Thilnd Suriyn Pulisher, Bngkok, Thilnd Vendrmini, ALA nd LC Trugo, 24 Phenolic compounds in cerol fruit (Mlpighi punicifoli L) J Brz Chem Soc, 15: Wng, H, G Co nd RL Prior, 1997 Oxygent rdicl soring of nthocynins J Agric Food Chem, 45: Wng, H, MG Nir, GM Strsurg, CY Chng, AM Booren nd JI Gry, 1999 Antioxidnt nd nti-inflmmtory ctivities of nthocynins nd their glycone, cynidin, from Trt cherries J Nt Prod, 62: Ymkw, T, S Kto, K Ishid, T Kodm nd Y Minod, 1983 Production of nthocynins y Vitis cells in suspension culture Agr Biol Chem, 47:

64 Journl of Applied Horticulture, 8(2): , July-Decemer, 26 Effect of different stlk lengths nd certin chemicl sustnces on vse life of gerer (Gerer jmesonii Hook) cv Svn Red Appl Journl MA Memn nd KM Dhi N M College of Agriculture, Deprtment of Horticulture, Nvsri, Gujrt Agriculturl University E-mil: si7@rediffmilcom Astrct An experiment ws crried out to determine the effect of different stlk lengths nd certin chemicl sustnces on vse life of gerer (Gerer jmesonii Hook) Cv Svn Red Twenty tretment comintions consisting of four chemicls viz, sucrose 4 %, sucrose 4 % + 8-HQC 25 ppm, sucrose 4 % + luminum sulphte 1 ppm, sucrose 4 % + citric cid 25 ppm, long with control (distilled wter) nd four lengths of stlk viz, 3, 4, 5, nd 6 cm The vse solution of sucrose 4% + 8-HQC 25 ppm nd stlk length of 6 cm, individully nd in comintion incresed fresh weight of flowers y promoting solution uptke These tretments re lso eneficil for improving the vse life of flowers nd useful life of flowers, opening of disc florets, with right, shining red colour nd freshness for longer durtion Key words: Gerer, Gerer jmesonii Hook, stlk length, chemicls, vse life, sucrose, citric cid, HQC Introduction Gerer elongs to the fmily Astercee, which consists of mny other importnt cut flowers nmely ster, dhli, chrysnthemum, gillrdi, zinni, mrigold etc (Bose nd Ydv, 1989) The genus Gerer, nmed in the honour of Germn nturlist, Trugott Gerer, consists of out 4 species of hrdy nd perennil flowering plnts, out of which only Gerer jmesonii Hook is under cultivtion It is ntive to Ntl nd Trnsvl nd is commonly known s Trnsvl disy, Brerton disy or Africn dsiy It is n importnt flower grown through out the world under wide rnge of climtic conditions Keeping qulity of flowers is ffected y internl nd externl fctors The internl fctors which re responsile for the keeping qulity of cut looms, re the rte of wter sorption nd trnspirtion Both these fctors gin depend on the reltive re of sorption nd the totl wter holding cpcity of the tissues After the flower is detched, the re of sorption is reduced drsticlly, wheres the rte of proportionte re for trnspirtion is much higher Therefore, unless something is done to reduce trnspirtion, the cut flower will wither in less time Respirtion is nother internl fctor tht ffects the life of the cut flower Besides, some environmentl fctors lso ffect cut flower life These re temperture, reltive humidity nd wind velocity Posthrvest hndling plys n importnt role in enhncing keeping qulity of flowers wherein efforts re mde to reduce stem plugging, restrict microil ctivity, dely flower senescence through provision of externl source of wter nd nutrients s required y the flower Mterils nd methods The experiment ws conducted t the PG Reserch Lortory, Deprtment of Horticulture, NM College of Agriculture, Gujrt Agriculturl University, Nvsri Cmpus, Nvsri Twenty tretment comintions consisting of four chemicls viz, sucrose 4 % (T 1 ), sucrose 4 % + 8-HQC 25 ppm (T 2 ), sucrose 4 % + luminum sulphte 1 ppm (T 3 ), sucrose 4% + citric cid 25 ppm (T 4 ) long with control (distilled wter) (T ) nd four lengths of stlk viz, 3 cm (L 1 ), 4 cm (L 2 ), 5 cm (L 3 ), nd 6 cm (L 4 ) were tried in Completely Rndomized Design with Fctoril concept All the tretments were repeted thrice The experimentl mteril were kept in glss ottles of 3 ml cpcity holding 25 ml tretment solution prepred in the lortory nd unch of three stlks of gerer (Cv Svn Red), hrvested t the stge when the outer two row of disc florets were perpendiculr to the stlk Totl sixty glss ottles nd one hundred nd eighty stlks, hving four different uniform lengths of 3, 4, 5 nd 6 cm were utilized The stlks were slightly recut t sl end t every 3 rd dy during experimenttion The dt on uptke of solution (ml) t 3 rd, 6 th nd 9 th dys, time tken (dys) to open 5, 75, nd 1 % disc florets, useful life of flowers (dys), vse life of flower (dys), recorded during the experiment were sujected to sttisticl nlysis The design with fctoril concept s descried y Pnse nd Sukhtme (1967) ws used The significnce of the tretment differences ws tested y F test on the sis of null hypothesis The pproprite stndrd error of men (SEm±) were clculted in ech cse nd the criticl differences (CD) t 5 per cent level of proility ws worked out to compre the two tretment effects Results The dt on solution uptke y gerer flower on third, sixth nd ninth dy re presented in Tle 1 The effect of different chemicls nd stlk length on solution uptke y gerer flower fter three, six nd nine dys were found to e significnt Among different chemicls, mximum uptke of solution (45 ml) t

65 148 Effect of different stlk lengths nd certin chemicl sustnces on vse life of gerer Tle 1 Effect of different chemicls nd stlk lengths (cm) on the solution uptke y the gerer flowers Tretments Solution uptke (ml) 3 rd dy 6 th dy 9 th dy Chemicls (T) T T T T T LSD (P=5) Stlk lengths (L) L L L L LSD (P=5) Zero vlue indicte tht these flowers hd completed their vse life efore ninth dy Tle 2 Interction effect of different chemicls nd stlk lengths (cm) on the solution uptke y the gerer flowers Tretments Solution uptke (ml) 3 rd dy 6 th dy 9 th dy T L T L T L T L T 1 L T 1 L T 1 L T 1 L T 2 L T 2 L T 2 L T 2 L T 3 L T 3 L T 3 L T 3 L T 3 L T 3 L T 3 L T 3 L LSD (P=5) Zero vlue indicte tht these flowers hd completed their vse life efore ninth dy third dy, (412 ml) t sixth dy nd t ninth dy (2394 ml) were oserved when flower stlks were kept in the solution of sucrose (4 %) + 8 HQC (25 ppm) (T 2 ) Among different stlk lengths, mximum mount of solution 4186, 3576 nd 1899 ml ws sored y gerer flower hving L 4 stlk length t third, sixth, nd ninth dy The interction effect of different chemicls nd stlk length on solution uptke y gerer flower on third dy (Tle 2) ws significnt Mximum solution uptke t third (4986 ml), sixth (464 ml) nd ninth dy (3756 ml) ws noticed when longest gerer flower (L 4 ) ws kept in solution of sucrose (4 %)+ 8-HQC (25 ppm) (T 2 L 4 ) The dt pertining to 5, 75 nd 1 % opening of florets reveled tht ll the tretments showed significnt difference (Tle 3) Among the different chemicls the highest time tken for opening of 5% disc florets (718 dys), 75 % disc florets (98 dys), 1% disc florets (149 dys) ws recorded when gerer stlks were kept in solution of sucrose (4 %)+ 8-HQC (25 ppm) (T 2 ) The interctions effect of different chemicls nd stlk lengths on time tken for opening of 5, 75 nd 1 % disc florets ws found to e significnt nd hve een presented in Tle 4 The dt indicted tht the mximum time tken for opening of 5% disc florets (783 dys), 75 % disc florets (137 dys), 1% disc florets (1218 dys) ws resulted when gerer stlk hving longest length were kept in solution of sucrose (4 %)+ 8-HQC (25 ppm) (T 2 L 4 ) The dt presented in Tle 5 on the effect of different chemicl nd stlk lengths on the useful life of gerer ws found to e significnt The dt presented in Tle 5 reveled Tle 3 Effect of different chemicls nd stlk lengths (cm) on the time tken to open 5, 75 nd 1 per cent disc florets of gerer Tretments Time tken to open (dys) 5 % disc florets 75% disc florets 1% disc florets Chemicl (T) T T T T T LSD (P=5) Stlk length (L) L L L L LSD (P=5) Tle 4 Interction effect of different chemicls nd stlk lengths (cm) on the time tken to open 5, 75 nd 1 per cent disc florets of gerer Tretments Time tken to open (dys) 5 % disc florets 75% disc florets 1% disc florets T L T L T L T L T 1 L T 1 L T 1 L T 1 L T 2 L T 2 L T 2 L T 2 L T 3 L T 3 L T 3 L T 3 L T 3 L T 3 L T 3 L T 3 L LSD (P=5)

66 Effect of different stlk lengths nd certin chemicl sustnces on vse life of gerer 149 Tle 5 Effect of different chemicls nd stlk lengths (cm) on useful life nd vse life of gerer flowers Tretments Chemicls (T) Useful life of flowers (dys) Vse life of flowers (dys) T T T T T LSD (P=5) Stlk lengths (L) L L L L LSD (P=5) Tle 6 Interction effect of different chemicls nd stlk lengths (cm) on useful life nd vse life of gerer flowers Tretments Useful life of flowers (dys) Vse life of flowers (dys) T L T L T L T L T 1 L T 1 L T 1 L T 1 L T 2 L T 2 L T 2 L T 2 L T 3 L T 3 L T 3 L T 3 L T 3 L T 3 L T 3 L T 3 L LSD (P=5) tht mong different chemicls, longest useful life of flower (987 dys) ws otined in sucrose (4 %)+ 8-HQC (25 ppm) (T 2 ) Among flower lengths, the highest useful life of flower (9 dys) ws recorded from L 4 (6 cm)the interction effect of different chemicls on useful life of flower ws lso found to e significnt (Tle 6) Longest useful life of flower (1169 dys) ws recorded when gerer cut flower hving mximum length L 4 (6 cm) were kept in solution of sucrose (4 %)+ 8-HQC (25 ppm) (T 2 L 4 ) The dt furnished in the Tle 5 reveled tht mong the different chemicls, the mximum vse life (1131 dys) ws otined y the use of sucrose (4 %)+ 8-HQC (25 ppm) (T 2 ) In cse of stlk length, the vse life of gerer incresed with increse in length The mximum vse life of stlk (117 dys) ws otined when stlk of gerer ws of 6 cm length (L 4 ) The interction effect of different chemicls nd stlk length on vse life of flower presented in Tle 6 ws found to e significnt Longer vse life (1365 dys) ws otined when gerer stlk of 6 cm length were kept in sucrose (4 %)+ 8-HQC (25 ppm) solution ( T 2 L 4 ) Discussion From the Tle 1 nd 2, it is ovious tht the solution uptke through stlk ws influenced y chemicls, stlk lengths nd their comintions The sorption of wter through stlk ws mximum t 3 rd, 6 th nd 9 th dy when stlks were kept in vse solution of sucrose (4 %)+ 8-HQC (25 ppm) Similr results were otined in gldiolus (De et l, 1996) nd in tuerose (Reddy et l, 1997) A eneficil effect of sucrose in sorption ws reported y De nd Brmn (1998) in tuerose nd of 8-HQC y Bhttchrjee (1993) in rose Other chemicls like luminum sulphte ws eneficil s it cidified the holding solution, which results in greter solution uptke y gerer Similr results were otined in gldiolus (Gowd nd Gowd, 199) nd in tuerose (Sini et l, 1997 nd De nd Brmn, 1998) Aluminum sulphte with sucrose in vse solution significntly influenced the wter uptke of the tuerose cut spike (Reddy nd Singh, 1996) The mximum solution uptke y cut flower stlks ws oserved in longest stlk of gerer t 3 rd, 6 th nd 9 th dy Similr result ws otined y Bhttchrjee (1993, 94) in rose Mximum solution uptke ws oserved under interction of 6 cm long stlk length nd sucrose (4 %)+ 8-HQC (25 ppm) thus longer stlk length hving more crohydrte nd HQC reduced stem lockge which results in increse in the solution uptke Results reveled tht time tken for opening of the disc florets ws significntly influenced y chemicls nd stlk lengths individully s well s y their comintion (Tle 3 nd 4) The mximum time tken to open the disc florets ws recorded in sucrose (4 %)+ 8-HQC (25 ppm) (T 2 ) solution According to Lrsen nd Cromorty (1967) nd Burdett (197) the microil growth re checked y the germicidl nd ctericidl properties of these chemicl sustnces nd therey were found effective for gldiolus (Ll et l, 199 nd Murli nd Reddy, 1991) Other chemicl tretments like sucrose (4 %) + luminum sulphte (1 ppm) nd sucrose (4 %) + citric cid (25 ppm) significntly delyed opening of disc florets over control In cse of stlk lengths, mximum time tken for opening of disc florets ws recorded for flowers hving longest stlk length of 6 cm (L 4 ) Interction effect of chemicl nd stlk length indicted tht mximum time tken for opening of disc florets ws oserved with the stlk hving mximum length nd kept in sucrose (4 %) + 8-HQC (25 ppm) solution Mximum useful life ws recorded in longest stlk length lone nd in comintion with chemicls like sucrose nd 8-HQC This my e due to higher crohydrte, thus promoting respirtion nd extending the longevity Similr results were otined in tuerose (De nd Brmn, 1998) Sucrose is min source of energy nd good respirtory sustrte for the mintennce of osmotic potentil while 8-HQC helped in controlling hrmful cteri nd prevented cteril plugging of wter conducting tissues nd there y incresed useful life of gerer stlk Mximum vse life ws otined with sucrose (4 %) +8-HQC

67 15 Effect of different stlk lengths nd certin chemicl sustnces on vse life of gerer (25 ppm) holding solution Similr results were recorded in tuerose (Singh nd Aror, 1995), rose (Msousky,1969 nd Bhttchrjee,1993) nd in chrysnthemum (Bht et l,1999) The incresed longevity of flower due to sucrose could e explined from the role of pplied sugrs in delying senescence nd promoting respirtion It delys the onset of excessive protein degrdtion s in gldiolus (Gowd nd Gowd,199 nd Merwe et l, 1986), tuerose (Pthk et l,1979); Mukhopdhyy,1982; De nd Brmn 1998 ) nd rose (Borochov et l,1976; Msousky,1969 nd Bhttchrjee, 1999) Sucrose is the min source of energy nd good respirtory sustrte for the mintennce of osmotic potentil in flower Kltler nd Steponkus (1976) concluded tht the min effect of pplied sugr in extending the longevity of flowers ws to mintin mitochondril structure nd function Sucrose could lso hve ntgonist effect on scisic cid in delying the senescence s oserved in rose (Borochov et l, 1976) nd crntion (Myk nd Dilley, 1976) Mrousky (1971) explined tht sugr improves the wter lnce in cut flowers, uptke of wter is incresed due to stomtl closure which reduces the trnspirtion rte The effect of 8-HQ components (8-HQC nd 8-HQS) in enhncing vse life of cut flowers hd een ttriuted to its nticteril property s noted y Serini nd Bnfi (1974) According to Ll et l (199) the chelting properties of the Quinoline compounds/esters proly chelted the metl ions of enzymes ctive in creting the stem lockge HQC lso cidifies the wter nd ffect the flower longevity Sucrose with HQS hs germicidl effect nd ws eneficil for prolonging the vse life of tuerose (Reddy et l,1997 ) The interction effect of chemicls nd stlk lengths ws lso found significnt The mximum vse life of the gerer ws recorded when gerer stlk hving longest length (L 4 ) kept in vse solution of sucrose (4 %)+ 8-HQC (25 ppm) solution (T 2 L 4 ) These fvourle effect on vse life of spike might e due to sufficient vilility of restorle sustrte nd 8-HQC which reduced microil ctivity nd incresed nd sorption of solution The results reveled tht the vse solution of sucrose 4% + 8- HQC 25 ppm nd stlk length of 6 cm, individully nd in comintion incresed fresh weight of flowers y promoting solution uptke These tretments re lso eneficil for improving the vse life of flowers nd useful life of flowers, opening of disc florets, with right, shining red colour nd freshness for longer durtion References Bht, A, SN Tripthi nd OP Sehgl, 1999 Effect of pulsing, pcking nd storge tretments on vse life of chrysnthemum cut flowers Adv Hort Forestry, 6: Bhttchrjee, SK 1993 Studies on post hrvest life of cut roses Indin J Hort, 5(2): Bhttchrjee, SK 1994 Post-hrvests life of cut roses s influenced y vrietl differences South Indin Hort, 42(5): Bhttchrjee, SK 1999 Evlution of different types of sugr for improving post-hrvest life nd qulity of cut roses Ann Agril Res, 2(2): Borochov, A, S Myk nd AH Hlevy, 1976 Comined effects of ABA nd sucrose on growth nd senescence of rose flowers Physiol Plnt, 36: Bose, TK nd LP Ydv, 1989 Commercil flowers Ny Prksh Clcutt Burdett, AN 197 The cuse of ent neck in cut roses J Amer Soc Hort Sci, 95(4): De, LC nd D Brmn, 1998 Post hrvest ehviour of cut tuerose spikes s ffected y chemicls JOrnmHort,1(2): De, LC nd D Brmn, 1998 Vse life of cut tuerose spikes s ffected y stge of hrvest, stlk length nd sucrose Oriss J Hort, 26(1): De, LC, SK Bhttchrjee nd RL Misr, 1996 Post hrvest life of pulsed gldiolus spikes s ffected y different chemicls J Ornm Hort, 4(1-2): Gowd, JVN nd VN Gowd, 199 Effect of clcium, luminum nd sucrose on vse life of gldiolus Crop Res, 3(1): Kltler, REL nd RL Steponkus, 1976 Fctors ffecting respirtion in cut roses J Amer Soc Hort Sci,11: Ll, SD, A Shh nd CC Pnt, 199 Effect of certin chemicl sustnces on vse life nd qulity of gldiolus cv Silver Horn Prog Hort, 22(1-4): Lrsen, FE nd RS Cromotry, 1967 Micro-orgnism inhiition y 8-HQC s relted to cut flower senescence Proc Amer Soc Hort Sci, 9: Mrousky, FJ 1969 Vsculr lockge, wter sorption, stomtl opening nd respirtion of cut Better Times roses treted with 9-Hydroxyquiroline citrte nd sucrose J Amer Soc Hort Sci, 94: Mrousky, FJ 1971 Inhiition of vsculr lockge nd incresed moisture retention in cut roses induced y ph, 8-Hydroxyquinoline nd sucrose J Amer Soc Hort Sci, 96: Myk, S nd DR Dilley, 1976 Effect of sucrose on response of cut crntion to kinetein, ethylene nd scisic cid J Amer Soc Hort Sci, 11(5): Merwe, JJ, Swrdt, Vnder, GH De nd L Durger, 1986 The effects of sucrose uptke from vse medium on the strch metolism of senescing gldiolus inflorescences South Africn Journl of Botny, 52(6): Mukhopdyy, TP 1982 Effect of chemicls on the florl development nd vse life of tuerose (Polinthes tueros L) vr Single South Indin Hort, 3(4): Murli, TP nd TV Reddy, 1991 Post-hrvest physiology of gldiolus flowers s influenced y colt nd sucrose Horticulture, New Technology nd pplictions, 63: 12 Pnse,VG nd PV Sukhtme, 1967 Sttisticl methods for Agril Workers ICAR puliction, New Delhi Pthk, S, MA Chudhuri nd SK Chtterjee, 1979 Effect of some germicides, hormones nd sugrs on longevity nd keeping qulity of tuerose Indin J Hort, 36: Reddy, BS nd K Singh, 1996 Effect of luminium sulphte nd sucrose on vse life of tuerose J Mhrstr Agric Univ, 21 (2): Reddy, BS, K Singh nd PM Gngdhrpp, 1997 Influence of 8-Hydroxyquionoline sulphte nd sucrose on post-hrvest physiology of tuerose cv Doule Krntk J Agric Sci, 1 (4): Sini, RS, R Ymdgni nd SK Shrm, 1994 Effect of some chemicls on the vse life of tuerose (Polinthes tueros L) cv Single South Indin Hort, 42 (6): Serini, G nd G Bnfi, 1974 Azione di sostnze conservntic ntiiotiche su flori Recisi Rivisl delt Qrto floro fuiticoltur Itlnn, 58: Singh, K nd JS Aror, 1995 Effect of 8-Hydroxyquionoline citrte, silver nitrte nd chrysl on vse life of cut chrysnthemum flower J Ornm Hort, 3 (1-2): 32-35

68 Journl of Applied Horticulture, 8(2): , July-Decemer, 26 Micropropgtion of Prthenocissus quinquefoli (L) from seedling explnts Appl Journl Yun Li, Dling Feng 1, Zhenyun Sun 2 nd Chen Niu 3 College of Biologicl Sciences nd Biotechnology, Beijing Forestry University, Beijing, 183, Chin 1 Current ddres : College of Life Science, Heei Agriculturl University, Boding, 711, Chin 2 Reserch Institute of Forestry, The Chinese Acdemy of Forestry, Beijing, 191, Chin Emil: sunzy@milforestryccn 3 Present ddress: Deprtment of Agronomy nd Horticulture, New Mexico Stte University, Ls Cruces, NM 883, USA Emil: niuc23@yhoocom Astrct Prthenocissus quinquefoli L ws successfully micropropgted through xillry ud multipliction from seedling explnts Shoot tips were isolted from seedlings nd cultured on B 5 medium supplemented with μm enzylminopurine (BA) nd 17 μm α-nphthlenecetic cid (NAA) to induce xillry uds The sprouted xillry uds formed multiple shoots when cultured on B 5 medium supplemented with 222 μm BA nd μm indole-3-utyric cid (IBA) The elongted shoots rooted in B 5 medium contining 49 μm IBA nd they survived from cclimtizing in soil to grow into helthy plnts Key words: Prthenocissus quinquefoli, xillry uds, micropropgtion, B 5 medium, enzylminopurine (BA), indole-3-utyric cid (IBA), indole-3-cetic cid (IAA), α-nphthlenecetic cid (NAA) Introduction Prthenocissus quinquefoli (L) Plnch (fmily Vitcee) commonly nmed s Virgini creeper, hs gined considerle ttention in ornmentl industries nd in lndscpe constructions ecuse of its climing hit nd rillint folige tht turns scrlet, crimson, or ornge in the fll ( com/ref/p/prth_qcfm) The creepers re tolernt of most soil conditions nd grow well under full sun to full shde However, P quinquefoli plnts do not grow well in cold climte, limiting its ppliction in res where the climte is cold yer-round P quinquefoli ws lso found to e symptomtic lterntive host of Xylell fstidios in hitts surrounding griculturl systems in Florid (Hopkins nd Adlerz, 1988) Therefore its common usge s n ornmentl nd wide distriution is thret in griculturl nd urn systems ecuse of potentil disese trnsmission to susceptile crop nd trees (Hopkins nd Adlerz, 1988, McElrone et l, 21) In vitro cultures hve long een ccepted for the production of virus-free plnts (Mellor nd Stce-Smith, 1977; Wng nd Hu, 1982; Tin et l, 1993) In vitro cultures re lso used in selecting nd screening of cultures tht exhiit genetic vrition, nd for tolernce to low or high tempertures (Tomes nd Swnson, 1982; Chleff, 1983; Plonen nd Buszrd, 1998; Mokotedi et l, 2) It is therefore highly desirle to use such iotechniques to improve nd ccelerte P quinquefoli reeding progress nd reduce long term progrm cost Furthermore, P quinquefoli show strong resistnce to phylloxer Grpe phylloxer (Phylloxer vitifolli Fitch) is serious risk to the viticulture nd wine industry (He, 21) This insect ttcks grpevine roots, slowly cusing decline in vine helth nd ultimtely destroying the grpevine Attempts mde to introduce the resistnce from P quinquefoli into grpe y trditionl reeding methods were unsuccessful due to species incomptiility (He, 21) Somtic hyridiztion hs the dvntge of overcoming species incomptiility nd improving plnt morphologicl trits or disese resistnce y DNA integrtion (Pndey et l, 1986; Tin et l, 22; Zimnoch-Guzowsk et l, 23), nd it could offer n lterntive to trnsferring the strong phylloxer resistnce of P quinquefoli to grpe cultivrs Thus, there is need to understnd the in vitro culture chrcteristics of P quinquefoli to enefit the successful ppliction of iotechnologicl pproches tht hve een used for plnt modifiction Tissue culture is sic tool in plnt iotechnology The successful nd efficient ppliction of tissue culture is highly dependent on the estlishment of relile regenertion system There were only two reports on in vitro study of P quinquefoli, which descried chitinse isoltion from P quinquefoli cell cultures (Bernsconi et l, 1987; Flch et l, 1993) To the est of our knowledge, no report is ville on in vitro regenertion of P quinquefoli The ojective of this study ws to investigte the menility of P quinquefoli for in vitro cultures The in vitro plntlets nd methods cn e susequently used s plnt source for the cold tolernt tretment nd other in vitro plnt improvements Mterils nd methods Estlishment of septic explnt: Prthenocissus quinquefoli (L) Plnch seeds were first wshed with running tp wter for five minutes nd soked in wter overnight The soked seeds were treted with 7% ethnol for 2 min After rinsing two times with sterile wter, the seeds were further surfce sterilized in 1% (w/v) sodium hypochlorite solution for 6 min, followed y four wshes in sterile wter The seeds were plced on B 5 (Gmorg et l, 1968) nd hlf strength Murshige nd Skoog medium (Murshige nd Skoog, 1962) All the medi used in this study

69 152 Micropropgtion of Prthenocissus quinquefoli (L) from seedling explnts contined 2% (w/v) sucrose nd were gelled with 5% gr The ph of the medi ws djusted to 6 prior to utoclving t 121 C for 2 minutes Seeds were germinted in light conditions with 16-h photoperiod of 4-5 µmol m -2 s -1 t 23±2 C The septic seedlings tht were otined fter 3 dys of culture were used s the source of explnts for the susequent experiments Axillry shoot initition nd multipliction of P quinquefoli: Shoot tips (include 1 cm hypocotyls nd two cotyledons) were excised from the 4 week septic seedlings nd were plced on shoot induction medi The effects of BA nd minerl composition on xillry shoot formtion were evluted y dding vrious concentrtion of BA (, 133, 222, 355, 887 nd 1774 µm) to B 5 or hlf strength MS medi supplemented with 1 µm NAA Ech tretment hd ten cultures nd ll experiments were repeted four times (totl of 4 explnts per experiment) The numer of xillry shoots produced per explnts nd percentge of explnts showing emergence of shoots were counted fter 4 weeks in culture For the multipliction of shoots, semisolid B 5 medium ws used The B 5 medium ws vriously supplemented with BA t different concentrtions ( µm) in conjunction with uxins (IAA, IBA nd NAA in rnge from 1 to 57 µm) Axillry shoots were excised from clusters nd sucultured t 3 dy intervls Multipliction rtes were clculted on the sis of the new xillry uds derived from one culture t the end of ech pssge Root initition nd trnsplnttion: After 5 weeks of suculture in multipliction medi, 2-3 cm long shoots with two to four leves were seprted from the explnts nd trnsferred to semisolid B 5 medium To study the effect of uxins on root initition, the medium ws vriously supplemented with μm IBA nd μm NAA Ech rooting tretment consisted of 1 shoots nd ws replicted three times, representing totl of 3 oservtions per tretment The percentge of shoots forming roots ws determined fter 3 weeks of culture Trnsfer to greenhouse conditions: Rooted plntlets on medi were plced in the greenhouse with nturl light rdition for one week Then the jr covers were opened nd the plnts were llowed to cclimtise for three dys The plntlets were removed from culture, rinsed thoroughly with wter to remove medi nd trnsferred to potting mixture (perlite : vermiculite 1:1) Individul plntlets were covered loosely with plstic wrp for one week Therefter, the plnts were grown in norml greenhouse nd nursery conditions Results nd discussion Initition of cultures: One hundred seeds were put on B 5 nd ½ MS medi for in vitro germintion The surfce-steriliztion procedures yielded 97-98% septic seed cultures Seeds strted germinting t dy nd grew to 3-4 cm long with fully extended cotyledons t 3 dys B 5 nd ½ MS medi were eqully effective for P quinquefoli seed germintion, 65% nd 68%, respectively All medi ecme rown fter 2 dys of culture due to the relese of color mterils from the seed cot To test the germintion effect of the rown medi, hlf of seeds were left in the sme rown medi nd hlf were trnsferred to fresh medi t one week, to llow these seeds fully relese the rown chemicls nd mke sure there ws no cteril or fungl contmintion There ws no difference in germintion frequency nd seedling growth during 3 dys of oservtion The results indicted tht color mterils relesed from the seed cot were not inhiitory for the seedling growth Axillry shoot initition nd multipliction of P quinquefoli: Shoot excised from hypocotyls were trnsferred to induction medi The B 5 medium ws superior to ½ MS for P quinquefoli xillry shoot induction, s 1% explnts cultured in B 5 medi supplemented with vrious plnt growth regultor (PGR) comintions induced xillry uds, produced more xillry uds per explnts nd grew longer xillry shoots compred with cultures in ½ MS medi (Tle 1) It ws noted tht high percentge of explnts (9-1%) cultured on sic B 5 or hlf strength MS medi without ny PGR produced n verge of only one xillry shoot ut ws weker thn PGR medi Among PGR tretments, those cultured on B µm BA + 1 µm NAA (BA: NAA 222:1) showed the est shoot elongtion performnce Though the xillry ud numer incresed with higher concentrtions (over 355 µm) of BA nd consistent NAA comintions, the shoot elongtion ws inhiited (Tle 1) Higher BA levels lso strongly inhiited shoot elongtion in grpe, which is from sme fmily s P quinquefoli (Lee nd Wetzstein, 199) Tle 1 Effects of BA nd minerl composition on xiliry ud induction of P quinquefoli shoots Medium type BA (µm) NAA (µm) Explnts producing xillry shoots (%) Axillry shoot length (cm±se) 9 1± ±36 1/2 MS ± ± ± ± ± ±26 B ± ± ± ±17 Ech tretment hd ten cultures nd ll experiments were repeted four times (totl of 4 explnts per experiment) The percentge of explnts showing emergence of shoots nd shoot length were counted within four weeks Shoot multipliction: Shoots induced from the ove cultures were excised nd trnsferred to semisolified B 5 medium supplemented with BA nd uxins for further prolifertion Different concentrtions of BA ( µm) nd NAA (1-54 µm) were tested in preliminry experiments Results indicted tht low concentrtion of BA (222µM) nd NAA (1 µm) in comintion were suitle for the new xillry ud induction nd growth High concentrtions of BA (133 µm) nd NAA (54 µm) in comintion reduced the percentge of explnts tht formed ud nd reduced the ud elongtion Only 7% explnts produced shoots nd the verge shoot growth ws 3 cm in one month under this culture condition In

70 Micropropgtion of Prthenocissus quinquefoli (L) from seedling explnts 153 tissue culture, where the lnce of cytokinin to uxin is key morphogenic fctor for otining mximum multipliction nd growth of the shoots (Skoog nd Miller, 1957), B 5 medium with 222 µm BA showed etter effects on multipliction when used lone in preliminry studies It ws then vriously supplemented with IAA, IBA nd NAA in rnge from 1µM to 57 µm The effects of uxins were determined fter 3 dys culture Of the vrious plnt growth regultor comintions tht were tested, B µm BA µm IBA or B µm BA + 1 µm NAA proved to e the est for P quinquefoli xillry shoot multipliction nd growth On these medi, over 93% explnts produced verge of two shoots, which grew more thn 16 cm in one month The percentge of explnts producing shoots ws reduced nd the shoot elongtion ws inhiited when higher concentrtions of uxins (IBA greter thn 148 µm nd NAA greter thn 27 µm) were used Compred with IBA or NAA, IAA ws less effective for P quinquefoli shoot induction nd growth, s indicted y lower percentge of shoot differentition (mximum 867%) nd slow shoot growth (mximum 12 cm) On verge, shoot multipliction rte of 2 fold every 3 dys ws mintined It is possile to otin higher shoot multipliction rte (3-4 fold) on B µm BA + 1 µm NAA, ut the elevted concentrtion of BA inhiited the elongtion of shoots nd possily lowered the recovery of shoots with rooting potentil in the susequent pssge ecuse of reduced shoot length, s is the cse of Anogeissus pendul nd A ltifoli culture (Sxen nd Dhwn, 21) Bsed on our oservtion of Jujue culture, the numer of shoots cn increse with every pssge nd ecome stle fter five to six sucultures (Luo et l, 1996) In grpe xillry shoot prolifertion study, Lee nd Wetzstein (199) lso oserved the rte of prolifertion ws slow during culture estlishment for the first 8 weeks, ut incresed rpidly therefter Hrge (21) reported tht reducing suculture frequency of Echince ngustifoli, E pllid nd E purpure from 4 to 2 weeks cn increse totl shoots produced per suculture from 28 to 239 fter 12 weeks We oserved slow growth nd yellow leves of P quinquefoli in vitro shoots when the suculture frequency ws extended from 3 dys to 45 dys It is possile to improve the P quinquefoli multipliction rte nd keep high shoot qulity y shortening the suculture time while mintining multiple suculture cycles The regenerted shoots longer thn 2 cm nd with pir of true leves were trnsferred to B 5 medium in the presence of IBA or NAA to initite roots P quinquefoli rooting frequency ws significntly ffected y plnt growth regultors IBA lone t 49 or 98 µm induced the highest percentge of rooting (1%) with 3 weeks of culture (Tle 2) The highest root numer per shoot ws induced t IBA concentrtion of 49 µm, with three to four vigorous terminl roots nd with lterl roots on them The roots emerged directly from the se of the shoots without cllusing Though rooting efficiency nd the root count per shoot ws similr when IBA ws incresed to 98 µm, cllus formtion ws oserved t this higher concentrtion IBA tretment The effects of incresing IBA concentrtion results in cllusing t the shoot se nd suppression of root elongtion ws reported in other plnt species Koroch nd coworkers (22) reported tht higher IBA rooted purple coneflower plntlets hd significntly lower survivl rtes fter trnsfer to the greenhouse compred to lower IBA tretments due to the sence of roots or n nomlous dventitious root system originting from cllus On the NAA (27-17 µm) rooting medium, the rooting frequency ws reltively low (6-767%), moreover, NAA induced root numer per shoot ws lower thn IBA tretment (verging 2 vs 3), clli formed t the shoot cut, nd some roots were thick nd lso differentited s cllus-like Only 76% of plntlets rooted in NAA survived fter cclimtiztion, while over 9% of plntlets rooted in IBA survived with sme tretment Our results showed tht IBA t 49 µm is idel for rooting of P quinquefoli Tle 2 Effect of uxin (IBA nd NAA) concentrtions on rooting of in vitro regenerted shoots of P quinquefoli Auxin (µm) Rooting (%) Performnce of root IBA (25) 8 ± 1 Less lterl roots IBA (49) 1 ± Norml, no cllus, with lterl roots IBA (98) 1 ± Slim root, thicker cllus NAA (27) 633 ± 153 Thick root NAA (54) 767 ± 116 Cllus NAA (17) 6 ± 265 Mny cllus Acclimtiztion nd trnsplnttion: Sixty-five regenerted plntlets were trnsferred into pots filled with perlite nd vermiculite (1:1) The survivl rte of these plntlets ws over 9% (3/33) following root initition with 49µM IBA (Fig 1) Phenotypiclly, ll the P quinquefoli plnts produced in this study ppered norml By contrst, only 75% (25/32) survivl rte ws chieved with NAA (54 µm) treted plntlets, which is significntly lower thn the IBA tretment The result is consistnt with previous reports tht dventitious roots originting from cllus hs deleterious effect on regenerted plnt trnsplnttion (Koroch et l, 22) Our results indicted tht P quinquefoli root initition tretment with IBA is superior to NAA y inducing more roust root systems directly from shoots In vitro multipliction of elite plnt species vi xillry ud prolifertion ws preferred in mny studies y voiding the tendency of undergoing somtic muttion (Lee nd Wetzstein, 199; Sxen nd Dhwn, 21; Shu et l, 23) Moleculr evidence showed tht plnts derived from xillry uds were clonlly uniform nd geneticlly stle (Shu et l, 23) We recently regenerted P quinquefoli plntlets from explnts of Fig 1 Well-estlished P quinquefoli plnts growing in pots