In Vitro Cell. Dev. Biol. Plant 42: , November December 2006 q 2006 Society for In Vitro Biology /06 $

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1 In Vitro Cell. Dev. iol. Plnt 42: , Novemer Deemer 26 q 26 Soiety for In Vitro iology /6 $18.+. DOI: 1.179/IVP26822 PHOTOSYNTHETIC ND CROHYDRTE STTUS OF ESY- ND DIFFICULT-TO-CCLIMTIZE SE OTS (UNIOL PNICULT L.) GENOTYPES DURING IN VITRO CULTURE ND EX VITRO CCLIMTIZTION CRMEN VLERO-RCM 1, MICHEL E. KNE 1 *, SNDR. WILSON 2, JOSEPH C. VU 3, JON NDERSON 3, ND NNCY L. PHILMN 1 1 Deprtment of Environmentl Hortiulture, Institute of Foo n griulturl Sienes, University of Flori, Ginesville, FL Deprtment of Environmentl Hortiulture, Institute of Foo n griulturl Sienes, University of Flori, IRREC, Fort Piere, FL Deprtment of gronomy, Institute of Foo n griulturl Sienes, University of Flori, Ginesville, FL (Reeive 17 Jnury 26; epte 21 ugust 26; eitor. M. Ree) Summry The photosyntheti n rohyrte sttus of n esy-to-limtize (EK 16-3) n iffiult-to-limtize (EK 11-1) genotype of Uniol pniult L. (se ots), ntive une speies of the southestern US, were evlute uring in vitro ulture n ex vitro limtiztion. Net photosyntheti rte ws eight times greter for EK 16-3 thn EK 11-1 plntlets fter ex vitro trnsfer. In vitro-proue leves were morphologilly similr to ex vitro-proue leves n exhiite similr photosyntheti ompetene. EK 11-1 plntlets exhiite greter trnspirtion rtes t the time of ex vitro trnsfer thn EK 16-3 plntlets. However, the smll mgnitue of this ifferene, lthough signifint, inite tht ontrol of wter loss ws proly not the min use for poor limtiztion of EK 11-1 plntlets. Crohyrte nlysis in vitro revele tht EK 16-3 plntlets utilize lef strh reserves more rpily thn EK 11-1 plntlets. Strh utiliztion orrelte with the evelopment of leves with expne lef les uring in vitro rooting in EK 16-3 plntlets. fter ex vitro trnsfer, oth genotypes exhiite signifint ereses of strh n solule sugr ontent in shoots n roots. However, the higher photosyntheti ility of shoots in EK 16-3 resulte in greter umultion of shoot solule sugrs thn EK 11-1 fter 2-wk ex vitro ulture. fter 6-wk in vitro rooting, there were signifintly higher hlorophyll n solule protein ontents, riulose 1,5-isphosphte roxylse (ruiso) n phosphoenolpyruvte roxylse tivities in EK 16-3 thn EK 11-1 shoots. These ifferenes lso orrelte with the evelopment of ntomil n morphologil lef fetures in EK 16-3 similr to those of greenhouse-proue leves. Key wors: miropropgtion; phosphoenolpyruvte roxylse; photosynthesis; ruiso. Introution Miropropgtion hs een use extensively for the rpi proution of mny plnt speies n ultivrs (Deergh n Zimmermn, 1991; Jeong et l., 1995; Hrtmnn et l., 22). However, espite its extrorinry potentil, this tehnology is still prolemti. One of the most importnt prolems is the poor survivl of plntlets following ex vitro trnsfer, uring limtiztion to greenhouse or fiel onitions (Pospíšilová et l., 1999). This prolem origintes from the poor evelopment of photosyntheti pity in vitro, whih hs een ttriute to the presene of sugr in the meium (Kozi, 1991; Pospíšilová et l., 1992; rigit et l., 22), low light n inequte CO 2 supply (Kozi n Iwnmi, 1988; De et l., 1993), n poor ontrol of wter loss use y high reltive humiity (RH) within the vessel (Desjrins, 1995; Estr-Lun et l., 21). These onitions *uthor to whom orresponene shoul e resse: Emil miropro@ ufl.eu n ultimtely influene plnt evelopment n photosyntheti performne ex vitro (Kozi, 1991; Preee n Sutter, 1991). In vitro ulture onitions frequently result in ltertions of mesophyll evelopment s well s hloroplst struture, nmely grn evelopment (Wetzstein n Sommer, 1982), whih my ffet the photosyntheti ility of plnts. t the iohemil level, low riulose 1,5-isphosphte roxylse/oxygense (ruiso) tivity (Grout, 1988) n high phosphoenolpyruvte roxylse (PEPC) tivity (Triques et l., 1997) re often enountere in C 3 speies. These onitions lso ontriute to low photosyntheti tivity (Sinh et l., 22). During limtiztion, in vitro-ulture plnts must go through trnsition from heterotropi or photomixotrophi moe to fully photoutotropi moe in the greenhouse. ommon strtegy of plnts uring ex vitro limtiztion is to use the rohyrte storge of in vitro-proue leves to over metoli emns of growing tissues (Piquers et l., 1998; Vn Huylenroek et l., 1998; Fuentes et l., 25). However, the funtion of in vitroproue leves uring limtiztion vries epening upon 572

2 PHOTOSYNTHETIC ND CROHYDRTE STTUS OF SE OTS 573 plnt speies. In some plnt speies, suh s Clthe (Clthe loiuse Ggnep. Mui Queen ), in vitro-proue leves hve limite photosyntheti ility n funtion s storge orgns whose energy reserves re onsume uring the initil ys of limtiztion (Vn Huylenroek et l., 1998). Furthermore, uliflower (rssi olere L.) or strwerry (Frgri nnss Duh. Kent ) leves exhiit greter respirtion thn photosyntheti rtes n their in vitro-proue leves senese rpily fter trnsplnttion ex vitro (Grout n ston, 1978; Grout n Millm, 1985). However, in other plnt speies, suh s pee lily (Spthiphyllum floriunum Shott. Petite ), in vitro leves re photosynthetilly ompetent n funtion similrly to greenhouseproue leves (Vn Huylenroek et l., 1998). Se ots (Uniol pniult L.) is perennil C 4 grss ntive to the southestern US n ommonly use for eh n une restortion n stiliztion (Wgner, 1964; rown n Smith, 1974). This speies is usully propgte y see. However, lterntive vegettive propgtion methos re neessry euse of the limittion of the nturl soures of plnts n sees (Hester n Menelssohn, 1987; hmn n Whitwell, 1995; urgess et l., 22, 25). Furthermore, onerns regring the use of unpte eotypes ollete from istnt lotions hve lso limite the olletion of plnt mterils. Consequently, miropropgtion protool ws evelope to mss-proue se ots genotypes from lolize sites (Philmn n Kne, 1994). When this protool ws pplie to multiple se ots genotypes, mirouttings of ifferent genotypes trnsferre ex vitro isplye iffering pities for limtiztion. Unerstning the resons for the low limtiztion pity of some se ots genotypes is neee to proue wie rnge of se ots genotypes effiiently using miropropgtion. In the present stuy, the photosyntheti ility n rohyrte sttus, s well s the tivity of some key enzymes in the ron fixtion proess of C 4 plnts, were ompre etween n esy-to-limtize se ots genotype n iffiult-to-limtize se ots genotype. Mterils n Methos Culture onitions. Estlishe n inexe in vitro shoot ultures of two se ots (U. pniult L.) genotypes, ollete from Egmont Key on the Flori Gulf Cost were use. These plnts were genotype using rnom mplifie polymorphi DN geneti nlyses (Rnmukhrhhi, 2), n hrterize s esy-to-limtize n iffiult-to-limtize (EK 16-3 n EK 11-1, respetively). Five se ots shoot lusters, eh onsisting of three shoots, 25 mm long, of EK 16-3 n EK 11-1 genotypes, were suulture into seprte Mgent G7 vessels (Mgent Corp., Chigo, IL, US) ontining 8 ml sterile Stge II meium. Stge II multiplition meium onsiste of Murshige n Skoog (MS) inorgni slts (Murshige n Skoog, 1962), supplemente with 87.6 mm surose,.56 mm myoinositol, 1.2 mm thimine HCl, 2.2 mm N 6 -enzylenine, n gelle with 8gl 21 TCe gr (PhytoTehnology Lortories, Shwnee Mission, KS, US). ll mei were juste to ph 5.7 with.1 N KOH prior to the ition of gr n utolving t kp n 1218C for 2 min. Cultures were mintine for 8 wk in growth hmer t 24 ^ 18C, 58 ^ 5% RH, 16-h photoperio provie y ool-white fluoresent lmps (Generl Eletri F2WT12 CW), n t 4 ^ 5 mmol m 22 s 21 photosyntheti photon flux (PPF) s mesure t ulture level. Susequently, the shoot lusters of eh genotype were suivie into single shoots n trnsferre to Stge III rooting meium. Stge III rooting meium onsiste of 8 ml sterile hlf-strength MS meium, supplemente with.56 mm myo-inositol, 1.2 mm thimine HCl, 87.6 mm surose, n 1 mm -nphthleneeti i, gelle with 8 g l 21 TCe gr n ontine in G7 vessels. Culture vessels ontine eight single mirouttings eh, n were mintine in ulture room t 22 ^ 28C, uner 16-h photoperio provie y ool-white fluoresent lmps (Generl Eletri F96T12 CW WM), t 1 ^ 5 mmol m 22 s 21 PPF, s mesure t ulture level. fter 6 wk, roote mirouttings were ple iniviully in 48-ell plug trys (eight six-elle loks, eh ell m; T.O. Plstis, In., Clerwter, MN, US) ontining orse vermiulite s supporting mteril n trnsferre to Stge IV greenhouse onitions. Plntlets were wtere s neee n Peters 2N 2P 2K liqui fertilizer (15 mg N l 21 ; The Sotts Compny, Mrysville, OH, US) ws pplie weekly. Greenhouse set points for ooling n heting were 24 n 228C, respetively, n nturl solr PPF rnge 9 12 mmol m 22 s 21 t noon. Photosynthesis stuies. Shoot lusters of the EK 11-1 n EK 16-3 se ots genotypes were ulture for 8 wk uner Stge II onitions s previously esrie. Susequently, single mirouttings were trnsferre to Stge III onitions for 6 wk, with 1 replite G7 vessels per genotype. ll roote mirouttings were trnsferre in 48-ell plug trys (eight six-elle loks, eh ell m; Summit Plstis In., Clerwter, OH, US) to greenhouse uner nturl solr PPF rnging etween 9 n 12 mmol m 22 s 21 uring the mesurements n y/night tempertures of 25/228C, respetively. Net photosyntheti rtes per lef re (P n ) were etermine with PP System Moel Cirs-1 (PP System Co., Lt, UK) without supplementl light soure n inlet CO 2 onentrtion fixe t 4 ^ 1 mmol mol 21. Mesurements were tken in full sun ner miy (1.m. to 12 p.m.) on newly forme n fully expne leves every week eginning the y fter estlishment ex vitro. Perent survivl ws sore every week uring Stge IV. Two plnts per plug try were mesure, ompiling t from 2 plnts per genotype. Dt were ollete weekly for 7 onseutive weeks. Photosynthesis enzymti stuies. Shoot lusters from EK 11-1 n EK 16-3 were ulture for 8 wk in G7 vessels uner Stge II onitions s previously esrie. There were four replite vessels per genotype, eh vessel ontining five plnts. Conurrently, nother set of se ots shoot lusters were ulture for 8 wk uner the sme onitions for shoot multiplition prior to trnsfer to Stge III onitions for 3, 6, or 9 wk. Eh tretment in Stge III onsiste of four replite vessels per genotype, eh vessel ontining eight mirouttings. Shoots of EK 11-1 n EK 16-3 ultures from Stges II n III in vitro n Stge IV ex vitro onitions were hrveste 3 h fter the eginning of the photoperio for ruiso n PEPC nlyses. Eh replite ollete from in vitro onitions onsiste of ll five shoot lusters per vessel from Stge II or ll eight lusters per vessel from Stge III, whih were immeitely ple in liqui N 2. During ex vitro Stge IV, eh replite ontine three plnts, whih were ple in liqui N 2 in the greenhouse. Susequently, ll replites were groun to fine power in mortr oole with liqui N 2 n ple in plsti vils in 288C freezer efore nlyses. For enzymti extrtion, powere shoot tissue (. 225 mg per smple) ws ple in glss mortr with 1.8 ml extrtion uffer ontining 1 mm iine (ph 8. t 258C), 1 mm MgCl 2,.1 mm EDT, 5 mm ithiothreitol (DTT), 1 mm isosorte, 2% PVP-4 (w/v), n.1% TX-1 (w/v). Lef tissue ws groun with extrtion uffer on ie for 2 min, trnsferre into two mirofuge tues, n entrifuge for 45 s..2-ml liquot of the superntnt ws inute (on ie for minimum of 5 min) with.1 ml 5 mm MgCl 2 plus.11 ml 2 mm NHCO 3 to otin fully rmylte ruiso ( tivte extrt ) prior to ssy of ruiso tivity. seprte liquot of the superntnt ws hel t room temperture for immeite ssy for PEPC. oth enzymes were ssye in totl volumes of.5 ml t 258C, in triplite, n ssys were omplete within 3 min from the strt of extrtions proeure with Hithi Moel U-2 Doule-em UV/VIS Spetrophotometer (Hithi Instruments, In., Dnury, CT, US). PEPC ws ssye spetrophotometrilly t 34 nm y following the reution of oxloeti i (O) y NDH in the presene of exess mlte ehyrogense (MDH) (shton et l., 199). The retion mixture ontine 1 mm iine (ph 8.), 1 mm MgCl 2,.1 mm EDT, 1 mm NHCO 3,5mM DTT, 2.5 U MDH, n.2 mm NDH. fter the ition of.1 ml extrt, stey seline ws estlishe n the retion ws initite y the ition of phosphoenolpyruvte to finl onentrtion of 5 mm. The liner erese in sorne ws reore over perio of 15 s. Totl ruiso tivity (fully tivte ruiso) ws ssye spetrophotometrilly se on the metho of Lilley n Wlker (1974). This enzyme-linke ssy ouples the tivity of ruiso with the oxition of NDH

3 574 VLERO-RCM ET L. using 3-phosphoglyeri phosphokinse n glyerlehye-3-phosphte ehyrogense extrte from rit musle (linking enzymes). The linking enzymes were purhse s mmonium sulfte preipittes. Sulfte ws remove prior to use either y eslting soluilize preipittes or y issolving the preipittes in 2% glyerol solution in uffer (Shrkey et l., 1991). The retion mixture for mesuring ruiso tivity ontine 1 mm iine (ph 8.), 2 mm MgCl 2,1mMEDT, 2 mm NCl, 1 mm NHCO 3,5mMDTT, 2.5 mm TP, 5 mm phosphoretine, 5 U retine phosphokinse, 5 U of eh of the linking enzymes,.2 mm NDH, n.6 mm riulose 1,5-isphosphte. fter stey seline sorne t 34 nm ws estlishe, the retion ws initite with.11 ml of tivte extrt. The liner erese in sorne resulting from the oxition of NDH ws reore over perio of 15 s. Chlorophyll ontent ws etermine using the metho esrie y rnon (1949). From eh tue ontining the rue extrt fter grining, n liquot of.1 ml ws trnsferre to two mirofuge tues, eh ontining.1 ml of wter. Susequently,.8 ml of 1% etone ws e in eh tue n inute in the rk for t lest 3 min in ie. fter 3 min of entrifugtion, the superntnt ws ollete n sorne ws mesure t 645 n 663 nm for totl hlorophyll etermintion. Totl solule protein (TSP) in the extrts ws quntifie y the ye-ining ssy of rfor (1976) using ovine serum lumin s stnr. Trnsmission eletron mirogrph stuies. Lef histologil ross setions from EK 11-1 n EK 16-3 mirouttings ulture in vitro in Stge III were otine to ompre unle sheth hloroplst ultrstruture. Trnsmission eletron mirosopy (TEM) ws use, for whih lef setions. 2 mm from the enter of the lef le were fixe in Trump s fixtive solution (MDowell n Trump, 1976). Fixtive infiltrtion ws hieve uner vuum for 2. Lef tissues were then rinse three times in phosphte uffer (ph 7.2), post-fixe in 1% uffere osmium tetroxie solution n then rinse in phosphte uffer, rinse three times in istille wter, n ehyrte in five-step sening ethyl lohol series (25, 5, 75, 95, n 1%), followe y ehyrtion in 1% etone. n enlo stin of 2% urnyl ette ws pplie etween the 75 n 95% steps of the ethyl lohol ehyrtion series. Lef setions were then emee in Spurr resin (Spurr, 1969). Ultrthin lef setions (7 nm) were ut from the enter prt of the lef le using Lei Ultrut ultrmirotome R (Lei Mirosopy n Sientifi Instruments, Deerfiel, IL, US), ollete on.35% form vr-ote opper gris, stine with methnoli urnyl ette n le itrte (Reynols, 1963). Setions were viewe on Hithi H7 trnsmission eletron mirosope (Hithi Sientifi Instruments) t 75 kv. Digitl mirogrphs were tken on iosn/digitl Mirogrph 2.5 (Gtn In., Plesnton, C, US) t n exposure level optimize for viewing the outer lyer n proesse with MEG View III/nlySIS 3.1 (Soft Imging System Corp., Lkewoo, CO, US). Crohyrte stuies. Plntlets from EK 11-1 n EK 16-3 se ots genotypes were ulture for 8 wk uner Stge II onitions, with 2 replite vessels per genotype, eh ontining five shoot lusters. Susequently, single shoots from eh genotype were ulture uner Stge III onitions for 3, 6, or 9 wk. Eh Stge III urtion tretment onsiste of five replite vessels per genotype, eh ontining eight single-shoot mirouttings. t week 8 of Stge II onitions, lusters of plntlets otine from five vessels were tken out of the meium, wshe, n pkge in luminum foil envelopes. Every pkge ontine three plntlets (su-smples), whih were ollete from the sme vessel. Smples were frozen in liqui N 2 n store t 288C prior to rying in 1-MR-TR freeze rier (The Virtis Compny, Griner, NY, US) for 7. Dry weights of eh su-smple were then reore. t weeks 3, 6, n 9 of Stge III onitions, ll plntlets from five vessels were ollete n proesse in the sme wy, this time seprting roots from shoots. prllel stuy ws omplete to nlyze the rohyrte sttus of oth genotypes uring Stge IV urtion. Plntlets from EK 11-1 n EK 16-3 se ots genotypes were ulture for 8 wk uner Stge II onitions, followe y 6 wk Stge III onitions n then trnsferre to Stge IV onitions for 4 wk. Five replite smples onsisting of three shoots or roots per genotype were ollete t weeks 2 n 4 of Stge IV n proesse s previously esrie. Proeures for totl solule sugr (TSS) extrtion were moifie following the esription y oersig n Negm (1985) n Miller n Lnghns (1989). Glss Psteur pipettes with glss wool plugs were loe with 5 mg of eh smple. Solule sugrs were extrte with 1.5 ml of methnol:hloroform:wter (MCW) [12:5:3 (v/v/v)] overnight. One hunre miroliters of mnnitol (1 mg ml 21 ) were e s n internl stnr to eh smple. One hour extrtions were repete two times with 1.5 ml MCW, followe y two itionl MCW (1.5 ml) rinses. Nnopure wter (3.5 ml) ws e to extrt prior to 2-min entrifugtion t 4 rpm. The queous phse ws remove n pplie to polyethylene olumns ontining 3 ml of 1:1 methnol:wter (MW) solution (v/v) n tion n nion exhnge resin (1 ml merlite IR-67 lyere with 1 ml Dowex 5-W, Sigm-lrih Co., St Louis, MO, US). Solule sugrs were elute n rinse two times with 1:1 MW prior to omplete evportion using RpiVp vuum evportor system (Lono Corp., Knss City, MO, US). The ry resiue ws resuspene in 1 ml high-pressure liqui hromtogrphy (HPLC)-gre wter n filtere through.45 mm memrne prior to HPLC injetion. TSSs (surose, gluose, n frutose) were nlyze using Wters 2695 High-Pressure Liqui Chromtogrph (Wters Tehnologil Corportion, Milfor, M, US) equippe with Wters 2414 refrtive inex etetor (Wters Tehnologil Corportion) n two onnete ior minex HPX-87C olumns (ior Lortories, Herules, C, US). Column n etetor tempertures were mintine t 8 n 58C, respetively. HPLC-gre wter ws use s the moile phse, t flow rte of.6 ml min 21. For strh nlysis, proeures were moifie s esrie y Hissig n Dikson (1979) n Miller n Lnghns (1989). The tissue resiue (remining in pipettes fter solule sugr extrtion) ws oven-rie overnight t 58C, suspene in 4 ml N-ette uffer (1 mm, ph 4.5), n kept in oiling wter th (98C) for 3 min. fter ooling to room temperture, 1. ml mylogluosise solution (from Rhizopus mol, Sigm- lrih Co.) [5 U per ssy in.1 M (ph 4.5) N-ette uffer] ws e to eh test tue to hyrolyze strh to gluose. Smples were inute for 48 h t 558C with osionl gittion. n liquot (1 ml) of eh smple (gluose hyrolyzte) ws trnsferre to len test tue n sujete to n enzyme ssy ontining gluose oxise (5 U ml 21 ) n peroxise (2 U ml 21 ). fter the ition of 2.2 N HCl (1. ml), sorne t 45 nm ws etermine using ekmn DU-64 spetrophotometer (ekmn Coulter In., Fullerton, C, US) n strh ontent ws lulte se on the regression eqution of the gluose lirtion line (. 1. mmol). For ex vitro nlyses, two replite su-smples were tken from eh tissue smple per genotype. These su-smples were verge to estimte strh n TSS ontents. Experimentl esigns n sttistil nlyses. ll experiments were rrnge in ompletely rnomize esigns. For photosynthesis mesurements, eh plnt per genotype ws onsiere replition. For rohyrte n enzymti nlyses, ll shoots or roots otine from eh vessel were onsiere replition. Min tretment effets n intertions were evlute using generl liner moel proeures evelope y Sttistil nlysis System (SS Institute In., 1999) n men seprtion ws evlute using Wller Dunn s test t P #.5. Results Photosyntheti n trnspirtion sttus ex vitro. The ility of plntlets to limtize to ex vitro onitions iffere signifintly etween genotypes. EK 16-3 plnts exhiite nerly 1% survivl fter 6 wk, wheres EK 11-1 plnts strte senesing fter 2-wk limtiztion, resulting in 29% survivl fter 6-wk ex vitro ulture (Fig. 1). Signifint ifferenes in P n were lso oserve uring initil plnt growth ex vitro, with P n vlues eing greter for EK 16-3 thn EK 11-1 plnts (Fig. 1). t week, ex vitro P n for EK 11-1 ws 1.9 mmol m 22 s 21, whih ws signifintly lower thn P n of EK 16-3 plnts. Surviving EK 11-1 plnts exhiite signifint inrese of P n over time tht ws not signifintly ifferent to the P n of EK 16-3 plnts fter 5 wk ex vitro ulture. Trnspirtion rtes of plnts t initil ex vitro trnsfer were signifintly higher for EK 11-1 thn EK 16-3 plntlets. fter 1-wk ex vitro ulture, trnspirtion rtes in oth genotypes inrese until fter week 3, when they erese (Fig. 1C).

4 PHOTOSYNTHETIC ND CROHYDRTE STTUS OF SE OTS 575 Ex vitro survivl (%) P n (µmol m 2 s 1 ) Trnspirtion rte (mol m 2 s 1 ) EK 11-1 EK Time in Stge IV (weeks) FIG. 1. Effet of in vitro ulture onitions on, ex vitro survivl;, ex vitro net photosyntheti rte per lef re (P n ); n C, ex vitro trnspirtion rte per lef re, of EK 11-1 n EK 16-3 genotypes uring Stge IV ulture. Mens ^ SE re shown (n ¼ 1). Crohyrte sttus in vitro n ex vitro. During weeks 3 9 of Stge III ulture, strh ontent in shoots ws greter in EK 11-1 thn EK 16-3 plntlets n oth genotypes exhiite stey erese of shoot strh with time (Fig. 2). fter 6 wk in Stge III, oth genotypes exhiite remrkle erese in strh ontent when trnsferre ex vitro to the greenhouse. y week 4 in Stge IV, shoot strh ontent of EK 11-1 plntlets ws 3.8 times lower thn tht of EK 16-3 plntlets. Trnsmission eletron mirogrphs (Fig. 3) of unle sheth ell hloroplsts inite ifferenes in the size n the istriution of strh grin hloroplsts with time n etween genotypes. While lrge strh grnules were foun fter 3 wk in Stge III, smll strh grins were oserve fter 6 n 9 wk in Stge III. Chloroplsts in EK 11-1 plntlets fter 3 wk in Stge III (Fig. 3) exhiite lrger numers of plstoglouli thn in EK 16-3 (Fig. 3D). Furthermore C t the sme time, thylkoi memrnes of EK 11-1 hloroplsts were seprte n ppere isrupte (Fig. 3). t 6 wk in Stge III, root strh ontent (Fig. 2) ws lower in oth genotypes ompre with shoot strh ontent (Fig. 2), n it ws not signifintly ifferent etween genotypes t 6 n 9 wk in Stge III ulture. Root strh ontent lso erese from 6 to 9 wk uner Stge III ulture onitions. Throughout Stge IV limtiztion, root strh ontent ws similr mong genotypes. t 2 wk in Stge IV, root strh ontent ws nerly eplete (13.2 n 6.5 times lower thn tht of EK 11-1 n EK 16-3, respetively, t week ). During Stge III, shoot solule sugrs (surose n hexose) inrese from weeks to 3 n then grully erese from weeks 3 to 9 (Fig. 4 C). t wk in Stge III (fter 8 wk in Stge II), there ws signifintly higher shoot surose ontent per ry weight in EK 16-3 thn EK 11-1 plntlets, wheres hexose n TSS were non-signifintly ifferent etween genotypes. lthough shoot surose, hexose, n TSS were similr mong genotypes t 3 n 9 wk in Stge III, t 6 wk, surose ontent ws 21% greter in EK 11-1 shoots thn EK 16-3 shoots. During Stge IV (fter 6 wk in Stge III), shoot solule sugrs erese from weeks to 2, n then remine stey from weeks 2 to 4 (Fig. 4 C). Furthermore, EK 16-3 plnts exhiite signifintly higher hexose n TSS ontents thn EK 11-1 plnts fter 2 n 4 wk ex vitro. During Stge III, root solule sugrs (surose n hexose) erese similrly mong genotypes etween weeks 6 n 9 (Fig. 5 C). Surose, hexose, n TSS ontents in roots were signifintly higher for EK 11-1 thn EK 16-3 plntlets, n erese fter 9 wk in Stge III in oth genotypes (Fig. 5 C). During Stge IV (fter 6 wk in Stge III), root surose n hexose of oth genotypes rmtilly erese within 2 wk, eing nerly eplete uring limtiztion. Chlorophyll n solule protein ontents. Totl hlorophyll ontent ws reltively low (.2 mg g 21 FW) throughout Stge III (EK 11-1) n for the first 3 wk of Stge III ulture (EK 16-3). Chlorophyll ontent of EK 16-3 then inrese signifintly fter 6 n 9 wk in Stge III (Fig. 6). During Stge IV (fter 6 wk in Stge III), hlorophyll levels of EK 16-3 plntlets generlly inrese with time. In EK 11-1 plntlets, the inrese in hlorophyll ws elye until fter plntlets were estlishe to greenhouse onitions (fter 4 wk in Stge IV). Chlorophyll ontent ws signifintly greter for EK 16-3 thn EK 11-1 plntlets fter 2 n 4 wk in Stge IV. Conversely, EK 11-1-limtize plnts (. 6wk in the greenhouse) exhiite higher hlorophyll levels thn EK 16-3 plnts. Totl solule protein (TSP) ontent in shoots of se ots genotypes ws lowest t the eginning of Stge III for oth genotypes n inrese with time, eing signifintly higher for EK 16-3 thn EK 11-1 plntlets fter 6 n 9 wk in vitro (Fig. 6). rpi inrese in TSP ws oserve in EK 16-3 plntlets uring the first 4 wk fter trnsfer to ex vitro onitions, with signifint erese fter plntlets were estlishe ex vitro (. 6 wk). EK 11-1 plntlets lso inrese their TSP ontent with time, eing higher fter plnts h limtize ex vitro. Photosyntheti enzyme sttus in vitro n ex vitro. During Stges III n IV, there were signifint ifferenes in PEPC tivity per grm fresh weight over time n etween genotypes (Fig. 7). EK 16-3 plntlets exhiite higher tivity of PEPC with time uner Stge III onitions tht ws lso signifintly higher

5 576 VLERO-RCM ET L. Shoot strh ontent (mg g 1 ry weight) Root strh ontent (mg g 1 ry weight) 14 EK11-1 EK T x Time in Stge III (weeks) T x Time in Stge IV (weeks) 2 4 FIG. 2., Shoot strh ontent n, root strh ontent of EK 11-1 n EK 16-3 genotypes uring in vitro Stge III (left) n fter mirouttings were roote for 6 wk, Stge III n trnsferre to Stge IV (right). Error rs inite SE (n ¼ 1). NOV nlysis is shown on top left orner of eh grph: T, time; G, genotype; NS, non-signifint; ** signifint t P #.1. Different letters on top of histors within eh ulture stge re signifintly ifferent oring to the Wller Dunn test t P #.5. thn EK 11-1 plntlets. oth genotypes inrese their PEPC tivities following ex vitro trnsfer. One the plntlets eme estlishe ex vitro (. 6 wk), oth genotypes h similr PEPC tivity. Differenes in ruiso tivity were lso oserve over time n etween genotypes uring Stge III n fter ex vitro trnsfer to Stge IV (Fig. 7). EK 11-1 plntlets exhiite lower ruiso tivity per grm fresh weight thn EK 16-3 plntlets, whih inrese fter 6 n 9 wk uner Stge III onitions. fter trnsferring plntlets to ex vitro onitions (following 6 wk Stge III), ruiso tivity i not signifintly inrese in EK 11-1 plntlets until they were estlishe ex vitro (. 6 wk). Conversely, ruiso tivity signifintly inrese in EK 16-3 plntlets fter 2wkex vitro ut it erese fter plnts were estlishe (.6 wk). The PEPC n ruiso tivities of mize (Ze mys L.) plnts ulture uner greenhouse onitions were ssye following the ientil nlytil proeures use for se ots, n PEPC/ruiso (P/R) rtio of 3.7 ws otine. Similrly, P/R rtios rnging from 3 to 5 were oserve in se ots ultures ex vitro (Fig. 7C). Conversely, lower P/R vlues were oserve when se ots were ulture uner Stge III onitions. EK 16-3 plntlets h higher P/R rtios thn EK 11-1 plntlets fter 9 wk in vitro, ut t p C s p s p s D E F t s p s p 3 weeks 6 weeks 9 weeks FIG. 3. Comprtive TEM of hloroplsts of C, EK 11-1 n D F, EK 16-3 genotypes fter (, D) 3 wk, (, E) 6 wk, n (C, F) 9 wk Stge III ulture onitions in unle sheth ells. r ¼ 2 mm. p, Plstoglouli; s, strh grnule; n t, thylkoi memrnes.

6 PHOTOSYNTHETIC ND CROHYDRTE STTUS OF SE OTS 577 Shoot surose ontent (mg g 1 DW) T x e e e EK 11-1 EK 16-3 T x G: * Shoot hexose ontent (mg g 1 DW) Shoot totl solule sugr ontent (mg g 1 DW) Time in Stge III (weeks) 2 4 Time in Stge IV (weeks) C FIG. 4., Shoot surose ontent;, shoot hexose ontent; n C, shoot totl solule sugr ontent of EK 11-1 n EK 16-3 genotypes uring in vitro Stge III (left) n fter mirouttings were roote for 6 wk, Stge III n trnsferre to Stge IV (right). Error rs inite SE (n ¼ 1). NOV nlysis is shown on top left orner of eh grph: T, time; G, genotype; NS, non-signifint; *,** signifint t P #.5, or.1, respetively. Different letters on top of histors within eh ulture stge re signifintly ifferent oring to the Wller Dunn test t P #.5. there were no signifint ifferenes etween genotypes fter trnsfer to Stge IV onitions. Photosyntheti enzyme nlyses expresse on sis of TSP inite tht PEPC tivity ws not ffete y Stge III urtion in EK 11-1 plntlets, ut inrese in weeks 6 n 9 of Stge III in EK 16-3 plntlets (Fig. 8). Plntlets of either genotype exhiite from 1.5 to 3.9 times greter PEPC tivities fter ex vitro limtiztion thn t the time of initil trnsfer (week ). Conversely, ruiso tivity per TSP i not vry from 6 wk in Stge III or fter ex vitro trnsfer in either genotype. itionlly, ruiso tivity per TSP ws signifintly higher in EK 16-3 fter 6 n 9wk in vitro n uring ex vitro limtiztion thn EK 11-1 plntlets (Fig. 8). Disussion Differenes in limtiztion pities etween se ots genotypes involve remrkle n ivergent evelopmentl n physiologil responses to in vitro ulture onitions. Low ex vitro survivl in the EK 11-1 genotype orrelte to in vitro evelopment of short leves hving short les with miniml le expnsion. In ontrst, EK 16-3 ultures proue elongte leves with expne les in Stge III y week 6. These leves were morphologilly similr to those proue ex vitro on limtize plnts. Physiologilly, these leves, like those in re rsperry (Ruus ieus L.) (Donnelly n Viver, 1984) n sin white irh [etul pltyphyll Szehuni (Shnei.) Reh.] (Smith et l., 1986), i not eteriorte rpily fter trnsplnttion n were photosynthetilly ompetent ex vitro. The P n in EK 16-3 leves t time of ex vitro trnsfer ws eight times greter thn EK 11-1 leves. Thus, there ws strong orreltion etween in vitro proution of se ots leves morphologilly similr to those proue ex vitro n photosyntheti ompetene. Given tht trnspirtion rtes were signifintly ifferent t the time of ex vitro trnsfer, initil poor ontrol of wter loss ex vitro most likely ontriute to lower EK 11-1 survivl. Initil ereses in trnspirtion rtes uring week 1 in oth genotypes possily reflet restrite wter uptke ue to limite root hir evelopment in vitro. fter week 1, the inrese in trnspirtion rtes of oth genotypes possily reflets the impt of the greter funtioning root

7 578 VLERO-RCM ET L. Root surose ontent (mg g 1 DW) Root hexose ontent (mg g 1 DW) G: * EK 11-1 EK 16-3 T x G: * Root totl solule sugr ontent (mg g 1 DW) Time in Stge III (weeks) T x 2 4 Time in Stge IV (weeks) C FIG. 5., Root surose ontent;, root hexose ontent; n C, root totl solule sugr ontent of EK 11-1 n EK 16-3 se ots genotypes uring in vitro Stge III (left) n fter mirouttings were roote for 6 wk, Stge III n trnsferre to Stge IV (right). Error rs inite SE (n ¼ 1). NOV nlysis is shown on top left orner of eh grph: T, time; G, genotype; NS, non-signifint; *,** signifint t P #.5 or.1, respetively. Different letters on top of histors within eh ulture stge re signifintly ifferent oring to the Wller Dunn test t P #.5. system omine with the ourrene of norml stomte funtion. Trnspirtion rtes mesure fter 6 wk ex vitro trnsfer were similr in oth genotypes n likely reflete mesurements of surviving plnts tht h eome limtize. However, ue to the smll mgnitue, leit signifint, ifferene in trnspirtion rtes etween genotypes t time of ex vitro trnsfer, we onlue tht ontrol of wter loss ws proly not the min use for poor ex vitro limtiztion. lthough high trnspirtion rtes my exerte the trnsition to ex vitro onitions, other physiologil limittions, inluing poor photosyntheti pity or limite rohyrte reserves uring limtiztion, h greter impt on survivl of EK 11-1 ex vitro. Strh umultion n utiliztion in plnt ells prior to the initition n progression of evelopmentl proesses, suh s shoot n root initition, hve een orrelte (Swrnkr et l., 1986; rn et l., 1994). The low initil strh levels in oth se ots genotypes, otine from 8-wk-ol Stge II ultures, inite tht the shoot lusters from whih mirouttings were erive, were strh eplete t the en of the multiplition stge. Strh ontent inrese rpily fter mirouttings were trnsferre to surose-ontining Stge III meium, prior to root emergene. Rpi umultion of strh ws lso oserve when egoni (egoni rex) explnts were trnsferre to meium ontining 87.6 mm surose prior the formtion of shoot-u primori (Mngt et l., 199). Greter eline in strh reserves in EK 16-3 over EK 11-1 fter 3 wk in vitro my hve resulte from the more rpi n erlier initition n growth of EK 16-3 roots n shoots. This oservtion is supporte y omprtive growth mesurements of oth genotypes uring Stge III ulture (Vlero-rm, 25). Our stuies inite tht se ots genotypes store strh n ontine solule sugrs in shoots n roots throughout Stge III. t the time of limtiztion, strh storge ws greter in shoots of EK 11-1 thn EK 16-3 plntlets, wheres shoot solule sugr ontent ws not signifintly ifferent etween genotypes. During limtiztion ex vitro, shoot strh n solule sugrs in oth genotypes were eplete, yet EK 16-3 mintine higher rohyrte levels thn EK 11-1 ex vitro. This ws ue, in prt, to the higher net photosyntheti rtes of EK 16-3 leves t time of ex vitro trnsfer. During limtiztion, EK 16-3 utilize two ron soures, nmely store rohyrte reserves n photossimiltes. In ontrst, EK 11-1 plnts epene primrily on store rohyrte reserves. Given tht strh levels were tully higher in shoots of the iffiult-to-limtize se ots genotype (EK 11-1) t the time of ex vitro trnsfer, low ex vitro survivl proly resulte from limite proution of photosynthetilly ompetent leves in vitro n susequent rpi epletion of energy reserves ex vitro. This is supporte y the very low P n in EK 11-1 plntlets uring the time of trnsfer to ex vitro onitions. Cpelles et l. (1991) esrie n inverse reltionship etween strh ontent in shoot hloroplsts n photosyntheti

8 PHOTOSYNTHETIC ND CROHYDRTE STTUS OF SE OTS 579 Chlorophyll ontent (mg g 1 FW) T x EK11-1 EK16-3 T x Totl solule protein (mg g 1 FW) T x >6 Time in Stge III (weeks) Time in Stge IV (weeks) FIG. 6., Chlorophyll ontent per shoot grm fresh weight n, TSP ontent per shoot grm fresh weight of EK 11-1 n EK 16-3 se ots genotypes uring in vitro Stge III (left) n fter mirouttings were roote for 6 wk, Stge III n trnsferre to Stge IV (right). Error rs inite SE (n ¼ 4). NOV nlysis is shown on top left orner of eh grph: T, time; G, genotype; NS, non-signifint; *,** signifint t P #.1, respetively. Different letters on top of histors n next to legen within eh ulture stge re signifintly ifferent oring to the Wller Dunn test t P #.5. rtes of in vitro rose (Ros multiflor L. Montse ) leves. itionlly, low photosyntheti rtes in vitro hve lso een ttriute to rohyrte umultion in the leves (zón- ieto, 1983). similr effet my our uring erly stges of se ots in vitro evelopment, when strh n solule sugr ontents in leves re high. t 3 wk in Stge III, EK 11-1 hloroplsts lso exhiite lrge numer of plstoglouli n seprtion of thylkoi memrnes. These fetures re often oserve in senesing leves (rntzen n rintis, 1975). Plstoglouli pprently funtion s extrlmellr pools for memrne lipis, utilize for memrne iosynthesis or for eposit fter memrne egrtion. In vitro leves usully serve s storge orgns to over metoli emns of growing tissues uring the initil ys of limtiztion (Cpelles et l., 1991; Vn Huylenroek n Deergh, 1996; Piquers et l., 1998; Vn Huylenroek et l., 1998; Fuentes et l., 25). Cpelles et l. (1991) lso onlue tht uring ex vitro limtiztion of rose, plnts with leves tht exhiite low photosyntheti rtes utilize strh reserves while reestlishing the photosyntheti pity of leves. However, in other speies, only leves proue ex vitro re photosynthetilly ompetent (Grout n Millm, 1985). Leves in EK 11-1 plnts exhiite low photosyntheti pity n utilize store strh reserves uring limtiztion. The susequent high ex vitro mortlity inite tht, in most EK 11-1 plnts, the respirtory emns were too high to llow restortion of photosyntheti pity in in vitro-proue leves or through proution of new leves. In these plnts, the llotion of energy resoures ppers to e irete mostly towrs mintenne of root iomss n nonlefy shoot proution. During Stge III ulture, strh ontent in roots of oth genotypes ws lower thn shoots n ws similrly eplete ex vitro. Solule sugr levels were higher in EK 11-1 thn EK 16-3 roots uring Stge III, ut those levels erese signifintly fter ex vitro trnsfer in oth genotypes. Surose in the meium is hyrolyze y the enzyme invertse tht is relese y mirouttings in response to wouning (De l Viñ et l., 1999; Sturm, 1999). Then roots uptke surose n hexose, whih re trnslote n store s strh in shoots n roots in vitro. In ontrst, uner ex vitro onitions, photosynthetilly tive leves re the soures of ron, where solule sugrs re proue n either store s strh reserves or trnslote into other sink orgns, suh s roots. Photossimiltes proue uring limtiztion in either genotype were trnslote n utilize for growth n evelopment, rther thn umulte s strh. This ws inite y the mintenne of low strh levels fter 2 n 4 wk ex vitro. The low photosyntheti pity of EK 11-1 in vitro-proue leves n e ttriute to the ltertion in the tivities of the two primry enzymes of CO 2 fixtion, nmely PEPC n ruiso, uring in vitro ulture n initil ex vitro limtiztion. Ruiso tivity in vitro hs een reportely reue y meium supplementtion with exogenous surose in C 3 speies, suh s strwerry (Hier, 1994; Desjrins, 1995) n plntin (Mus v. CEMS 3/4) (rgón et l., 25). This my, in turn, use feek inhiition of photosynthesis euse of low phosphte or high sugr-phosphte onentrtion in the ytosol, oth known to ffet the regenertion of the ruiso sustrte RuP. itionlly, C 3 plnts exhiit inrese respirtion, resulting in inrese PEPC tivity (Hier n Desjrins, 1994). Inrese PEPC tivity hs lso een reporte in the C 4 speies Flveri ientis, when fe with

9 58 VLERO-RCM ET L. PEP roxylse tivity (µmol g 1 FW min 1` ) Ruiso tivity (µmol g 1 FW min 1 ) T x T x EK 11-1 EK 16-3 T x T x PEPC/Ruiso rtio T x C >6 Time in Stge III (weeks) Time in Stge IV (weeks) FIG. 7., Ruiso tivity per shoot grm fresh weight;, PEPC tivity per shoot grm fresh weight; n C, PEPC/ruiso rtio of EK 11-1 n EK 16-3 se ots genotypes uring in vitro Stge III (left) n fter mirouttings were roote for 6 wk, Stge III n trnsferre to Stge IV (right). Error rs inite SE (n ¼ 4). NOV nlysis is shown on top left orner of eh grph: T, time; G, genotype; NS, non-signifint; ** signifint t P #.1. Different letters on top of histors within eh ulture stge re signifintly ifferent oring to the Wller Dunn test t P # mm exogenous surose in the ulture meium (Furnk et l., 1997). In ontrst, ruiso tivity per TSP i not signifintly hnge in either se ots genotype fter ex vitro trnsfer. Furthermore, erese PEPC tivity per TSP use y in vitro onitions ws oserve in oth genotypes. Similr results hve een oserve in plntin (rgón et l., 25). Ex vitro P/R rtios for se ots verge 3.6, whih ws omprle to the P/R rtio we otine with mize. However, in vitro P/R rtios were lowere, possily y the reue PEPC tivity mesure. During weeks 6 n 9 of Stge III ulture, the tivities of PEPC n ruiso n the ontents of hlorophyll n TSP were signifintly greter in EK 16-3 thn EK Histologil setions of in vitro-proue leves of EK 11-1 n EK 16-3 (Vlero-rm, 25) inite tht fter 6 wk in Stge III, EK 16-3 leves exhiite more typil Krntz ntomy of C 4 plnts thn EK 11-1 leves. fter 6 wk in Stge III, the fixtion of CO 2 y PEPC n the CO 2 onentrting mehnism in EK 16-3 might hve reue some of the ineffiy of ruiso uner the stress use y in vitro onitions, enling the mintenne of photosynthesis (Roríguez et l., 23). Likewise in sugrne (Shrum offiinrum L.) plnts, typil C 4 speies (Crfts-rnner n Slvui, 22), photosynthesis ws tolernt of reltively high environmentl stress. itionlly, in vitro sugrne leves exhiite miniml Krntz ntomy ompre with the more typil Krntz ntomy exhiite y ex vitro leves (Roríguez et l., 23). This inrese in enzymti tivity, hlorophyll n solule protein ontent fter 6 wk in Stge III orreltes with the initil erese of strh n solule rohyrtes in shoots of oth genotypes uring the sme time perio, n with the formtion of the first fully expne leves with typil Krntz ntomy in EK 16-3 (Vlero-rm, 25). These inrese levels of hlorophyll n TSP ontent n inrese enzymti tivities re initive of the evelopment of photosyntheti hrteristis in EK 16-3 fter 6 wk in Stge III. Furthermore, EK 16-3 plntlets trnsferre ex vitro fter 6 wk in Stge III exhiite 1% survivl. Conversely, EK 11-1 plnts exhiite limite lef elongtion n expnsion uring Stge III ulture (Vlero-rm, 25), whih orrelte with lower PEPC n ruiso tivities, n low hlorophyll n TSP ontents in vitro ompre to EK 16-3 plnts. This limite evelopment of photosyntheti fetures in vitro resulte in 3% survivl fter 6 wk ex vitro onitions.

10 PHOTOSYNTHETIC ND CROHYDRTE STTUS OF SE OTS 581 PEP roxylse tivity (µmol mg 1 TSP min 1 ) T x e e e e EK 11-1 EK T x T: NS T x G: * Ruiso tivity (µmol mg 1 TSP min 1 ) e e e >6 Time in Stge III (weeks) Time in Stge IV (weeks) FIG. 8., PEPC tivity per shoot milligrm totl solule protein (TSP) n, ruiso tivity per shoot milligrm TSP of EK 11-1 n EK 16-3 se ots genotypes uring in vitro Stge III (left) n fter mirouttings were roote for 6 wk, Stge III n trnsferre to Stge IV (right). Error rs inite SE (n ¼ 4). NOV nlysis is shown on top left orner of eh grph: T, time; G, genotype; NS, nonsignifint; *,** signifint t P #.5 or P #.1, respetively. Different letters on top of histors n next to legen within eh ulture stge re signifintly ifferent oring to the Wller Dunn test t P #.5. Conlusions Lower photosyntheti pity (P n ) of EK 11-1 thn EK 16-3 t time of trnsfer to ex vitro onitions ws orrelte with reue survivl. In ition to lower photosyntheti pity ex vitro, other ftors, inluing initilly higher trnspirtion rtes n insuffiient rohyrte reserves to filitte the trnsition from in vitro to ex vitro onitions, further ontriute to poor ex vitro survivl in EK Crohyrte nlyses inite rpi umultion of strh n sugrs in plntlets for the first 3 wk following trnsfer to Stge III meium in oth genotypes. These rohyrtes were use for root n shoot initition n growth uring the lter Stge III ulture perio. Crohyrte reserve utiliztion in EK 16-3 uring the lte Stge III ulture perio ws greter thn EK 11-1, n resulte in lower strh n solule sugr reserves thn EK 11-1 t the time of ex vitro trnsfer. fter 6 wk in Stge III, there were signifintly greter inreses in PEPC n ruiso tivities, n in hlorophyll n TSP ontents in EK 16-3 thn EK 11-1 plntlets. These ifferenes orrelte with the evelopment of ntomil n morphologil lef fetures similr to those in the greenhouseproue leves (Vlero-rm, 25). Stge III in vitro ulture onitions ifferently ffete the growth n evelopment of photosyntheti pity of se ots genotypes. Those genotypes ple of eveloping photosynthetilly ompetent leves, while storing rohyrtes in vitro, n overome the energy emns to fee respirtion requirements of ells, s well s to fee growth requirements of ifferent orgns uring limtiztion. Diffiult-to-limtize se ots genotypes oul possily enefit from moifitions of the in vitro ulture onitions, suh s ltertion of the environmentl ulture onitions or the meium omponents, for improve ex vitro limtiztion. knowlegments This projet ws evelope uner the guine of the Flori Se Grnt College Progrm n fune from NO, Deprtment of Commere, Grnt No. N16RG The uthors woul like to thnk Crolyn rtusk for her sttistil expertise, Chris Wilson for his HPLC instrumenttion, n Kren Kelley n Fre ennett in the Eletron Mirosopy Core Lortory t the UF, for their guine n ssistne in histologil stuies. Referenes rgón, C. E.; Eslon, M.; Cpote, I.; Pin, D.; Cejs, I.; Roriguez, R.; Cñl, M. J.; Snovl, J.; Roels, S.; Deergh, P.; Gonzlez-Olmeo, J. Photosynthesis n ron metolism in plntin (Mus ) plntlets growing in temporry immersion ioretors n uring ex vitro limtiztion. In Vitro Cell. Dev. iol. Plnt 41:55 554; 25. rigit, L.; González,.; Sánhez Tmés, R. Influene of CO 2 n surose on photosynthesis n trnspirtion of tiini eliios explnts ulture in vitro. Physiol. Plnt. 115: ; 22. rnon, D. I. Copper enzymes in isolte hloroplsts; polyphenol oxises in et vulgris. Plnt Physiol. 24:1 15; rntzen, C. J.; rintis, J. M. Chloroplst struture n funtion. In: Govinjee, e. ioenergetis of photosynthesis. New York: emi Press; 1975: shton,. R.; urnell, J. N.; Furnk, R. T.; Jenkins, C. L. D.; Hth, M. D. Enzymes of C 4 photosynthesis. Meth. Plnt iohem. 3:39 72; 199. zón-ieto, J. Inhiition of photosynthesis y rohyrtes in whet leves. Plnt Physiol. 73: ; hmn, G. R.; Whitwell, T. Nursery proution of Uniol pniult (southern se ots). HortTehnology 5: ; 1995.

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