Oak somatic and gametic embryos maturation is affected by charcoal and specific aminoacids mixture

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1 Ann. For. Si. 67 (21) 25 INRA, EDP Sienes, 21 DOI: 1.151/forest/2998 Avilble online t: Originl rtile Ok somti n gmeti embryos mturtion is ffete by hrol n speifi minois mixture Betriz Pintos 1,JoseA.Mnzner 2 *, M. Angeles Bueno 1 1 Forest Bioteh Lb., INIA-CIFOR, A-6 Km 7,5, 284 Mri, Spin 2 Tehnil University of Mri (UPM), ETSI Montes, Ciu Universitri, 284 Mri, Spin (Reeive 4 Februry 29; epte 18 August 29) Keywors: tivte hrol / mino is / ork ok / gmeti embryo / plntlet limtion Mots-lés : hrbon tif / ies minés / hêne-liège / embryon gmétique / limttion e plntule Abstrt Development of both somti n gmeti embryogenesis hs mny pplitions in lonl forestry n geneti improvement, for instne s mss-propgtion of genetilly improve plnts n proution of pure lines through ouble-hploi plnt regenertion from gmeti embryos. The gol of this work ws to improve growth, mturtion n plntlet regenertion of ork ok (Querus suber L.) embryos from both somti n gmeti origin. Ativte hrol promote signifint inrese in growth in terms of reltive size n weight of both somti n ouble-hploi embryos, s well s more effiient ontrol of seonry somti embryogenesis uring evelopment. A signifint intertion ws lso observe with mino i nutrition. While some mino is (i.e., glutmine, rginine or sprgine) i not show signifint ifferenes with the ontrols, mixture of these three mino is or gmm mino butyri i stimulte embryo growth. The highest survivl rte uring limtion of plntlets from both somti n ouble-hploi origin ws obtine when the embryos h been previously ulture on bsl meium with 3% surose n 1% tivte hrol for two months t 4 C n germinte on meium supplemente with 6-benzylminopurine n inole-3-butyri i. We obtine more thn 9 ork ok plntlets limte from severl embryogeni lines, with high survivl rte, emonstrting tht this methoology is pplible for lrge sle plntlet proution. We lso report the first regenertion of ouble-hploi plntlets in ork ok. Résumé L mturtion es embryons gmétiques et somtiques e hêne est ffetée pr le hrbon et le mélnge minoies spéifiques. Le éveloppement es eux embryogenèses gmétique et somtique e nombreuses pplitions ns l foresterie lonle et l méliortion génétique, pr exemple en tnt que propgtion en msse e plnts génétiquement méliorés et l proution e lignées pures pr régénértion e plnts hploïes oublés embryons gmétiques. L objetif e e trvil étit méliorer l roissne, l mturtion et l régénértion es plntules es embryons e hêne-liège (Querus suber L.) es eux origines gmétique et somtique. Le hrbon tif fvorisé une ugmenttion signifitive e l roissne en termes e tille et e pois es embryons hploïes oublés et somtiques, insi qu un ontrôle plus effie e l embryogenèse somtique seonire u ours u éveloppement. Une intertion signifitive églement été observée ve l nutrition minoie. Alors que ertins ies minés ( est-à-ire, l glutmine, l rginine ou l sprgine) ne montrent ps e ifférenes signifitives ve les témoins, un mélnge e es trois ies minés ou l ie butyrique mino gmm stimulé l roissne e l embryon. Le tux e survie le plus élevé u ours e l limttion es plntules origine à l fois somtique et hploïe oublé, été obtenu lorsque les embryons ont été uprvnt ultivés sur milieu e bse à 3 % e shrose et e 1 % e hrbon tif pennt eux mois à 4 C et ont germé sur un milieu omplété pr l 6-benzylminopurine et l ie inole-3-butyrique. Nous vons obtenu plus e 9 plntules e hênes-lièges limtés issus e plusieurs lignes embryogéniques, ve un tux e survie élevé, e qui émontre que ette méthoe est pplible pour l proution à grne éhelle e plntules. Nous nnonçons églement l première régénértion e plntules hploïes oublés e hêne-liège. * Corresponing uthor: josentonio.mnzner@upm.es Artile publishe by EDP Sienes

2 Ann. For. Si. 67 (21) 25 B. Pintos et l. 1. INTRODUCTION Cork ok (Querus suber L.) is forest speies of high eologil vlue in mny Meiterrnen ountries. In ition, the ork is rw mteril of eonomi vlue for the wine inustry n mny other prouts ontributing to the rurl evelopment of the res where this tree is ntive. Nevertheless, its long life spn n irregulr see rops, the iffiulty of see onservtion n of vegettive reproution re serious rwbks for the use of the lssil geneti improvement progrms in this speies. Somti embryogenesis hs been teste for lrgesle plnt propgtion of rnge of tree speies (Bueno et l., 1992;Hernnezet l.,23; Lelu-Wlter n Pâques, 29; Mnzner et l., 1993; Prk,22; Sutton, 22). Nevertheless, embryo ormny n immturity problems neee to be overome. Osmotium (high surose onentrtion) n hilling tretments h been teste in orer to promote germintion of ork ok somti embryos (Fernánez-Guijrro et l., 1995; Grí-Mrtín et l., 21) n ytokinin pplition inrese pex elongtion of germinting ork ok somti embryos (González-Benito et l., 22; Hernánez et l., 23b). Aifferent pproh is bse on the proution of pure lines through ouble-hploi plnt regenertion from gmeti embryos inue in nther ulture (Bueno et l., 1997). In wooy ngiosperms, gmeti embryogenesis hs been inue either by nther ulture, mirospore ulture or prthenogenesis in bout 8 speies, e.g., Mlus omesti (Höfer, 24), Populus spp. (Blursson et l., 1993; Deutsh et l., 24), et. By this pproh, the umbersome metho of repete bkrossings is hieve in one step. Problems of ientifition of the ellulr origin n genotypi omposition of nther-erive embryos were overome through the use of moleulr mrkers, i.e., enzymes n rnom mplifie polymorphi DNA (RAPD; Bueno et l., 2) nsimple sequene repets (SSR; Gomez et l., 21), by ploiy level stuies through flow ytometry (Bueno et l., 23; Loureiro et l., 25), n by ytologil nlysis of erly mirospore ivisions (Pintos et l., 25). All these methos orroborte the gmeti origin n the geneti vribility of the nther embryosfrom ifferent hploi ells (mirospores). Finlly, ork ok hploi embryos were trete with ntimitoti gents in orer to obtin ouble-hploi plntlets (Pintos et l., 27). Little ttention hs been evote to lte mturtion n germintion. During mturtion, ell expnsion n storge substne umultion tkes ple. The orret storge of reserve substnes is ruil for fuelling the germintion proess (Merkle et l., 1995). Embryo weight inrese is n initor of embryo qulity n pre-requisite for suessful germintion (Grí-Mrtin et l., 25). Our objetive ws to evelop n effiient metho for plntlet regenertion of ork ok (Querus suber L.) either from somti or nther-erive hploi embryos. In the present rtile, we stuie the effet of substnes suh s tivte hrol n mino is on the growth of ork ok embryos, n the role of plnt growth regultors in the stimultion of germintion. The most suessful results obtine with somti embryos were then suessfully pplie to ouble-hploi embryos from nther origin. 2. MATERIAL AND METHODS 2.1. Plnt mteril Brnhes bering tkins were ollete every week in My, uring the flowering perio of Querus suber L., from 1 selete trees, five of them lote in Extremur (Western Spin) n five in Mri. Immture orns were ollete every two weeks, from the sme selete trees, uring the perio of fruit evelopment, from lte July to the beginning of September. Immture orns n tkins, between.5 n 1 m in length, were ollete n surfe-sterilize by immersion in 7% (v/v) ethnol for 3 s n in 2% (v/v) soium hypohlorite (35 g L 1 tive hlorine) with rop of Tween 2 for 2 min followe by three rinses in sterile istille wter of 1 min eh. Immture zygoti embryos n nthers were isolte, issete n ulture uner septi onitions Culture mei Bsl ulture meium for both somti n nther erive orkok embryos ontine full mronutrients (Sommer et l., 1975, meium 1), mirominerls n oftors (Murshige n Skoog 1962), 3% (w/v) surose n ws soliifie with.8% (w/v) gr (Bueno et l., 1992). This bsl meium will be esignte SM1X throughout the text. Meium ontining ouble onentrtion of both mronutrients (Sommer et l., 1975) n mirominerls n oftors (Murshige n Skoog, 1962), 15% (w/v) surose n soliifie with.8% (w/v) gr, will be esignte SM2X throughout the text. All mei were juste to ph = 5.6 fter ition of gr n before utolving t 1 tm (12 C) for 2 min. In ll experiments, plnt growth regultors n tivte hrol re e to the meium before utolving. Amino is re e to the meium fter utolving by filter-steriliztion (.22 μ). Meium ws ispense in 9 m imeter sterile Petri ishes (25 ml per ish) Somti embryo inution from zygoti embryos Cork ok immture zygoti embryos were ulture on SM1X meium supplemente with.5 mgl 1, 2,4-ihlorophenoxyeti i (2,4-D) (inution meium) for one month. Then embryos were trnsferre to SM1X meium supplemente with 5 mg L 1 glutmine (Gln) without 2,4-D Hploi embryo inution from nther ulture Cork ok nthers bering mirospores t the uninulete stge were pretrete by hilling t 4 C for mximum of 7 n then were ulture on SM1X supplemente with 1% (w/v) tivte hrol. Anther ultures were subjete to het shok tretment t 33 C for 5 in the rk (Bueno et l., 1997). One month lter, hploi embryogeni msses emerge from insie the nthers n were subjete to ntimitoti tretments Double-hplois obtine by ntimitoti tretments Oryzlin (Duhef r )1μM in 1% i-methyl-sulfoxie (DMSO) or miprophos methyl (Fluk r )1μM in 3% etone were use in 25p2

3 Ok somti n ouble-hploi embryo germintion Ann. For. Si. 67 (21) 25 vitro to inue hromosome oubling. All the solutions were sterilize by ultrfiltrtion (.22 μ). Hploi embryogeni msses emerging from insie the nthers n ontining initil trnsluent globulr embryos were immerse into these ntimitoti solutions for 48 h in the rk t 24 ± 1 C. Then, they were subulture on SM1X meium supplemente with 5 mg L 1 Gln. Double-hploi inution rte ws evlute two months lter (Pintos et l., 27) Effet of mino is n tivte hrol on embryo growth Immture trnsluent embryos t the otyleonr stge, bout 3 4 mm in size n pproximte fresh weight of bout 3 mg, either from somti origin or from nther-erive ouble-hplois were ulture in 9 m imeter Petri ishes ontining 25 ml SM1X meium (ontrol) or supplemente with ifferent mino is: Gln, rginine (Arg), sprgine (Asn), gmm-mino butyri i (GABA) or mixture of the three mino is Gln, Arg n Asn (3.5 mm eh). In the se of somti embryos, ll mino i tretments were either ombine with or without 1% tivte hrol. Double-hploi embryos from six ifferent prent trees were subjete to the sme mino i tretments n the ontrol, but only in ombintion with 1% tivte hrol. Then, both ultures (i.e. from somti or nther-erive origins) were subjete to temperture of 25 ± 1 C in rkness. For utomte sizing of embryos, the projete re of eh embryo ws mesure with n imge nlyzer (Imge Tool R ). Prior to their estblishment in the ifferent mino i n tivte hrol tretments, eh embryo ws weighe in septi onitions n size using n utomte proeure to pture the projete re of eh embryo (Imge Tool R ). One month lter, both prmeters were mesure n orreltions fitte with the sttistil pkge SPSS 1.. (SPSS, 1999). Embryo mturtion ws ssesse by two inies, the reltive inrement in projete re (RAI), n the reltive weight inrement (RWI): RAI = (A f A i ) / A i (1) where A i is the embryo initil projete re n A f is the finl projete re. RWI = (W f W i ) / W i, (2) where W i is the initil weight n W f is the finl weight. For this experiment, 2 somti embryos were ulture in eh of the 12 tretments (the ontrol n five mino i tretments, with or without tivte hrol). The ifferent embryo lines were homogeneously istribute mong tretments. The experiment ws repete three times, thus totl of 72 embryos ws use. In the se of ouble-hplois, lso 2 embryos were ulture in eh of the ontrol n five mino i tretments, n the experiment ws repete three times, thus totl of 36 embryos were use Effet of the ombintion of ifferent plnt growth regultors on the germintion of somti n ouble-hploi embryos Cork ok embryos either from somti or ouble-hploi origin, were ulture on SM1X meium supplemente with 1% tivte hrol for one month. When they rehe pproximtely 1.5 to 2 m in length n 7 1 mg fresh weight, they were trnsferre to SM2X meium supplemente with 1% tivte hrol nstoret4 C in rkness for two months strtifition. Then they were imbibe in sterile istille wter, insie 1 ml tubes for 24ht4 C in rkness. Germintion of somti embryos ws inue in SM1X meium supplemente with ifferent ombintions of 6-benzylminopurine (BAP;.5,.1,.25 n.5 mgl 1 ) with inole-3-butyri i (IBA,.1 mgl 1 ), nphthleneeti i (NAA,.1 mgl 1 ), or gibberelli i (GA 3,.1 mgl 1 ) n ontrol tretment without plnt growth regultors. The tretments to inue germintion of ouble-hploi embryos were ombintions of BAP (.5,.1,.25 mg L 1 ) with IBA (.1, 1 mg L 1 ) n ontrol tretment without plnt growth regultors. In both somti n ouble-hploi embryos, 5 iniviuls were ulture per tretment uner 16-h photoperio (1 μmol m 2 s 1 ) n 25 ± 1 C temperture, n the experiment ws repete three times Effet of the onentrtion of the strtifition meium on the germintion of somti n ouble-hploi embryos Cork ok embryos either from somti or ouble-hploi origin were mture on SM1X meium supplemente with 1% tivte hrol for one month. When they rehe pproximtely 1.5 to 2 m in length n 7 1 mg fresh weight, they were trnsferre to two ifferent strtifition mei: either SMIX or SM2X meium supplemente with 1% tivte hrol n store t 4 Cinrkness for two months. Then they were imbibe in sterile istille wter, insie 1 ml tubes for 24 h t 4 C in rkness. Germintion of somti embryos ws inue in SM1X meium with the surose onentrtion erese to 1.5% (w/v), the gr onentrtion inrese to 1% (w/v), n supplemente with.5 mg L 1 BAP n.1 mgl 1 IBA, uner 16-h photoperio (1 μmol m 2 s 1 ) n 25 ± 1 C temperture. For the experiment with somti embryos, totl of somti embryos were ulture on SM2X meium n 2 38 somti embryos were ulture on SM1X meium. In the se of ouble-hploi embryos, two hunre embryos were ulture per tretment n the experiment ws repete three times Alimtion of ork ok plntlets Plntlets proue uring the two previous germintion experiments were extrte from the germintion test tubes tking re not to mge the roots. Agr ws wshe with tp wter n the plntlets were trnsferre to 1 ml nursery pots fille with pet: perlite: vermiulite (1:1:1). Then they were subjete to preventive tretment with PREVICUR R fungiie (Propmorb 1.8 gl 1 ). The reltive humiity of the limtion room ws lose to 1% using fog system. Photoperio ws set to 16 h light (1 μmol m 2 s 1 )n8h rkness n temperture to 25±1 C. There ws grul reution in reltive humiity uring the next ys. After two or three weeks, the plntlets were trnsferre to the greenhouse. In the seon yer of nursery, the plntlets were trnsferre to 2 L pots with pet: vermiulite (3:1). The survivl rte ws lulte s the rtio between the number of survive plntlets one yer fter trnsfer to the 2 L pots n the totl mount of plntlets proue in the lbortory n trnsferre to 1 ml nursery pots. 25p3

4 Ann. For. Si. 67 (21) 25 B. Pintos et l Sttistis The effets of tivte hrol n mino is on embryo growth were ompre by nlysis of vrine (Generl Liner Moel of the SPSS 1.. sttistil pkge; SPSS, 1999). Mens were ompre with Dunn s multiple rnge test t the 5% signifine level. For sttistil lultions of the germintion (i.e., elongtion of both embryo root n shoot), survivl n limtion rtes of the ifferent experiments, expresse s perentges, log-liner moel ws fitte using Chi-squre test. Asymptoti stnr errors of the prmeter estimtes were ompute by the Delt metho (Lee, 1977). The rtio of the log-liner prmeter estimte to its stnr error ws use to obtin the frequeny signifine level. RAI (month -1 ) b f e f f f A 3. RESULTS 3.1. Effet of mino is n tivte hrol on somti embryo growth Forty one somti embryo lines were obtine from the ten selete trees. The results obtine in reltive size inrese re shown in Figure 1A. Signifint ifferenes in growth were ue to the presene of tivte hrol in the ulture meium. Reltive growth in projete re of the embryos ulture in tivte hrol without mino is verge 871% per month. In ontrst, the growth of embryos subjete to the ontrol tretment (no mino is n no tivte hrol) ws only 167% per month. The ition of mixture of three mino is (Gln, Arg n Asn) or GABA proue signifint inrese of embryo size s ompre to the ontrol without tivte hrol. For instne, the ombintion of the three mino is without tivte hrol promote 57% inrement per month, while when this tretment ws ombine with the presene of tivte hrol, growth inrese to 733% per month. Similr results were obtine for fresh weight reltive growth (Fig. 1B). Growth in weight ws signifintly higher in embryos ulture on meium ontining tivte hrol (1736% per month), where embryos rehe size of 1.3 m in length n n pproximte fresh weight of.7 g (Fig. 2,A1 n A2), thn in the ontrol (55% per month), where finl fresh weight ws bout 25 mg (Fig. 2, B1nB2).Also,in the tretment with mixture of three mino is (Gln, Arg n Asp) without tivte hrol, the fresh weight inrement (1 598% per month) ws signifintly higher thn the ontrol. GABA, Gln n Asn tretments lso inue signifintly higher RWI thn the ontrol (Fig. 1B). Amino is promote embryo growth in the bsene of tivte hrol but none of the mino i tretments improve embryo growth over the no-mino i ontrol in the presene of tivte hrol (Fig. 1B). Furthermore, we observe tht in the presene of tivte hrol n without mino is, ork ok somti embryos i not show repetitive embryogenesis, while in the bsene of tivte hrol, repetitive embryogenesis ppere t the bse of mny embryos (t not shown). RWI (month -1 ) Ativte hrol Ativte hrol Without tivte hrol Without tivte hrol TREATMENTS Control Gln+Arg+Asn GABA Gln Arg Asn Figure 1. Effet of mino is glutmine (Gln), rginine (Arg), sprgine (Asn), γ-mino-butyri i (GABA), 3.5 mm eh, mixture of the three mino is Gln, Arg n Asn (3.5 mm eh) n the ontrol with or without 1% tivte hrol on ork ok somti embryo growth per month. (A) Reltive size inrement in projete re per month (RAI, month 1 ). (B) Reltive fresh weight inrement per month (RWI, month 1 ). Tretments with ifferent letters re sttistilly signifint t the 5% level Effet of mino is on ouble-hploi embryo growth Results on the reltive projete re inrement re shown in Figure 3A. No signifint ifferenes were foun mong tretments. In generl, ouble-hploi embryo growth is slightly lower thn tht of somti embryos. For instne, the ontrol meium n tht of Arg inue 734 n 723% b B 25p4

5 Ok somti n ouble-hploi embryo germintion Ann. For. Si. 67 (21) 25 RAI (month -1 ) 1 9 A Figure 2. Growth of ork ok somti embryos. (A1) Initil size of somti embryo ulture on meium with tivte hrol (+AC), one month fter embryogenesis inution. (A2) Finl spet of somti embryo ulture for one month on meium with tivte hrol (+AC). (B1) Initilsizeofsomtiembryo ulture onmeium without tivte hrol ( AC), one month fter embryogenesis inution. (B2) Finl spet of somti embryo ulture for one month on meium without tivte hrol ( AC). RWI (month -1 ) B per month, respetively, in ouble-hploi embryos, while in somti embryos, RAI rise to 871 n 826% in the sme tretments, respetively (Fig. 1A). Agin, fresh weight reltive inrement ws not signifint mong tretments (Fig. 3B). In this se, mei supplemente either with Gln or Arg or the ontrol showe similr fresh weight inrement, i.e., 1 424, n 1 45% per month, respetively. Vlues re slightly less thn in the se of somti embryos (bout 16%, Fig. 1B) Effet of the ombintion of ifferent plnt growth regultors on the germintion of somti n ouble-hploi embryos The tretment with BAP (between.25 n.5 mg L 1 ) n IBA (.1mgL 1 ) inue signifintly higher germintion rte (from 38.3 to 43.3%) thn those of BAP + NAA (25 32%) or the ontrol without PGRs (Fig. 4). The ombintion of.1 mgl 1 GA 3 with BAP use 7% embryo nerosis n ws exlue from the nlysis. The higher BAP onentrtion (.5 mgl 1 ) ombine with IBA reue root growth n stimulte shoot prolifertion. Double-hploi embryos trete with.5 mg L 1 BAP n.1mgl 1 IBA germinte t signifintly higher rte, 18.7%, thn the other tretments (Tb. I) TREATMENTS Control Gln+Arg+Asn GABA Gln Arg Asn Figure 3. Effet of mino is glutmine (Gln), rginine (Arg), sprgine (Asn) n γ-mino-butyri i (GABA), 3.5 mm eh, n the ontrol with 1% tivte hrol on ork ok gmeti embryo growth per month. (A) RAI: reltive size inrement in projete re per month (month 1 ). (B) RWI: reltive fresh weight inrement per month (month 1 ) Effet of the onentrtion of the strtifition meium on the germintion of somti n ouble-hploi embryos Somti embryos strtifie on SM1X meium supplemente with 1% tivte hrol n store t 4 Cin rkness for two months showe the highest germintion rte (57.2%) in omprison with those strtifie on SM2X meium supplemente with 1% tivte hrol (Fig. 5). In the se 25p5

6 Ann. For. Si. 67 (21) 25 B. Pintos et l. Germintion (%) * * * BAP (mg L -1 ) * IBA.1 mg L -1 NAA.1 mg L -1 Figure 4. Germintion rte (%) of ork ok somti embryos trete with 6-benzyl-minopurine (BAP,,.5,.1,.25 n.5 mgl 1 ) ombine with IBA (.1 mgl 1 ), NAA (.1 mgl 1 ) or without uxin (ontrol). Tretments mrke with n sterisk (*) were sttistilly signifint t the 5% level. Tble I. Germintion rte (%) of ork ok ouble-hploi embryos trete with 6-benzyl-enine (BAP,,.5,.1 n.25 mg L 1 ) ombine with IBA (.1, 1 mg L 1 ) or without uxin (ontrol). Tretments mrke with n sterisk (*) were sttistilly signifint t the.5 level. Tretment Germintion (%).25 mg L 1 BAP +.1mgL 1 IBA mgL 1 BAP +.1 mgl 1 IBA 15.5 mg L 1 BAP +.1mgL 1 IBA 18.7*.1mgL 1 BAP + 1mgL 1 IBA 9.7.1mgL 1 BAP 11 Control (no PGRs) 6.3 GERMINATION RATE (%) b of ouble-hploi embryos, the germintion rte ws very low ( %) n no signifint ifferenes were observe between both meium onentrtions Alimtion of ork ok plntlets The survivl rte of limte plntlets from both somti n ouble-hploi embryos whih were strtifie on SM1X meium with 1% tivte hrol for two months t 4 C ws signifintly higher thn the survivl rte of plntlets oming from embryos strtifie on SM2X meium (Fig. 6). Differenes lso were observe between the survivl rte of plntlets from somti n ouble-hploi origin, whih were 77.96% n 13.96% respetively in the best strtifition tretment (SM1X meium). A totl of 99 limte plntlets from somti embryos n 14 from ouble-hploi embryos (Fig. 7), previously ulture in SM1X supplemente with 1% tivte hrol for one month t 25 C n strtifie for two months t 4 C, were obtine. When the SM2X meium ws use Somti embryos SM2X + 1%AC Double-hploi embryos SM1X + 1%AC Figure 5. Germintion rte (%) of ork ok somti n oublehploi embryos previously ulture on strtifition meium: SM2X+ 1% tivte hrol (AC); SM1X + 1% tivte hrol (AC). Tretments with ifferent letters re sttistilly signifint t the 5% level. uring strtifition, 685 plntlets from somti n five from ouble-hploi embryos were obtine. 4. DISCUSSION 4.1. Effet of mino is n tivte hrol on embryo growth Embryo growth is ritil step for the evelopment in vitro n germintion of somti embryos (Lelu n Lbel, 1994; Muri n Mnzner, 23). During mturtion, ell 25p6

7 Ok somti n ouble-hploi embryo germintion Ann. For. Si. 67 (21) 25 Alimte plntlets (%) b Somti embryos SM2X + 1% AC Double-hploi embryos SM1X + 1% AC Figure 6. Alimtion rte (%) of ork ok plntlets from either somti or ouble-hploi embryos previously ulture on strtifition meium: SM2X + 1% tivte hrol (AC); SM1X + 1% tivte hrol (AC). Tretments with ifferent letters re sttistilly signifint t the 5% level. Figure 7. Double-hploi plntlets limte in the greenhouse. expnsion n umultion of reserves tke ple (Merkle et l., 1995), whih implies both size n weight inrement. In ork ok, this hs been signifintly promote by the effet of tivte hrol e to the ulture meium. Nevertheless, in some ses growth inhibition hs lso been reporte (Friborg n Eriksson, 1975; Friborg et l., 1978), lso with embryos from nther origin (Johnsson, 1983). The positive effet of 1% tivte hrol on stopping seonry somti embryogenesis n promoting somti embryo growth n lter germintion hs been observe in other wooy speies, suh s Txus brevifoli (Chee, 1996) nlrix mrshlinsii (Von Aerks et l., 22).Grolletl.(22) lsoshowe the benefiil effet of.5% tivte hrol on Mnihot esulent somti embryos. Our results obtine with the growth of ork ok somti embryos were pplie to ouble-hploi embryos from nther origin. The positive effet of tivte hrol on reltive growth ws ssume n the oublehploi embryos were subjete to the mino i tretments, mintining the presene of 1% tivte hrol in the ulture meium. In vitro explnts exue inhibitory phenoli ompouns to the ulture meium tht my be sorbe by tivte hrol (Friborg et l., 1978), n its rkening effet my ttenute the tivity of phenol oxiising enzymes (Linington, 1991). Also, tivte hrol bsorbs 5-hyroxi-methyl-2- furlehie (HMF), seonry prout of surose utolving whih reues plnt growth (Ro n Pttbirmn, 199). Wetherhe et l. (1978) emonstrte tht.3% tivte hrol bsorbs HMF n stimultes explnt growth n inues 2 to 3% inrement in plntlet number. Another hypothesis for the benefiil effet of tivte hrol is the bsorption of uxins liberte by the embryos to the meium uring their evelopment, whih my interfere with embryo morphology n germintion (Merkle et l., 1995). In Querus suber L., we observe signifint inrese of both embryo size n fresh weight ue to tivte hrol, s well s better ontrol of repetitive embryogenesis in mture embryos. This phenomenon, lle utoembryony, is frequent in speies suh s ork ok, n is unesirble uring mturtion n lter stges. This problem hs been etete in relte Querus speies (Chlup, 199). The ition of bsisi i (ABA) to the mturtion meium prtilly solves tht problem, like in Q. ilex L. (Muri n Mnzner, 24) ninq. suber L. (Grí-Mrtin et l., 25). Amino is promote signifint fresh weight inrement, exept for Arg. In greement with this results, Lelu-Wlter n Pâques (29) use 1.45gL 1 Gln for hybri lrh somti embryogenesis, n Gleeson et l. (24) foun tht proline stimulte growth of embryogeni ultures of lrh (Lrix leptoeurope Dengler), sitk sprue (Pie sithensis (Bong.) Crr.) n ok (Querus robur L.) in stress onitions. Furthermore, the ombintion of mino is n tivte hrol gve the best results, similrly to those obtine in Cuumis stivus L. with Gln, Arg, Asn, glyine n isteine 1 mm together (Ashok n Murthy, 24) Germintion of somti n ouble-hploi embryos We hve observe in our experiments tht the ork ok somti embryos previously strtifie in meium with higher minerl nutrients n surose onentrtions (SM2X) germinte t lower rte thn those previously strtifie in meium with the lower surose onentrtion (SM1X). Both somti n ouble-hploi embryos previously ulture in the lower 25p7

8 Ann. For. Si. 67 (21) 25 B. Pintos et l. onentrtion meium (SM1X) were more suessful in the limtion phse thn those embryos oming from the SM2X meium. Our results hve shown tht ytokinin ombine with uxins improve oorinte germintion in ork ok, onfirming previous reserh (González-Benito et l., 22). Germnà n Chinone (23) use1mgl 1 GA 3 n.1 mg L 1 NAA for Citrus lementin Hort., obtining 96% germintion. Blursson et l. (1993) obtine ventitious shoots from nther embryos of Populus trihorp with 2.5 μm BAPn.5 μm NAA n roote them with.25 μm IBA. In ontrst, Deutsh et l. (24) germinte embryos obtine from isolte mirospores of Populus nigr previously ulture on meiumwith1mgl 1 BAP n.2 mg L 1 NAA Alimtion of ork ok plntlets There re few reports on the effetive limtion of plntlets from somti or gmeti embryogenesis in the genus Querus. Gings n Lineberger (1989) only limte three plntlets of Q. rubr, whih ie six months lter. Ishii et l. (1999) trnsferre 1 plntlets from somti embryos of Q. serrt, eight of whih survive. Chlup (199) obtine 78% survivl from 7 Q. robur n 62 from Q. petre plntlets. Kim et l. (1997) proue33plntletsofq. utissim, 2 of whih were trnsplnte into pots, 12 survive in the greenhouse n eight in the fiel. Hernánez et l. (23b) trnsferre to ex vitro onitions totl of 73 plntlets of Q. suber from lef explnts, but six months lter only 33 survive in the fiel. Deutsh et l. (24) obtine plntlet regenertion perentges lower thn 1.2% in Populus nigr mirospore ultures. Germnà n Chinone (23) estblishe 54 oublehploi plntlets of Citrus lementin Hort. from nther ultures. Our results of 99 ork ok plntlets limte from multiple-line somti embryogenesis, with high survivl rte, show tht this methoology is pplible for lrge sle plntlet proution. Nevertheless, ouble-hploi plntlet regenertion is still hllenging tsk, possibly beuse the geneti lo tht is expresse in the ouble-hploi mteril mke them weker thn heterozygous plntlets from somti origin. Aknowlegements: This work ws supporte by projet RTA C2-2. Dr Pintos ws reipient of PhD grnt of the Ntionl Institute of Agronomi Reserh (INIA). REFERENCES Ashok K.H.G. n Murthy H.N., 24. Effets of sugrs n mino is on nrogenesis of Cuumis stivus L. Plnt Cell Tissue Orgn Cult. 78, Blursson S., Nørgr J.V., Krogstrup P., n Anersen S.B., Mirospore embryogenesis in nther ulture of three speies of Populus, n regenertion of ihploi plnts of Populus trihorp. Cn. J. For. Res. 23: Bueno M.A., Astorg. R., n Mnzner J.A., Plnt regenertion through somti embryogenesis in Querus suber. Physiol. Plnt. 85: Bueno M.A., Gómez A., Bosiu M., Mnzner J.A., n Viente O., Stress inue formtion of hploi plnts trough nther ulture in ork-ok (Querus suber L.). Physiol. 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