Interaction between sucrose and ph during in vitro culture of Nephrolepis biserrata (Sw.) Schott (Pteridophyta)

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1 At ot. rs. (): 9-3. Intertion etween surose nd ph during in vitro ulture of Nephrolepis iserrt (Sw.) Shott (Pteridophyt) Sndr Terez Amrósio nd Ntoniel Frnklin de Melo, Reeived: April 5, 3. Aepted: April, RESUMO (Interção d srose e níveis de ph durnte o ultivo in vitro de Nephrolepis iserrt (Sw.) Shott (Pteridophyt)). O presente trlho relt o efeito de diferentes vlores de ph e onentrções de srose n propgção in vitro de Nephrolepis iserrt. Frondes otids sseptimente prtir do ultivo de estolões form inoulds em meio semi-sólido MS, suplementdo om, 5, 3, 5 e g.l - de srose e ph justdo pr 3, 5, 7 e 9, em rrnjo ftoril. Form vlidos: número e omprimento ds frondes, número de pins, omprimento e diâmetro d rque e iomss fres e se d prte ére. Em gerl, houve iniição d regenerção de rotos e folhs em todos os trtmentos de ph n usêni de srose. Por outro ldo, presenç de srose no meio induziu 5,5 e 3,5 rotos por explnte no ph 5 e 7, respetivmente. Conentrções entre 5 e 5g.L - de srose umentrm o omprimento e o diâmetro ds folhs e o ph 9 não fetou o peso folir seo, sendo indequdo pr o desenvolvimento de novs frondes. Os esporófitos jovens form sumetidos, om suesso, o proesso de limtção. Plvrs-hve: pteridófits, plnts ornmentis, ultur de teidos ABSTRACT (Intertion etween surose nd ph during in vitro ulture of Nephrolepis iserrt (Sw.) Shott (Pteridophyt)). The present work reports the effet of different ph nd surose onentrtions on in vitro propgtion of Nephrolepis iserrt. Fronds septilly otined from stolon segment ulture were ultivted in MS semi-solid medium supplemented with, 5, 3, 5 nd g.l - surose nd ph djusted to 3, 5, 7 nd 9, in ftoril design. Frond numer nd length, pinne numer, rquis length nd dimeter, fresh nd dry mtter weight were mesured. Inhiition of shoot nd lef regenertion ws oserved in ll the ph tretments in the sene of surose. On the other hnd, when surose ws dded to the medium, the shoot numer inresed, rehing the mximum verge vlues of 5.5 nd 3.5 shoot per explnt t ph 5 nd 7, respetively. Surose onentrtions from 5 to 5g.L - inresed lef length nd dimeter nd the ph 9 did not ffet the dry mtter weight, nd ws still not dequte for development of new fronds. Young sporophytes were suessfully limted. Key words: ferns, ornmentl plnts, tissue ulture Introdution In the sporophyti phse, pteridophytes ommonly present lrge nd omplex fronds, with elorte leves, whih, esides filitting spore dispersion nd supporting sexul reprodution for the spore presene, lso provide exellent ornmentl qulities (Jones 97). Therefore, the pteridophytes fronds re known worldwide due to their ornmentl vlue, nd severl studies hve een onduted for optimizing in vitro onditions, not only to ontriute for the propgtion of ornmentl speies in ommeril sle, ut lso to understnd the speies iology (Fernández et l. 999). Among the pteridophytes speies used s ornmentl foliges in ommeril nd residentil environments, we n find those of the genus Nephrolepis (Morton 95; Zimmermn & Jones 99). It is highly distintive genus hrterized y undnt stolons, disrtiulte, inequilterl nd usully entire pinne, hydthodes in the upper surfe of the pinne nd spores with reltive thin nd tuerulte-rugose perispore (Tryon & Tryon 9). Severl diffiulties imposed y speifiity of fern life yle interfere on in vitro ultivtion protool estlishment (Morini ). Aording to Stokes (9), in vitro propgtion of pteridophytes speies n require prolonged periods of time, whih sometimes might extend to severl yers, so tht the frond nd plnt size n reh the uniformity demnded y the mrket of ornmentl plnts. During in vitro regenertion of ferns speies, some ftors ffet the multiplition effiieny nd mny spets hve een exmined, suh s explnt donor soure (Atmne et l. ; Whittier & Brggins ), nutrient type (Pdhy Emrp Semi-Árido, C. Postl 3, CEP 53-97, Petrolin, PE, Brsil Corresponding Author: ntoniel@pts.emrp.r

2 Amrósio & Melo: Intertion etween surose nd ph during in vitro ulture of Nephrolepis iserrt (Sw.) Shott... & Meht 9; Fernández et l. 999) nd physil ulture onditions (Lin & Yng 999). Sporophyte frgments hve een used s explnts soure, whih re usully the most prtil nd vile form of miropropgtion of ornmentl speies in ommeril sle (Bertrnd et l. 999). Sine the 97 s, in vitro multiplition protools of Nephrolepis speies hve een reported. Loesher & Alreht (979) oserved the effet of environmentl, nutritionl nd hormonl onditions on in vitro orgnogenesis of N. exltt, vr. ostoniensis, using juvenile segments of stolon. Lê (93) reported the influene of surose onentrtions on sporophyte propgtion of N. exltt (L.) Shott using gmetophyti tissue s explnt soure, while Psqul et l. (99) propgted the sme fern y shoots, in order to study in vitro multiplition under the effet of surose nd minerl slt ssoition. Higuhi et l. (97) desried the effet of growth regultors nd surose on histologil differentition nd fresh mtter weight of N. ordifoli Prsel. The ssoition of these vriles with ph is importnt euse it ould influene the multiplition effiieny, estlishment nd qulity of the propgted sporophytes (Hndrek 99; Koedm et l. 99; Pevlek-Kozlin 99). In the present work four ph tretments nd five levels of surose onentrtions were studied, in order to evluted in vitro indution, regenertion nd estlishment of N. iserrt sporophytes. Mteril nd methods The mteril studied ws otined from ultivted plnts nd identified s Nephrolepis iserrt (Sw.) Shott (Dvlliee/Filiopsid) (Tryon & Tryon 9). The plnts were mintined in the Experimentl Glsshouse of Emrp Semi-Árido, Petrolin-PE, for snitry tretment. Initilly, young sporophytes ultivted in glsshouse, in vses with sustrte (orgni mtter nd vermiulite :), were treted with solution ontining Benomyl (.5%), Nuvron (mg.l - ) nd Extrvon (mg.l - ) solution one week. Stolon segments were olleted, wshed in wter ontining -3 drops of ommeril detergent, nd re-wshed in running distilled wter. The disinfetion proess onsisted of immersion in 7% lohol (v/v) for - minutes, followed y minutes of immersion in % sodium hypohloride solution nd rinsed in sterile distilled wter. A hundred stolon segments were ut into piees of m length, pled in MS semi-solid medium (Murshige & Skoog 9) supplemented with 3g.L - surose, with ph djusted to 5.9. Juvenile sporophytes were otined fter 9 dys. Single shoots initited from juvenile sporophytes were used s explnts for the experimentl ssys, s previously desried (Byrne & Cponetti 99). Under sterile ondition, explnts were trnsferred to MS semi-solid medium supplemented with ftoril omintions of surose t, 5, 3, 5 nd g.l - nd ph djusted to 3, 5, 7 nd 9. Eh tretment onsisted of four replites nd ws mintined in growth hmer t 5 ± ºC nd h light photoperiod. All medi were solidified with 7g.L - gr nd utolved t ºC, kg.m - pressure, for minutes. After utolving, the finl ph shifted downwrds round.. After 5 dys of ultivtion, frond nd pinne numers, frond length, rhis dimeter, nd fresh nd dry mtter weight of eril prts were evluted. The Dunn test (p.5) ws used for evluting the dt otined with different surose onentrtions nd ph levels fter ANOVA ssy (Sttsoft In 997). Plnts from in vitro ulture were trnsferred to plsti oxes (veg-ox), using sustrte omposed y vermiulite, snd nd orgni mtter in the rtio ::, previously sterilized. The limtiztion ws rried out in glsshouse t n verge temperture of 7±ºC, 5% reltive humidity nd 5-5µmol.m -.s - light intensity. Results nd disussion The sporophyte snitry stte ws onsidered ritil ftor for in vitro ultivtion. Explnts with endophyti miroorgnisms displyed growth nd differentition into primry leves, ut the development ws interrupted round the th dy fter inoultion. This kind of explnt ws not vile for estlishment or regenertion experiments. However, new shoots from helth explnts were otined to 5 dys fter inoultion. There ws % of shoot regenertion nd % showed oth nerosis nd ontmintion. Aording to Morini (), the min prolem for estlishment of in vitro propgtion protools in pteridophytes is the presene of miroorgnism in the plnts, hindering the disinfetion proess nd ffeting their survivl. Frond nd pinne numers nd fresh mtter weight were signifintly ffeted y the intertion

3 At ot. rs. (): 9-3. Tle. Effets of surose nd ph on frond numer, pinne numer nd fresh mtter weight of 5 dy-old Nephrolepis iserrt ultivted in vitro. ph Surose Frond Pinne Fresh weight (g.l - ) numer numer (g) Mens followed y the sme letter in the sme olumn do not differ t 5% level y the Dunn s test. - Vlues not oserved. etween surose onentrtion nd ph (Tle ). Regrding frond nd pinne numers, stisftory results were otined with the inrese of surose onentrtion in ll ph tretments. The inrese of surose onentrtion signifintly improved frond prodution. Mximum vlues of 5.5 nd 3.5 fronds per explnt were otined, with g.l - surose t ph 5 nd 7, respetively. Surose t 5g.L - nd ph 5 lso inresed the fresh mtter weight. On the ontrry, n inrese in surose onentrtion redued the frond indution in N. exltt (Psqul et l. 99). It ws lso reported tht surose onentrtions higher thn 5g.L - used derese in the formtion of new prothllis olonies in this speies (Lê 93). Length, dimeter nd dry mtter weight of eril prts were not signifintly ffeted y the intertion etween surose onentrtion nd ph. In this se, the Figure shows dt nlyzed seprtely, euse ph nd surose levels in ANOVA were signifint. For simpliity, however, we hve presented the grphs with levels pooled (Figure ). Responses were otined t surose onentrtion of 3 nd 5g.L - regrding frond length, rhis dimeter nd dry mtter weight. The sene or higher surose onentrtion (g.l - ) signifintly redued these prmeters. Dt suggested inhiition of plnt development t ph 9, in whih redution of frond length nd rhis dimeter ws lso oserved. Similr response ws oserved for dry mtter weight, lthough sttistil differenes were not oserved mong ph levels 3, 5 nd 9. Thus, pronouned growth responses were otined t ph 5 nd 7. This intense growth is possily due to inrese in ell division with susequent frond prodution, s reported y Pevlek-Kozlin (99) in Pltyerium ifurtum. In this speies, neutrl or id ph stimultes in vitro frond prodution. Under nturl onditions, ph lso ppers to hve seletive effet in the estlishment of mny ornmentl speies (Symonds et l. ). Some pteridophytes hve een reported to prefer lkline soils (Hndrek 99), wheres others re idiphilous (Koedm et l. 99) or yet generlists (Rnl 995). Under in vitro onditions, the medium ph my hve signifint effets on growth nd differentition of ells, lthough there re mny reports showing tht ells in ulture n signifintly lter the ph of the externl medium through differentil uptke of nutrients nd/or through H + fluxes (Minoh 97). Some ftors, suh s limited photosyntheti tivity due to in vitro onditions, my explin the signifint derese of regenertion rte under surose sene. However, high surose level in the ulture medium might result in hlorophyll degrdtion nd reflet on the frond development (Fernández et l. 999). Despite of in vitro plnt regenertion n e suessful without surose, its presene usully promotes signifint inrese of new regenerted strutures, sine in the indution phse, the ron soure umulted in the plnt tissue is enough to strt the development (Llonde et l. 999). Menwhile, during the development phse, lrge nd ville ron mounts re neessry for struturl growth, differentition nd development. In generl, surose in medium n serve s n osmoti gent nd s ron nd energy soure. Mny ultures, espeilly emryo nd some shoot ultures, require medium with high osmoti potentil (Tremly & Tremly 995). In the present study, ll in vitro regenerted plnts did not show ny oservle morphologil normlities. During limtiztion proess, the regenerted sporophytes produed new fronds quite well distriuted in the pex, nd show more intense green olor when ompred to the originl sporophyte soure. In onlusion, the results desried here provided effiient pprohes for in vitro multiplition of N. iserrt.

4 Amrósio & Melo: Intertion etween surose nd ph during in vitro ulture of Nephrolepis iserrt (Sw.) Shott... Frond length (mm) Frond length (mm) Rhis dimeter (mm).,.,.,.,,.,.,.,.,.,.,.,.,.,.,., Rhis dimeter (mm) Dry weight (mg),.,.,.,.,.,.,.,.,.,.,.,.,.,.,., Dry weight (mg) ph Surose (g.l - ) Figure. Influene of surose nd ph on frond length, rhis dimeter nd dry mtter weight of Nephrolepis iserrt plnts ultivted in vitro for 5 dys. The dt were nlyzed seprtely euse only ph nd surose levels in ANOVA were signifint. Sme letter indite no differene t 5% level y the Dunn's test.

5 At ot. rs. (): Aknowledgments The uthors re very thnkful to Mri Terez Atíde, for her tehnil ssistne; to Adrin Myumi Yno-Melo, for the sttistil nlyses nd help in the mnusript; to CNPq (Pro. 339/9-) nd Emrp Semi-Árido, for their finnil support. Referenes Atmne, N.; Blervq, N.; Mihux-Ferriere, N. & Vsseur, J.. Histologil nlysis of indiret somti emryogenesis in the Mrh Clumoss Lyopodiell inundt (L.) Holu (Pteridophytes). Plnt Siene 5: Bertrnd, A.M.; Aluerne, M.A.; Fernández, A.; González, A. & Sánhez-Tmés, R In vitro orgnogenesis of Polypodium mrium. Plnt Cell, Tissue nd Orgn Culture 57: 5-9. Byrne, T. & Cponetti, J.D. 99. Morphogenesis in three Cultivrs of Boston Fern II. Amerin Fern Journl (): -. Fernández, H.; Bertrnd, A.M. & Sánhez-Tmés, R Biologil nd nutritionl spets involved in fern multiplition. Plnt Cell, Tissue nd Orgn Culture 5: -. Hndrek, K.A. 99. Growth of ferns in soil-less medi, s ffeted y ph, iron nd lium/mgnesium rtio. Sienti Hortiulture 5(-): 5-. Higuhi, H.; Amki, W. & Suzuki, S. 97. In vitro propgtion of Nephrolepis ordifoli Prsel. Sienti Hortiulture 3: 5-3. Jones, O.L. 97. Enylopedi of ferns: An introdution to fern, their struture, iology, eonomi importne, ultivtion nd propgtion. British Museum, : -. Koedm, N. Busher, P. & Speyroek, D.V. 99. Aidiphily in pteridophytes: ssessment of the role of root tionexhnge properties. Journl of Plnt Nutrition 5(): 5-9. Llonde, S.; Boles, E.; Hellmnn, H.; Brker, L.; Ptrik, J.W.; Frommer, W.B. & Wrd, M.F The dul funtion of sugr rriers: trnsport nd sugr sensing. The Plnt Cell (): Lê, L. 93. Essi de multiplition de Nephrolepis exltt pr ulture in vitro de tissu gmétophytique. Revue Suisse de Vitiulture, Aroriulture et Hortiulture 5(3): 9-9. Lin, B. & Yng, W Blue light nd sisi id independently indue heterophyllous swith in Mrsile qudrifoli. Plnt Physiology 9: 9-3. Loesher, N.H. & Alreht, C.N Development in vitro of Nephrolepis exltt vr. ostoniensis runner tissues. Physiologi Plntrum 7(): 5-5. Minoh, S.C. 97. ph of the medium nd the growth nd metolism of ells in ulture. Pp. 5-. In: J.M. Bong & D.J. Durzn (eds.). Cell nd tissue ulture in forestry. Mrtinus Nijhoff Pulishers. Morini, S.. In vitro ulture of Osmund reglis fern. Journl of Hortiulturl Siene & Biotehnology 75(): 3-3. Morton, C.V. 95. Oservtions on ultivted ferns. V. The speies nd forms of Nephrolepis. Amerin Fern Journl : -7. Murshige, T. & Skoog, F. 9. A revised medium for rpid growth nd iossys with too tissue ultures. Physiologi Plntrum 5: Psqul, M.; Ishid J.S. & Hoshid, E. 99. Influêni de diferentes onentrções de srose e sis mineris sore multiplição in vitro de Nephrolepis exltt - um smmi ornmentl. Pesquis Agropeuári Brsileir 9(): -. Pdhy, M.A. & Meht, A.R. 9. Propgtion of fern (Nephrolepis) through tissue ulture. Plnt Cell Reports : -3. Pevlek-Kozlin, B. 99. Effets of surose nd gr onentrtion nd medium ph on stghorn fern [P. ifurtum (Chr.) C. Cv.] shoot multiplition. Hortiulturl Siene : -. Rnl, M.A Esteleimento de pteridófits em mt mesófil semideídu do estdo de São Pulo.. Nturez dos sustrtos. Revist Brsileir de Biologi 55(): Stokes, P. 9. Hrdy ferns. Interntionl Plnt Propgtors Comined Proeedings 3: Symonds, W.L.; Cmpell, L.C. & Clemens, J.. Response of ornmentl Eulyptus from idi nd lkline hitts to potting medium ph. Sienti Hortiulture : -3. Sttsoft In Sttisti for Windows. Computer Progrm Mnul. Tuls. Tremly, L. & Tremly F.M Mturtion of lk sprue somti emryos: Surose hydrolysis nd resulting osmoti pressure of the medium. Plnt Cell, Tissue nd Orgn Culture : 39-. Tryon, R.M. & Tryon, A.F. 9. Ferns nd llied plnts, with speil referene to tropil Ameri. Springer- Verlg. Whittier, D.P. & Brggins, J.E.. Oservtions on the mture gmetophyte of Phylloglossum (Lyopodiee). Amerin Journl of Botny 7: 9-9. Zimmermn, R.H & Jones J.B. 99 Commeril miropropgtion in North Ameri. In: P.C. Deergh & R.H. Zimmermn (eds.). Miropropgtion: Tehnology nd Applition. Kluwer Ad. Versão eletrôni do rtigo em