Romnin Biotehnologil Letters Vol. 15, No.2, Supplement, 2010 Copyright 2010 University of Buhrest Printed in Romni. All rights reserved ORIGINAL PAPER In vitro ulture medium nd explnt type effet on llogenesis nd shoot regenertion in two genotypes of ornmentl strwerry Astrt 12 Reeived for pulition, Deemer 20, 2009 Aepted, Ferury 5, 2010 ANCA NICOLETA SUTAN *, AUREL POPESCU *, VALENTINA ISAC ** * University of Pitesti, Fulty of Sienes, Deprtment of Biology nd Hortiulture, Pitesti, Romni ** Reserh Institute for Fruit Growing, Pitesti, Romni Corresponding uthor: An Niolet Sutn, University of Pitesti, Fulty of Sienes, Deprtment of Biology nd Hortiulture, Trgul din Vle, no. 1, 110040 Pitesti, Romni, tel.: 0040 348 453100, fx: 0040 348 453123, e-mil: nsutn@yhoo.om As some of the ornmentl vrieties of strwerry otined from Frgri x Potentill rosses re lking the ility to form runners, their in vitro propgtion is dependent on either diret or indiret orgnogenesis. The influene of ulture medium omposition nd explnt type were investigted in two genotypes of ornmentl strwerry, Pink Pnd nd Serent, respetively, in order to estlish n effiient protool for regenertion y indiret orgnogenesis. Aiming to good rte of llogenesis nd shoot regenertion, the effet of different omintions nd onentrtion of growth regultors (2,4-D, IBA, nd BAP) dded in ulture medi (either MS or LF) were evluted with lef nd petiole explnts. It ws found tht the highest frequeny of explnts forming llus hve een indued in oth vrieties investigted on the LF sl medium ontining 0.5 mg/l or 1.0 mg/l 2.4-D nd, respetively, 3.0 mg/l BAP. A mximum of 100% lef explnts, nd 92% petiole explnts formed lli hving hrteristis of those regenerting shoots in Serent vriety. Similrly, mximum of 92% petiole explnts formed llus in Pink Pnd intergeneri vriety. Key words: llus indution, genotype, explnt type, ulture medium, plnt growth regultors. Introdution Sexul omptiility of Potentill plustris with some Frgri speies (Niemirowiz-Szzytt, [13]; Syegh nd Hennerty, [17]), llowed the ourene of lrge rnge of Frgri x Potentill intergeneri hyrids, omining the ornmentl vlue given y the euty of their flowers nd prolonged lossoming seson (My - Otoer) with prodution of edile fruits. Beuse the geneti limittions ssoited with high heterozygosity nd polyploidy whih hmper the trditionl reeding methods, the lonl propgtion of intergeneri hyrids Frgri x Potentill provides n dvntge for the multiplition of these elite plnts without sexul reomintion. Cllogenesis nd regenertion of llus ulture vi shoot orgnogenesis seem to e highly dependent of numer of ftors, suh s rtio of uxins nd ytokinins in the ulture medium (Dewitte nd Murry [4]), the genotype (Lndi nd Mezzetti [7]), nd the soure of the explnts used. Different ultivrs of strwerry hve shown lrge vriility in the differentition ompetene of their somti tissue, nd mny workers hve reported the use of different omintions of growth regultors for shoot regenertion from different types of explnts (Shrt et l. [18]; Pssey et l. [14]; Zho et l. [23] ; Yonghu et l. [21]; Bisws [2]). The ojetive of this study ws to find the most pproprite type of explnt nd to investigte the speifi nutrient requirements for otining llus tissue from lef nd petiole explnts in Pink Pnd nd Serent intergeneri hyrids derived from Frgri x
ANCA NICOLETA SUTAN, AUREL POPESCU, VALENTINA ISAC Potentill rosses, in order to estlish n highly effiient protool for lrge sle propgtion of new vrieties of ornmentl strwerry developed through reeding nd seletion. Mterils nd methods Plnt mteril. Pink Pnd nd Serent genotypes from the Frgri Germplsm Colletion of the Institute for Fruit Growing, Pitesti, Mrineni, Romni were ultured in vitro strting from either meristems or self-pollintion derived seeds, on LF sl medium. Lef disks nd petiole segments olleted from six weeks old in vitro plntlets, preultured for two weeks on LF sl medium ontining 0.5 mg/l BAP, 0.5 mg/l IBA nd 0.2 mg/l GA 3 (Sorvri et l. [19]), were used s explnts. Cllus indution experiments. Cllus indution medi onsisted of Murshige nd Skoog [11] nd Lee nd Fossrd [8] sl medi, eh of them supplemented with vrious omintions of plnt growth regultors, totl of six different omintion for eh vriety nd explnt type, respetively, eing tested (Tle 1). Dextrose, t onentrtion of 40 g/l, ws used s ron soure in ll ulture medi. Tle 1. Comintion nd onentrtion of plnt growth regultors dded in sl medi in order to indue llus formtion. Cllus indution medi ode Bsl ulture medi Comintion nd onentrtion of plnt growth regultors (µm) 2,4 - D IBA BAP 1 MS, LF 2,7-1.3 2 MS, LF 4.5-1.3 3 MS, LF 4.5-2.2 4 MS, LF - 2.5 1.3 5 MS, LF - 4.9 1.3 6 MS, LF - 4.9 2.2 Both the lef explnts (0.3-0.5 mm dimeter) nd petiole segments (0.3-0.5 mm) were rndomly pled onto the ulture medium. The lef explnts were pled with their xil surfe in ontt with the medium. After two weeks of inution in the drk, in the growth hmer t the temperture of 22-24 C, the ultures were mintined under photoperiod of 16 hours light/8 hours drkness nd light intensity of out 40 µmol m -2 s -1. The numer of explnts forming lli ws sored fter 45 dys of ulture for oth vrieties. Shoot orgnogenesis. Cllus ultures initited from the lef nd petiole-derived lli were mintined on the sme medium whithout suulturing them, until the shoots indution. The numer of shoots formed per llus ws determined fter five months in ulture under 16 hour light photoperiod. Experimentl design nd sttistil nlysis. The rte of llogenesis (%) ws determined s the rtio of the numer of explnts tht developed lli to the totl numer of explnts. To void mjor sttistil errors, ll of the experimentl tretment (two genotypes x two type of explnts x two type of sl medi x six growth regultors omintions) ws performed with five replites (in onil flsks with 30 ml of ulture medium, losed with otton-wool ungs nd tinfoil) of six somti explnts. Anlysis of vrine ws used to nlyse the effets nd intertions of the four min ftors (genotype, sl medium, omintion nd onentrtion of PGR nd explnt types) on indution of llogenesis. Sttistil nlysis of the dt otined with Pink Pnd nd Serent vrieties respetively, on sl medi ontining different onentrtions of uxins for llus indution, were performed using Dunn s Multiple Rnge test, t p < 0.05 nd Independent Smple T- Romnin Biotehnologil Letters, Vol. 15, No. 2, Supplement (2010) 13
In vitro ulture medium nd explnt type effet on llogenesis nd shoot regenertion in two genotypes of ornmentl strwerry Test, working with Sttistil Pkge for the Soil Siene (SPSS) sttistil softwre (version. 16.0). Results The llogeni response ws indued fter 3 weeks of in vitro ulture, in response to IBA or 2.4-D nd BAP dded in MS nd LF si medi. These lli were formed ll over the surfe of the lef explnts nd were predominntly initited t the ut edges of the petiole segments, diretly in ontt with the ulture medium. It is known tht plnt hormones re present in higher quntities fter wounding nd re involved in ell prolifertion t the wound site (Khl [6]). Cllus ultures exhiited pink pigmenttion of nthoynins, in oth lef nd petiole explnts of Serent vriety. Effet of genotype. Signifint differenes on llus indution nd prodution ws oserved etween the two genotypes of ornmentl strwerry. Irrespetive of the si ulture medium, plnt growth regultors omintion nd onentrtion, or explnt type, the rte of llogenesis ws found to e, generlly, higher in Serent thn in Pink Pnd vriety. Differenes ws less pronouned when petiole explnts were ultured on MS si medium or indited etter performne of Pink Pnd vriety on LF medium supplemented with IBA nd BAP (Fig.1). Numer of explnts forming llus / totl numer of explnts (%) 140 120 100 80 60 40 20 0 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 Lef explnt Petiole explnt Lef explnt Petiole explnt M&S L&F Pink Pnd" Serent" Fig. 1. The influene of genotype on the llogenesis ility of lef nd petiole explnts (rs represent stndrd devition; letters ove the olumns indite perentges signifintly different from eh other, p<0.05). Effet of the ulture medium omposition. Considering oth types of somti explnts, ulture medium omposition signifintly influene llus indution in ornmentl strwerry genotypes investigted. The highest rtes of llus indution ws 100% from lef explnts nd 92% from petiole explnts (Fig. 3), respetively, in LF sl medium, ontining 2.7 µm or 4.5 µm 2.4-D nd 1.3 µm BAP (Tle 1), inditing more dequte omposition of nutrients to the in vitro regenertion requirements of this intergeneri hyrid. Thus, in Pink Pnd vriety, the rte of llogenesis ws 44% for lef explnts nd 60% for petiole explnts, respetively, in medi with the sme onentrtion of 2.4-D nd BAP. In Serent vriety, the ility of lef nd petiole explnts to produe llus ws higher on ulture medi 14 Romnin Biotehnologil Letters, Vol. 15, No. 2, Supplement (2010)
ANCA NICOLETA SUTAN, AUREL POPESCU, VALENTINA ISAC ontining low onentrtions of 2,4-D nd BAP. As shown in Figure 1, n inhiition of llogenesis ourred for oth types of explnts on medi with n inresed BAP onentrtion (2.2 µm, insted of 1.3 µm). The plnt growth regultors hd signifint effets on the indution of llus formtion in Pink Pnd vriety. The highest overll perentge of lef explnts forming llus (92%) ws indued in LF sl medium with low onentrtions of IBA (2.5 µm) nd BAP (1.3 µm). Surprisingly, the lef explnts filed to form llus on MS sl medium supplemented with the ove mentioned omintion of growth regultors. However, the sme omintion of growth regultors promoted llus formtion from the petiole explnts on oth LF nd MS medi, with frequeny of 84% nd 80%, respetively. When dded to the MS sl medium, the omintion of 2.2 µm BAP nd 4.9 µm IBA (Fig. 2) promoted llus prolifertion in oth lef nd petiole explnts from Serent vriety, while only petiole explnts were indued to form llus in Pink Pnd vriety. Numer of explnts forming llus / totl numer of explnts (%) 140 120 100 80 60 40 20 0 Lef explnt Petiole explnt Lef explnt Petiole explnt Lef explnt Petiole explnt Lef explnt LF MS LF MS Serent Pink Pnd Petiole explnt 1 2 3 4 5 6 Figure 2. The effet of ulture medium on the llogenesis ility of lef nd petiole explnts (rs represent stndrd devition; letters ove the olumns indite perentges signifintly different from eh other, p<0.05). Effet of explnt types. Sttistil nlysis reveled signifint differenes in llus indution frequeny not only etween the two genotypes, ut lso etween somti tissues used s explnts. Serent vriety showed the highest perent of lef explnts (100%) forming llus on LF sl medium. Petiole explnts proved to e the explnts type with the highest perentge of llus formtion when ultured on MS sl medium, for oth vrieties of ornmentl strwerry investigted (Fig. 3). Shoot orgnogenesis. Compring with llus indution nd prolifertion, regenertive ility of tissue-derived lli ws reltively low in Serent vriety. Moreover, lli formed from explnts of Pink Pnd vriety were unle to regenerte shoots on medi used in this study. Shoots regenertion ws otined in Serent vriety only from those explnts whih previously showed high frequeny of llus formtion (Fig 1). The frequeny of shoot regenertion from lef-derived lli ultured on LF sl medium supplemented with 2.5 µm IBA nd 1.3 µm BAP (36%) ws signifintly higher ompred to those ultured on MS sl medium supplemented with 4.9 µm IBA nd 1.3 µm BAP (22%). Shoot regenertion ws indued lso, ut t low frequeny (10%), when petiole explnts were ultured on LF Romnin Biotehnologil Letters, Vol. 15, No. 2, Supplement (2010) 15
In vitro ulture medium nd explnt type effet on llogenesis nd shoot regenertion in two genotypes of ornmentl strwerry sl medium, supplemented with 2.5 µm IBA nd 1.3 µm BAP. For this genotype, the llus indution frequeny nd plnt regenertion ility ppered to e in n inverse reltionship. Tle 2. Perentges of lef nd petiole explnts showing shoot formtion in Serent vriety of intergeneri hyrid Frgri x Potentill Cv. Serent Bsl ulture Explnt type forming shoots (%) PGR omintions medium Lef explnts Petiole explnts LF 2.5 µm IBA + 1.3 µm BAP 36 ± 2.3 10 ± 1.1 MS 4.9 µm IBA + 1.3 µm BAP 22 ± 1.6 - Dt re expressed s mens ± SE. Mens in the sme olumn tht re followed y different letters re signifintly different (p<0.05) using Dunn s Multiple Rnge Test. Numer of explnts forming llus / totl numer of explnts (%) 140 120 100 80 60 40 20 0 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 Pink Pnd Serent Pink Pnd Serent MS Lef explnt 5 6 LF Petiole explnt 1 2 3 4 5 6 (rs represent stndrd devition; letters ove the olumns indite perentges signifintly different from eh other, p<0.05). Fig. 3. The ility to form llus of lef nd petiole explnts s influened y the intertion etween genotype nd ulture medium omposition. Disussion Plnt genotype, explnt types, sl ulture medium nd PGR omintions nd onentrtions, signifintly influened the frequeny of llus formtion nd shoot regenertion, respetively, in Serent nd Pink Pnd vrieties. In terms of the numer of explnts whih formed llus, the petiole explnts gve etter response thn the lef explnts. Mny other uthors reported tht oth llus indution nd shoot orgnogenesis were dependent on the explnt soure (Pssey [14]; Denth [3]). However, our results proved tht is no orreltion etween llus formtion ility nd shoot formtion pity, s in Serent vriety, whose lef explnts exhiited low ility to form llus, ut lef-derived lli regenerted shoots t reltively high perentge. The etter regenertive ility of lef explnts n e ttriuted to the size of explnts. In this respet, Pierik [16] reported tht lrger explnts sometimes regenerte esier tht smller ones, nd tht the lrger explnts produe more shoots in vitro. This study showed tht sl ulture medium nd plnt growth regultors types, onentrtions nd omintions re nother key ftors regulting llus indution nd shoot 16 Romnin Biotehnologil Letters, Vol. 15, No. 2, Supplement (2010)
ANCA NICOLETA SUTAN, AUREL POPESCU, VALENTINA ISAC orgnogenesis. Cllus indution frequeny ws ffeted y onentrtion of BAP nd the effet of uxins on llus indution ws genotype speifi. In low onentrtion 2,4-D hs een shown to e the most effetive for llus indution in Serent vriety, irrespetive of explnt type ultured. As hve een reported, 2,4-D indue llus formtion in vriety of speies (M nd Xu [9], Tho et l. [20]). In ontrst, IBA dded in low onentrtions in ulture medium, ws most effetive in stimulting llus formtion, in Pink Pnd vriety. These oservtions were similr to those noted y other utors (Nehr et l. [12] ; Brelo et l. [1] 1998; Gruhl et l. [5]) Presumly, mintinne of tissue-derived lli in ulture without their trnsfer to the fresh medium my e the reson for the low regenertion frequeny in Pink Pnd nd Serent vrieties, respetively, or even the lk of regenertion in the se of lef-derived lli. On the other hnd, while IBA in omintion with BAP stimulted shoot orgnogenesis in Serent intergeneri hyrid of Frgri x Potentill, 2,4-D hs proven inpproprite for shoots regenertion in oth investigted genotypes of ornmentl strwerry. Moreover, nthoynin prodution does not influene llus indution in Serent vriety, ording with Mori et l. [10]. Genotype ws proven to e ritil ftor for indiret shoot orgnogenesis in intergeneri hyrids Frgri x Potentill. There were distint differenes in llus formtion ility nd shoots regenertion frequeny etween the two ornmentl strwerry vrieties investigted. Signifint vritions in llus formtion nd shoot regenertion ility of the different genotypes hve een previously reported in strwerry (Brelo et l. [1]; Pssey et l. [14]; Lndi nd Mezzetti [7]). Considering tht speifi genes re involved in shoot orgnogenesis (dedifferentition, quisition of ompetene nd indution), s reported y Phillips [15], it is likely tht, in some genotypes, genes involved in shoot orgnogenesis my e suppressed due to inpproprite ulture ondition. REFERENCES 1. M. Brelo, I. El-Mnsouri, J.A. Merdo, M. Quesd, F.P. Alfro, Regenertion nd trnsformtion vi Agroterium tumefiens of the strwerry ultivr Chndler. Plnt Cell Tiss. Org. Cult., 54: 29-36 (1998). 2. M.K. Bisws, U.K. Roy, R. Islm, M. Hossin, Cllus ulture from lef lde, nodl, nd runner segments of three strwerry (Frgri sp.) lones. Turk. J. Biol., 33: 1-6 (2009). 3. C.S. Denth, Strwerry sepl: Another explnt for thidizuron-indued dventitious shoot regenertion. In Vitro Cell. Dev. Biol. Plnt, 41: 671-676 (2005). 4. W. Dewitte, J.A. Murry, The plnt ell yle. Anuu. Rev. Plnt. Biol., 54: 235-264 (2003). 5. A. Gruhl, M. Korin, E. Żurwiz, Conditions of trnsformtion nd regenertion of Induk nd Elist strwerry plnts. Plnt Cell Tiss. Org. Cult., 79: 153-160 (2004). 6. G. Khl, Wound repir nd tumor indution in higher plnts. In: T. Akzw & H. Imsei (eds). The New Frontiers in Plnts Biohemistry, Jpn Sientif. So. Press. Tokyo nd Mrtinus Nijhoff/Dr. W. Junk Pul., The Hgue, 1983, pp. 193-216. 7. L. Lndi, B. Mezzetti, TDZ, uxin nd genotype effets on lef orgnogenesis in Frgri. Plnt Cell Rep., 25: 281-288 (2006). 8. E.C.M. Lee, R.A. de Fossrd, Some ftors ffeting multiple ud formtion of strwerry (Frgri x nnss Duh.) in vitro. At Hort., 78: 187-196 (1977). 9. G. M, W. Xu, Indution of somti emryogenesis nd dventitious shoots from immture leves of ssv. Plnt Cell Tiss. Org. Cult., 70: 281-288 (2002). 10. T. Mori, I.M. Skur, M. Sekim, S. Furuski, Use of uxin nd ytokinin to regulte nthynin prodution nd omposition in suspension ultures of strwerry ells. J. Si. Food Agri., 65: 271-276 (1994). 11. T. Murshige, F. Skoog, A revised medium for rpid growth nd iossys with too tissue ultures. Physiol. Plntrum, 15(3): 473-497 (1962). 12. N.S. Nehr, C. Stushnoff, K.K. Krth, Diret shoot regenertion from lef diss. J. Am. So. Hort. Si., 114: 1014-1018 (1989). Romnin Biotehnologil Letters, Vol. 15, No. 2, Supplement (2010) 17
In vitro ulture medium nd explnt type effet on llogenesis nd shoot regenertion in two genotypes of ornmentl strwerry 13. K. Niemirowiz-Szytt, The result of intergeneri pollintion of Frgri x nnss Duh. nd Frgri virginin Duh. y Potentill speies. At So. Bot. Pol., 53: 443-454 (1984). 14. A.J. Pssey, K.J. Brrett, D.J. Jmes, Adventitious shoot regenertion from seven ommeril strwerry ultivrs (Frgri x nnss Duh.) using rnge of explnt types. Plnt Cell Rep., 21: 397-401 (2003). 15. G.C. Phillips, In vitro morphogenesis in plnts - reent dvnes. In Vitro Cell. Dev. Biol. Plnt, 40: 342-345 (2004). 16. R.L.M. Pierik, In vitro ulture of higher plnts s tool in the propgtion of hortiulturl rops. At Hort., 226:25-40 (1998). 17. A.J. Syegh, M.J. Hennerty, Intergeneri hyrids of Frgri nd Potentill, At Hort., 348: 151-154 (1993). 18. J.G. Shrt, M.J. Slentijn, F.A. Krens, Tissue speifi expression of the ß-gluuronidse reporter gene in trnsgeni strwerry (Frgri x nnss) plnts. Plnt Cell Rep. 21: 313 319 (2002). 19. S. Sorvri, S. Ulvinen, T. Hieternt, H. Hiirslmi, Preulture medium promotes diret shoot regenertion from miropropgted strwerry lef disks. HortSiene, 28(1): 55-57 (1993). 20. N.T.P. Tho, Y. Ozki, H. Okuo, Cllus indution nd plntlet regenertion in ornmentl Alosi. Plnt Cell Tiss. Org. Cult., 89: 83-90 (2003). 21. Q. Yonghu, Z. Shnglong, S. Asghr, Z. Lingxio, Q. Qioping, C. Kunsong, X. Chngjie, Regenertion mehnism of Toyonok strwerry under different olor plsti films. PlntSi., 168:1409-1424 (2005). 22. 22. M.H. Yu, Cllus indution nd differentition from lef explnts of different speies of the genus Bet. Crop Si., 29: 205-209 (1989). 23. Y. Zho, Q. Liu, R.E. Dvis, Trnsgene expression in strwerries driven y heterologous phloem-speifi promoter. Plnt Cell Rep., 23:224 230 (2004). 18 Romnin Biotehnologil Letters, Vol. 15, No. 2, Supplement (2010)