Abstract. Introduction

Transcription

1 In vitro propgtion of Dmsk Roses 85 In vitro propgtion of three Dmsk Roses essions Bhreh Allhverdi Mmghni, Mhlgh Ghornli 2*, Mohmmd Hssn Assreh,nd As Ghmri Zre Reserh Institutes of Forests nd Rngelnds, Tehrn, Irn 2 Deprtment of Biology, Islmi Azd University, Gorgn Brnh, Gorgn, Irn Astrt An in vitro propgtion protool hs een developed for three superior Dmsk rose essions using xillry uds of mture 5 yers-old plnts. Effets of ulture medium nd plnt genotype were evluted using split plot design sed on ompletely rndomized design with three replitions. Surfe steriliztion ws rried out with 0.% HgCl 2 for five minutes. The pproprite sesons for olleting explnts were summer nd utumn. Endogenous ontmintion ws eliminted y efotxim ntiioti. Shoot growth rte nd shoot index showed etter performnes on MS thn on WPM medium. Thidiuzuron (TDZ) in omintion with 6-enzylminopurine (BAP) indued signifintly higher numer of shoots per explnts thn the most optimum BAP tretment lone. The highest level of shoot multiplition rte (5.9) ws reorded t omintion of 5 mgl - BAP nd 0. mgl - TDZ. Type nd onentrtion of uxins did not hve signifint effets on shoot multiplition nd shoot length. Between vrious ytokinins, BAP ws more effetive thn kinetin on shoot multiplition. There ws no onsistent response y oth shoot multiplition rte nd genotype to different onentrtions on growth regultors. Two essions were rooted on the medium supplement with 0. nd 0.2 mgl - nphtlene eti id respetively. Keywords: miropropgtion; multiplition rte; ulture medium; Plnt Growth Regultors (PGR); Ros dmsen Mill Allhverdi Mmghni, B., Ghornli, M., Assreh, M. H., Ghmri Zre, A., 200., In vitro propgtion of three Dmsk Roses essions. Irnin Journl of Plnt Physiology (2), Introdution Dmsk Rose (Ros dmsen Mill.) is n importnt romti plnt signifint from eonomi point of view in Irn nd some other ountries suh s Bulgri. Besides romti nd ornmentl vlues of R. dmsen, its mediinl properties espeilly nti-depression effets re worth onsidertion. *Corresponding uthor. E-mil ddress: mghornli@gorgniu.ir Reeived: July, 200 Aepted: Septemer, 200 Phytohemil ompounds hve een isolted from this speies whih hve ntimiroil nd ntifungiidl hrhteristis. Mhmood et l., (996) hve reported tht wter nd methnol extrts of R. dmsen exhiited moderte nti-hiv tivity. Trditionlly, rose plnts hve een propgted on rootstoks nd through utting method (Roert nd Shum, 2003). The onventionl propgtion of this speies ws ssoited with vrious prolems suh s limittion of stok plnts nd prolonged

2 86 Irnin Journl of Plnt Physiology, Vol (), No (2) prodution time (Skirvin et l., 990) nd low dventitious root formtion on uttings. Tissue ulture of R. hyrid ultivrs nd Ros speies hs eome n lterntive method of propgtion (Arnold et l., 992; Tei-Aghdei et l., 2005). Miropropgted roses hve the potentil to e ommerilized in ll mrket spets inluding pot prodution, grden nd lndspe plnts s well s for ut flower prodution (Roert nd Shum, 2003). Ex vitro plnts proved to e superior in vigor, plnt rhiteture nd flower prodution (Roert nd Shum, 2003). Reently, miropropgtion through mture explnts ws reported in different speies viz wild herry (Prunus vium L.) (Dukovi, 2006), Teton grndis (Shirin et l., 2005), Alnus glutinos (Lll et l., 2005), Ceder (Cedrus tlnti nd C. lini) (Renu-Mortet et l., 2005), Hgeni yssini (Feyiss et l., 2005) nd Ai mngium (Monteuuis, 2004). But in R. dmsen Mill., miropropgtion ws onduted through juvenile explnts (Jrzdeh nd Khush-Khui, 2005; Kfi et l., 2004). Kumr et l., (200) on the other hnd, used mture ushes in their study. They onluded tht the ulture initited on medium supplement with TDZ exhiited more shoot prolifertion nd growth thn the medium ontining BAP (Kumr et l., 2005). Generlly, growth nd development of miropropgted plntlets depends on ftors suh s mroelement omposition, totl slt strength of the medium, vitmin mixture used, sugr onentrtion, growth regultor omposition nd so on (Arnold et l., 995). The im of this work ws to determine the effets of ulture medium nd omintion of vrious plnt growth regultors on shoot prolifertion of mture explnts nd root regenertion of three elite Irnin R. dmsen Mill essions. Mteril nd methods Plnt mteril nd steriliztion Twigs of M6 (Kshn), G (Est Azeryjn) nd G2 (West Azeryjn) essions were olleted from mture elite plnts tht were grown t experimentl field of Biotehnology group, Reserh Institute of Forests nd Rnglnds, Tehrn, Irn (Tle ). Shoot explnts with nodule setion, fter removing of leves nd thorns were exposed to running tp wter (h) nd dipped in lohol (70%, 30 s). Then, different methods of surfe steriliztion were used. The explnts were surfe sterilized y immersion in 0.% HgCl 2 for five minutes nd rinsed three times in sterile distilled wter. This surfe steriliztion method ws used in different sesons to find the most pproprite seson for olleting explnts. Shooting stge The resulted explnts were trnsferred into glss vil (one explnt per vil) ontining 0 ml MS medium. After one month (30 d), five equl shoots were pled in MS ulture medium ontining 50 ml. First, the effet of ulture medi, MS (Murshige nd Skoog, 962) nd WPM (M Cown nd Sellmer, 982) ontining.5, 2.5, 5 mgl - BAP on shoot multiplition (shoot numer per explnts) nd shoot length were studied. Then, the effet of BAP (5 mgl - ) omintion with 0. nd 0. mgl - s well s IAA (0. nd 0.5 mgl - ) nd IBA (0. nd 0.5 mgl - ) ws Tle The properties of three seletive Irnin essions of Ros dmsen Mill Aesions Code Lotion of Growth Importnt Trit (Origin) M6 Isfhn High perentge of essentil oil G Est Azeryjn High qulity of essentil oil G2 West Azeryjn Long flowering period Tei-Aghdei et l. (2005), Tei-Aghdei et l. (2005), Tei-Aghdei et l.( 2004)

3 In vitro propgtion of Dmsk Roses unontminted explnt (%) M6 G G Spring Summer Autumn Winter Hrvesting seson of explnts Fig. I. Effet of explnts hrvesting seson on ontmintion perentge in three seletive essions studied. Finlly, three levels of BAP (2, 2.5 nd 5 mgl - ) nd Kin (2, 2.5 nd 5 mgl - ) lone nd their omintion y dding TDZ (0.05 mgl - ) nd IBA (0. mgl - ) on shoot prolifertion nd shoot length in three ession were exmined. For improved rooting, BAP onentrtion redued to.5, nd 0.5 mgl - in onseutive suultures. Rooting stge The shoots were trnsferred to rooting regenertion medium, modified MS medium ontined /2, /3 nd /4 onentrtions of mronutrients, surose s well s full onentrtion of mironutrient supplemented with different onentrtions of NAA (0., 0.2, 0.5 nd mgl - ). In order to derese leves nd shoot tip nerosis dditionl vitmin omplex, ontining iotin ( mgl - ), lium pntothente (0.5 mgl - ), rioflvine (0.5 mgl - ), nd foli id (0.5 mgl - ) were used. The ntiioti nd vitmin omplex were sterilized with filter sterile. Then ntiioti nd vitmin were dded to the medium fter utolved nd ooled medium. The ph of medium ws djusted to 5.7 ±0. y dding NOH. The vessels were inuted in growth room with 6h light t 25 C. Eh jr ws used s plot. Tle 2 Comprison of multiplition rte, shoot length (m), green, yellow nd neroti leves perentge on MS nd WPM medi in three seletive Irnin essions Multiplition rte Shoot length (m) Green leves (%) Yellow leves (%) Neroti leves (%) esions M G G Culture medium MS WPM In eh olumn the sme letters re not signifintly different t P 0.05 y using Dunn's multiple rnge test.

4 88 Irnin Journl of Plnt Physiology, Vol (), No (2) multiplition rte BAP onentrtion (mg/l) Shoot lenght (m) BAP5mg/l BAP2.5mg/l BAP.5mg/l BAP onentrtion Fig. II. Effets of BAP lone (5, 2.5 nd.5 mgl - ) on shoot multiplition (up) nd shoot length (down) in M6 ession of R. dmen Mill Sttistil nlyses The experiment ws done using split plot design sed on ompletely rndomized design with three replitions, where the effets of two ftors suh s ulture medium nd genotype were studied. The effet of one ftor (e.g. growth regultor) ws exmined in ompletely rndomized design. Eh tretment ws replited three times with five explnts in eh plot. Anlysis of vrine rried out y SAS softwre nd mens were ompred y using Dunn's multiple test.

5 In vitro propgtion of Dmsk Roses multiplition rte mg/l IBA 0.5mg/l IBA 0.mg/l IAA 0.5mg/l IAA 0.0mg/l TDZ+ 0.mg/l IBA Plnt growth regultor tretment 0.5mg/l IBA 0.mg/l IAA 0.5mg/l IAA.8.6 Shoot lenght (m) mg/l IBA 0.5mg/l IBA 0.mg/l IAA 0.5mg/l IAA 0.mg/l IBA Plnt growth regultor tretment 0.5mg/l IBA 0.mg/l IAA 0.5mg/l IAA Fig. III. Effets of TDZ (0. nd 0.0 mgl - ), IAA nd IBA (0. nd o.5 mgl - ) onentrtion on shoot multiplition (up) nd shoot length (down) in M6 ession. In ll tretments BAP t dose of 5 mgl - ws used.

6 90 Irnin Journl of Plnt Physiology, Vol (), No (2) Results Summer nd utumn were the pproprite seson for hrvesting explnts. High levels of ontmintion were oserved in spring nd winter exept for G2 (Fig. I). Surfe steriliztion did not eliminte endogenous ontmintion present in the internl tissues. Bteril ontmintion ws eliminted using efotxime with no negtive effet on shoot multiplition. The explnts hd higher shoot multiplition, shoot length nd etter green leves on MS thn WPM medium (Tle 2). Effet of genotypes on shoot multiplition, nd green nd neroti leves ws lso signifint (Tle 2). M6 ession showed the highest shoot multiplition thn essions G nd G2. In ontrst, the most neroti leves were oserved on G2 ession (Tle 2). The qulity of shoots deresed when the suulture period prolonged in WPM. This phenomenon ws ler in M6 genotype. Adding lium in the form of lium pntotente, deresed hlorosis of leves nd shoot tips. During the estlishment stge, shoots were sprouted from xillry uds. After one month, one shoot with 5 to 6 ompound leves ws oserved on most of the explnts. These shoots were trnsferred to fresh medium. Cholorosis followed y nerosis of old leves nd low multiplition rte were the two prolemti phenomen t the end of first the suulture. To solve the prolem, different omintions of growth regultors were exmined. In the first step, high level of BAP enhned shoot prolifertion nd redued shoot length (Fig. II). Adding TDZ to the medium ontining BAP promoted essory nd xillry ud formtion (Figs. 3 nd 5). The highest shoot prolifertion (5.9) ws otined t omintion of 5 mgl - BAP nd 0. mgl - TDZ (Fig. 3). Type nd onentrtion of uxin hd little effet on shoot multiplition nd shoot length. The highest multiplition rte ws otined in 0. mgl - IBA, 5 mgl - BAP nd 0. mgl - TDZ. In ontrst, mximum shoot length ws oserved in the medium with 0.5 mgl - IAA nd 5 mgl - BAP nd 0.0 mgl - TDZ (Fig. III). Shoot multiplition nd shoot length were vried with genotypes nd BAP s well s Kin onentrtion. M6 ession needed 2 mgl - BAP nd mgl - Kin for mximum shoot multiplition nd shoot length (Fig. IV). The other two essions, G nd G2, hieved the highest prolifertion nd length t 2.5 mgl - BAP nd 2.5 mgl - Kin. Kinetin lone hd deleterious effet on shoot multiplition on three essions (Fig. V-). The result of rooting indited tht root regenertion of shoots t in vitro ondition were diffiult. 25% of shoots in G ession were rooted on modified /3 MS medium supplemented with 0. mgl - NAA (Fig. 6-). On the other hnd, root regenertion of G2 ession ws found on modified /2 MS medium y dding 0.2 mgl - NAA (Fig. VI ). Disussion Development of in vitro propgtion proedures for rpid multiplition of rose ultivrs is of gret importne. Two possile strtegies for eliminting endogenous ontmintion re disrding ontminted explnts nd using ntiiotis. Cefotxim is n ntiioti ommonly used in plnt tissue ulture. It hs low eukryoti toxiity nd is effetive t low doses (Mthis nd Boyed, 986). Humr nd Ords (999) hve reported tht efotxim hd nontoxi effet on stone pine otyledons nd ws signifintly improved forming uds in the surfe of otyledons. Nerosis followed y hlorosis of leves nd shoot tip nerosis were oserved in ultured shoots my e ttriuted to lium defiieny, in this work. Roerts nd Shum (2003) indited some type of roses require more lium slts s lium defiieny my led to shoot tip nerosis, hyperhydrtion of stem nd lef tissues. They showed tht dding lium in the multiplition medium signifintly inresed shoot multiplition rtes nd stem length. Applition of lium pntotente omplex my enhne shoot qulity nd root regenertion. Clium regulted ellulr proess suh s ell division nd elongtion (Ghornli nd Blr, 2003). Arnold et l., (995) hve suggested tht lium nd potssium hve role in uxin sorption. Neroti lef perentge ws higher in WPM medium ttriuted to lower totl nitrogen ontent. Poor shoots growth of Ros hyrid L. on

7 In vitro propgtion of Dmsk Roses multiplition rte M6 G G mg/l BAP 2.5mg/l BAP 2mg/l BAP+ 2mg/l Kin Plnt growth regultor tretment 2.5mg/l Kin 5mg/l Kin Shoot lenght (m).5 M6 G G mg/l BAP 2.5mg/l BAP 2mg/l BAP+ 2mg/l Kin Plnt growth regultor tretments 2.5mg/l Kin 5mg/l Kin Fig. IV. Effets of BAP nd kinetine on shoot multiplition (up) nd shoot length (down) in three seletive Irnin essions WPM medium hs lso een reported (Vn der Slm et l., 994). In ddition to lower ontent of nitrogen, some enefiil elements, suh s olt were not found in WPM medium. Adding TDZ nd BAP to medium enhned xillry shoot prolifertion. This result ws in greement with Aer fremnni (Kerns nd Meyer, 987), Frxinus merin L. (Nvrrete et l., 989) Pyrus ommunis L. (Singh nd Bhti,

8 92 Irnin Journl of Plnt Physiology, Vol (), No (2) 988) nd Vitis rotundifoli Mihx, (Sudrsono nd Godly, 99). TDZ is the most potent ytokinin for tissue ulture of woody speies. Comintion of TDZ nd other plnt growth regultors n e more effetive thn using them lone (Huettemn nd Preee, 993). However multiplition rte were different for severl ultivrs of Ros. They lso suggested tht ertin roses require t lest initilly longer ulture period efore eptle prolifertion ourrene nd re independent of BAP onentrtion (Arnold et l., 992). Also () Fig. V. () In vitro shoot multiplition in M6; () Using kinetin fter suulturing TDZ omined with kinetin hd no effets on shooting. It seems tht Kinetin hd inhiitory effet on oth shoot multiplition nd TDZ tivity. Kinetin hd inhiitory effet on shoot multiplition when it ws used lone (Tles 2, 3, nd 4). Although uds in estlishment stge whih were inoulted on medium ontining kinetin sprouted, they did not survive during trnsferring. High levels of BAP hd no signifint effet on multiplition rte of G nd G2 essions. In ontrst, inresing BAP lone triggered high multiplition rte in M6 ession. The result is in greement with the study reported y Arnold et l., (992) who hve pointed out tht the effets of BAP on () onsistent with this study, Tei-Aghdei et l., (2007) hve emphsized genotype nd growth regultors s key ftors for in vitro ulture of R. dmsen. Diffiulties in rooting regenertion of Dmsk rose were onsidered y other uthors (Jrzdeh nd Khush-Khui, (2005) nd Kfi et l., (2004). Using TDZ with high onentrtion of BAP deresed dventitious root formtion on miroshoots. BAP n e umulted in plnt tissue t high onentrtion in the form of onjugtes, intive gluosides, s well s physiologilly tive riosides nd riotides (Podwyszynsk, 2003). Huettmn nd Preee (993) suggested tht rooting of miroshoots were diffiult euse of rry over effet from () () Fig. VI. Rooting in G2 () nd G () essions on modified MS medium supplement with 0.2 nd 0. mgl - NAA, respetively

9 In vitro propgtion of Dmsk Roses 93 ytokinin. Podwyszynsk (2003) suggested tht the prolem with root formtion of some rose genotypes my result from indequte endogenous level of uxin nd other growth regultors, phenolis or enzymes. Low rooting ttriuted to mture explnts might hve some inhiitory mterils (Podwyszynsk, 2003). The result indites there were different responses mong esions for rooting. Hsegw (980) suggested tht there is differene in rooting requirements nd responses to ulture ondition, s well s in rooting pity (Podwyszynsk, 2003). Therefore, it is reommended tht vritions mong esions of Dmsk rose e determined to otin optimum yield. For propgtion of superior Dmsk rose esions, further studies re needed to identify ftors enhning rooting nd susequent limtiztion of plntlet to greenhouse ondition. Aknowledgement This work ws rried out t Biotehnology Reserh Group in Reserh Institute of Forest nd Rngelnds, Tehrn, Irn. The Authors would like to thnk Mrs. Shekofeh Shhrzd for lrtory ssistne nd Dr. Tei-Aghdei for vlule omments on the mnusript. Referenes Arnold N. P., Binns, M. R., Brthkur,N., Clouter D.C., 992. A study of growth regultor nd time of plntlet hrvest on the in vitro multiplition rte of hrdy nd hyrid te Rose, Journl of Hortiulture Siene, 67 (6): Arnold N.P., Binns, M.R., Clouter D.C., 995. Auxin, Slt onentrtion nd their intertion during in vitro Rooting of Winter-hrdy nd hyrid te Roses. Hortsiene, 30(7): Durkovi, J. J., Rpid miropropgtion of mture wild herry. Biologi Plntrum. 50 (4): Feyiss, T. M., Wlnder, M. L., Negsh, L., Miropropgtion of Hgeni yssini: multipurpose tree. Plnt Cell, Tissue nd Orgn Culture, 80 (2): Ghornli, M., Blr, M., Minerl Nutrition in Plnt. Teher Trining University Pulisher, Tehrn, 355p. Hsegw, P.M., 980. Ftor ffeting shoot nd root initition from ulture rose shoot tip. Journl of Amerin Soiety for Hortiulturl Siene, 5: Huettemn, C.A., Preee, J. E., 993. Thidizuron: A potent ytokinin for woody plnt tissue ulture, Plnt Cell Tissue nd Orgn Culture, 33: Humr J.M., Ords, R.J., 999. The toxiity of Antiiotis nd heriides on in vitro Adventitious Shoot formtion on Pinus pine L. Cotyledones, In vitro Cellulr nd developmentl iology plnt. 35: Jrzdeh Z., Khush-Khui, M., Ftor ffeting tissue ulture of dmsk Rose (Ros dmsen Mill.), Sienti Hortiulture 05: Kfi, M., Nikht, A., Blr, M., Mirmsoomi, M., Effet of plnt growth regultors on in vitro growth hrteristis of Dmsk rose (Ros dmsen Mill.) Irnin Journl of Siene nd Tehnology of Hortiulture, 5 (3): Kerns, H. P., Jr Meyer, M. M., 987. Diligene finds the hemil key to miropropgting new mple. Amer. Nurserymn 65 (): Kumr A., Sood, A., Plni, U.T., Gupt, A.K., Plni, L.M., 200. Miropropgtion of Ros dmsen Mill from mture ushes using tidizuron. The Journl of Hortiulture Siene nd iotehnology, : Lll, S., Mndegrn, Z., Roerts, A. V., Shoot multiplition in ultures of mture Alnus glutinos, Plnt Cell Tissue nd Orgn Culture, 83 (30): Mhmood N., Piente, S., Pizz C., Burke A., Khn, A.I., Hy, A.J., 996. The nti-hiv tivity nd mehnisms of tion of pure ompounds isolted from Ros dmsen Mill., Biohemistry nd Biophysies Reserh Communition, 229 (): Mrelis-Vn Aker C.A.M., Sholten, H.J., 995. Develpment of xillry uds of rose in vitro. Sienti Hortiulture, 63: Mthis R. J., Boyd, L. A., 986 Cefotxime stimultes llus growth, emryogenesis nd regenertion in hexploid red whet (Tritium estivum L. em Thell). Plnt Siene.46: M own, B. H., Sellmer, J.C., 982. Medi nd physil environment 4-7. In: Bong nd Durzoon, Cell nd Tissue Culture in foresty. Vol. Generl priniple nd iotehnology. Mrtinus Nijhoff pulishers, pordreht 422.

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