Use of multinodal explants for micropropagation of Leucadendron Safari Sunset

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1 Instituto Ncionl de Investigción y Tecnologí Agrri y Alimentri (INIA) Spnish Journl of Agriculturl Reserch (3), Avilble online t ISSN: X eissn: Use of multinodl explnts for micropropgtion of Leucdendron Sfri Sunset E. Suárez 1 *, J. F. Pérez-Frncés 1 nd J. A. Rodríguez-Pérez 2 1 Deprtmento de Biologí Vegetl. Fcultd de Frmci. Universidd de L Lgun (ULL) L Lgun (Tenerife). Spin 2 Deprtmento de Ingenierí, Producción y Economí Agrri. Escuel Técnic Superior de Ingenierí Agrri. Universidd de L Lgun (ULL) L Lgun (Tenerife). Spin Abstrct Leucdendron Sfri Sunset is one of the most vluble Protecee cultivr, due to its economic importnce s cutflower. In vitro tissue culture of this cultivr nd others my offer mssive cloned plnt production tht permit to stisfy the mrket demnd. Multinodl explnts of Leucdendron Sfri Sunset were estblished in vitro on hlf strength Murshige nd Skoog medium supplemented with 2.2 µm benzylminopurine (BAP). Vrious ntioxidnt tretments were used to reduce the presence of phenolic compounds in culture. No importnt differences in growth nd multipliction between these tretments were detected during in vitro estblishment nd multipliction, but explnts cultured on medium with 1 g L 1 of polyvinylpyrrolidone (PVP) developed higher number of buds in the estblishment phse. Prolifertion of xillry shoots ws chieved with 0.88 µm BAP, being slightly higher on medium with 2.2 µm BAP lthough the buds were deformed, showing bnorml leves. The use of phytgel in this phse incresed prolifertion rte nd shoot growth. Low percentges rooting were obtined when microcuttings were cultured on the sme bsl medium supplemented with severl indole-3-butyric cid (IBA) concentrtions. The best rooting percentge (37.5%) ws obtined with 44.1 µm IBA. This work shows tht micropropgtion of L. Sfri Sunset is possible with multinodl explnts, lthough is necessry to optimize the protocol for commercil pplictions. Additionl key words: ntioxidnt tretment, in vitro tissue culture, ornmentl plnts, phenolic compounds, Protecee. Resumen Empleo de explntos multinodles pr l micropropgción de Leucdendron Sfri Sunset Leucdendron Sfri Sunset es uno de los cultivres más importntes de Protecee debido su importnci económic como flor cortd. El cultivo in vitro de este cultivr y otros, puede ofrecer un producción msiv de clones que permit stisfcer l demnd del mercdo. Se estblecieron in vitro explntos multinodles de L. Sfri Sunset en un medio Murshige y Skoog, con los mcronutrientes l mitd de su concentrción, y bencilminopurin (BAP) 2,2 µm. Pr reducir l presenci de compuestos fenólicos en los cultivos, se empleron diferentes trtmientos ntioxidntes. No se observron diferencis importntes en cunto l crecimiento y multiplicción de ls yems, entre los ntioxidntes empledos durnte el estblecimiento y multiplicción in vitro, sin embrgo, se obtuvo un myor número de yems (25 ± 2,82) en los explntos sembrdos en un medio con 1 g L 1 de polivinilpirrolidon (PVP). L multiplicción de ls yems se logró con BAP 0,88 µm. L ts de proliferción fue ligermente superior l dicionr l medio de cultivo BAP 2,2 µm, pero en este medio se desrrollron yems deformes, con hojs normles. El empleo de fitgel incrementó l ts de multiplicción y crecimiento de los vástgos. Se obtuvieron bjos porcentjes de enrizmiento l cultivr los microesquejes en el medio bsl suplementdo con diferentes concentrciones de ácido indol-3-butírico (IBA). El mejor porcentje de enrizmiento (37,5%) se obtuvo con IBA 44,1 µm. Este trbjo muestr que es posible l micropropgción de L. Sfri Sunset prtir de explntos multinodles, unque es necesrio optimizr el protocolo con fines comerciles. Plbrs clve dicionles: compuestos fenólicos, cultivo in vitro de tejidos, plnts ornmentles, Protecee, trtmiento ntioxidnte. * Corresponding uthor: ensurez@ull.es Received: ; Accepted: Abbrevitions used: BAP (benzylminopurine), GA 3 (giberellic cid), IBA (indole-3-butyric cid), NAA (nphthlenecetic cid), PVP (polyvinylpyrrolidone), 1/2 MS (hlf-strength mcronutrient slts).

2 Micropropgtion of Leucdendron Sfri Sunset 791 Introduction Sfri Sunset (L. slignum L. lureolum), is n ornmentl plnt widely cultivted for cut flower. It is the most cultivted Leucdendron on worldwide bsis. First bred in New Zelnd, it expnded rpidly to other zones. It is present in ll the prote producing regions: New Zelnd, Isrel, Austrli, Hwii, Cliforni, Chile, Equdor, South Afric, Zimbbwe, Azores nd Mdeir mong others. Presently, millions of florl stems re sold t flower uctions in Hollnd, being extensively used by florists for florl rrngements (bskets, centrepieces, etc.) nd bouquets. It hs drk green leves nd its terminl brcts present luminous red during summer, drkening with the course of the seson to dopt drk wine-red colour. Owing to its fst growth nd erly entrnce in production it hs become highly profitble cultivr. Its propgtion by stem cuttings is esy, but it is frequently difficult to stisfy the demnd for plnts using conventionl methods of vegettive propgtion. Micropropgtion could be vlid lterntive to solve this problem, s well s to provide the indispensble plnt helth gurntee for vegettive mteril exchnge t n interntionl level. The need to estblish protocols for the micropropgtion of different types of protes hs been supported by the IPA (Interntionl Prote Assocition) t the Interntionl Prote Conference held in 2006 in Sn Diego, Cliforni. Although diverse uthors hve studied the micropropgtion of some protes (Hu nd Wng, 1983; Ben- Jcov nd Jcobs, 1986; Seelye et l., 1986; Rugge et l., 1989; Kuniski, 1989, 1990; Bunn nd Dixon, 1992,b; Wtd et l., 1992,b; Pérez-Frncés et l., 1995; Rugge, 1995; Dis Ferreir et l., 2003; Wu nd du Toit, 2004), results hve not been totlly stisfctory, s t present there is still no firm tht specilises in mrketing in vitro produced prote plnts. A previous report by Pérez-Frncés et l. (1995) showed the effect of some fctors on in vitro multipliction of this cultivr. In this study, the use of benzylminopurine (BAP) improved bud prolifertion rtes. The ddition of nphthlenecetic cid (NAA), indole-3-butyric cid (IBA) or gibberellic cid (GA 3 ) to the prolifertion medium contining BAP did not significntly ffect the formtion of new buds per explnt or their length. The presence of ctivted chrcol in the medium clerly inhibited bud prolifertion nd n MS medium (Murshige nd Skoog, 1962) t hlf strength produced the best response. Dis Ferreir et l. (2003) worked with the sme cultivr, obtining the best results on modified MS medium contining scorbic cid nd combintion of BAP nd GA 3 when shoot tips were used for estblishment. They obtined only 20% elongted plnts with explnts from the tender prts of the shoots. In the present pper, we report method for micropropgting Leucdendron Sfri Sunset from multinodl (2-3 node) explnts. These re good option for this technique, since they provide 2-3 buds per explnt. Mteril nd methods Estblishment Explnts were removed from ctively growing terminl shoots of plnts growing in greenhouse nd used to initite in vitro culture. Shoots 20 cm in length were dipped into 2 g L 1 Benlte solution (50% Benomyl) for 1 hour. The clen mterils were given quick (5 s) rinse in 70% ethnol, followed by wshing in distilled wter. These were then surfce-sterilized in 5% sodium hypochlorite solution with one drop L 1 of Tween 80 (surfctnt) under vcuum for 15 min, followed by three wshes in sterilized distilled wter. Before plnting segments in the culture medium, they were dipped into different ntioxidnt solutions to diminish excessive oxidtion. Multinodl segments (3-5 cm length with 2-3 nodes) were plced in bsl MS medium (Murshige nd Skoog, 1962) contining hlf-strength mcronutrient slts (1/2 MS), 3% sucrose, 7 g L 1 gr, nd 100 mg L 1 myo-inositol. The 1/2 MS medium ws supplemented with plnt growth regultors nd the ntioxidnt substnces nd djusted to ph 5.7 before utoclving. Antioxidnt tretments were used in the estblishment phse to reduce explnt oxidtion: combintion of citric (1,500 mg L 1 ) nd scorbic cid (100 mg L 1 ) (Pérez-Frncés et l., 1995) or polyvinylpyrrolidone (PVP) (1 g L 1 ), employed in different wys: T1 (scorbic nd citric cid solution shken for 30 min before culture), T2 (T1 followed by drkness for three dys fter culture), T3 (T1 then subculture every three dys during the first week of culture), T4 (scorbic nd citric cid included in the medium), P1 (PVP solution shken s in T1), P2 (P1, then treted s in T2), P3 (P1 then subcultured s in T3), P4 (s T4 but with PVP), O (s T2 without ntioxidnts), SF (subcultured s in T3 without ntioxidnts). The cultures were mintined for 8 weeks during which they were trnsferred once

3 792 E. Suárez et l. / Spn J Agric Res (2010) 8(3), ) b) medium supplemented with NAA (37.8, 43.2, 48.6, 54, 59.4 µm) or IBA (34.3, 39.2, 44.1, 49, 53.9 µm) or ii) dipped in solution of IBA (0.73, 2.45, 4.90 mm) or NAA (0.81, 2.70, 5.40 mm) for 5 s nd then cultured on 1/2 MS medium without growth regultors. At lest 24 tubes were rised for ech tretment nd ll the experiments were repeted three times. Number of bud-breks were recorded t weekly intervls. Results were nlysed sttisticlly by nlysis of vrince, using the SPSS sttisticl softwre pckge (version 17.0). Stndrd error of the men ws clculted nd is indicted by ± sign. Results Figure 1. Multinodl explnts. ) Explnt A: explnts where petioles were cut off leving 1-2 mm petioles. b) Explnt B: explnts where petioles were totlly removed. fter 4 weeks of incubtion. Twenty millilitres of medium ws dispensed into ech ( mm) glss tube nd utoclved t kp nd 121 C for 20 min. Explnts were divided in two groups (Fig. 1). Leves of group A explnts were cut off leving 1-2 mm petioles, wheres leves of explnts B were totlly eliminted. During estblishment, explnts were inserted into the bsl medium supplemented with 2.2 µm BAP. Cultures were mintined t 25 ± 2 C, in culture room illuminted with cool white fluorescent tubes (110 µmol m 2 s 1 ) on shelves during the 16 h photoperiod. Within 2 weeks, xillry buds sprouted nd 2 weeks lter, buds were excised nd subcultured in multipliction medium. At lest 24 tubes were rised for ech tretment nd the experiment ws repeted two times. Multipliction Multipliction ws crried out using BAP t two concentrtions (0.88 µm nd 2.2 µm) nd two gelling gents: 0.7% gr (gr-gr/gum-gr, Sigm Chemicl, St. Louis, Mo. A1296) nd 0.25% phytgel (Sigm). Multipliction rtes were recorded during the first four subcultures. Rooting For rooting, microcuttings ( cm length) from the multipliction phse were cultured on i) 1/2 MS Estblishment When multinodl segments from green shoots were used, 66.7% of explnts sprouted buds on medium with PVP nd BAP 2.2 µm, producing pproximtely two buds per explnt. Tble 1 shows the men vlues of buds developed 4 weeks fter culture for ech tretment. Explnts A developed higher number of buds fter ll tretments ssyed, nd men of 25 buds sprouted when PVP ws included in the medium (P4), in comprison with the 7 buds developed by explnts B during the sme tretment. The highest number of buds (10) produced by explnts B ws obtined when explnts remined Tble 1. Buds men developed four weeks fter culture with ech ntioxidnt tretment in ech explnt type C (control, without ntioxidnt tretment), T1 (scorbic nd citric cid solution shken for 30 min before culture), T2 (T1 followed by drkness for three dys fter culture), T4 (scorbic nd citric cid included in the medium), P1 (PVP solution shken s in T1), P4 (s T4 but with PVP), O (s T2 without ntioxidnts), SF (subcultured every three dys during the first week of culture without ntioxidnts). Explnts A (leves were cut off leving 1-2 mm petioles), Explnts B (leves were totlly eliminted) Tretment Explnt A Explnt B C 21 ± 5.65 b 4 ± 4.24 b T1 18 ± 2.82 b 4.5 ± 2.12 b T2 20 ± 5.65 b 7.5 ± 3.5 b T4 17 ± 5.65 b 8 ± 0.0 b P ± b 10 ± 1.14 b P2 25 ± 2.82 b 6 ± 4.24 b O 11.5 ± ± 3.82 Vlues in ech column followed by different letter re significnt different t P = 0.05%.

4 Micropropgtion of Leucdendron Sfri Sunset 793 ) 5 5 b) Multipliction rte Multipliction rte BAP concentrtion (mg L 1 ) 0 Agr Gellificnt gent Phytgel Figure 2. Effect of ) different BAP concentrtions nd b) gellificnt gent on the multipliction rte of Leucdendron Sfri Sunset. Mens nd stndrd devitions for independent vribles nd ANOVA tests. The sme letters indicte no significnt differences t P = 0.05%. 30 min in PVP solution before being cultured in the medium (P1), lthough it ws very low in comprison with the buds produced by explnts A (23 buds). All ntioxidnt tretments used were effective for explnt oxidtion nd necrosis control, but no importnt differences between them were detected. Nevertheless, some observtions suggested tht explnts smller thn 30 mm in length presented greter browning symptoms, but sttisticl dt re insufficient to verify this. Multipliction The buds with gretest development during the estblishment phse were isolted from the explnts ) c) d) b) nd subcultured on multipliction medium consisting of 1/2 MS medium nd two different BAP concentrtions (2.2 µm nd 0.88 µm). Multipliction rtes obtined were low in both cses nd no significnt differences were identified between them (Fig. 2). However, shoot cultures growth with BAP 2.2 µm showed stunted growth with smll nd bnorml leves. Moreover these buds presented more symptoms of necrosis thn buds obtined on 1/2 MS medium supplemented with 0.88 µm BAP, which showed norml development (Fig. 3-b). Though the multipliction rtes did not lter for the different ntioxidnt tretments used (Tble 2), they did depend on the subculture number, showing significntly decline in the third subculture (Tble 2). With the use of phytgel s gelling gent, multipliction rtes continued to be low (Fig. 2b), nd no significnt differences were observed in the multipliction rtes in comprison with the buds multiplied in medium gelled with gr. However, buds obtined with phytgel showed stronger growth in both BAP concentrtions without hyperhydricity symptoms nd presented less symptoms of browning (Fig. 3c-d). Tble 2. Effect of different tretments nd subcultures on the multipliction rtes of L. Sfri Sunset. Multipliction rtes mens obtined by Duncn s test Tretment Multipliction rte Multipliction Subcultures rte C P b O b Figure 3. Multipliction of Leucdendron Sfri Sunset : ) nd b) in medium gelled with gr, ) 0.8 µm BA nd b) 2.2 µm BA; c) nd d) in medium gelled with phytgel, c) 0.8 µm BA, d) 2.2 µm BA. Sme letters within column indicte no significntly differences t P = 0.05%. C: control. P1: PVP solution for 30 min on shrking before culture. O: drkness during three dys fter culture.

5 794 E. Suárez et l. / Spn J Agric Res (2010) 8(3), Tble 3. Rooting percentges obtined with different indole-3-butyric cid (IBA) nd nphthlenecetic cid (NAA) concentrtions Growth Concentrtion Rooting regultor (µm) (%) IBA NAA Rooting When microshoots (1.5-3 cm length) were cultivted in 1/2 MS rooting medium contining NAA or IBA, fter 4 weeks in the estblishment phse nd 16 weeks in multipliction phse (4 subcultures), IBA gve better results thn NAA (Tble 3). Root induction ws preceded by cllusing t the bse of the shoot on IBA concentrtions higher thn 34.3 µm. On 1/2 MS medium with 49 µm IBA, which proved to be the best, 37.5% of shoots formed roots. Roots ppered fter 4 weeks nd the mximum response ws observed fter 7 weeks, reching between mm in length (Fig. 4). Roots obtined with IBA concentrtions bove 49 µm showed very little growth (1-2.5 mm in length). No rooting ws seen when microshoots were dipped in n IBA solution nd then cultured on 1/2 MS medium without growth regultors. The ddition of ctivted chrcol to the medium did not improve the rooting percentge or root length. Discussion Use of multinodl explnts provided more thn one bud per explnt nd reduced browning. Dis Ferreir et l. (2003) studied three types of Sfri Sunset explnts: shoot tips (5-10 mm), explnts from the green prt of the shoots nd explnts from the remining lignified shoots. They chieved 65% bud elongtion from shoot tips but only 20% from explnts from the green prt of the shoots, in n MS medium including combintion of growth regultors. They did not obtin Figure 4. A rooted microcutting of Leucdendron Sfri Sunset on 1/2 MS medium with 44.1 µm IBA. positive results from the oldest explnts. Similr results were found by Rugge et l. (1989), who reched higher percentge of sprouted buds using multinodl green explnts thn with semi-lignified nd lignified explnts. Our results were better when the explnt leves were cut off leving 1-2 mm petioles, thn with explnts without petioles. This could be due to excessive phenols production in the zone tht produced dmge on the xillry meristems. In previous studies with members of the Protecee, the utiliztion of explnts with nd without lef remnnts gve rise to differences. In mny of them, uthors used explnts with 1-5 mm of lef (Ben-Jcob nd Jcobs, 1986; Wtd et l., 1992b; Rugge, 1995), though others used explnts without remins of lef (Seelye et l., 1986; Kuniski, 1989), nevertheless only Pérez-Frncés et l. (2001b) compred the development of xillry buds in these two types of explnts with Leucdendron discolor, with n inferior response in explnts with 1-3 mm of petiole thn with the other type. The need for study on the ction of ntioxidnt tretments on the in vitro culture of this plnt rises from the Protecee being rich in phenolic compounds (Mln, 1992; Wtd et l., 1992b; Rugge, 1995; Wu

6 Micropropgtion of Leucdendron Sfri Sunset 795 nd du Toit, 2004). After the dissection of explnts, these compounds my be oxidized by different enzymes (polyphenoloxidses nd tyrosinses) which re synthesized nd/or liberted due to injury during excision nd steriliztion of tissues (Torres, 1989). Oxidtion products re known to inhibit the enzymtic ctivity, browning the tissues nd drkening the medium. Drkening cn be reduced by limiting the production of phenolic compounds, modifying the redox potentil, inctivting the phenolses, nd reducing the ctivity of the phenolse nd/or the vilbility of substrte (Hu nd Wng, 1983). In severl reports concerning Protecee, the use of n ntioxidnt solution is necessry in some phses of culture: citric nd/or scorbic cid (Pérez-Frncés et l., 1995; Dis Ferreir et l., 2003; Wu nd du Toit, 2004), PVP (Pérez-Frncés et l., 2001,b), or BAP pplied to the entire mother plnt t 200 mg L 1. This ltter significntly reduced browning nd promoted bud sprouting in vitro (Rugge, 1995; Pérez-Frncés et l., 2001b). It is lso possible to pply n etioltion tretment to new ctively growing shoot on the mother plnt (Wtd et l., 1992b). In our present study, we evlute the effect of different ntioxidnt tretments on explnt oxidtion, using PVP nd n scorbic nd citric cids solution combined with drkness nd frequent subculture. The PVP bsorbs the phenolic substnces so they cnnot be oxidized by the polyphenoloxidses, wheres the citric nd scorbic cid prevent oxidtion of the phenolic compounds. Explnt incubtion in drkness in the initil period nd the frequent trnsfer of explnts into fresh medium were the other tretments ssyed. No differences in tissue oxidtion were observed between the different tretments. Results during the multipliction phse were similr to those reported by Pérez-Frncés et l. (1995); nevertheless Dis Ferreir et l. (2003) obtined higher multipliction rtes, lthough they used combintion of different growth regultors. Buds obtined in medium with 2.2 µm BAP browned rpidly due to their poor development. There re cses in which the effect of the BAP t certin concentrtions inhibits bud elongtion, cusing browning. To diminish this, the BAP concentrtion cn be reduced in this phse, or be combined with n uxin or gibberellin to eliminte the inhibitory effect of BAP on bud growth (Orelln Pérez, 1998). In our work, using lower BAP concentrtion ws sufficient to promote bud development nd reduce the risk of browning. Multipliction rtes obtined were slightly higher but not significntly different when phytgel ws used s gr substitute, moreover these buds were longer. Similr results in Sfri Sunset using gelling gent were obtined by Pérez-Frncés et l. (1995). Any differences observed my be result of the higher purity of phytgel nd unidentified impurities in the gr (Shrm nd Rmmurthy, 2000), which could contribute to inhibiting shoot growth (Hu nd Wng, 1983). There re some reports of phytgel ddition improving the multipliction phse in different plnts (Sdt nd Hennerty, 2002; Thimmppih nd Sdhn, 2002). The mjor problem in our study ws the low rooting percentge obtined. Rooting in vitro my be reduced due to long period in culture (Norton nd Norton, 1986), moreover, the presence of cytokinin in the medium during the estblishment nd multipliction phses (Torres, 1989; Peddboin et l., 2006) cn lso negtively ffect rooting. This cn be incresed using high cytokinin concentrtions (Figueiredo et l., 2001). In our cse, presence of BAP during the estblishment nd multipliction phses could hve reduced the rooting rtes. In conclusion, the present work shows tht micropropgtion of Leucdendron Sfri Sunset is possible with multinodl explnts, lthough for commercil pplictions it will be necessry to optimize the protocol nd study the fctors tht ffect rooting in this cultivr fter reltively long period in culture. Acknowledgement The uthors wish to thnk Dr. Mrí Teres Rmos Domínguez (Dpto. Estdístic, Investigción Opertiv y Computción, Universidd de L Lgun) for her excellent ssistence with the sttisticl nlysis. References BEN-JAACOV J., JACOBS G., Estblishing Prote, Leucospermum nd Serruri in vitro. Act Hort 185, BUNN E., DIXON K.W., In vitro propgtion of the rre nd endngered Greville scpiger (Protecee). HortScience 24(3), BUNN E., DIXON K.W., 1992b. Micropropgtion of Stirlingi ltifoli (Protecee), n importnt cut flower from Western Austrli. Hort Science 27(4), 368. DIAS FERREIRA C., DIAS J.D., CANHOTO J.M., In vitro propgtion of Leucdendron lureolum L. slignum cv. Sfri Sunset : ultrstructurl nd n-

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