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Available online at www.notulaebotanicae.ro

Not. Bot. Hort. Agrobot. Cluj 37 (2) 2009, 261-263

Print ISSN 0255-965X; Electronic ISSN 1842-4309

Notulae Botanicae Horti AgrobotaniciCluj-Napoca

In Vitro Flowering from Cultured Nodal Explants of Rose (Rosa hybrida L.)

Kantamaht KA NCHANAPOOM1) , Nonlapan POSAYAPISIT1) , Kamnoon KANCHANAPOOM2)

1)Center or Genomics and Bioinormatics Research, Tailand; kantamaht.k@psu.ac.th.2) Prince o Songkla University, Department o Biology, Faculty o Science, Hat Yai, Songkhla,

90112 Tailand; kamnoon_k@yahoo.co.th (corresponding author)

Abstract

Roses are one o the worlds most important ornamentals or a long time and are most oen used or ornamental, medicinal andaromatic purposes. Te study reports in vitro multiple shoot ormation and ower induction oRosa hybrida L. cv. Red Masterpiecewith maximum number o 5 shoots per explant on MS medium supplemented with 3 mg/l BA and 1 mg/l kinetin, ollowed by ower

induction on MS medium containing 2 mg/l BA or 9 weeks. Te shoots readily rooted on MS medium devoid o growth regulators.Shoots cultured under various photoperiods did not ower. Rooted plantlets were hardened and established in pots with 100% survival.

Keywords: in vitro owering, micropropagation, nodal culture,Rosa hybrida, subculture time

Introduction

Roses are one o the worlds most important ornamen-tals or a long time. Tey belong to the Rosaceae and are

grown world wide as cut owers, potted plants and homegardens. Rose is an important perennial ower shrub orvine o the genus Rosa, within the amily Rosaceae thatcontains over 100 species and comes in a variety o colors.

Te switch rom vegetative stage to reproductive stageo growth is one o the most critical events in the lie oa plant. An in vitro owering system is considered to be aconvenient tool to study specic aspects o owering and

whole mechanisms o the reproductive process such asoral initiation, oral organ development and oral senes-cence (Goh, 1992). Several successul attempts to induce invitro owering o roses have been reported (Vu et al., 2006;

Wanget al., 2002). Te application o cytokinins, photo-period and subculture time to promote in vitro owering iswell documented in many plant species including roses(Vuet al., 2006; Wanget al., 2002).

Te study aimed to develop a protocol or in vitro ow-ering oRosa hybrida L. cv. Red Masterpiece with aim to

provide convenience to study specic aspects o oweringand mechanism o reproduction o this important rose cul-tivar.

Materials and methods

Plant materials

Te Rosa hybrida L. cv. Red Masterpiece was used inthe experiment. Nodal explants containing lateral buds o

actively eld-grown Red Masterpiece roses were cut into3 cm length segments and washed in running water to re-

move dirt. Tese segments were surace disinested using70% ethanol or 15 sec and then immersed in 20% (v/v)o commercial laundry bleach CloroxM solution (5.25%NaOCl) containing 2 drops o ween-20 emulsier to aid

wetting or 20 min. Tereaer, the sterilized explants wererinsed 2-3 times with sterile distilled water. Te explants

were trimmed to 1 cm long pieces beore transer to theculture medium.

Medium preparation and culture conditionsMS (Murashige and Skoog, 1962) salts supplemented

with 3% sucrose and 0.82 %agar (MermaidM) was usedto prepare the culture medium. Te pH o all media wasadjusted to 5.8 with 1 NNaOH or 1 NHCl beore auto-claving at 1.05 kg/cm2, 121C or 20 min. Cultures weremaintained at 251C air temperatures in a culture room

with a 16-h light photoperiod unless otherwise stated.Each treatment contained 20 capped glass jars with 20 mlo regeneration medium and each jar (115 ml capacity)contained 1 cm long one explant with a culture cycle o3 weeks. Tereaer, the regenerating explants were sub-cultured to resh medium or developing green and normaladventitious shoots were rooted on growth regulators reeMS medium. Aer development o adequate length oshoots and roots, the plantlets were transerred to 330 ml

screw-topped jars containing sterile vermiculite or 2 weeksor hardening of.

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had no signicant efect on shoot multiplication and thephotoperiod did not inuence in vitro owering (ab.2). Te age o mother plants inuenced ower induction(Demeulemeester and DePro, 1999) thereore, it is pos-sible that the explants used in this study remained at the

vegetative phase. ab. 3 shows the efect o subculture timeon ower induction in vitro. Aer the 3-week-intervals orthree consecutive subcultures, the percentage o in vitroowering was 50%. Te ower buds were small; however,the owers were normal. It seems that a period o nine

weeks in culture was appropriate or owering in the pres-ent study. Wanget al. (2002) stated that there has been noreport on the efect o subculture time on in vitro ower-ing and the subculture time could substantially afect invitro owering.

o establish complete rose plants, regenerated shootswere excised and transerred to MS medium without

growth regulators. Roots that developed on this mediumwere thick, long and brous. wo weeks o rooting was ad-

Inuence o BA and kinetin on in vitro oweringo test the efect o BA (N6-benzyladenine) and kine-

tin on in vitro owering, the nodal explants were asepti-cally cultured on MS medium containing 0, 1, 2, or 3 mg/lBA or kinetin singly or in combination.

Inuence o photoperiod on in vitro oweringTree photoperiods (light/dark cycle) i.e. 12/12, 16/8

and 8/16 were used to monitor in vitro owering underan illumination o 20 mol m-2s-1 photosynthetic photonux density provided by cool-white uorescent light.

Inuence o subculture timeExplants were subcultured to resh MS medium sup-

plemented with 2 mg/l BA every 3 weeks resulted in 3, 6,9, 12, and 15 weeks-interval.

Statistical analysisAll experiments were conducted on three diferent

days. Analysis o variance was done using standard statis-tical techniques and the diference between means wascompared using ukeys test atp0.05.

Results and discussion

Multiple shoots were recorded on all explants aer 3weeks o culture, on MS medium containing various con-centrations o BA and kinetin. Variable number o shoots

was recorded on all culture media. Maximum number

o 5.30.7 shoots per explant (p0.05) was recorded onMS medium containing 3mg/l BA in combination with1 mg/l kinetin (ab. 1). Tis suggests that in this cultivarrequired cytokinins to prolierate high number o shoots

per explant without intervening callus stage. Previous re-search shows on other rose species regarding developmento multiple shoot on regeneration media containing BAand IBA (Khosh-Khui and Jabbarzadeh, 2007; Kumar et

al., 2001) or BA and NAA (Dreahl et al., 2007; Vu et al.,2006; Wang et al., 2002) with intervening callus phase.Tereore MS medium containing 3 mg/l BA and 1 mg/lkinetin was considered as optimal or shoot prolieration.Tese in vitro regenerated shoots were urther multipliedon this medium by successive subculture, aer every three

weeks.In vitro owering was observed on MS medium con-

taining 2 mg/l BA aer 9 weeks o culture (ab. 1). Teowers had normal petals and sepals at opening (Fig. 1).Initially the ower was pale red in color becoming red inthe later stages o growth. BA is widely used or in vitroowering in many roses (Dobres et al., 1998, Wanget al.,2002, Vu et al., 2006) and a number o other plant species(Lin et al., 2004, aylor et al., 2005). Te application ocytokinins induces molecular changes associated with theoral transition (Bernier et al., 2002).

Efect o photoperiod on individual shoots was re-corded aer 4 weeks o culture. Length o photoperiod

ab. 1 Efect o diferent combinations o BA and kinetin onmultiple shoot ormation and in vitro owering oRosa hybridaL. cv. Red Masterpiece

MS medium

BA(mg/l)

Kinetin(mg/l)

Number o shootsper explant(Mean SE)

Explantsproducing

ower(%)

0 0 1.0 1.1d

0

1 0 1.9 0.2bcd 0

2 0 2.8 0.3bcd 5

3 0 2.5 0.3bcd 0

0 1 3.0 0.3bc 0

0 2 2.4 0.3bcd 0

0 3 1.4 0.2cd 0

1 1 1.4 0.1cd 0

1 2 2.1 0.2bcd 0

1 3 1.0 0.0d 0

2 1 4.7 0.5a 0

2 2 3.7 0.3ab 0

2 3 4.8 0.7a 0

3 1 5.3 0.7a 0

3 2 2.6 0.3bcd 0

3 3 2.0 0.3bcd 0

Te dierent letters within column show signifcant

dierence analyzed by ukeys test atp0.05.

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equate beore transplanting to polystyrene pots contain-ing soil mixture (1 sand: 1 manure: 1 decayed leaves). Invitro-derived plants did not display any phenotypic varia-tion during subsequent vegetative development.

Conclusions

A micropropagation system or Rosa hybrida cv. RedMasterpiece has been worked out utilizing nodal ex-

plants. BA and subculture time have a promoting efect onin vitro ower initiation. Although 50% o owering wasobserved, more reliable culture regimes or in vitro owerneed to be elucidated.

Acknowledgement

Tis research was nancially supported by Facul-ty o Science, Prince o Songkla University,Hat Yai, Song-khla, Tailand.

References

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Dobres, M., L. Williams and R. Gail (1998). Micropropagationo rose plants. United States patent 5,843,782 December 1,

1998.Dreahl, A., M.G. Quoirin and F. L. Cuquel (2007).

Micropropagation oRosa hybrida cv. Vegas via axillarybuds. Acta Horticulturae. 751:407-411.

Goh, C. J. (1992). Studies on owering in orchids-A reviewand uture directions. Proceeding o Nagoya InternationalOrchid Show92 (NIOC), pp 44-49.

Khosh-Khui, M. and Z. Jabbarzadeh (2007). Efects o severalvariables on in vitro culture o Damask Rose (Rosa damascenaMill.). Acta Horticulturae. 751:389-393.

Kumar, A., A. Sood, U. . Plani, A. K.Gupta and L. M. S. Plani(2001). Micropropagation oRosa damascena Mill. rommature bushes using thidiazuron. Journal o HorticulturalScience and Biotechnology. 76:30-34.

Lin, C. S., C. C. Lin and W. C. Chang (2004). Efecto thidiazuron on vegetative tissue-derived somaticembryogenesis and owering o bamboo Bambusa edulis.Plant Cell, issue and Organ Culture. 76:75-82.

Murashige, . and F. Skoog (1962). A revised medium orrapid growth and bioassays with tobacco tissue cultures.Physiologia Plantarum. 15:473-497.

aylor, N. J., M. E. Light and J. Van Staden (2005). In vitroowering oKniphofa leucocephala: inuences o cytokinins.

Plant Cell, issue and Organ Culture. 83:327-333.Vu, N. H., P. H. Anh and D. . Nhut (2006). Te role o sucroseand diferent cytokinins in the in vitro oral morphogenesiso rose (hybrid tea) cv. First Prize. Plant Cell issue andOrgan Culture. 87:315-320.

Wang, G. Y., M. F. Yuan and Y. Hong (2002). In vitro owerinduction in roses. In Vitro Cellular and DevelopmentalBiology- Plant. 38:513518.

ab. 2 Efect o diferent photoperiod on multiple shootormation oRosa hybrida L. cv.Red Masterpiece

Photoperiod(light/dark)

Number o shoots per explant(Mean SE)

12/12 2.6 0.4

16/8 2.8 0.48/16 2.0 0.2

Fig. 1. In vitro owering o the Rosa hybrida L. cv. Red Mas-terpiece shoots cultured on MS medium supplemented with2mg/l BA or 9 weeks.

ab. 3 Efect o subculture time on in vitro owering inRosahybrida L.cv. Red Masterpiece

Subculture period(weeks)

Explants ower in vitro(%)

3 0

6 0

9 50

12 0

15 0