In vitro studies on Dracaena fragrans cv. Massangeana revealed that young stem segments were capable of

proliferating shoots on agar-solidified Murashige and Skoog (MS) basal medium containing different combinations

and concentrations of BAP and NAA. Highest percentage of explants forming shoots was obtained on medium

supplemented with 3.0 mgll BAP and 0.1 mgll NAA. The highest number ofshoots per explant occurred on medium

containing 2.0 mgll BAP only. Highest percentage of callus formation and highest mean fresh weight of callus from

young stem segments were achieved on MS medium supplemented with 1.0 mgll2,4-D. Adventitious rooting occurred

after transferring excised shoots onto a hormone-free MS medium. Rooting was 100% for shoots derived from media

with 0-2.0 mg/l BAP and a relatively low concentration of NAA (0.1 mg/l)

Abdul Aziz, Maheran

Abdul Rashid, Azmi

Ooi, H. L.

Universiti Putra Malaysia Institutional Repository

Pertanika J. Trap. Agric. Sci. 19(2/3): 123-127 (1996)ISSN: 0126-6128© Penerbit Universiti Pertanian MalaysiaIn Vitro Responses of Dracaena fragrans cv. Massangeanato Growth RegulatorsMAHERA A. AZIZ, H.L. 001 1 and A.A. RASHIDDepartment of Agronomy and HorticultureFaculty of AgricultureUniversiti Pertanian Malaysia43400 UPM Serdang, Selangor, Malaysia1Hits Enterprise (Malaysia) Sdn Bhd19 ]alan SSj8B, Subang ]aya47000 Petaling ]aya, Selangor, MalaysiaKeywords: Dracaena fragrans, Murashige and Skoog (MS) m.edium, HAP, NAA, 2,4-D, shootformation, callus, rootingABSTRAKKajian in vitro ke atas Dracaena fragrans cv. Massangeana mendapati segmen batang muda berupayamembentuk pucuk diatas media pepejal Murashige dan Skoog (MS) yang mengandungi berbagai kombinasi danparas BAP dan NAA. Peratus eksplan membentuk pucuk paling tinggi diperolehi pada medium yang ditambahdengan 3.0 mgjl BAP dan 0.1 mgll NAA. Jumlah pucuk se eksplan paling tinggi berlaku pada medium yangmengandungi 2.0 mgll BAP sahaja. Peratus pembentukan kalus dan min berat basah kalus dari segmen batangmuda adalah paling tinggi pada medium MS yang dibekalkan dengan 1.0 mgll 2,4-D. Akar adventitius terbentukselepas pucuk dialih ke medium MS tanpa hormon. Pengakaran adalah 100% bagi pucuk yang dialih dari mediumyang mengandungi 0-2.0 mgll BAP dan kepekatan NAAyang rendah (0.1 mgll).ABSTRACTIn vitro studies on Dracaena fragrans cv. Massangeana revealed that young stem segments were capable ofproliferating shoots on agar-solidified Murashige and Skoog (MS) basal medium containing different combinationsand concentrations of BAP and NAA. Highest percentage of explants forming shoots was obtained on mediumsupplemented with 3.0 mgll BAP and 0.1 mgll NAA. The highest number ofshoots per explant occurred on mediumcontaining 2.0 mgll BAP only. Highest percentage ofcallus formation and highest meanfresh weight ofcallus fromyoung stem segments were achieved on MS medium supplemented with 1.0 mgll2,4-D. Adventitious rooting occurredafter transferring excised shoots onto a hormone-free MS medium. Rooting was 100% for shoots derivedfrom mediawith 0-2.0 mgll BAP and a relatively low concentration of NAA (0.1 mgll).INTRODUCTIONDracaenas are woody monocotyledonsbelonging to the family Agavaceae. Theyare popular foliage ornamentals in tropicaland temperate regions and are highlydesirable as indoor plants and for outdoorlandscaping. Dracaena fragrans cv. Massan-geana, characterized by its sword-shapeddark green leaves with a yellow striperunning along the centre, is among thedracaena cultivars fast gaInIng the atten-tion of commercial growers. However,propagation by conventional methods isslow and therefore production of clonalplants by tissue culture will be useful tomeet the increasing demand for plantingmaterial. Establishment of a plant regen-eration system through direct organogenesisor via callus is also a prerequisite to furtherin vitro genetic manipulation of theMAHERAN A. AZIZ, H.L. 001 AND A.A. RASHIDcultivar. Tissue culture procedures havebeen described for D. godseffiana (Millerand M urashige 1976) , D. marginata cv.Tricolor (Chua et al. 1981), D. deremensiscv. Warneckii (Debergh 1975), D. congestaand D. invisa (Debergh and Maene 1981)and green-foliage D. fragrans (Dragan1989). There is no report on D. fragranscv. ·Massangeana. The present paper aimsto assess the in vitro response of D. fragrans cv.Massangeana to various growth regulators.MATERIALS AND METHODSVigorously growing shoots taken from 1-2year-old plants, with leaves removed, werewashed under running tap water for 30min. They were surface-sterilized for 20min in 10% vjv Clorox with a few drops ofTween 20 emulsifier, and rinsed in fivechanges of sterile distilled water. Thematerial was further immersed in 5% vjvClorox for 2 min, rinsed five times withsterile distilled water and finally cut intosegments 3-4 mm thick. Explants werecultured individually on 10 ml of Mura-shige and Skoog (MS) (1962) medium with3.0% sucrose, 0.7% Bacto-agar and theappropriate combinations and concentra-tions of auxin andjor cytokinin. For shootinduction, 0-0.3 mgjl a-napthaleneaceticacid (NAA) in combination with 0-30 mgj1 benzylaminopurine (BAP) was used. Forthe initiation and proliferation of callus,stem explants were cultured on MSmedium containing 0.5-2.0 mgjl 2,4-di-chlorophenoxy-acetic acid (2,4-D), and onMS medium containing 0.5 mgjl BAP in'combination with 1.0 and 2.0 mgjl indole-butyric acid (IBA). Shoots which attained5-15 mm in height were separated andcultured on a hormone-free MS medium topromote root formation and shoot elonga-tion. All cultures were incubated under a16-hour photoperiod using white fluores-cent tubes (Philips, TLD 36wj54) at anirradiance of 65 uE m-2 S-1 and a tempera-ture of 27 ± 1°C. The experiments werearranged in a completely randomizeddesign with three replications for eachtreatment and four explants per replicate.Each experiment was repeated three times.In vitro regenerated plantlets were removedfrom culture vessels, washed thoroughly toremove traces of nutrient medium andplanted in small pots containing a mixtureof vermiculite and sand (1: 1). For the firstweek of transfer the plantlets were coveredwith plastic perforated with small holes tomaintain a high humidity. Plantlets weresubsequently transferred to larger pots andplaced outdoors.RESULTSEffect of BAP and NAA on Shoot FormationWithin 20 days of culture, adventitiousbuds were induced at the cut surface ofstem segments placed on various combina-tions of BAP and NAA. In most explants,moderate callus formation preceded budformation. On medium supplemented witha higher level of BAP (3.0 mgjl) butwithout NAA, adventitious buds wereinduced directly from the explants withoutan intervening callus. Shoots developedfrom the adventitious buds by week 7.Table 1 summarizes the effect of BAPand NAA on shoot formation from stemexplants of D. fragrans cv. Massangeanaafter 8 weeks in culture. Shoot formationoccurred in all treatments including thecontrol. In treatments containing 0.1 mgjlNAA the percentage of shoot formationincreased with increasing levels of BAP,with 3.0 mgjl BAP producing the highestpercentage of explants with shoots (88.8 % ).The highest number of shoots per explant(1.44 ± 0.10) occurred on medium contain-ing 2.0 mgjl BAP but without NAA·.Highest mean length of shoots (3.68 ±1.47 mm) was also attained on the samemedium (Table 1).124 PERTA IKA J. TROP. AGRIC. SCI. VOL. 19 NO. 2/3, 1996IN VITRO RESPONSES OF DRACAENA FRAGRANS CV. MASSANGEANA TO GROWTH REGULATORSTABLE 1Effect of BAP and NAA on shoot formation from stem segments of Dracaena fragranscv. Massangeana at week 8BAP NAA Exp1ants Number of shoots Mean length(mg/1) (mg/1) with shoots per explant of shoots(%) (mm)0 0 44.4 0.77 ± 0.29 1.94 ± 0.631.0 0 22.2 0.22 ± 0.10 1.99 ± 1.262.0 0 77.7 1.44 ± 0.10 3.68 ± 1.473.0 0 22.2 0.22 ± 0.10 0.33 ± 0.190 0.1 44.4 0.44 ± 0.10 1.21 ± 0.551.0 0.1 55.5 0.88 ± 0.22 2.47 ± 0.672.0 0.1 66.6 0.66±0.19 2.22 ± 0.483.0 0.1 88.8 0.88 ± 0.11 1.44 ± 0.480 0.3 22.2 0.55 ± 0.10 0.33 ± 0.191.0 0.3 66.6 0.66 ± 0.01 1.77 ± 0.482.0 0.3 55.5 0.55 ± 0.22 1.10 ± 0.613.0 0.3 66.6 0.66 ± 0.01 1.66 ± 0.57Values given are ± standard errorsTABLE 2Callus induction on stem exp1ants of Dracaenafragrans cv. Massangeana at week 6Concentration Explants with Mean fresh Response(mg/l) callus (%) weight of callus(g)0.5 2,4-D 77.7 0.075a fc1.0 2,4-D 88.8 0.135a hfn2.0 2,4-D 44.4 0.082a fcn0.5 BAP +1.01BA 77.7 0.095a hcn0.5 BAP +2.01BA 55.5 0.046a hnValues with a similar letter in a column indicate no significant difference at 5% probabilitylevel.Key to Table: h = hard, f = friable, c = chlorophyllous, n = non-chlorophyllousEffect of 2,4-D and Combinations of BAP andIBA on Callus Initiation and ProliferationResponse of stem explants after six weeks ofculture on media with different concentra-tions of 2,4-D, and combinations of BAPand IBA is shown in Table 2. Callus wasinitiated at the cut surface of explants byweek 2 and was obtained in all treatments.Highest percentage of explants with callusand the highest mean fresh weight of callusPERTANIKA J. TROP. AGRIC. SCI. VOL. 19 NO. 2/3, 1996 125MAHERAN A. AZIZ, H.L. 001 AND A.A. RASHIDwere obtained on medium with 1.0 mg/12,4-D. Mean fresh weight of callus pro-duced did not differ significantly among thetreatments. The callus produced on me-dium with 1.0 mg/l 2,4-D were either hardor friable and non-chlorophyllous. At ahigher level of 2,4-D (2.0 mg/l) callusproduced were friable, mostly yellowish,slow growing and gradually turned brownafter the third week of culture. MS mediumwith 0.5 mg/l BAP and 1.0 mg/l IBAproduced callus which were hard andwhich turned green after 6 weeks inculture. Shoot differentiation from calluswas observed in both media containing 0.5mg/l BAP with 1.0 mg/l IBA and 0.5 mg/lBAP with 2.0 mg/l IBA by the fifth week ofculture.RootingIn vitro proliferated shoots excised andtransferred to hormone-free MS mediumrooted by week 2. Table 3 shows the effectof BAP and NAA concentrations (usedpreviously to induce the shoots) on rooting.Shoots derived from media with 0.1 mg/lNAA and BAP 0-2.0 mg/l showed 100%rooting on transfer to hormone-free MSmedium. Rooting was also 100% withshoots derived from a previously hor-mone-free medium (control). Shoots in-duced on media with 1.0-2.0 mg/l BAP anda higher NAA concentration (0.3 mg/l)showed a lower rooting response in thehormone-free MS medium. It is alsoevident from Table 3 that roots generallytend to be shorter as their numberincreases.DISCUSSIONShoot formation from stem explants ofD. fragrans cv. Massangeana occurred inall combinations of BAP and NAA tested,including the control. An increasing trendin shoot formation was attained when 0.1mg/l NAA was added to the increasingBAP levels, indicating a stimulatory effectof the auxin at a relatively low concentra-tion. In media without NAA and in mediasupplemented with a higher level (0.3 mg/l)of NAA, shoot formation was variable. Inall treatments except in a medium with aTABLE 3Rooting response of shoots on hormone-free MS mediumPrevious treatment % of Mean number Mean lengthshoots with of roots of rootsBAP NAA roots per plantlet (mm)(mg/l) (mg/l)0 0 100.0 1.33 ± 0.33 15.17 ± 5.291.0 0 33.3 2.00 ± 0.01 9.00 ± 0.012.0 0 50.3 0.50 ± 0.40 17.50 ± 4.283.0 0 66.3 1.00±0.10 26.50 ± 5.300 0.1 100.0 1.00 ± 0.10 16.67 ± 5.691.0 0.1 100.0 2.00 ± 0.57 11.33 ± 3.812.0 0.1 100.0 1.66 ± 0.01 19.50 ± 1.893.0 0.10 0.31.0 0.3 50.0 1.33 ± 0.66 18.33 ± 9.272.0 0.3 33.3 2.00 ± 0.01 10.50 ± 0.103.0 0.3Values given are ± standard errors- indicates no shoots were transferred126 PERTANIKA J. TRap. AGRIC. SCI. VOL. 19 NO. 2/3, 1996IN VITRO RESPONSES OF DRACAENA FRAGRANS CV. MASSANGEANA TO GROWTH REGULATORShigher BAP concentration (3.0 mg/l) andwithout NAA, moderate callus formationpreceded shoot formation. The productionof shoot and callus even on the hormone-free medium strongly implies the presenceof endogenous hormones within the stemtissues of D. fragrans cv. Massangeana. Easeof shoot and callus formation from stemexplants in comparison to leaf and shoot tipexplants has been reported in D. deremensiscv. Warneckii (Debergh 1975).Dracaenas are among the few mono-cotyledons which possess a cambium intheir stems (Zimmermann and Tomlinson1970; Esau 1977). The occurrence of thecambium may have facilitated shoot andcallus induction from stem exp1ants ofD. fragrans cv. Massangeana.In the study on callusing ability of stemsegments of D. fragrans cv. Massangeana,2,4-D alone or combinations of BAP andIBA were effective in stimulating callusformation. Mean fresh weight of callus didnot differ significantly among the treat-ments, but the percentage of callus forma-tion varied. The gradual browning of callusobserved on 2.0 mg/l 2,4-D indicated thedeleterious effect of the auxin at thisconcentration. Other researchers havereported a similar effect of 2,4-D at 2.0mg/l and above on other dracaena speciesand cultivars investigated (Debergh 1975;Chua et at. 1981; Dragan 1989). Debergh(1975) showed the addition of 1.0 or 2.0mg/l kinetin to 2.0 mg/l 2,4-D couldneutralize the effect of 2,4-D, resulting ina more organized type of callus.Rooting of in vitro shoots of D. fragranscv. Massangeana was achieved on ahormone-free MS medium. Successful root-ing of other dracaena species required theinclusion of low levels of auxins in therooting medium such as IBA (0.1-2.0 mg/l)or NAA (0.1-1.0 mg/1) (Debergh 1975;Chua et at. 1981; Dragan 1989), or using alow salt MS medium (Debergh and Maene1989). This implies a species-specific root-ing behaviour of dracaena species under invitro conditions. It is also evident that arelatively low supplement of auxin (0.1 mg/1 NAA) to BAP (0-2.0 mg/l) in the shootinduction and proliferation medium has astimulatory effect on rooting of in vitroshoots of D. fragrans cv. Massangeanawhen placed on a hormone-free medium.The present study demonstrates thetotipotent capacity of stem segments ofD. fragrans cv. Massangeana to regenerateplantlets in vitro.REFERENCESCHUA, B.D., J.T. KUNISAKI and Y. SAGAWA.1981. In vitro propagation of Dracaena margi-nata cv. Tricolor. HortScience 16(4): 494.DEBERGH, P. 1975. Intensified vegetative multi-plication of Dracaena deremensis. Acta Horti-culturae 54: 83-92.DEBERGH, P. and L.J. MAE E. 1981. A schemefor commercial propagation of ornamentalplants by tissue culture. Scientia Horticulturae14: 335-345.DEBERGH, P. and L.J. MAE E. 1989. Cordylineand dracaena. In Handbook of Plant CellCulture, Ornamental Species, ed. P.V. Ammir-ato, D.R. Evans, W.R. Sharp and Y.P.S.Bajaj, p. 337-351. New York: McGraw-Hill.DRAGAN, V.V. 1989. In vitro propagation of greenfoliage Dracaena fragrans Ker. Plant Cell Tissueand Organ Culture 17: 13-19.ESAU, K. 1977. Anatomy of Seed Plants, p. 317-318.New York: Wiley.MILLER, L.R. and T. MURASHIGE. 1976. Tissueculture propagation of tropical foliage plants.In vitro 12(12): 797-813.MURASHIGE, T. and F. SKOOG. 1962. A revisedmedium for rapid growth and bioassays withtobacco tissue cultures. Physiologia Plantarum15: 473-497.ZIMMERMANN, M.H. and P.B. TOMLINSON.1970. The vascular system in the axis ofDracaena fragrans (Agavaceae). 2. Distributionand development of secondary vascular tissue.J. Arnold Arb 51: 478-491.(Received 18 January 1995)(Accepted 30 August 1996)PERTANIKA J. TROP. AGRIC. SCI. VOL. 19 NO. 2/3, 1996 127

In vitro responses of dracaena fragrans cv. massangeana to growth regulators

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In vitro responses of dracaena fragrans cv. massangeana to growth regulators

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