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HORTSCIENCE 27(5):450-452. 1992.

In Vitro Propagation ofHeliconia

psittacorum by Bud CultureM.J. Nathan, C.J. Goh, and P.P. Kumar

Department of Botany, National University of Singapore, Lower KentRidge Road, Singapore 0511

Additional index words. multiple shoots, rhizome, tissue culture, micropropagation

Abstract. A protocol was developed for in vitro propagation ofHeliconia psittacorum

L.f. by culture of terminal and axillary buds of rhizomes. Cultures were initiated onmodified Murashige and Skoog (MS) medium containing 40 M BA, 150 ml coconut

water/liter, 30 g sucrose/liter, and 2 g Gelrite/liter. Shoot multiplication was achievedon the above medium without coconut water, but supplemented with 10 M BA. Shootswere rooted on MS basal medium and successfully acclimated to greenhouse conditions.

Chemical names used:N- (phenylmethyl)-1H- purin-6-amine (BA).

The genusHeliconia L. is represented by150 species of tropical plants that consti-tute the family Heliconiaceae (Donselman andBroschat, 1986; Keng, 1978). Generally,members of the Heliconiaceae are vegeta-tively propagated using rhizomes (Broschatand Donselman, 1983b). The inflorescencesare terminal, brightly hued, and can be eithererect or pendulous depending on the species,

bearing two or more boat-shaped bracts aris-ing from a central axis (Broschat and Don-selman, 1983a).

Vegetatively propagated rhizomes ofHel-iconia spp. cannot now be imported into sev-eral countries without stringent quarantinecontrols because of the discovery of strainsofPseudomonas solacearum (Smith) Smithin diseased plants grown from rhizomes of

Heliconia spp. directly imported from Ha-waii (Akiew et al., 1990).

Tissue culture has been used as a rapidmeans of vegetative propagation in the or-namental horticulture industry, enabling theproduction of disease-free propagules that can

be readily disseminated (Chu, 1985). Cur-rently, such methods are not available for thepropagation ofHeliconia spp. Hence, thepresent study was undertaken to develop

procedures for in vitro propagation ofH.psittacorum from excised axillary and ter-minal buds. Pretreatments of the explantsource and manipulation of the componentsof the culture medium were carried out tooptimize micropropagation.

Preparation of explants. Shoot explantswere obtained from developing rhizomes ofH. psittacorum growing under field condi-

tions (Fig. 1A). Roots and leaf bases of therhizomes were removed before being washed

Received for publication 26 June 1991. Acceptedfor publication 9 Dec. 1991. We thank the man-agement of Jurong Birdpark, Singapore, for pro-viding plant materials and Ian M. Turner for adviceon statistical analysis. The cost of publishing this

paper was defrayed in part by the payment of pagecharges. Under postal regulations, this papertherefore must be hereby marked advertisement

solely to indicate this fact.

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in tap water. The rhizomes were blotted dryand stored in polyethylene bags for 0, 1, 3,5, or 7 days at room temperature (25 2C).Tissue blocks, 1 cm

3, containing either the

apical bud or the prominent axillary bud wereexcised and washed in running tap water for15 min, rinsed in 80% ethanol for 30 sec,and immersed for 15 min in a 0.8% NaOClsolution with continual agitation. Followingsurface sterilization, buds were rinsed twicewith sterile distilled water for 5 min eachtime. The outer tissues of each bud werefurther trimmed to 3 mm3 and used as ex-plants (Fig. 1B). Before culture, explants wereagain surface-sterilized with a 0.4% NaOClsolution for 10 min and rinsed with steriledistilled water as above.

Culture conditions. The culture mediumused was Murashige and Skoog (1962) in-organic salts supplemented with the follow-ing (in mgliter

-1): thiamine-HCl, 0.5; myo-

inositol, 100; sodium hydrogen phosphate,170; adenine sulphate, 80; and sucrose,30,000. Gelrite, 2 gliter

-1(Merck & Co.,

Rahway, N.J.), was used as the solidifyingagent. The combination noted was desig-

nated as the basal medium. Coconut water(150 mlliter

-1), BA, and 1H- indole-3-acetic

acid (IAA) were incorporated into this me-dium as required. The coconut water (liquidendosperm) used in these experiments wasobtained from 6- to 7-month-old green co-

conuts (Cocos nucifera L. Malayan Tall).It was deproteinated by boiling, then filteredthrough one sheet of Whatman 4 filter paper(Whatman Intl., Maidstone, England) andstored at -20C. The pH of the media wasadjusted to 5.7 0.1, and the media wereautoclaved for 20 min at 121C.

Cultures were established in 150-ml bo-rosilicate test tubes with 15 ml of mediumand one bud per tube. Multiplication androoting of shoots was carried out in MagentaGA-7 containers (Sigma Chemical Co., StLouis) with 60 ml of the appropriate mediumand four shoots per container. All cultureswere incubated at 26 1C with a 16-h pho-

toperiod provided by cool-white fluorescentlamps. The lamps provided a photosyntheticphoton flux of 60 molm

-2s

-1at the level

of the explants as measured with a LI-CORLI-185B quantum/radiometer/photometer (LI-COR, Lincoln, Neb.). Shoots were subcul-tured at 3-week intervals for the first 2 to 3months and at 6-week intervals during shootproliferation.

All studies were conducted with eight to12 replicates per treatment, with at least twoindependent experiments of a completelyrandomized design. Percentage data weresubjected to arcsin transformation before one-or two-way analysis of variance (ANOVA)where indicated, using STATGRAPHICSsoftware (Statistical Graphics Corp., Rock-ville, Md.).

Establishment of buds in culture. Buds wereplaced on basal medium containing coconutwater; BA at 0, 10, 20, 40, or 80 M; andIAA at 0, 1, or 10 M in factorial combi-nation (Table 1). Treatments with BA stim-ulated swelling of the buds in the first 4 weeksof culture. Color of the buds changed fromcreamy white to green within 7 to 14 days.In the absence of BA, bud development wasarrested. With increasing concentrations ofBA from 10 to 40 M, the buds enlarged.However, at 80 M BA the buds remained

green with no enlargement. The majority ofbuds on medium with 40 M BA and 0 MIAA continued to enlarge. There was no ob-servable difference in buds on 40 M BAand varying concentrations of IAA. Two-wayANOVA indicated that at all levels of BA,

the concentrations of IAA tested had no sig-nificant effect on survival (range 50% to 84%)and enlargement of the buds (Table 2). Also,there was no significant interaction betweenBA and IAA at the levels tested. The max-imum number growing cultures (53%) wererecorded at 40 M BA at the end of 6 weeksof incubation (Table 1). This treatment yieldeda significantly better response than either 10or 20 M BA (1

2= 15.56 and 13.57, re-

spectively, at P < 0.001). Since ANOVAindicated no significant effect of IAA on budenlargement (response), data within eachconcentration of BA were pooled before thechi-square test. After 8 weeks of culture, ex-plants from all treatments were transferredto medium containing 40 M BA.

The maximum number of buds suitable forculture was obtained from rhizomes storedfor 7 days in polyethylene bags (Table 3).No fungal or bacterial infection was ob-served during storage of the rhizomes, andcontamination in vitro was the lowest (33%)in cultures established from these buds ascompared to buds from rhizomes stored forless than 1 day (>90% contamination). Al-though the cut edges turned brown, the rhi-zomes remained turgid up to 7 days in storage.Preliminary studies indicated that storage inexcess of 9 days under the same conditions

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resulted in excessive browning and desic-cation of the rhizomes. Storage of rhizomesbefore explanting also enhanced bud sur-

vival. After 6 weeks in culture, 64% of budsfrom rhizomes stored for 7 days respondedwell in the initiation medium containing 40M BA, in contrast to