Plant Cell, Tissue and Organ Culture 70: 259–268, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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In vitro culture of Cedrela fissilis Vellozo (Meliaceae)

Eduardo da Costa Nunes, Carolina Volkmer de Castilho, Fabio Netto Moreno & Ana MariaViana∗Departamento de Botânica, Universidade Federal de Santa Catarina, 88040-900 - Florianopolis, Santa Catarina,Brazil (∗requests for offprints; Fax: +48-3319258; E-mail: amarna@floripa.com.br)

Received 6 February 2001; accepted in revised form 27 December 2001

Key words: acclimatization, callus culture, rooting, shoot culture

Abstract

An efficient micropropagation protocol was developed for Cedrela fissilis (Meliaceae) using nodal segments fromjuvenile origin for axillary shoot proliferation. Shoot proliferation was significantly affected by salt formulation,explant origin and 6-benzyladenine concentration. Maximum multiplication rates (6–7 new plants were producedin the second subculture cycle per single cotyledonary node cutting) were achieved on Murashige and Skoogmedia supplemented with 1.25–5.0 µM 6-benzyladenine. Addition of α-naphthaleneacetic acid to these mediacaused significant inhibition on shoot proliferation and growth and stimulated callus formation. High frequencycallus initiation and synergistic effects on callus growth were achieved on Murashige and Skoog medium sup-plemented with 6-benzyladenine at either 1.25, 2.5 or 5.0 µM combined, respectively, with 2.5, 1.25–5.0 or5.0 µM α-naphthaleneacetic acid. Rooting was achieved, after 10–12 days, with 87–100% of the node cuttingson half strength Murashige and Skoog medium either without growth regulators or supplemented with 2.5 µMindole-3-butyric acid. Regenerated plants were successfully acclimatized on sterilized sand, for 21 days, but forfurther plant development the sand:soil (1:1) mixture was the best substrate. The survival rate of plantlets underex vitro conditions was 100% after 3 months. The optimized micropropagation and callus culture protocols offerthe possibility to use the organ/cell culture techniques for vegetative propagation, cryopreservation and secondarymetabolism studies.

Abbreviations: BA – 6-benzyladenine; IAA – indole-3-acetic acid; IBA – indole-3-butyric acid; NAA – α-naphthalene acetic acid; 2,4-D – dichlorophenoxyacetic acid; AR – Anderson Rhododendron; WPM – Lloyd andMcCown; SH – Schenk and Hildebrandt; W – White; B5 – Gamborg B5; KM – Kao and Michayluk; PGRs – plantgrowth regulators

Introduction

Cedrela fissilis Vellozo (Meliaceae) is a valuable, fastgrowing timber tree species of the endangered AtlanticForest (South Brasil). Its wood has excellent multipleproperties and has a generalized use in the furniture,navy and construction industries. An oil with aro-matic and medicinal properties is obtained from itsdistilled wood (Reitz et al., 1979). Like several mem-bers of the Meliaceae, it is a potentially very valuableplantation species but damage by shoot borers is themain constraint to its large scale utilization in refor-estation programs (Haines, 1994). It is attacked by

Hypsipyla grandella Zeller (Lepidoptera, Pyralidae)and Oncideres sp. (Cerambicydae) and a suitable con-trol method for it has not yet been found. According toMaruyama et al. (1989), alternative methods includingsilviculture control and tree-improvement programshave been considered.

The few studies available on in vitro culture of thegenus Cedrela are on micropropagation by shoot tipculture, in vitro conservation and organogenesis byhypocotyl segment culture of C. odorata (Maruyamaet al., 1989; Maruyama et al., 1997a,b; Cerdas etal., 1998). Therefore, the development of in vitroculture systems for C. fissilis will be the basis to en-

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sure the introduction of genes for insect resistance,clonal propagation of selected resistant clones and theapplication of biotechnological approaches for ger-mplasm conservation, synthetic seed technology usingin vitro derived vegetative propagules and secondarymetabolism studies.

The aim of the work reported here was to developefficient and reproducible methods for shoot initiation,rooting, acclimatization and callus culture initiation ofC. fissilis from juvenile seedling stock explants.

Materials and methods

Plant material

The experiments were conducted with juvenile stockoriginated from seedlings produced under sterile con-ditions. Seeds originating from São Paulo State wereharvested from selected trees by Instituto Florestal deSão Paulo (Brasil) and held at 5◦C until use.

Seed sterilization and germination

Seeds were surface sterilized in commercial bleach(2.5% active chlorine) with 2–3 drops of Tween 20for 75 min, rinsed three times for 10 min in steriledistilled water and cultured on Murashige and Skoogbasal medium (MS, Sigma Co., USA) (Murashige andSkoog, 1962) supplemented with 2% (w/v) sucroseand 0.2% (w/v) Phytagel (Phytagel, Sigma Co., USA).The pH values were adjusted to 5.8 before autoclavingat 121 ◦C for 18 min. The cultures were incubated at25 ◦C under 16-h photoperiod and photon flux of 20–25 µmol m−2 s−1 at plant level, supplied by PhilipsTDL fluorescent light tubes. These culture conditionswere used in all the experiments mentioned belowunless otherwise stated.

Establishment of shoot cultures

To test the effects of BA and NAA on the estab-lishment of shoot cultures, cotyledonary node cut-tings (0.8–1.0 cm) obtained from 30-day-old seedlingsaseptically grown in the light were cultured on MSmedium supplemented with 2% (w/v) sucrose, 0.2%(w/v) Phytagel and combinations of different concen-trations (0, 1.25, 2.5 and 5.0 µM) of these PGRs. Inthe experiments to assess the effect of explant originand culture media on shoot culture initiation, cotyle-donary node cuttings and epicotyl cuttings consistingof the most distal epicotyl node and the apical bud,

were excised from 30-day-old seedlings asepticallygrown in the light and cultured on MS, SH (Schenkand Hildebrandt, 1972), KM (Kao and Michayluk,1975), AR (Anderson, 1978), W (White, 1963), WPM(Lloyd and McCown, 1981) or B5 (Gamborg et al.,1968) medium supplemented with 2% (w/v) sucrose,0.2% (w/v) Phytagel and 2.5 µM BA. There were 10–15 replicates per treatment and the experiment wasrepeated twice. Data of frequency of shoot formation,number of shoots initiated per explant and shoot lengthwere recorded after 30 or 45 days, depending on theexperiment.

Rooting

In evaluations of the ability of different auxins to sup-port root initiation single node cuttings excised fromshoots produced in the multiplication medium were in-oculated on MS medium supplemented with 2% (w/v)sucrose, 0.2% Phytagel and either 2,4-D, IBA, NAAor IAA present at 0, 1.25, 2.5 or 5.0 µM. To as-sess the separate effects of different basal media MS,B5, SH, W or WPM salts were supplemented with5.0 µM IBA. In the experiments to test the effect ofMS concentrations, either full or half strength MS (1/2MS) basal medium was supplemented with 2% (w/v)sucrose, 0.2% Phytagel and 0, 1.25, 2.5 or 5.0 µMIBA. There were 10–30 replicates per treatment. Dataof frequency of rooting, number of roots initiated perexplant and root length were recorded after 35 days.

Acclimatization

For the experiments on acclimatization, 30-day-old re-generated plants rooted on MS medium were removedfrom the test tubes, washed free of Phytagel and thentransferred to seed trays containing steam-sterilizedmedium grade river sand covered with a PVC film, forassessment during 12 weeks of survival and growth.After the first week the PVC film was gradually re-moved and the plants were exposed to the culture roomatmosphere (average 70% RH). After 21 days theywere transferred to 250 ml plastic pots containing a1:1 mixture of sand and soil. At all stages of acclimat-ization, the plants were kept in the culture room, underidentical culture conditions to those used for the tissueculture studies. Three replicates having 15 microplantseach were used per treatment.

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Establishment of callus cultures

Cotyledonary node cuttings (0.8–1.0 cm) obtainedfrom 30-day-old seedlings aseptically grown in thelight were used for callus initiation. In experimentsto test the effects of BA and NAA, the MS mediumwas supplemented with 2% (w/v) sucrose, 0.2% (w/v)Phytagel and combinations of different concentrations(0, 1.25, 2.5 and 5.0 µM) of these PGRs. There were10–15 replicates per treatment. Data of frequency ofcallus formation and callus fresh weight were recordedafter 45 days.

Statistical analysis

Significance of the treatment effects was determinedby analysis of variance employing a completely ran-dom design. Variations among treatment means wereanalysed by the Least Significant Difference (LSD)test at the 95% probability level. All experiments wereconducted at least twice and analysed data are meansof independent experiments.

Results

Seed germination and initial seedling growth

Seed germination was initiated after 3–4 days andreached maximum 95% within 10–12 days in culture.After 4 weeks of in vitro growth, the seedlings reacheda height of approximately 8.2±1.0 cm, root length of8.6±1.2 cm, 0.505±0.1 g fresh weight, with one coty-ledonary node, two epicotyl nodes, one apical bud and3.1±0.5 leaves.

Shoot initiation and multiplication

Shoots were produced in 73–100% of the culturesin all treatments except when 1.25–2.5 µM BA werecombined, respectively with 2.5 and 2.5–5.0 µM NAA(data not shown). In some treatments, callus prolif-eration was observed at the base of the two axillarybuds and the shoots regenerated were a combinationof axillary and adventitious shoots (Figure 1A). Fig-ure 2 shows the results of shoot proliferation fromcotyledonary node cuttings on BA and/or NAA. Thebest results were obtained with 1.25–2.5 µM BA (2.6–2.7 shoots per explant). In all treatments when NAAwas used alone and in the control without PGRs, themaximum number of shoots formed per explant was

Table 1. The effect of basal media salt formulation supple-mented with 2.5 µM BA on the frequency of morphogenicresponses and shoot initiation from cotyledonary and epicotylnode cuttings of Cedrela fissilis after 30 days

Explant Basal Cultures Number Average

medium with of shoot

shoots shoots/ length

(%) explant (cm)

Cotyledonary Node MS 100aa 3.2ab 1.7ab

SH 92 a 2.3 b 0.6 b

KM 85 a 2.2 b 1.2 ab

AR 58 c 1.3 bc 1.0 ab

W 64 c 1.7 bc 0. 3 c

WPM 100 a 2.6 ab 1.3 ab

B5 100 a 2.2 b 0.7 b

Epicotyl Node MS 99 a 2.7 ab 1.1 ab

SH 100 a 1.1 c 0.5 b

KM 100 a 1.8 bc 0.6 b

AR 78 b 1.9 bc 0.6 b

W 57 c 1.0 c 0.3 c

WPM 90 a 2.2 b 1.1 ab

B5 60 b 1.3 bc 0.5 b

aValues shown were proportions obtained from 3 replicates of12–15 cultures. bValues (N= 10–20) within a single columnfollowed by a similar letter were not significantly differentaccording to the LSD at the 5% level.

1.8. The strongest inhibition effects in the sprout-ing of axillary buds were observed in combinationsof 1.25 µM BA and 5.0 µM NAA, 2.5 µM BA and2.5–5.0 µM NAA, and 0.0 µM BA and 2.5–5.0 µMNAA. The highest number of nodes per explant (5.5),which could then be excised for further multiplicationand production of new microplants, was achieved at2.5 µM BA. Significant inhibition in the number ofnodes per explant (<2.0) was observed in combina-tions of 1.25–2.5 µM BA and 5.0 µM NAA (Figure 3).Shoot length was best when 5.0 µM BA was used with2.50 µM NAA (Figure 4). This culture condition pro-duced significantly higher shoot elongation (>3.0 cm).Shoot growth was drastically inhibited at 5.0 µM NAAand in combinations of either 1.25, 2.5 or 5.0 µM BA,respectively with 5.0, 2.5–5.0 or 5.0 µM NAA.

Table 1 shows that some tissue culture media sup-plemented with 2.5 µM BA had variable effects onmorphogenesis and growth of cotyledonary and epi-cotyl node cuttings. Shoot initiation was observed in85–100% of the cultures initiated either from cotyle-donary nodes on MS, SH, KM, WPM and B5 or fromepicotyl nodes on MS, SH, KM and WPM. Rhizogen-

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Figure 1. Shoot cultures of Cedrela fissilis originated from cotyledonary node cuttings. (A) Note the callus and the new buds formed at the baseof both axillary buds, after 45 days. (B) Rooted shoot on plain MS medium after 15 days of transferring. (C) Acclimatized micropropagatedplants. (D) Calli from cotyledonary node cuttings produced on MS medium supplemented with BA and NAA. Note the globular aspect andadventitious roots. Bars = 1 cm.

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Table 2. The effect of auxins on rooting of in vitro raisednode cuttings of Cedrela fissilis after 35 days

Growth Concentration Rooting Number of

regulators (µM) (%) roots/explant

2,4-D 0.00 40ca 1.0 cb

1.25 20 d 1.5 b

2.50 20 d 1.0 c

5.00 0 ∗

IBA 0.00 45 c 1.0 c

1.25 60 b 1.5 b

2.50 55 c 1.8 b

5.00 85 a 1.9 b

NAA 0.00 65 b 1.0 c

1.25 10 d 5.0 a

2.50 25 d 6.5 a

5.00 45 c 2.0 b

IAA 0.00 40 c 1.0 c

1.25 50 c 1.5 b

2.50 60 b 1.5 b

5.00 50 c 2.8 b

a Values shown were proportions obtained from 3 replicatesof 10 cultures. bValues (N = 30) within a single columnfollowed by a similar letter were not significantly differentaccording to the LSD at the 5% level. ∗ Treatment which didnot form roots.

esis was completely inhibited at all salt formulationstested. Cotyledonary and epicotyl node cuttings cul-tured on MS and WPM supported, after 30 days,the best shoot elongation (1.1–1.7 cm) and maximumnumber of shoots produced per explant (2.7–3.2).However, cotyledonary node cuttings produced 6.0–7.0 nodes per explant, after 60–90 days on MS, re-spectively, while epicotyl node cuttings produced anaverage of 4.0 nodes per explant in the same period(Figure 5).

Rooting

Table 2 shows that rooting was completely inhibitedby 5.0 µM 2,4-D. The highest rooting percentage(85%) was achieved with 5.0 µM IBA, while the max-imum number of roots (5–6) per explant was producedon 1.25–2.5 µM NAA. Data presented in Table 3 in-dicate that some tissue culture media supplementedwith 5.0 µM IBA had variable effects on the percent-age of rooting and root length. Although maximumrhizogenesis was observed in 75–80% of the cultures

Table 3. The effect of basal media salt formulation sup-plemented with 5.0 µM IBA on rooting of in vitro raisednode cuttings of Cedrela fissilis after 35 days

Basal Rooting No. of Average

medium (%) roots/explantt root

length (cm)

MS 80 aa 2.5 ab 2.0 bb

B5 75 a 2.0 a 1.5 b

SH 75 a 2.5 a 1.7 b

WPM 60 b 2.0 a 2.0 b

W 60 b 2.5 a 0.5 a

aValues were proportions obtained from 3 replicates of12 cultures. bValues (N = 12) within a single column fol-lowed by a similar letter were not significantly differentaccording to the LSD at the 5% level.

on MS, B5 and SH there were no significant effects inthe numbers of roots (2.0–2.5) produced per explantand root elongation (1.5–2.0 cm), except on the Wmedium.

Root initiation either on full strength or halfstrength MS supplemented with different concentra-tions of IBA, was evident after 4–6 days of culturein all treatments except in the absence of growth reg-ulators and on half strength MS with 5.0 µM IBA(Figure 1B). Data in Table 4 shows that after 10days of culture rooting percentages were significantlyhigher (73–100%) in all treatments except on fullstrength MS without PGRs (53%). However, no dif-ferences between the treatments were observed after35 days of culture when the maximum rooting per-centages (87–100%) were achieved. The numbers ofroots per explant was not affected by the treatmentsbut the mean root length (8.5–10.0 cm) was signific-antly higher on full strength MS supplemented with1.25–2.5 µM IBA. In the controls without PGRs, theaverage root length varied from 5.3–6.5 cm independ-ently of the MS concentration. On half strength MSsupplemented with 2.5 µM IBA the maximum rootingratios (100%) occurred at the 10th day of culture andon half strength MS in the absence of PGRs (87%),at the 12th day. On full strength MS either withoutPGRs or supplemented with 1.25 µM IBA the max-imum rooting percentages (93%) were reached within18–35 days (Table 4).

Hardening of regenerated plants

Regenerated plants were successfully acclimatized(Figure 1C). One hundred percent of the plantlets sur-vived hardening on river sand for 25 days and for

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Figure 2. The effect of BA and NAA on shoot organogenesis from cotyledonary node cuttings of Cedrela fissilis after 45 days. Different lettersdenote statistical differences between treatments.

Figure 3. The effect of BA and NAA on number of nodes produced per explant from cotyledonary node cuttings of Cedrela fissilis after 45days. Different letters denote statistical differences between treatments.

another 90 days after transfer to potting medium con-sisting of three different substrates. However, differ-ences were detected in growth parameters accordingto the substrates. After 90 days acclimatization mi-croplants grown in river sand:soil mixture showedsignificantly higher fresh weight (11.4 mg), dry weight

(2.6 mg), shoot length (19.8 cm), number of leaves(12.7) and number of nodes (13.7) (Table 5).

Callus culture

The efficiency of callus initiation was 100% in most ofthe treatments (data not shown). The lowest rates (70–82%) of callus initiation were observed in explants

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Figure 4. The effect of BA and NAA on shoot length produced from cotyledonary node cuttings of Cedrela fissilis after 45 days. Differentletters denote statistical differences between treatments.

Figure 5. Number of nodes produced per explant of Cedrela fis-silis initiated either from cotyledonary (continuous line) or epicotyl(dotted line) node cuttings on MS supplemented with 2.5 µM BA.Different letters denote statistical differences between treatments.

cultured on 1.25–5.0 µM NAA (data not shown). Interms of callus growth, BA at 1.25, 2.5 and 5.0 µMin combination, respectively, with either 2.5, 1.25–5.0or with 5.0 µM NAA, supported significant increase infresh weight (759.0–811.0 mg per explant) (Figure 6).These results were in contrast to the treatments whereeither 1.25–5.0 µM BA (262.0–569.0 mg per explant)or 1.25–5.0 µM NAA (154.0–204.0 mg per explant)were used separately. The calli were friable and de-pending on the treatment showed globular structuresand/or rhizogenesis (Figure 1D).

Discussion

Multiplication of woody species through the sequen-tial subculture of axillary bud explants has beenachieved for several forest tree species, although someof the multiple shoots arising from the culture maybe adventitious in origin (Le Roux and van Staden,1991). The method has been applied successfully toolder trees; however, the best results for most specieswere achieved from young seedling material. Multi-plication rates for hardwood species are low, 5–10propagules per culture cycle, in comparison to othermicropropagation approaches, but even a multiplic-ation factor of this order can account for rates ofmillions per year for many species (Haines, 1994). ForC. fissilis, shoot multiplication occurred as a result ofthe release of axillary buds from apical dominance byBA in the culture medium. Originally there are onlytwo axillary buds per explant. In some treatments, calliproliferated and adventitious buds were produced atthe base of the axillary buds. As a result the multiplic-ation rate increased to 6–7-fold after 60–90 days in thesecond subculture cycle, as the subculture of each ofthe single node cuttings produced a new plant. Sim-ilar multiplication rates have been reported for otherMeliaceae species. Maruyama et al. (1989) reporteda micropropagation system for C. odorata from ju-venile seedlings, through the culturing of shoot tips,affording a 3-fold multiplication rate per month and

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Table 4. The effect of MS concentration and IBA on the frequency (%) of rooting of invitro raised node cuttings of Cedrela fissilis, after 10, 18, 35 days, and on the number ofroots/explant and average root length, after 35 days

Basal IBA Rooting Number of Average

medium (µM) (%) roots/explant root length

10th day 18th day 35th day (cm)

MS 0.00 53 ca 87 aa 93 aa 2.4 ab 5.3 cb

1.25 73 abc 80 a 93 a 2.1 a 10.0 a

2.50 73 abc 93 a 93 a 2.1 a 8.5 ab

5.00 78 abc 92 a 92 a 2.7 a 6.8 bc

1/2 MS 0.00 73 abc 87 a 87 a 2.9 a 6.5 bc

1.25 87 ab 93 a 93 a 2.2 a 6.6 bc

2.50 100 a 100 a 100 a 2.8 a 6.8 bc

5.00 67 b 93 a 93 a 2.6 a 5.8 bc

aValues shown were proportions of 3 replicates of 15 cultures. bValues (N = 10–20) withina single column followed by a similar letter were not significantly different according tothe LSD at the 5% level.

Table 5. Ex vitro growth of acclimatized microplants of Cedrelafissilis after 90 days

Substrate Fresh Dry Shoot No. of No. of

weight weight length leaves/plant nodes/plant

(g) (g) (cm)

Sand 0.5 ca 0.1 ca 3.2 ba 5.4 ca 6.4 ca

Sand/Soil 11.4 a 2.6 a 19.8 b 12.7 a 13.7 a

Soil 8.2 b 1.9 b 19.1 a 10.5 b 11.5 b

aValues (N = 12) within a single column followed by a similarletter were not significantly different according to the LSD at the5% level.

Lee and Rao (1988) reported a 6-fold multiplicationrate with nodal segments from Swietenia macrophyllaseedlings.

The effectiveness of BA in promoting axillary budgrowth in C. fissilis is well documented for otherwoody species. In the reports on micropropagationof C. odorata by Maruyama et al. (1989) and Cer-das et al. (1998) only BA was tested and in the latterthe explants failed to respond organogenically to thisPGR. According to Maruyama et al. (1989) the micro-propagation of C. odorata from juvenile seedlings waspossible by culturing the shoot tips on WPM supple-mented with 1.25 µM BA. For Swietenia macrophyllaLee and Rao (1988) reported a protocol where nodalsegments from seedlings were successfully culturedon MS medium supplemented with 10–50 µM BAand Maruyama and Ishii (1999) showed that 10 µMBA was also effective in inducing multiple shoots

from shoot tips. In the work reported here, cotyledon-ary node cuttings were used to initiate shoot culturesand the proliferative capacity of this explant dependedon the medium formulation, being superior on MSmedium. Shoot development in terms of number ofnodes/explant was inferior when epicotyl-derived ex-plants were used and was affected by the slow growthand senescence after 30 days.

The cytokinin-induced axillary bud prolifera-tion from nodal segments of Daphne gnidium(Thymeleaceae) (bay laurel) was significantly in-creased by the auxin IAA and in some species a lowconcentration of auxins can promote growth of axil-lary buds by couteracting the inhibitory effects of highcytokinin concentrations on shoot elongation (Gavidiaet al., 1996). Similar results were obtained for shootcultures of C. fissilis when shoot length was promotedby the combination of 5.0 µM BA and 2.5 µM NAA.In other treatments when NAA was used at 5.0 µMalone or in combination with BA it strengthened apicaldominance at the expense of shoot proliferation.

Successful micropropagation of many woody spe-cies is frequently limited by their recalcitrance to formadventitious roots. In the work reported here, max-imum rooting percentages (87–100%) were achievedwithin 10–12 days on half strength MS either PGR freeor supplemented with 2.5 µM IBA.

The interaction of culture media, in terms of nu-trient, ionic strength, PGR type and concentrationand the physiological status of explants, may ex-plain the results in the different culture conditions

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Figure 6. The effect of BA and NAA on callus growth of Cedrela fissilis initiated from cotyledonary node cuttings after 45 days. Differentletters denote statistical differences between treatments.

tested. The differences observed in rooting in the dif-ferent media tested may be due to the differences inammonium:nitrate rates and total ionic strength (Mc-Cown and Sellmer, 1987). The MS and WPM mediahave similar ammonium:nitrate rates but the total ionicstrength of WPM is 45% of MS while W mediahas even lower total ionic strength and no nitrate orammonium.

The results on rooting are in contrast to those ob-tained for other Meliaceae species when the period oftime required for maximum rooting is considered. Thebest rooting percentage for C. odorata (73%) was ob-tained on half strength WPM using 2.5 µM IBA after30 days (3.7 roots per explant; 6.1 cm root length).Combinations of 2.5 µM IBA and 0.27 µM NAA werenecessary to achieve 90% rooting (4.2 roots per ex-plant; 1.3 cm root length) (Maruyama et al., 1989).Rooting of Swietenia macrophylla was achieved inhalf strength WPM medium containing 2.5 µM IBAalone or in combination with 0.25 µM NAA or inPGR free medium and 50% rooting rate was achievedafter 2 months (Maruyama and Ishii, 1999). Theseauthors also observed that the increase of NAA con-centration caused significant increase in the numberof roots per explant, similarly to C. fissilis, when in-creasing NAA levels up to 2.5 µM caused an increasein the roots/explant, but a subsequent decrease. Theyalso reported successful acclimatization proceduresfor Swietenia macrophylla but using a method in-

cluding irrigation of the regenerated plants with plantnutrient solution.

There was a promotive synergistic effect of BAand NAA on callus growth. The calli were appropriatefor the development of cell suspension cultures andare currently being studied concerning their morpho-genic potential. C. fissilis is also a species of medicinalimportance and, therefore, the development of cellsuspension and root cultures will be required as apotential strategy for phytochemical and pharmacolo-gical studies (Viana et al., 1999).

Conclusions

These are the first successful attempts to establish con-sistent micropropagation and callus culture protocolsfrom juvenile seedlings of C. fissilis. The results onshoot multiplication show that BA was indispensablefor the sprouting and multiplication of axillary budsof cotyledonary node cuttings. This technology mayprovide a valuable tool in a tree improvement program,although studies of micropropagating adult trees willbe essential. The callus culture protocols describedabove will help to foster the studies on secondarymetabolites and the establishment of regeneration andgenetic transformation systems.

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Acknowledgements

The authors thank the Conselho Nacional de Desen-volvimento Científico e Tecnológico (CNPq/Brasil)for the fellowship held by FN Moreno.

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