Plant Cell, Tissue and Organ Culture 66: 167–173, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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In vitro regeneration of Acacia mangium via organogenesis

Deyu Xie & Yan Hong∗Institute of Molecular Agrobiology, 1 Research Link, The National University of Singapore, Singapore 117604(∗requests for offprints; Fax: +65-872-7007; E-mail: hongy@ima.org.sg)

Received 23 August 2000; accepted in revised form 2 April 2001

Key words: micropropagation, regeneration, thidiazuron

Abstract

Plant regeneration of Acacia mangium was achieved through organogenesis in callus cultures. Calli were inducedfrom five types of explants (embryo axes and cotyledons of mature zygotic embryos as well as leaflets, petioles andstems of seedlings) of A. mangium on MS (Murashige and Skoog, 1962) basal medium containing 9.05 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 13.95 µM kinetin (KT). Green or green purple compact nodules containingclusters of meristematic centers were induced in these calli after transfer to MS basal medium containing 1.14–22.75 µM thidiazuron (TDZ) and 1.43–2.86 µM indole-3-acetic acid (IAA). A combination of 4.55 µM TDZ and1.43 µM IAA promoted the highest percentage of calli to form nodules, in 8–11% of calli derived from cotyledons,embryo axes, leaflets or petiole and in 4% of calli derived from stems. Twenty-two percent of the nodules formedadventitious shoots on MS basal medium containing 0.045 µM TDZ. Shoots were elongated on MS mediumcontaining 0.045 µM TDZ supplemented with 7.22 µM gibberellic acid. The medium containing 10.75 µM NAAand 2.33 µM KT promoted rooting of 10% of the elongated shoots. Plantlets grew up well in the green house.

Abbreviations: 2,4-D – 2,4-dichlorophenoxyacetic acid; 6-BA – 6-benzylaminopurine; Asn – L-asparagine; CH– casein enzymatic hydrolysate; GA3 – gibberellic acid; Gln – L-glutamine; IAA – indole-3-acetic acid; KT –kinetin; MS – Murashige and Skoog, 1962; NAA – α-naphthaleneacetic acid; Pro L – proline; TDZ – 1-phenyl-3-(1,2,3-thiadiazol-5-yl) Urea (thidiazuron); Vc – vitamin C (L-ascorbic acid)

Introduction

Acacia, a leguminous genus in the family Mimosacea,contains more than 1200 species in tropical and sub-tropical regions (Simmons, 1987). Acacia mangiumWilld. is a multipurpose, fast growing tropical legumetree. An adult tree is up to 30 m tall and its bole is oftenstraight to over half the total height. A. mangium hashigher short fibre quality than other raw pulp sourceslike Eucalyptus, reed and wheat straw (Paavilainen,1998). It also allows denser plantation than specieslike Eucalyptus (Johansson, 1998). Because of its highquality fibre and high biomass yield, it is a preferredchoice of wood source for the pulp industry. It wasestimated that by 2004, the Asia Paper and Pulp groupwould obtain all its wood from plantations consist-ing mainly of A. mangium (Bayliss, 1998). By 1996

already 123 000 hectares of land had been plantedwith A. mangium, indicating the economic value ofthis species. A. mangium is also increasingly usedfor reforestation and soil rehabilitation of degradedland in many regions of Malaysia, India and Indonesia(Widiarti and Alrasjid, 1987). A. mangium self- andcross-pollinates and interspecific pollination with Aca-cia auriculiformis was reported (Sedgley et al., 1992;Sornsathapornkul and Owens, 1999). These reproduc-tion characteristics create a large diversity which isdisadvantageous to commercial propagation and plant-ation through seeds. Therefore, clonal propagationof superior trees will be of great importance for A.mangium plantations. Plant regeneration has been re-ported for a few Acacia species, such as A. catechuregeneration via somatic embryogenesis (Rout et al.,1995) and A. auriculiformis regeneration through or-

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ganogenesis (Rao and Prasad, 1991). Shoot propaga-tion (Ahmad, 1991; Galiana et al., 1991; Bhaskar andSubhash, 1996) and the isolation of protoplast fromsterile seedlings (Toshihiro and Sonoko, 1999) havebeen reported for A. mangium. However, there areno reports on in vitro regeneration. In this paper, wereport regeneration of A. mangium through organo-genesis in calli cultures from five types of explantstested.

Materials and methods

Plant materials

Mature seeds were collected from a natural grove ofA. mangium trees of 20–30 m in height at the Sci-ence Park Drive of Singapore. Seeds were treated with98% (v/v) H2SO4 for 1–2 min and washed with tapwater five times. Treated seeds were sterilized with70% (v/v) ethanol for 2–3 min and washed five timeswith sterile double deionized water (ddH2O). Seedswere further sterilized with 0.1% HgCl2 for 6 minand washed five times with sterile ddH2O followed bysterilization again in 30% Clorox� (containing 5.25%sodium hypochlorite) for 6 min and washed five timeswith sterile ddH2O. Zygotic embryos were asepticallyisolated from seeds and used for further experiments.

Media preparation and culture conditions

All media were adjusted to pH 5.8 with sterile 1 NKOH after autoclaving at 121◦C for 25 min. Plantgrowth regulators were filter sterilised with a 0.2 µmmembrane and added to the media after autoclaving.All cultures were maintained under warm white fluor-escent lights at an irradiance of 26 µmol s−1 m−2 witha 16-h photoperiod at 28◦C.

Callus induction

Zygotic embryos were germinated on MS basal me-dium (Murashige and Skoog, 1962) containing 30 gl−1 sucrose and solidified with 0.25% (w/v) phytagel.

Embryo axes, 0.15–0.2 cm long, and cotyledonscut into 0.3 × 0.4-cm pieces were used as explants.Leaflets, petioles and stems were excised from 50-day-old seedlings. Leaflets were cut into 0.3 × 0.5-cmpieces. Petioles and stems were cut into 0.5–0.8-cmlong segments. Cotyledon pieces, embryo axes, leaf-let pieces, petiole and stem segments were inoculatedon MS basal medium containing 9.05 µM 2,4-D and

13.95 µM KT, 100 mg l−1casein enzymatic hydrolys-ate (CH), 100 mg l−1ascorbic acid (vitamin C, Vc),150 mg l−1glutamine (Gln), 150 mg l−1asparagine(Asn), 150 mg l−1 proline (Pro) and 30 g l−1sucroseand solidified with 0.3% (g l−1) phytagel. Leaf pieceswere cultured with the abaxial side touching the me-dium.

Induction of adventitious nodules and buds

Calli derived from petioles were used to optimize plantgrowth regulator (PGR) combinations for induction ofadventitious bud differentiation. Calli were culturedfor 2 months on MS basal medium containing differentcombinations of TDZ (0–91µM) and IAA (1.43–2.86µM) (Table 1); 6-BA (4.44, 8.88, 22.22 µM) and IAA(1.43, 2.86 µM); 6-BA (2.22, 4.44, 8.88, 13.33 µM)and NAA (0, 0.54, 2.69, 5.38µM); KT (13.95 µM)and NAA (2.69, 5.38 µM). All media were supple-mented with 100 mg l−1CH, 100 mg l−1Vc, 150 mgl−1Gln, 150 mg l−1Asn, 150 mg l−1 Pro and 30 g l−1

sucrose and were solidified with 0.3% (w/v) phytagel.Each treatment was conducted with 110 pieces of calli(about 0.1 g fresh weight for each) and repeated twice.Ten pieces of calli were inoculated onto 50 ml of me-dium in 100 × 25-mm petri dishes. The percentageof calli that produced green or green purple compactnodules was calculated. The best medium was furtherused to compare the efficiencies of adventitious buddifferentiation in calli from all five types of explants.

Adventitious shoot induction

The nodules, which were induced from petiole de-rived callus on the medium containing 4.55 µM TDZand 1.43 µM IAA, were cultured for two months onMS basal medium containing 0–4.55 µM TDZ, 100mg l−1CH, 100 mg l−1Vc, 150 mg l−1Gln, 150 mgl−1Asn, 150 mg l−1 Pro and 30 g l−1sucrose andsolidified with 0.35% (w/v) phytagel. Each treatmentwas conducted with ninety nodular pieces (about 0.1 gfresh weight for each) and repeated twice. Ten nodularpieces were inoculated onto 50 ml of each mediumcontained in 100 × 25-mm petri dishes. The percent-age of the nodules that formed shoots with pinnateleaves was calculated for each medium. The optimalmedium was used to induce shoots from nodules thatwere formed in calli derived from leaflets, cotyledons,embryo axes and stems.

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Adventitious shoot elongation and rooting

Clusters of adventitious shoots with pinnate leaveswere cultured for 2 months on MS basal mediumcontaining 0.045 µM TDZ, 7.22 µM GA3, 100 mgl−1CH, 100 mg l−1Vc, 150 mg l−1Gln, 150 mgl−1Asn, 150 mg l−1Pro and 30 g l−1 sucrose and so-lidified with 0.35% (w/v) phytagel. Shoots of 2–3 cmin length were then excised and cultured for 1 monthon 1/2 MS basal medium containing 10.75 µM NAA,2.33 µM KT, 100 mg l−1CH, 100 mg l−1Vc, 150 mgl−1Gln, 150 mg l−1Asn, 150 mg l−1 Pro and 20 gl−1sucrose and solidified with 0.35% (w/v) phytagel.Rooted shoots were transferred onto 1/2 MS basal me-dium containing 20 g l−1 sucrose and solidified with0.35% (w/v) phytagel.

Transfer to soil

After formation of at least 10 lateral roots, plantletswere transplanted into pots with peat and white sand(3:1, v/v) and maintained in a growth chamber underday-light type fluorescent lights at an irradiance of 52µmol s−1m−2 with a 16-h photoperiod at 28◦C. Onemonth later, plantlets were transferred to the greenhouse.

Histological analysis

The nodules were fixed in 2.5% (v/v) glutaraldehydein 50 mM sodium phosphate buffer (pH 7.2) overnightat room temperature, then dehydrated through agraded ethanol series (20, 30, 50, 70 and 90%) sequen-tially for 20 min three times at each stage and finallythrough 100% ethanol for 30 min three times. Tissueswere embedded in plastics embedding medium (Leicaplastic embed Kit) and were sectioned at a thickness of5–8 µm. Sections were stained in 0.025% (w/v) tolu-idine blue O for 1 min, dried at 42◦C, mounted withDPX (BDH), and examined under a light microscopy(Leica).

Statistical analysis

Analysis of variances (one-way ANOVA, Motulsky1995) was used to test if there are significant differ-ences between means obtained with different treat-ments or with different calli derived from differentexplants at the 5% level of significance (p=0.05).Means followed by the same letter are not significantlydifferent from each other.

Table 1. Effects of TDZ and IAA on nodule induction in petiolederived callus

TDZ IAA Percentage of callus producing nodules (%)

(µM) (µM) Mean ± SE

0 1.43 0

0.045 1.43 0

0.1 1.43 0

1.14 1.43 2.85±0.79b

4.55 1.43 9.00±1.32a

9.1 1.43 3.75±2.50b

22.75 1.43 2.56±0.46b

91 1.43 0

0 2.86 0

0.045 2.86 0

0.1 2.86 0

1.14 2.86 0

4.55 2.86 7.33±0.5a

9.1 2.86 2.34±1.34b

22.75 2.86 1.56±0.67b

91 2.86 0

The average values of three independent repeated experiments(each with 110 pieces of calli) are given. Means followed bythe same letter are not significantly different from each other atp=0.05.

Results and discussion

Callus formation

MS basal medium containing 9.05 µM 2,4-D and13.95 µM KT promoted 100% callus formation fromall five types of explants (Figure 1A,D). Calli inducedfrom all five types of explants were friable, loose andwhite-yellowish (Figure 1A,D). The same medium didnot induce any adventitious bud differentiation fromcallus.

Nodule formation from callus

Formation of green or green-purple compact nodulesfrom calli indicated the initiation of adventitious buddifferentiation. The highest percentage of calli produ-cing nodules was observed in calli that were culturedfor at least 40 days on MS basal medium containing4.55 µM TDZ and 1.43 µM IAA (Table 1 and Figure1B,E). Calli from all five types of explants producedthe nodules (Figure 1B,E,G) that were distinct fromthe original friable loose white-yellowish calli (Figure1A,D). Histological sections showed that the noduleshad clusters of meristematic centers (Figure 1L).

The effects of different combinations of plantgrowth regulators on nodule induction were studied

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Figure 1. Acacia mangium regeneration through organogenesis from callus. Callus induction was conducted on the medium containing 9.05µM 2,4-D and 13.95 µM KT. Adventitious bud differentiation was induced on the medium containing 4.55 µM TDZ and 1.43 µM IAA.Shoot formation was induced on the medium containing 0.045 µM TDZ. Shoots were rooted on the 1/2 MS basal medium containing 10.75µM NAA and 2.33 µM KT. (A–C) Regeneration from callus derived from petioles: (A) callus induction (green parts are the parts of explantsnot completely dedifferentiated into calli, bar = 1.5 cm); (B) induction of nodules (bar = 0.5 cm); (C) shoot formation (bar = 1.0 cm). (D–F)Regeneration from callus derived from leaflets: (D) callus induction (bar = 1.0 cm); (E) induction of nodules (bar = 0.5 cm); (F) shoot formation(bar = 1.0 cm). (G–K) Regeneration from callus derived from stems: (G) nodules (bar = 1.5 cm) cultured for shoot induction; (H) shoots (bar =1.5 cm); (I) shoots rooting (bar = 2.0 cm); (J) a plantlet before being transplanted into a pot; (K) plants growing in pots. (L) Histological sectionof nodules obtained from callus derived from petioles showing clusters of meristematic centres (bar = 20 µm).

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Table 2. Comparison of nodule induction in calliderived from five types of explants

Callus source from explants Percentage of callus

producing nodules (%)

Mean ± SE

Leaflet 8.13±5.04a

Petiole 9.00±1.32a

Stem 4.17±0.84b

Cotyledon 10.83±5.84a

Embryo axis 10.23±0.49a

The average values of three independent repeated ex-periments (each with 110 pieces of calli) are given.Means followed by the same letter are not signific-antly different from each other at p=0.05.

with calli derived from petioles. Among all the me-dia tested, the combinations of 1.14–22.75 µM TDZand 1.43–2.86 µM IAA induced the compact nodulesfrom callus cultures (Table 1). The medium containing4.55 µM TDZ and 1.43 µM IAA promoted the highestpercentage of calli derived from petioles (9%) to formnodules (Table 1). Changes of TDZ concentration inmedia from 0.0 to 91.0 µM significantly affected theformation of nodules in calli and TDZ at the level of4.55 µM was most efficient (Table 1). The combin-ation of 4.55 µM TDZ and 1.43 µM IAA inducednodules from calli derived from leaflets (Figure 1E),stems (Figure 1G), cotyledons and embryo axes (Table2). Using concentrations of 0.01–0.4 µM (0.0022 –0.088 mg l−1) TDZ was recommended for in vitro re-generation of woody plants (Lu, 1993), but the mediacontaining 0.045–0.1 µM TDZ failed to induce nod-ules and adventitious buds in our experiments (Table1). These results suggest that different species have adifferent requirement for TDZ.

The combination of 4.44 µM (1.0 mg l−1) 6-BAand 2.69 µM (0.5 mg l−1) NAA or 4.44 µM (1.0mg l−1) 6-BA alone promoted adventitious bud forma-tion from callus in A. auriculiformis regeneration (Raoand Prasad, 1991). However, the same combination of4.44 µM 6-BA and 2.69 µM NAA or other combin-ations of 2.22–13.33 µM 6-BA and 0–5.38 µM NAAfailed to induce nodules in A. mangium callus (datanot shown). Rout et al. (1995) reported A. catechuregeneration via somatic embryogenesis on mediumcontaining 13.9 µM KT and 2.7 µM NAA, but com-binations of 13.95 µM KT and 2.69–5.38 µM NAAdid not induce nodules in A. mangium callus (datanot shown). The different responses of three Acaciaspecies to different plant growth regulators may be

Table 3. Effects of different TDZ con-centrations on shoot induction from nod-ules.

TDZ Percentage of nodules producing

shoots with pinnate leaf (%)

(µM) Mean ± SE

0 0

0.045 22.26±13.66a

0.1 7.03±4.83a,b

0.23 5.93±2.89b

1.14 0

2.27 0

4.55 0

The average values of three independ-ent repeated experiments (each with 90pieces of nodules induced from petiolederived callus on the medium contain-ing TDZ 4.55 µM and IAA1.43 µM).Means followed by the same letter are notsignificantly different from each other atp=0.05.

due to variations of their genetic background. Ahmad(1991) reported that 2.22 µM (0.5 mg l−1) 6-BA wasthe most efficient in A. mangium micropropagation.In our experiment, neither 2.22 µM 6-BA alone northe combinations of 2.22–22.22 µM 6-BA with 0–5.38 µM NAA or 1.34–2.86 µM IAA induced nodulesfrom callus (data not shown).

The efficiencies of nodule induction were com-pared in calli derived from five types of explants onMS medium containing 4.55 µM TDZ and 1.43 µMIAA (Table 2). The percentage of nodule formationwas nearly 11% in callus obtained from cotyledons,followed by callus obtained from embryo axes (nearly10%) and less efficient for petioles, leaflets, and stemsderived calli (Table 2). The efficiencies of noduleformation in calli obtained from cotyledons, embryoaxes or petioles were significantly higher (p<0.05)than in calli obtained from stems. Similarly, variationsin efficiency of regeneration among different explantswere reported for Eucalyptus grandis × E. urophyllahybrid (Cid et al., 1999) and Helianthus smithii Heiser(Laparra et al., 1997).

Adventitious shoot induction

The induction media (with 1.14–22.75 µM TDZ and1.43–2.86 µM IAA) promoted formation of secondarynodules instead of shoots during subculture. There-fore, a different step for shoot induction was neces-sary. The nodules began to form adventitious shoots

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with pinnate leaves after culture for 40 days on me-dia supplemented with 0.045–0.23 µM of TDZ (Table3 and Figure 1C,F,H). The medium containing 0.045µM TDZ promoted the highest percentage (nearly22%) of nodules to form adventitious shoots (Table 3).Each cluster of adventitious shoots contained an aver-age of five shoots. These results agreed with the reportthat low TDZ concentrations (0.01–0.4 µM) were ef-fective for in vitro shoot induction in woody plants(Lu, 1993). The efficiency of shoot formation fromthe nodules decreased with higher concentrations ofTDZ in the medium (Table 3). The media containing1.14–4.55 µM TDZ only promoted formation of sec-ondary nodules. This is in line with the report that highconcentrations of TDZ (1.14–4.55 µM) inhibit shootformation (Lu, 1993). Similarly, Kim et al. (1997)reported that media supplemented with the lower con-centration of 0.45 µM TDZ promoted the most shootformation.

Shoot elongation and rooting

Adventitious shoots elongated slowly on the mediumcontaining 0.045 µM TDZ. Efficient shoot elongationwas achieved by transferring clusters of adventitiousshoots with pinnate leaves onto medium containing0.045 µM TDZ supplemented with 7.22 µM GA3.In 2 months, shoots elongated to 2–3 cm long andformed new pinnate leaves. Effects of GA3 to stim-ulate adventitious shoot development and elongationwere documented in many plant regeneration sys-tems, such as in vitro shoot proliferation of cassava(Bhagwat et al., 1996), shoot regeneration of Pas-siflora foetida (Hicks et al., 1996) and regenerationof Elaeagnus angustifolia (Economou and Maloupa,1995).

The rooting of elongated shoots was induced on1/2 MS basal medium containing 10.75 µM NAA and2.33 µM KT. The frequency of elongated shoots form-ing roots was 10±2.4% after culture for 30 days (Fig-ure 1I), which is comparable to rooting of A. mangiumshoots with IBA (Galiana et al., 1991). Plantlets weretransferred onto 1/2 MS basal medium until at least 10lateral roots had formed (Figure 1J), then they weretransferred into pots containing peat and white sand(3/1, v/v) (Figure 1K). More than 200 plantlets aregrowing well in the growth chambers and green housewithout visual abnormalities.

Acknowledgement

We acknowledge that the research was funded bygrants from the National Science and TechnologyBoard, Singapore.

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