Plant Cell, Tissue and Organ Culture 66: 167173, 2001. 2001 Kluwer Academic Publishers.

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167

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 induced from ve types of explants (embryo axes and cotyledons of mature zygotic embryos as well as leaets, petioles and stems of seedlings) of A. mangium on MS (Murashige and Skoog, 1962) basal medium containing 9.05 M 2,4dichlorophenoxyacetic acid (2,4- D) and 13.95 M kinetin (KT). Green or green purple compact nodules containing clusters of meristematic centers were induced in these calli after transfer to MS basal medium containing 1.14 22.75 M thidiazuron (TDZ) and 1.432.86 M indole-3-acetic acid (IAA). A combination of 4.55 M TDZ and 1.43 M IAA promoted the highest percentage of calli to form nodules, in 811% of calli derived from cotyledons, embryo axes, leaets or petiole and in 4% of calli derived from stems. Twenty-two percent of the nodules formed adventitious shoots on MS basal medium containing 0.045 M TDZ. Shoots were elongated on MS medium containing 0.045 M TDZ supplemented with 7.22 M gibberellic acid. The medium containing 10.75 M NAA and 2.33 M 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 -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 subtropical regions (Simmons, 1987). Acacia mangium Willd. is a multipurpose, fast growing tropical legume tree. An adult tree is up to 30 m tall and its bole is often straight to over half the total height. A. mangium has higher short bre quality than other raw pulp sources like Eucalyptus, reed and wheat straw (Paavilainen, 1998). It also allows denser plantation than species like Eucalyptus (Johansson, 1998). Because of its high quality bre and high biomass yield, it is a preferred choice of wood source for the pulp industry. It was estimated that by 2004, the Asia Paper and Pulp group would obtain all its wood from plantations consisting mainly of A. mangium (Bayliss, 1998). By 1996 already 123 000 hectares of land had been planted with A. mangium, indicating the economic value of this species. A. mangium is also increasingly used for reforestation and soil rehabilitation of degraded land in many regions of Malaysia, India and Indonesia (Widiarti and Alrasjid, 1987). A. mangium self- and cross-pollinates and interspecic pollination with Acacia auriculiformis was reported (Sedgley et al., 1992; Sornsathapornkul and Owens, 1999). These reproduction characteristics create a large diversity which is disadvantageous to commercial propagation and plantation through seeds. Therefore, clonal propagation of superior trees will be of great importance for A. mangium plantations. Plant regeneration has been reported for a few Acacia species, such as A. catechu regeneration via somatic embryogenesis (Rout et al., 1995) and A. auriculiformis regeneration through or-

168 ganogenesis (Rao and Prasad, 1991). Shoot propagation (Ahmad, 1991; Galiana et al., 1991; Bhaskar and Subhash, 1996) and the isolation of protoplast from sterile seedlings (Toshihiro and Sonoko, 1999) have been reported for A. mangium. However, there are no reports on in vitro regeneration. In this paper, we report regeneration of A. mangium through organogenesis in calli cultures from ve types of explants tested. 13.95 M KT, 100 mg l1 casein enzymatic hydrolysate (CH), 100 mg l1 ascorbic acid (vitamin C, Vc), 150 mg l1 glutamine (Gln), 150 mg l1 asparagine (Asn), 150 mg l1 proline (Pro) and 30 g l1 sucrose and solidied with 0.3% (g l1 ) phytagel. Leaf pieces were cultured with the abaxial side touching the medium. Induction of adventitious nodules and buds Calli derived from petioles were used to optimize plant growth regulator (PGR) combinations for induction of adventitious bud differentiation. Calli were cultured for 2 months on MS basal medium containing different combinations of TDZ (091M) and IAA (1.432.86 M) (Table 1); 6-BA (4.44, 8.88, 22.22 M) and IAA (1.43, 2.86 M); 6-BA (2.22, 4.44, 8.88, 13.33 M) and NAA (0, 0.54, 2.69, 5.38M); KT (13.95 M) and NAA (2.69, 5.38 M). All media were supplemented with 100 mg l1 CH, 100 mg l1 Vc, 150 mg l1 Gln, 150 mg l1 Asn, 150 mg l1 Pro and 30 g l1 sucrose and were solidied 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 medium in 100 25-mm petri dishes. The percentage of calli that produced green or green purple compact nodules was calculated. The best medium was further used to compare the efciencies of adventitious bud differentiation in calli from all ve types of explants. Adventitious shoot induction The nodules, which were induced from petiole derived callus on the medium containing 4.55 M TDZ and 1.43 M IAA, were cultured for two months on MS basal medium containing 04.55 M TDZ, 100 mg l1 CH, 100 mg l1 Vc, 150 mg l1 Gln, 150 mg l1 Asn, 150 mg l1 Pro and 30 g l1 sucrose and solidied with 0.35% (w/v) phytagel. Each treatment was conducted with ninety nodular pieces (about 0.1 g fresh weight for each) and repeated twice. Ten nodular pieces were inoculated onto 50 ml of each medium contained in 100 25-mm petri dishes. The percentage of the nodules that formed shoots with pinnate leaves was calculated for each medium. The optimal medium was used to induce shoots from nodules that were formed in calli derived from leaets, cotyledons, embryo axes and stems.

Materials and methods Plant materials Mature seeds were collected from a natural grove of A. mangium trees of 2030 m in height at the Science Park Drive of Singapore. Seeds were treated with 98% (v/v) H2 SO4 for 12 min and washed with tap water ve times. Treated seeds were sterilized with 70% (v/v) ethanol for 23 min and washed ve times with sterile double deionized water (ddH2O). Seeds were further sterilized with 0.1% HgCl2 for 6 min and washed ve times with sterile ddH2 O followed by sterilization again in 30% Clorox (containing 5.25% sodium hypochlorite) for 6 min and washed ve times with sterile ddH2 O. Zygotic embryos were aseptically isolated from seeds and used for further experiments. Media preparation and culture conditions All media were adjusted to pH 5.8 with sterile 1 N KOH after autoclaving at 121C for 25 min. Plant growth regulators were lter sterilised with a 0.2 m membrane and added to the media after autoclaving. All cultures were maintained under warm white uorescent lights at an irradiance of 26 mol s1 m2 with a 16-h photoperiod at 28 C. Callus induction Zygotic embryos were germinated on MS basal medium (Murashige and Skoog, 1962) containing 30 g l1 sucrose and solidied with 0.25% (w/v) phytagel. Embryo axes, 0.150.2 cm long, and cotyledons cut into 0.3 0.4-cm pieces were used as explants. Leaets, petioles and stems were excised from 50day-old seedlings. Leaets were cut into 0.3 0.5-cm pieces. Petioles and stems were cut into 0.50.8-cm long segments. Cotyledon pieces, embryo axes, leaflet pieces, petiole and stem segments were inoculated on MS basal medium containing 9.05 M 2,4-D and

169 Adventitious shoot elongation and rooting Clusters of adventitious shoots with pinnate leaves were cultured for 2 months on MS basal medium containing 0.045 M TDZ, 7.22 M GA3 , 100 mg l1 CH, 100 mg l1 Vc, 150 mg l1 Gln, 150 mg l1 Asn, 150 mg l1 Pro and 30 g l1 sucrose and solidied with 0.35% (w/v) phytagel. Shoots of 23 cm in length were then excised and cultured for 1 month on 1/2 MS basal medium containing 10.75 M NAA, 2.33 M KT, 100 mg l1 CH, 100 mg l1 Vc, 150 mg l1 Gln, 150 mg l1 Asn, 150 mg l1 Pro and 20 g l1 sucrose and solidied with 0.35% (w/v) phytagel. Rooted shoots were transferred onto 1/2 MS basal medium containing 20 g l1 sucrose and solidied with 0.35% (w/v) phytagel. Transfer to soil After formation of at least 10 lateral roots, plantlets were transplanted into pots with peat and white sand (3:1, v/v) and maintained in a growth chamber under day-light type uorescent lights at an irradiance of 52 mol s1 m2 with a 16-h photoperiod at 28C. One month later, plantlets were transferred to the green house. Histological analysis The nodules were xed in 2.5% (v/v) glutaraldehyde in 50 mM sodium phosphate buffer (pH 7.2) overnight at room temperature, then dehydrated through a graded ethanol series (20, 30, 50, 70 and 90%) sequentially for 20 min three times at each stage and nally through 100% ethanol for 30 min three times. Tissues were embedded in plastics embedding medium (Leica plastic embed Kit) and were sectioned at a thickness of 58 m. Sections were stained in 0.025% (w/v) toluidine blue O for 1 min, dried at 42 C, mounted with DPX (BDH), and examined under a light microscopy (Leica). Statistical analysis Analysis of variances (one-way ANOVA, Motulsky 1995) was used to test if there are signicant differences between means obtained with different treatments or with different calli derived from different explants at the 5% level of signicance (p=0.05). Means followed by the same letter are not signicantly different from each other.
Table 1. Effects of TDZ and IAA on nodule induction in petiole derived callus TDZ (M) 0 0.045 0.1 1.14 4.55 9.1 22.75 91 0 0.045 0.1 1.14 4.55 9.1 22.75 91 IAA (M) 1.43 1.43 1.43 1.43 1.43 1.43 1.43 1.43 2.86 2.86 2.86 2.86 2.86 2.86 2.86 2.86 Percentage of callus producing nodules (%) Mean SE 0 0 0 2.850.79b 9.001.32a 3.752.50b 2.560.46b 0 0 0 0 0 7.330.5a 2.341.34b 1.560.67b 0

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

Results and discussion Callus formation MS basal medium containing 9.05 M 2,4-D and 13.95 M KT promoted 100% callus formation from all ve types of explants (Figure 1A,D). Calli induced from all ve types of explants were friable, loose and white-yellowish (Figure 1A,D). The same medium did not induce any adventitious bud differentiation from callus. Nodule formation from callus Formation of green or green-purple compact nodules from calli indicated the initiation of adventitious bud differentiation. The highest percentage of calli producing nodules was observed in calli that were cultured for at least 40 days on MS basal medium containing 4.55 M TDZ and 1.43 M IAA (Table 1 and Figure 1B,E). Calli from all ve types of explants produced the nodules (Figure 1B,E,G) that were distinct from the original friable loose white-yellowish calli (Figure 1A,D). Histological sections showed that the nodules had clusters of meristematic centers (Figure 1L). The effects of different combinations of plant growth 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 9.05 M 2,4-D and 13.95 M KT. Adventitious bud differentiation was induced on the medium containing 4.55 M TDZ and 1.43 M IAA. Shoot formation was induced on the medium containing 0.045 M TDZ. Shoots were rooted on the 1/2 MS basal medium containing 10.75 M NAA and 2.33 M KT. (AC) Regeneration from callus derived from petioles: (A) callus induction (green parts are the parts of explants not completely dedifferentiated into calli, bar = 1.5 cm); (B) induction of nodules (bar = 0.5 cm); (C) shoot formation (bar = 1.0 cm). (DF) Regeneration from callus derived from leaets: (D) callus induction (bar = 1.0 cm); (E) induction of nodules (bar = 0.5 cm); (F) shoot formation (bar = 1.0 cm). (GK) 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 section of nodules obtained from callus derived from petioles showing clusters of meristematic centres (bar = 20 m).

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Table 2. Comparison of nodule induction in calli derived from ve types of explants Callus source from explants Percentage of callus producing nodules (%) Mean SE 8.135.04a 9.001.32a 4.170.84b 10.835.84a 10.230.49a Table 3. Effects of different TDZ concentrations on shoot induction from nodules. TDZ Percentage of nodules producing shoots with pinnate leaf (%) (M) Mean SE 0 0.045 0.1 0.23 1.14 2.27 4.55 0 22.2613.66a 7.034.83a,b 5.932.89b 0 0 0

Leaet Petiole Stem Cotyledon Embryo axis

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

with calli derived from petioles. Among all the media tested, the combinations of 1.1422.75 M TDZ and 1.432.86 M IAA induced the compact nodules from callus cultures (Table 1). The medium containing 4.55 M TDZ and 1.43 M IAA promoted the highest percentage of calli derived from petioles (9%) to form nodules (Table 1). Changes of TDZ concentration in media from 0.0 to 91.0 M signicantly affected the formation of nodules in calli and TDZ at the level of 4.55 M was most efcient (Table 1). The combination of 4.55 M TDZ and 1.43 M IAA induced nodules from calli derived from leaets (Figure 1E), stems (Figure 1G), cotyledons and embryo axes (Table 2). Using concentrations of 0.010.4 M (0.0022 0.088 mg l1 ) TDZ was recommended for in vitro regeneration of woody plants (Lu, 1993), but the media containing 0.0450.1 M TDZ failed to induce nodules and adventitious buds in our experiments (Table 1). These results suggest that different species have a different requirement for TDZ. The combination of 4.44 M (1.0 mg l1 ) 6-BA and 2.69 M (0.5 mg l1 ) NAA or 4.44 M (1.0 mg l1 ) 6-BA alone promoted adventitious bud formation from callus in A. auriculiformis regeneration (Rao and Prasad, 1991). However, the same combination of 4.44 M 6-BA and 2.69 M NAA or other combinations of 2.2213.33 M 6-BA and 05.38 M NAA failed to induce nodules in A. mangium callus (data not shown). Rout et al. (1995) reported A. catechu regeneration via somatic embryogenesis on medium containing 13.9 M KT and 2.7 M NAA, but combinations of 13.95 M KT and 2.695.38 M NAA did not induce nodules in A. mangium callus (data not shown). The different responses of three Acacia species to different plant growth regulators may be

The average values of three independent repeated experiments (each with 90 pieces of nodules induced from petiole derived callus on the medium containing TDZ 4.55 M and IAA1.43 M). Means followed by the same letter are not signicantly different from each other at p=0.05.

due to variations of their genetic background. Ahmad (1991) reported that 2.22 M (0.5 mg l1 ) 6-BA was the most efcient in A. mangium micropropagation. In our experiment, neither 2.22 M 6-BA alone nor the combinations of 2.2222.22 M 6-BA with 0 5.38 M NAA or 1.342.86 M IAA induced nodules from callus (data not shown). The efciencies of nodule induction were compared in calli derived from ve types of explants on MS medium containing 4.55 M TDZ and 1.43 M IAA (Table 2). The percentage of nodule formation was nearly 11% in callus obtained from cotyledons, followed by callus obtained from embryo axes (nearly 10%) and less efcient for petioles, leaets, and stems derived calli (Table 2). The efciencies of nodule formation in calli obtained from cotyledons, embryo axes or petioles were signicantly higher (p<0.05) than in calli obtained from stems. Similarly, variations in efciency of regeneration among different explants were reported for Eucalyptus grandis E. urophylla hybrid (Cid et al., 1999) and Helianthus smithii Heiser (Laparra et al., 1997). Adventitious shoot induction The induction media (with 1.1422.75 M TDZ and 1.432.86 M IAA) promoted formation of secondary nodules instead of shoots during subculture. Therefore, a different step for shoot induction was necessary. The nodules began to form adventitious shoots

172 with pinnate leaves after culture for 40 days on media supplemented with 0.0450.23 M of TDZ (Table 3 and Figure 1C,F,H). The medium containing 0.045 M TDZ promoted the highest percentage (nearly 22%) of nodules to form adventitious shoots (Table 3). Each cluster of adventitious shoots contained an average of ve shoots. These results agreed with the report that low TDZ concentrations (0.010.4 M) were effective for in vitro shoot induction in woody plants (Lu, 1993). The efciency of shoot formation from the nodules decreased with higher concentrations of TDZ in the medium (Table 3). The media containing 1.144.55 M TDZ only promoted formation of secondary nodules. This is in line with the report that high concentrations of TDZ (1.144.55 M) inhibit shoot formation (Lu, 1993). Similarly, Kim et al. (1997) reported that media supplemented with the lower concentration of 0.45 M TDZ promoted the most shoot formation. Shoot elongation and rooting Adventitious shoots elongated slowly on the medium containing 0.045 M TDZ. Efcient shoot elongation was achieved by transferring clusters of adventitious shoots with pinnate leaves onto medium containing 0.045 M TDZ supplemented with 7.22 M GA3 . In 2 months, shoots elongated to 23 cm long and formed new pinnate leaves. Effects of GA3 to stimulate adventitious shoot development and elongation were documented in many plant regeneration systems, such as in vitro shoot proliferation of cassava (Bhagwat et al., 1996), shoot regeneration of Passiora foetida (Hicks et al., 1996) and regeneration of Elaeagnus angustifolia (Economou and Maloupa, 1995). The rooting of elongated shoots was induced on 1/2 MS basal medium containing 10.75 M NAA and 2.33 M KT. The frequency of elongated shoots forming roots was 102.4% after culture for 30 days (Figure 1I), which is comparable to rooting of A. mangium shoots with IBA (Galiana et al., 1991). Plantlets were transferred onto 1/2 MS basal medium until at least 10 lateral roots had formed (Figure 1J), then they were transferred into pots containing peat and white sand (3/1, v/v) (Figure 1K). More than 200 plantlets are growing well in the growth chambers and green house without visual abnormalities. Acknowledgement We acknowledge that the research was funded by grants from the National Science and Technology Board, Singapore. References
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