Introduction

The Acacia group (Leguminosae family, Mimosaceae subfamily) comprises more than 1250 known species of shrubs and trees (Simmons 1987), which are dispersed widely in tropical and subtropical regions of Australia, Africa, Asia, and South America (Beck and Dunlop 2001). Most species of Acacia have hard trunks and can be used as firewood, railway crosstie, pit timbers, a source of pulpwood production, furniture, farm tools, and especially as sawdust in polypropylene bags for mushroom farming. In the era of global environmental change, the pseudoleaf of Acacia trees evolved into the state of preventing water evapotranspiration and forms a self-protection mechanism. It adapts to the tropical soil in arid and humid areas, so that it can exist in extreme atmospheric conditions (Palmberg 1981). Because of its numerous and vigorous roots, it can hold onto soil and increase the soil fertility by symbiotic relationship with rhizobia and mycorrhizal fungi, which help improve the nitrogen deficiency of poor ridge land; withstand strong wind, drought, and poor soil quality; reduce soil erosion; and contribute to the stability of sand dunes (Skolmen 1986). Acacia trees are used for reforestation to reclaim wasteland and improve soil health, or as timber and shelter which are important for sustainable development of arid and tropical regions (Gantait et al. 2018).

The Acacia tree is naturally a cross-pollinated species. Only by grafting, cutting, layering, or tissue culture of asexual propagation can it have a great potential for clonal propagation of superior genotypes. The Acacia species have been given due importance in tree tissue culture owing to their proven wasteland reclamation ability, and ecological and economic significance. Micropropagation offers a rapid means to produce clonal planting stock for afforestation, woody biomass production, and conservation of elite germplasm. Generally, it is difficult to induce regeneration of mature woody plants in vitro. There were previously some reports on the development of plantlets from callus raised through shoot tip, cotyledonary buds, and nodal explant from new germination young seedlings in Acacia species (Mathur and Chandra 1983; Mittal et al. 1989; Darus 1990; Huang et al. 1994; Nandwani 1995; Nangia and Singh 1996; Vengadesan et al. 2000; Xie and Hong 2001a, b; Quoirin 2003; Rout et al. 2008; Monteuuis et al. 2013). According to the results of these studies, most of the explants were obtained from young seedlings with the best regeneration ability. However, the trunk character of young seedlings from a cross-pollinated woody tree is not known, which is different from obtaining explants from the elite mature plants or from young seedlings for micropropagation. When tissue culture is applied to the mature woody plants, there is a great difference in juvenile characters (Hackett 2011). Therefore, it is necessary to establish a culture system from mature plants as a source of explants and capable of efficient plant regeneration, to be widely used in the promotion of elite trees identified from their adult-stage phenotype. In vitro micrografting can also be an alternative for woody plant asexual propagation (Palma et al. 1996), but it is very time-consuming and is mostly used for special purposes, such as virus removal from contaminated elite mature mother stock plants.

One prominent aspect of the establishment of mother stock of woody perennials in microculture is “stabilization.” The most obvious signs showing the stabilization stage are the successful disinfection, no lethal browning, and seasonal dormancy of the explants (McCown 2000; Chung and Chen 2018). When seedlings germinated in aseptic culture are used as explants, there is no contamination problem as well as no juvenility problem. However, the explants of mature woody plants grown in the field often have serious contamination and browning to overcome. The studies of melon, petunia, tobacco, and cauliflower examined 0 to 2.0 mL L−1 range of PPM™ (Plant Preservative Mixture) concentrations added in the first culture medium which was found to be optimal for controlling contamination without causing a reduction (Compton and Koch 2001; Miyazaki et al. 2010; Rihan et al. 2012). Browning of explants from woody plant species often was a problem, too. The aseptic culture system was established to overcome browning by using activated carbon (Thomas 2008) , vitamin C (Ko et al. 2009), PVP (Saxena and gill 1986; Reustle and Natter 1994), and citric acid (Huang et al. 2012), which was also the beginning step for a successful micropropagation (Singh et al. 1993).

In addition, previous studies on Acacia reported that the evaluation of appropriate cytokinin, such as BA (Skolmen and Mapes 1976), TDZ (Huetteman and Preece 1993; Xie and Hong 2001a, b; Vengadesan et al. 2003), 2ip (Banerjee 2013), and kinetin (Nangia and Singh 1996), was carefully selected to break the apical dominance, avoid browning, and promote lateral bud proliferation, while GA was used to promote shoot elongation (Vengadesan et al. 2000, 2003). Finally, it is necessary to harden the plantlets in vitro in a proper way before transplanting, so as to facilitate the survival of plantlets transplanted.

There is no report on the micropropagation of Acacia confusa from mature trees. In the current study, in vitro cultures were initiated from the nodal sections taken from the adult plants in the field. The disinfection and juvenility of the explants, browning of tissues, and seasonal effect of materials will be the key factors for success. In this study, dormant buds from nodal segments of mature Acacia trees were used as explants for micropropagation and cultures were carried in different months of a year. The fact is that explants were derived from a large number of new vigorous branches after hard pruning; the value of explants can be used not only as the maintenance of elite trees for genetic fixation, but also as the rapid asexual propagation and the conservation of endangered tree species.

Materials and Methods

Source, juvenile treatment, and nodal section disinfection

The elite trees of Acacia confusa used in this study spanned 15 y in age and were collected at the Chungpu stations of Taiwan Forest Research Institute and Da-Yeh University in Taiwan. For harvesting more juvenile explants for in vitro culture, the main stem of an adult tree was cut to 50 to 100 cm in height that would induce new shoots growing close to the ground. Explants were taken once every mo for in vitro culture to identify the time (month) on culture response.

Nodal sections were obtained by removing the leaves from new branches of field-grown A. confusa, washed with detergent for 3 times, put into a 150-mL flask containing RO water, and shaken with rotary shaker at 250 rpm for 2 h. These nodal sections were again shaken by hand in 50 mL of 70% ethanol adding 1 drop of acetic acid for 30 to 45 s. Finally, the explants were disinfected while swirling in 100 mL 20% Clorox™ (sodium hypochlorite 5.25%, Auckland, CA) bleach solution containing 1 drop of polyoxyethylene 20-sorbitan monolaurate (Tween 20) and with 40 kHz intensity of ultrasonic treatment for 15 min, then rinsed three times in sterile distilled water for 1 min each time.

PPM™ treatments in nodal culture

Following the disinfection described above, the explants were inoculated on culture medium with or without 2.0 mL L−1 PPM™. The inoculated basal medium was that of Murashige and Skoog (MS; Murashige and Skoog 1962) with 3.2 g L−1 polyvinylpyrrolidone (PVP, Acros Organics, Geel, ANR), 30 g L−1 sucrose (Sigma-Aldrich, St. Louis, MO), and 7.0 g L−1 phytoagar (Sigma-Aldrich, St. Louis, MO) adjusted to a pH of 4.8 with NaOH and HCl. PPM™ treatments were grouped into four tests (Table 1). Half a liter of medium was dispersed into a glass bottle and covered with plastic cap before autoclaving at 121 °C and 124 kPa for 15 min. Medium (25 mL) was dispersed into a 90 mm × 20 mm sterilized plastic Petri dish. Five explants were cultured in one Petri dish. Each treatment consisted of 50 explants. Cultures were first kept in dark at 25 ± 1 °C for 10 d. The disinfection tests were repeated four times. Percentage of sterilization of explants was calculated.

Table 1 Using PPM™ to control contamination on Acacia confusa nodal explants after surface disinfection
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Nodal section culture of mature A. confusa trees

In this process, the nodal sections of sterilized explants were cut into approximately 1-cm segments. The explants were cultured on basal media that were MS (Murashige and Skoog 1962), 1/2 MS, or Woody Plant Medium (WPM; Lloyd and McCown 1980) containing various combinations of growth regulators including 0.5, 1.0, 2.0, 4.0, and 6.0 mg L−1 of BA (Koch-Light Limited, Cambridge, UK); 0.5, 1.0, and 2.0 mg L−1 of TDZ (Chem Service, West Chester, PA); 0.5, 1.0, and 2.0 mg L−1 of 2iP (Phyto Technology Lab., Shawnee Mission, KS); 1.0 mg L−1 kinetin (Koch-Light, Buckinghamshire, UK); 12, 50, and 100 mg L−1 of AS (Sigma-Aldrich); 0.1, 0.25, 0.5, 1.0, and 1.5 mg L−1 of IBA (Koch-Light); or 4.0, 5.0, 8.0, 10, and 12 mg L−1 of GA (Koch-Light) (as shown in Table 2). All the testing media have the adjunctive 3.2 g L−1 PVP, 30 g L−1 sucrose (Sigma-Aldrich), 2.0 mL L−1 PPM™, and 7.0 g L−1 phytoagar, adjusted to a pH of 4.8 with NaOH and HCl. Twenty-five mL of medium was dispersed into each 90 mm × 20-mm plastic Petri dish after autoclaved at 121 °C and 124 kPa for 15 min. Each Petri dish was inoculated with 5 explants and there were 10 culture dishes in each treatment group. All of culture dishes were placed in the incubator (16-h light/8-h dark). According to the above disinfection and culture methods, the nodal section samplings of adult plants began in August 2019 and ends in July 2020 to test the effects of different times of the year in the field.

Table 2 Necrosis and survivals in nodal culture of mature Acacia confusa trees at the initial establishment stage with different growth regulator combination
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Effects of growth regulators on shoot proliferation

After the aseptic culture of nodal explants was successfully established, the proliferation medium provided the axillary shoots for the next rooting stage. The explants were inoculated on MS medium supplemented with testing three types of cytokinin TDZ, BA, and AS in combination with IBA and glutamine (as shown in Table 3). Each culture bottle contained 50 mL medium in a 125-mL Erlenmeyer flask and inoculated 3 responsive explants. The number of explants with regenerated shoots and the number of newly formed axillary buds from the shoot on each responsive explant were recorded after 3 wk of culture. The regenerated shoots were subcultured at 45-d intervals onto medium of the same growth regulator composition for each test.

Table 3 Effects of the basal medium containing various combinations of growth regulators and glutamine for nodal culture of mature Acacia confusa trees
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The percentage of the explants with shoots and the average number of axillary bud were calculated as per following formulae:

$$Percentage of explants with shoot \left(\%\right)=\left[\left(number of explants with shoot\right)/\left(total number of explants inoculated\right)\right]\times 100$$
$$Average number of axillary buds per responsive explant=\left(sum of the number of axillary buds in each explant\right)/\left(number of explants with shoot\right)$$

Microcutting rooting trials

Well-grown shoots (3 to 5 cm in length) were collected as microcutting from proliferating shoot masses. Microcuttings were moved to 1/2 MS basal medium supplemented with 3.2 g L−1 PVP, and treated with either IBA or NAA (0.5, 1.0, and 2.0 mg L−1) in combination with or without 100-g L−1 banana pulp. After the culture medium was finally prepared, the 30 g L−1 sugar and 7.0 g L−1 agar are added at a pH 4.8 for rooting. Medium of 25 mL in a 100-mL glass tube was inoculated with 1 microcutting for rooting.

Statistical analysis

All experiments were performed in a completely randomized design. Data were subjected to analysis of variance using Statistical Product and Service Solutions (SPSS). Mean separation was achieved using one-way ANOVA analysis; ρ < 0.05 indicated significant difference.

Results and Discussion

Explant sources and disinfection procedure

Most woody crop in vitro cultures are first established using shoot culture approaches where the stimulation of axillary buds from shoot tips is the prime objective (Chung and Chen 2018). The nature of the explant used considerably influences its multiplication. In the cases of mature A. confusa trees, many nodal section explants can be obtained, and the in vitro operation is convenient. However, clonal regeneration by somatic embryogenesis or organogenesis is still difficult for many woody species and is often limited to the use of juvenile explants (Bonga et al. 2010; Hackett 2011) which are derived mostly from in vitro seed germination. The problems of field-grown explants are contamination, necrosis, season, and juvenility. In this study, the main stem of an adult tree was severely pruned to induce new shoots emerging close to the ground (Fig. 2a, b). Hard cutting back of main stem of a mature tree often induces the invigoration and possible rejuvenation, which can promote the regeneration ability of propagules (Hartman et al. 2014).

According to the general disinfection procedure (70 to 75% ethanol and 20% Clorox™), the contamination of A. confusa nodal segments had not been completely controlled and the follow-up experiments could not be carried out. We analyzed the effects of sodium hypochlorite and PPM™ on surface sterilization. No shoots were produced, either because of contamination of the explants, or loss of explants as a result of using 75% ethanol together with longer treatment of sodium hypochlorite. Results from this study showed that 2.0 mL L−1 PPM™ was an effective agent for controlling the contamination in A. confusa micropropagation system. After 10 d of culture, the control group not treated with PPM™ but by general disinfection procedure resulted in only 43.7% of surface sterilization. However, the PPM™ treatments showed high efficiency of more than 97.0% sterilization effect of explants from field-grown plants (Table 1).

The determination of the suitable concentration is an essential requirement for using PPM™ since the high concentrations can be harmful to plant cells (Rihan et al. 2012). It seems that there is a relationship between phenolic-like component exudation and the concentration of PPM™ used since there were no signs of the presence of these compounds when PPM™ concentrations were higher than 0.25 mL L−1 (Rihan et al. 2012). This accorded with Chamandoosti (2010) who mentioned that there was a relationship between chemical composition of the medium and phenolic compound production. The influence of PPM™ on plant regeneration depends on the plant species. Experiments of melon, petunia, tobacco, and cauliflower examined a 0 to 2.0 mL L−1 range of PPM™ concentrations when using it in the initial culture medium which was found to be optimal for controlling the contamination without causing a reduction (Compton and Koch 2001; Miyazaki et al. 2010; Rihan et al. 2012).

Table 1 shows there is no significant difference among the three treatment groups NPPM, PPM, and PPPM (with or without PPM™). The results indicated that it was not necessary to soak explants in PPM™ solution before culturing, but only 2.0 mL L−1 PPM™ added into the medium resulted in low contamination. Explants with low contamination rate were also derived from soaking in a 50% PPM solution (500 mL L−1 PPM™) for 10 min without adding PPM™ to the culture medium. But the PPM™ concentration used in this method is much higher and less economical. Therefore, the results revealed that adding 2.0 mL L−1 PPM™ in the medium was enough to achieve good sterilization of the explants (Table 1).

Mother stock establishment in in vitro culture

When the sterile explants from field-grown nodal segments were cultured and examined, over 85.0% of explants remained green for 2 wk and gradually turned brown and died later when cultured on medium with combinations of BA, TDZ, 2iP, and kinetin. However, the combinations of BA, AS, IBA, and GA media were effective for decreasing the necrosis and provided with good growth response (Table 2). There are many factors affecting browning of explants, such as plant species and genotypes, physiological status, damage from sterilization, medium composition, culture conditions, and seasonal effect. The addition of browning inhibitors can alleviate the toxicity of phenolic substances and achieve the purpose of protecting explants. For example, activated charcoal and polyvinylpyrrolidone (PVP) are able to adsorb phenolic substance; ascorbic acid can be absorbed by the explants and have anti-oxidation effect (Saxena and Gill. 1986; Reustle and Natter 1994; Ko et al. 2009). A report on the research of Cinnamomum kanehirae in Taiwan demonstrated the browning of explants occurred after long-term culture could be reduced by adding 3.2 g L−1 PVP to the medium (Chang et al. 2002). In our study of A. confusa, PVP was used as anti-browning agent. It was also combined with different cytokinins and GA to induce outbreak of dormant buds to form multiple shoots (Table 2; Fig. 2c). In addition to the establishment of a stable mother stock in vitro, the purpose of mass reproduction was achieved in this study.

The nodal segments of mature A. confusa trees had the best growth response on MS medium supplemented with 4.0 to 6.0 mg L−1 BA, 0.1 to 1.5 mg L−1 IBA, 10 mg L−1 GA, and 12 mg L−1 AS (Table 2) resulting in 3 to 6 new buds on each node (Fig. 2c). Girijashankar (2011), in a survey of 18-mo-old A. auriculiformis trees, reported that MS medium with 2.0 mg L−1 BA and 0.1 mg L−1 NAA supported high survival rate of explants, and nodal culture could induce 76% of shoot regeneration. Zhang et al. (1995) reported that shoot induction on MS basal medium supplemented with 10 μM BA and 0.5 μM IBA was obtained through culturing A. auriculiformis and A. mangium from 4-y-old mother trees, and the shoot regeneration rate of these two species was 66.7% and 55.0%, respectively. However, in our preliminary study, 15 y aged mature A. confusa trees were sampled and cultured without hard pruning. The survival rate of explants could not reach more than 5% in testing media (the data is not presented). After hard pruning (cutting the main stem down to 1 m above ground), the survival rate of the explants from new growth was increased to nearly 25% (Table 2) on medium with best nutrient combination, and the probability of axillary bud induction was about 19% in the initial culture (Fig. 1). The age of donor tree for explants may be counted on the different response in shoot regeneration in vitro. When the basal medium was concerned, 1/2 MS or WPM was used instead of MS medium (Table 2), the sprouting of new shoots showed 2 wk later than that of MS basal medium in this study. The differences in micropropagation in relation to various Acacia species, different physiological status of donor trees, or different applied basal medium were reviewed by Gantait et al. (2018).

Figure 1.
figure 1

Percent of axillary bud induction from monthly sampling of mature Acacia tree nodal culture in 2019 to 2020

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Seasonal effect on survival and regeneration of explants

In woody plants, nodal explants from seedlings had higher multiplication rates than the explants from adult plants. Cultures initiated at the beginning of May produced the highest percentage of shoot forming explants and multiplication rate in Quercus euboica (Kartsonas and Papafotiou 2007). In addition to seasonal bud dormancy, predetermined and limited periods of seasonal shoot growth and a marked change in growth characteristics as the plants progress from the juvenile to adult phases of their life cycle all complicate responsiveness and predictability in vitro. Therefore, in this experiment, explants taken from adult A. confusa trees for culture had been treated with rejuvenation by manipulating donor plants with hard pruning in advance (Fig. 2a, b).

Figure 2.
figure 2

(a) The mature mother tree of A. confusa treated by strong cutting. The height of finger point was about 1.5 m from soil, and the diameter of the branch was about 2 cm. (b) The red mark shows that the rejuvenated shoots were growing out. (c) The establishment of a stable mother stock in vitro. (d) The combinations of 1 mg L−1 BA, 0.5 mg L−1 TDZ, 12 mg L−1 AS, and 500 mg L−1 glutamine could significantly improve the proliferation of adventitious buds. (e, f) Shoots generated in vitro with a length 3 to 5 cm would be excised to microcutting. (g) In vitro rooting. (h) Transplantation to soil

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For explants of mature A. confusa trees obtained in different seasons, inoculated on MS medium supplemented with 4.0 mg L−1 BA, 0.1 mg L−1 IBA, 10 mg L−1 GA, 12 mg L−1 AS, and 3.2 g L−1 PVP, growth response was mainly concentrated in the warm period of summer from May to July; the regeneration frequency of axillary buds ranged from 15.8 to 18.5%. Figure 1 also showed that axillary buds could be induced in August and December with a gradually decreased frequency to 9.5% and 6.8%. The outbreak of dormant buds could not be induced without GA addition (Table 2). In addition to the selection of medium and seasonal factors, the juvenile character of explants (Fig. 2 a, b) is the key to the sprouting of dormant buds on nodal segments. Chang et al. (2002) also mentioned seasonal effect that shoot tips taken in spring season gave the highest survival and multiplication rate on in vitro culture of Cinnamomum kanehirae.

Shoot multiplication

The explants with in vitro response were subcultured in MS medium supplemented with three types of cytokinin TDZ, BA, and AS in combination with IBA and glutamine (as shown in Table 3). Table 3 indicates that the explants cultured in MS medium with 1.0 mg L−1 BA, 0.5 mg L−1 TDZ, 12 mg L−1 AS, and 500 mg L−1 glutamine initiated primarily clumpy and dwarf buds with approximately 2.9 axillary buds per responsive explant after 3 wk of culture. The number of shoots derived was significantly different at ρ < 0.05 by one-way ANOVA analysis. Only in the combinations of BA and IBA did the proliferation of axillary buds show obviously elongated shoots. The results showed that MS basal medium with the abovementioned plant growth regulators and glutamine affected the number of buds formed on subculturing nodal explant of A. confusa (Table 3). The regenerated shoots from nodal explant were transferred at a 45-d interval onto the same medium and growth regulator composition (Fig. 2d, Fig. 3).

Figure 3.
figure 3

Effects of adventitious bud multiplication on subculturing medium after 45 d. The treatment groups indicated by the horizontal axis were in accordance with the medium code in Table 3. Different letters indicate statistically significant differences. The bar line represents the standard deviation of samples

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Figure 3 shows that bud proliferation was significantly higher when explants were cultured on medium containing 0.5 mg L−1 TDZ compared to 1.0 mg L−1 (Table 3). Adding glutamine to the culture medium increased the number of shoots. Glutamine supports the growth of cells that have high energy demands and synthesizing large amounts of proteins and nucleic acids. It is an alternative energy source for rapidly dividing cells and cells that use glucose inefficiently (Greenwell and Ruter 2018). Higher percentage of nitrogen may improve the growth of plantlets and embryogenesis of cells (Ogita et al. 2001). In this study, 500 mg L−1 glutamine was added to the medium containing BA, TDZ, and AS at the same time; the multiplication of axillary buds decreased with the increase of BA concentration. This may be due to the fact that BA, TDZ, and AS are cytokinins, and excessive cytokinin can inhibit the lateral bud proliferation (Huetteman and Preece 1993). In this study, 12 kinds of medium for inducing axillary bud regeneration were tested. The results showed that the combinations of 1.0 mg L−1 BA, 0.5 mg L−1 TDZ, 12 mg L−1 AS, and 500 mg L−1 glutamine could significantly improve the proliferation of multiple shoots (Fig. 2d; Fig. 3).

Most of the studies in tissue culture of Acacia were used juvenile explants such as embryo, cotyledon, stem segment, and hypocotyl of seedling (Beck and Dunlop 2001; Vengadesan et al. 2002; Quoirin 2003; Gantait et al. 2018), and that only a few reports use materials from larger trees. Huang et al. (2012) reported that A. mangium 3- to 5-y-old stem segments had 20 to 30% shoot regeneration opportunities. Vengadesan et al. (2003) developed an in vitro propagation protocol using nodal explants from a 10-y-old ‘elite’ tree of A. sinuata. Maximum shoot proliferation was achieved from nodal explants collected during the December to March time frame. MS medium supplemented with 8.9 μmol BA, 2.5 μmol TDZ, and 135.7 μmol AS (60 d after inoculation) had the best induction effect on shoot proliferation (Vengadesan et al. 2003). The above report is not exactly similar to our current results on A. confusa which showed best seasonal effect in May to July, and next in December. The concentration of cytokinin used in culturing A. sinuata was approximately equal to 2.0 mg L−1 BA, 0.5 mg L−1 TDZ, and 50 mg L−1 AS, which was not comparable to our data derived from culturing A. confusa. In the current study, it is demonstrated that 500 mg glutamine and 3.2 g L−1 PVP added to the subculturing medium promote the multiple shoot proliferation and resist browning (Fig. 2d).

Plantlets rooting in vitro and transplanting to soil

Shoots generated in vitro with a length of 3 to 5 cm were excised and cultured on 1/2 MS medium supplemented with IBA or NAA (Fig. 2e, f) for rooting in vitro. Of the two auxins tested for their ability to induce the root growth, MS medium supplemented with 1.0 mg L−1 IBA supported the best rooting response with 72.2% rooting frequency and an average of 3.5 roots per plant and 4.2 cm in root length within 6 wk (Fig. 2g). However, when the concentration of NAA or IBA was 2 mg L−1, callus was induced at the basal part of the shoots. In A. confusa culture, adding 100 g L−1 banana pulp did not induce root formation, but banana pulp was effective for rooting in orchid plants (Souza et al. 2013).

In vitro rooted plantlets were hardened by moving culture glass tubes next to a window where oblique sunshine was available for more than 4 wk. Hardened plantlets were removed from the culture medium, washed thoroughly in water, and transferred to clay pots that contained a mixture of peat moss, vermiculite, and perlite in a 1:1:1 ratio. The potted plantlets were successfully acclimatized in a moist and shady place and eventually transferred to a greenhouse. Around 90% of the plantlets survived and initiated new growth after 2 mo (Fig. 2h).

Conclusion

The micropropagation system described above is a commercially viable method for propagating a mature ‘elite’ tree of A. confusa. This study shows that PPM™ can be an effective agent for controlling the contamination of explants. MS medium containing 4.0 mg L−1 BA, 0.1 mg L−1 IBA, 10 mg L−1 GA, and 12 mg L−1 AS and the adjunctive 3.2 g L−1 PVP is sufficient for the establishment of mother stock in in vitro culture. Explants with shoots induced on the node in the first culture were subcultured on MS medium with the combination of 1.0 mg L−1 BA, 0.5 mg L−1 TDZ, 12 mg L−1 AS, and 500 mg L−1 glutamine for mass propagation of A. confusa plantlets in vitro.

In conclusion, (1) hard pruning of mature mother trees should be applied first to induce rejuvenated shoots for the explants, (2) donor plants should be kept away from pathogenic organisms to reduce contamination of explants, (3) seasonal effect for a successful micropropagation should be determined in the different areas, and (4) PPM™ and PVP in the initial culture medium are necessary for a clean and viable nodal culture of Acacia grown in the field.