Establishment of seed germination and micropropagation protocols for Senegalia nigrescens, a potential anti-virulent species

ABSTRACT

This study describes seed germination and micropropagation of Senegalia nigrescens, an economic, medicinal and nitrogen-fixing species of South Africa. Seeds of S. nigrescens were subjected to pre-sowing treatments including soaking in cold water for 24 h, sulphuric acid (H₂SO₄) or hydrochloric acid (HCl) for 4, 8 and 12 min and mechanically scarifying seeds before sowing on Petri plates with wet filter paper. Mechanically-scarified seeds were also sown aseptically on a filter paper bridge, plain agar, ½ or full strength Murashige and Skoog (MS) basal medium. Single nodal explants from MS-derived seedlings were grown on MS media supplemented with 0.0, 0.5, 1.0 and 2.0 mg L⁻¹ of benzylaminopurine (BAP) or kinetin (KIN) and a combination of 0.5 mg L⁻¹ of BAP and KIN to investigate shoot multiplication. No significant differences were observed in the number of shoots produced across all treatments. However, the treatment containing 1.0 mg L⁻¹ KIN produced a significantly higher shoot length (14.17 ± 5.20 mm) than 0.5, 1.0 and 2.0 mg L⁻¹ BAP (7.67 ± 3.87, 6.75 ± 2.93 and 8.70 ± 3.56 mm, respectively). The highest rooting rate (16.7%) was obtained on ¼ strength MS supplemented with either indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) at concentrations 1.0 and 0.5 mg L⁻¹, respectively. Rooted plantlets were successfully acclimatised with a 66.7% survival rate. The findings from this study would be of great benefit for the commercial propagation of S. nigrescens.

KEYWORDS:

• Acacia
• dormancy
• pre-sowing
• in vitro regeneration
• scarification

Introduction

The continuous increase in the global human population accompanied by rapid industrialisation has contributed to climate change over the years. Afforestation, a major climate change control system (Zomer et al. 2008) is being developed as part of South Africa's effort towards ensuring environmental sustainability. With over one million hectares of land currently serving commercial forestry, South Africa's agro-forestry industry now contributes about 2.3% to the Gross Domestic Product (GDP) of the nation (Greyling et al. 2015) via the cultivation and harvesting of exotic timber species like Pinus, Eucalyptus and Acacia (now called Senegalia) (Van Der Zel 1997). Although the genus Acacia, which belongs to the family Fabaceae and sub-family Mimosoideae, has about 1500 species across the globe (Dyer 2014), only 61 of these are African which were recently re-classified under the genus Senegalia among which is Senegalia nigrescens (previously Acacia nigrescens) (Kyalangalilwa et al. 2013). S. nigrescens represents a nodulating member of the genus, known for its nitrogen-fixing activity, a property that significantly contributes to improved agriculture and land rehabilitation (Brockwell et al. 2005).

The establishment of forest species can be achieved either through sexual or asexual propagation. Sexual propagation via seeds allows for genetic diversity and is a relatively cheaper means of propagation than asexual methods (Hartmann et al. 2011). Nevertheless, in the Senegalia species, the limitations of seed progeny variability and delays in radicle emergence (Nyamukuru et al. 2014) make the asexual approach more attractive. Asexual propagation involves regenerating new plants from vegetative organs like buds, stems, leaves and roots, either in vitro or in the field, and can mass-produce ‘pure lines’ within a short time. With this approach, forest products, including medicinally-important compounds, can be commercially harvested more effectively.

S. nigrescens is a deciduous, pod-bearing, white-flowered species. The tree is characterised with a thick stem bark having knobs equipped with small black hooked thorns (Fornara 2005). S. nigrescens is widely distributed across South Africa through Zimbabwe up to Tanzania. It is a medium-to-tall (6 to 30 m in height), slow growing tree (Dharani, 2006). Its hardness, drought- and termite-resistance properties make it suitable for use as mine props and fence posts (Howell et al. 2002). Its roots and stem bark are traditionally used for the treatment of snake bite, toothache (Dharani 2006) and dysentery (Chauke et al. 2015). S. nigrescens wood is also used as fuel as it burns with severe heat yielding long-lasting coal (Liengme 1983).

A series of flavonoids have been previously reported from the heartwood of S. nigrescens (Fourie et al. 1972; Malan 1993). Additionally, our recent phytochemical study on the leaves, stem bark and roots of this species yielded novel bioactive compounds pointing to the plant's potential as an anti-bacterial and anti-quorum sensing agent (Bodede et al. 2018). The economic importance, nitrogen-fixing properties and reservoir of biologically active constituents with anti-virulent potential suggest a higher demand for S. nigrescens in the near future. Moreover, species of Senegalia are known for delayed, uneven germination due to seed coat-imposed dormancy (Rasebeka et al. 2014). It thus becomes imperative to investigate a sustainable multiplication method for this multi-purpose species. Micropropagation studies within the genus Acacia have been widely documented (Beck et al. 1998; Beck and Dunlop 2001; Dhabhai et al. 2010; Shahinozzaman et al. 2012). Nevertheless, there is no report to date on the micropropagation of S. nigrescens. Therefore, the aims of this work were to find the most effective pre-sowing treatment to promote S. nigrescens seed germination and to utilise the resulting in vitro seedlings for micropropagation studies of the plant.

Materials and methods

Pre-sowing treatment and seed germination

S. nigrescens seeds were purchased from Silver Hill Seeds (Cape Town, South Africa) in February 2015. The seeds were preserved in air-tight containers under ambient temperature in the dark before the start of the experiment. The pre-sowing treatments included soaking seeds in cold water for 24 h, H₂SO₄ or HCl for 4, 8 and 12 min each and mechanically scarifying seeds using commercial sandpaper (60 Grit). After each treatment, the seeds were thoroughly rinsed under tap water before sowing onto 9 cm Petri plates with wet filter paper. Twelve seeds were used for each treatment unless otherwise stated. Seeds were considered germinated when radicle emergence was visible (Tobe et al. 2000). The final germination percentage was recorded after 2 weeks.

In vitro germination

Mechanically-scarified seeds were subjected to surface sterilisation by first rinsing in a solution of sterile distilled water (SDW) and Tween 20® for 3 min. These were immediately transferred into 70% ethanol (EtOH) for 1 min and then rinsed with two lots of SDW. The seeds were then placed into a solution of 3.5% (v/v) sodium hypochlorite (NaOCl) for 10 min and finally rinsed 3 times with SDW. Surface sterilised seeds (10 seeds per treatment) were sown on different substrates and nutrient media viz. filter paper bridge, plain agar, ½ or full strength Murashige and Skoog (MS) (Murashige and Skoog 1962) basal medium.

Shoot multiplication

Two-week-old, single nodal explants (1.5 cm long) were aseptically excised from full strength MS-derived seedlings, rinsed in 3.5% (v/v) NaOCl for 30 s, followed by SDW (3 rinses, 30 s per rinse) after which the bleached edges were trimmed before transferring onto fresh MS media supplemented with benzylaminopurine (BAP) (0.0, 0.5, 1.0 or 2.0 mg L⁻¹) or kinetin (KIN) (0.0, 0.5, 1.0 or 2.0 mg L⁻¹) or a combination of 0.5 mg L⁻¹ BAP and 0.5 mg L⁻¹ KIN. The carbon source and hardening agents were 3% (w/v) sucrose and 1.0% (w/v) agar, respectively, while the culture vessels were 10 × 4 cm glass bottles with 3 explants in each. Each treatment had four replicates. The pH of all media was adjusted to 5.8 ± 0.1 before autoclaving at 121°C and 1.2 kg cm⁻² pressure for 20 min. All cultures were maintained at 25°C under diffuse white light of 55 µmol m⁻² s⁻¹ with an 18 h photoperiod for 6 weeks after which the number of shoots, shoot length, height of plantlet, percentage of explants forming callus and percentage of explants with contamination were recorded. The multiple shoots were then aseptically excised into 1.5 cm nodal segments and elongated for 1 week on plain MS under the same growth conditions here-mentioned.

Rooting and acclimatisation

Shoots (2-3 cm long) from elongated in vitro plantlets were cultured on different rooting media which comprised ½ or full strength MS supplemented with indole-3-acetic acid (IAA) (0.5, 1.0, and 2.0 mg L⁻¹, with or without 0.1 mg L⁻¹ BAP) or indole-3-butyric acid (IBA) (0.5 or 1.0 mg L⁻¹, with or without 0.1 mg L⁻¹ BAP). Six week-old rooted plantlets were then rinsed with SDW to remove agar from the roots before being carefully transferred into flower pots (height = 85 mm, top diameter = 100 mm) containing potting soil, watered once-off with ¼ strength liquid MS and then maintained at 25 °C under diffuse white light of 5 µmol m⁻² s⁻¹ with an 18 h photoperiod in a growth room for 14 days. The pots were covered with plastic bags for the first 7 days and, thereafter, the bags were perforated to decrease humidity for another 7 days. The plantlets were watered when the soil appeared dry. Thereafter, they were transferred to a greenhouse, where they were watered daily (for 2 min), morning and evening, by an automatic watering system. The % survival was recorded after 4 weeks.

Statistical analyses

All statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS, Version 23, IBM Corporation, Cornell, New York). Data were subjected to one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test to determine if there were significant (P < 0.05) differences among the means.

Results and discussion

Germination response of S. nigrescens to pre-sowing and substrate treatments

The final germination percentages (FGPs) after each pre-sowing and substrate/nutrient media treatment are presented in Table 1. Overall, the success rate for the germination of S. nigrescens across all pre-sowing treatments was high (83.33 ± 28.97–100.00 ± 0.00%). The highest FGP (100%) was recorded for mechanically-scarified seeds, sown on filter paper and on full MS. This result agrees with previous findings in another Acacia species i.e. mechanical scarification gave the best result (100% germination) in A. polyacantha (Missanjo et al. 2014). However, when soaking S. nigrescens seeds in H₂SO₄ for 4, 8 and 12 min in the present study, FGPs of 83.33 ± 28.97, 91.67 ± 14.43 and 98.33 ± 2.89% (not significantly different), respectively, were obtained, which contrast to a previous report on the same species where the maximum germination percentage achieved was only 30% from seeds pre-soaked in H₂SO₄ for 3 min (Rasebeka et al. 2014). Therefore, longer H₂SO₄ soaking times enhance S. nigrescens seed germination. Alternatively, the increase in FGP in the present study could be an effect of the geographical origin of the seeds which is known to influence certain gene expression in either delaying or speeding up dormancy breakage, as was observed with the ecotypes of Arabidopsis thaliana (Vaistij et al. 2013).

Table 1. Effect of pre-sowing treatments and substrate/nutrient media type on the final germination percentage (FGP) of S. nigrescens after 2 weeks of growth.

Treatment category	Treatment type	Final germination %
*Pre-sowing	Cold Water 24 h	91.67 ± 14.43^a
	H2SO4 4 min	83.33 ± 28.87^a
	H2SO4 8 min	91.67 ± 14.43^a
	H2SO4 12 min	98.33 ± 2.89^a
	HCl 4 min	83.33 ± 14.43^a
	HCl 8 min	91.67 ± 10.41^a
	HCl 12 min	91.67 ± 5.77^a
	Mechanical Scarification	100.00 ± 0.00^a
**Substrate/nutrient media	Filter paper bridge	61.13 ± 9.64^b
	Plain agar	80.57 ± 14.16^ab
	½ MS	80.57 ± 17.33^ab
	Full MS	100.00 ± 0.00^a

*Seeds were sown on filter paper in Petri plate. **Mechanically scarified seeds were used. The FGPs with the same alphabet are not significantly different by Tukey's Post Hoc Test at p < 0.05 within the column.

Having established mechanical scarification as the best pre-sowing treatment for S. nigrescens, mechanically-scarified seeds were surface-sterilised as before and sown on different substrates and nutrient media (Table 1). Seeds cultured on full strength MS resulted in 100% FGP as was obtained from the original mechanical scarification treatment although this was not significantly different from the FGPs on plain agar (80.57 ± 14.16%) or ½ strength MS (80.57 ± 17.33%). Only seeds sown on the filter paper bridge had a significantly lower FGP (61.13 ± 9.64%) than the seeds sown on full strength MS. The 100% FGP obtained from full strength MS suggests that optimal germination is obtainable when seeds of S. nigrescens are sown on media with higher concentrations of macro- and micro-nutrients, However, for cost-effectiveness, the results show that plain agar can be utilised for commercialisation purposes. This study has shown that hard seededness in Acacia can be overcome by mechanical and chemical pre-sowing treatments, thereby allowing for water imbibition by the embryo (Rasebeka et al. 2014) which in turn promoted germination.

Shoot multiplication

Bud break was observed within a week and shoots were well developed after 4 weeks. The response of S. nigrescens nodal explants to the cytokinins BAP and KIN are presented in Table 2. The number of shoots produced across the seven treatments was in the range of 2.58 ± 1.31 - 4.50 ± 1.62, with no significant differences amongst the values. It has been widely reported that BAP is more effective in in vitro shoot organogenesis than KIN for Acacia species (Mittal et al. 1989; Galiana et al. 1991; Dewan et al. 1992; Beck et al. 1998; Khalafalla and Daffalla 2008; Rout et al. 2008; Shahinozzaman et al. 2012) but in the present study, there were no significant differences between KIN and BAP shoot multiplication treatments, or when they were used in combination. Since the number of shoots obtained from the plant growth regulator (PGR)-free medium was not significantly different from those obtained in the PGR treatments, it is likely that S. nigrescens contains endogenous PGRs whose concentrations meet the plant's need to induce shoots. This is in agreement with other reports which show that hormone-free media produced multiple shoots in some Acacia species (Jones et al. 1990; Zhang et al. 1995; Beck et al. 2000).

Table 2. Effect of BAP and KIN on shoot multiplication, plantlet height, callus formation and fungal contamination in S. nigrescens nodal explants after 6 weeks of in vitro culture.

Treatment (mg L^−1)	No. of shoots	Shoot length (mm)	Height of plantlet (mm)	% explants forming callus	% contaminated explants
Plain MS (control)	3.92 ± 1.78^a	11.75 ± 3.14^ab	20.42 ± 5.05^abc	16.70 ± 0.39^d	0.00 ± 0.00^c
BAP 0.5	3.25 ± 1.76^a	7.67 ± 3.87^bc	16.75 ± 5.31^c	33.30 ± 0.49^c	16.70 ± 0.39^b
BAP 1.0	2.58 ± 1.31^a	6.75 ± 2.93^c	14.75 ± 3.77^c	0.00 ± 0.00^e	25.00 ± 0.45^a
BAP 2.0	2.90 ± 1.45^a	8.70 ± 3.56^bc	20.30 ± 7.13^abc	50.00 ± 0.53^b	0.00 ± 0.00^c
KIN 0.5	3.17 ± 1.75^a	11.50 ± 5.71^abc	20.50 ± 10.26^abc	25.00 ± 0.45^c	16.67 ± 0.39^b
KIN 1.0	3.67 ± 1.87^a	14.17 ± 5.20^a	27.42 ± 9.76^a	50.00 ± 0.52^b	25.00 ± 0.45^a
KIN 2.0	3.08 ± 1.31^a	11.25 ± 3.08^abc	21.67 ± 6.61^abc	16.67 ± 0.39^d	0.00 ± 0.00^c
BAP 0.5 + KIN 0.5	4.50 ± 1.62^a	12.08 ± 2.81^ab	29.58 ± 9.33^a	66.67 ± 0.49^a	33.30 ± 0.49^a

Note: BAP - benzylaminopurine, KIN - kinetin. Values are represented as mean ± standard deviation (where n = 12). Values with similar letters are not significantly different to one another by Tukey's Post Hoc Test at p < 0.05. (Shoot length: length of longest shoot; Height of plantlet: distance between the longest root and shoot tip. Both measurements were taken after 6 weeks of culture).

PGRs significantly influenced the shoot length and height of plantlets in some treatments (Table 2). KIN at 1.0 mg L⁻¹ produced a significantly higher shoot length (14.17 ± 5.20 mm) than BAP at 0.5 mg L⁻¹ (7.67 ± 3.87 mm), 1.0 mg L⁻¹ (6.75 ± 2.93 mm) and 2.0 mg L⁻¹ (8.70 ± 3.56 mm). Likewise, KIN at 1.0 mg L⁻¹ produced significantly higher plantlet height (27.42 ± 9.76 mm) than BAP at 0.5 mg L⁻¹ (16.75 ± 5.31 mm) and 1.0 mg L⁻¹ (14.75 ± 3.77 mm). Although KIN has been successfully used for shoot development in some plant species when used singly (Hesar et al. 2011; Jerzy et al. 2013; Abu-Romman et al. 2015) or combined with other PGRs (Shaheenuzzaman et al. 2011; Tolera et al. 2014), this was not the same for most Acacia species where BAP, used singly or in combination with other PGRs, was found to be superior to KIN for shoot development (Galiana et al. 1991; Girijashankar 2011; Rathore et al. 2014). Species within the same genus, or different genera, often exhibit variations in their morphogenic response to the same PGRs due to differences in genotype, explant type and culture conditions which in turn modifies the mechanisms of action of the PGRs (Gaspar et al. 1996).

In vitro shoot regeneration from nodal explants is sometimes characterised by the formation of callus around the basal end of the explant due to the effect of the auxin accumulation in this plant part (Marks and Simpson 1994). Such masses of undifferentiated cells sometimes inhibit desirable morphological differentiation of the plantlet (Mittal et al. 1989; Nanda et al. 2004; Dhabhai et al. 2010) although they also represent platforms on which indirect organogenesis of plant organs can be obtained (Vengadesan et al. 2003). Even though some treatments resulted in up to 66.67 ± 0.49% explants forming callus at the basal ends in the present study (Table 2), the callus did not inhibit the induction of multiple shoots from the axillary bud and no evidence of indirect organogenesis was observed. The combination of BAP and KIN in this study resulted in the highest percentage of explants forming callus, which is in agreement with other reports on Acacia (Ortiz et al. 2000; Xie and Hong 2001; Rathore et al. 2012).

There was no record of fungal contamination in S. nigrescens explants cultured on plain MS, 2.0 mg L⁻¹ BAP and 2.0 mg L⁻¹ KIN (Table 2). Furthermore, there were no significant differences in the percentage contaminated explants between BAP or KIN treatments of the same concentration (16.70 ± 0.39% in BAP and KIN at 0.5 mg L⁻¹ and 25.00 ± 0.45% in BAP and KIN at 1.0 mg L⁻¹). The factors contributing to the occurrence of fungal contaminants in S. nigrescens, like other woody species, is yet to be fully understood. Different surface sterilisation methods have been employed for Acacia species (Gantait et al. 2016). Sterilisation involving dipping explants in 70% EtOH for 30–90  s followed by 0.1% (w/v) mercuric chloride (HgCl₂) for 5–10  min and repeated washing in sterile water, are the most common. However, HgCl₂ is known to be environmentally hazardous (Saha 1972) thus, more environmentally-friendly alternatives are being sought for tissue culture application. In the present study, the sequential and time-controlled use of Tween 20®, 70% EtOH, 3.5% NaOCl and SDW effectively removed surface contaminants, as there was no record of fungal contamination in the seeds of S. nigrescens germinated in vitro. However, as seen in Table 2, fungi were observed in some treatments during in vitro shoot multiplication. This may be attributed to endogenous fungal pathogens, which to date, are still a subject of concern in Acacia micropropagation (Gantait et al. 2016).

Rooting and acclimatisation

In vitro shoots of S. nigrescens were cultured on media which contained different MS salt strengths and varying concentrations of IAA, IBA and BAP, singly or combined (Figure 1). The rooting response was low, the highest percentage of rooted explants (16.7%) was obtained on ¼ strength MS supplemented with either 1.0 mg L⁻¹ IAA or 0.5 mg L⁻¹ IBA. This contrasts with reviews on micropropagation in the genus Acacia which showed that rooting was more successful using ½ strength MS supplemented with various concentrations of PGRs (Beck and Dunlop 2001). In other rooting studies among the Acacia species, ½ strength MS was used with very low auxin (IAA and IBA) concentration (Galiana et al. 1991) or hormone free (Girijashankar 2011). When using ½ strength MS in the present study, rooting was only observed when combined with 0.5 mg L⁻¹ IBA (8.3%). The results imply that reduced MS strength is required for rooting in S. nigrescens similar to Acacia catechu in which the best rooting (60%–80%) was achieved on ¼ strength MS but supplemented with higher auxin concentration (3 mg L⁻¹ IAA) (Kaur et al. 1998). The choice of the concentration range (0–2 mg L⁻¹) for rooting PGRs in this study was based on literature which mostly use PGRs of concentrations between 0 and 1 mg L⁻¹ in Acacia species (Gantait et al. 2016). Few reports exist on Acacia species using PGR concentration above 2 mg L⁻¹ for rooting (Gantait et al. 2016). More studies are therefore needed to fully understand the requirements for rooting in S. nigrescens. Acclimatisation was achieved on potting soil. The controlled exposure of plantlets to hardening conditions resulted in a 66.7% survival rate of S. nigrescens.

Figure 1. Effect of MS strength and PGRs on the in vitro rooting of S. nigrescens after 6 weeks of culture.

To the best of our knowledge, this work has described the first report on the micropropagation of S. nigrescens. Mechanical and chemical pre-sowing treatments were effective for overcoming the barrier of hard seededness in the germination of S. nigrescens. Different PGR types and concentrations used in this work had no significant effect on shoot multiplication but significantly influenced the shoot length and plantlet height while the best rooting was achieved using ¼ strength MS in the presence of either 1.0 mg L⁻¹ IAA or 0.5 mg L⁻¹ IBA. The survival rate for the acclimatisation stage was 66.7%. Further studies are necessary for the optimisation of the rooting stage of this species and more insight is needed to eliminate endogenous fungal contamination in vitro. However, the germination, shoot multiplication and acclimatisation protocols reported in this study provide valuable information for subsequent works in the regeneration of S. nigrescens, necessary for large-scale harvesting of the plant's products and its pharmacologically-active compounds by plant-based drug developers.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Notes on contributors

Olusola Bodede is a Doctoral graduate of the University of KwaZulu-Natal whose Ph.D. research focused on the phytochemical investigation and tissue culture studies of selected South African medicinal plants.

Shakira Shaik is an academic in the School of Life Sciences at the University of KwaZulu-Natal. Her research focuses on the micropropagation of South African medicinal plants.

Roshila Moodley is an academic in the School of Chemistry & Physics at the University of KwaZulu-Natal. She is a natural products chemist and is interested in the phytocompounds derived from medicinal plants.