Development of an in vitro regeneration system for Heinsia crinita (Afz.) G. Taylor via direct induction of shoot proliferation from explants

Abstract

Objective

The African bush apple (Heinsia crinita) is a highly valued orphan shrub that supports the nutritional and natural medicine needs of many sub-Saharan African communities. However, the crop remains poorly conserved and without any known genetic improvement. Accordingly, the current study sought to develop for the first time, an in vitro regeneration system based on direct shoot proliferation from its stem and hypocotyledonary explants using combinations of two cytokinins (benzyl adenine – BA, thidiazuron – TDZ) and the auxin (naphthalene acetic acid (NAA), in Murashige and Skoog (MS) medium.

Results

Combinations of BA and NAA effectively induced multiple shoot formation from stem and hypocotyledonary explants of the crop. The most effective treatment (1.0 mg/L BA + 0.1 mg/L NAA) induced an average of 13.77 and 30.32 shoots per responsive hypocotyl and stem explants, respectively. Combinations of TDZ and NAA were less effective in promoting shoot induction in the explants at the concentrations tested compared to BA and NAA combinations. Hypocotyledonary explants achieved complete plant regeneration without multiple shoot formation in a hormone-free MS medium. In vitro shoots regenerated from both stem and hypocotyledonary explants were 100% successfully rooted on a half-strength hormone-free medium, and acclimatized to produce H. crinita plants with over 90.0% efficiency.

Introduction

African bush apple is one of the orphan crops of African origin with immense potential to fight hunger and poverty. Like most orphan crops, it adapts well to its endemic environments and is usually harvested from the wild. It is also semi-cultivated as a vegetable mostly valued for its aromatic leaves used in local soups. Research findings have also shed light on its medicinal value including  in vitro neuroprotective potentials, anti-plasmodial activity, protection against diabetes-induced electrolyte imbalance and haematological disorders and modulatory effect against induced oxidative stress [1,2,3,4].

However, despite its dietary and medicinal usefulness, the crop faces an increasing threat of extinction due to the sustained loss of natural forests, the paucity of conservation strategies [5], the complete absence of genetic breeding efforts, and the concomitant decline in the traditional knowledge about its cultivation and use. The only research effort toward the conservation of this crop via domestication was a basic study [5] that evaluated how seed source can affect important parameters such as seed germinability and early seedling growth. The African bush apple is normally propagated by seed but loss of seed viability is often a major concern. The plant would thus benefit greatly from biotechnological interventions through the use of tools such as micropropagation that enable large-scale production of progeny plants with similar genetic identity to the parent plant [6]. As the most developed and optimized biotechnological tool [7], micropropagation would serve as an important conservation strategy for H. crinita via vegetative propagation, and also an essential prerequisite for its biotechnological breeding via genetic transformation or genome editing [8].

Our preliminary findings on H. crinita explants’ responsiveness to the plant growth regulators, benzyl adenine (BA) and naphthalene acetic acid (NAA), showed stem explants as good candidates. Following up on the indicated study, this study used stem and hypocotyledonary explants of the crop to develop an efficient  in vitro regeneration system via direct multiple shoots proliferation.

Materials and methods

Sterilization of plant material and in vitro germination of H. crinita seedlings

Mature fruits of H. crinita were obtained from a field in which semi-domesticated fruiting plants in Calabar, Cross River State, Nigeria were cultivated. The fruits were dried at room temperature for 14 days; the seed clusters were extracted, soaked in running tap water, and filtered several times to remove the embedding material. The individual seeds released were sterilized by gentle shaking in a solution of 1% sodium hypochlorite (stock contains 5% active chlorine) and one drop of Tween-20, for 10 min at room temperature. The seeds were subsequently rinsed seven times with sterile distilled water, and plated on Murashige and Skoog (MS) medium containing 1% sucrose, 0.5 g/L 2-(N-morpholino) ethane sulfonic acid (MES), and 0.8% agar, pH 5.7 in Petri dishes (ø = 90 mm, depth = 20 mm). The treated seeds were subjected to 4 °C cold treatment for 2 days before incubating at 22 °C. Germination started after 10 days.

Multiple shoots regeneration from stem and hypocotyl explants of H. crinita

Regeneration experiments were conducted using 4-week-old stem and 7-day-old hypocotyledonary explants (Supplementary Fig. 1) of H. crinita. One thousand H. crinita seeds were germinated in vitro, and the seedlings served as the source of stem explants. Stem segments (5 mm, without leaves, petioles, and apical buds) were cultured on MS medium containing 3% sucrose, 0.8% agar and supplemented with either BA (0.5, 1.0, 1.5, 2.0 mg/L) in combination with NAA (0.1, 0.2, 0.5, 1.0 mg/L), or TDZ (0.022, 0.044, 0.110, 0.220 mg/L) in combination with NAA (0.002, 0.004, 0.010, 0.020 mg/L). Control treatments contained no plant growth regulators. The stem segments were placed flat on the culture medium and subcultured on fresh media bi-weekly. A total of 30 explants (10 per replicate treatment) were evaluated for shoot regeneration after 6 weeks. Data was taken on the percentage of responsive stem explants (number of explants producing at least one shoot divided by the total number of explants cultured, multiplied by 100), and the number of shoots per responsive stem explant.

For hypocotyledonary explants, one thousand H. crinita seeds were germinated in vitro. Seven-day-old hypocotyledonary explants (5 mm length) were cultured on MS medium containing 3% sucrose and 0.8% agar, supplemented with either 1.0 mg/L BA + NAA (0.1, 0.2, 0.5, 1.0 mg/L) or TDZ (0.022, 0.110, 0.220 mg/L) + NAA (0.002, 0.004, 0.010 mg/L). Explants were inoculated flat on the culture medium and subcultured on fresh media every 2 weeks. A total of 30 hypocotyledonary explants (10 per replicate treatment) were evaluated for shoot regeneration after 4 weeks. Data was taken on the percentage of responsive hypocotyls (number of hypocotyl explants producing at least one shoot divided by the total number of hypocotyls cultured per treatment, multiplied by 100), and the number of shoots per responsive hypocotyl explant.

Rooting of shoots on hormone-free medium and acclimatization of plantlets

Microshoots regenerated from stem and hypocotyl explants cultured on MS media supplemented with 1.0 mg/L BA + 0.1 mg/L NAA combination were randomly selected and excised. The excised shoots (30 each from stem and hypocotyl explants) were cultured for 30 days in Petri dishes (ø = 100 mm, depth = 55 mm) containing half-strength hormone-free MS medium supplemented with 1.5% sucrose and 0.8% agar, pH 5.8. Data was taken on the percentage of rooted shoots after 30 days of culture.

Acclimatization of rooted shoots was done following the protocol of [9] with modifications (shoots were not immersed in 1 g/L benomyl solution, and the medium volume used to water the plants was halved). The plantlets were transferred to nursery pots (ø = 101.6 mm, depth = 88.9 mm) containing sterile vermiculite, and watered every 3 days with 10 mL of half-strength hormone-free MS medium without sucrose. The pots were covered with transparent polythene and kept in a growth room at 25 °C, 65% relative humidity, and 16:8 h photoperiod. The polythene covers were perforated every 2 days and completely removed after a 21 days acclimatization period to obtain hardened plantlets. Plant acclimatization success rate (%) was compared between 30 plantlets obtained from each of both stem and hypocotyl explants.

Results and discussion

In vitro shoot regeneration from stem and hypocotyl explants in BA + NAA medium

Stem explants’ responsiveness to shoot induction ranged from 16.67 to 86.67%, and was higher in medium containing higher ratios of BA to NAA. The average number of shoots produced per responsive stem explant differed significantly (p < 0.05) across treatments and was highest (30.32) on MS medium + 1.0 mg/L BA + 0.1 mg/L NAA (Table 1). Generally, more shoots were induced in BA + NAA treatments with higher ratios of BA: NAA while stem explants cultured on hormone-free MS medium produced the least number of shoots (1.24). With hypocotyl explants; the percentage of explant responsiveness ranged from 46.67 to 90.00%. The number of shoots per responsive explant also differed significantly (p < 0.05). Increasing concentrations of NAA at fixed BA concentration (1.0 mg/L) reduced hypocotyl explants’ responsiveness to shoot formation as well as the number of shoots per responsive explant and shoot induction was completely inhibited at NAA concentration of 1.0 mg/L (Fig. 1). Treatment with 1.0 mg/L BA + 0.1 mg/L NAA produced the highest number of shoots (13.77 shoots per explant), followed by 1.0 mg/L BA + 0.2 mg/L NAA (7.73 shoots), then 1.0 mg/L BA + 0.5 mg/L NAA (3.90 shoots). An average of 1.00 shoot per explant was produced in the control treatment and did not differ significantly from 0.00 shoot produced under 1.0 mg/L BA + 1.0 mg/L NAA treatment (Table S1).

Table 1 In vitro shoot induction from 4-weeks old H. crinita stem explants after 6 weeks culture at 26℃ in MS medium supplemented with either N-Benzyladenine (BA) or thidiazuron (T) in combination with naphthalene acetic acid (NAA)

Fig. 1

H. crinita hypocotyl explants’ response to culture on MS medium supplemented with BA/TDZ + NAA. a Regeneration of multiple shoots from 7-day-old hypocotyl explant after 4 weeks culture on MS medium supplemented with 1.0 mg/L BA + 0.1 – 1.0 mg/L NAA (i-v) at 26℃. b Complete plant regeneration from 7-day-old hypocotyl explants after 4 weeks culture on MS medium supplemented with different levels of TDZ + NAA (i-v) at 26℃

In the scientific literature about in vitro regeneration, the roles of cytokinins in enhancing cellular divisions and hence the promotion and development of new shoots from somatic tissues are well known. For example, high cytokinin: auxin concentration ratios have induced multiple shoots directly or indirectly from shoot tips of sesame [10] and hypocotyl explants of Cannabis sativa L [8]. Our results follow a similar pattern as high cytokinin (BA): auxin (NAA) combinations promoted the induction of more shoots from stem and hypocotyl explants of H. crinita compared to treatments with no hormone and those with lower ratios of BA: NAA. The hypocotyl explants in particular showed spontaneous regeneration of both shoots and roots thus completing plant regeneration on a hormone-free medium similar to previous independent reports [8, 11] with hypocotyls of Cannabis sativa and Passiflora setacea, respectively. The levels and balance of endogenous hormones within an explant is among the factors that could contribute to the rate of shoot induction [12], and this could be responsible for the spontaneous shoots and roots formation on H. crinita hypocotyls in hormone-free medium. The achievement of complete plant regeneration from hypocotyls of H. crinita on a hormone-free medium clearly promises a faster route to micropropagation of the crop. However, for a larger scale production of plants to be achieved, there would be a need to regenerate more shoots per hypocotyl explant via the inclusion of a high ratio of exogenous cytokinin: auxin in the culture medium as demonstrated in this study.

In vitro shoot regeneration from stem and hypocotyl explants in TDZ + NAA medium

On TDZ + NAA medium, the responsiveness of stem explants ranged from 46.67 to 86.67%, with the control treatment showing the least responsiveness (46.67%) while the remaining 16 TDZ + NAA treatments showed no significant variation at p > 0.05 (Table 1). A range of 1.31– 3.26 shoots were produced by the responsive explants cultured in media containing TDZ + NAA. There were significant differences (p < 0.05) among the treatments in the number of shoots per responsive explant. The highest shoot number per responsive explant (3.26) was produced in T0.22 + N0.002, followed by T0.11 + N0.010 treatment which produced 2.38 shoots. The other treatments were comparable with the control treatment in the average number of shoots (Table 1). With hypocotyl explants, 43.33 – 86.67% responded positively to shoot induction on TDZ + NAA medium. Explants on hormone-free medium had the least responsiveness (43.33%) but the number of shoots induced per responsive explant did not differ significantly (p > 0.05) across all treatments as each had an average of one (Fig. 1).

In a previous report [13], TDZ facilitated efficient micropropagation of woody species at low concentrations. However, in this study, TDZ was ineffective, at the concentrations tested, for stimulating multiple shoot induction from H. crinita stem and hypocotyl explants. Previous studies have shown the use of TDZ for the direct induction of shoots in both herbaceous and woody species including hypocotyls of Jatropha curcas [14] and Plukenetia volubilis [15]. A major difference however is that the current study evaluated TDZ at a maximum concentration of 0.22 mg/L which is about 2.3 times and 4.5 times weaker than were used for J. curcas and P. volubilis respectively. Thus, it is possible that an increased TDZ concentration could trigger a higher proliferation of shoots from H. crinita explants.

Rooting of microshoots and ex vitro acclimatization of plants

Microshoots derived from stem and hypocotyl explants were cultured on hormone-free MS medium and rooting was analyzed in both treatments after 30 days. All (100%) of the cultured shoots, irrespective of explant source, were successfully rooted (Table S2). A complete regeneration system in H. crinita using stem and hypocotyl explants is presented in Fig. 2. Auxins, especially indole-3-butyric acid (IBA), are known to promote the effective rooting of microshoots and are typically used in most rooting experiments [16]. However, endogenous hormones especially auxins present in the microshoots are also known to play a role in stimulating root formation and could be responsible for the achievement of high rooting efficiency of H. crinita microshoots under hormone-free conditions. Our results on rooting efficiency are higher than 75% and 30% reported for hormone-free rooting of S. tomentosa [16] and Aloe vera [17] microshoots. Results of ex vitro acclimatization of rooted H. crinita microshoots on sterile vermiculite watered with half-strength MS medium are comparable to 100% success achieved in tomato with a similar acclimatization strategy [9]. A combination of vermiculite and sugar-free medium was also effectively used in the in vitro acclimatization of wasabi plants [18].

Fig. 2

In vitro regeneration cycle of Heinsia crinita using hypocotyl and stem explants. From seed germination through explant derivation to plant acclimatization, 7-day-old hypocotyl and 4-week-old stem explants would take about 98 and 119 days respectively to produce acclimatized plantlets

Conclusion

Multiple shoots proliferation from stem and hypocotyl explants of H. crinita could be achieved via culture on MS medium containing cytokinin + auxin combinations, especially at 1.0 mg/L BA + 0.1 mg/L NAA. These microshoots could be maintained in cultures as a conservation strategy or mass-produced via micropropagation after rooting and ex vitro acclimatization.

Limitations

At the time of the research, the authors could not extend preliminary regeneration experiments to TDZ for pre-determining its effective concentration range that would promote shoot regeneration due to seed viability issues that affected overall germination percentage and reduced the amount of available explants. For the same reason, regeneration experiments using hypocotyls were not as expansive as with stem explants. There was also no means at the time to carry out a histological confirmation of the origin of regenerated microshoots to confirm whether they were a result of direct organogenesis.