Synthetic seed propagation of the therapeutic-honey plants Leptospermum polygalifolium and L. scoparium (Myrtaceae)

Keywords: adventitious rooting, alginate encapsulation, callus, cytokinin, mānuka honey, methylglyoxal, propagation, tissue culture.

The antibacterial and wound-healing activity of mānuka honey is derived primarily from the compound methylglyoxal (MGO) (Cooper et al. 2001; Irish et al. 2011). This compound is produced gradually in honey from dihydroxyacetone (DHA), which occurs at high concentrations in floral nectar of the New Zealand mānuka tree, Leptospermum scoparium Salisb. (Stephens et al. 2010; Norton et al. 2015; Cokcetin et al. 2016; Grainger et al. 2016). The natural distribution of L. scoparium extends into south-eastern Australia; in addition, some other Australian Leptospermum species such as L. polygalifolium J.R.Forst. & G.Forst. are sources of nectar with high levels of DHA (Irish et al. 2011; Williams et al. 2018). In 2021, New Zealand exported 9800 tonnes of mono-floral and multi-floral mānuka honeys, worth US$259 million (MPI (Ministry for Primary Industries) 2021). Demand for therapeutic Leptospermum honey with high MGO levels exceeds global supply (Spiteri et al. 2017; Hermanns et al. 2020), and so, there has been a drive to establish nectar plantations of Leptospermum species that produce high levels of DHA (Williams et al. 2018; Clarke and Le Feuvre 2021; Grunennvaldt et al. 2022). Plantation establishment from seedlings has been challenging because Leptospermum seed are often difficult to extract from the fruit or to germinate (Shipton and Jackes 1986; Lyne and Crisp 1996; Battersby et al. 2017).

Vegetative propagation is widely used as an alternative approach for producing plants that are difficult to germinate from seed or have a limited seed supply (Trueman et al. 2007; Cochrane et al. 2021; Offord et al. 2021). Vegetative propagation has mostly involved the use of cuttings (Pijut et al. 2011; Wendling et al. 2014a; Stuepp et al. 2017), tissue culture (George 1993; Trueman et al. 2018; Somerville et al. 2021) or grafting (Trueman et al. 2014; Wendling et al. 2014b). Tissue culture can provide hundreds of L. polygalifolium or L. scoparium nursery stock plants from a single seed in <12 months, and from these, 88–93% of L. polygalifolium cuttings and 65–76% of L. scoparium cuttings successfully form roots (Darby et al. 2021a, 2021b). Nonetheless, the potential exists to bypass delays in producing stock plants and harvesting cuttings by developing more rapid methods for plant production.

Synthetic seeds have been used as a highly efficient vegetative method to propagate and distribute plant germplasm (Gantait et al. 2015; Rihan et al. 2017; Kulus 2019; Sharma et al. 2019). Synthetic seeds are artificially encapsulated miniature cuttings that provide the functions of a natural seed but, like cuttings, remain true-to-type to their donor plant (Cheruvathur et al. 2013; Gantait and Mitra 2019). Both unipolar structures (e.g. axillary buds and apical shoot tips) and bipolar structures (e.g. somatic embryos with a root apical meristem and a shoot apical meristem) can be encapsulated as synthetic seeds (Hung and Trueman 2011a; Bhatia and Bera 2015; Rihan et al. 2017). An advantage of synthetic seeds over traditional approaches for producing plantlets is their capacity for convenient storage and transport to other laboratories and plant nurseries (Hung and Trueman 2012a, 2012b; Hung and Dung 2015; Naz et al. 2018). Nothing was known of the capacity for Leptospermum species to produce viable shoots and roots from synthetic seeds; however, synthetic seed methods have been developed for other Myrtaceae taxa from the genera Acca, Corymbia and Eucalyptus (Watt et al. 2000; Cangahuala-Inocente et al. 2007; Rai et al. 2008; Hung and Trueman 2012a).

Synthetic seeds of Myrtaceae have been constructed using 3% sodium alginate in the encapsulation solution, followed by transfer to 100 mM CaCl₂ (Watt et al. 2000; Rai et al. 2008; Hung and Trueman 2012a, 2012b). The encapsulation solution usually includes full-strength Murashige and Skoog (MS) medium (Watt et al. 2000; Hung and Trueman 2012a, 2012b). Optimal shoot regrowth from a high percentage of synthetic seeds of Psidium guajava or Corymbia torelliana × C. citriodora occurs on half- or full-strength MS medium without the addition of plant hormones such as cytokinins (Rai et al. 2008; Hung and Trueman 2012a). Cytokinins such as benzyladenine (BA) are often incorporated in tissue culture media to promote axillary bud growth or callus formation (George 1993; Hung and Trueman 2010, 2012c; Trueman et al. 2018). Although BA reduces the percentage of synthetic seeds with shoot emergence in both P. guajava and C. torelliana × C. citriodora (Rai et al. 2008; Hung and Trueman 2012a), it increases the average number of shoots emerging from each of the emergent synthetic seeds of C. torelliana × C. citriodora (Hung and Trueman 2012a). BA also inhibits adventitious rooting (Cangahuala-Inocente et al. 2007; Trueman and Richardson 2007; Rai et al. 2008; Trueman et al. 2018), and so it is usually excluded from media when root emergence is required to form a plantlet with both shoots and roots.

The objective of this study was to assess the amenability of L. polygalifolium and L. scoparium shoots to encapsulation and propagation as synthetic seeds. Specifically, the study aimed to determine the extent to which shoot and root emergence from encapsulated shoots are affected by the presence of BA in the emergence medium and by the nutrient strength of the encapsulation medium. The first hypothesis was that BA would decrease the percentage of synthetic seeds with shoot emergence, and prevent root emergence, but would increase the number of emerged shoots from each of the emergent synthetic seeds. The second hypothesis was that half-strength MS medium in the encapsulation solution would result in lower shoot and root emergence than full-strength MS medium.

Nodal explants were sourced from three clones of L. polygalifolium (P1, P6 and P11) and two clones of L. scoparium (S6 and S12) that had been proliferated in vitro (Darby et al. 2021b). The shoots were raised by germinating seed for 3–4 weeks, initiating shoots for 5 weeks, and then proliferating shoots on hormone-free, full-strength MS medium (Murashige and Skoog 1962) (PhytoTechnology Laboratories, Shawnee Mission, KS, USA) with 87.6 μM sucrose for four passages of 7, 8, 8 and 4 weeks, respectively. The shoots were maintained at 28°C under a 16-h photoperiod with irradiance of ∼100 μmol m⁻² s⁻¹ (Darby et al. 2021b).

After 4 weeks in the fourth proliferation passage, nodal explants were cut to ∼4–5 mm length before encapsulation. Two encapsulation solutions were prepared, comprising either full-strength MS medium (FMS) or half-strength MS medium (HMS), each of which contained 3% (w/v) low-viscosity sodium alginate (Sigma-Aldrich, St. Louis, MO, USA). Synthetic seeds were constructed by placing the explants into the liquid encapsulation solution for 10 min. A 10-mL disposable pipette with a 7-mm-diameter opening was used to draw up a single explant with encapsulation solution, which was then dispensed into a beaker containing 100 mM CaCl₂ solution. This caused the globule of encapsulation solution to form a skin that encapsulated the explant within the alginate matrix. The synthetic seeds (Fig. 1a) were retrieved by decanting off the CaCl₂ solution after 30 min and washing three times with sterile distilled water. They were placed on sterile paper for ∼5 min to remove excess liquid. In total, 100 synthetic seeds in encapsulation matrix with FMS and 50 with HMS were created for each of the five clones.

Fig. 1.  Encapsulation and synthetic seed germination of Leptospermum polygalifolium and L. scoparium: (a) freshly constructed synthetic seeds of L. polygalifolium; (b) synthetic seed of L. polygalifolium with single emergent shoot; (c) synthetic seed of L. polygalifolium with two emergent shoots; (d) synthetic seed of L. scoparium that has formed adventitious roots and shoots. Scale bars: (a) 10 mm, (b–d) 5 mm.

Two emergence media were prepared, one without BA and the other containing 2.2 μM BA. Both consisted of full-strength MS medium with 2% sucrose, pH 5.8, solidified with 0.8% (w/v) agar (Bacto Laboratories, Liverpool, NSW, Australia). Emergence medium was placed in 375-mL jars (50 mL per jar) and autoclaved at 104 kPa and 121°C for 15 min. For each clone, 50 synthetic seeds per treatment were placed onto emergence medium in 10 replicate jars, with each jar containing five synthetic seeds. The three treatments were prepared in a partial design to test the two study hypotheses: (1) FMS in the encapsulation solution without BA in the emergence medium (i.e. the control); (2) FMS in the encapsulation solution with 2.2 μM BA in the emergence medium (i.e. the treatment for testing the first hypothesis); and (3) HMS in the encapsulation solution without BA in the emergence medium (i.e. the treatment for testing the second hypothesis) (Table 1). The synthetic seeds were maintained at 28°C under a 16-h photoperiod with a photosynthetic photon flux density of 100 μmol m⁻² s⁻¹ emitted from cool-white, fluorescent tubes. Shoot emergence (Fig. 1b, c) and root emergence (Fig. 1d) from each synthetic seed were recorded after 4 weeks.

Table 1.  Encapsulation and emergence treatments used in the production and germination of Leptospermum polygalifolium and L. scoparium synthetic seeds.

Data for percentage of synthetic seeds with emerged shoots from the only two successful treatments for L. polygalifolium were analysed by t-test after confirming normality of the distributions. Data for percentage of synthetic seeds with emerged shoots from all three treatments for L. scoparium were analysed by analysis of variance (ANOVA), followed by Tukey’s honestly significant difference (HSD) test when differences were detected among the means. Data for number of emerged shoots per emergent synthetic seed of both species were analysed by nested ANOVA (with synthetic seeds nested into jars), followed by Tukey’s HSD test when differences were detected among the means. Data were analysed using SPSS Statistics Ver. 26 (IBM, Armonk, NY, USA). Differences between means were regarded as significant at P ≤ 0.05. Means are presented with standard errors.

Shoots emerged in 24% (±4%) to 94% (±4%) of L. polygalifolium nodes that were encapsulated in FMS solution (Fig. 2a–c). The use of 2.2 μM BA in the emergence medium increased the percentage of synthetic seeds with emergent shoots from 58% (±16%) to 94% (±4%) in L. polygalifolium clone P6 (Fig. 2b) but did not significantly affect shoot emergence percentages in the other two L. polygalifolium clones (Fig. 2a, c). BA induced shoot emergence through callus in 60% (±16%) and 40% (±16%) of synthetic seeds of clones P1 and P6, respectively (Fig. 2a, b). Shoots emerged in 100% of L. scoparium synthetic seeds that were encapsulated in FMS solution and germinated on medium lacking BA (Fig. 2d, e). BA reduced shoot emergence to 54% (±8%) in L. scoparium clone S12 but did not affect shoot emergence significantly in S6 (Fig. 2d, e). BA induced shoot emergence through callus in 8% (±5%) and 26% (±11%) of synthetic seeds of clones S6 and S12, respectively (Fig. 2d, e).

Fig. 2.  Effect of Murashige and Skoog (MS) medium strength in the encapsulation solution (FMS, full MS; HMS, half MS) and benzyladenine (BA) concentration (0 or 2.2 μM) in the emergence medium on shoot emergence from synthetic seeds of: (a–c) Leptospermum polygalifolium (L. poly) clones P1, P6 and P11; and (d, e) L. scoparium (L. scop) clones S6 and S12. Data are means + s.e. for total shoot emergence; means with different letters within a clone are significantly different (t-test for P1, P6 and P11; ANOVA and Tukey’s HSD test for S6 and S12; P ≤ 0.05; n = 10–12).

Shoots did not emerge from L. polygalifolium nodes that were encapsulated in HMS solution (Fig. 2a–c). The use of HMS in the encapsulation solution reduced shoot emergence significantly in L. scoparium clone S12, from 100% to 54% (±8%), but did not affect the shoot emergence percentage significantly in S6 (Fig. 2d, e).

The average number of shoots emerged per germinated synthetic seed ranged from 1.2 (±0.1) to 1.7 (±0.1) in L. polygalifolium, with the number not affected significantly by the presence of BA in the emergence medium (Fig. 3a–c). However, BA increased the average number of emergent shoots in the L. scoparium clones from the baseline of 1.0 to 1.5 (± 0.1) in S6 and 1.2 (± 0.1) in S12 (Fig. 3d, e).

Fig. 3.  Effect of Murashige and Skoog (MS) medium strength in the encapsulation solution (FMS, full MS; HMS, half MS) and benzyladenine (BA) concentration (0 or 2.2 μM) in the emergence medium on the number of emerged shoots per emergent synthetic seed of: (a–c) Leptospermum polygalifolium (L. poly) clones P1, P6 and P11; and (d, e) L. scoparium (L. scop) clones S6 and S12. An asterisk (*) signifies a treatment that had no emerged shoots. Data are means + s.e.; means with different letters within a clone are significantly different (nested ANOVA and Tukey’s HSD test; P ≤ 0.05; n = 12–56).

In both species, roots emerged only from nodes that were encapsulated in FMS solution and germinated on medium lacking BA (Fig. 4). Roots emerged in 26% (±11%) to 57% (±8%) of these L. polygalifolium synthetic seeds and in 100% of these L. scoparium synthetic seeds. All of these synthetic seeds also formed shoots.

Fig. 4.  Effect of Murashige and Skoog (MS) medium strength in the encapsulation solution (FMS, full MS; HMS, half MS) and benzyladenine (BA) concentration (0 or 2.2 μM) in the emergence medium on the percentage of synthetic seeds with root emergence in: (a–c) Leptospermum polygalifolium (L. poly) clones P1, P6 and P11; and (d, e) L. scoparium (L. scop) clones S6 and S12. Data are means presented with s.e. (n = 10–12).

This study has demonstrated that two high-value species for producing therapeutic honey, L. polygalifolium and L. scoparium, are highly amenable to propagation as synthetic seeds using a simple formulation of hormone-free, full-strength MS medium in both the encapsulation solution and emergence medium. This technique may be useful for accelerating the establishment of nectar plantations by providing a more effective method to convert in vitro shoots into plantlets.

Addition of the cytokinin BA to the emergence medium increased the percentage of synthetic seeds with emergent shoots in one L. polygalifolium clone but decreased the percentage with emergent shoots in one L. scoparium clone. These results provide support from one of the two L. scoparium clones for the first hypothesis that BA would decrease the percentage of synthetic seeds with shoot emergence, as it does with synthetic seeds of P. guajava and C. torelliana × C. citriodora (Rai et al. 2008; Hung and Trueman 2012a). However, BA stimulated multiple-shoot formation in some synthetic seeds of L. scoparium, providing support for the hypothesis that BA would also increase the number of emerged shoots from each of the emergent synthetic seeds. BA stimulated some explants of both species to form shoots through callus, consistent with its effects in conventional tissue cultures of L. polygalifolium (Darby et al. 2021b) and eucalypt species (Trueman and Richardson 2007; Hung and Trueman 2010, 2011b; Trueman et al. 2018). Therefore, BA-treated synthetic seeds have the capacity to produce very large numbers of shoots if they are transferred subsequently to shoot-proliferation medium (Darby et al. 2021b). BA-free emergence medium might be employed when a germplasm collection of only a few synthetic seeds from each of many L. scoparium clones is transferred between laboratories and it is important to maximise the percentage of synthetic seeds with at least one emergent shoot (Hung and Trueman 2012a). On the other hand, emergence medium containing BA might be employed when many synthetic seeds of only a few clones are transferred, but mass-proliferation is required from each clone (Hung and Trueman 2012a).

Benzyladenine prevented adventitious rooting in Leptospermum synthetic seeds, confirming part of the first hypothesis of the study. BA also prevents adventitious rooting in synthetic seeds of Acca sellowiana and P. guajava (Cangahuala-Inocente et al. 2007; Rai et al. 2008) and in conventional tissue cultures of L. polygalifolium and L. scoparium (Darby et al. 2021b). Therefore, use of BA would not be recommended when synthetic seeds are being used to form plantlets for direct transfer to ex vitro conditions (Hung and Trueman 2012a; Hung and Dung 2015; Naz et al. 2018). High frequencies of root emergence were obtained from L. polygalifolium and L. scoparium synthetic seeds without the use of rooting hormones such as indole-3-butyric acid. This auxin is required for root emergence from many synthetic seeds of C. torelliana × C. citriodora (Hung and Trueman 2012a), although it is often not required for root emergence from conventional explants (George 1993; Trueman et al. 2007, 2018). Instead, a simple method, using full-strength MS medium without auxins or cytokinins in either the encapsulation solution or emergence medium, allowed 26–57% of L. polygalifolium and 100% of L. scoparium synthetic seeds to form complete plantlets.

The use of half-strength MS medium in the encapsulation solution was insufficient for shoot emergence from L. polygalifolium synthetic seeds and for root emergence from either L. polygalifolium or L. scoparium synthetic seeds. Shoot emergence did occur on many L. scoparium explants that were encapsulated in HMS solution, albeit at reduced frequency in one of the clones, but these all failed to form adventitious roots. The results confirm the second hypothesis that half-strength MS medium in the encapsulation solution would decrease shoot and root emergence compared with full-strength MS medium. They demonstrate that nodal explants can be highly sensitive to the nutrient concentrations in the encapsulation solution despite having been immersed in encapsulation solution for only 10 min. Nutrients from the solution are presumably absorbed by explants during immersion, and residual nutrient solution may also remain on the surface of the explants when they are dispensed into CaCl₂ to form the synthetic seeds. Synthetic seeds of other Myrtaceae taxa have been encapsulated previously using either nutrient-free solution (Cangahuala-Inocente et al. 2007; Rai et al. 2008) or full-strength MS solution (Watt et al. 2000; Hung and Trueman 2012a, 2012b). However, the present results show that full-strength MS solution is required for shoot emergence from L. polygalifolium clones and is optimal for shoot emergence across L. scoparium clones. The nutritional status of in vitro shoots and nursery cuttings also affects the capacity to form adventitious roots in many Myrtaceae species (Woodward et al. 2006; Cunha et al. 2009; Trueman et al. 2013; Oberschelp and Gonçalves 2016). The present results indicate that the nutritional status of encapsulated explants can, similarly, affect the adventitious rooting capacity of synthetic seeds.

The plantlet conversion frequencies from synthetic seeds were higher than the previously obtained frequencies of 31% and 44% from unencapsulated explants of L. polygalifolium and L. scoparium, respectively (Darby et al. 2021b). However, that study reported plantlet conversion after a period of acclimatisation under nursery conditions. Plantlets of both species can be acclimatised readily to the nursery (Darby et al. 2021b), but further research is required to assess whether ex vitro synthetic seedlings acclimatise more easily than conventional plantlets, including the potential to convert synthetic seeds directly into plantlets in potting mix rather than aseptic culture medium. Synthetic seeds of the eucalypt C. torelliana × C. citriodora and the mahogany Khaya senegalensis have been converted directly into plantlets under nursery conditions, with conversion success of 46–90% and 42–80%, respectively, using organic-compost potting mixes (Hung and Trueman 2011a, 2012a). Further research is also warranted to determine the potential storage duration of synthetic seeds under minimal-growth conditions. Synthetic seeds of C. torelliana × C. citriodora and K. senegalensis can be stored at 14°C in darkness for at least 12 months, with very high frequencies of shoot emergence (92–100% and 71–98%, respectively) and excellent shoot development (Hung and Trueman 2012b). Synthetic seeds potentially provide an efficient means for germplasm batch-storage and propagule distribution to overcome some of the bottlenecks in producing stock plants and cuttings from in vitro explants.

In conclusion, L. polygalifolium and L. scoparium synthetic seeds are highly suitable for mass propagation and germplasm distribution, using a simple formulation of hormone-free, full-strength MS medium in both the encapsulation solution and the emergence medium. The synthetic seed methods developed here could be coupled with the highly successful methods developed recently for in vitro shoot proliferation, ex vitro cutting propagation, and nursery-plant cultivation of the same species (Darby et al. 2021a, 2021b; Grunennvaldt et al. 2022). Alternatively, they could be developed further to allow direct conversion of synthetic seeds into plantlets under nursery conditions (Hung and Trueman 2011a, 2012a; Hung and Dung 2015). These methods can facilitate the mass production of L. polygalifolium and L. scoparium plants for establishing nectar plantations, which will assist greatly in meeting the demand for high-value therapeutic Leptospermum honey.

The data that support this study will be shared upon reasonable request to the corresponding author.

The authors declare no conflicts of interest.

Ian Darby was the recipient of a USC Postgraduate Scholarship.