Propagation of Polygonatum macranthum (Maxim.) Koidz. from immature seeds using a new sterilization procedure

Abstract

Natural seed germination is difficult to achieve in numerous plant species of wide economic importance. The germination of Polygonatum macranthum seeds takes as long as one and a half years under natural conditions. In addition, propagation by rhizome is also extremely slow in this species. Therefore, the natural propagation of P. macranthum through seeds or rhizome is not efficient. In this study, an efficient in vitro propagation system for P. macranthum from immature seeds with seed coat was developed, using a new surface sterilization protocol that utilized a low concentration of hypochlorite. In vitro germination was achieved at a rate of 30% within 9 weeks after inoculation on 1/2 MS medium. Shoot explants from seedlings were successfully cultured on 1/2 MS medium. Supplementation of the 1/2 MS medium with cytokinin 6-benzylaminopurine (BAP) facilitated efficient propagation by microrhizome. An efficient propagation rate of 1.3 microrhizomes per shoot in an 8-week culture period could be achieved by using a concentration of 1 mg l⁻¹ BAP. During 4 weeks of acclimatization, 88% of shoots were rooted and started to grow into juvenile plants. After about 16 weeks in the field, 13% of the acclimatized plants showed viable growth and healthy regenerating shoots. The cultivation system demonstrated in this study can be used to propagate P. macranthum.

Polygonatum macranthum is one of the rarest Polygonatum species native to Japan. It is an economically important species because of its nutritive and medicinal properties (Takagi 2001a). The plant is commonly known as Solomon’s seal and is considered to be important in traditional Chinese medicine. Young shoots are edible and are known to have the best taste of all Polygonatum species (Takagi 2001a), which is perhaps why it is known as “mountain asparagus”. It grows to a height of 80–130 cm in single stands (Figure 1A) and bears pale green tubular flowers beneath the stem (Ohwi 1965). The plant is distributed mainly in mountainous and cold regions. The species can be cultivated without fertilization on appropriate soils with a simple sunshade (made of black mesh and about 2 m in height), and can be successfully grown without substantial cultivation management requirements. Longer growth period and the absence of efficient propagation means are the main problems in the cultivation of P. macranthum. Its seeds are characterized by “double dormancy” in which the first leaf emergence occurs only after two winter seasons in order to overcome radicle and epicotyl dormancy. Moreover, the species also exhibits rhizome dormancy, requiring a winter season before starting the subsequent growth cycle (Takagi 2001a, b, 2005). If a single stem is cut off, it would only be restored after a dormant year. Furthermore, it becomes impossible for the plant to store nutrients for propagation in the following year and the rhizomes become smaller and cannot produce shoots and a new rhizome (Figure 1B). Therefore, P. macranthum is characterized by extremely low natural propagation rates. Alternating cold and warm stratification was suggested to be effective for reducing the time required for natural seed germination of P. macranthum from 19.5 to 9 months (Takagi 2001a). Rhizome dormancy had been broken by application of colder conditions, leading to shortening of the lengthy growth cycle of the species by a few months (Takagi 2005). To date, an efficient propagation and cultivation of this species has not been established. In vitro propagation might play a major role in overcoming extremely slow natural propagation of plant species with medicinal and other economically important properties. In the present study, an efficient micropropagation system of P. macranthum has been successfully established. All in vitro culture experiments were carried out in Nagaoka University of Technology. Freshly harvested unripe fruits and rhizomes of P. macranthum were collected from the nursery of the Niigata Agricultural Research Institute during the summer of 2016. Immature seeds with seed coat (Figure 2A) were obtained. Rhizomes were sliced to ca. 0.5 cm thickness in order to obtain rhizome explants. Ten explants of each tissue (immature seeds with seed coat, and rhizome explants) were subjected to a 1 min dip in 70% ethanol and subsequently treated by the following two surface sterilizing treatments; (1). Explants were initially treated with 1% (w/v) Sirvip™ solution (Vitroplants, Japan) for 1 h. They were subsequently treated with 0.07% (w/v) calcium hypochlorite solution for 30 min, followed by a brief rinse with a fresh 1% (w/v) Sirvip™ solution. Explants were inoculated on the medium with no further rinsing by sterilized distilled water; (2). Explants were treated with sodium hypochlorite at two different concentrations (1% and 2% (w/v)) for 10 min. After each treatment, the explants were rinsed with sterilized distilled water for 3 times. The sterilized explants were inoculated onto the solid half strength of MS (1/2 MS) basal medium (Murashige and Skoog 1962) prepared with 3% sucrose and 0.8% agar. For all the experiments, the pH of the medium was adjusted to 5.8. Upon inoculation, the explants were kept in total darkness at a constant temperature of 20°C for 1 week. And then, these were transferred into light culture condition, under a 16 h photoperiod using cool white fluorescent light, and 70–90 µM m⁻² s⁻¹ light intensity, at 20°C. Immature seed explants of P. macranthum with seed coat that were surface-sterilized with Sirvip™ germinated in vitro (Figure 2B) in 9 weeks, which was significantly faster than its lengthy germination time in nature. The efficiency of seed germination was 30% (Table 1). Germination could not be achieved when the explants were surface-sterilized using a conventional surface sterilizer, sodium hypochlorite at 1% and 2% (w/v). However, all three surface sterilization treatments resulted in a 100% decontamination rate. Surface sterilization is a crucial step during any in vitro propagation procedure, because it eliminates the microorganisms that are attached to the explant surface and ensure proper growth of the explant in vitro. Sirvip™ is a surface-sterilizer, and in this research, it was supplemented by a lower concentration of hypochlorite during surface sterilization of P. macranthum explants. It could be inferred that the strong oxidizing nature of sodium hypochlorite at both investigated concentrations affected the viability of immature seed explants of P. macranthum. Even though sodium hypochlorite is among the most common surface sterilants for seed explants along with mercuric chloride, hydrogen peroxide and antimicrobial agents (Ahmadi et al. 2012; Bai and Qu 2001; Barampuram et al. 2014; Schuller et al. 1989), its strong oxidizing nature can be harmful to certain explant tissues. Cell membranes of cotyledons were reported to have been injured by surface sterilization with sodium hypochlorite (Kaneko and Morohashi 2003). However, the tissues of P. macranthum seed explants with seed coat were left undamaged during surface sterilization with Sirvip™, as it was only supplemented with a hypochlorite of a concentration as low as 0.07%. The effective surface sterilization activity of Sirvip™ could be attributed to its antifungal and antibacterial constituents; natamycin, oxolinic acid, and captan (Valle Arizaga et al. 2019). Natamycin is an effective antifungal agent for seed explants (Pengfei et al. 2013). Oxolinic acid is a known bactericidal organic acid (Barry et al. 1984), and captan is a broad-spectrum fungicide of whose main mechanism of action includes numerous physiological and morphological changes to fungal cells including disruption of respiration through enzymatic changes (Lukens 2013). It could further be suggested that exposure of the immature seed explants with seed coat to a hypochlorite concentration of 0.07% as a post treatment to Sirvip™ rendered efficient germination of P. macranthum under in vitro conditions. Conditional exposure to lower hypochlorite concentrations has been proposed to favor seed germination (Miyoshi and Masahiro 1998). Owing to the effective nature of the antimicrobial constituents of Sirvip™ and its combination with lower concentrations of hypochlorite, the surface sterilization treatment resulted in successful seed germination. Successful propagation was achieved by subsequent culturing of the shoots obtained from the seedlings, on 1/2 MS medium (Figure 2D). Rhizome explants of P. macranthum that were surface-sterilized using Sirvip™ achieved only rooting, at a rate of 10% (Table 1, Figure 2C) even though no contamination was observed in any of the explants. Surface sterilization of rhizome explants using sodium hypochlorite at 1% and 2% (w/v) resulted in 100% decontamination, but rooting or shoot growth could not be achieved. As a considerable amount of rhizome tissue was exposed to sodium hypochlorite during surface sterilization, the explants could have had a higher mortality because of its strong oxidizing nature. In order to evaluate the effect of 6-benzylaminopurene (BAP) and 1-naphthaleneacetic acid (NAA) on microrhizome formation, ten shoots of P. macranthum derived from sub culturing the original shoots from immature seeds, were separately inoculated on 1/2 MS medium supplemented with BAP (0.1 to 1.0 mg l⁻¹) and, NAA (0.01 to 0.1 mg l⁻¹ with constant BAP concentration of 1.0 mg l⁻¹) respectively. Supplementing the 1/2 MS medium with cytokinin BAP at the concentration of 1.0 mg l⁻¹ resulted in the highest propagation rate of 1.3 microrhizomes per shoot in the 8 week (Table 2, Figure 3A), which was significantly different from that in the control and in the treatment with lower concentrations of BAP (0.1 mg l⁻¹). Shoot multiplication required a long time (16–24 weeks) when the basal medium only comprises of 1/2 MS. Favorable effects of BAP on bulblet and pseudo-bulblet formation were reported in Leucojum (Stanilova et al. 1994) and Lilium species (Nhut 1998) respectively. Bulblets are an efficient means of propagation because of their nature as propagules (Askari et al. 2018). During our study, microrhizome formation was not observed in P. macranthum shoots grown on phytohormone free 1/2 MS medium. It could be concluded that BAP at a concentration of 1.0 mg l⁻¹ was effective in inducing the formation of new microrhizomes and thereby facilitating propagation. NAA was not found to be effective for the propagation of P. macranthum by microrhizome (Table 3). Obtained microrhizomes were separated from each other and were inoculated on 1/2 MS medium. They were kept in total darkness at 15°C temperature for breaking microrhizome (bulblet) dormancy and were subsequently incubated at 20°C with a 16-h photoperiod. The microrhizomes grew into individual shoots with rooting during 8 weeks (Figure 3B). Well-grown young P. macranthum plantlets with several leaves and roots (Figure 4A) were transplanted to a sterilized soil of Hinode-sand (Hinode company, Japan) and gradually acclimatized for 4 weeks, under screen house conditions at a temperature of 25°C. A survival rate of 88% was achieved during acclimatization under screen house conditions. Regeneration of new shoots was apparent during this period (Figure 4B). They were subsequently transferred to the Niigata Agricultural Research Institute for evaluating field growth in a nursery bed with sunshade during the spring-summer season. After approximately 4 months, during the spring-summer season in the field with sunshade (made of black mesh and about 2 m in height), each plant generated several new microrhizomes and shoots (Figure 4C). Under natural conditions, if seeds were planted during the autumn, the seedlings of P. macranthum will begin their growth cycle in the spring of the second year (Takagi 2001a). A rhizome generates a single stand per growth cycle (Figure 1B) under natural conditions. Although only 13% of the investigated individual plants were successfully grown, the viability of these plants was significantly high owing to their multiple shoots and microrhizomes. It could be assumed that the absence of a cold period during plant growth in this experiment (in contrast to the natural growth cycle) led to a low percentage of plants achieving field growth. Our results on the in vitro germination of immature seeds with seed coat using surface sterilization supplemented by lower hypochlorite concentration, efficient in vitro propagation facilitated by BAP, and successful acclimatization followed by achieving field growth present a new cultivation system for the rare plant species P. macranthum.

Figure 1. Polygonatum macranthum (A) at maturity bearing fruits, (B) rhizome producing only one new rhizome per growth cycle.

Figure 2. The in vitro propagation of P. macranthum was established. (A) Unsterilized immature seeds of P. macranthum. (B) Germinated immature seed of P. macranthum. (C) Rhizome explant with in vitro rooting. (D) Shoots developed upon sub culturing of initial shoots from germinated immature seeds.

Table 1. Germination and root generation efficiency of P. macranthum explant types under different surface sterilizing treatments.

Surface sterilizer	Germination efficiency of immature seed explants (%)	Root generation efficiency of rhizome explants (%)
1% (w/v) Sirvip*	30	10
1% (w/v) sodium hypochlolrite**	0	0
2% (w/v) sodium hypochlolrite**	0	0

*Explants were initially dipped in 70% ethanol for one minute. They were subsequently treated with 1% (w/v) Sirvip™ solution for 1 h, followed by a 30-min dip in 0.07% (w/v) calcium hypochlorite solution. After a brief rinse with a fresh 1% (w/v) Sirvip™ solution, explants were directly inoculated on phytohormone free 1/2 MS basal medium, **Explants were initially dipped in 70% ethanol for 1 min. They were subsequently treated with sodium hypochlorite (at concentrations of 1% (w/v) and 2% (w/v)) for 10 min. After each treatment, the explants were rinsed with sterilized distilled water for 3 times before inoculating them on phytohormone free 1/2 MS basal medium, n=10.

Table 2. Effect of BAP on microrhizome formation.

BAP concentration (mg l−1)	Mean No. of microrhizomes (at 8 weeks)
0	0b
0.10	0.27b
0.50	0.82ab
1.00	1.30a

BAP concentration was varied on 1/2 MS basal medium. Mean values denoted with the same letter are not significantly different from each other at p≤0.05, n=11.

Figure 3. The effect of 6-benzylaminopurene (BAP) on regeneration of microrhizomes in P. macranthum. (A) Regeneration of microrhizomes on 1/2 MS medium with 1 mg l⁻¹ BAP. (B) Shoot growth and rooting of microrhizomes separated from each other.

Table 3. Effect of NAA on microrhizome formation.

NAA concentration (mg l−1)	Mean No. of microrhizomes (at 8 weeks)
0	1.33a
0.01	0.64a
0.05	0.75a
0.10	0.88a

NAA concentration was varied on 1/2 MS basal medium containing a BAP concentration of 1.00 mg l⁻¹. Mean values denoted with the same letter are not significantly different from each other at p≤0.05, n=11.

Figure 4. Cultivation of P. macranthum. (A) In vitro propagated P. macranthum plantlets. (B) Acclimatized juvenile plant with a new shoot. (C) Multiple shoots and new microrhizomes of an established plant in the field.