In Vitro Propagation of Peumus boldus Molina Using a Temporary Immersion System

Abstract

Peumus boldus Mol. (boldo) is a Chilean endemic tree species, characteristic of the region’s sclerophyllous forests. Increasing demand for its leaves and bark, valued for their antioxidant properties, has contributed to declining populations of this species in its natural habitat. This decline is further exacerbated by low natural germination rates and anthropogenic pressures. To support conservation efforts, efficient mass propagation protocols are needed. This study pioneered the use of temporary immersion systems (TIS) for the in vitro propagation of boldo, successfully developing a novel mass propagation protocol. To optimize the in vitro propagation of boldo shoots using the temporary immersion system (TIS), various immersion durations were tested. While a 3 min immersion resulted in a high proliferation rate (10.8), it also induced shoot vitrification, a detrimental physiological disorder. However, reducing the immersion duration to 2 min successfully prevented vitrification while still achieving a satisfactory multiplication rate of 7.2. The shoots reached an average length of 6.1 to 6.4 cm with 6-benzylaminopurine (4.44 μM). Ex vitro rooting was achieved in 75.4% of shoots treated with 1476 μM indole-3-butyric acid (IBA) across all tested substrates. The plantlets subsequently acclimatized with a survival rate between 77.1% and 83.3%.

1. Introduction

Peumus boldus Mol. (P. boldus) is an evergreen tree endemic to the sclerophyllous forests of Chile. Its range extends from Tongoy Bay (30°20′ S) in the Coquimbo region south to the Damas River in Osorno (41°20′ S) [1,2,3]. Approximately 70% of the P. boldus population is concentrated in the Mediterranean forests of central Chile, between 30° and 37° S latitude [4]. This slow-growing tree can reach heights of up to 20 m and possesses leathery, dark green leaves with a rough upper surface and a smooth, light yellowish-green underside, it is also notably aromatic [3,5,6]. Currently, there is a growing interest in this species, mainly due to the medicinal properties of its leaves, as they promote digestion, act as a diuretic and help resolve liver, and gallbladder disorders. The bark also contains valuable bioactive compounds, including alkaloids, flavonoids, essential oils, and potent antioxidants [7,8,9,10,11,12,13]. However, P. boldus populations are threatened by habitat loss driven by climate change, agriculture, overexploitation for boldine production, and deforestation from land-use change and fire. Wildfires are the leading cause of mortality for both P. boldus and the Chilean Mediterranean forest [14,15]. The natural regeneration of P. boldus is hindered by low seed germination rates and slow seedling establishment. Conventional propagation methods using cuttings and seeds have limited success, with reported rates between 16% and 44%, respectively [16,17,18,19,20]. Micropropagation techniques have been explored using semi-solid culture media. Nodal explants cultured with 4.44 μM 6-benzylaminopurine (BAP) achieved an average proliferation rate of 4.5 shoots per explant, with 80% rooting success and 100% survival after acclimatization [21]. Other studies using BAP at 2.46 and 4.92 μM produced four shoots per explant, while epicormic shoots required 1.11–3.33 μM BAP for an optimal response [22,23]. To date, there are no published reports on the micropropagation of P. boldus in liquid culture media. This study explored the use of temporary immersion systems (TIS) for the mass propagation of P. boldus. TIS utilizes two containers: one for the culture medium and the other for the plant material. An automated system with a solenoid valve and air compressor controls the periodic immersion of plant material in the liquid medium. This setup allows for the precise control of immersion duration and frequency [24,25].

The objective of this research was to develop a protocol for the mass propagation of P. boldus using a TIS approach.

2. Materials and Methods

Nodal segments (2 cm long) with one axillary bud were obtained from stock plants maintained in the Propagation Laboratory at the Pontificia Universidad Católica de Valparaíso, Quillota, Chile. Following the protocol in semi-solid culture medium described by our research group [21], one hundred explants were randomly selected and established on Murashige and Skoog (MS) (Sigma-Aldrich, St. Louis, MO, USA) basal medium [26] supplemented with 1 g L⁻¹ polyvinylpyrrolidone (PVP) (Sigma-Aldrich, Darmstadt, Germany), 0.75 μM thiamine (Sigma-Aldrich, Darmstadt, Germany), and 30 g L⁻¹ sucrose (Sigma-Aldrich, St. Louis, MO, USA). The pH was adjusted to 5.8 ± 0.1 prior to the addition of 6.5 g L⁻¹ agar (Algas Marinas S.A., Santiago, Chile) [21]. The medium was autoclaved at 121 °C for 15 min. The disinfection used was a 1% sodium hypochlorite solution (Comercial Vimaroni S.A., Quilpué, Chile), which was supplemented with the antioxidants ascorbic acid (Merck KGaA, Darmstadt, Germany) (2838.97 μM) and citric acid (Comercial Vimaroni S.A., Quilpué, Chile) (2602.49 μM), along with 1 mL of Tween 20 (Loba Chemie Pvt. Ltd, Mumbai, India). The mixture was shaken continuously for 10 min. This was followed by a series of five rinses with sterile distilled water under a laminar flow hood [21]. Cultures were maintained for 8 weeks in a growth chamber at 25 ± 1 °C under a 16 h photoperiod with a light intensity of 400–700 nanometers (nm) (PPFD 9.96 μmol m⁻² s⁻¹) (fluorescent lamps Philips TL-D 36W/54 brand) (Shanghai, China). In this way, the plant material that was used in the following tests was homogenized.

2.1. In Vitro Multiplication Using a Temporary Immersion System (TIS)

Boldo shoots (2 cm) derived from the established in vitro cultures were used for the TIS experiment. The effects of immersion duration (1, 2, and 3 min) and immersion frequency (every 12 or 24 h) on in vitro shoot proliferation were evaluated. Cultures were grown in 2000 mL glass bottles (VMGLASS 3.3, Cerrillos, Chile) containing 10 boldo shoots each. The culture medium consisted of MS basal salts and vitamins (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 4.44 μM BAP (Sigma-Aldrich, St. Louis, MO, USA) and 30 g L⁻¹ sucrose (Sigma-Aldrich, St. Louis, MO, USA) (pH 5.7). The medium (200 mL per vessel) was held in 1000 mL containers connected to the culture vessels via an automated TIS system. Cultures were maintained in a growth chamber at 25 ± 1 °C under a 16-h photoperiod with a light intensity of 400–700 nm (PPFD 9.96 μmol m⁻² s⁻¹) (fluorescent lamps Philips TL-D 36W/54 brand) (Shanghai, China).

In the case of this study, it was decided to develop a temporary immersion system, which consisted of two 2000 mL bottles, one containing the culture medium and the second the boldo shoots. These bottles were joined by sterilized silicone hoses of 6 mm internal diameter and 0.2 μM filters connected to an air compressor; the air flow moved towards the container with the plants and these remained submerged in the medium. The air flow allowed the culture medium to bubble so that the explants were oxygenated, and then the process was reversed, that is, the solenoid valve was opened and the medium was returned to the original tank by air pressure (Figure 1). This system connected to an automated irrigation system, where the duration of each immersion and the frequency were controlled. Figure 2 shows a photograph of the system used, showing the two upper sections intended to contain the TIS, the bottles, filters, hoses, and solenoid valves.

2.2. Ex Vitro Rooting

Shoots (4 cm in length) obtained from the TIS experiment were used for the ex vitro rooting trials. The base of each shoot was trimmed and disinfected with 1.8 g L⁻¹ Benomyl (Nufarm Chile, Ltda., Vitacura, Santiago). To stimulate root formation, shoots were treated with four concentrations of indole-3-butyric acid (IBA) (Sigma-Aldrich, St. Louis, MO, USA): 0, 492, 984, and 1476 μM. For each treatment, it was kept for 30 s in the respective concentrated hydroalcoholic solutions to stimulate root formation. Treated shoots were then transplanted into 200 cc containers filled with one of four different substrate mixtures: perlite (Harbolite Chile S.A., Quilicura, Chile, vermiculite (Protekta Ltda., Quillota, Chile), peat moss (Stender GmbH, Schermbeck, Germany), or a peat:perlite:vermiculite mix (1:1:1 v/v/v). Each plant was covered with a transparent 200 cc container to maintain humidity. The plantlets were maintained in a growth chamber at 25 ± 1 °C under a 16 h photoperiod with a light intensity of 400–700 nm (PPFD 9.96 μmol m⁻² s⁻¹) (fluorescent lamps Philips TL-D 36W/54 brand) (Shanghai, China).

The rooting percentage (defined as the presence of at least one root 1 cm in length) was determined at 45 days. The experiment followed a completely randomized design with a 4 x 4 factorial arrangement (4 IBA concentrations × 4 substrates). Percentages were natural-logarithm-transformed. Each treatment consisted of 10 explants with 3 replicates. Data were analyzed using a two-way ANOVA and analysis of components of variance. Tukey’s test (p ≤ 0.05) was used to determine significant differences between treatment means using Minitab 19 statistical software (Minitab Inc., State College, PA, USA).

2.3. Ex Vitro Acclimatization

Rooted boldo plantlets from the ex vitro rooting stage were used for the acclimatization trials. Plantlets were transferred to containers with the same type of substrate as used in the rooting experiment (perlite, vermiculite, peat moss, or a 1:1:1 peat moss–perlite–vermiculite mix). For each substrate, 10 rooted plantlets were randomly selected and transferred to one of two acclimatization conditions:

• Cold Greenhouse: Plantlets were placed on a bench with basal heating (25 °C) within a cold greenhouse. Temperatures inside the greenhouse fluctuated between 18 and 30 °C.
• Growth Chamber: Plantlets were maintained in a growth chamber at 25 ± 1 °C under a 16 h photoperiod with a light intensity of 400–700 nm (PPFD 9.96 μmol m⁻² s⁻¹) (fluorescent lamps Philips TL-D 36W/54 brand) (Shanghai, China).

The transparent containers covering the plantlets were gradually removed in four discrete steps over a four-week period to reduce relative humidity. After four weeks, the containers were removed completely.

The survival rate (%) of the acclimatized boldo plantlets was recorded at 40 days. The survival rate was calculated as the percentage of live plants relative to the total number of acclimatized plants. This experiment followed a completely randomized design with a 4 × 2 factorial arrangement (4 substrates × 2 acclimatization conditions). Percentages were natural-logarithm-transformed. Each treatment consisted of 10 plants with 3 replicates. Data were analyzed using a two-way ANOVA and analysis of components of variance. Tukey’s test (p ≤ 0.05) was used to determine significant differences between treatment means using Minitab 19 statistical software (Minitab Inc., State College, PA, USA).

3. Results

3.1. In Vitro Multiplication Using a Temporary Immersion System (TIS)

The interaction between both factors (immersion duration and immersion frequency) did not have a significant impact either on shoot proliferation or on shoot length (p ≥ 0.05) (

Table 1. Effect of immersion duration on the proliferation rate and shoot length of P. boldus.

Immersion Duration	Proliferation Rate	Shoot Length (cm)
1 min	3.2 ± 0.3 c *	6.1 ± 0.3 a *
2 min	7.2 ± 0.2 b	6.4 ± 0.3 a
3 min	10.8 ± 0.3 a	4.0 ± 0.2 b
F Immersion Duration	p = 0.000	p = 0.000
F Immersion Frequency	p = 0.510	p = 0.861
F Immersion Duration × Immersion Frequency	p = 0.441	p = 0.620

* Different letters indicate significant differences according to Tukey’s test (p ≤ 0.05).

). Similarly, the immersion frequency treatments did not have a significant effect on these parameters (p ≥ 0.05). However, both the proliferation rate and shoot length were significantly affected by the immersion duration (p ≤ 0.05) (Table 1).

The 3 min immersion duration yielded the highest shoot multiplication rate at 10.8 shoots per explant. However, this treatment also induced vitrification in the shoots (Figure 3C). In contrast, reducing the immersion duration to 2 min produced a lower, but still substantial, multiplication rate of 7.2 shoots per explant while avoiding vitrification (Figure 3B). The shortest immersion duration tested, 1 min, resulted in the lowest multiplication rate of 3.2 shoots per explant (Figure 3A).

Shoot length was also influenced by the immersion duration. The 1 min and 2 min immersion treatments yielded shoots with average lengths of 6.1 cm and 6.4 cm, respectively. In contrast, the 3 min immersion duration produced significantly shorter shoots, with an average length of only 4 cm (Table 1).

3.2. Ex Vitro Rooting

The interaction between the factors (type of substrate (perlite, vermiculite, peat moss, and a 1:1:1 v/v/v combination of peat moss, perlite, and vermiculite) and the concentration of IBA (0, 492, 984, and 1476 μM)) did not have a significant effect on the ex vitro rooting of boldo shoots (p ≥ 0.05) (

Table 2. Effect of various IBA concentrations on the ex vitro rooting percentage of P. boldus shoots.

Treatment	IBA Concentration	% Rooting
IBA1	0	17.4 ± 3.1 d *
IBA2	492 μM	36.4 ± 4.3 c
IBA3	984 μM	47.1 ± 4.1 b
IBA4	1476 μM	75.4 ± 4.1 a
F Substrate		p = 0.349
F IBA		p = 0.000
F Substrate × IBA		p = 0.697

* Different letters indicate significant differences according to Tukey’s test (p ≤ 0.05).

). Importantly, the IBA concentration emerged as the sole factor that had a significant influence on rooting success (p ≤ 0.05) (Table 2).

A rooting percentage of 75.4% was achieved when the shoots were treated with 1476 μM IBA, regardless of the substrate used (Figure 4). Lower IBA concentrations of 984 μM and 492 μM resulted in rooting percentages of 47.1% and 36.4%, respectively.

3.3. Ex Vitro Acclimatization

As shown in

Table 3. Effect of different combinations of substrates and acclimatization systems on the survival (%) of P. boldus shoots during the acclimatization stage. Different letters indicate significant differences according to Tukey’s test (p ≤ 0.05).

Substrate	Acclimatization Treatment	% Acclimatization
Perlite	Bench with basal heat	77.1 ± 4.1 a
Perlite	Growth chamber	77.1 ± 4.2 a
Vermiculite	Bench with basal heat	80.0 ± 3.1 a
Vermiculite	Growth chamber	83.3 ± 4.3 a
Peat moss	Bench with basal heat	80.0 ± 3.2 a
Peat moss	Growth chamber	80.0 ± 3.0 a
Peat moss, perlite, and vermiculite (1:1:1 v/v/v)	Bench with basal heat	80.0 ± 3.1 a
Peat moss, perlite, and vermiculite (1:1:1 v/v/v)	Growth chamber	80.0 ± 3.0 a
F Substrate		p = 0.740
F Acclimatization condition		p= 0.800
F Substrate x Acclimatization condition		p = 0.977

and Figure 5, the various combinations of substrate type and acclimatization treatment did not significantly affect the survival of boldo plantlets. Survival rates ranged from 77.1% to 83.3% across all treatment combinations. The substrate types tested were perlite, vermiculite, peat moss, and a mixture of peat moss, perlite, and vermiculite (1:1:1 v/v/v). The acclimatization treatments were a cold greenhouse with basal heat (25 °C; ambient temperature 18–30 °C) and a growth chamber (25 ± 1 °C, 16:8 h light/dark photoperiod, 400–700 nm light intensity [PPFD 9.96 μmol m⁻² s⁻¹]).

4. Discussion

The results of this study demonstrate the advantages of using a temporary immersion system over semi-solid media for the propagation of boldo. Notably, the TIS approach yielded higher in vitro shoot proliferation rates. This system likely provides improved nutrient absorption and promotes greater plant uniformity compared to semi-solid culture.

However, a major challenge associated with TIS for many plant species is the issue of hyperhydricity, a physiological disorder that can arise from the use of liquid culture media [27,28].

In the case of boldo, the highest proliferation rate of 10.8 shoots per explant was achieved with a 3 min immersion duration and an immersion frequency of every 12 or 24 h. However, these shoots exhibited hyperhydricity, likely due to the liquid culture medium. The problem of hyperhydricity has been reported in various woody plant species, including eucalyptus, blueberry, walnut, pistachio, and apple, among others [29,30,31,32,33,34].

Hyperhydricity is directly related to water content and water potential; therefore, immersion frequency and duration play an important role in explant vitrification [35]. For example, it has been reported that decreasing the immersion frequency from every 8 h to every 14 or 16 h improved proliferation and reduced vitrification in pistachio [32].

Another factor to consider is the availability of free air space for in vitro plants. Improved shoot growth has been reported in TIS, likely due to the increased availability of O₂ (oxygen) and the simultaneous decrease in the concentration of CO₂ (carbon dioxide) and C₂H₄ (ethylene) provided by the fresh air supply during immersions [36]. Although there are limited studies on this factor, the positive results observed in boldo suggest that this variable should be considered in future studies on this and other woody species.

Previous research on boldo achieved a proliferation rate of 3.2 and an average shoot length of 5.9 cm using 4.44 μM BAP in a semi-solid MS culture medium [21]. In comparison, the current study using the TIS approach demonstrated a significant increase in both proliferation and shoot length. Specifically, a proliferation rate of 7.2 was obtained with a 2 min immersion duration and a frequency of every 12 or 24 h. Furthermore, average shoot lengths of 6.1 to 6.4 cm were achieved with 1 and 2 min immersion durations and frequencies of every 12 or 24 h.

Another variable considered in this study was ex vitro rooting. A 75% rooting percentage was achieved when using 1476 μM IBA, regardless of the substrate (peat moss, perlite, vermiculite, or a 1:1:1 v/v/v peat:perlite:vermiculite mix). This contrasts with the 85% in vitro rooting reported by [21], who used MS medium supplemented with 9.84 μM IBA. This is probably because, in this case, the rooting was carried out ex vitro, together with acclimatization.

Acclimatization success rates ranged from 77% to 83%, demonstrating a high level of effectiveness, particularly when compared to previous research that used cuttings or seeds, which achieved success rates of only 16% to 44% [16,19,20].

5. Conclusions

This research successfully developed a mass propagation protocol for Peumus boldus using a novel approach—the temporary immersion system. The use of TIS improved both the shoot length and proliferation rate compared to previous reports. Furthermore, the shoots rooted successfully with the application of IBA, and subsequent acclimatization achieved a high survival rate of 83%.

This study determined that the complete in vitro culture period, from initial explant to acclimatized plantlet, takes approximately 7 to 9 months. Notably, this protocol can yield 7.2 plants from a single nodal segment with one axillary bud. The effectiveness of this TIS-based protocol offers a valuable opportunity for the large-scale multiplication of this threatened, endemic species. It presents a new strategy for the propagation and sustainable conservation of P. boldus, as well as potentially other woody species facing conservation challenges within the Chilean sclerophyllous forest.