Cultivation of Siberian Motherwort Plants (Leonurus sibiricus L.) in In Vitro Culture

Abstract

In this study, an attempt was made to micropropagate the Siberian motherwort (Leonurus sibiricus L.) in in vitro cultures. The aim of this study was to compare methods of sterilization of seeds, which served as a source of initial explants to induce in vitro culture and to multiply shoots from explants isolated from them. The primary explants in the experiment were seeds. The seeds were placed on ½ MS medium without PGR and enriched with GA₃ at a concentration of 1 mg∙dm⁻³. The addition of GA₃ to the medium stimulated their germination. Sodium hypochlorite (NaClO) and 70% ethanol (C₂H₅OH) were used to sterilize the seeds. NaClO was used for proper sterilization in concentrations: 0.0% (1), 1.5% (2), 2% (3), and 2.5% (4) for 11 min. As secondary explants in the experiment, single-node fragments of sterile seedlings derived from sterile seeds were used, which were placed on MS medium, with the addition of plant growth regulators (PGR) from the group of auxins and cytokinins. The largest number of sterile and live Siberian motherwort seedlings was obtained in the variant in which 2.5% NaClO was used for proper sterilization of the seeds. To optimize the procedure of in vitro micropropagation, the axillary shoots of Leonurus sibiricus L. were transferred during the third passage onto nine different combinations of MS medium: PGR-free medium, with different concentrations of BAP (6-benzylaminopurine) (0.0, 2.0, 3.0, 4.0, 5.0 mg∙dm⁻³) and BAP (0.0, 2.0, 3.0, 4.0, 5.0 mg∙dm⁻³) with NAA (1-naphthaleneacetic acid) (1.0 mg∙dm⁻³). The largest number of shoots (9.62) forming was obtained on the medium supplemented with 4 mg∙dm⁻³ BAP and 1 mg∙dm⁻³ NAA, while the longest shoots were on the medium without PGR. Induction of rhizogenesis of Siberian motherwort plants in in vitro cultures was achieved on MS medium with the addition of 0.5 mg∙dm⁻³ IAA (indole-3-acetic acid). At the stage of adaptation to ex vitro conditions, high plant survival of 90% was achieved by using a solution with MS salts (25%) for irrigation.

1. Introduction

Siberian motherwort (Leonurus sibiricus L.), commonly known as Marihuanilla, is a herbaceous plant belonging to the Lamiaceae family, comprising 236 genera and 6900–7200 species. There are approximately 20 different species within the Leonurus genus. Siberian motherwort is native to Russia, Mongolia, and China [1]. It is a rare herbal plant in Poland. The herbal raw material of Siberian motherwort is the entire plant, with a particular emphasis on the flowers.

Siberian motherwort owes its healing properties to the content of biologically active substances belonging to alkaloid groups in its tissues. One of them is leonurine, which is a psychoactive ingredient [2,3]. Additionally, another alkaloid isolated from the Leonurus genus is stachydrine. The presence of monoterpenoids, sesquiterpenoids, diterpenoids, triterpenoids, iridoids, flavonoids, sterols, phenylpropanoids, and cyclic peptides was also found [2]. Other chemical substances included in Siberian motherwort are diterpene lactones (leonotinin), labdanum-type diterpenes (leosibirin), and polyphenol (chlorogenic acid and caffeic acid) [4]. L. sibiricus has been used in Eastern medicine for centuries due to the presence of bioactive substances. In traditional Chinese medicine, this plant is used medicinally for impotence and gynecological problems. It is used as a diuretic. It can also be used to treat rheumatic fever and arthritis [5]. Moreover, motherwort has antibacterial, anti-inflammatory, and antioxidant properties. L. sibiricus has allelopathic properties, and it can also be used in agriculture as a bioherbicide [2,6,7,8,9,10,11,12]. This species is also valued as a melliferous plant [13].

A commonly employed technique in biotechnology for the mass propagation of plants, including medicinal species, is micropropagation [14]. This involves the use of plant tissue and cell culture techniques to produce propagation material. However, there is limited information in the literature regarding the micropropagation of Siberian motherwort. In the micropropagation process, plants are propagated vegetatively in controlled and sterile laboratory conditions on artificial substrates (media). The simplest and most frequently used method of micropropagation is to stimulate the growth of lateral buds. Explants in this type of culture may be shoot tips, single axillary buds, nodal fragments of shoots, fragments of shoots with a leaf, and an active axillary bud [15]. Micropropagation allows for quick cloning of medicinal plants, which can result in obtaining high-quality seedlings in a short time. The use of this technique is particularly desirable in the case of herbs that are subject to the requirements of standardization of raw materials for the pharmaceutical industry [16].

The aim of the research was to optimize the micropropagation of Siberian motherwort plants from seeds. The explants used in the experiment were single-node fragments of seedlings, which were placed on Murashige and Skoog medium (MS) [17], with the addition of plant growth regulators (PGR) from the group of auxins and cytokinins: NAA (naphthyl-1-acetic acid) and BAP (6-benzylaminopurine) in various concentrations and combinations. In this research, the micropropagation technique using nodal segments with axillary buds was adopted, as this method is able to guarantee the genetic stability of new plants [18].

2. Materials and Methods

2.1. Plant Material

Siberian motherwort (L. sibiricus) seeds were collected in August 2021 in the city of Bydgoszcz (53°07′15.6″ N 18°00′23.1″ E), Poland, from a collection of 10 plants of unknown provenance maintained at the Department of Biotechnology, the Bydgoszcz University of Science and Technology. The seeds were manually taken out for germination.

2.2. Sterilization and Germination of Seeds

The experiment was carried out in March 2022 The starting material for the research were seeds of the Siberian motherwort (L. sibiricus), which came from plants from our own collection. Four sterilization variants were used. In the first stage, all seeds were subjected to preliminary sterilization, which consisted of immersion for 60 s in 70% ethyl alcohol (C₂H₅OH) (Chempur, Piekary Śląskie, Poland). The second stage included proper sterilization, in which each group was immersed in the following concentrations of sodium hypochlorite (NaClO) (Warchem, Zakręt, Poland): 0.0% (1), 1.5% (2), 2% (3), and 2.5% (4) with the addition of 2 drops of Tween 20 detergent (Sigma-Aldrich, Burlington, MA, USA). This treatment lasted 11 min. The control sample consisted of seeds that were disinfected only with 70% C₂H₅OH. The final stage consisted of rinsing the seeds three times in sterile and double-distilled water.

Sterile Siberian motherwort seeds were individually inserted into the nutrient medium. This activity was performed under sterile conditions. The experiment used ½ MS (Sigma-Aldrich, Burlington, MA, USA). The medium without growth regulators (PGR-free medium) and with the addition of gibberellic acid GA₃ (Sigma-Aldrich, Burlington, MA, USA) in the amount of 1 mg∙dm⁻³ was used. The medium was solidified with a 0.8% agar (Biomaxima, Lublin, Poland). The pH of the medium was set to 5.7 and sterilized in an autoclave using the following parameters: pressure, 0.1 MPa; temperature, 121 °C; and time, 20 min.

The prepared medium was poured into glass test tubes, which were placed in baskets with 24 compartments. In each sterilization variant, 24 samples (seeds) were used. Each treatment consisted of two replicates. The baskets with the nutrient solution and seeds were moved to the growth room with controlled growth conditions: 16 h light/8 h darkness, i.e., 16 h photoperiod, light intensity approx. 40 µmol·m⁻²·s⁻¹ (daylight lamps of 40 W) (PILA, Piła, Poland) emitting daylight, temperature 25 ± 2 °C. Observations of the number of germinated seeds in each used variant were made every week for a period of 4 weeks.

2.3. Shoot Propagation

After approximately 4 weeks of seedling growth, explants were isolated from them, which were single-node sections of shoots. Seedlings were taken out of the test tubes, their leaves were removed, and then the shoot was cut into 1 cm nodal sections. These activities were carried out in a chamber with a laminar flow of sterile air using sterile instruments (Figure 1).

Isolated single-node explants were placed on MS medium without PGR (passages I and II). In order to stimulate lateral buds located in the leaf axils, the multiplied shoots were divided and transferred into MS medium without PGR and MS medium enriched with BAP and NAA in various combinations and concentrations (passage III). After 6 weeks from the second passage, the third passage was performed, in which axillary shoots were isolated and inoculated into 9 variants of MS media with different concentrations and combinations of BAP (0.0; 2.0; 3.0; 4.0; 5.0 mg∙dm⁻³) and NAA (1.0 mg∙dm⁻³) (Sigma-Aldrich, Burlington, MA, USA). Within each combination, 50 explants were transferred onto the specific growth medium, and the experiment was repeated two times.

Prepared media were poured into 25 mL Erlenmeyer flasks and sterilized in an autoclave, which was set to the following parameters: temperature, 121 °C; pressure, 0.1 MPa; and time, 20 min. Three explants were placed in each flask. In vitro plant cultures were carried out in a growth room under conditions similar to those during seed germination. In each variant, 50 explants were inoculated into the medium. After 6 weeks of plant growth, the percentage of explants with axillary shoots, the number of axillary shoots per explant, and their length were measured and the percentage of explants with developing callus tissue was calculated.

2.4. Root Formation and Growth of Plants Under In Vivo Conditions

After 6 weeks of culture, the developed axillary shoots were divided and placed in jars with MS and MS medium with the addition of IAA auxin (indolyl 3-acetic acid) (Sigma-Aldrich, Burlington, MA, USA) in the amount of 0.5 mg∙dm⁻³. After 4 weeks, the shoots rooted in vitro were planted into plastic pots filled with sterile substrate (horticultural substrate and perlite in a 3:1 ratio). To reduce transpiration, the plants were covered with glass jars. A total of 40 rooted shoots were placed under a 16 h photoperiod and at a temperature of 24 °C. In order to increase the effectiveness of the acclimatization process, 20 plants were watered with an aqueous MS salt solution (25%) and another 20 with tap water (0%). After 28 days of adaptation to ex vitro conditions, the plants were moved to the greenhouse.

2.5. Statistical Analysis

Data on the effect of PGR on the number and length of shoots and roots obtained from nodal explants of Siberian motherwort were subjected to analysis of variance in a completely random design. Single-factor analysis of variance (ANOVA) was performed using the Statistica^® 13.1 package. The significance of differences (LSD—lowest significant difference) was evaluated using the Tukey single confidence intervals for the significance level of p ≤ 0.05. The arithmetic mean values are shown in tables ± standard deviation.

3. Results and Discussion

3.1. Sterilization and Germination of Seeds

The aim of the first stage of the experiment was to obtain healthy and sterile Siberian motherwort seedlings (Figure 1), which served as a source of single-node explants for subsequent experimental phases. For the proper sterilization of Siberian motherwort seeds, the most frequently used chemical compound for disinfection of plant material was selected—sodium hypochlorite (NaClO)—in the following concentration variants: 0% (1); 1.5% (2); 2% (3); and 2.5% (4). Most often, chlorine-based compounds are used for the proper sterilization of plant explants, which include sodium hypochlorite, calcium hypochlorite, chloramine, ACE, and mercury chloride. Due to the fact that this factor acts not only on microorganisms but also on plant cells, it is necessary to determine the optimal duration of proper sterilization in the compound used [19,20,21].

Taking into account the number of germinated seeds, the development of sterile and live, undamaged seedlings, and fungal and bacterial contamination, it was found that the fourth sterilization variant was the most effective (Table 1). In variant 4, in which sodium hypochlorite at a concentration of 2.5% was used for proper sterilization, the largest number of sterile and live seedlings was obtained on average on ½ MS medium without PGR and ½ MS with the addition of 1 mg∙dm⁻³ GA₃ (Figure 2). The average number was 18 (37.5%). This variant also had the least amount of fungal and bacterial contamination (10.5%).

At the germination stage, adding 1 mg∙dm⁻³ of GA₃ to the medium stimulated the germination of Siberian motherwort seeds. Similar results were obtained by Devi et al. (2012) [22], concluding that GA₃ added to MS medium stimulated the seed germination of bamboo plants (Dendrocalamus giganteus Munrow) and Khuat et al. (2022) [23] observed a positive effect on the germination of cardamom seeds (Amomum tsao-ko Crevost and Lemarié). The positive effect of GA₃ on seed germination is related to the fact that gibberellins are growth stimulants necessary for the germination of seeds of many plant species, and their activity is manifested in breaking seed dormancy and accelerating this process [24].

3.2. Shoot Propagation

The explants used to initiate the in vitro culture of Siberian motherwort were single-node sections of shoots with buds in the leaf axils. According to Koziara (2002) [19], young seedlings can be used as a source of explants to initiate in vitro culture because they have very good regenerative properties. According to the authors of Kanwar and Kumar (2008) [25] and Assim (2008) [26], the use of this type of explant for plant micropropagation makes it possible to obtain daughter plants that are genetically identical to the mother plant. This fact results from the same histological and genetic structure as the shoot apical meristems. Nodal segment explants are less likely to contribute to somaclonal variation because plant regeneration usually occurs without an intermediate callus stage [27].

All culture media promoted shoot formation. The high content of cytokinins and low amount of auxin used in the experiment promoted the micropropagation of plants in in vitro cultures. According to Beyl (2011) [28], cytokinins derived from adenine (BAP, KIN) abolish the dominance of the apical bud and, at the same time, stimulate the growth of axillary shoots from axillary buds. According to Pasternak and Steinmacher (2024) [29], the main effect of cytokinin in in vitro tissue culture is shoot induction, which occurs through the induction and maintenance of auxin biosynthesis. Auxin is an essential hormone responsible for all processes in plant tissue culture. This phytohormone is responsible for both the morphogenesis of shoots and roots.

The highest number of shoots, although not statistically proven, was obtained on the MS medium with the addition of BAP at a concentration of 4 mg∙dm⁻³. In the presented experiment, a mean shoots number obtained on MS medium enriched only with BAP at a concentration of 4 mg∙dm⁻³ and BAP at a concentration of 4 mg∙dm⁻³ with NAA at a concentration of 1 mg∙dm⁻³ resulted in 9.37 and 9.62 units, respectively (Table 2, Figure 3). In many studies, the authors have proven that adding cytokinins to the medium increases the efficiency and regeneration of shoots in in vitro cultures. Plant species in which the influence of cytokinins on the formation of shoots has been confirmed include Thymus serpyllum L. [30], Hemidesmus indicus L. [31], Stevia rebaudiana [32,33], Chrysanthemum [34], and Vaccinium corymbosum [35].

These results confirm that cytokinins have important physiological functions. They stimulate cell division, activate RNA synthesis, stimulate protein synthesis, and influence enzyme activity. Application to the medium-high concentrations of cytokinins resulted in limiting the growth of shoots and leaves and supporting the formation of meristematic clusters [36].

As observed during the in vitro culture of Siberian motherwort, when the concentration of BAP was increased, the shoot number also increased up to an optimum concentration of 4 mg∙dm⁻³, beyond which the number of shoots declined to 9.19 and 8.93 per explant at 5 mg∙dm⁻³ (Table 2). These results are in accordance with those obtained by Lobna et al. (2008) [36] They noticed that increasing cytokinin concentration from 1.0 to higher concentrations (6.0 mg∙dm⁻³) generally had a depressive effect on the morphogenesis characteristics of Paulownia kawakamii. According to Kim et al. (2023) [37], the shoot induction culture responses in media containing BA exhibited consistent performance across all concentrations. However, it was at low cytokinin concentrations that the number of buds and nodes increased. The highest number of shoots and nodes per explant in grapevine in vitro culture was achieved with media containing 2.0 μM BA. These values were higher than BA at 8.0 μM. However, it should be noted that the optimal types and concentrations of cytokinins for in vitro propagation vary among different plant species [38,39].

The longest shoots of Siberian motherwort were recorded on explants growing on an MS control medium without PGR (3.5 cm). The shortest ones were observed in plants grown on MS substrates with the addition of 5 mg∙dm⁻³ BAP (1.38 cm) and 5 mg∙dm⁻³ BAP with 1 mg∙dm⁻³ NAA (1.43) (Table 2). A similar effect was observed by Abdulla et al. (2010) [40] regarding the Ficus anastasia plant height, which was negatively affected by the increase in cytokinin concentration. However, the control plant was longer in length than any of the cytokinin treatments in combination with auxin. In the process of micropropagation of Arbutus andrachne, Bertsouklis and Papafotiou (2009) [41] found that cytokinins were the least effective as they could not induce elongation of the shoots.

On media enriched with 1 mg∙dm⁻³ NAA, slightly longer shoots are observed compared to the media with the same amount of cytokinin BAP. This result can be explained by the fact that auxin induces shoot elongation processes. The process of shoot elongation is related to the acidification of the cell wall caused by exogenous auxin, which loosens the cell wall and increases the uptake of water and potassium, leading to an increase in cell length [27,42]. According to Alvarenga et al. (2015) [43] and Clapa and Hârța (2021) [44], auxin also is important as a signal for cell division and differentiation. This growth regulator also plays an important role in apical dominance. Segmentation of the shoot into single-node sections with buds in the leaf axils consequently leads to interruption of apical dominance.

In all variants of media with the addition of BAP and NAA, callus tissue was formed on the cut surface of the explants. The highest callus content (100%) was recorded in the variants in which BAP was used at a concentration of 3, 4, or 5 mg∙dm⁻³, used alone or in combination with auxin (NAA). Lower callus content was obtained in plants grown in the substrate with the addition of BAP in the amount of 2 mg∙dm⁻³ (75%) or with NAA in the amount of 1 mg∙dm⁻³ (76%). According to sources, from the point of view of the efficiency of shoot multiplication, callus is a negative phenomenon in direct organogenesis due to the fact that it competes with shoots for nutrients and adventitious shoots may be formed from it, which may result in somaclonal variability [45].

3.3. Root Formation and Growth of Plants Under In Vivo Conditions

In the experiment conducted with Siberian motherwort, induction of rhizogenesis in in vitro cultures was achieved on MS medium without PGR and MS medium with the addition of IAA auxin in the amount of 0.5 mg⸱dm⁻³ (Table 3). The percentage of rooted plants was 100% in both cases. Statistical analysis revealed that plants formed more roots on the MS medium with the addition of IAA compared to the control medium (PGR-free medium).

According to Pasternak and Steinmacher (2024) [29], there are two key factors for successful rooting: a high rate of auxin biosynthesis in new shoots and a high rate of auxin flux from these shoots through the vessel. Exogenous phytohormones serve as a tool for regulating various processes in the plant tissue culture; however, plant morphogenesis is regulated exclusively by endogenous hormones, and exogenous hormones can only serve as modulators of their action. The results of the experiment are consistent with the results of other authors who, after enriching the MS medium with IAA, observed that this exogenous auxin stimulated root formation in other plant species such as S. rebaudiana [46], Brassica oleracea [47], Dahlia [48], and Helichrysum arenarium L. Moench [49]. Al-Amin et al. (2009) [50], conducting experiments on the influence of auxins on the rooting process of Musa spp., obtained the best results in terms of root number after adding IAA and IBA auxins to the medium. In the case of micropropagation of H. arenarium, Sawilska and Figas (2006) [51] concluded that the shoots root well in a medium without growth regulators. Similar observations were also made by Tomaszewska-Sowa and Figas (2011) [52] in their research on micropropagation of the cup plant (Silphium perfoliatum L.). According to Tomaszewska-Sowa and Figas (2011) [52] and Anbazhagan et al. (2010) [53], many plant species form roots on media without the addition of growth regulators, while enriching the medium with exogenous auxins IAA, NAA, or IBA can accelerate the rhizogenesis process.

Based on observations, it was shown that at the stage of adaptation to ex vitro conditions, the survival rate in the group of plants watered with tap water alone was 70%, while among the plants watered with a solution with MS salts was 90% (Figure 4, Figure 5 and Figure 6). A similar effect was also observed by Jitendra et al. (2012) [54] and Tomaszewska-Sowa et al. (2015) [55] during the acclimation of S. rebaudiana plants, and Figas et al. (2016) [49] conducted research on the acclimatization of H. arenarium. The positive result of using an aqueous MS salt solution can be explained by the fact that the presence of exogenously applied ions in the aqueous salt solution weakened the effect of stress factors, thus influencing the higher survival of acclimatized plants.

4. Conclusions

Obtaining sterile seedlings from seeds makes it possible to obtain mother and reproductive material free from diseases and pests. Moreover, this method allows to obtain a large amount of material in a short time without degradation of the natural environment. The largest number of sterile and live Siberian motherwort (L. sibiricus) seedlings was obtained in the variant in which 70% ethanol and 2.5% sodium hypochlorite were used for proper sterilization of the seeds. The addition of GA₃ to the medium stimulated their germination. Among the tested combinations of BAP and NAA, the use turned out to be the best in the micropropagation process 4 mg∙dm⁻³ BAP and 1 mg∙dm⁻³ NAA (9.62). Induction of rhizogenesis of Siberian motherwort plants in in vitro cultures was achieved on MS medium with the addition of 0.5 mg∙dm⁻³ IAA. At the stage of adaptation to ex vitro conditions, high plant survival of 90% was achieved by using a solution with MS salts (25%) for irrigation. Due to the high number of axillary shoots of the Siberian motherwort, which were formed from axillary buds located in the leaf axils of single-node shoot sections, the quick ability to root the acquired shoots, and the easy acclimatization plants, the method described in the publication can be used for the production of plants on a commercial scale. In further stages of research, it is necessary to develop an efficient sterilization and micropropagation protocol for vegetative parts of plants.

Author Contributions

Conceptualization, A.F.; methodology, A.F.; software, A.F. and M.T.-S.; validation, A.F. and M.T.-S.; formal analysis, A.F. and Z.G.; investigation, A.F. and M.T.-S.; resources, A.F., M.T.-S. and Z.G.; data curation, A.F. and M.T.-S.; writing—original draft preparation, A.F., M.T.-S. and Z.G.; writing—review and editing, A.F. and M.T.-S.; visualization, A.F. and M.T.-S.; supervision, A.F.; project administration, M.T.-S.; funding acquisition, A.F. and M.T.-S. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Data will be made available upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures and Tables

Figure 1. A Siberian motherwort (L. sibiricus) seedling grown from a seed in a test tube, in in vitro culture (A); in a Petri dish during isolation of single-node explants (B); single-node explants grown in vitro (C).

Figure 2. Effectiveness of chemical sterilization with sodium hypochlorite (NaClO).

Figure 3. The axillary shoots and callus tissue in the cultivation of Siberian motherwort (L. sibiricus) in in vitro cultures (passage III) on MS medium with the addition of BAP and NAA.

Figure 4. The effect of using a water solution of MS salts (25%, v/w) for irrigating the plants of Siberian motherwort (L. sibiricus) during acclimatization to ex vitro.

Figure 5. Siberian motherwort (L. sibiricus) plants during acclimatization to ex vitro.

Figure 6. Siberian motherwort (L. sibiricus) plant obtained as a result of micropropagation after 28 days of acclimatization (A); plant of Siberian motherwort (L. sibiricus) in the flowering phase (B,C).

Table 1

The effect of chemical sterilization of Siberian motherwort (L. sibiricus) seeds with sodium hypochlorite (NaClO) on sterility, seed germination, and seedling development on ½ MS medium without PGR and ½ MS medium with the addition of 1 mg∙dm⁻³ GA₃.

VariantSterilization	SterilizingSubstance	OutputNumber of Seeds (pcs) *	Sprouted Seeds (pcs)		Sterile and Live Seedlings (pcs)		Contaminations(pcs)
			½ MS	½ MS+ 1 GA3	½ MS	½ MS+ 1 GA3	½ MS	½ MS+ 1 GA3
IControl	70% C2H5OH	48 (2 × 24)	0	0	0	0	48	48
II	1.5% NaClO	48 (2 × 24)	8	28	8	26	24	8
III	2.0% NaClO	48 (2 × 24)	10	22	10	14	10	4
IV	2.5% NaClO	48 (2 × 24)	10	26	10	26	10	0

* pcs stands for the number of seeds (samples).

Table 2

The influence of BAP and NAA on the development of axillary shoots and callus tissue in the cultivation of Siberian motherwort (L. sibiricus) in in vitro cultures (passage III).

Plant GrowthRegulator(mg∙dm−3)		Mean Number of Explants Forming Shoots (%)	Mean Number of Explants Forming Callus Tissue (%)	Mean NumberofShoots/Explant	MeanAxillary ShootLength (cm)
BAP	NAA
0.0	0.0	50 (100)	0 (0)	4.47 ± 1.54	3.50 ± 1.76
2.0	0.0	50 (100)	37 (75)	4.35 ± 1.49	2.28 ± 1.28
2.0	1.0	50 (100)	38 (76)	4.07 ± 2.24	2.39 ± 1.31
3.0	0.0	50 (100)	50 (100)	5.22 ± 2.25	1.98 ± 0.78
3.0	1.0	50 (100)	50 (100)	5.48 ± 2.51	2.20 ± 0.97
4.0	0.0	50 (100)	50 (100)	9.37 ± 1.51	1.72 ± 0.65
4.0	1.0	50 (100)	50 (100)	9.62 ± 1.92	1.99 ± 0.96
5.0	0.0	50 (100)	50 (100)	9.19 ± 2.03	1.38 ± 0.48
5.0	1.0	50 (100)	50 (100)	8.93 ± 1.56	1.43 ± 0.38
	LSD0.05	-	-	1.350	0.527

Results are mean ± SD (standard deviation); LSD—the lowest significant difference (Tukey’s confidence half-interval) at p ≤ 0.05; BAP—6-benzylaminopurine (cytokinin), NAA—1-naphthaleneacetic acid (auxin).

Table 3

Root formation in Siberian motherwort (L. sibiricus) on the tested variants of media after 9 weeks of culture.

Medium	Mean Number of Explants	Mean of Explants Forming Roots (%)	Number of Root Bundles (cm)	Mean Number of Rootsper Explant
MS + 0.5 mg∙dm−3 IAA	20	100	6.69 ± 1.35	11.62 ± 3.34
MS + 0 PGR (control)	20	100	6.88 ± 2.15	9.50 ± 2.45
LSD0.05	-	n.s.	n.s.	1.520

Results are mean ± SD (standard deviation); results are mean ± SD (standard deviation); LSD—the lowest significant difference (Tukey’s confidence half-interval) at p ≤ 0.05; n.s.—not significant; IAA—indole-3-acetic acid (auxin); MS—Murashige and Skoog medium (1962).

References

1. Li, J.; Niu, L.; Huang, H.; Li, Q.; Xie, C.; Yang, C. Anti-inflammatory labdane diterpenoids from the aerial parts of Leonurus sibiricus. Phytochemistry; 2024; 217, 113927. [DOI: https://dx.doi.org/10.1016/j.phytochem.2023.113927] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37956887]

2. Zhang, R.H.; Liu, Z.K.; Yang, D.S.; Zhang, X.J.; Sun, H.D.; Xiao, W.L. Phytochemistry and pharmacology of the genus Leonurus: The herb to benefit the mothers and more. Phytochemistry; 2018; 147, pp. 167-183. [DOI: https://dx.doi.org/10.1016/j.phytochem.2017.12.016]

3. Huang, L.; Xu, D.Q.; Chen, Y.Y.; Yue, S.J.; Tang, Y.P. Leonurine, a potential drug for the treatment of cardiovascular system and central nervous system diseases. Brain Behav.; 2021; 11, pp. 1-10. [DOI: https://dx.doi.org/10.1002/brb3.1995] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33300684]

4. Suwatronnakorn, M.; Issaravanich, S.; Palanuvej, C.; Ruangrungsi, N. Standardization of Leonurus sibiricus L. aerial part and capillary electrophoresis quantitative analysis of its leonurine content. J. Adv. Pharm. Technol. Res.; 2021; 12, pp. 291-297. [DOI: https://dx.doi.org/10.4103/japtr.JAPTR_243_21] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34345610]

5. Ciocarlan, N. Some aspects of introduction new medicinal plants in the Botanical Garden of ASM. Conservation of Plant Diversity; Gradina Botanica: Chișinău, Moldova, 2012; pp. 283-290.

6. Wieczfinska, J.; Sitarek, P.; Kowalczyk, T.; Pawliczak, R. Leonurus sibiricus root extracts decrease airway remodeling markers expression in fibroblasts. Clin. Exp. Immunol.; 2020; 202, pp. 28-46. [DOI: https://dx.doi.org/10.1111/cei.13481] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32562256]

7. Lee, M.J.; Lee, H.S.; Park, S.D.; Moon, H.I.; Park, W.H. Leonurus sibiricus Herb Extract Suppresses Oxidative Stress and Ameliorates Hypercholesterolemia in C57BL/6 Mice and TNF-α Induced Expression of Adhesion Molecules and Lectin-Like Oxidized LDL Receptor-1 in Human Umbilical Vein Endothelial Cells. Biosci. Biotechnol. Biochem.; 2010; 74, pp. 279-284. [DOI: https://dx.doi.org/10.1271/bbb.90582] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20139626]

8. Islam, A.K.M.M.; Suttiyut, T.; Anwar, M.P.; Juraimi, A.S.; Kato-Noguchi, H. Allelopathic Properties of Lamiaceae Species: Prospects and Challenges to Use in Agriculture. Plants; 2022; 11, 1478. [DOI: https://dx.doi.org/10.3390/plants11111478] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35684250]

9. Rolim de Almeida, L.F.; Delachiave, M.E.A.; Marques, M.O.M. Composition of the essential oil of rubim (Leonurus sibiricus L.—Lamiaceae). Rev. Bras. Plantas Med.; 2005; 8, pp. 35-38.

10. Rolim de Almeida, L.F.; Delachiave, M.E.; Sannomiya, M.; Vilegas, W.; Campaner dos Santos, L.; Mancini, E.; De Feo, V. In vitro allelopathic potential of Leonurus sibiricus L. leaves. J. Plant Interact.; 2008; 3, pp. 39-48. [DOI: https://dx.doi.org/10.1080/17429140701749906]

11. Sitarek, P.; Rijo, P.; Garcia, C.; Skała, E.; Kalemba, D.; Białas, A.J.; Szemraj, J.; Pytel, D.; Toma, M.; Wysokińska, H. et al. Antibacterial, anti-inflammatory, antioxidant, and antiproliferative properties of essential oils from hairy and normal roots of Leonurus sibiricus L. and their chemical composition. Oxidative Med. Cell. Longev.; 2017; 2017, 7384061. [DOI: https://dx.doi.org/10.1155/2017/7384061]

12. Sayed, M.A.; Imam, R.; Siddiqui, M.N.; Raihanun-Nabi, S.M.; Aktar, S.; Das, S.R. Allelopathic activity of Leonurus sibiricus L. on seed germination and seedling growth of wheat and identification of 4-hydroxybenzoic acid as an allelochemical by chromatography. Pak. J. Bot.; 2016; 48, pp. 1189-1195.

13. Klimont, K.; Bulińska-Radomska, Z.; Górka, J. Możliwość wykorzystania wybranych roślin miododajnych do rekultywacji terenów po eksploatacji siarki. Pol. J. Agron.; 2013; 12, pp. 17-25.

14. Ekiert, H. Farmaceutyczne aspekty biotechnologii roślin. Część I. Wprowadzenie—Metodyka i główne kierunki badawcze. Farm. Pol.; 2009; 65, pp. 69-77.

15. Baby, G.; Rafeekher, M.; Soni, K.B.; Kumari, P.I.; Reshmi, C.R.; Sheena, A.; Rahman, A.M. Advances in Micropropagation Techniques for Aquascaping Plants: A Comprehensive Review. Arch. Curr. Res. Int.; 2024; 24, pp. 14-22. [DOI: https://dx.doi.org/10.9734/acri/2024/v24i11944]

16. Thiem, B.; Kikowska, M. Zapewnienie jakości roślin leczniczych rozmnażanych w kulturach in vitro. Herba Pol.; 2008; 54, pp. 168-178.

17. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant.; 1962; 15, pp. 473-497. [DOI: https://dx.doi.org/10.1111/j.1399-3054.1962.tb08052.x]

18. Martins, M.; Sarmento, D.; Oliveira, M.M. Genetic stability of micropropagated almond plantlets, as assessed by RAPD and ISSR markers. Plant Cell Rep.; 2004; 23, pp. 492-496. [DOI: https://dx.doi.org/10.1007/s00299-004-0870-3] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15372197]

19. Koziara, Z. Porównanie metod odkażania nasion eukaliptusa kłującego (Eucalyptus gunnii Hook.) wysiewanych w kulturach in vitro. Zesz. Probl. Postęp. Nauk Rol.; 2002; 483, pp. 119-124.

20. Luwańska, A.; Wielgus, K.; Szalata, M.; Lipiński, D.; Zeyland, J.; Słomski, R. Optymalizacja warunków regeneracji in vitro szałwii lekarskiej. Biul. Inst. Hod. Aklim. Rośl.; 2013; 270, pp. 133-142. [DOI: https://dx.doi.org/10.37317/biul-2013-0010]

21. Szot, P.; Parzymies, M.; Poniewozik, M. Rozmnażanie storczyków w kulturach tkankowych. Ann. Hortic.; 2015; 25, pp. 33-51.

22. Devi, W.S.; Bengyella, L.; Sharma, G.J. In vitro seed germination and micropropagation of edible bamboo Dendrocalamus giganteus Munro using seeds. Biotechnology; 2012; 11, pp. 74-80. [DOI: https://dx.doi.org/10.3923/biotech.2012.74.80]

23. Khuat, Q.V.; Kalashnikova, E.A.; Kirakosyan, R.N.; Nguyen, H.T.; Baranova, E.N.; Khaliluev, M.R. Improvement of In Vitro Seed Germination and Micropropagation of Amomum tsao-ko (Zingiberaceae Lindl.). Horticulturae; 2022; 8, 640. [DOI: https://dx.doi.org/10.3390/horticulturae8070640]

24. Binenbaum, J.; Weinstain, R.; Shani, E. Gibberellin Localization and Transport in Plants. Trends Plant Sci.; 2018; 23, pp. 410-420. [DOI: https://dx.doi.org/10.1016/j.tplants.2018.02.005] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29530380]

25. Kanwar, J.K.; Kumar, S. In vitro propagation of Gerbera—A review. Hortic. Sci.; 2008; 35, pp. 35-44. [DOI: https://dx.doi.org/10.17221/651-HORTSCI]

26. Assim, M.; Khawar, K.M.; Özcan, S. In vitro micropropagation from shoot meristems of Turkish cowpea (Vigna unguiculata L.) CV. Akkiz. Bangladesh J. Bot.; 2008; 37, pp. 149-154. [DOI: https://dx.doi.org/10.3329/bjb.v37i2.1723]

27. De-Souza, R.; Adams, C.R.; de-Melo, R.C.; Guidolin, A.F.; Michel, A.; Coimbra, J.L.M. Growth Regulators and Their Reflection on Different Hop Genotypes Cultivated under in vitro Conditions. Braz. J. Biol.; 2022; 82, e242596. [DOI: https://dx.doi.org/10.1590/1519-6984.242596]

28. Beyl, C.A. PGRs and their use in micropropagation. Plant Tissue Culture, Development, and Biotechnology; Trigiano, R.N.; Gray, D.J. CRC Press: Boca Raton, FL, USA, Taylor & Francis Group: London, UK, 2011; pp. 33-56.

29. Pasternak, T.P.; Steinmacher, D. Plant Growth Regulation in Cell and Tissue Culture In vitro. Plants; 2024; 13, 327. [DOI: https://dx.doi.org/10.3390/plants13020327]

30. Khajuria, A.K.; Bisht, N.S.; Bhagat, N. In vitro organogenesis and plant regeneration of Thymus serpyllum L.: An important aromatic medicinal plant. Vitr. Cell. Dev. Biol.-Plant; 2020; 56, pp. 652-666. [DOI: https://dx.doi.org/10.1007/s11627-020-10094-9]

31. Pathak, A.R.; Joshi, A.G. Regeneration of Hemidesmus indicus (L.) R. Br. using in vitro nodes: An alternative method for efficient multiplication of shoots. Not. Sci. Biol.; 2021; 13, 10831. [DOI: https://dx.doi.org/10.15835/nsb13210831]

32. Ahmed, M.B.; Salahin, M.; Karim, R.; Razvy, M.A.; Hannan, M.M.; Sultana, R.; Hossain, M.; Islam, R. An efficient method for in vitro clonal propagation of a newly introduced sweetener plant (Stevia rebaudiana Bertoni) in Bangladesh. Am.-Eurasian J. Sci. Res.; 2007; 2, pp. 121-125.

33. Doliński, R.; Jabłońska, E. Mikrorozmnażanie stewii (Stevia rebaudiana Bert.) z eksplantatów węzłowych izolowanych z roślin wytworzonych in vitro. Agron. Sci.; 2015; 70, pp. 13-24. [DOI: https://dx.doi.org/10.24326/as.2015.4.2]

34. Kulus, D. Zastosowanie kultur tkankowych w mikrorozmnażaniu chryzantem. Nauka Niejedno Ma Imię; Flizikowski, J. Wyd. UTP: Bydgoszcz, Poland, 2014; pp. 63-72.

35. Kharel, P.; Creech, M.R.; Nguyen, C.D.; Vendrame, W.A.; Munoz, P.R.; Huo, H. Effect of explant type, culture medium, and BAP concentration on in vitro shoot development in highbush blueberry (Vaccinium corymbosum L.) cultivars. Vitr. Cell. Dev. Biol.-Plant; 2022; 58, pp. 1057-1065. [DOI: https://dx.doi.org/10.1007/s11627-022-10299-0]

36. Lobna, S.; Taha, M.M.; Ibrahim, S.; Farahat, M.M. A Micropropagation Protocol of Paulownia kowakamii through in vitro culture technique. Aust. J. Basic Appl. Sci.; 2008; 2, pp. 594-600.

37. Kim, S.H.; Zebro, M.; Jang, D.C.; Sim, J.E.; Park, H.K.; Kim, K.Y.; Bae, H.M.; Tilahun, S.; Park, S.M. Optimization of Plant Growth Regulators for In Vitro Mass Propagation of a Disease-Free ‘Shine Muscat’ Grapevine Cultivar. Curr. Issues Mol. Biol.; 2023; 45, pp. 7721-7733. [DOI: https://dx.doi.org/10.3390/cimb45100487]

38. Kurepa, J.; Smalle, J.A. Auxin/Cytokinin Antagonistic Control of the Shoot/Root Growth Ratio and Its Relevance for Adaptation to Drought and Nutrient Deficiency Stresses. Int. J. Mol. Sci.; 2022; 23, 1933. [DOI: https://dx.doi.org/10.3390/ijms23041933]

39. Sato, M.; Hosokawa, M.; Doi, M. Somaclonal Variation Is Induced de Novo via the Tissue Culture Process: A Study Quantifying Mutated Cells in Saintpaulia. PLoS ONE; 2011; 6, e23541. [DOI: https://dx.doi.org/10.1371/journal.pone.0023541]

40. Abdulla, A.E.; Malki, H.; Elmeer, K. Influence of auxin and cytokinine on in vitro multiplication of Ficus anastasia. Afr. J. Biotechnol.; 2010; 9, pp. 635-639.

41. Bertsouklis, K.F.; Papafotiou, M. In vitro propagation of Arbutus andrachne L. Acta Hortic.; 2009; 813, pp. 477-480. [DOI: https://dx.doi.org/10.17660/ActaHortic.2009.813.63]

42. Iacuzzi, N.; Salamone, F.; Farruggia, D.; Tortorici, N.; Vultaggio, L.; Tuttolomondo, T. Development of a New Micropropagation Protocol and Transfer of In vitro Plants to In Vivo Conditions for Cascade Hop. Plants; 2023; 12, 2877. [DOI: https://dx.doi.org/10.3390/plants12152877] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37571031]

43. Alvarenga, I.C.A.; Pacheco, F.V.; Silva, S.T.; Bertolucci, S.K.V.; Pinto, J.E.B.P. In vitro culture of Achillea millefolium L. Quality and intensity of light on growth and production of volatiles. Plant Cell Tissue Organ Cult.; 2015; 122, pp. 299-308. [DOI: https://dx.doi.org/10.1007/s11240-015-0766-7]

44. Clapa, D.; Hârța, M. Establishment of an Efficient Micropropagation System for Humulus lupulus L. cv. Cascade and Confirmation of Genetic Uniformity of the Regenerated Plants through DNA Markers. Agronomy; 2021; 11, 2268. [DOI: https://dx.doi.org/10.3390/agronomy11112268]

45. Ferreira, M.d.S.; Rocha, A.d.J.; Nascimento, F.d.S.; Oliveira, W.D.d.S.; Soares, J.M.d.S.; Rebouças, T.A.; Morais Lino, L.S.; Haddad, F.; Ferreira, C.F.; Santos-Serejo, J.A.d. et al. The Role of Somaclonal Variation in Plant Genetic Improvement: A Systematic Review. Agronomy; 2023; 13, 730. [DOI: https://dx.doi.org/10.3390/agronomy13030730]

46. Laribi, B.; Rouatbi, N.; Kouki, K.; Bettaieb, T. In vitro propagation of Stevia rebaudiana [Bert.]—A non caloric sweetener and antidiabetic medicinal plant. Int. J. Med. Arom. Plants; 2012; 2, pp. 333-339.

47. Kamińska, M.; Sliwinska, E. Effective micropropagation of kale (Brassica oleracea convar. Acephala var. sabellica): One of the most important representatives of cruciferous crops. Plant Cell Tissue Organ Cult.; 2023; 153, pp. 601-609. [DOI: https://dx.doi.org/10.1007/s11240-023-02497-4]

48. Fatima, B.; Usman, M.; Ashraf, T.; Waseem, R.; Ali, M.A. In vitro shoot regeneration from cotyledon and hypocotyl explants of Dahlia cultivars. Pak. J. Agric. Sci.; 2007; 44, pp. 312-316.

49. Figas, A.; Tomaszewska-Sowa, M.; Sawilska, A.; Keutgen, A.J. Improvement of in vitro propagation and acclimation of Helichrysum arenarium L. Moench. Acta Sci. Pol. Hortorum Cultus; 2016; 15, pp. 17-26.

50. Al-Amin, M.D.; Karim, M.R.; Amin, M.R.; Rahman, S.M.A.N.; Mamun, A.N.M. In vitro micropropagation of banana. Bangladesh J. Agric. Res.; 2009; 34, pp. 645-659. [DOI: https://dx.doi.org/10.3329/bjar.v34i4.5840]

51. Sawilska, A.; Figas, A. Micropropagation of Helichrysum arenarium (L.) Moench. Proceedings of the International Conference Biotechnology; Česke Budějovice, Czech Republic, 15–16 February 2006; Scientific Pedagogical Publishing: New York, NY, USA, 2006; pp. 721-723.

52. Tomaszewska-Sowa, M.; Figas, A. Optimization of the processes of sterilization and micropropagation of cup plant (Silphium perfoliatum L.) from apical explants of seedlings in in vitro cultures. Acta Agrobot; 2011; 64, pp. 3-10. [DOI: https://dx.doi.org/10.5586/aa.2011.041]

53. Anbazhagan, M.; Kalpana, M.; Rajendran, R.; Natarajan, V.; Dhanavel, D. In vitro production of Stevia rebaudiana Bertoni. Emir. J. Food Agric.; 2010; 22, pp. 216-222. [DOI: https://dx.doi.org/10.9755/ejfa.v22i3.4891]

54. Jitendra, M.; Monika, S.; Ratan, S.D.; Priyanka, G.; Priyanka, S.; Kiran, D.J. Micropropagation of an Anti diabetic Plant—Stevia rebaudiana Bertoni, (Natural Sweetener) in Hadoti Region of South-East Rajasthan, India. Int. J. Biol. Sci.; 2012; 1, pp. 37-42.

55. Tomaszewska-Sowa, M.; Figas, A.; Sawilska, A.K. A non caloric sweetener Stevia rebaudiana Bertoni-tissue cultured plantlets for organic farming and home gardening. Infrastruct. Ecol. Rural. Areas; 2015; 4, pp. 1261-1273. [DOI: https://dx.doi.org/10.14597/infraeco.2015.4.3.091]