Introduction

Phytotherapy is becoming increasingly popular around the world and is a major area of natural medicine. According to reports from the World Health Organization approximately 80% of people in the world use natural methods to complement conventional treatment (Lachowicz and Oszmiański 2019). Moreover, the demand for substances of natural origin that have a wide spectrum of biological activity is still growing (Krychowiak et al. 2014). Therefore, plants that can synthesize potent secondary metabolites are of interest to science and industry.

Reynoutria japonica Houtt. (syn. Fallopia japonica (Houtt.) Ronse Decr.; Polygonum cuspidatum Sieb. & Zucc.) called Japanese knotweed is a plant belonging to the family Polygonaceae. This herbaceous perennial geophyte is native to eastern Asia (Stachowicz et al. 2021). R. japonica is naturally rich in many secondary metabolites belonging to the group of phenolic compounds, like stilbenes, quinones, lignans, flavonoids, coumarins or phenolic acids, and it was used in natural Asian medicine for over 1800 years (Bozin et al. 2017). These metabolites are synthesized in plants as a part of their defense system in response to environmental factors (Labudda et al. 2020a; Muszyńska et al. 2021). On the other hand, because of their properties, they also belong to substances used in medicine (Makowski et al. 2020).

R. japonica is included in Chinese Pharmacopoeia, where it is believed to treat nervous system disorders, bronchitis, high blood pressure, and jaundice (Wang et al. 2022b). It has been proved, that Japanese knotweed has anticancer (Zhang et al. 2019; Kim et al. 2019) and antioxidant activity (Bozin et al. 2017), as well as may be applied in the prevention of the circulatory and digestive systems diseases (Liu et al. 2019), and in respiratory infections (Wang et al. 2022b). It is considered that the rhizome and roots of Japanese knotweed are the richest in phenolic compounds with a broad range of biological activity (Lachowicz and Oszmiański 2019). Therefore, the majority of available research is focused on phytochemistry and biological activity of underground parts of R. japonica– rhizome and roots (Bozin et al. 2017). Moreover, until now, the only research available on Japanese knotweed was performed with the use of plants grown in their natural environment (collected from uncontrolled conditions). Thus, the content of biologically active secondary metabolites in plants may be biased because of different collection locations. Therefore, the application of controlled conditions during the cultivation of Japanese knotweed seems to be a reasonable solution for obtaining plant material with a repeatable phenotype.

Japanese knotweed was introduced in 1825 to Europe and after, in the late nineteenth century, to North America as an ornamental plant, as well as a fodder and erosion control plant. Over time, it has become one of the most invasive plants on these continents (Zubek et al. 2022). Today, R. japonica, with two other representatives of this botanical genus, Reynoutria sachalinensis and Reynoutria x bohemica, is included in the top 100 world’s most dangerous invasive alien species being a huge threat to native ecosystems (Vidican et al. 2023). This plant can rapidly colonize various environments and predominantly grows over secondary anthropogenic habitats and areas polluted by industry, but less frequently in natural niches, like forest edges or river valleys. The invasion strategy of Japanese knotweed in various habitats is related to its ability to synthesize a large number of biologically active compounds that influence the behavior and/or composition of soil microbial communities, as well as allelopathic compounds synthesis (Stefanowicz et al. 2017, 2021; Zubek et al. 2022; Soln et al. 2022).

As far as Japanese knotweed appears to be a promising source of secondary metabolites with potential application in the pharmaceutical industry its acquisition from natural sites is impossible. R. japonica is found in polluted habitats, often contaminated with heavy metals and other toxic elements (Vidican et al. 2023). Lerch et al. (2022) showed the ability of R. japonica to accumulate Cd, Cu, Zn, and Ni and to distribute those elements from the roots to the stem and leaves. Rahmonov et al. (2014) reported the presence of Pb and Cr in the tissue of Japanese knotweed collected from various sites. Valorization of plant biomass for industrial purposes harvested from the places of Japanese knotweed natural occurrence threatens its pollution with toxic elements (Lerch et al. 2022).

Considering that the content of secondary metabolites in the tissues of wild plants depends on variable environmental factors, and at the same time wild plants can accumulate substances harmful to health, plant material from natural sites is not suitable for industrial use. Thus, it seems necessary to make its acquisition independent of populations occurring in the natural environment. Derivation of Japanese knotweed tissue cultures will, therefore, allow for cultivation in strictly controlled conditions, where the obtained plants will accumulate valuable secondary compounds at a constant level. Moreover, the introduction of knotweed to in vitro cultivation will allow the future use of biotechnological tools, such as elicitation or genetic transformation, aimed at increasing the content of phenolic compounds in its tissues. Therefore, in this study, for the first time, controlled in vitro conditions were used for the cultivation of R. japonica plants.

Plant tissue culture technology gives the possibility to produce valuable and standardized plant material. Plants grown in in vitro conditions are free from contaminants that occur in the natural environment and have an even phenotype (Grzegorczyk-Karolak et al. 2024). Many authors reported effective plant biomass production by micropropagation (Grzegorczyk-Karolak et al. 2019; Kunakhonnuruk et al. 2019; Makowski et al. 2023). Depending on the plant genus and the aim of cultivation various modifications of traditional agar-solidified medium for tissue cultures may be used. Cultivation of plants in liquid media with rotary shaking (agitated cultures) or in temporary immersion bioreactors may lead to increased biomass and/or secondary metabolite production (Kikowska et al. 2020; Makowski et al. 2023). Such platforms are efficient for scaling up the production of plant-derived compounds. Therefore, developing Japanese knotweed cultivation methods in in vitro conditions can provide pollution-free plants, rich in secondary metabolites of a medicinal nature. We hypothesized, that due to better distribution and availability of nutrients in agitated cultures and in bioreactors, plant biomass accumulation may increase as compared to culture in agar-solidified medium. Also, mechanical stimulation of agitated culture, as well as periodic flooding in bioreactors may induce stress reactions in plants, which in turn results in increased levels of phenolic compound production in R. japonica tissue.

The aim of this study was to: (1) introduce, for the first time, R. japonica into in vitro culture conditions, (2) evaluate its growth and secondary metabolites production in agar-solidified medium, agitated cultures and in temporary immersion bioreactor Plantform™, compared to plants cultivated in soil (ex vitro conditions). The research will enable the selection of the best conditions for growing knotweed in vitro to obtain secondary metabolites.

Materials and methods

Plant material for tissue culture initiation

The primary explants for the R. japonica tissue culture initiation were side buds located on knotweed rhizomes. Buds from the intact plants were collected in March 2021 in Kraków, Poland, occurred on the banks of the Rudawa River (50°03’35.5"N, 19°54’03.7"E). Buds were used as explants to establish and stabilize shoot cultures in in vitro conditions.

Shoot culture initiation

The explants were surface disinfected according to the method described by Makowski et al. (2023), with modifications. Briefly, buds were washed in tap water with Tween 20 (Sigma Aldrich, Germany). Then, explants were treated with 70% (v/v) ethanol for 30 s. In the next step, buds were surface sterilized with 5.25% calcium hypochlorite for 20 min. Next, sterile explants were rinsed three times in sterile distilled water and placed in 250 mL flasks with 50 mL of ½ MS medium solidified with agar (0.8%) (Murashige and Skoog 1962). The medium had no growth regulators and contained 3% of sucrose. pH of medium was 5.8, adjusted prior autoclaving. Flasks with initial buds were placed in the growing room at 21 ± 2 °C, under white fluorescence light characterized by a photosynthetic photon flux density (PPFD) of 50 µmol×m-2×s-1 and a photoperiod of 16 h/8 h light/dark cycle. After 3–4 weeks newly grown shoots of R. japonica started to appear on initial buds. Shoots were cut off and subcultured to fresh medium with the same composition as described above. Shoots rooted spontaneously without addition of plant growth regulators. Plants were subcultured every 4–5 weeks and served as an initial material for the further experiments.

In vitro and ex vitro cultivation of R. japonica

Plants in each applied system: in solid medium culture, agitated culture, temporary immersion bioreactors and in ex vitro conditions were cultivated at 21 ± 2 °C under white fluorescence light with a PPFD of 100 µmol×m-2×s-1 and a photoperiod of 16 h/8 h light/dark cycle.

Cultures in solid medium (SM)

In this experiment two-leaf plants from previously obtained cultures were subcultured to 250 mL Erlenmeyer flasks (5 explants per flask) with 50 mL of ½ MS medium solidified with 0.8% agar, without growth regulators, containing 3% sucrose, with pH 5.8 (adjusted prior to autoclaving). Explants were weighted and measured. After 5 weeks of cultivation plants were collected for further analysis. The experiment consisted of 15 flasks (n = 15).

Cultures in liquid medium with rotary shaking– agitated cultures (AC)

For AC of R. japonica initiation, two-leaf plant explants placed in 250 mL Erlenmeyer flasks (5 plants per flask) with 50 mL of liquid ½ MS medium, without growth regulators, containing 3% sucrose, with pH 5.8 (adjusted prior to autoclaving). Explants were weighted and measured. Cultures were placed on a rotary shaker (Phoenix RS-LS 20, DanLab, Poland) with continuous mode at 140 rpm. After 5 weeks plants were collected for further analysis. The experiment consisted of 15 flasks (n = 15).

Cultures in temporary immersion bioreactors (TIB)

TIB cultures were grown in a Plantform™ temporary immersion system (Plant Form, Sweden). 15 explants from previously obtained starter cultures were transferred to each bioreactor. One bioreactor contained 500 mL of liquid ½ MS medium, without growth regulators, containing 3% sucrose, with pH 5.8 (adjusted prior to autoclaving). Explants were weighted and measured. The immersion and aeration period in the bioreactors was programmed in the following cycle: 30 min of immersion, 20 min of gravitational fall of the medium, and 10 min of aeration. After 5 weeks of cultivation plants were harvested for further analysis. The experiment consisted of 5 bioreactors (n = 5).

Plants cultivated in ex vitro conditions (ex vitro)

For this experiment, 4 weeks-old, rooted and healthy plants obtained from in vitro cultures from solid medium were transferred to the pots with peat and perlite (3:1) for the acclimatization to ex vitro conditions (one plant in one pot). Plants were weighted and measured before transfer. Pots with R. japonica were placed in growing chamber with initial humidity 90%. The humidity was decreased 10% each week to final humidity 50%. After 4 weeks plants were fully acclimatized to ex vitro conditions. The experiment consisted of 20 pots with plants (n = 20).

Preparation of plant material for biochemical analysis

After the experiment with different cultivation systems plants were harvested, and fresh biomass of shoots and roots (separately) was weighted separately from each flask/bioreactor/pot. Also, photo documentation and the observation of plants’ health status and plants’ morphology was performed. Plants were packed in paper envelopes and freeze-dried for 24 h. Dry shoots and roots from each experimental conditions were weighed and homogenized to the powder. Plant’s material was stored in plastic falcons in -20 °C.

Shoot length and multiplication

To estimate shoot length shoots of plants from each experimental conditions were measured with ruler. Also, the number of shoots grown on one explant in in vitro conditions and one acclimatized plant to ex vitro conditions were calculated.

Growth index (GI) and dry weight (DW) estimation

Estimation of GI was performed according to Tokarz et al. (2018). The GI was calculated according to the following formula: GI [%] = (FW2 - FW1)/FW2 × 100, where FW1 is the fresh weight of the plants at the beginning of the experiment and FW2 is the final fresh weight. Determination of DW content in shoots and roots of R. japonica plants from each experimental condition was performed according Tokarz et al. (2020). The DW content in the plant tissue was calculated according to the following formula: DW [%] = DW2 × 100/FW2, where DW2 is the dry weight after freeze-drying.

Determination of phenolic compounds presence in R. japonica tissue

Extraction procedure

Plant extracts were prepared according to the procedure described by Makowski et al. (2021b), with modifications. For the preparation of methanolic extracts, 100 mg of homogenized lyophilized tissue powder were weighed separately for shoots and roots, from each tested cultivation system. The tissue samples were subjected twice to extraction with 6 mL of 80% methanol (HPLC-grade) by sonication in an ultrasonic bath (POLSONIC 2, Poland) for 30 min, 21 ± 2 °C. Sample for each research object (shoots/roots from each applied cultivation systems) was prepared in 5 biological repetitions (n = 5). The obtained extracts were centrifuged (10000 RPM), and then filtered through sterilizing syringe filters (0.22 μm, Millex®GP, Millipore) prior to HPLC analysis to the 1,5 mL vials.

Phytochemical analysis

To estimate the accumulation of phenolic compounds in plant tissue high-pressure liquid chromatography with a diode array detector (DAD-HPLC) was used. The quantitative analyses of selected secondary metabolites in the extracts were conducted with a validated method, using an apparatus of Merck-Hitachi (LaChrom Elite) with a DAD L-2455 detector and on a Purospher RP-18 (250 × 4 mm; 5 μm, Merck, Germany) column according to former established and validated method according to Sułkowska-Ziaja et al. 2017 and Szopa et al. 2020. The flow rate was 1 mL×min− 1, the temperature was set to 25 °C, and the injection volume was 10 µL. The detection wavelength was set to 254 nm. The mobile phase consisted of A—methanol, 0.5% acetic acid in ratio 1:4, and B—methanol (v/v). The gradient program was as follows: 0–20 min, 0% B; 20–35 min, 0–20% B; 35–45 min, 20–30% B; 45–55 min, 30–40% B; 55–60 min, 40–50% B; 60–65 min, 50–75% B; and 65–70 min, 75–100% B, with a hold time of 15 min. Identification was performed by comparison to the retention times and UV spectra of phenolic compounds: caftaric acid (y = 1E + 08x-13409; R2 = 0.9903; RT = 5.52 min), protocatechic acid (y = 1E + 078x + 159880; R2 = 0.9991; RT = 6.98 min), chlorogenic acid (y = 4E + 07x-45223; R2 = 0.9997; RT = 11.67 min), caffeic acid (y = 6E + 07x-169512; R2 = 0.9996; RT = 16.96 min), ferulic acid (y = 6E + 07x + 30835; R2 = 0.9983; RT = 36.91 min), epigallocatechin (y = 3E + 06x-30719; R2 = 0,9999; RT = 7.53 min), catechin (y = 6E + 06x + 54243; R2 = 0.9991; RT = 8.51 min), epicatechin gallate (y = 3E + 07x-148672; R2 = 1.0; RT = 15.43 min), epicatechin (y = 6E + 06x-48481; R2 = 0.9994; RT = 19.03 min), isoquercetin (y = 9E + 07x + 1113000; R2 = 0.9999; RT = 45.42 min), trifolin (y = 8E + 07x + 583711; R2 = 0.9994; RT = 49.25 min) avicularin (y = 1E + 08x-1E + 09; R2 = 0.9995; RT = 49.40 min), quercitrin (y = 7E + 07x-813339; R2 = 0.9979; RT = 49.90 min), and apigenin (y = 6E + 07x-1E + 06; R2 = 0.9999; RT = 64.65 min) (acquired from Sigma-Aldrich Co., Germany). The above-mentioned compounds were presented in the chromatogram of the extract from R. japonica shoots cultivated in vitro (Fig. 1). The quantification was performed based on the calibration curves method. Samples were prepared and analyzed in five replications. The results were expressed in mg × 100 g− 1 DW.

Fig. 1
figure 1

Example DAD-HPLC (λ = 254 nm) chromatogram of extract from Reynoutria japonica shoots cultivated in in vitro conditions. Identified compounds: 1 - caftaric acid, 2 - protocatechic acid, 3 - epigallocatechin, 4 - catechin, 5 - chlorogenic acid, 6 - epicatechin gallate, 7 - caffeic acid, 8 - epicatechin, 9 - ferulic acid, 10 - isoquercetin, 11 - trifolin, 12 - avicularin, 13 - quercitrin, 14– apigenin

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Sum of phenolic compounds (SPC) calculation

SPC was calculated by summing up the content of all tested phenolic derivatives: protocatechic acid, chlorogenic acid, caffeic acid, ferulic acid, caftaric acid, catechin, epigallocatechin, epicatechin gallate, epicatechin, isoquercetin, quercetin, avicularin, trifolin and apigenin. Results were expressed as mg × 100 g− 1 DW.

Statistical analysis

A one-way analysis of variance (ANOVA) was performed to determine significant differences between means during statistic elaboration of growth index of plants. Two-way analysis of variance (ANOVA) was performed to determine significant differences between means during the statistical elaboration of the rest presented parameters: dry weight, the sum of phenolic compounds, accumulation of phenolic derivatives, as well as the sum of phenolic acids, the sum of catechin derivatives and the sum of flavonoids (Tukey post-hoc test at p < 0.05). STATISTICA 12.0 (StatSoft Inc., Tulsa, OK, USA) was used to conduct statistical analyses. The principal component analysis (PCA) was performed with the use of Past software (Hammer and Harper 2001) to show global differences and/or similarities between culture types and between plant parts (shoots and roots) based on the sum of phenolic acids, catechin derivatives, and flavonoids. Before PCA analysis, the data were normalized by calculating their log10 values.

Results

Biometric parameters and plant morphology

An important parameter in the evaluation of plant viability cultivated in liquid media is the lack of abnormal morphology and hyperhydricity (Kikowska et al. 2020). In this study, after 5 weeks of R. japonica cultivation in all tested conditions (SM, AC, TIB, ex vitro), the plants showed no signs of stress or hyperhydricity (Figs. 2, 3 and 4). Furthermore, plants harvested from ex vitro conditions (Fig. 4) and temporary immersion bioreactors (Fig. 3) had bigger leaf area and shape than those from SM and AC (Fig. 2).

Fig. 2
figure 2

Reynoutria japonica plants after 5 weeks of cultivation in solid (left) and agitated (right) culture (A, B). Scale bar = 1 cm

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Fig. 3
figure 3

Reynoutria japonica plants after 5 weeks of cultivation in temporary immersion bioreactor Plantform™ (A, B). Scale bar = 1 cm

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Fig. 4
figure 4

Reynoutria japonica plants after 5 weeks of cultivation in soil (ex vitro) (A, B). Scale bar = 1 cm

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Moreover, in TIB and ex vitro conditions length of shoots was larger than in SM or AC (Table 1). TIB conditions led to the highest shoot multiplication, having the biggest number of shoots per one explant (Table 1).

Table 1 Shoot length [cm] and number of shoots per explant of Reynoutria japonica in: solid culture, agitated culture, temporary immersion bioreactor Plantform™ and soil (ex vitro). Different letters indicate statistical significance of means acc. Two-way ANOVA, post hoc Tuckey test at p < 0.05, SD—standard deviation. Statistical analysis performed for each of the tested parameters separately
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The obtained results demonstrated that the growth index of plants cultivated in AC, TIB, and ex vitro conditions increased significantly 1.5-, 1.8- and 2.7-fold as compared to SM, respectively (Fig. 5).

Fig.5
figure 5

Growth index [%] in Reynoutria japonica plants after 5 weeks of cultivation in: solid medium (SM), agitated culture (AC), temporary immersion bioreactor Plantform™ (TIB) and soil (Ex vitro). Upper case letters indicate statistical significance of means acc. one-way ANOVA, post hoc Tuckey test at p < 0.05; the bar represents the standard deviation

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In presented research DW was measured separately for shoots and roots of R. japonica from each experimental condition. The obtained results showed that shoots from AC, as well as shoots and roots from ex vitro conditions had increased dry weight content by 1.4; 1.7; 1.5-fold, respectively, as compared to SM (Fig. 6).

Fig. 6
figure 6

Dry weight [%] content in Reynoutria japonica shoot and root after 5 weeks of cultivation in: solid medium (SM), agitated culture (AC), temporary immersion bioreactor Plantform™ (TIB) and soil (Ex vitro). Upper case letters indicate statistical significance of means acc. one-way ANOVA, post hoc Tuckey test at p < 0.05; the bar represents the standard deviation

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Sum of phenolic compounds

In the current study the concentration of total phenolic compounds content in in vitro and ex vitro propagated R. japonica plants was evaluated and expressed as a sum of phenolic compounds (SPC) estimated with the use of DAD-HPLC method (Fig. 7). The highest SPC was obtained for shoots from AC (3188.1 mg × 100 g− 1 DW). Shoots from TIB had significantly lower SPC than shoots from AC, with a value of 2638.0 mg × 100 g− 1 DW. Interestingly, a similar amount of SPC was found in shoots of plants cultivated in SM and in ex vitro conditions as well as roots obtained in AC culture (2312.4, 2149.3, and 2123.4 mg × 100 g− 1 DW, respectively). The lowest SPC was reported for roots of R. japonica cultivated in ex vitro conditions (785.70 mg × 100 g− 1 DW). The level of phenolic compounds in the roots grown on SM (1830.5 mg × 100 g− 1 DW) was higher by 16% than in the roots of plants grown in TIB (1569.3 mg × 100 g− 1 DW) (Fig. 7).

Fig. 7
figure 7

Sum of phenolic compounds [mg × 100 g− 1 DW] in Reynoutria japonica shoot and root after 5 weeks of cultivation in: solid medium (SM), agitated culture (AC), temporary immersion bioreactor Plantform™ (TIB) and soil (Ex vitro). Upper case letters indicate statistical significance of means acc. one-way ANOVA, post hoc Tuckey test at p < 0.05; the bar represents the standard deviation; DW– dry weight

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The variable presence of phenolic acids, catechins and other flavonoids in R. japonica in vitro cultures

Using the DAD-HPLC method qualitative and quantitative analysis of extracts from Japanese knotweed plants was performed. In the analyzed samples from in vitro conditions, the same qualitative composition of phenolic derivatives was found, as in the plants cultivated in soil. The presence of 5 phenolic acids: ferulic, caftaric, chlorogenic, protocatechic and caffeic acid was confirmed (Table 2). Moreover, the presence of 9 flavonoids, including 4 catechines: catechin, epigallocatechin, epicatechin gallate and epicatechin (Table 3), as well as other flavonoids: isoquercetin, quercetin, avicularin, trifolin and apigenin was determined (Table 4).

Table 2 Accumulation of phenolic acids [mg × 100 g-1 DW] in Reynoutria japonica shoot and root cultivated in: solid culture (SM), agitated culture (AC), temporary immersion bioreactor Plantform™ (TIB) and soil (ex vitro). Different letters indicate statistical significance of means acc. Two-way ANOVA, post hoc Tuckey test at p < 0.05, SD—standard deviation, DW—dry weight. Statistical analysis performed for each of the tested parameters (the content of a given compound and the sum) separately
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Table 3 Accumulation of catechin derivatives [mg × 100 g-1 DW] in Reynoutria japonica shoot and root cultivated in: solid culture (SM), agitated culture (AC), temporary immersion bioreactor Plantform™ (TIB) and soil (ex vitro). Different letters indicate statistical significance of means acc. Two-way ANOVA, post hoc Tuckey test at p < 0.05, SD—standard deviation, DW—dry weight. Statistical analysis performed for each of the tested parameters (the content of a given compound and the sum) separately
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Table 4 Accumulation of flavonoids [mg × 100 g-1 DW] in shoot and root of Reynoutria japonica cultivated in: solid culture (SM), agitated culture (AC), temporary immersion bioreactor Plantform™ (TIB) and soil (ex vitro). Different letters indicate statistical significance of means acc. Two-way ANOVA, post hoc Tuckey test at p < 0.05, SD—standard deviation, DW—dry weight. Statistical analysis performed for each of the tested parameters (the content of a given compound and the sum) separately
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Shoots produce more phenolic acids than roots, while the highest sum of phenolic acids was obtained in shoots from ex vitro plants (1135.8 mg × 100 g− 1 DW) (Table 2). Among all compounds from this group the dominant in shoots and roots (excluding roots of plants in soil) was caftaric acid. AC and TIB increased accumulation of ferulic acid in shoots, with the concentration 66.8 and 70.0 mg × 100 g− 1 DW, respectively. Interestingly, the highest level of ferulic acid was obtained for roots from ex vitro conditions (146.4 mg × 100 g− 1 DW). Production of chlorogenic acid reached the highest level in shoots from ex vitro conditions (93.1 mg × 100 g− 1 DW) and roots from SM (92.7 mg × 100 g− 1 DW). Protocatechic and caffeic acids were accumulated more efficiently in roots, than in shoots. SM was the most effective platform for protocatechic acid accumulation in roots, with concentration of 14.3 mg × 100 g− 1 DW, while 35.4 mg × 100 g− 1 DW of caffeic acid was found in roots from TIB (Table 2).

The highest sum of catechin derivatives was obtained in shoots from AC (2054.3 mg × 100 g− 1 DW), and then for the roots from the same type of culture (1744.7 mg × 100 g− 1 DW). Moreover, AC plants were characterized by high content of catechin, epigallocatechin and epicatechin in shoots: 647.6, 729.5 and 523.2 mg × 100 g− 1 DW, respectively. In roots, 670.0 mg × 100 g− 1 DW of catechin were accumulated in AC, while 387.9 and 375.1 mg × 100 g− 1 DW of epigallocatechin from SM and AC, respectively. Epicatechin gallate was found at the highest level in roots for AC (216.2 mg × 100 g− 1 DW) and TIB (224.8 mg × 100 g− 1 DW) and epicatechin in roots from AC (483.3 mg × 100 g− 1 DW) (Table 3).

In vitro conditions: SM, AC and TIB led to an increased sum of flavonoids in shoots (425.0, 465.7 and 497.2 mg × 100 g− 1 DW, respectively), compared to ex vitro plants. The highest concentration of flavonoids in roots was obtained from AC and TIB (172.0 and 100.0 mg × 100 g− 1 DW). SM and AC conditions promoted synthesis of isoquercetin in shoots, while AC and TIB in roots. The highest quercetin level (269.2 mg × 100 g− 1 DW) was found in shoots from TIB. The highest avicularin yield was observed in shoots from AC and TIB (16.5 and 15.2 mg × 100 g− 1 DW), trifolin in shoots from AC (17.1 mg × 100 g− 1 DW), and apigenin from SM, AC and TIB (173.5, 175.4, 176.0 mg × 100 g− 1 DW) (Table 4).

Variable influence of cultivation systems on R. japonica secondary metabolites production

To verify the general patterns of phenolic compounds distribution in R. japonica roots and shoots cultivated in different conditions (SM, AC, TIB, ex vitro) a principal component analysis (PCA) was performed (Fig. 8). By combining the data of sums of phenolic acids, sums of catechin derivatives and sums of other flavonoids (8 objects x 3 variables), as much as 80.3% (PC1) and 18.14% (PC2) of the data variability among plant parts (shoots vs. roots) and growing conditions could be explained. Moreover, the variances of the variables describing the content of other flavonoids and phenolic acids were mainly explored by PC1, while the variabilities of catechin derivatives content was mainly explained by the PC2. The analysis clearly discriminates shoots and roots of R. japonica. Moreover, the content of phenolic compounds is more similar between different in vitro cultivation methods (SM, AC, TIB) than in soil (ex vitro) (Fig. 8). Interestingly, the roots’ phenolic compounds content from SM and TIB is almost identical, contrary to shoots from the same conditions. This may suggest that roots were exposed to similar stress conditions in SM and TIB, while for shoots it was different. It seems that the content of phenolic compounds was affected the most by AC growing conditions, which may be considered the most stressful for the plant (constant shaking, oxygen limitation). Indeed, in this condition, the production of phenolic compounds was the largest both among tested shoots and roots (Fig. 7). Since the growth index and dry weight of the plants from AC conditions were like TIB conditions (Figs. 5 and 6), it can be considered that growing R. japonica plants in liquid culture with rotary shaking is the most promising when looking at the number of secondary metabolites it produces.

Fig. 8
figure 8

Comparison of plant cultivation method effect on the content of phenolic acids, catechin derivatives and other flavonoids in roots (black dots) and shoots (grey squares) as measured with principal component analysis. Solid medium (SM), agitated culture (AC), temporary immersion bioreactor Plantform™ (TIB) and soil (Ex vitro)

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Discussion

Invasive plant species are one of the most important problems in environment protection and preservation of the natural habitats. On the other hand, many of invasive plant species have unique adaptational strategies, often based on the ability to synthesize biologically active secondary metabolites in their tissue (Stefanowicz et al. 2021). Therefore, they can be a source of valuable compounds with medical potential. This is why plant biotechnology looks up for novel strategies to propagate invasive species in separation from the natural environment, in stable, controlled conditions to produce plant biomass rich in plant-derived chemicals. To the best of our knowledge this is the first report about application of micropropagation in R. japonica plants cultivation. The present work focuses on biomass accumulation and synthesis of secondary metabolites from the group of phenolic acids, catechins, and other flavonoids in Japanese knotweed tissue harvested from different cultivation modes, including cultures in solid medium (SM), agitated cultures (AC), temporary immersion bioreactor (TIB) and plants cultivated in soil (ex vitro conditions). Creation of R. japonica tissue culture is also the first necessary step to enable further research that will aim to increase the synthesis of the secondary metabolites in its tissues with the use of biotechnological tools.

Application of liquid medium and shaking technology, as well as bioreactors as the modifications of traditional agar-solidified, stationary plant cultures may be a chance for increasing the yield in medical plant cultivation (Makowski et al. 2020; De Carlo et al. 2021; Hwang et al. 2022). Many authors reported possible changes in growth effectivity and dry weight accumulation in plants depending on the method of cultivation in in vitro conditions (Moreira et al. 2013; Malik et al. 2016; Kunakhonnuruk et al. 2019; Szopa et al. 2019). Tissue cultures in liquid medium with rotary shaking were reported as an effective mode of cultivation for plant biomass production for Dionaea muscipula, Hypericum performatum and Knautia sarajevensis (Makowski et al. 2020; Kwiecień et al. 2015; Karalija et al. 2017). Nevertheless, some studies indicate liquid systems as the unfavorable conditions for plant tissues multiplication due to limited oxygen exchange and/or hydromechanical stress (Savio et al. 2012; Grzegorczyk-Karolak et al. 2015). Pontechium maculatum plants grown in liquid medium with rotary shaking presented a lower growth index than plants grown on agar-solidified medium (Makowski et al. 2023). Furthermore, Scutellaria alpina cultivated in stationary and agitated liquid medium did not derive shoots in the research of Grzegorczyk-Karolak et al. (2017). Effectiveness of plant propagation as an agitated culture relates to the plant’s sensibility to constant immersion and possible oxygen limitation (Savio et al. 2012), as well as mechanical stimulation during shaking (Makowski et al. 2020). R. japonica naturally occurs in wet habitats, like river valleys or forest edges, which may explain its good adaptation to liquid culture conditions (Rahmonov et al. 2014). Both methods of cultivation connected with liquid medium (AC and TIB) stimulated biomass growth and shoot multiplication compared to SM. Moreover, the increased growth index of plants from AC compared to SM may relate to better availability of nutrients in liquid medium. What is more, in TIB, medium flow during the flooding and aeration phase may contribute to a better distribution of medium ingredients thus making them more available to plants (Kunakhonnuruk et al. 2019). Similarly, increased growth of Schizandra chinensis was obtained in Plantform™ bioreactors by Szopa et al. (2019). The best proliferation and fresh weight yield for sundew were observed for its growth in the bioreactor system as reported by Kunakhonnuruk et al. (2019).

In our previous study conducted on P. maculatum, plants obtained from TIB, and ex vitro conditions had different morphology (bigger leaves and callus tissue) than plants cultivated in SM and AC (Makowski et al. 2023). Maliński et al. (2019) also observed a bigger leaf and shape of Lychnis flos-cuculi acclimatized to ex vitro conditions. In the presented study, R. japonica plants showed a bigger leaf shape in TIB compared to SM. It may be related to a bigger volume of the container, larger medium volume per one bioreactor (0.5 L) and ethylene elimination during the aeration phase (Bello-Bello et al. 2010). In turn, the highest growth of plants in soil (acclimatized to ex vitro conditions) is probably related to the effective photosynthesis process (Soni et al. 2021).

One of the important parameters of plant growth and development is the dry weight (DW) accumulation (Isah et al. 2018). This study showed increased DW accumulation for shoots in AC and whole plants (shoot and root) from ex vitro conditions. Interestingly, a study on D. muscipula plants cultivated in liquid medium with rotary shaking did not show differences in DW accumulation when compared to plants grown in SM (Makowski et al. 2020). Moreover, in another study, only shoots of P. maculatum plants cultivated ex vitro had increased DW content as compared to plants cultivated in SM, AC, and TIB (Makowski et al. 2023). Similarly, Kunakhonnuruk et al. (2019) reported an increase in DW accumulation in Drosera communis plants grown in a temporary immersion system compared to a semi-solid medium and permanent-immersion system. The higher DW of Japanese knotweed shoots obtained in AC is probably related to the acclimation strategy to constant mechanical stimulus (during shaking). However, increased DW of ex vitro plants may be related to cell wall rebuilding during the acclimation process and increased photosynthesis activity (Salgado Pirata et al. 2022).

For the first time, the content of phenolic compounds in shoots and roots of Japanese knotweed from in vitro conditions was evaluated and compared to plants cultivated in soil. According to Wang et al. (2022b) rhizomes and roots of R. japonica were reported in Chinese Pharmacopoeia as a potent source of secondary metabolites with a broad range of biological activity, used in traditional Chinese medicine. Interestingly, our research showed that shoots of R. japonica plants from in vitro conditions contain between 25 and 68% more phenolic compounds than roots, depending on the growing conditions (Fig. 7). Moreover, the type of cultivation method significantly affected the content of phenolic compounds in plant organs. The sum of phenolic compounds was highest in Japanese knotweed from AC. P. maculatum plants grown in liquid medium with rotary shaking presented an increased total phenolic content in shoots when compared to SM, while the highest value of total phenolic accumulation was obtained in shoots from TIB, and ex vitro cultivated plants (Makowski et al. 2023). Similarly to R. japonica cultures, the lowest accumulation of total phenols was observed in P. maculatum roots in ex vitro plants. On the other hand, Eryngium alpinum intact plant roots had higher levels of phenolic compounds than those cultivated in tissue cultures (Kikowska et al. 2020). Similarly, Szopa et al. (2019) reported that leaves of S. chinensis intact plants accumulate more phenolics than plants obtained during in vitro cultivation. On the other hand, Salvia viridis shoot culture appeared as a more effective source of phenolic compounds than plants from ex vitro conditions (Grzegorczyk-Karolak et al. 2019). Phenolic compounds accumulate in plant tissue in response to stress factors (Caretto et al. 2015). An increase in the synthesis of secondary metabolites in R. japonica observed in AC and TIB may be induced by mechanical and/or flooding stress. Also, generally higher values of phenolic metabolites in aboveground parts of examined plants compared to underground parts may relate to the fact, that Japanese knotweed in tissue cultures, as well as acclimatized to ex vitro conditions (cultivated in pots with soil) did not produce the rhizomes. Moreover, our study presents data obtained from the experiment on plants cultivated in ex vitro conditions, but in the controlled identical environment as for the cultivation of plants in in vitro conditions. Other available literature data focused on the phytochemistry of Japanese knotweed were obtained from the plants grown in the field/natural environment (Lachowicz and Oszmiański 2019).

Cultivation of medical plant species in in vitro systems may lead to changes in secondary metabolite composition in comparison to plants from ex vitro conditions (Muraseva and Kostikova 2021). Nevertheless, the results from presented experiments (SM, AC, TIB, ex vitro) confirmed the presence of phenolic acids and flavonoids that were previously described in shoots and roots/rhizomes of R. japonica obtained from the field environment, which were reported by Bozin et al. (2017), Lachowicz et al. (2019), Lachowicz and Oszmiański (2019), Stefanowicz et al. (2021), Stefanowicz et al. (2022).

The level of phenolic acid concentration in R. japonica was dependent on the cultivation system, as well as the type of the plant organ. The highest concentration of phenolic acids was accumulated in shoots from ex vitro conditions, where the dominant derivative– caftaric acid obtained a concentration 981.2 mg × 100 g− 1 DW. Lachowicz and Oszmiański (2019) studied the content of phenolic compounds in leaves, stalks, and roots of R. japonica collected from field plants. They reported the following concentrations of caftaric acid: 440.0, 20.0 and 10.0 mg × 100 g− 1 DW in leaves, stalk and roots, respectively. Phenolic acids are synthesized in plants through the shikimic acid by phenylpropanoid pathway. The crucial reaction of phenylalanine conversion into trans-cynnamic acid is catalyzed by phenylalanine ammonia lyase (PAL) (Kumar and Goel 2019). PAL activity, and hence, phenolic acid synthesis relates to stress responses, as well as to primary and secondary metabolism (Labudda et al. 2020b). Plants cultivated in ex vitro conditions have increased activity of primary and secondary metabolism pathways due to being completely autotrophic, compared to the plants from in vitro conditions. Effective photosynthesis derives resources for secondary compound synthesis, but on the other hand, together with cellular respiration produce reactive oxygen species (ROS). Phenolic acids may reduce ROS and prevent oxidation of lipid membranes. AC and TIB conducted to 1.25- and 1.31-fold increase in the sum of phenolic acids in shoots, compared to SM. Similarly, TIB stimulated phenolic acid production in P. maculatum shoots (Makowski et al. 2023), while agitated culture of E. alpinum synthesized more phenolic acids than SM plants (Kikowska et al. 2020).

Phytochemical analysis allowed for quantification of nine flavonoids in examined extracts. Four of them: catechin, epigallocatechin, epicatechin gallate and epicatechin belong to the catechin derivatives and constitute an important group of secondary metabolites characteristic of Japanese knotweed (Lachowicz et al. 2019). Both shoots and roots of R. japonica obtained from each cultivation method were reach in catechins (Table 3). Three of them, catechin, epigallocatechin, and epicatechin, were dominant relative to epicatechin gallate. The most popular raw material, famous for its high content of catechin derivatives, is green tea. The health-promoting properties of green tea result from the catechins it contains (Musial et al. 2020). We can hypothesize that the presence of various catechins in R. japonica tissue causes several of its biological activities. Lachowicz et al. (2019) studied the concentration of catechins in the leaves and rhizomes of R. japonica plants collected from the garden. They reported 145.4 and 8.10 mg × 100 g− 1 DW of catechin in leaves and rhizome, respectively. In the same study epicatechin content in leaves was 329.58 and in rhizome 74.14 mg × 100 g− 1 DW. Our results indicate the advantage of growing R. japonica in in vitro conditions, because of higher catechin synthesis from 87 to 183% in shoots and from 142 to 229% in roots. The most favorable growing conditions AC and TIB increase catechin content probably through stress induction. Mechanical stimulation during agitating, as well as temporary flooding in bioreactors, may induce the defense response in plants, manifested by increased secondary metabolite synthesis (Makowski et al. 2023).

The content of the rest of the flavonoids identified and analyzed in Japanese knotweed is, above all, determined by the plant organ. The shoots of R. japonica accumulated more flavonoids than the roots (Table 4). Nevertheless, cultivation methods used for plant propagation in the experiment also affected flavonoid content. Stefanowicz et al. (2021) reported the following concentrations of quercetin in leaves, stem, and roots/rhizomes of R. japonica collected from the field: 63.0, 68.0, 220.0 mg × kg− 1 DW, while in this study quercetin level in TIB shoots obtained 269.2 mg × 100 g− 1 DW. The obtained results showed that controlled in vitro conditions stimulate flavonoid synthesis in Japanese knotweed plants.

Conclusions

We can conclude that the most suitable methods for the cultivation of Japanese knotweed rich in phenolic metabolites are agitated cultures and temporary immersion bioreactors Plantform™. These modes of cultivation accelerate the growth of plants as compared to traditional cultures on solid medium, probably because of the better availability of nutrients from liquid media. Moreover, agitated and bioreactor cultures accumulate more phenolic compounds, than plants from solid medium and ex vitro conditions. Increased synthesis of secondary metabolites is probably the response to stress induced by shaking of agitated cultures and flooding (hypoxia stress) in bioreactors. We can conclude that R. japonica plants obtained through in vitro propagation are a rich source of biologically active polyphenols and may be used as a safe material for the propagation of plants for pharmacological use.