Linnaea borealis L. var. borealis—In Vitro Cultures and Phytochemical Screening as a Dual Strategy for Its Ex Situ Conservation and a Source of Bioactive Compounds of the Rare Species

Linnaea borealis L. (Twinflower)—a dwarf shrub in the Linnaeeae tribe of Caprifoliaceae family—is distributed across the Northern Hemisphere. By means of this study, a reliable protocol for efficient micropropagation of uniform L. borealis L. var. borealis plantlets has been provided for the first time; callus culture was also established. Different initial explants, types of cultures, media systems, and plant growth regulators in Murashige and Skoog (MS) media were tested. Agitated shoot cultures in the liquid media turned out to be the best system for the production of sustainable plant biomass. After stabilization of the callus lines, the highest growth index (c.a. 526%) was gained for callus maintained on MS enriched with picloram. TLC and UHPLC-HESI-HRMS analysis confirmed the presence of phenolic acids and flavonoids, and for the first time, the presence of iridoids and triterpenoid saponins in this species. Multiplication of L. borealis shoot culture provides renewable raw material, allowing for the assessment of the phytochemical profile, and, in the future, for the quantitative analyses and the studies of the biological activity of extracts, fractions, or isolated compounds. This is the first report on in vitro cultures of traditionally used L. borealis rare taxon and its biosynthetic potential.


Introduction
Linnaea borealis L. (Twinflower), a creeping dwarf shrub, was Linnaeus's favorite plant and became his symbol. This taxon was formerly assigned to the Caprifoliaceae family; however, it was transferred to the family of Linnaeaceae [1,2]. A more recent classification has ascribed twinflower to the Linnaeeae tribe of Caprifoliaceae s.l. [3]. There are the three subspecies recognized within L. borealis, which morphologically differ from each other, namely L. borealis var. borealis in Eurasia, L. borealis var. Americana, and L. borealis var. longiflora-both in North America [3,4]. Twinflower has circumboreal distribution, across the Northern Hemisphere, occurring from Scotland and northern Europe through Russia to Siberia, northern Asia to Kamchatka and Japan, northern China and Mongolia, and from Alaska and Canada to Greenland. The main distribution of twinflower in under controlled conditions and affect secondary metabolites production in the medicinal species. Furthermore, in vitro cultures allow to avoid problems related to the collection, transport, and storage of plant material, which is available all year round. Moreover, micropropagation provides renewable raw plant material, allowing for the assessment of the phytochemical profile, quantitative analyses, and studying the biological activity of extracts, fractions, or isolated compounds [15][16][17].
The aim of the current work was to develop an efficient protocol of Linnaea borealis L. var. borealis micropropagation by the development of axillary buds and multi-shoot culture, as well as induction of callus from various plant explants and optimization of growth of callus on the different media. The study also aimed at the multiplication of in vitro plants for the preliminary phytochemical assessment for comparison with intact plants. Additionally, the study of propagation was undertaken not only for twinflower conservation, but also for the further ornamental and pharmacological application. So far, there has been no report available on L. borealis var. borealis in vitro cultures and detailed phytochemical screening.

Results
In present study, the efficient micropropagation protocol of Linnaea borealis var. borealis was established using the method of stimulation of new buds from pre-existing meristems (nodal segments or shoot tips with apical meristems). The influence of the type of the plant explant, hormonal supplementation in the medium, and culture system (solid or liquid) on shoot multiplication were estimated (Tables 1-4). In the next step, the effect of the auxin type/combined auxins on L. borealis shoot rooting was evaluated ( Table 5). As the artificial conditions of in vitro systems may trigger the so-called somaclonal variation in the propagated plantlets, therefore it was necessary to monitor the genetic fidelity of the clones (Table 6).

Shoot Multiplication
The presence of BAP (first-generation synthetic cytokinin) in the multiplication medium had a particularly positive impact on the development of new shoots from explants in the form of multi-shoots. In turn, the addition of kinetin (also artificial cytokinin) to the multiplication medium was important for the development of new shoots from the nodal fragments of stems. Double shoots, shoot segments with one node, and single shoots with the apical meristem were the best explants for multiplication of shoot biomass. The media with the high cytokinin content were crucial for the development of new shoots. The highest number of new shoots (7.63 ± 0.73) was obtained from double shoots on the solid medium supplemented with BAP (2.0 mg/L), IAA (0.1 mg/L) and GA 3 (Table 1). Micro-shoots were morphologically proper, not vitreous, and did not form callus at the base; leaves exhibited a vivid green color ( Figure 1). In the subsequent experiment, explants with a different number of shoots (single, or a cluster of double or triple shoots) were placed on the selected solid medium-BAP (1.0 mg/L), IAA (0.1 mg/L) and GA 3 (1.0 mg/L). A double shoot indicated to be the best explant for multiplication. The highest number of new micro-shoots per explant was 10.89 ± 0.55 and 5.37 ± 0.26 per shoot ( Figure 1, Table 2). Admittedly, double and triple shoots grown in the liquid medium proved to be the best material for multiplication of new shoots (their number increased up to about 40); double and single shoots increased the most in length (by about 150%). Therefore, the results of the study have suggested that double shoots are the best explants for liquid culture to obtain a large number of shoots that grow significantly in length, as they generally contribute to the greatest increase in plant biomass. The authors believe that a large number of new shoots is associated with rinsing the whole explant in the liquid medium, which may affect initiation and the development of buds forming new lateral shoots. Well-expanded leaves of cultured shoots had correct morphology and did not show any changes ( Figure 1, Table 3). Summarizing the results of a series of experiments, the introduction of L. borealis L. var. borealis into in vitro cultures enabled rapid clonal multiplication. Homogeneous plant material was obtained by stimulating the development of axillary buds on the MS medium variant wit BAP 1.0 mg/L + IAA 0.1 mg/L + GA 3 1.0 mg/L. The most efficient multiplied shoot biomass was obtained in agitated liquid culture (Table 4).

Shoot Rooting
It was extremely difficult to produce rootlets in the experiment, as twinflower is a dwarf shrub. The first roots were observed after two weeks of culture; after another four weeks, roots were well developed. In vitro-multiplied shoots formed vigorous roots of a white color with frequency of about 30-100%, depending on the applied medium. The highest percentage of root induction of in vitro multiplied shoots was obtained for shoots grown on the solid MS medium with higher concentrations of IBA alone or in combination with IAA. Additionally, these media were advantageous for the number of emerging roots, most of which, about 19 roots per shoot, appeared on the following media: MS + IBA 4.0 mg/L and MS + IBA 2.0 mg/L + IAA 1.0 mg/L. The least preferred media for rooting of shoots were those that lacked IBA and those in which IAA was present at a low concentration. Micro-shoots underwent direct root formation, without a callus phase on all the tested media ( Figure 1, Table 5).

Genome Size Estimation
Micropropagation of L. borealis var. borealis was performed through meristematic tissues; however, the genome size estimation was necessary to ensure that plant material is homogeneous. The 2C DNA content of leaves in control seedlings was 1.778 pg and it was similar in multiplied shoots (Table 6; Figure 2). No statistical differences were detected. Table 6. The nuclear DNA content in leaves of Linnaea borealis var. borealis obtained from seedlings and micropropagated plantlets.

Callus Induction and Maintenance
The influence of the type of the plant explant and hormonal supplementation in the solid medium on callus induction and proliferation were estimated (Table 7).
Leaves, internode stems, and roots of L. borealis var. borealis previously multiplied in vitro were used as explants in the experiment. Callus was observed in all the three types of explants, usually at the point of the organ cut. Formation of callus on leaves lasted from four to eight weeks, while on fragments of stems and roots, it took six to 12 weeks. Callus forming on plant explants was light yellow and milky. It was passaged every five weeks until stabilized (VII, VIII, IX passages). As the observations showed, the mean callus induction from leaves was 39.37%, from stems-37%, and from roots-45.5%. After stabilization of the callus lines, the highest growth index gain was characteristic of callus culture maintained on MS medium enriched with picloram 2.0 mg/L ( Figure 3, Table 7).

UPLC-HESI-HRMS Phytochemical Screening
UPLC-MS/MS analysis demonstrated in the studied material, namely leafy shoots of intact plants, shoots and roots of micropropagated plantlets, and callus biomass, the diversity of chemical compounds with known pharmacological activity (Figures 4 and S1-S4, Table 8) [18][19][20][21]. The results indicated the presence of the four major groups of secondary metabolites in the studied plant material of L. borealis L. var. borealis. The presence of iridoid and triterpene compounds, apart from phenolic acids and flavonoids, has been detected for the first time. UPLC-MS/MS analysis in the negative and positive ion mode indicated the presence of 30 phenolic (including benzoates and hydroxycinnamic acid derivatives) and 19 flavonoid compounds. Thirteen iridoid compounds have been detected for the first time in this species. Two organic acids (quinic and pantothenic acid) were annotated in the profile. Additionally, 14 triterpenoid saponins were confirmed with the use of mass spectrometry (Table 8).           In this study, the profile of hydroxycinnamic acid derivatives was mainly presented by esters of quinic acid with caffeic, coumaric, and ferulic acids. Four caffeoylquinic acids were recognized as trans-3-caffeoylquinic, trans-5-caffeoylquinic, trans-4-caffeoylquinic, and cis-5-caffeoylquinic. As reported by Ramabulana et al. [22], isomers of caffeoylquinic were recognized by the characteristic fragmentation pattern following at m/z value 191.0551 (C 7 [23]. The fragmentation spectra of these precursor ions show the loss of hexose (−162.0527, C 6 H 10 O 5 ) and fragmentation of aglycone. The compound 29 was putatively described as grandifloroside in comparison with its MS/MS spectra and exact mass. Moreover, two [M − H] − ions m/z 585.2195 at 7.54 and m/z 583.2036 at 7.98 min were marked as unknown terpene glycosides. Several triterpene saponins were found in the plant extracts. These compounds were recorded as macranthoidin A, akebiasaponin D, loniceroside C, bourneioside B, cauloside C, alpha-hederin, and cauloside A. Furthermore, three free aglycones were recognized with the use of the exact mass. These compounds could be annotated as hederagenin (15.14 min), gypsogenin (15.66 min), and oleanonic acid (17.32 min). Four compounds were marked as unknown saponins.

Discussion
To the best of the authors' knowledge, this study has shown L. borealis introduction into in vitro cultures and the development of the micropropagation protocol, as well as establishment of callus induction and maintenance for the first time.
Micropropagated plants and biomass from the other types of in vitro cultures can be an alternative source of secondary metabolites with the biological activity, which is particularly important for rare or unavailable in nature taxa, for example, due to the species protection status. Biotechnological production of biomass of such species facilitates the phytochemical and biological research without destroying their natural environments. Plant in vitro cultures can provide the sufficient quantity of high quality uniform biomass under controlled conditions [15,16,24].
Qi et al. [25], in their work on micropropagation of the species also belonging to Caprifoliaceae family, i.e., Lonicera edulis, indicated that in vitro propagation of multi-shoots promotes rapid multiplication of plant material and can be used in mass reproduction. The authors obtained the most efficient shoot propagation on a similar MS medium with the addition of BAP (1.0 mg/L) and IBA (0.2 mg/L) [25]. Similarly, in in vitro shoot cultures of Lonicera caerulea var. kamtschatica Pojark., the presence of BAP also enhanced production of new micro-shoots; however, its high concentration (2.0 mg/L) resulted in callus formation at the base of shoots, which is an undesirable feature for the homogeneity of the plant material [26]. This cytokinin at a lower concentration (0.1 mg/L) influenced significant growth of Lonicera periclymenum L. [27]. It is widely known that BAP positively affects the development of axillary buds, especially when combined with auxin of much lower concentration [28][29][30]. In the present study, multiplied shoots of L. borealis were not vitreous, regardless of the concentration of cytokinin, which is a desirable feature for the quality of plant material. Nonetheless, in the work of Dziedzic [26], it was observed that the media supplemented with a higher concentration of BAP (2.0 mg/L) brought a high percentage (even 36%) of vitrified shoots in L. caerulea var. kamtschatica. In a protocol of other micropropagated shrubs from Caprifoliaceae, namely Kolkwitzia and Weigela, the employment of BAP with a low concentration of auxin was preferred for the stimulation of the development of new buds [31,32].
Shoots cultured in the liquid media are a good source of biomass for the phytochemical and biological studies. Shoot biomass of L. borealis grew because not only horizontal fragments of stems (which came in contact with the solid medium in the solidified culture systems), but also the nodal segments in vertical stems, poured with the liquid medium, were exposed to the medium nutrients and phytohormones. Agitation clearly promoted shoot growth in liquid cultures of some other species, for example, Eryngium alpinum [17], Lychnis flos-cuculi [33], Scutellaria alpina [34], and Schisandra chinensis (Turcz.) Baill. [35]. Interestingly, shoots of the studied L. borealis had correct morphology, while agitated shoots from the liquid media of many plant species cultured in vitro were characterized by abnormality and hyperhydricity, for example, E. alpinum [17] or S. alpina [34].
Micro-shoots of woody plants are usually difficult to grow roots [36]. The use of MS medium supplemented with IAA (5.0 mg/L) and IBA (2.0 mg/L) was dictated by the report on shoot rooting of the woody species, L. caerulea var. kamtschatica, grown in in vitro cultures. The same types and concentrations of two auxins were used in rooting of L. borealis shoots; however, MS medium was used in this study. In the research of Dziedzic [26], Woody Plant Medium (WPM) was applied. Moreover, satisfactory rooting was also obtained for other honeysuckle plants [36,37].
Explants from shoot cultures and plants of L. borealis from in vitro culture were used for callus induction; these were leaves, apical fragments of rootlets, and internodal fragments of stems. The highest percentages of responses were obtained from roots, while the most intense callus development was observed on leaves. The differentiation in callus induction from distinct plant explants was affected by the type and age of an explant [38].
The purpose of the preliminary phytochemical analysis was to investigate whether in vitro plant cultures of L. borealis var. borealis are capable of biosynthesis of secondary metabolites. Multiplied shoots obtained from in vitro cultures produced secondary metabolites analogous to those produced by plants growing in the wild. The analysis of the biomass extracts of callus cultures revealed that callus produced phenolic acids, but was not able to biosynthesize flavonoids. The reason for this inability may be the type of a nutrient solution and a concentration of plant growth regulators used in the medium. The results of other authors indicated that callus cells of various plant species usually do not produce flavonoids, but are able to synthesize phenolic acids [28,39].
The analysis with the use of HPLC/MS has allowed to observe the presence of iridoid compounds and triterpenoid saponins in the European species L. borealis var. borealis for the first time. The Lonicera genus as well as L. borealis species belong to the family of Caprifoliaceae s.l., hence, there was a high probability of finding iridoids in biomass of the species studied [40]. Compounds annotated as loganin, secoxyloganin, secologanin, secologanin, loganic acid, and morroniside were present in Lonicera species-L. morrowii, L × bella, and L. tatarica [40]. The phytochemistry of Lonicera was previously investigated due to the importance of various species in traditional pharmacopeias, and the genus contains a class of secondary compounds, iridoid and secoiridoid glycosides, with the established economic importance [40]. According to Jensen et al. [41], secologanin and morroniside-iridoids of VI group (simple secoiridoids), sweroside-iridoid of VII group and loganin-iridoid of X group are characteristic of the family of Caprifoliaceae.
Several triterpene saponins were found in the studied extracts of L. borealis. Few triterpenoid compounds were formerly reported in closely related Lonicera species. These compounds were annotated as hederin-type triterpenoid saponins macranthoidin A found in L. confuse [42], akebiasaponin D and cauloside C determined in L. macranthoides [43], as well as loniceroside C and cauloside A revealed in L. japonica [44,45]. Moreover, lupine-type triterpenoid saponin-bourneioside B present in the studied L. borealis-was previously detected in L. bournei [46].
The presence of a larger number of phenolic acids and flavonoids similarly to the study of Glennie [11] was detected. In present study, the profile of hydroxycinnamic acid derivatives was mainly presented by esters of quinic acid with caffeic, coumaric, and ferulic acids. The second group of phenolic compounds were flavonoids. This group was represented by glycoconjugates of quercetin, kaempferide, kaempferol, luteolin, and apigenin, which were partially identified by Glennie in American varieties of L. borealis [11]. Research manuscripts reporting large datasets that are deposited in a publicly available database should specify where the data have been deposited and provide the relevant accession numbers. If the accession numbers have not yet been obtained at the time of submission, please state that they will be provided during review. They must be provided prior to publication.

Shoot Multiplication on Solid Media
In the first experiment, the authors of the study tried to find out whether node segments or shoot tips with apical meristems (both of 2-2.5 cm with 3-4 nodes) were more favorable explants for shoot multiplication in relation to double shoots (a cluster consisting of 2 short shoots). To determine beneficial conditions for shoot multiplication, the media were supplemented with BAP (0.5-2.0 mg/L) or kinetin (Kin 1.0 mg/L, 2.0 mg/L), IAA (0.1 mg/L), and gibberellic acid (GA 3 1.0 mg/L). Multiplication of shoots was replicated three times for each hormonal treatment, using at least 10-20 explants.
The second experiment was dedicated to verify how many new shoots may be obtained as a result of shoot multiplication within eight weeks. For this purpose, single shoots as well as multi-shoots (with two or three shoots), obtained from the initially multiplied shoots, were transferred to the solid MS medium with the selected concentrations of PGRs, that is BAP (1.0 mg/L), IAA (0.1 mg/L) and GA 3 (1.0 mg/L). Due to a particularly small size of a plant, explants used for the experiments were not only single shoots, but also clusters of two or three micro-shoots. Multiplication of shoots was replicated three times using at least 10-20 explants.
After a few subcultures, the multiplication rates were recorded by determination of the number of new micro-shoots that proliferated from the initial explant.

Shoot Multiplication in Liquid Media (Agitated Cultures)
Single shoots as well as multi-shoots (with two or three stems) about 2-3 cm long with 5-6 nodes, obtained from developed shoot cultures on the solid media, were transferred to the liquid MS medium with the selected concentrations of PGRs, namely BAP (1.0 mg/L), IAA (0.1 mg/L), and GA 3 (1.0 mg/L); 100-cm 3 Erlenmeyer flasks with 10 cm 3 of medium were used for shoot biomass production (the ratio of glass volume to medium volume was 10:1). Cultures were maintained on a rotary shaker (110 rpm). After eight weeks of culture, the number of new shoots per explant and the shoots length index (LI) were measured. At least 10 explants were used for multiplication of shoots. The initial (L0) and the final (LX) lengths of cultured shoots were measured. The length increase index [LI] was calculated according to the following formula: LI = [(LX − L0)/L0] × 100%.

Shoot Rooting
An attempt at rooting of clusters of several shoots was made. Multi-shoots (3-4 shoots) 3 to 5 cm in length were used. Explants were kept in 70-cm 3 glass tubes containing 15 cm 3 of the solidified medium (1/4 MS-salts reduced to 25%, 1/2 MS-salts reduced to 50%, MSfull strength of mineral salts) without auxins or supplemented with IAA (0.5-4.0 mg/L) or indole-3-butyric acid (IBA 0.5-5.0 mg/L), or the combination of both auxins. Shoots were grown under the same light and temperature conditions as shoot cultures obtained via clonal propagation. After eight weeks, the number and the length of induced roots were observed.

Callus Induction and Maintenance
Fragments of stems, leaves, and roots of micropropagated plantlets were used for callus initiation. Explants were transferred to 150-cm 3  Callus induced on explants was removed and transferred to a new medium of the same PGRs composition. Callus cultures were passaged every five weeks. In order to stabilize culture, fragments that were developing well were selected during the passage. After the morphological evaluation, the callus lines with the fastest growth were selected and the rate of biomass growth was calculated for the three following passages (VII, VIII, IX). The initial (FW0) and the final (FWX) weights of the callus lumps were measured. The growth index (GI) was calculated after three passages during three consecutive subcultures according to the following formula: GI = [(FWX − FW0)/FW0] × 100%. The flasks were stored in a darkroom. The following MS media were used in the experiment of callus stabilization: supplemented with Pic 1.0 mg/L or Pic 2.0 mg/L as well as with 2,4-D 2.0 mg/L and NAA 0.5 mg/L.

Flow Cytometry
Leaves of seedlings and in vitro shoots multiplied on MS medium, which was optimal for micropropagation, were used for the nuclear DNA content estimation. Petunia hybrida P × Pc6 (2.85 pg/2C) Marie and Brown, 1993) served as an internal standard. The samples were prepared as previously described [48], by simultaneous chopping of leaves of a sample and an internal standard in 1.0 mL of cold nuclei isolation buffer [49] supplemented with 1% (v/v) polyvinylpyrrolidone (PVP-10), propidium iodide (PI; 50 µg/cm 3 ) and ribonuclease A (50 µg/cm 3 ). For each sample, at least 5000 nuclei were analyzed directly after preparation, using the CyFlow Ploidy Analyzer flow cytometer (Sysmex Partec). Histograms were analyzed using the CyView 1.6 computer program. The analyses were replicated three times for each plant material. The coefficient of variation (CV) of G0/G1 peak of L. borealis ranged from 3.51 to 5.11%. The nuclear DNA content was calculated using the linear relationship between the ratio of the G0/G1 peak positions L. borealis/Petunia on a histogram of fluorescence intensities.
The Aquity UPLC (Waters, Milford, MA, USA) with the high resolution Orbitrap mass spectrometer (Thermo Fischer, Bremen, Germany) were used for the analysis of the methanolic extract of Linnea borealis. Two microliters of the sample were injected onto the BEH C13 column (1.7 µm of diameter, 2.1 × 150 mm) and separated with the use of 0.1% formic acid (LC-MS grade, Fluka) in ultrapure water (solvent A, MiliQ, Merck, Darmstadt, Germany) and acetonitrile (solvent B, LC-MS grade, Merck) at the flow rate of 0.300 µL/min and column temperature of 50 • C. The gradient program consisted of 2% B, 12 min-30% B, 15 min-98% B, 17 min-98% B, 18 min-2% B, and isocratic 2% B to 20 min. The PDA detector was operated at 250, 270, 330, and 360 nm of the wavelength. The heated electrospray ion source (HESI-II) settings were the following: capillary voltage −2.5 kV (negative), +3.5 kV (positive), sheath gas flow-35, auxiliary gas flow-10, sweep gas flow-3 arbitrary units, ion transfer tube temperature-400 • C, auxiliary gas heater temperature 350 • C, and S-lens RF level-50. The spectra were recorded at mass resolution of 70,000 FWHM in m/z range of 150-1500 and at 200 ms maximum injection time for full-MS scans and resolution of 17 500 FWHM, and at 50 ms maximum injection time for data dependent MS2 scans (top 5).

Statistical Analysis
The collected biotechnological data were subjected to a one-way analysis of variance (ANOVA) followed by Duncan's POST-HOC test. ANOVA and the subsequent Student's test were used for the flow cytometric results analysis. A two-sided p-value of 0.05 was applied to declare statistical significance. All the analyses were conducted employing STATISTICA v. 13 (StatSoft, Inc., Kraków, Poland, 2015).

Conclusions
The introduction of the rare and protected species L. borealis L. var. borealis into in vitro cultures enabled rapid clonal multiplication. This study demonstrated the influence of the type of the plant explant, hormonal supplementation in the medium and culture system on shoot multiplication and root development. The most efficient multiplied shoot biomass may be obtained from numerous lateral buds developed from multi-node stem segments in agitated liquid culture, in accordance with the biology of this species. A vigorously growing callus also ensures a good source of plant biomass and may be useful to obtain cell suspension culture in the future. In vitro technique can be used as a nondestructive approach for producing secondary metabolites from the homogenous biomass of medicinally important plants.
The preliminary phytochemical studies confirmed the presence of phenolic acids and flavonoid compounds in the species and have demonstrated the presence of iridoids and triterpenoid saponins for the first time. The chemical profile of European twinflower suggests the potential uses of both plant material from intact plants and biomass from in vitro cultures as a source of bioactive compounds with the confirmed pharmacological activity. Multiplied biomass with the profiled phytochemical composition of valuable secondary metabolites could be used for the biological studies of pharmacological interest.
Collection of in vitro plantlets may be also concerned as an ex situ conservation strategy for this rare European taxon.
Supplementary Materials: The following are available online. Figure S1: 2-D chromatogram of Linnaea borealis var. borealis extract of leafy shoots from natural sites. Figure S2