Establishment and triterpenoid production of Ocimum basilicum cambial meristematic cells

The application of plant suspension culture to produce valuable compounds, such as the triterpenoids oleanolic acid and ursolic acid, is a well-established alternative to the cultivation of whole plants. Cambial meristematic cells (CMCs) are a growing field of research, often showing superior cultivation properties compared to their dedifferentiated cell (DDC) counterparts. In this work, the first-time establishment of O. basilicum CMCs is demonstrated. DDCs and CMCs were cultivated in shake flasks and wave-mixed disposable bioreactors (wDBRs) and evaluated regarding triterpenoid productivity and biomass accumulation. CMCs showed characteristic small vacuoles and were found to be significantly smaller than DDCs. Productivities of oleanolic and ursolic acid of CMCs were determined at 3.02 ± 0.76 mg/(l*d) and 4.79 ± 0.48 mg/(l*d) after 19 days wDBR cultivation, respectively. These values were consistently higher than any productivities determined for DDCs over the observed cultivation period of 37 days. Elicitation with methyl jasmonate of DDCs and CMCs in shake flasks resulted in increased product contents up to 48 h after elicitor addition, with the highest increase found in CMCs at 232.30 ± 19.33% (oleanolic acid) and 192.44 ± 18.23% (ursolic acid) after 48 h. For the first time, cambial meristematic cells of Ocimum basilicum were established and cultivated in a disposable bioreactor system. These cells outperform dedifferentiated cells of the same organism regarding productivity.


Introduction
The perennial herb Ocimum basilicum (family Lamiaceae), commonly known as basil, is valued worldwide for its culinary uses, but its applications go well beyond that. Basil and its essential oil are often used in traditional medicine (Irondi et al. 2016;Ezeani et al. 2017;Bae et al. 2020), cosmetics (Vivas Castaño et al. 2016;Volpe et al. 2018;Yeşil et al. 2020), and pharmacology (Sestili et al. 2018;Zhan et al. 2020). Among the pharmacologically interesting compounds found in basil are linalool (Medeiros Venancio et al. 2016), rosmarinic (Kwon et al. 2019), oleanolic (OA; Qamar et al. 2020) and ursolic acid (UA; Arshad Qamar et al. 2010;Kümmritz et al. 2014). The latter are triterpenoids, a class of isoprenoid-based secondary metabolites. OA and UA were shown to possess antiinflammatory (Kashyap et al. 2016), antitumoral (Pięt and Paduch 2018), hepatoprotective (Gutiérrez-Rebolledo et al. 2016) and antioxidant (Srinivasan et al. 2020;Guo et al. 2020) properties among others. These properties make oleanolic and ursolic acid interesting candidates for clinical application, which could result in high future demand. The cultivation of whole plants for valuable substances is impractical from both economic and ecological stances because of long growth periods, huge crop areas and risk of crop loss. By utilizing plant cell culture, specific cell types can be used for production of valuables in a more controllable environment (Ramachandra Rao and Ravishankar 2002). Basil cell suspension culture Communicated by Christophe Hano.

Electronic supplementary material
The online version of this article (https ://doi.org/10.1007/s1124 0-020-01942 -y) contains supplementary material, which is available to authorized users. was established in the past for the production of rosmarinic acid (Kintzios et al. 2003) and more recently for phenolics and anthocyanins (Nazir et al. 2019) as well as oleanolic and ursolic acid (Pandey et al. 2019).
An interesting type of plant cell culture are cambial meristematic cells (CMC), which are derived from vascular cambium. These cells were described to grow at a faster rate, aggregate less and accumulate more product than dedifferentiated cells (DDCs) of the same plant (Ochoa-villarreal et al. 2015). CMCs were described for Taxus cuspidata (Lee et al. 2010), Catharanthus roseus (Moon et al. 2015;Zhu et al. 2018) and Tripterygium wilfordii (Song et al. 2019) among others, but not yet for O. basilicum.
The application of a suitable bioreactor system is crucial for plant cell cultivation in respect to biomass accumulation, cell viability and product formation (Eibl and Eibl 2008;Valdiani et al. 2019). While stirred-tank reactors are routinely used for plant cell culture (Arias et al. 2017;Pérez-Hernández et al. 2019), the mechanical stirring poses a source of hydrodynamic stress for the plant cells, decreasing cell viability during the process (Takeda et al. 1994). Alternatives without mechanical stirring are found in bubble column, airlift or some types of disposable bioreactors. Wave-mixed disposable bioreactors (wDBR) provide mass and energy transfer by a gentle rocking motion, reducing shear stress on the cells (Eibl and Eibl 2008). This reactor type was used in the past for cultivation of Nicotiana tabacum (Terrier et al. 2007;Raven et al. 2015), Hordeum vulgare (Ritala et al. 2008) and Malus domesticus (Schürch et al. 2008).
A popular means to enhance secondary metabolite formation in plant cell culture is known as elicitation (Giri and Zaheer 2016;Thakur et al. 2019). The application of an elicitor triggers stress or defence related responses in plant cells (Narayani and Srivastava 2017). Methyl jasmonate (MeJa) is a signal molecule involved in plant defence. When applied to plant cell culture, the production of secondary metabolites can be increased, for example rosmarinic acid in O. basilicum DDCs (Pandey et al. 2019) or flavonoids in Pueraria candollei (Udomsin et al. 2020).
In this study, CMCs of O. basilicum are described for the first time. Their productivity of the triterpenoids oleanolic and ursolic acid is described in shake flasks as well as wDBRs and compared to DDCs of the same organism. Additionally, eliciting effects of MeJa on triterpenoid production of CMCs are investigated in shake flasks.

Establishment of CMC cell culture
Cell culture was established from commercially available O. basilicum plant. Young shoots were cut and surface sterilized by incubation in 0.56 mM ascorbic acid solution for 90 min, followed by 1 min incubation in 70% (v/v) ethanol and incubation in 1% (v/v) sodium hypochlorite for 20 min. Shoots were rinsed in sterile deionized water for 5 min after each sterilization step. Afterwards, shoots were quartered along the shoot axis and incubated on agar plates containing 30 g/l sucrose, 4.4 g/l Linsmaier-Skoog (LS) media, 4 g/l plant agar and 1 mg/l 2,4-dichlorophenoxyacetic acid. Subcultivation on the same medium was performed at a 2 week interval. Light microscopy was performed biweekly to check for the emergence of new cells in the cambial layer. When cells emerged, they were scraped off and transferred to agar plates for strain maintenance (see below). Cell morphology was routinely inspected with a light microscope (Nikon Eclipse NI, Düsseldorf, Germany).

Strain maintenance and pre-culture
CMCs were maintained on agar plates containing LSmedium (30 g/l sucrose, 4.4 g/l LS-media, 1 g/l MES and 1 mg/l 2,4-D, pH adjusted to 5.7 with sodium hydroxide) with an addition of 5.5 g/l plant agar. Original DDC culture was established from a commercial basil plant (unpublished results). LS-medium for DDCs additionally contained 1 mg/l 6-furfurylaminopurine. Subcultivation of agar plates was performed every 4 weeks. All agar plates were kept in an incubator (Binder, Tuttlingen, Germany) at 28 °C in darkness. To establish precultures, 1 g/l cell wet weight (CWW) of DDCs or CMCs were transferred into 500 ml shake flasks containing 50 ml LS-medium and incubated in an incubation shaker (Multitron, Infors GmbH, Einsbach, Germany) for 14 days at 28 °C, 120 rpm and 2.5 cm eccentricity in darkness. Additional homogenization of the cultured cells was not necessary, since all cultures were friable.

Cultivation in shake flasks
To assess growth and productivity in 500 ml shake flasks over time, 1 g/l CWW DDCs or CMCs from precultures were incubated in 50 ml LS-medium in an incubation shaker for up to 37 days at 28 °C and 120 rpm in darkness. Flasks were capped with cellulose plugs. Whole flasks were harvested by vacuum filtration starting after 4 days in a 3 daysrhythm and frozen at -20 °C until further use (n = 2).

Cultivation in disposable bioreactors
To assess productivity in wDBRs, a BIOSTAT RM 20 basic rocker (Sartorius AG, Göttingen, Germany) with 2 l Flexsafe RM basic SC bags was used. 1 g/l CWW DDCs or CMCs from precultures were incubated in their respective liquid media for 37 days at 7° tilt, 20 rpm, 50 ml/min aeration with ambient air and 28 °C in darkness. 1 ml samples containing 1 3 medium and biomass were drawn in a 3 days-rhythm starting after 4 days. Biomass was separated from media by centrifugation at 14,000 rpm for 5 min and frozen at −20 °C until further use.

Neutral red staining
50 mg cells from agar plates were incubated in 1 ml 0.005% (w/v) neutral red solution for 3 min. Suspensions were then centrifuged twice for 2 min at 14,000 rpm. Supernatant was discarded each time and cells were resuspended in 1 ml phosphate buffer (pH 7.2). All staining steps were performed in darkness. Suspended cells were pipetted on a microscopy slide and observed under a light microscope.

Determination of cell size
50 mg DDCs or CMCs from agar plates were suspended in 1 ml of the respective liquid media. Suspended cells were pipetted on a microscopy slide and observed under a light microscope. Six images were recorded from each of ten slides for both cell types (n(DDC) = 204, n(CMC) = 282).

Elicitation
To investigate the effects of elicitation on triterpenoid production, 10 g/l DDCs or CMCs from precultures were incubated in 500 ml shake flasks containing 50 ml of their respective liquid media with an addition of 200 µM MeJa dissolved in 100% ethanol at 28 °C, 120 rpm and 2.5 cm eccentricity in darkness. Triplicates of whole flasks and one control flask were harvested after 24 h, 48 h, and 72 h.

Triterpenoid extraction and analysis
Frozen biomass was freeze-dried (Christ, Osterode, Germany) at −20 °C and 1.03 mbar for 24 h. Triterpenoids were extracted by grinding 50 mg dry biomass in a mortar with 250 mg sea sand (particle size 0.1 -0.315 mm) and 750 µl ethanol for 2 min. Samples were collected in 15 ml reaction vessels by washing the mortar with another 750 µl of ethanol and stored at −20 °C until further use. Samples were prepared for analysis using a polytetrafluoroethylene filter (pore size 0.22 µm). Reversed-phase high performance liquid chromatography analysis was done on a Waters alliance 2695 with Waters PDA detector 2998 at isocratic flow (0.3 ml/min), 20 µl injection volume and 55 °C column temperature. The mobile phase consisted of methanol with 0.1% formic acid in a ratio of 92:8. A Supelco Discovery HS C18, 5 µm, 250 mm x 4.6 mm column was used. UV spectra were obtained using a detection wavelength of 250 nm. External standards of OA and UA were used for a seven-point calibration.

Statistical analysis
Values are presented as means including standard deviation unless otherwise noted. Shapiro-Wilk test was used to determine normality of data sets. Mann-Whitney U Test was used to determine significant differences between data sets; p < 0.001 is indicated by triple asterisk.

Establishment of O. basilicum CMCs and their morphology
The emergence of new cells in the vascular cambium of O. basilicum explants could be observed with a light microscope as early as 7 days after preparation. Emergent cells were carefully scraped off and incubated on solid medium. CMCs were morphologically identified by their small size and abundance of small vacuoles (Fig. 1a). Abundance of small vacuoles and generally small cell size compared to DDCs from the same species are distinguishing features widely described for CMCs (Lee et al. 2010;Song et al. 2019;Zhou et al. 2015).To confirm the observed structures as small vacuoles in CMCs, they were stained with neutral red, which penetrates the cell wall and membranes, accumulating in vacuoles (Fig. 1c). O. basilicum DDCs were also stained this way, confirming the absence of smaller vacuoles (Fig. 1b). Neutral red has been used for decades as a reliable stain for plant vacuoles (Timmers et al. 1995;Dubrovsky et al. 2006;Kaur et al. 2018) due to the ion trap mechanism, during which neutral red is protonated in acidic pH and loses its ability to pass membranes. Since the pH of the plastid stroma is generally in neutral or alkaline ranges (Su and Lai 2017) and thylakoid lumen acidity is light-dependent, neutral red accumulation in plastids is unlikely. Additionally, cells were kept in the dark during cultivation and staining, making the generation of a proton gradient across the thylakoid membrane unlikely. To confirm this, neutral red stained CMCs were observed with confocal laser scanning microscopy, where no overlap of neutral red fluorescence and chlorophyll a autofluorescence was found in vacuolar structures (Fig. S1, denoted by arrows). Cell size of DDCs were determined to spread around a median of 88.05 µm (Fig. 1d,

CMC growth and productivity compared to DDCs
CMC growth was investigated in shake flasks over a period of 37 days (Fig. 2a). CMCs exhibited exponential growth between 16 and 25 days after inoculation, starting and ending 3 days earlier than DDCs (Fig. 2a, Fig. S2). Growth of CMCs continued until the last day of the cultivation period, reaching 203.02 ± 21.47 g CWW /l after 37 days of cultivation. DDC CWW peaked at 261.55 ± 228.68 g/l after 28 days but declined to 55.97 ± 0.72 g CWW /l after 37 days (Fig. S2), possibly due to cell death and lysis. While nutrient depletion cannot be ruled out as a reason for this decline, it was determined that sugars were not fully depleted at this time (data not shown). CWW concentrations are in the range of previously reported CWW values for DDCs of other plant species, for example Panax ginseng (Lian et al. 2002) and Tribulus terrestris (Khandy et al. 2017). Considering the extreme variation of DDC CWW between 22 and 31 days, CMCs appear to be superior to DDCs in biomass accumulation.
Productivity of oleanolic and ursolic acid of CMCs in shake flasks increased until 19 days after inoculation, where both peaked at 1.73 ± 0.49 mg/(l*d) and 2.73 ± 0.53 mg/ (l*d), respectively (Fig. 2a). The productivity then declined until the end of the cultivation period, reaching 0.74 ± 0.46 mg/(l*d) (oleanolic acid) and 0.62 ± 0.25 mg/ (l*d) (ursolic acid). While the variability of the productivities also increases for both triterpenoids towards the end of the cultivation, this is not considered problematic because productivities do not reach previously detected levels within their variability. The apparent relationship between cell growth and triterpenoid productivity is not yet described for O. basilicum cells, but a similar behaviour was reported in Salvia officinalis, where highest triterpenoid content preceded highest biomass and dropped off afterwards (Bolta Fig Haas et al. 2014). To visualize the differences between DDCs and CMCs at 19 days cultivation, productivities in both shake flasks and wDBR were observed in more detail (Fig. 2b). In shake flasks, oleanolic acid productivity of DDCs was found at 0.17 ± 0.20 mg/(l*d) and ursolic acid productivity at 0.04 ± 0.04 mg/(l*d) after 19 days. At that timepoint, CMC productivities of oleanolic and ursolic acid were 9.9-fold and 68.3-fold higher, respectively. Cultivation of DDCs in wDBR resulted in productivities of oleanolic and ursolic acid of 0.22 ± 0.01 mg/(l*d) and 1.00 ± 0.19 mg/ (l*d) after 19 days, respectively. CMC productivities in shake flasks were still 7.8-fold (oleanolic acid) and 2.7-fold (ursolic acid) higher. At 19 days cultivation, the data indicates that CMCs are superior to DDCs in terms of productivity. Cultivation of CMCs in wDBR revealed even higher productivities for both triterpenoids, which were found at 3.02 ± 0.76 mg/(l*d) for oleanolic and 4.79 ± 0.48 mg/(l*d) for ursolic acid. Compared to CMC productivities in shake flasks, both oleanolic and ursolic acid productivities were increased 1.75-fold, indicating that cultivation of O. basilicum CMCs in wDBR is favourable. This is in line with a previous report, where cultivation of O. basilicum DDCs in a wDBR system lead to increased rosmarinic acid content (Kintzios et al. 2004). Conversely, oleanolic and ursolic acid content could not be increased when O. basilicum DDCs were cultivated in a stirred-tank reactor (Pandey et al. 2019). The indication for better productivities of CMCs in wDBR cultivation was confirmed by productivities obtained over the time courses of all cultivations (Fig. 2c, d). In a direct comparison, after 25 days, productivities of oleanolic acid determined in wDBR cultivation converged with those from shake flask cultivation within standard deviation margin. The productivities of ursolic acid determined in wDBR cultivation stayed above those from shake flasks over the rest of the cultivation period. Productivities of CMCs were higher than those of DDCs in both shake flasks and wDBR at most time points. Exceptions to this were found after 28 days of cultivation, where the highest productivity for ursolic acid in DDCs in wDBR was found at 2.03 ± 0.08 mg/(l*d). This value was 2.18-fold higher than ursolic acid productivity of CMCs in shake flasks at 28 days. In wDBR at 28 days, ursolic acid productivity of CMCs was still 1.77-fold higher. This observed pattern continued until the end of the cultivation period. Regarding oleanolic acid, no such exception was found. These findings are consistent with previous reports on CMCs. In Taxus cuspidata CMCs, paclitaxel yield was found to be eightfold higher in CMCs than in DDCs (Lee et al. 2010). Tripterygium wilfordii CMCs also yielded at least twofold higher terpenoid contents than DDCs (Song et al. 2019). Across most cultivations in this work, productivities of oleanolic acid were found to be lower than of ursolic acid, which is consistent with previous reports on O. basilicum (Pandey et al. 2019). An exception was found in DDCs in SF, where both productivities were around the same level over the cultivation period, but generally very low. There was no indication for a possible cultivar influence on the triterpenoid levels of CMCs and DDCs, as was reported for other metabolites in whole basil plants before (Kwee and Niemeyer 2011;Flanigan and Niemeyer 2014), when comparing the triterpenoid levels of six Ocimum lines established in our lab to those of their parent plants (data not shown).
For the first time, O. basilicum cells were cultivated in a wDBR. In a previous study, the rosmarinic acid production of O. basilicum DDCs was shown to be increased when cultivated in a disposable airlift reactor (Kintzios et al. 2004). In general, wDBR systems were used in the past for plant cell suspensions of various uses, often yielding satisfactory results (Eibl et al. 2009). Oleanolic and ursolic acid production were not investigated before in wDBRs. Considering the negative influence of hydrodynamic stress on plant cells, most pronounced by stirring (Kieran et al. 1997), it appears reasonable to favour non-stirred cultivation systems like wave wDBRs. However, it was recently postulated that careful adjustment of stirring speed may enhance limonoid production in Azadirachta indica cell culture (Villegas-Velásquez et al. 2017). During cultivation of CMCs in a stirred tank reactor at 120 rpm stirring speed, the highest productivities of oleanolic and ursolic acid were determined at 2.26 mg/ml*d and 2.10 mg/ml*d after 13 days cultivation, respectively (Fig. S3). These values were 1.33fold (oleanolic acid) and 2.36-fold (ursolic acid) lower than the highest productivities obtained in wDBR cultivation. In summary, productivities were shown to improve when cultivating CMCs over DDCs. Among the investigated cultivation strategies, wDBR currently appears to be the best strategy to obtain high productivities in CMCs.

Elicitation
To investigate a potential increase in product contents, elicitation with 200 µM MeJa was performed in DDCs and CMCs in SF (Fig. 3). Highest increase from control in oleanolic and ursolic acid concentration in DDCs was found after 48 h incubation at 169.16 ± 19.91% and 184 ± 38.27%, respectively. In CMCs, highest increases from control were found after 48 h incubation as well at 232.30 ± 19.33% for oleanolic acid and 192.44 ± 18.23% for ursolic acid, both of which were higher than increases found in DDCs. A strong positive effect of MeJa elicitation on product contents of both metabolites is thus indicated. In contrast to these findings, a recent study reported only a slight increase in oleanolic acid concentration and a decrease in ursolic acid concentration in O. basilicum under similar conditions (Pandey et al. 2019). In whole plants, however, MeJa elicitation led to increases in both triterpenoids (Misra et al. 2014). Other studies reported increased rosmarinic acid contents following MeJa elicitation in O. basilicum cultured cells (Pandey et al. 2015) and whole plants (Kim et al. 2006). After 72 h incubation with MeJa, a decrease of both triterpenoid concentrations was determined in CMCs at 91.13 ± 28.92% for oleanolic acid and 89.05 ± 24.76% for ursolic acid. This decrease indicates a detrimental effect of prolonged incubation with MeJa. While high elicitor concentration was reported to decrease product concentration in Linum album (Baldi et al. 2010), prolonged exposition alone did not decrease metabolite content below control levels in Withania somnifera (Sivanandhan et al. 2013) and Gingko biloba (Kang et al. 2009). Secretion of triterpenoids into the surrounding media could not be detected. Despite this, MeJa elicitation appears to be an efficient tool to increase product content in O. basilicum DDCs and CMCs.

Conclusion and outlook
The establishment and cultivation of a CMC line from O. basilicum could be successfully demonstrated for the first time. Characteristic morphological features of CMCs, their relatively small size (median: 57.66 µm) and abundance of small vacuoles, were found to be consistent with previous works. Productivities of DDCs and CMCs were elucidated in shake flasks and wDBR. The highest productivities of the valuable triterpenoids oleanolic and ursolic acid were achieved in CMC cultivation in a wDBR after 19 days at 3.02 ± 0.76 mg/(l*d) for oleanolic and 4.79 ± 0.48 mg/(l*d) for ursolic acid, outperforming DDC productivities at least 1.77-fold. The findings provide a novel basis upon which the cultivation strategy for CMCs can be expanded and optimized. One enhancement is elicitation, which was shown to nearly double oleanolic and ursolic acid contents in shake flask experiments. Long-term suspension cultures of Ocimum cells are currently under investigation. The presence of triterpenoid glycosides was not shown in cultured Ocimum cells before and would also be interesting to elucidate. Future experiments should also compare process engineering parameters between stirred and wave-mixed cultivation systems. O. basilicum CMCs thus possess a great perspective for future applications as productive systems for valuable triterpenoids.
Funding Open Access funding enabled and organized by Projekt DEAL. Parts of this work (establishment of CMC cell line) has been funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)-Project-ID 172,116,086 -SFB 926.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
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