Species-specific secondary metabolites from Primula veris subsp. veris obtained In Vitro adventitious root cultures: an alternative for sustainable production

Primula veris subsp. veris L. is a perennial herbaceous and medicinal plant species the roots and flowers of which are a source of valuable pharmaceutical raw materials. The plant tissues are used to produce expectorant and diuretic drugs due to their high content of triterpene saponins and phenolic glycosides. Underground roots of P. veris can be obtained only through a destructive process during the plant’s harvesting. In the present study, an in vitro adventitious root production protocol was developed as an alternative way of production, focused on four species-specific secondary metabolites. Root explants were cultured in Murashing & Skoog liquid medium supplemented with 5.4 µM α-naphthaleneacetic acid, 0.5 µM kinetin, L-proline 100 mg/L, and 30 g/L sucrose, in the dark and under agitation. The effect of temperature (10, 15 and 22 ◦C) on biomass production was investigated. The content of two flavonoid compounds (primeverin and primulaverin), and two main triterpene saponins (primulic acid I and II) were determined after 60 days of culture and compared with 1.5-year-old soil-grown plants. The accumulated content (mg/g DW) of bioactive compounds of in vitro adventitious roots cultured under 22 ◦C was significantly higher than the other two temperatures of the study, being 9.71 mg/g DW in primulaverin, 0.09 mg/g DW in primeverin, 6.09 mg/g DW in primulic acid I, and 0.51 mg/g DW in primulic acid II. Compared to the soil-grown roots (10.23 mg/g DW primulaverin, 0.28 mg/g DW primeverin, 17.01 mg/g DW primulic acid I, 0.09 mg/g DW primulic acid II), the in vitro grown roots at 22 ◦C exhibited a 5.67-fold higher content in primulic acid II. However, primulic acid I and primeverin content were approximately three-fold higher in soil-grown roots, while primulaverin content were at similar levels for both in vitro at 22 ◦C and soil-grown roots. From our results, tissue culture of P. veris subsp. veris could serve not only for propagation but also for production of species-specific secondary metabolites such as primulic acid II through adventitious root cultures. This would therefore limit the uncontrolled collection of this plant from its natural environment and provide natural products free from pesticides in a sustainable wa

Effects of paclobutrazol and chlormequat on growth and flowering of lavender

Growth retardants allowed production of more uniform, compact, flowering plants for commercial use of lavender (Lavandula stoechas). Paclobutrazol at 200, 400 and 600 mg.L-1 (ppm) was sprayed in a single or double application. Chlormequat was applied at 4000, 6000 and 8000 mg.L-1 in single, double or triple spray applications repeated every 13 days. Paclobutrazol reduced lateral shoot elongation and plant height, increased the number of nodes within lateral shoots, but delayed time to anthesis. In contrast, chlormequat reduced plant height with no effect on flowering. Paclobutrazol potentially may be used commercially on lavender at rates of 200 to 400 mg.L-1 in single or double applications. The same result may be achieved with chlormequat by using 4000 to 6000 mg.L-1 in three or more applications

Rose productivity and physiological responses to different substrates for soil-less culture

Cultivation of roses in various soil-less media was studied with the aim to identify the optimum soil condition for rose production. Madelon roses grafted on rootstock of Rosa indica var. major were transplanted to polyethylene bags containing zeolite and perlite (at ratios of 25z:75p, 50z:50p, 75z:25p and 100z:0p, v/v) in a climate-controlled greenhouse. Net photosynthesis (Anet), stomatal conductance (gs) and water use efficiency (WUE) of roses were followed for 5 months. Flower production and quality were recorded in three flowering flushes during a 5-month period. Analysis of variance of repeated measurements showed that even though the overall Anet did not differ among treatments (average 18.7 μmol m−2 s−1), trends in Anet seasonality for roses in 25z:75p substrate differed significantly from those in 50z:50p, 75z:25p or 100z:0p. Stomatal conductance did not show any significant seasonality or trends in response to substrate mixtures, averaging 0.89 mol m−2 s−1. Water use efficiency was significantly lower for roses in 25z:75p than in 100z:0p mixtures (1.8 ± 0.15 and 2.0 ± 0.13 μmol m−2 s−1 CO2 /mmol m−2 s−1 H2 O, respectively). Cumulative production of rose plants did not differ among substrate mixtures. Productivity significantly differed among flower stem classes. Stem class I (\u3e70 cm) and class V (≤30 cm) exhibited the least production, contributing to only 7.6 and 3.7% of the total production, respectively. The highest productivity was observed in classes III (51–60 cm) and IV (31–50 cm), contributing to the bulk of productivity (68.4%). Class II contributed a 20.3% of the production. Results showed that zeolite and perlite acted as inert materials. Zeolite did not exert any positive effect on productivity, in contrast to what has been reported in literature recently. Use of perlite resulted in a little improvement in photosynthesis, however this improvement was not reflected by a significant increase in production

Non-polar secondary metabolites and essential oil of ex situ propagated and cultivated Sideritis syriaca L. subsp. syriaca (Lamiaceae) with consolidated identity (DNA Barcoding): towards a potential new industrial crop

Over the past decade, perennial plants of the genus Sideritis L. have attracted great scientific interest and they have become the subject of several studies, including the European Union's herbal monograph. The present study aimed to explore the identity of a biotype of S. syriaca subsp. syriaca documented with DNA barcoding, investigated its macro- and micro- propagation and to study the chemical profile of the dichloromethane extract, as well as of the Essential Oil (EO) of the ex situ propagated and cultivated plant material. An improved in vitro propagation protocol, as well as pioneer effective propagation with cuttings (both at commercially acceptable rates) are provided for a DNA-barcoded biotype of Sideritis syriaca L. subsp. syriaca (malotira, Cretan mountain tea) which is a single-island endemic plant of Crete (Greece), with declining wild populations due to over collection. From the aerial parts of ex situ propagated and cultivated material (harvested in July) two phytosterols (β-sitosterol and stigmasterol), two ent-kaurene diterpenes; siderol and eubotriol, as well as one methylated flavone (xanthomicrol) have been isolated. This is the first time that eubotriol has been isolated from this taxon. The structures of the isolated compounds have been established by spectroscopic means and the complete Nuclear Magnetic Resonance (NMR) assignments for eubotriol are presented. Moreover, the essential oil was obtained by hydrodistillation of the plant material during the flowering stage and was analyzed by Gas Chromatography-Mass Spectrometry (GC–MS). Forty-four compounds were identified. The monoterpene hydrocarbons constituted the major fraction of the essential oil (34.2%), while β-phellandrene (18.5%) and kaur-15-ene (or ent-kaur-15-ene; 17.3%) were the main compounds. In conclusion, the above results document the first necessary steps taken for the ex situ conservation and sustainable exploitation of a new industrial crop with promising potential. © 2020 Elsevier B.V

Endophytic Bacteria From the Roots of the Medicinal Plant Alkanna tinctoria Tausch (Boraginaceae): Exploration of Plant Growth Promoting Properties and Potential Role in the Production of Plant Secondary Metabolites

Alkannin and shikonin (A/S) are enantiomeric naphthoquinones produced in the roots of certain plants from the Boraginaceae family such as Lithospermum spp. and Alkanna spp. They possess antimicrobial, anti-tumoral and wound healing properties. The production of secondary metabolites by Alkanna tinctoria might be influenced by its endomicrobiome. To study the interaction between this medicinal plant and its bacterial endophytes, we isolated bacteria from the roots of wild growing Alkanna tinctoria collected near to Athens and Thessaloniki in Greece. Representative strains selected by MALDI-TOF mass spectrometry were identified by partial 16S rRNA gene sequence analysis. In total, 197 distinct phylotypes of endophytic bacteria were detected. The most abundant genera recovered were Pseudomonas, Xanthomonas, Variovorax, Bacillus, Inquilinus, Pantoea, and Stenotrophomonas. Several bacteria were then tested in vitro for their plant growth promoting activity and the production of cell-wall degrading enzymes. Strains of Pseudomonas, Pantoea, Bacillus and Inquilinus showed positive plant growth properties whereas those of Bacteroidetes and Rhizobiaceae showed pectinase and cellulase activity in vitro. In addition, bacterial responses to alkannin and shikonin were investigated through resistance assays. Gram negative bacteria were found to be resistant to the antimicrobial properties of A/S, whereas the Gram positives were sensitive. A selection of bacteria was then tested for the ability to induce A/S production in hairy roots culture of A. tinctoria. Four strains belonging to Chitinophaga sp., Allorhizobium sp., Duganella sp., and Micromonospora sp., resulted in significantly more A/S in the hairy roots than the uninoculated control. As these bacteria can produce cell-wall degrading enzymes, we hypothesize that the A/S induction may be related with the plant-bacteria interaction during colonization. © Copyright © 2021 Rat, Naranjo, Krigas, Grigoriadou, Maloupa, Alonso, Schneider, Papageorgiou, Assimopoulou, Tsafantakis, Fokialakis and Willems

Cutting-edge analytical technologies for the comprehensive metabolic profiling of Alkanna tinctoria roots cultured in greenhouse conditions

The use of plants containing naphthoquinone derivatives Alkannins & Shikonins (A/S) by humans dates back to ancient times. In recent decades, the use of A/S has seen a resurgence and A/S have risen to a pivotal role as pharmaceutical and cosmeceutical agents, since they possess strong wound healing, antimicrobial, anti-inflammatory, tissue regenerative and antitumor properties. It is thus crucial to enhance the biosynthesis of bioactive A/S in Alkanna tinctoria plants, that naturally produce high amounts of these metabolites [1]. In the frame of “MICROMETABOLITE” EU H2020 project, we have optimized a workflow for the metabolic profiling of A. tinctoria roots, cultured in the greenhouse from plants obtained by in vivo shoot cuttings. A fast and reliable extraction procedure was achieved for comprehensive profiling and identification of A/S and other metabolites biosynthesized in the roots. The aim of this work was to determine the growth stage with peak A/S production, while simultaneously obtaining additional information on the root metabolome. A combination of UHPLC-HRMS and NMR was used for metabolite identification, HPLC was utilized for reliable quantitation of A/S and the extracts were subjected to chiral HPLC analysis [2] for determination of the enantiomeric A/S ratio. Different A/S derivatives and other metabolites were identified in plant roots using UHPLC-HRMS and NMR. Six A/S derivatives and total A/S were quantified using HPLC-DAD. From six vegetation stages of A. tinctoria grown under greenhouse conditions, fruiting period was found to peak A/S production (1% wt/wt of root), while the enantiomeric alkannin/shikonin ratio remained constant (93.7%)