Micropropagation of Salvia wagneriana Polak and hairy root cultures with rosmarinic acid production

Abstract Salvia wagneriana Polak is a tropical species native to Central America, well adapted to grow in the Mediterranean basin for garden decoration. Micropropagation has been assessed from axillary shoots of adult plants using a Murashige and Skoog basal medium, with the addition of 1.33-μM 6-benzylaminopurine for shoot proliferation; the subsequent rooting phase occurred in plant growth regulator-free medium. The plants were successfully acclimatised with high survival frequency. Hairy roots were induced after co-cultivation of leaf lamina and petiole fragments with Agrobacterium rhizogenes and confirmed by PCR. The establishment and proliferation of the selected HRD3 line were obtained in hormone-free liquid medium and the production of rosmarinic acid (RA) was evaluated after elicitation. The analysis of RA was performed by LC-ESI-DAD-MS in the hydroalcoholic extracts. The addition of casein hydrolysate increased the RA production, whereas no enrichment was observed after the elicitation with jasmonic acid. Graphical abstract


Introduction
Salvia wagneriana Polak (subgenus Calosphace; section Cardinales) is native to Guatemala and Costa Rica, and shows characteristic showy rosy-pink flowers (Clebsch & Barner 2003). It is well adapted to grow in the Mediterranean region where it is used for its ornamental value as in the garden design, amenity planting and pot plant production.
Three new clerodane diterpenoids, hardwickiic acid and 1,10-dehydrosalviarin were extracted from the surface exudates of the aerial part of S. wagneriana plants grown in open field (Bisio et al. 2004). These last two compounds exhibited good insecticidal and antifeedant activities. Thus, this sage species may represent a new promising source of secondary metabolites and its in vitro manipulation could support a fast propagation for ornamental purposes but also favour the production of important bioactive compounds.
Various trials to improve secondary metabolite production in in vitro tissue cultures of Lamiaceae species have been reported, since the biotechnological approach represents a good method to develop the controlled production of valuable natural metabolites . In vitro undifferentiated cell cultures (callus and cell suspensions) and/or organ cultures (hairy roots, HR) were established for several Salvia species. Rosmarinic acid (RA), ursolic acid and lithospermic acid were produced by calli or cell suspension cultures in S. officinalis, S. fruticosa. and S. miltiorrhiza (Karam et al. 2003;Barberini et al. 2013;Haas et al. 2014). RA is one of the most common secondary metabolites found in several Salvia spp., (Petersen & Simmonds 2003) and for this reason it is a good marker for the evaluation of Lamiaceae secondary metabolism.
The transformation by wild-type Agrobacterium rhizogenes strains has been used to induce HR and enhance the production of secondary metabolites in many plant species . HR utilisation for the production of diterpenoids and triterpenoids was described in S. sclarea (Kuzma et al. 2008), while in S. officinalis for RA biosynthesis (Grzegorczyk et al. 2006). The hairy root tissue induction is a valuable method for the production of secondary metabolites, even if these are scarsely produced in the starting explant (e.g. Glycyrrhiza glabra Asada et al. 1998). The use of elicitors influences the secondary metabolism and an enhancement of RA and lithospermic acid B was observed after treatment of S. milthiorriza HR with methyl jasmonate (Xiao et al. 2009), while the HR of the same species showed a significant decrease in RA when endophytic bacteria were added (Yan et al. 2014).
In the frame of an European project for the valorisation of sage species, a screening of the possibility to obtain hairy roots from somatic tissues of several Salvia species has been carried out. The aim of this work was to explore the in vitro performance of S. wagneriana, an emerging species known in gardens for its ornamental value, in order to supply propagation material for its use as ornamental plant. S. wagneriana was tested for multiple use cultures and to induce the HR formation to evaluate the biosynthetic activity. The HR biomass production and its RA content after casein hydrolysate addition and elicitation with jasmonic acid were monitored.

Micropropagation of S. wagneriana
S. wagneriana was typically multiplied by cuttings in spring season, the only period that guarantees the achievement of 70% rooting (C. Cervelli pers. comm.). Therefore, the in vitro proliferation represents an ideal tool for a fast propagation useful for the ornamental pot plant production. A comprehensive investigation of the potential in vitro manipulation of S. wagneriana was undertaken, thereby allowing the authors to set up an easy micropropagation protocol for this species. The proposed protocol of explant surface sterilisation permitted to obtain 65% of aseptic explants. An adaptation phase to in vitro condition of 4 weeks was sufficient to induce the emergence of new buds. Shoot propagation was then assessed using MS basal medium alone or with the addition of BA (1.33 μM). In the total absence of growth regulators, shoots were able to elongate reaching on average 7.42-cm in height (Table S1) and develop roots (94.04%). The addition of BA to the medium assured a better multiplication rate (3.32-cm shoots per explants), although the shoots were shorter (3.54 cm) than those grown in the hormone-free medium. These shoots showed a notable ability to develop roots and the rooting percentage reached 87.37% (Table S1). Rooted plants were  transferred directly to the acclimatisation stage without further in vitro treatments. A mean acclimatisation value of 80% was observed after 50 days (data not shown), thus the plants were successfully transferred to pots. The use of MS medium added with BA determined a good shoot proliferation rate for a rapid clonal propagation.

Hairy roots induction and development
Hairy root lines were produced after the inoculation of leaf lamina or petiole fragments with two A. rhizogenes strains, the 15834 ATCC and 1855 NCPPB. After 15 days of inoculation, root primordia developed from the explants were treated with both A. rhizogenes strains. After 60 days, the petioles showed a greater percentage of putative HRs (35%) than those obtained from the leaf fragments (22%) ( figure 1(A)), even though this percentage was comparable with that obtained from S. miltiorrhiza leaves using the same Agrobacterium strain (Hu & Alfermann 1993). Therefore, the leaves of S. wagneriana were not considered as the ideal organ for the transformation. However, the ability of petiole transformation was different between the two strains; after 30 days of incubation, the percentage of putative transformed fragments, evaluated by the emergence of roots, induced by the strain 15834, was lower than that induced from strain 1855. After 60 days of culture, an opposite behaviour was observed since the tissues inoculated with the 15834 strain reached the highest value (31%, figure 1(B)).
These putative transformed roots were cut from the mother tissues and grown separately. Eleven single putative transformed lines were isolated from petioles inoculated with the 15834 strain. The transformed status of hairy root fast-growing lines was ascertained by PCR analysis with specific primers of A. rhizogenes T-DNA rolC and virC1 genes. rolC band of 514 bp was present in 8 of the 11 putative HR lines of S. wagneriana induced by 15834 A. rhizogenes strain (lines 2-12, figure 1(C)). This band was absent in the control plant (lane 1, figure 1(C)). The amplification band of the virC1 gene was detected only in the A. rhizogenes samples (data not shown), confirming that the root lines are truly transformed and not simply contaminated. The rolC positive HR lines were cloned and the HRD3 line was selected for its fast growth and transferred in the liquid medium to induce a consistent biomass proliferation. The fresh weight growth curve (figure 2(A)) showed a classical lag phase (8 days) and a further clearly defined exponential phase with a peak on day 28, whereas the dry weight exhibited a later steep exponential phase from day 28 to day 35. The HR growth started with the cell distension, followed by an increase in dry matter, as already observed in Chinese sage (Shi et al. 2007).

RA determination
It is well known that RA is a typical active constituent of many Lamiaceae spp. and it is related to the total antioxidant potential of sage and rosemary extracts (Matsingou et al. 2003). for this reason, RA was chosen as marker compound to monitor the HR biomass productivity of S. wagneriana on the basis of its higher stability to temperature, light and air exposure in comparison to carnosol (CRN) and carnosic acid (CA) (okamura et al. 1994;Mulinacci et al. 2011). No references are reported in the literature on the RA content in the wild S. wagneriana plants til now, so the aim of this work was to evaluate if the HR cultures could be able to produce this important antioxidant constituent. The in vitro HR and the correspondent liquid media samples were analysed immediately after the extraction step in order to avoid any degradation process of the active compounds. furthermore, electrospray ionisation mass spectrometry (ESI-MS) was chosen because of its soft ionisation, which allows the production of ions, even for labile compounds such as CA and CRN (Table S2). However, RA resulted the main target compound for the quali-quantitative analysis by LC-DAD-ESI-MS of the HR extracts. The LC-DAD-ESI-MS screening pointed out the presence of RA in the HRD3 line, while it was not found in the correspondent liquid culture media. No levels of CRN and CA were detected in each analysed samples. figure S2 shows the HRD3 line extract chromatogram spiked with an artificial standard solution of markers (100 μg/ml) in order to point out their different time elutions. However, our chemical analysis done on aerial wild whole plants showed only traces of RA (data not shown); this confirmed some results obtained with other sage species (e.g. S. milthiorriza) that the hairy root tissue induction is a valuable method for the production of secondary metabolites, even though not sufficiently produced in the starting plant.
Some new secondary metabolites of S. wagneriana were already isolated and determined in this plant (Bisio et al. 2004), but they were absent in HR samples and liquid media. Nevertheless, the present paper demonstrated for the first time that RA is a constituent of the hydroalcoholic extract of S. wagneriana HR, although in low amount. The selected HRD3 line showed 173-μg/g fW of RA content. Thus, the line was treated with jasmonic acid (JA) and casein hydrolysate (CH) separately to enhance the RA production ( figure 2(B)). A different RA content was detected according to the elicitation treatments applied. The use of 3.3-mg/L JA promoted a significant increase in the concentration of RA (213-μg/g fW). However, the application of a double-strength concentration of JA reduced the RA production. Interesting results were obtained with casein hydrolysate (CH) treatment because the RA content at both concentrations tested (CH200 and CH400) was twofold higher than control (491-and 466-μg/g fW, respectively; figure 2(B)).
The elicitation approach confirmed its efficacy as a system to increase RA yield. Xiao et al. (2009) demonstrated an evident increase in RA driven by methyl jasmonate in S. milthiorriza and in cell culture of Mentha x piperita by JA (Krzyzanowska et al. 2012). Casein hydrolysate is actually not considered as an elicitor, whereas it is a source of organic nitrogen, calcium, phosphate and different amino acids (Hong et al. 2012) and could be considered an enhancer of metabolism. The effect of CH on the enhancement of secondary metabolite content has been reported in cell culture of several species and in HR cultures (Brodelius et al. 1989;Bais et al. 2002).

Conclusions
The in vitro manipulation of S. wagneriana is reported in this paper for the first time showing the opportunity of a fast propagation for ornamental purposes. It is well known that hairy root cultures induced by A. rhizogenes are able to produce fast and great biomass and simultaneously biosynthesise secondary metabolites more than normal roots. The choice of RA as marker compound to monitor the HR biomass productivity indicates that S. wagneriana HR cultures are able to produce good amounts of this secondary metabolite especially after casein hydrolysate addition. further biotechnological studies are requested to better define the ability of S. wagneriana to produce other bioactive compounds.