1. Introduction
Salvia bulleyana Diels, commonly named
Zi Danshen, is one of the endemic Chinese
Salvia species that overgrows the northwest hillsides of Yunnan Province [
1]. Its roots have been used as an equivalent of Danshen (dried and micronized roots of
Salvia miltiorrhiza) [
2]. In traditional Chinese medicine, the phrase
Zi Danshen means “to have healing properties”, and the plant is used locally to soothe irritability and insomnia [
2]. It is also known as an effective herbal agent in coronary heart disease, and liver and kidney dysfunctions [
2]. Two main classes of secondary metabolites have been isolated from the roots of
S. bulleyana: phenols and tanshinones [
2,
3]. The main compounds of the raw material are two polyphenolic acids: rosmarinic acid (RA) and salvianolic acid K (SAK) [
2,
4,
5,
6].
Many studies have confirmed that RA has strong antioxidant properties [
7]. Due to the current consumer concerns about the safety of synthetic preservatives, plant extracts rich in polyphenols such as RA are increasingly being used to extend the shelf life of food and cosmetics [
8]. RA has already been investigated (with positive results) as an antioxidant in many food models, such as beverages [
9], dairy products [
10], processed meat [
11], or edible oils [
12]. Moreover, it has been found to demonstrate bacteriostatic and bactericidal activities, making it an even more promising candidate to be a natural preservative [
13,
14]. An additional advantage of RA is its health-promoting properties confirmed in many in vitro and in vivo models. The antiradical potential of RA together with antiinflammatory activity by inhibiting various enzymes, including lipoxygenase, cyclooxygenase, nitric oxide synthase, or myeloperoxidase, play a key role in its protective cellular effects [
15,
16]. The compound was found to decrease inflammatory cytokine expression by modulating key adipogenic transcription factors [
17]. RA can also prevent and treat atopic dermatitis and allergies [
18] and has demonstrated therapeutic potential against some cancers, e.g., colon, skin, or breast cancer [
18] by a multidirectional mechanism, i.e., by inhibiting proliferation and migration and inducing apoptosis [
19].
Due to these properties, the demand for RA and its industrial importance is growing. However, its content in the roots of various sage species rarely exceeds 1% of dry weight and is often many times lower [
20]. Additionally, environmental fluctuations strongly affect plant growth and the phytochemical profile, resulting in qualitative and quantitative variations in secondary metabolites [
21]. Meanwhile, plant biotechnology can offer alternative methods for obtaining valuable plant material with a more stable constituent profile and without degrading the environment. In vitro cultures are independent of geographical factors and seasons, allow for the elimination of biological contaminants (bacteria, fungi, viruses, parasitic insects), and offer efficient production in a shorter period of time than in traditional field conditions [
22]. Moreover, in vitro cultures offer the possibility to increase the biosynthesis of bioactive compounds by simple strategies such as selecting highly-productive lines, modifying a medium’s components, or changing the physical conditions of the culture (temperature, lighting) [
23].
One of the most studied and promising in vitro plant systems are hairy roots. They are characterized by having a high stability, and selected clones may demonstrate rapid growth in a medium without regulators [
24]. In our previous research, we established a procedure for obtaining hairy root cultures of
S. bulleyana that are rich in phenolic acids including RA [
25] and might be developed into a novel promising source of those compounds. For this to happen, further improvement in the culture’s productivity should be performed by optimization of its growth conditions.
As a multicriteria decision making process, optimization of a culture’s performance is a complex task. Preferably, the optimal culture should be characterized by superior growth parameters and maximum production of all the important phytochemicals. However, it might not always be possible to maximize all the criteria simultaneously, as conditions promoting growth might not be ideal for metabolite production and vice versa. To facilitate the decision making in such cases, many statistical/mathematical tools have been developed. Attempts have also been made to use artificial intelligence models and optimization algorithms in order to improve plant tissue culture [
26]. The methods are becoming more promising for modeling and optimizing complex systems to achieve better results in less time; however, despite their potential, their use is still limited due to complex definition terms and computational algorithms. Such complexity is not always required, and, if possible, simpler tools that allow satisfactory results to be obtained should be applied. A very simple tool that can be used for the selection of the best option from several available variants based on a couple of chosen parameters is the technique for order of preference by similarity to ideal solution (TOPSIS). It allows a solution to be selected from the set of alternatives that is closest to the theoretical ideal best option and farthest from the theoretical worst option. Among the advantages of the method are its objectivity, rationality, and simple computation process [
27]. The method has been successfully applied in many areas of industry [
27]; however, its potential in plant biotechnology has so far been underestimated.
The aim of this study was to optimize for the first time the growth conditions of the previously selected clone of S. bulleyana hairy roots as a novel source of phenolics. The study examines the influence of different media compositions and concentrations, and vitamin and sucrose contents, as well as the effects of various light conditions on secondary metabolite accumulation and the growth of the transformed root culture. In ambiguous cases, TOPSIS analysis was applied to identify the optimal conditions, which is a newly emerging approach in biotechnological studies. Finally, the research included a detailed analysis of culture growth and the production of bioactive compounds every five days over a 50-day growth cycle to determine the optimal harvest time.
3. Discussion
The present work is a follow-up to previous research, in which an efficient methodology for obtaining S. bulleyana hairy roots was established. From the obtained clones, the one characterized by the highest biomass and phenol accumulation (clone C4) was taken for a further optimization process. In the present study, we examined for the first time the effects of selected culture conditions, in terms of basal media, and vitamin and sucrose concentrations, as well as light treatment, on improving productivity, with the aim of optimizing polyphenol production in the hairy roots of S. bulleyana.
Contemporary in vitro plant cultures used defined media with specified concentrations of individual macro- and microelements and vitamins to obtain reproducible results. The present study used four popular basal media for root cultivation, both with full (MS, WP, B5, SH) and half macro- and microelement contents (½MS, ½WP, ½B5 and ½SH).
The best results were revealed for
S. bulleyana roots cultivated in SH basal media (SH and ½SH). These media have also been found to be particularly advantageous for the hairy roots of
Levisticum officinale, Pimpinella anisum, and
Angelica gigas [
28,
29,
30,
31]. Both SH and ½SH are characterized by the lowest calcium (Ca
2+) content among the used media. Calcium is an essential element for plant metabolic processes, although its high level could result in phosphate precipitation, the disruption of related metabolic pathways, and interference with Mg
2+ function [
31]. These media also contain higher levels of micronutrients such as cobalt and iodine in comparison to the others but lower levels of molybdenum and zinc. It is possible that changes in calcium, molybdenum, and zinc ion concentration may have a greater influence on changes in secondary metabolite production in the
S. bulleyana culture rather than plant growth. For instance, the ½B5 medium demonstrated half the accumulation of root biomass of SH, despite having similar calcium, molybdenum, and zinc contents.
Moreover, the SH media were distinguished by an extremely low ratio of NH
4+ to NO
3− ions. Nitrogen in SH media is supplied primarily in the form of NO
3−, and NH
4+ ions account only for about 10% of the total amount of nitrogen ions supplied, while that value is 30–40% in the case of the other media used in this study. Some researchers suggest that low total nitrogen content in the medium is beneficial for the growth of hairy roots; such conclusions have been reported for cultures of the medicinal plants
Anisodus acutangulus [
32],
Gmelina arborea [
33] a and
Salvia viridis [
34]. Meanwhile, SH media contain an intermediate content of total nitrogen compared to other media used; thus, the nitrate to ammonium ratio in the medium seems to have a more significant effect on
S. bulleyana roots than the total amount of nitrogen. George et al. [
31] reported that root growth is often promoted by NO
3− and depressed by NH
4+. Thus, root culture could prefer media containing no NH
4+, or very little. Additionally, the NO
3−/NH
4+ balance significantly influences the pH of the medium: the use of ammonium ions before nitrates acidifies the medium. Consequently, media with higher NH
4+ content could inhibit culture growth to a greater degree. Other researchers also suggest that a high ammonium ion level in the medium can depress the uptake of some metal ions, such as calcium, magnesium, and potassium, which in turn could restrict the nutrient flow into the plant tissue and lower the biomass yield [
35]. Some studies, similarly to the present results, also indicated that ammonium ions are necessary for culture growth; however, only a small amount is needed for optimal hairy root growth and further increases in their content resulted in a decrease in the DW of hairy roots [
36,
37].
Additionally, SH and ½SH media are rich in vitamins; in particular, they contain 5–10 times higher amounts of nicotinic acid compared to the other media used in this experiment. These media are also distinguished by a 10-times higher content of inositol. Despite being a carbohydrate (hexitol), inositol is not regarded as a carbohydrate source for in vitro culture, but rather as a vitamin-like growth enhancer. In particular, inositol promotes cell and protoplast division, may have a role in the uptake, storage, and utilization of ions, as well as possibly participating in stress responses or cell-to-cell communication [
38]. Inositol is also important for the storage and transport of auxins, which control the growth of plant tissues. Although it is not itself a hormone, it may be responsible for controlling the functionality of the phytohormones. Inositol has been hypothesized to form conjugates with auxins, thus allowing their safe storage and/or transport. Moreover, these conjugates regulate the availability of active auxins for physiological responses as needed [
39].
The vitamin content appears to have an important effect on the biomass and polyphenol accumulation in the hairy roots of S. bulleyana. Therefore, the study also estimated the effect of medium vitamin concentration on the culture efficiency. Hence, SH and ½SH media were prepared with half and quarter vitamin concentrations, as well as without vitamins. Despite reducing the vitamin content to half or a quarter, concentrations of nicotinic acid and inositol in SH media were still higher than in the other media used in the experiment, and this change did not adversely affect the growth of the culture. However, in the SH medium, reducing the vitamin content resulted in a gradual decrease in the content of bioactive compounds in hairy roots. In contrast, in the ½SH medium, reducing the vitamin level to half stimulated the production of most polyphenolic acids; only a complete lack of these nutrients drastically reduced the level of secondary metabolites, similar to the SH medium. Thus, ½SH medium with half of the vitamin content demonstrated the greatest accumulation of secondary metabolites in the cultures. In this case, the productivity was 924.4 mg/L for TPC, 741.8 mg/L for dominant RA, and 76.6 mg/L for SAK.
Another parameter tested for the root growth optimization was the sugar content. The choice of carbon source plays an important role in the growth, development, and production of hairy roots and other in vitro cultures [
40,
41]. Sugar is believed to increase metabolite production in some species due to elevated levels of osmotic stress [
42]; however, excessive osmotic pressure could disturb the exchange of components and water between the medium and plant tissues and inhibit their growth. Weremczuk-Jeżyna et al. [
41] found 3% to be the optimal sucrose concentration for growth and RA production in
Dracocephalum forrestii roots. However, other studies found lower (1%) or higher (4%) concentrations to be optimal for the accumulation of biomass and secondary metabolites in transformed roots [
40,
43]. It can be seen that no specific sucrose concentration is preferable in all cases. Therefore, the present study examined the effects of four different sucrose concentrations (2%, 3%, 4%, 5%).
It was found that media with 2% and 3% sucrose supported the TPC in
S. bulleyana root culture. Of these, 3% sucrose resulted in a significantly higher accumulation of dry mass, resulting in more efficient productivity of most phenolic acids. However, the levels of SAK and RAH significantly increased in the presence of higher sucrose concentrations (4 and 5%). With regard to RAH, as in the case of other secondary metabolites with a sugar moiety, sucrose enrichment may have increased the glycosidation process. Similar results were also reported for anthocyanin production in
Panax sikkimensis cultures [
44].
LED treatments have also been reported to enhance the biosynthesis of bioactive metabolites in vitro [
45,
46]. It was observed that light could regulate the expression of genes responsible for the biosynthesis of the phenylpropanoid pathway enzymes that synthesize polyphenolic acids [
46,
47]. The presence of blue light significantly increased the PAL (phenylalanine ammonia-lyase) activity in strawberry fruits [
46], the initial step enzyme of the polyphenol pathway, as well as other enzymes, including shikimate dehydrogenase, tyrosine ammonia-lyase, cinnamate-4-hydroxylase, 4-coumarate/coenzyme A ligase, chalcone synthase, dihydroflavonol-4-reductase, and flavanone-3-β-hydroxylase. Blue light has been found to stimulate the production of polyphenolic acids in some
Lamiaceae species, such as
Ocimmum basilicum,
Agastache rugose, or
Dracocephalum forrestii [
45,
48,
49].
However, in the present study, all types of LED treatment inhibited secondary metabolite biosynthesis in
S. bulleyana roots compared to roots cultivated in the dark. This is by no means an unusual finding as root growth naturally takes place in the absence of light. Transformed root cultures are also usually cultivated in the dark, and light conditions have little effect on their growth and metabolite accumulation. Such observations were reported in the case of
Dracocephalum moldavica [
50] and
Salvia officinalis [
51]. Different LED treatments also showed no significant effect on the biomass accumulation and content of salvianolic acid B in hairy roots of
Salvia miltiorrhiza, while only a slight increase in RA levels was observed for those roots exposed to certain types of mixed light, e.g., blue with red, far-red, and green light [
52]. In addition, light can even act as a stress factor, inhibiting the growth of root cultures, as observed for
S. bulleyana culture. Similarly, the
Rhaponticum carthamoides and
Pimpinella anisum hairy roots cultivated under light or photoperiod conditions demonstrated impaired growth in comparison to those kept in darkness [
29,
53].
Finally, based on our detailed analysis of growth and secondary metabolite production, we can propose an optimal harvest time for the plant material. During the 50-day growth period, the fresh weight of the hairy root culture of
S. bulleyana increased 14-fold and the dry weight 16-fold, reaching a maximum on days 40–50. The highest total phenol content including RA level was achieved during the stationary phase. A study of
Conodopsis pilosula hairy roots achieved maximum biomass on day 50 of culture, and, similarly to the present study, the highest secondary metabolite content was reached during the stationary phase [
54]. In addition, in
Dracocephalum forrestii and
S. viridis culture, the greatest polyphenolic acid accumulation was also recorded in the stationary phase [
34,
41].
Ultimately, the optimized
S. bulleyana hairy roots accumulated 93.6 mg TPC/g DW during 40 days of cultivation; this value was more than twice that obtained in the same plant material before optimization (39.6 mg/g DW) [
25] and four times higher than in the roots of field-grown plants in the second year of cultivation [
4]. Furthermore, the accumulation of RA in the optimized
S. bulleyana culture on day 40 (70 mg/g DW) was almost twice as high as was found for clone C4 before optimization [
25] and eight times higher compared to the roots of the mother plant [
4]. This amount of RA was many times higher than noted in the roots of 26 other field-grown species of Chinese sage used as Danshen [
2]; it was also higher than observed in the roots of some other
Lamiaceae species appreciated for their high RA content, such as lemon balm, mint, rosemary, and oregano [
55]. Only a few papers have reported similarly high RA content in other optimized transformed root cultures [
50,
56,
57,
58].
The presented modifications of the cultivation conditions also allowed for the increased accumulation of other phenolic acids in the transformed roots of
S. bulleyana. The optimization resulted in a seven-fold increase in the production of SAE, an eighteen-fold increase in RAH, and a two-fold increase in MR in comparison to the original unmodified roots [
25], although the maxima for individual metabolites were not reached on the same growth day. On the other hand, SAK was the only compound whose level, even after optimization, was slightly lower than in the roots of the field plant [
4]. However, also for this compound, the selection of appropriate cultivation conditions and harvest time increased its content several times. It is also noteworthy that the
S. bulleyana culture grown in vitro requires a much shorter time to harvest (40 days vs. 2 years), does not degrade the crop during root harvesting, and is free from microbial and other environmental contaminations that are frequently encountered in the field-cultivated plants.
It was also demonstrated during the study, that TOPSIS analysis is a suitable and convenient tool for facilitating the optimization of growth conditions of in vitro plant cultures. By selecting a couple of the most relevant optimization aims, it is possible to indicate an objective solution approaching the ideal scenario as closely as possible. Thus, this approach might be recommended for a wider application in biotechnological studies.