Obtaining Cell Cultures of Medicinal Plants

*Torkwase Emmanuella Bulya,Tatiana V. Glukhareva and Elena G. Kovaleva*

### **Abstract**

*In vitro* propagation of medicinal plants has been incorporated into producing healthy plants that are beneficial to humanity. Some basic principles and factors tend to influence the cultivation process, thus, causing this method of plant propagation to be adapted owing to the importance and benefits surrounding this method. The main objective of this research work was to obtain cell cultures of medicinal plants of *Cichorium intybus, Stevia rebaudiana Bertoni, Monarda citriodora, and Rhodiola krylovii*. In obtaining the cell cultures of these medicinal plants, some steps need to be followed. In this research, the effect of different methods of sterilisation/cultivation of plant seeds and explants were evaluated using two different media compositions, observable differences between sterile and non-sterile plant seedlings of *C. intybus, Monarda citriodora, and Rhodiola krylovii.* The effect of growth regulator (Kinetin) and non-growth regulator (Kinetin) on the cell cultures was observed in solid and liquid media; the dry and wet weight was determined for a callus of Chicory grown in cell suspension culture. All results were presented on tables and charts.

**Keywords:** medicinal plants, callus, solid media, liquid media, suspension culture, *Cichorium intybus*, *Stevia rebaudiana Bertoni*, *Monarda citriodora*, *Rhodiola krylovii*

#### **1. Introduction**

Plant cell cultivation based on the *in vitro* process is not general for medicinal species. Therefore, many questions are yet to be dealt with regarding the cultivation and quality assessment of the plants produced via the *in vitro* method of cultivating these plant materials, such as seeds, callus, seedlings, and hairy roots. Plant cell culture is a technology that investigates some of the conditions that promote cell division and other growth factors in *in vitro* conditions, and it is considered a valuable tool in both primary and applied studies and commercial applications [1, 2]. It has been demonstrated in previous studies that factors, such as irrigation and nutrient status, affect the chemical profile and composition of plants. Since the understanding is limited of the mechanisms and activity of herbal formulations to combat disease, quality assessment is often reduced to quantifying one or a few compounds. Herbal formulations are, however, very complex, and only a few selected compounds, can be optimal to determine the changes in the chemical profile and composition of cultivated plants [3].

Medicinal plant cultivation employs basic materials, such as substrate, small mineral elements, water, and light, to grow without stress compared to animal rearing. Environmental conditions and climatic conditions can harm plants growing in an outdoor setting. Despite the changes caused by environmental factors, such as elevated temperature, high humidity, and others, thus, to avoid repeatability and uniformity across the experiment, it is necessary to define and control specific growth conditions by growing plants indoors for research [4].

The progressiveness of subsistence culturing of plants used for commercial trading in the area of medicinal plants has assumed rise to an increase in the rate at which these medicinal plants are harvested from wild habitats [5]. When harvested to an extent, every plant can be exposed to annihilation, although medicinal tree species are most vulnerable to harvesting as they are slow-growing, slow- reproducing and many have specific habitat requirements that limit their distribution [6]. Thus, plants withering owing to harvesting are, therefore, not readily replaced. For centuries the sustainable use of medicinal plants was facilitated by several indirect control methods and some intentional management practices. Some of these practices became unused as development and alteration in traditional healing practices were experienced.

Plant cell culture is a technique that investigates the conditions that influence cell division and genetic regeneration *in vitro* propagation, and it is considered an essential tool in both applied and fundamental studies, additionally as in commercial application [1, 2]. Presently, the facilities for *in vitro* cell cultures are found to be applicable in each plant biology laboratory as a helpful tool for various purposes since tissue culture has turned into a fundamental asset for modern biotechnology, from the critical biochemical aspects to the massive propagation of selected individuals. There are five main areas where *in vitro* cell cultures are being currently applied and can be recognised: As a model system for essential plant cell physiology aspects, generation of genetic modified fertile individuals, large-scale propagation of elite materials, preservation of imperilled plant species, and metabolic engineering of fine chemicals [7].

In addition, *in vitro* culture is a technique that involves the replication of new cells, tissues, and organs derived wholly from the mitotic cell division, consequently generating cloned cells, tissues, and individuals, viz., with identical genetics to the mother plant. Application of *in vitro* culture techniques to effectively produce secondary metabolites, basically plant-derived medicinal compounds, has its main advantages as follows:


#### **1.1 Importance of plant tissue culture techniques**

Plant tissue culture techniques can be widely used as biotechnological tools for basic and applied purposes. Thus, ranging from investigating plant developmental

#### *Obtaining Cell Cultures of Medicinal Plants DOI: http://dx.doi.org/10.5772/intechopen.104650*

processes, functional gene studies, commercial plant micro-propagation, and the generation of transgenic plants with explicit industrial and agronomical traits. Then, plant breeding and crop improvement, virus elimination from infected materials to render high-quality, healthy plant material, preservation, and conservation of germplasm of vegetatively propagated plant crops. And save plant species that are endangered by external factors, such as the environment. Additionally, plant cell and organ cultures are interested in producing secondary metabolites of industrial and pharmaceutical interest. Modern technologies, such as genome-editing ones combined with tissue culture and Agrobacterium tumefaciens infection, are currently promising alternatives for the precise genetic manipulation of attractive agronomical or industrial traits in crop plants [7].

#### **1.2 Basic principles of plant cell culture**

There are some basic principles of plant cell culture, which are as follows: (1) Select an appropriate explant from a healthy and vigorous plant, (2) eradicate microbial infection of an explant from the surface, (3) inoculate the explant in an adequate culture medium, and (4) provide the explant in culture with the suitable controlled ecological conditions. In the case of *in vitro* regenerated plants, they are subjected to acclimatisation process in the greenhouse before the transference to *ex vitro* conditions. *In vitro* clonal propagation is one of the most current extended commercial applications of tissue culture [9]. These principles are contingent on the part of the plant that is cultured; we can refer to them as cell culture (gametic cells, cell suspension, and protoplast culture), tissue culture (callus and differentiated tissues), and organ culture (any organ, such as zygotic embryos, roots, shoots, and anthers, among others). Each type of culture is used for different basic biotechnological applications [9].

While demand for a consecutive increase in the supply of medicinal herbs to accelerate the evacuation of natural and artificial resources, enhancing various medicinal plants in domestication, adaptation, and cultivation can emerge as an essential strategy for facing the increasing request. Overall, the tendency is towards a more significant proportion of cultivated material in all countries [10]. Most of the national and international companies worldwide, such as over-the-counter markets, the mass and niche markets, and many herb companies, choose cultivated plants because cultivated material could be confirmed as 'organic' or 'biodynamic' [11].

#### **1.3 Practical significance of medicinal plants**

In the advanced world of medicine, medicinal plants are essential as raw materials for essential drugs, although synthetic drugs and antibiotics brought about a revolution in controlling different diseases. On the contrary, these synthetic drugs are not accessible to millions of individuals, and some research has shown that thousands of plants contain potent antioxidant compounds, especially phytochemicals and vitamins, as a result of the redox properties they possess and the effect they have to quench singlet oxygen reactive species and tendency to chelate metals [12, 13]. Those living in remote places depend on traditional healers they know and trust. The judicious utilisation of medicinal herbs can even cure deadly diseases that have long defied synthetic drugs evaluated via *in vitro* assays and by *in vivo* supplementation of human and animal models [14]. Some medicinal plants predominately have similar characteristics and components. The likes of Chicory (C. intybus), Stevia (*S. rebaudiana*

*Bertoni), Bee Balm/Cambridge Scarlet (Monarda cititrodora), Rhodiola krylovii*, *and Hedysarum coronarium* are found to have some standard bioactive components (see **Table 1**).

#### *1.3.1 Chicory (*C. intybus*)*

Medicinal plants are predominantly used as food supplements. Chicory (C. intybus) is from the Asteraceae family, a biennial/perennial herbaceous plant, and the stems and leaves are usually eaten as salads. At the same time, the roots are taken as quasi coffee after roasting since there is a similarity between the taste of Chicory to coffee taste, and it is free from caffeine [15]. In medicinal applications, the whole sectors of the Chicory plant have been used, owing to its vital bioactive compounds, such as chicoric acid, vitamins, flavonoids, phenols, sesquiterpene lactones, and fructose polymer inulin, which can act as a sweetener and pre-biotic ingredient [16, 17].

Previous studies have proposed that chicory inulin enriched with oligo-fructose improves calcium absorption and promotes bone maker in the intestines of healthy post-menopausal women [18]. Supplements of chicory help reduce iron overload and aid the proper functioning of the liver [19]. Chicory inulin has a health-enhancing mechanism potential that can disrupt the activity of gut microbiota when it is enriched, and there is a change in its composition [20]. Research has shown that increased intestinal gram-negative bacteria load in persons with diabetes can be linked to the issue of higher lipopolysaccharide (LPS) production, a structural compound in gram-negative bacteria [21]. Hence, data obtained by Landmann et al [22] indicate that chicoric acid can decrease acute alcohol-induced steatosis in mice via induction of iNOS and iNOS-dependent signalling cascades in the liver when it is altered.

#### *1.3.2 Stevia (*S. rebaudiana *Bertoni)*

The stevia plant has a variety of species that are rich in taste; *S. rebaudiana* is a sweetener plant that is from the genus family Asteraceae and is referred to sweet leaf, sweet weed, honey leaf, or sweet herb. It is the sweetest when compared to other


#### **Table 1.**

*Common characteristics of some medicinal plants.*

#### *Obtaining Cell Cultures of Medicinal Plants DOI: http://dx.doi.org/10.5772/intechopen.104650*

Stevia species. Nevertheless, it has been identified around the globe to have a sweeter glycoside called stevioside that was the primary isolate from it; there are different other sweetness related phytochemicals, such as stevioside, rebaudioside A, B, C, D, E, and glucoside A, which has also been isolated from the leaves of *S. rebaudiana* [23, 24].

#### *1.3.3 Bee balm/*Cambridge scarlet *(*Monarda citriodora*)*

*M. citriodora* or *Monarda didyma*, also known as bee balm or Cambridge scarlet, is a herbaceous plant from the subgenus family Cheilyctis and from Aristatae, which originated from North America and was naturalised in Europe. These plant species have been cultivated as garden plants, food, and medicine [25].

Essential oil is a vital constituent of this plant and has been studied by many researchers due to individual species of Monarda [26].

The essential oils obtained from the leaves and flowers of *M. didyma* L. and *M. citriodora* L. cultivated in France compared to others grown in different countries had the same quantitative differences. Thus, the data collected by Collins et al. [27] show a significant component known as linalool, which was found in both flowers and leaves ranging from 64.5% to 74.2%, respectively. In the flowers, y- terpinene was 5.3% and the leaves had 0.9%. The levels of P-cymene in the flowers were 11.0%, which was higher than the composition of p-cymene in leaves at 2.1% [28].

#### *1.3.4* Rhodiola krylovii

Rhodiola is a genus plant called Hong Jing Tian; Crassulaceae comprises over 200 species. Thus, the approximated number of species is 20, which include: *Rhodiola krylovii, Rhodiola rosea, Rhodiola alterna, Rhodiola crenulata, Rhodiola quadrifida, Rhodiola sachalinensis, Rhodiola sacra, and Rhodiola brevipetiolata* [29]. These medicinal plants are cultivated in the Himalayan belt, Tibet, China, and Mongolia. Nonetheless, they are also cultivated in Europe and North America, and these plant products are sold in the market as dietary supplements [30, 31].

Rhodiola plant varies in species, and studies carried out on varieties of Rhodiola have shown salidroside to be available in all the species of the Rhodiola genus. At the same time, rosavin (rosavin, rosin, rosarian) are compounds with a certain amount of *R. rosea* L. [32]. Rhodiola plant, particularly *R. rosea*, is often grown and used in Eastern Europe and Asia to promote physical and mental health. It is cast-off as a traditional medicine for nervous system stimulation to cure depression and fatigue, improve work output, and avoid high-altitude illness, mountain malhypoxia, and anoxia [33]. In contrast, it is used in Russia and Mongolia to treat chronic illness and weakness resulting from pathogenic infection [34]. *R. rosea* has been proven to have cardiovascular protection effects [35, 36]. In addition, the Rhodiola capsule displays anti-depressive potency in patients with depression when administered in dosages of either 0.3 or 0.6 g/day over 12 weeks. Rhodiola capsule tends to improve the eminence of life and clinical symptoms. The high doses of Rhodiola capsules are better than the lower doses [37]. Extensive efforts have been put to cultivate this plant [38].

#### *1.3.5* H. coronarium

*H. coronarium L* is a perennial forage legume plant usually called French honey suckle, sulla, or sulla clover, and it is a bushy, herbaceous perennial or biennial that typically grows to 3'feet tall with a short life span. The plant is native to Northern

Africa (Algeria (N.), Morocco, Tunisia) and Europe Southwestern Europe, such as Spain. It is a plant that has been cultured as a domestic plant in the 18th century in Southern Italy and as a biennial crop for hay making, grazing, and ensiling [39]. The genus *Hedysarum L.* is approximately made up of 100 species. These are widely spread from the temperate region to boreal regions of the Northern Hemisphere. The plants of this genus occur in numerous habitats, such as deserts or seashores, alpine and arctic meadows, and stony grasslands [40, 41].

Medicinal plants can be protected through increased regulation, and the introduction of sustainable wild harvesting methods, a more viable long-term substitute is to increase domestic cultivation of medicinal plants. Cultivation also opens the possibility of using biotechnology to solve problems inherent in the production of herbal medicines. These include species misidentification, genetic and phenolic variability, variability and instability of extracts, toxic components, and contaminants. Cultivation offers the opportunity to optimise yield and achieve a uniform, high-quality product. However, the prospective cultivator of medicinal plants must make the difficult decision of which species to grow in what is a rapidly shifting and fashionprone market [42]. Plant-specialised metabolites, also known as secondary metabolites in opposition to so-called primary ones, represent a massive reserve of bioactive compounds amenable for many human applications. Among them, polyphenols are particularly desirable in food crops due to their numerous health benefits, notably their antioxidant properties [43].

The World Health Organisation has valued that over 80% of the world's population in developing countries depend primarily on herbal medicine for basic healthcare needs [38]. However, the use of herbal medicines in developed countries is also growing, and 25% of the UK population takes herbal medicines regularly. Approximately two-thirds of the 50,000 different medicinal plant species in use are collected from the wild, and, in Europe, only 10% of medicinal species used commercially are cultivated [38]. There is concern about decreasing numbers, loss of genetic diversity, local extinctions, and habitat degradation. Well-known species threatened by wild harvesting include *Arctostaphylos uva-ursi* (bearberry), *Piper methysticum*, and *Glycyrrhiza glabra* (liquorice). Thus, between 4000 and 10,000 medicinal species might now be endangered [38, 44].

The main bioactive compounds in medicinal and aromatic plants are secondary metabolites, abiotic stress such as water deficit stress, which tends to have a more significant effect on the medicinal plants'secondary metabolites, and biologically active substances [45]. Phenolic compounds are the essential constituents in the cell defence system against free radicals in abiotic and biotic stresses and are involved in various plant processes, such as growth and reproduction [46]. Results of many studies [45–47] have shown the influences of reduced irrigation or water deficit stress on active substances of medicinal and aromatic crops.

The *in vitro* culturing of medicinal plants depends on the explant and the interaction of the medium. Hence, agar, as a conventional gelling agent, has been reported to have several drawbacks that negatively affect culture growth and differentiation. The gradual uptake of nutrients in the solid medium may lead to lower nutrient availability to the plants. Hence, a reduction in growth rate [48, 49] reported that an agarsolidified medium has lower water availability and uptake by the plants than a liquid medium. This lower uptake of nutrients could explain the lower rate of development of plantlets in solid media compared to liquid media [50]. However, the use of phytagel has not been widely reported. The liquid medium was discovered to cause more roots, nodes, and leaves in the plantlets to sprout than the solid medium; in

#### *Obtaining Cell Cultures of Medicinal Plants DOI: http://dx.doi.org/10.5772/intechopen.104650*

addition, the liquid medium is cheaper than the solid medium and more economical to use than the solid medium for potato *in vitro* micro-propagation [51].

On the contrary, the factors that affect the number of polyphenols in plant tissues depend on the age or genetic traits of the plant and many external factors, such as microorganism and pest infestation, environmental factors (temperature, humidity, and moisture), which depend on altitude and time of harvest [52–54].

#### **1.4 Composition of culture media for growing medicinal plants**

*In vitro* micro-propagation comes along with plantlet acclimatisation and growth in the greenhouse or the field, which is a prospect that can be incorporated in the product of secondary plant metabolites, especially in rare or endangered species, and in those difficult to propagate [55] and slow to grow [56]. The culture media is made up of minerals; micro (Mn, Zn, Cu, B, Fe, and Mo) and macronutrients (P, K, H, Mg, S, N, and Ca), and vitamins (B1 (thiamin), B6 (nicotinic acid pyridoxine)), growth hormones/regulators/stimulators (auxins, cytokinin, gibberellins, and abscisic acid), and agar (Bacto and Purified) for solid media.

#### *1.4.1 Solid media*

Sucrose 3 g, solution of iron chelate 0.5%, macronutrients 5 ml, micronutrients 0.1 ml, vitamins 0.1 ml, Kinetin 0.1 ml (c = 1 mg/ml), 1-naphthalene acetic acid (NAA) 0.4 mg/ml or 0.25 ml, agar 0.9 g, distilled water 100 ml according to Murashige and Skoog, 1962 method for media composition of 100 ml.

#### *1.4.2 Liquid media*

Sucrose 3 g, solution of iron chelate 0.5%, macronutrients 5 ml, micronutrients 0.1 ml, vitamins 0.1 ml, Kinetin 0.1 ml (c = 1 mg/ml), 1-naphthalene acetic acid (NAA) 0.1 ml, dichlorophenoxyacetic acid 0.1 ml, distilled water 100 ml according to Murashige and Skoog, 1962 method for media composition of 100 ml.

Furthermore, to find high-frequency adventitious shoot regeneration for similar genotypes, adequate concentrations, and a combination of growth regulators, such as auxins and cytokinin (Kinetin, zeatin, and thidizuron-N-Phenyl, N-1,2,3 thiadiazol-5 urea), should be controlled in this regard. Since the type of plant tissue and concentrations of plant growth regulators in plant cell culture can meaningfully affect the growth morphogenesis of plants [57] Liquid culture system is a critical step to enhance the multiplications rates of shoots produced *in vitro* [58]. The main properties of cytokinin include releasing lateral bud dormancy and stimulating cell division [59].

#### **1.5 Vital components of medicinal plants**

Plant growth regulators, including zeatin and thidiazuron (TDZ), and physical, chemical, and biological factors can affect the morphogenesis or organogenesis of plants. Shoot regeneration and development will vary among lingonberry clones [60]. Although most medicinal products are conventional, including those containing molecules derived from medicinal plants, in this case, isolated from the whole, contain a single PDMC as a chemical marker of reference. Cinnamon is an illustrative example of these two treatments: While the use of the cinnamon bark as an infusion to treat infectious diseases characterises the use of the medicinal plant; its primary, secondary metabolite, cinnamaldehyde, which is isolated from the bark, proved to be an efficient antimicrobial agent [61] and can be used as a conventional medicament, similar to other types of synthetic antimicrobials.

#### *1.5.1 Chicory (*Cichicorium intybus L*.)*

Its species and use categorise Chicory; thus, industrial Chicory, also known as *C. intybus* L. var. sativum, is among the family of the Asteraceae, which is extensively used to produce inulin in South Africa, northern Europe, India, and Chile (Street et al., 2013). Although, the comprehension of the bioactive compounds that undergo synthesis by the biochemical pathways is farfetched [62]. Chicory as an essential plant contains four primary polyphenols: Caftaric, Chlorogenic, Isochlorogenic, and chicoric acids that are prominent in the type of *C. intybus* found in Nord-Pas-de-Calais, France [63]. Caffeic esters in Chicory have been portrayed to have antioxidant properties and potential therapeutic properties, such as anti-diabetic properties [64–66]. One of the essential phenolic compounds in Chicory is chicoric acid, also known as diacetyl tartaric acid; it is used to treat AIDS; thus, it serves as an anti-AIDS agent [67]. The root pulps of Chicory constitute a significant by-product of inulin producing industries and are used as feeds for animals. Extracts from chicory pulps contain a high quantity of pectin, a polysaccharide widely used as a gelling agent, stabiliser, and thickening agent in food [68]. Inulin is a soluble fibre that develops naturally in the chicory plant and has powerful medicinal benefits for human health, it controls and lowers fat, sugar, and calorie in the body, thus giving a tasty appeal, and it can be described as a natural fructan that tends to provide nutritional and health benefits when modified with oligo-fructose than when it is in a pure form [69].

#### *1.5.2* S. rebaudiana Bertoni

*S. rebaudiana* is referred to as a medicinal plant owing to its natural sweet attribute. This natural resource is recommended for millions of diabetic patients as part of their daily intake since it serves as a natural sweetener and contains substances that promote wellness. Also, the leaves of Stevia comprise flavonoids, antioxidants, alkaloids, water-soluble chlorophylls, xanthophyll, water-soluble inert oligosaccharides, free sugars, amino acids, essential oils, trace elements, vitamins, hydroxycinnamic acids (Caffeic), and polyphenols. Thus, the low-calorie diterpenoid steviol glycosides found in the leaves of Stevia give a sweet taste that is almost 300 times sweeter than usual sucrose [70–72]. Steviol glycosides from the leaves of Stevia are rapidly being developed into an essential ingredient for the food industries for use as sweetener and flavour garnish. The biochemical constituents contained in the plant are of benefit to the pharmaceutical industry [73].

#### *1.5.3* M. citriodora *(bee balm/*Cambridge scarlet*)*

Monarda plant contains an essential oil rich in phenolic monoterpenes, which differs according to its taxon and region of cultivation, and this was shown for medicinal plants. Thus, numerous species, such as *Monard didyma L.* (*M. citriode)* and *Monard fifistulosa* L., are used as medicinal, flavouring, and ornamental plants due to their composition. The hybrid crossing between these species may lead to dynamic hybrids with elevated decorative value and high essential oil contents [74–76]. The Monarda leaves and flowering stems contain water infusions that possess diuretic,

#### *Obtaining Cell Cultures of Medicinal Plants DOI: http://dx.doi.org/10.5772/intechopen.104650*

anti-helminthic, carminative, expectorant febrifuge, stimulant, and rubefacient properties, which help in the treatment of colds, headaches, and reduce insomnia, also acts as a stomach agent. The plant also possesses strong antifungal activity [77].

#### *1.5.4* Rhodiola krylovii

*Rhodiola krylovii* is a medicinal plant with several compositions, such as polyphenols, which include; flavonoids, tyrosol, pro-anthocyanidins and cinnamyl alcohol glycosides, organic acids, essential oils, sugars, fats, alcohols, and proteins [78]. Several types of research have demonstrated that the main composition of the Rhodiola plant is tyrosol and salidroside. These compounds possess anticancer bioactivities, antifatigue, antidepressant, antioxidant, adapt genic, anti-inflammatory, and antinociceptive, modulate immune function and prevent cardiovascular, neuronal, liver, and skin disorders [79]. Due to their bioactivity, Rhodiola plant extracts, such as tyrosol and salidroside, tend to stop ageing and attenuate age-related diseases in humans and animals [80].

#### *1.5.5* H. coronarium

*H. coronarium* (Sulla or French honeysuckle) is typified mainly by high percentages of Norisoprenoids as breakdown products of carotenoids, controlled by vomifoliol. Hence, the other main compounds include 3-hydroxy-4-phenylbutan-2 one and methyl syringate. These compounds are extractable natural volatiles and semi-volatiles distinct compounds, such as a small number of terpenes, norisoprenoids, benzene derivatives, aliphatic compounds, and Maillard reaction products, in the extracts signifies that *H. coronarium* is rather distinctive as compared to the other kinds of honey of the genus that gas chromatography and mass spectro photometer have chemically studied. However, specific markers of the honey botanical origin have not been discovered. In addition, the results obtained show that *H. coronarium* has the potential for a chemical characterisation since reliable data have been obtained so far by using high-performance liquid chromatography and headspace solid-phase micro-extraction in the initial study of *H. coronarium* natural volatiles that is extractable from Sardinian sulla honey samples. Including a variety of distinctive norisoprenoids, benzene derivatives, aliphatic compounds, and Maillard reaction products, but only a few terpenes were found [81, 82].
