**1. Introduction**

For over a century, plant tissue culture technology has been an important tool in crop improvement and development: producing disease-free plant material [1], obtaining transgenic plants [2,3], breaking dormancy, and micropropagating elite plants with highly desirable chemotype [4], thereby leading to more uniform plant production [5,6]. This is the technology for conserving in vitro germplasm of elite [7,8], rare, and endangered plant species [9–11], implementing breeding programs for innumerous crops as well as encapsulated seeds [12], and studying plant biosynthesis through cell and root cultures [12,13], the interaction between endophytes and the hostplant [14,15].

High-demand plants face great challenges: Depletion of species diversity due to overharvesting and environmental pollution affecting natural populations are strong factors that support the argument for cultivating rare and elite high-yielding medicinal plants. In addition, the cultivation of medicinal plants is the most effective way of addressing the gap between supply and demand. Breeding studies are necessary both to develop pharmaceutical plants as crops and to scale up their production [16]. Still, few success stories about breeding medicinal plants such as *Artemisia annua* L. exist. Because micropropagation is the tool of producing clones—especially with high-yielding chemotypes—for industrial purposes, it solves this target-breeding problem. Moreover, as the *Echinacea* study [17] showed, micropropagation's demonstrated ability for mass selection suggests that together with bioassays it could form part of an overall strategy to screen elite phenotype lines.

**Citation:** Moraes, R.M.; Cerdeira, A.L.; Lourenço, M.V. Using Micropropagation to Develop Medicinal Plants into Crops. *Molecules* **2021**, *26*, 1752. https:// doi.org/10.3390/molecules26061752

Academic Editors: Muhammad Ilias and Dhammika Nanayakkara

Received: 24 February 2021 Accepted: 19 March 2021 Published: 21 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Micropropagation is an in vitro technology of rapidly multiplying elite plants using modern plant tissue culture methods. It is well-known for its applications in the agro, horticultural and forestry industries, this review focuses on a less-commonly known area which is on medicinal plants and the need to develop them as medicinal crops. Li et al. [18] defined pharmaceutical crops in three distinct categories: 1) crops for the production of small therapeutic molecules (STMs), 2) standardized therapeutic extracts (STEs), and 3) large therapeutic molecules (LTMs). In addition, this review also examines micropropagation of functional food plants to ensure their development as crop.

### **2. Pharmaceutical Crops for Production of Small Therapeutic Molecules (STM)**

Drug discovery programs and the formation of knowledge of different pharmacological classes of pharmaceuticals owe much to traditional medicine in countries such as China and India [19,20]. Some natural compounds are extracted or used as templates for synthesis or as a precursor for the semi-synthesis (e.g., paclitaxel, artemisinin, podophyllotoxin, cannabinoids, galantamine, vinca alkaloids, atropine, ephedrine, digoxin, morphine, quinine, reserpine, tubocurarine etc.). Many of these compounds provide therapeutic relief for several major illnesses including cancer, Alzheimer, malaria, high blood pressure, fever, and anxiety. As researchers confirm the medicinal utility of these natural resources, they suffer depletions with the increased demand.

According to McChesney et al. [20], pharmaceutical natural substances require considerations beyond supply and demand: the establishment of successful production systems must be sustainable, environmentally safe, reliable, and affordable. Thus, the development of medicinal crops is a key factor to obtaining a commercially viable source of medicinal biomass for the pharmaceutical industry. In fact, non-stable supply sources could lead to bottlenecks that limit potentially beneficial products. For example, researchers pointed to insufficiency in the biomass supply of anti-cancer pharmaceutical ingredients such as podophyllotoxin and paclitaxel, as the major limiting factor at phase III clinical trials, which led to overharvesting of the natural resources of *Podophyllum emodi* Wall ex Royle in India [21] and *Taxus baccata* L. in Europe [22].

Given the shortage of biomass supply limiting clinical phase III trials of paclitaxel and podophyllotoxin, several laboratories engaged in different research approaches that included bioprospecting studies searching for alternate sources with high yields of the active compounds [23–25]. Clippings of cultivated *Taxus* sp. became the reliable source for production of paclitaxel [18], and Sisti et al. [26] reported methods of semi-synthesis using abundant intermediates for production of paclitaxel. Majada et al. [27] reported a procedure to obtain high-yielding *T. baccata* plantlets by screening micropropagated juvenile seedlings that accumulate 10-deacetyl baccatin III. The selected genotypes of *T. baccata* grow faster and contain high taxene content.

For its part, podophyllotoxin is the starting compound for semisynthesis of etoposide and teniposide, two potent DNA topoisomerase cancer drugs utilized in the treatment of small lung and testicular cancers, lymphomas/leukemias and the water-soluble etoposide phosphate, also known as etopophos (Figure 1). To assure podophyllotoxin supply, a buffer extraction procedure using leaf biomass of mayapple plants provides a sustainable alternative source [28]. Later, we published a survey and a database of high-yielding podophyllotoxin colonies [29,30] and an in vitro propagation protocol of Podophyllum peltatum L. to rapidly produce podophyllotoxin-rich plantlets [5].

**Figure 1.** Structures of (−)-podohyllotoxin present in *Podophyllum* sp and its commercial chemotherapeutic derivatives.

*Artemisia annua* L. is the source of artemisinin, an endoperoxide sesquiterpene lactone that is very difficult to synthesize, precursor of a common anti-malarial drug (Artemether). Artemisinin production comes from cultivated plants selected for their high artemisinin content [18]. Selection of genotypes with high artemisinin concentration in wild populations resulted in lines containing up to 1.4 percent artemisinin (on dry leaves basis). The leading commercial source, 'Artemis,' exhibited extensive variation of metabolic and agronomic traits; artemisinin content on a μg/mg dry basis for individual plants ranged 22-fold, plant fresh weight varied 28-fold, and leaf area ranged 9-fold [31].

While Ferreira and Janick [32] found that the in vitro production of artemisinin will never be commercially feasible, Wetzetein et al. [33] suggested that cultivation of micropropagated high-yielding artemisinin plants with levels above 2% and improved agronomic traits (high leaf area and shoot biomass production) may reach productivity of 70 kg/ha artemisinin using a crop density of 1 plant m<sup>−</sup>2. We include in Table 1 examples of pharmaceutical plant species classified as small therapeutic molecules STM's (18) and their micropropagation protocols to produce elite clones for higher yields. Taxol® is another success story. According to McChesney et al. [20], the path from the discovery to a pharmaceutical drug requires a viable production system (cultivation, harvest, extraction, purification and isolation) where every step of a natural product must be systematically evaluated. Micropropagation of the superior source (chemotype or variety of the species) may help

to produce biomass with a high and consistent concentration of the natural product or a precursor of the natural product that can be converted economically by semi-synthesis to the final bulk active product.

**Table 1.** Commercial sources of pharmaceuticals often used in therapies of several illnesses that are micropropagated [17].


#### **3. Phytomedicine Crops for the Production of Standardized Therapeutic Natural Products**

Herein, we describe the category of phytomedicinal crops similar to what Li et al. [18] reports regarding pharmaceutical crops for production of standardized therapeutic extracts (STEs). Additionally, we relate examples wherein micropropagation proves useful as a method for ensuring the stability of biomass supply of phytomedicines by allowing breeders to select phytomedicinal crops with an eye towards maintaining genetic consistency and the sustainability of wild plant population.

Also known as botanical drugs, herbal remedies, and herbal medicines, phytomedicines are classified in the United States as dietary supplements according to the specific claim as described in the Dietary Supplement Health and Education Act (DSHEA) of 1994 [18]. In Europe, the phytomedicines are standardized and certified medicinal products and in Asia they have a status of traditional medicine. Phytomedicinal crops relate to the cultivation of medicinal species by which a mixture of multiple active compounds commercialized as standardized products. Usually, phytomedicines are evaluated for quality as the means to ensure safety, as complex mixtures of secondary compounds, to maintain consistency is fundamental to their efficacy. Thus, authenticity and uniformity and well-defined cultivation practices and postharvest processes are essential to certify safety and efficacy. Govidaraghavan and Sucher [44] reinforce that herbal productions must follow good agricultural and collection practice (GACP), good plant authentication and identification practice (GPAIP), good manufacturing practice (GMP) before and during the manufacturing process, guided by analytical tools, and micropropagation is an important tool in ensuring uniformity and consistency in open pollinated crops.

As of today, the majority of phytomedicines are still harvested from the wild, which causes habitat destruction, genetic diversity loss, as well as ingredient mislabeling, variability and contamination. In Brazil, products are sourced from the wild, as well as from cultivation in agroforest or in small gardens. They are chosen without proper guide from health-care professionals because medical schools do not include in their curriculum the disciplines of phytomedicines or phytotherapy. In 2016 the Brazilian Health Regulatory Agency, ANVISA, officially recognized twenty-eight medicinal plant species as herbal drugs and published their monographs [45] in the first edition (Memento). The monographs are a complete therapeutic guide of phytomedicinals reviewed and accepted by ANVISA as therapies used in SUS, the public health system of Brazil. The majority of the phytomedicinals included in this first edition, was introduced to Brazil by immigrants and later became part of traditional use especially by the rural communities.

The increased consumption of phytomedicine offers an opportunity to develop medicinal plant production systems as crop. Conventional plant breeding may improve agronomic traits in association with molecular markers aiding crop development. The greatest obstacles for such a program remain predicting which extracts remain active, specifically resembling all the medicinal properties described in the ones harvested in the wild [46]. In this context, micropropagation may produce clones that could be screened using bioassays to assure bioactivity. Moraes et al. [17] used tissue culture techniques to produce *Echinacea* sp. clones and later screened those using human monocytes assays to identify high and low activity. The immune response between the two selected clones after field cultivation due to bacterial endophytes was the same [47]. The selection procedure using in vitro propagation techniques, genetic markers, and bioassay work are approaches for selection of elite germplasm [17].

Micropropagation allows one to mass generate plants with genetically identical chemotype for cultivation purposes. Reinhard [48] suggested that different chemotypes in Cat's Claw (*Uncaria tometosa* (Willd. ex Schults) DC) might have different healing properties: tetracyclic oxindole alkaloid acting on the central nervous system, and the pentacyclic oxindole alkaloid affecting the immune system. The immunological effect of both alkaloid mixtures is antagonistic and therefore may be unsuitable for therapy. For Reinhard [48], the production of safe and efficacious Cat's Claw phytomedicinal requires chemical identification prior to harvesting and perhaps even before the cultivation.

Micropropagation also allows one to select plants based on the chemical profile in order to standardize a particular chemotype. Morais et al. [49] reported that the chemical composition of *Lippia sidoides* Cham. (syn. *Lippia origanoides)* varied according to cultivation sites. Thymol is the major component of essential oil extracted from crops grown in northeast Brazil [50–52], whereas carvacrol is the major component present in *L. sidoides* harvested from Lavras, Minas Gerais [53] and 1.8-cineole, isoborneol, and bornyl acetate in São Gonçalo do Abaeté, Minas Gerais, Brazil. Standardized essential oil of *L. sidoides* is recommended for topical applications on skin, mucous membranes, mouth, throat and vaginal washings as antiseptic [45]. According to Santos et al. [53], genotypes regulate chemical polymorphism thymol and carvacrol. Phenotypical variation is likely to influence biological properties and the type of industrial application. Planting thymol or carvacrol clones ensured a high-quality biomass for safe and efficacious products [54].

Finally, micropropagation proves useful to reduce consumption pressure on potentially threatened wild populations [55]. For example, bark extraction of barbatimão to produce phytomedicine has depleted genetic diversity of *Stryphnodendron polyphythum* Mart. natural resources. The bark of this Brazilian tree is widely utilized as a woundhealing phytomedicine with anti-inflammatory, antioxidant and antimicrobial activities. Souza-Moreira et al. [55] showed that proanthocyanidins present in the bark are responsible for its healing properties. França et al. [10] published an efficient micropropagation protocol to produce barbatimão plantlets, while Correa et al. [56] defined the conditions for in vitro germplasm conservation to reduce pressure on its threatened status. Table 2 includes in vitro propagation protocols to produce healthy plantlets for cultivation purposes, thus aiding the development of phytomedicinal crops.

As the above paragraphs state, micropropagation can provide an effective technique to those seeking to mold a supply chain of a product, in order to ensure the genetic homogeneity of plant clones, chemical profile, and finally sustainability of those plants harvested in the wild.


**Table 2.** Micropropagation protocols of medicinal plants considered phytomedicine by the Brazilian Regulatory Agency (ANVISA).
