In Vitro Propagation and Secondary Metabolite Production from Medicinal Plants: Current Trends (Part 1)

Plants in addition to primary metabolites produce secondary metabolites which are of immense pharmaceutical importance and other industrial uses. Secondary metabolites are produced due to the stress experienced by plants in response to external triggers/agents like elicitors. Elicitation involves two types of elicitors namely biotic and abiotic. Elicitors have a vital role in plant tissue culture as these improve secondary metabolite content in cultures. Other culture conditions including volume and types of medium, duration, etc., also affect the yield of alkaloids. Extensive research has been carried out for the enhanced level of alkaloids in in vitro cultured plants. Various common elicitors used in media are methyl jasmonate (MeJA), yeast extract (YE), fungal extract, ions from various salts like CdCl2, heavy metal ions, and ionic, nonionic radiations, etc. The fungal cell wall components oligosaccharides and peptides have also been used as elicitors for the induction/enhancement of secondary metabolites in plant cell/organ cultures. The influence of sample representation of biotic and abiotic elicitors, i.e., YE, Aspergillus flavus, MeJA, CdCl2 , CaCl2 , has been discussed taking a few medicinals and oil yielding plants from authors’ laboratory. A direct link of stress with elicitors including plant growth regulators (PGRs) has been established showing over accumulation of proline, protein, SOD, APX and other antioxidant enzyme activity with increased levels of elicitation. Increasing demand forces researchers to conduct further investigation in this area for the production of phyto-compounds and even for viable commercial exploitation.

The present study investigated the difference in the phytoconstituents in the methanolic extract of mother and tissue cultured plants of Scaevola taccada (Gaertn). Roxb., an important medicinal plant of the Goodiniaceae family. An efficient protocol was established to rapidly multiply S. taccada using nodal explants. The explants were cultured on MS medium supplemented with different concentrations of BAP (0.5 mg/l, 2.5 mg/l, 5.0 mg/l, 10.0 mg/l), IAA (1.0 mg/l), Kinetin (1.0 mg/l), ascorbic acid (100 mg/l) and citric acid (25 mg/l). The maximum number of multiple shoots were obtained in MS medium supplemented with BAP (5.0 mg/l) in combination with Kinetin (1.0 mg/l) and additives ascorbic acid (100 mg/l) and citric acid (25 mg/l). Subculturing multiple shoots at periodic intervals of every 4 weeks produced the maximum number of shoots. The in vitro generated shoots were rooted in half-strength MS medium supplemented with IBA (0.5,1.0,1.5,2.0,2.5) mg/l NAA (0.5,1.0,2.0,2.5) mg/l. Among these, the highest root induction was obtained in IBA (1.5 mg/l) and NAA (0.1 mg/l). The rooted plantlets were transferred to pots containing a mixture of vermiculite and perlite for acclimatization for three weeks. The plants were hardened in a greenhouse and planted in open fields. Phytochemical analysis shows the methanolic extracts of the tissue cultured plants produced more bioactive compounds having various pharmaceutical importance than the mother plant.

The anti-neoplastic herb, Catharanthus roseus (L.) G. Don (Apocynaceae), is a high-value, low-volume medicinal herb, which is the focus of global attention in view of being the source of terpenoid indole alkaloids (MIAs). MIAs are one of the largest classes of phyto-alkaloids, and many of them are sources of important pharmaceutical products. C. roseus is known to harbour more than 130 different bioactive MIAs that make it an interesting plant, finding use in several traditional and modern medical therapies. The remarkable presence of cellular and subcellular compartmentations for the synthesis and storage of MIAs allows the accumulation of these medicinally important MIAs in leaves (viz. vindoline, catharanthine, vinblastine, vincristine) and stem and roots (viz. tabersonine, ajmalicine, reserpine, serpentine, vindoline, catharanthine, horhammericine, leurosine, lochnerine). Out of them, any medicinally active MIAs found in Catharanthus roseus, vinblastine and vincristine are special since they possess anticancerous properties, along with ajmalicine and serpentine, which possess antihypertensive properties. However, the low plant yield and nonavailability of alternative chemical synthesis methods have increased their demand and market cost. In the research era of more than three decades, a plethora of studies have been carried out on C. roseus to explore, understand, explain, improve and enhance the Homo/Heterologous biosynthesis of MIAs. Metabolic engineering (ME) and synthetic biology are two powerful tools that have played and contributed majorly to MIAs studies. This chapter concentrates mainly on the efforts made through metabolic engineering and synthetic biology of MIAs in plant and microbial factories in the last three decades.

Climate change conditions affect plant growth, net primary productivity, photosynthetic capability, and other biochemical functions that are essential for normal metabolism. The stimulation of biosynthesis of secondary metabolites is an important strategy developed by plants to cope with adverse environmental conditions. Many of these metabolites display a wide array of biological and pharmacological properties (e.g., antioxidant, anti-inflammatory, antiproliferative, anti-allergic, antiviral, and antibacterial) and, thus, have valuable applications as pharmaceuticals, agrochemicals, cosmetics, fragrances, and food additives. The aim of this review is to present an overview of the impact of abiotic stress factors in the biosynthesis of secondary metabolites by in vitro cultures. Our literature survey showed that plant tissue culture has been an effective tool to understand plant response to abiotic stresses, such as drought, salinity, temperature, nutrient deficiency, or exposure to ultraviolet radiation, which is of particular interest in the actual scenario of climate change conditions. Furthermore, this technique appears as an environmentally friendly alternative for the production of high-value secondary metabolites for many applications. 

Trichomes are specialised epidermal outgrowth that is present on the aerial parts of plants. On the basis of morphological and cellular variation, they are categorized into non-glandular trichomes (NGTs) and glandular trichomes (GTs). NGTs are known to be involved in the protective and defensive roles that attribute to provide structural and chemical corroboration to form specialized groups of secondary metabolites. GTs are specialized micro-organs that are considered factories for the biosynthesis of a considerable amount of different classes of bioactive metabolites. Conventionally these glandular and non-glandular trichomes are known for their protective roles against different biotic and abiotic stresses. Recently, they have attracted the interest of various researchers as a specialized organ for the production of various bioactive molecules of high pharmaceutical and commercial values. The major groups of secondary metabolites such as terpenoids, flavonoids, phenylpropanes, methyl ketones, acyl sugars and defensive proteins are reported in the trichomes of different plant species. However, the conception of the molecular regulation of their biosynthesis, storage and distribution during the development of trichomes is scattered. This review compiles structural and functional aspects of GTs and NGTs along with the molecular mechanism regulated for the production of secondary metabolite in these specialized organs. In addition, the role of several bio-physical parameters that affect the trichome biochemistry, which either directly or indirectly influence the biosynthesis of secondary metabolite, will also be focussed. The systemized knowledge of trichome biology, secondary metabolite pathway modulation and metabolic engineering at one platform will be helpful to explore recent advances in the field of trichome engineering in many medicinally important plants. 

Plants are an important source of natural products for health care throughout the globe. Recent trends show an abrupt increase in the demand for medicinal plants due to their cost-efficiency, safety, and potency. The medicinal properties of the plants are attributable to the presence of secondary metabolites, which accumulate as the natural defense against herbivory and other interspecies defenses. Along with their medicinal uses, secondary metabolites are also used in flavorings, agrochemicals, fragrances, bio-pesticides, and food additives. The demand for secondary metabolites is mainly expedited through the collection of medicinal plants from the wild. This has provided an impetus for overharvesting medicinal plants from the wild, and many of them are threatened. The accumulation of secondary metabolites in medicinal plants is limited, and therefore diverse strategies for improving the production of secondary metabolites are a priority. Biotechnological applications, especially plant tissue culture techniques, offer a viable alternative for obtaining secondary metabolites. Along with the optimization of growth media and culture conditions, the role of plant growth regulators is vital in enhancing biomass and secondary metabolite accumulation in the culture medium. The present chapter demonstrates the types and uses of plant growth regulators with a focus on the application of plant growth regulators for the production of secondary metabolites from medicinal plants.

An indigenous reddish-brown landrace rice of the indica variety known as Hassawi rice (<i>Oryza Sativa</i> L.) is cultivated in Saudi Arabia. This rice variety has both nutritive and non-nutritive bioactive components that have therapeutic potential and promote favorable metabolic profiles. Hassawi rice has health advantages that should be further investigated, especially for the treatment of diabetes and obesity. There is a direct need for the conservation and improvement of this important germplasm source. Breeding efforts are limited, although a couple of hybrids were developed. Biotechnology approaches offer effective tools for crop genetic improvement. In this direction, in vitro regeneration of this crop has been developed that enabled the evaluation of abiotic stress factors. Furthermore, recent genomic studies revealed that Hassawi rice harbors novel alleles for salinity tolerance. This chapter reviews the research carried out on Hassawi rice in relation to nutritional and health benefits as well as secondary metabolites bioactivity and progress made on in vitro culture and genomics.

This study demonstrates a rapid, economic and efficient plantlet regeneration protocol for an exotic ornamental and medicinal plant  Allamanda cathartica L. by using shoot tip explants. Interaction of various PGRs (mT, IAA, IBA or NAA) and sucrose was tested in MS medium to obtain maximum shoot regeneration from shoot tip explants. mT (3.0 µM) + NAA (0.5 µM) + 4% sucrose was found to be an optimum combination for maximum shoot proliferation with 20.80 mean shoot number and 7.60 cm mean shoot length after 12 weeks of culture based on 93.20% responsive explants. Microshoots (4-5 cm) showed maximum rhizogenic response as they produced 4.20 mean root number with 4.90 cm root length after 4 weeks of culture on ½ MS medium when supplemented with 0.5 µM NAA. Well-developed rooted plantlets were acclimatized successfully with a 96% survival rate. The primary phytochemical screening of aqueous leaf extract in the regenerants revealed the presence of proteins, carbohydrates, lipids, phenols, proteins, and saponins. Quantification of phytochemical constituents showed that the amount of phenols was highest, followed by lipids, proteins, carbohydrates, saponins and alkaloids in the micropropagated plants. These phyto-constituents are known to cure numerous ailments.

Pharmaceuticals such as alkaloids, terpenoids, steroids, saponins, monoterpenes, flavonoids and amino acids are now being produced using plant cell culture technologies. The standardization of plant metabolite processing technologies using in vitro cultures assists in the understanding of their biosynthesis and accumulation biology. The development of metabolites in plant cell cultures is affected by a number of factors, including physical, chemical, nutritional and genetic factors. The controlled production of plant metabolites in cell cultures is a viable alternative not only for reducing pressure on the natural habitats of plant species but also for providing year-round conditions for metabolite production. Exposure of cultured cells to biotic and abiotic elicitors increased the production of plant metabolites. Hairy root induction has recently been discovered to be effective in the production of metabolites synthesized in various parts of plants.

Treatment of microbial infections has become more challenging with the evolution of antibiotic resistant microbes and indiscriminate use of antibiotics. Several phytochemicals have shown potential inhibitory action against such microbes. These antimicrobials have shown their efficacy in treating such infections. These natural products also played significant role in restoration of activity of less effective antibiotics, when used in combination with antibiotics. But still, scientists are facing some major challenges in using such metabolites for medicines- there is urgent need to explore more plants showing microbial inhibition activity, plant products from field grown plants are not sufficient to meet the growing demand and purification of antimicrobial compounds, so that dosage for patients can be finalized. Tissue culture has emerged as great technology not only in the conservation of such medicinal plants but it provides major application for the production of secondary metabolites. Various micropropagules such as calli, in vitro cultures, and cell suspensions have shown their potential for the production of pharmaceutically active compounds similar to mature plants. Production of such phytochemicals can be enhanced by manipulating media supplements, culture conditions and elicitations. As, in nature production of antimicrobials is the result of interaction between the plants and microbes, therefore, such interaction can be provided to in vitro cultures by biotic elicitation. In vitro production of antimicrobial compounds has been reported in many plants such as Ricinus communis, Calendula officinalis, Abrus precatorius, etc. Thus, plant tissue culture paves an efficient and feasible method of production of such natural compounds as an alternative of antibiotics.

Plants are active biochemical factories of a vast group of secondary metabolites (SMs) and these SMs are indeed a basic source of various commercial pharmaceutical drugs. From the prehistoric time, plants have been used for therapeutic resolutions. Medicinal and aromatic plants are the biogenic pond of diverse forms of SMs, which results in their overexploitation. There is an increasing need for the natural phytochemicals from plants for sustainable and economical value forces their mass production through in vitro plant tissue culture (PTC) methods. A vast quantity of medicinal plants and their metabolites have been developed by in vitro culture techniques in a small time period related to conventional methods. In vitro plant cell cultures assist in a potential role in the commercial production of SMs. The novel prime practices of in vitro techniques facilitate transgenic cultures and enlighten the understanding lane of regulation and expression of biosynthetic pathways. SMs have composite chemical alignment and are created in response to different forms of stress to accomplish various physiological tasks in the plant host system. They are immensely utilized in pharmaceutical industries, dietary supplements, cosmetics, fragrances, dyes, flavors, etc. SMs are also termed specialised metabolites, secondary products, toxins or natural products; these are basically organic compounds produced by plants and are not directly involved in the growth and development of the plant. Instead, they usually intervene with ecological interactions and conceivably produce selective support for the plant host by increasing its survivability or productivity. Few SMs are specific for a narrow set of plant species within a phylogenetic group. SMs habitually play a vital role in the defense systems of plants against herbivory and other interspecies defences. Human beings uses SMs mainly for medicines, pigments, flavourings and recreational drugs. Prolonged use of these SMs in several industrial areas still needs to be focused to enhance the fabrication by using in vitro PTC practices and optimizing their largescale fabrication using bioreactors. The present book chapter intends to highlight the rationale of the in vitro production of SMs from medicinal plants and their progress in the modern epoch for the mass production facts toward the step of commercial and economical forte.

Over the past few years, there has been a tremendous global shift of preference toward herbal medicine because of its affordability, accessibility, efficacy, and lesser side effects. The pharmacological and healing properties of the herbs are due to the presence of a wide array of secondary metabolites. These metabolites are biosynthesized through defined pathways and stored in various parts of the plant, like leaf, root, rhizome, bark, and floral parts. In recent years due to the growing realization of the pharmaceutical properties of medicinal plants, they have been subjected to indiscriminate exploitation. Further, the lack of agrotechnology in many cases and the nonavailability of broad genetic diversity provide impediments to their largescale cultivation and improvement. This situation has created a huge gap between the demand and supply of medicinal plants all over the world. Hence, rapidly propagating high valued medicinal plants through unconventional technologies is warranted and will provide high dividends to farmers and the herbal industry. Further, generating large-scale healthy, genetically uniform plants with defined chemical content will facilitate pre-clinical and translational studies. Therefore, efforts in the development of robust in vitro propagation systems for herbal plants can address the core concern of their conservation and large-scale utilization. Studies on cell suspension, hairy root culture, and genetic transformation have provided the desired impetus in metabolic engineering and enhanced their commercial value. The present article highlights some of these developments and provides a futuristic perspective on the subject.