In Vitro Propagation and Secondary Metabolite Production from Medicinal Plants: Current Trends (Part 2)

Senna alata is an ethnomedicinal plant. The crude extracts of the plants are said to have a large number of medicinal properties due to their phytochemicals. In the present study, we made an attempt to isolate and screen the phytochemical constituents present in the species. In order to determine the bioactive constituents present in S. alata, and the effect of drying on the loss of bioactive constituents, studies on a set of pharmacognostical parameters were conducted on seeds, shade and sun-dried leaves of S. alata as per US pharmacopeia and WHO guidelines. The results of the present studies showed the presence of various important bioactive molecules that are responsible for the medicinal properties of the species. The phytochemical analysis of seed extracts revealed the presence of alkaloids, flavonoids, tannins, saponins, anthraquinones, resins and glycosides in all the extracts, while coumarins, phenols, terpenoids, phlobatannins and quinines are completely absent in all the seed extracts. Preliminary phytochemical investigations from shade and sun-dried leaf extracts showed alkaloids, flavonoids, anthraquinones, saponins, glycosides and tannins in high amounts in all the extracts, resins and phenols are present in moderate amounts. Terpenoids and phlobatannins are present only in fresh leaf extracts. Studies were also conducted on the physicochemical and organoleptic properties of leaves of S. alata that help in the identification and standardization of the leaf extracts for manufacturing of plant-based drugs of S. alata.

Plants are an immense source of phytochemicals with therapeutic effects and are widely used as life-saving drugs, and other products of varied applications. Plant tissue culture is a unique technique employed under aseptic conditions from different plant parts called explants (leaves, stems, roots, meristems, etc.) for in vitro regeneration and multiplication of plants and synthesis of secondary metabolites (SMs). Selection of elite germplasm, high-producing cell lines, strain enhancements, and optimization of media and plant growth regulators may lead to increased in vitro biosynthesis of SMs. Interventions in plant biotechnology, like the synthesis of natural and recombinant bioactive molecules of commercial importance, have attracted attention over the past few decades; and the rate of SMs biosynthesis has increased manifold than the supply of intact plants, leading to a quick acceleration in its production through novel plant cultures. Over the years, the production of SMs in vitro has been enhanced by standardising cultural conditions, selection of high-yielding varieties, application of transformation methods, precursor feeding, and various immobilization techniques; however, most often, SM production is the result of abiotic or biotic stresses, triggered by elicitor molecules like natural polysaccharides (pectin and chitosan) that are used to immobilize and cause permeabilization of plant cells. In vitro synthesis of SMs is especially promising in plant species with poor root systems, difficulty in harvesting, unavailability of elite quality planting material, poor seed set and germination, and difficult to propagate species. Thus, the present article reviews various biotechnological interventions to enhance commercially precious SMs production in vitro.

Withania somnifera (L.) Dunal, commonly known as ashwagandha or Indian ginseng, is an important medicinal plant that belongs to the family Solanaceae. Ashwagandha has been used from time immemorial in different systems of medicine and extensively used in the Indian system of medicine, and there is discussion of this plant in different ayurvedic scripts like Charaka samhita, Ashtanga sangraha, etc. The plant is extensively used for anti-aging and general well-being, and also has anti-cancer potential. Ashwagandha is also known for its antioxidant, anti-inflammatory, and other therapeutic activities. In the recent days of Covid-19, the plant has been extensively used as an immunostimulant. The plant has great potential for its raw materials, especially for the extraction of bioactive molecules like withanolide-A, withaferin-A, withasomniferin, withanone, etc. The conventional mode of propagation could not meet the required commercial demand for either the pharmaceutical industries or the traditional practitioners. The conventional method of obtaining biomass is influenced by a large number of environmental factors, where biomass quality and quantity of bioactive molecules have shown variation. To overcome this, biotechnological approaches such as plant tissue culture techniques have been established for large-scale cultivation using micropropagation and also other techniques like a callus and cell suspension culture, shoot culture, adventitious root culture, and hairy root culture have been extensively used for in vitro production of bioactive molecules from ashwagandha. With the advent of metabolic engineering, biosynthetic pathway editing has made it possible to obtain higher yields of desired metabolites. The present chapter focuses on the in vitro propagation, biosynthesis of withanolides, and tissue culture strategies for obtaining high biomass and metabolites. The chapter also focuses on different elicitation strategies, metabolic engineering approaches, and the development of elite germplasms for improved metabolite content. The chapter also identifies research lacunas that need to be addressed for the sustainable production of important bioactive molecules from ashwagandha.

Plants are indispensable for the preservation of human life. They supply us with oxygen, food, fuel, and shelter while also holding a crucial role in disease treatment, such as cancer, diabetes, and tumors. Medicinal plants are harnessed across various cultures and nations as medicinal precursors. In today's era, biotechnological methods like tissue culture are vital for selecting, multiplying, and conserving medicinal plant genotypes. Regeneration under in vitro conditions notably enhances the production of high-quality plant-based medicines. Plant tissue culture techniques offer a unified approach for producing standardized phytopharmaceuticals, yielding consistent plant material for physiological characterization and active phytoconstituent assessment. While many medicinal plants are successfully regenerated under in vitro conditions, there are certain species that continue to be cultivated in soil, with their large-scale development through micropropagation remaining uncommon. The micropropagation technique employed for cloning these medicinal plants involves the utilization of various concentrations of plant growth regulators within a media variant (MS 1962). The process of plant regeneration is achieved through both organogenesis and embryogenesis, facilitated by the supplementation of auxins and cytokinins. In this context, this chapter provides a concise overview of the integrated micropropagation culture system designed for the effective propagation of medicinally significant specimens.

Aloe vera (L.) Burm. f. is a medicinal plant that has gained widespread interest due to the distinctive biological activities associated with its biologically active phytocomponents. To combat the difficulties caused by microbe resistance, it is urgently necessary to investigate potent antimicrobials as a natural alternative to synthetic chemicals. This challenging task is attracting a lot of interest from the scientific community worldwide. The previous antimicrobial results of A. vera indicated its broad spectrum to treat a variety of infectious diseases, which will support the development of new herbal antimicrobial agents and avoid the side effects of conventional antibiotics as well as preserve the fruit quality and extend the shelf-life of various vegetables and fruits To take advantage of the prospective uses of this plant, the current review offers insight into the phytochemical composition, and its production-limiting factors, antimicrobial and antioxidant properties, as well as the promising use of A. vera in postharvest fruit-coating. 

Oroxylum indicum (L) Kurz is a medicinal forest tree with therapeutically active principles owing to its anticancer, anti-inflammatory, antimicrobial, antiulcer, anti-arthiritic, and anti-angiogenic properties and known to be employed in ayurveda, Unani and folk medicine. Due to the possession of biologically active constituents, the tree is uprooted for the isolation of phytoconstituents and preparation of drugs from different parts of a tree and is over-exploited by pharmaceutical industries. Hence the tree is becoming an endangered species. In view of the above, this medicinally important tree species needs conservation and also thorough study on its medicinal properties. In vitro culture methodologies have to be employed for large-scale production and to know the importance and the activity of various chemical components of this valuable medicinal tree, as this knowledge plays a vital role in the conservation and synthesis of active principles with specific activity to treat various ailments. The present review focuses on the published data on conservation and also phytochemical studies of O. indicum to highlight the traditional usage of this tree species in various health disorders and also to conserve the tree using various in vitro culture techniques for its large-scale production.

Ocimum basilicum is a well-known, economically important therapeutic plant that belongs to the family Lamiaceae. Basil is marvelous in the environment as the complete plant has been used as a conventional remedy for domestic therapy against numerous illnesses since ancient times. O. basilicum exhibited interesting biological effects due to the presence of several bioactives such as eugenol, methyl eugenol, cineone and anthocyanins. O. basilicum possesses antimicrobial, antiinflammatory, hepatoprotective, hypoglycemic, immunomodulator, antiulcerogenic, antioxidant, chemomodulatory and larvicidal activities. The oil of this plant has been found to be valuable for the cure of wasp stings, snakebites, mental fatigue, and cold. The demand of this multipurpose medicinal plant is growing day by day due to its economic importance, pharmacological properties and its numerous uses in cooking and folk medicine. Thus seeing the exciting biological activities of O. basilicum, micropropagation could be a fascinating substitute for the production of this medicinal plant because numerous plantlets can be achieved in fewer times with the assurance of genetic stability. An overview of the current study showed the use of the plant tissue culture technique for micropropagation, which is very beneficial for duplicating and moderating the species, which are problematic to regenerate by conventional methods and save them from extinction. 

Plants are interesting natural resources that have had a close association with mankind since their existence. Their utility ranges from simple food, fodder, varied commercial and industrial products, and above all, as efficacious medical agents to cure various human health ailments. Amongst this vast reservoir of natural economical wealth, Rhubarb (Rheum Linn; Family: Polygonaceae), a perennial herb represented by about 60 extant species occurring across Asian (mostly restricted to China) and European countries, is one of the oldest and best-known medicinal plant species which finds extensive use in different traditional medical systems. Over the past several decades, and owing to the pharmacological efficacy of Rhubarb, the plant species has been subjected to different natural and anthropogenic pressures in the regions of its occurrence, rendering it threatened. In this context, the present chapter provides the basic account of Rhubarb while giving a gist of its therapeutic potential vis-à-vis major bio-active secondary chemical constituents. Additionally, the focus has been given to the in vitro production system of this wondrous drug for its sustainable conservation and meticulous utilization while highlighting various attributes of the technique of tissue culture such as somatic embryogenesis, cell suspension cultures, hairy roots, etc. , as projected potential approaches for desirable benefits from the genus Rheum.

Trivrit [Operculina turpethum (L.) Silva Manso], belonging to the family Convolvulaceae, is a perennial, herbaceous and creeping vine. It is a medicinal plant which is widely used in traditional systems of Indian medicine. The roots, undamaged bark, stem and leaves possess immense medicinal properties and are used in the treatment of various ailments, including bronchitis, skin diseases, tuberculosis, cough, asthma, rheumatism, jaundice, ulcer, gastrointestinal disturbances, etc. The plant is enlisted as threatened species in different states of India, particularly in Odisha, due to indiscriminate destruction of forests, shrinkage of natural habitats, and unsustainable harvesting and collection for medicinal uses. Thus, there is an urgency for its protection and conservation. To scale up the production of O. turpethum, aiming at its conservation, micropropagation can be an alternative in order to circumvent the limitations of conventional propagation of the plant. Keeping this in view, an efficient protocol for plant regeneration of O. turpethum by axillary shoot proliferation from nodal segments was optimized. Multiple shoots were induced from mature nodal explants by axillary shoot proliferation on Murashige and Skoog’s (1962) (MS) medium augmented with different types and concentrations of plant growth regulators. The highest number of shoots (13.3) proliferated on MS + 3.0 mg/L meta-Topolin. In vitro regenerated shoots were rooted on ½ MS medium containing 0.5 mg/L indole-3- butyric acid. In vitro regenerated plants with well-developed roots were successfully acclimatized in the small pots containing sterile garden soil and sand (1:1), followed by transfer to the large pot containing garden soil. Finally, plants were successfully established in the field. The biochemical fidelity, in terms of secondary metabolites, was checked for tissue culture raised-field established plant vis-à-vis mother plant.

Tea(Camellia sp.) is a non-alcoholic drink consumed across the globe. Upon consumption, it provides refreshment and enormous health benefits. Tea possesses antioxidant compounds which prevent human health from several diseases and disorders as well. Micropropagation and somatic embryogenesis are two distinct cell and tissue culture methods which have been utilized for a long time for the production of secondary metabolites having economical and industrial values. Micropropagation is a clonal propagation method accomplished by selection of explants and establishment of culture in basal media followed by shoot multiplication, development of callus, rhizogenesis, hardening and acclimatization by transferring plantlets from the laboratory to an open environment in the greenhouse or in the field. Somatic embryogenesis is the development of embryos from somatic cells, not from the zygotic cells. It consists of induction, multiplication, development and maturation of the embryo. Globular, heart and torpedo, these three distinguishable developmental stages are visible in somatic embryogenesis. Numerous genes associated with cell division, organ formation and specific cellular processes related to somatic embryogenesis have been identified. Tea possesses several secondary metabolites which have versatile functions. Caffeine, theobromine and theophylline are typical secondary metabolites which impart characteristic taste and flavour to tea. In addition, polyphenols, catechins, proanthocyanin and flavonoids act as antioxidant compounds and possess several health benefits. Various cell and tissue culture methods have been adopted for the biosynthesis of secondary metabolites on laboratory and industrial scales. These methods can be adopted on a larger scale, from experimental laboratory investigation to the industrial setup for the discovery of novel metabolic compounds for their potential applications as medicines and in commercial sectors.

In recent times, natural herbal products/biomolecules are gaining immense impetus, over modern synthetic allopathic medicines, for curing serious human ailments as the former are proving their better efficacy, causing no or minimum side effects. Consequently, many pharmaceutical industries are coming forward for exploring novel drugs based on medicinal plants. Cullen corylifolium (L.) Medik., a well-known traditional medicinal herb of China and India, is extensively used in Ayurvedic medicine to cure several skin diseases such as psoriasis, leprosy and leucoderma. Besides, it also has properties like antioxidant, anti-cancer, antiinflammatory, hepatoprotective, anti-diabetic, anti-mycobacterial, and anti-helminthic due to the occurrence of a number of important furanocoumarins and isoflavonoids. Furanocoumarins and isoflavonoids are biosynthesized via the phenylpropanoid pathway in the plant parts of C. corylifolium and are extensively used as anticancerous agents. The prominent marker compounds occurring in C. corylifolium are psoralen, genistein and daidzein produced mainly in the green seeds. These are highly expensive and occur in very low amounts. In vitro cell, tissue and organ culture can be used as an alternative, controllable, sustainable and eco-friendly tool for rapid multiplication of cells for the synthesis and elicitation of bioactive compounds. In addition, various strategies such as precursors feeding, hairy root culture, biotic and abiotic elicitors, cell suspension cultures, cloning and overexpression of genes involved in biosynthetic pathways of secondary metabolites. are also available for the enhancement of bioactive secondary metabolites. The present review aims at the screening of high-yielding elite plant parts, biosynthetic pathways of psoralen, daidzein and genistein, and various strategies employed for their elicitation and isolation in C. corylifolium. 

Pelargonium is one of the most recognized aromatic herbs due to its wide distribution around several countries and its perfumery and aromatherapy properties. The present chapter aims at exploring the current scientific study on the various species of Pelargonium along with its significance. The essential oil of Pelargonium contains more than 120 monoterpenes and sesquiterpenes obtained from the steam distillation of herbaceous parts. Citronellol, geraniol, rhodinol, 6, 9 –guaidiene, and 10-epi-γ eudesmol are the principal components responsible for its oil quality. Traditionally, propagation of pelargonium is done through cuttings from its mother plant material. However, the tissue culture approach is one of the reliable techniques for propagation and conservation, not influenced by environmental conditions. More likely, tissue culture approaches used are somatic embryogenesis, callus culture, direct regeneration, meristem culture, and hairy root culture. Transcriptome analysis has also been carried out in Pelargonium graveolens to understand the metabolic pathway. In order to accomplish the maximum oil production and better geranium varieties through genetic engineering, Agrobacterium mediated transformation systems have been developed. These standardised genetic transformation procedures were used to over-express, silencing, and heterologous expression of desired genes in Pelargonium to understand the outcome and succeed with enhanced essential oil production with better quality for the ultimate benefit.