Micropropagation of Medicinal Plants

 Overexploitation, climate change, and pressure from invasive species are threatening the diversity of medicinal plants; a few of them are extinct or in the endangered category. The mass multiplication of some medicinal plants outside their natural habitat affected the biochemical diversity of the plants, thereby decreasing their medicinal value. Hence, micropropagation of high-yielding, elite genotypes was preferred over time to conserve the species and meet the pharmaceutical needs. Although micropropagation was promising, the diversity and quantity of bioactive compounds of the in vitro plants were not comparable to those of their counterparts in nature. The in vitro plants, challenged with a plethora of biotic and abiotic stresses, were poorly acclimatized, with abject survival. During the last few decades, the role of endophytes with their mechanisms in enhancing growth, development, and stress tolerance has been proven among field-grown plants. In consequence, the role of endophytes in micropropagation is gaining prominence to address the vulnerability, acclimatization, and enhanced bioactive compounds of tissue culture plants. This approach of the use of competent endophytes is known as biotization. This chapter brings together the current status, possibilities, and limitations of the most promising biotization of medicinal plants. Biotization of endophytes in micropropagation is a potential tool for the production of medicinal plants with enriched bioactive compounds with improved therapeutic effects.

Tylophora indica (Burm.f.) Merrill is one of the most commonly used medicinal plants with bioactive alkaloid-rich secondary metabolites. This plant is used to treat asthma, dysentery, whooping cough, rheumatic pains, jaundice, and cancer. Rapid exploitation of this plant in natural habitats and poor regeneration methods, which are not in pace with those of destruction, make tissue culture methods a viable option to be used as a method of conservation. In the present chapter, tissue culture protocols have been reported till now as the best viable means in the rapid multiplication of T. indica. Sterilization protocols, callus induction and somatic embryogenesis methods, and direct and indirect organogenesis used by different researchers in mass propagation and acclimatization are given in detail. The present chapter gives an insight into the hormones needed and the response of the explants, which will be helpful for those who want to propagate this medicinal plant under in vitro conditions.

Withania somnifera, commonly known as 'Indian ginseng', is a highly important and valuable medicinal plant in traditional family medicine, containing a variety of medicinal bioactive molecules for over 3,000 years. Various medicinal properties of plants are attributed to steroidal lactones (withanolides) present in plants. Its commercial cultivation is hampered by low seed viability and germination rates. Tissue culture techniques can play an important role in the preservation, clonal propagation, and qualitative improvement of this medicinal plant. In vitro shoot differentiation and micropropagation of W. somnifera from various small excised explants such as hypocotyl and cotyledon leaves, shoot tips, nodes and internodes. Optimal normal growth, reproduction, and development of W. somnifera through the in vitro processes of morphogenesis of many tissues may differ in several different plants based on key plant nutrient requirements. The current review provides a comprehensive study on the development of W. somnifera tissue culture research activity. It also discusses the medicinal properties of this plant.

Aloe vera is a medicinal plant with several properties and is used in pharmaceutical, medicinal, biomaterials, food, and cosmetic industries. This plant is associated with hot climates and dry habitats, with plantlet production based on lateral shoot propagation, an expansive and slow method, insufficient to meet the increasing demand of the industry. Therefore, the development of a suitable Aloe vera micropropagation protocol is crucial, keeping the genetic integrity and providing largescale plantlets production. Nevertheless, parameters like source of plant tissue, surfacesterilization process, culture medium conditions and plant growth regulator concentration can affect the morphogenic response process. Since all parameters are defined to obtain the best performance, the micropropagation protocol is suitable to be used commercially, providing mass production of Aloe vera plantlets with high quality. 

Phyllanthus amarus Schum. & Thonn. is a medicinal plant belonging to the family Phyllanthaceae. It is used in Siddha, Homeopathy, Unani and Ayurvedic medicines. This plant is used for treating skin diseases, diabetes, fever, diarrhea, gastric problems and migraine. It also acts as an anticonvulsant, anti-inflammatory, analgesic, antiamnesic, antimicrobial, nephroprotective, antiviral, hepatoprotective, anticancer, and anti-atherosclerotic agent. It grows as a weed, but because of its high medicinal value, this plant was propagated in vitro, so as to meet the market demands and for rapid propagation. In the present review, detailed work done till now is discussed, with different methods of sterilization, and hormones used for shoot, callus and root induction.

Hedychium, a tropical to subtropical Asian genus with about 100 species, has various medicinal and horticultural uses. There is a high rate of exploitation and disappearance of its species from natural habitats. Additionally, habitat loss and natural calamities should speed up the erosion of this plant species. Micropropagation is considered a multiplication and conservation strategy for medicinal plants. Micropropagation in Hedychium is very scanty, and protocols have been developed only for less than 20 species so far. Hedychium coronarium and H. spicatum are wellstudied species in vitro among the micropropagated species. It is interesting that micropropagations through protocorm-like bodies were achieved in H. coronarium. The selection of explants and their axenic development in vitro is the major hurdle in micropropagation. Cotyledonary nodes, shoot tip or shoot tip meristems from axenically germinated seeds, rhizome buds, rhizome meristem, and zygotic embryos were the explants commonly used for the micropropagation of Hedychium. Various in vitro methods such as somatic embryogenesis, direct organogenesis and indirect organogenesis, multiplication through microrhizome induction, and propagation through protocorm-like bodies were frequently tried for the successful micropropagation of this genus.

Jatropha curcas plants are a rich source of several natural components, the great majority of which are utilized for human health and the treatment of various ailments. J. curcas is a perennial plant in the family Euphorbiaceae that is gaining commercial significance due to its various industrial and medical uses. The purpose of this study was to improve the micropropagation conditions of J. curcas by using single node explants using the tissue culture technique.

Moringa oleifera Lam. tree is considered a miracle tree due to its nutrientrich profile. This plant has been widely cultivated throughout the world for its medicinal and nutritional benefits. Moringa plant contains a significant amount of various phytochemicals such as alkaloids, flavonoids, saponins, tannins, and phenolics that are responsible for their various medicinal, nutritional, and other applications. The use of moringa in pharmacological drugs not only increases cultivation but also increases the over-exploitation of this tree. As the conventional methods of moringa reproduction are not much effective, we require additional alternative strategies to multiply moringa plants. Micro-propagation is considered an effective method to produce a large number of transplants within a short time period. This chapter explores the micro-propagation approaches of M. oleifera together with its nutritional profile and biological activities. 

Oldenlandia umbellata is an important medicinal herb distributed in the tropics used in the formulation to treat asthma, bronchitis, bronchial catarrh, snake bite, and many infectious diseases. The mature roots of O. umbellata are also known as a source of the natural dye anthraquinone (AQ), the second largest group of textile dye. However, extraction of the dye contained in the roots of this plant may pose a threat to its survival in its natural habitat. This chapter explores the scope and relevance of micropropagation of medicinally and economically significant O. umbellata, thereby saving this species in the wild and scaling up dye production through in vitro means. In vitro propagation efforts of this herb were made through axillary bud proliferation using benzyl adenine and by adding an auxin transport inhibitor, quercetin. A somatic embryo-based propagation system was also established through an in vitro starvation method. Based on available methods, O. umbellata can be efficiently propagated and conserved superior germplasm by applying the most suitable in vitro propagation methods. 

The article presents the developed protocol for the propagation of tuberous orchids and their organs. The protocol allows for the cultivation of materials for reintroduction, restoration of disturbed populations and biomedical research. A phytochemical analysis of this plant group was conducted for the first time, confirming their medicinal properties. Micropropagation was employed to achieve this objective. The study focused on nine species of tuberous orchids from the Orchidaceae family: Dactylorhiza traunsteineri, D. maculata, D. fuchsii, D. incarnata, D. urvilleana, D. baltica, Gymnadenia conopsea, G. conopsea f. gigantea and Orchis militaris. The results demonstrated that these species contain chemical substances with potential physiological activities. For example, squalene exhibited positive effects such as antifungal, anticancer, antibacterial, antioxidant, and others. Additionally, other identified chemical substances demonstrated antiproliferative and proapoptotic activities against colon cancer, as well as antibacterial, anti-inflammatory, antioxidant, antitumoral, and antifungal properties.

The use of cutting-edge biotechnological methods such as in vitro propagation enables the large-scale production of disease-free plant material, rapid cloning, and conservation of the elite genotype within a very short period. Additionally, the technique has enormous potential for the production of pharmacologically significant secondary metabolites and plant-based medicines of high quality. Operculina turpethum (L.) Silva Manso is an important medicinal plant of the family Convolvulaceae and is used to treat several health ailments. Overexploitation and inadequate conservation strategies have put the plant on the verge of extinction. This chapter provides a concise overview of the current status of the endangered medicinal plant Operculina turpethum with special attention given to the in vitro propagation and conservation of the immense medicinal plant.

Eryngium foetidum L. is an important medicinal and aromatic plant of the family Apiaceae. The plant is extensively used in traditional medicine and for culinary purposes. The essential oil of the plant has very high economic value in both national and international markets due to its application in the pharmaceutical, cosmetics, and perfumery industries. The plant is generally propagated through seeds. However, due to low seed viability, the plant is restricted to certain regions, which in turn hinders the commercial application of the medicinal plant. Therefore, quick and mass multiplication of the plant is needed, which may be accomplished by micropropagation. This is necessary in order to satisfy the ever-increasing demand of the pharmaceutical and cosmetic industries. In this chapter, a variety of methods for micropropagation have been explained, each of which utilises a different component of the plant as an explant. 

Garlic (Allium sativum L.) is the most often used medicinal plant and the second most commonly used Allium species after onion. It belongs to the Alliaceae family. Garlic originated in Central Asia and is currently cultivated all over the world. Garlic is rich in bioactive components and is used in various medicinal uses. Garlic has a greater concentration of sulfur-containing compounds, which contribute to its pungent odour. The major phytoconstituents of garlic are alliin, allicin, ajeones (oilsoluble organosulfur compounds); water-soluble organosulfur compounds such as Sallyl cysteine (SAC), metabolites allyl mercaptan (AM), allyl methyl sulphide (AMS), and S-allyl-mercapto cysteine (SAMC). Due to its bioactive components, garlic has various pharmacological properties, including anticancer, antidiabetic, antiinflammatory, antioxidant, and antibacterial action. Garlic micropropagation is feasible due to its widespread use and robust pharmacological activity. Micropropagation of garlic, which includes meristem culture or shoot tip culture, is reported to have various advantages, including the ability to create disease-free plant material, develop a higher number of desired plants, enhance the bioactive of garlic, and improve crop quality. This chapter briefly summarizes garlic's bioactive components, their pharmacological actions, the role of micropropagation in garlic, and its application.

Glycyrrhiza glabra belongs to the family Fabaceae and is commonly called licorice. It is an important medicinal plant in Europe, China, and the Mediterranean. The plant's therapeutic value is also mentioned in Ayurveda and Siddha. Licorice is cultivated for commercial purposes in many parts of the world because of its economic value and demand. It is used as a flavoring agent in juices, candies, soft drinks, and beverages because of its characteristic taste and smell. In addition, it is regarded as a sweetener and thirst quencher. Licorice contains phytochemicals, and the most abundant compounds are glycyrrhizic acid, anethole, liquiritigenin, isoliquiritin, pinocembrin, and licoflavanone. The plant is a good source of antioxidants and exhibits anti-inflammatory, antimicrobial, antiviral, anti-diabetic, and anti-cancer activity. Even though it has many health-benefiting features, consuming high amounts of licorice can lead to hypertension, hypokalemia, and congestive heart failure. Due to its high demand, good medicinal value, and poorly developed cultivation strategy, researchers are focusing on different aspects of the in vitro propagation of the plant. Studies have revealed that micropropagation of licorice has improved the level of secondary metabolites and high antioxidant properties. Thus, this chapter focuses on the propagation method of licorice, primarily focusing on micropropagation. Moreover, it also highlights the phytochemistry and important pharmacological activity of Glycyrrhiza glabra. 

Plants and plant materials have long been employed in traditional herbal remedies. In recent decades, natural resource demand has surged by 8–15% each year in Asia, North America, and Europe. Vetiver is a plant that produces essential oils and is widely used in perfumery, cosmetics, and herbal medicine. The utilisation of controlled conditions can help overcome traditional cultivation challenges and may be used to modify phenotypic variance in the concentration of bioactive chemicals. The goal is to improve extract potency, minimise toxin levels, and improve extract consistency and predictability. In this review, an attempt has been made to present in vitro propagation approaches used in vetiver. 

Actinidia deliciosa, commonly known as Kiwifruit (Chinese gooseberry), belongs to the family Actinidiaceae. The edible and fleshy fruit has gained popularity over the past few decades owing to its high nutritive value, and medicinal and potential curative properties. The fruit is rich in vitamin C, folate, vitamin E, dietary fibers, antioxidants, enzymes, phytonutrients, etc. The presence of actinide in Kiwis helps in regulating gastric abnormalities, hypertension, cardiovascular inflammation, hemostatic disorder, and abnormal glucose metabolism, and prevents cancer. Consequently, the fruit holds a considerable market value that has led to the establishment of industrial organizations comprising growers and distributors. For the purpose of fulfilling the constant market demands, it is crucial to maintain quality standards, timely production, and an abundance of planting material. This chapter discusses the various in vitro propagation methods, including diverse and detailed approaches for both the direct and indirect organogenesis for large-scale production of good-quality kiwi plants, along with ex vitro hardening and acclimatization processes. It is apparent that the plant tissue culture techniques can be suitably applied for the mass production of kiwi fruit, while other in vitro manipulations and further biological research are needed to improve the field performance and post-harvest life of the fruit and its plant.

Pterocarpus marsupium Roxb. is one of the important plants of the Fabaceae family and is present in different regions of the world. It is greatly valued for its medicinal properties and has often been used for medical purposes. It was observed that P. marsupium contains numerous phytochemical components, such as glycosides, proteins, cardiac glycosides, terpenoids, alkaloids, carbohydrates, and flavonoids. Due to overexploitation, the natural population of P. marsupium is declining steadily, because of which it is required to be cultivated on a larger scale. The conventional propagation methods of P. marsupium are time-consuming processes, and the plant is not easy to propagate through seeds because of its low germination percentage. Hence, to overcome the problem related to conventional propagation and to reduce the destruction of plants in wild habitats, tissue culture functions as an important tool to conserve the plant. The tissue culture practice is extremely useful to meet the rising demands of the people because it gives a significant number of elite genotype progenies within a limited time and without seasonal dependence.

Balanites aegyptiaca belongs to the family Zygophyllaceae, present in tropical countries of the world. This plant is well known for its medicinal properties. B. aegyptiaca contains numerous phytochemical components such as glycosides, proteins, steroids, terpenoids, alkaloids, and flavonoids. Due to overexploitation, the natural population of plants is declining in the wild. Also, conventional propagation of the plants is not sufficient in terms of the production and the number of the plants. Therefore, to reduce the problem associated with traditional propagation and production of plants on a larger scale, in vitro propagation is the most suitable approach. During in vitro propagation, a sufficient number of elite genotype progenies within a limited time period and without seasonal dependence are produced.

Ruta graveolens L., a multipurpose perennial herb, belongs to the family Rutaceae. It is a native of the Mediterranean region and is commonly known as Garden Rue or Herb of Grace. From time immemorial, Rue has been known for its rich aromatic and medicinal properties. More than 120 compounds of different classes of natural products, such as acridone alkaloids, coumarins, essential oils, flavonoids and furanoquinolines, have been isolated from R. graveolens. Having a vast range of secondary metabolites, this plant has been used worldwide for several therapeutic usages. The essential oil obtained from the distillation of the entire plant has several therapeutic values. The entire plant is used as an abortifacient, anthelmintic, antispasmodic, carminative, emmenagogue, expectorant, haemostatic, ophthalmic, and rubefacient. Besides pharmaceutical applications, this plant is used in cosmetics and food items. Ripened fruits are used as condiments and leaves are used to make pickles. Several effective protocols for micropropagation have been developed by several researchers. Due to its vast usage, the plant is disappearing in the wild. Conventional propagation methods do not meet the market demands. Hence, there is an urgent need to shift to in vitro methods for quick and genetically elite plant production. In this chapter, detailed protocols for in vitro propagation methods are discussed.