Micropropagation of Medicinal Plants

The genus Cissus Linn. belongs to the Family Vitaceae (formerly Ampelidaceae) and comprises about 350 species distributed all over the world, having rich phytochemicals with medicinal as well as commercial value. This genus is a storehouse of large varieties of phytochemicals such as alkaloids, flavonoids, and phytosterols, making this genus pharmaceutically important. Some species contain high quantity of calcium ions in their stem extract, which is possibly responsible for their bone healing activity. In vitro propagation of plantlets provides the opportunity to conserve endangered species as well as to use the beneficial species without disturbing their natural habitat. The present review comprises in vitro protocols used to conserve the species, exploit and enhance useful metabolites. The whole plant, parts and metabolites isolated from in vitro cultures of Cissus species may be used further for pharmaceutical purposes.

Juglans regia L., commonly called walnut, is a nutrient-rich fruit. Besides many therapeutic properties, the plant is highly valued for its timber, which along with the fruit-nut, fetches a very high demand in the domestic and international markets. The ever increasing demand for these plant products is not being sufficed by the existing supplies. This is owing to the fact that conventional methods of walnut propagation are time and space-consuming and show limited responsiveness. Walnut cuttings are also difficult to root, making large-scale propagation a challenge. Consequently, walnut micropropagation has become extremely important to ensure rapid mass production of selected cultivars in a small space, and for an indefinite time period. The tissue-cultureraised products are robust, disease-free, and have desirable characteristics. The aim of this chapter is to compile information on tissue culture studies on Juglans regia with a special focus on the latest developments in the field. The chapter covers various pathways employed for the in vitro propagation of walnut, hardening, and acclimatization of tissue culture raised plantlets to ensure better quality, quantity, and sustainability of walnut trees to meet the demand of the growing global population. 

A tropical and subtropical Asian genus called Alpinia is used for both horticultural and medicinal purposes. Species having ornamental uses are now distributed widely all over the world. Different species of Alpinia are widely used in traditional medicine for treating many diseases. Several Alpinia species have now been experimentally demonstrated to have medicinal properties. Excess trade of many species of Alpinia, such as A. calcarata, A. galanga etc., as well as habitat loss and urbanization demands its mass propagation. Therefore, one of the best methods for its mass propagation and conservation is micropropagation. In vitro studies of medicinal taxa such as A. calcarata, and A. galanga and ornamental species such as A. purpurata has been well established. Different in vitro approaches such as direct organogenesis, callogenesis and indirect organogenesis, somatic embryogenesis (SEs), and multiplication using inflorescence buds were generally tried for the successful micrpropagation of different species of Alpinia. Genetic and phytochemical fidelities of the in vitro raised plants were also studied in many instances to enhance the commercial use of it.

Zingiber officinale, belonging to the family of Zingiberaceae, is commonly known as ginger and is commercially grown as a spice and for culinary purposes. It is a potential Ayurvedic herb with many medicinal properties. A small section of the plant's rhizome is widely used for micropropagation. Besides rhizome explants, callus induction, shoot induction, and meristem culture are used to propagate the plant. For the production of ginger's pest-resistant and disease-free planting material, micropropagation is regarded as the best method. Various classes of bioactive entities, such as flavonoids, alkaloids, glycosides, phenols, tannins, terpenoids, steroids, saponins, and oils, have been identified in the plant. Phenolic bioactives such as gingerols and shogaols are primarily responsible for their therapeutic properties. Various pharmacological activities have been investigated in ginger. This review concentrates on different advanced methods for ginger propagation, especially micropropagation.

Atropa, a Solanaceae member, contains many active chemical compounds such as atropine, saponins, polyphenols, scopolamine and hyoscyamine. Because of the presence of these active principles, endangered species Atropa acuminata and Atropa belladonna have been indiscriminately exploited in traditional medicine for treating various disorders and thus Atropa acuminata has become an endangered species in some regions. Due to the threat of extinction, low seed germination and seedling survival rate, there is a need for conservation through efficient micropropagation protocols. In this regard, the current chapter is focused on micropropagation methods/protocols developed by various researchers using various explants of Atropa acuminata and Atropa belladonna and their responses to different media compositions with respect to direct and indirect organogenesis in vitro, as the technique of in vitro regeneration has played a pivotal role in the mass multiplication of many plant species.

Aegle marmelos (L.) Corr. is a plant of religious and medicinal importance in India. All of its plant parts have been reported to possess medicinal uses due to the presence of various phytoconstituents. Looking at its perspectives, Aegle is successfully propagated in vitro, primarily through organogenesis, using numerous explants. Efficient micropropagation is ensured by proper sterilization, preparation of explants, and use of antioxidants to avoid media browning. Various factors that affect the regeneration rate include season of explant collection, explant origin, phenological growth stage, concentration and combination of Plant Growth Regulators (PGR), culture media composition, and addition of additives to the media to enhance the micropropagation rate. The present review chapter compiles numerous reports of the effective micropropagation of A. marmelos and factors that affect the rate of micropropagation.

 Artemisia absinthium L. or ‘wormwood’, commonly known as ‘Dawna’, is a small perennial herb with a dark fragrance due to glandular trichomes present all over the plant. Medicinal properties of A. absinthium are known in most of Asia, South America, and Europe. Essential oil, along with other phytoconstituents, like flavonoids, phenolic acids, tannins, and lignans, imparts medicinal potential to this species. It revealed antibacterial, antitumor, antimalarial, antioxidant, anthelmintic, antipyretic, antidepressant, antiulcer, antiprotozoal, hepatoprotective, neurotoxic and neuroprotective action. Due to its wide range of disease curing potential, A. absinthium germplasm is always under the pressure of overexploitation and loss of habitat. To cope with the higher industrial demand of this plant, the use of biotechnological techniques related to micropropagation can provide the best alternative. In vitro propagation using any explants has been extensively studied for the conservation of its plant genetic resources. Other micropropagation methods, such as callus culture, cell suspension, and organogenesis, have been adapted with the aim of secondary metabolite extraction and artemisinin enhancement. Modern biotechnological tools such as Agrobacterium-mediated gene transformation are mainly applied to hairy root and shoot cultures to optimize the biosynthesis of artemisinin. The present review throws light on various biotechnological studies carried out on A. absinthium, presenting the respective outcomes. 

Bacopa monnieri (L.) Wettstein is a medicinal herb from the family Plantaginaceae widely known as ‘water hyssop’ or ‘brahmi’. The therapeutic potential of plants is due to the presence of many bioactive secondary metabolites, majorly brahmine, herpestine, alkaloids, and saponins (bacosides), which are responsible for pharmacological effects including neuroprotective, hepatoprotective, gastroprotective, antioxidant, anti-inflammatory, and antimicrobial properties. Vegetative cultivation of Bacopa on a large scale has its limitations due to the lack of viability of seeds during propagation and the unpredictable nature of the production of phytochemicals for commercial purposes, which can be overcome by tissue culture mechanism. Over the past few decades, many studies on the tissue culture of Bacopa in establishing a standardized protocol were reported. This chapter deals with de novo organogenesis of the root and shoot along with the callus induction and somatic embryogenesis from different explants of B. monnieri on MS basal nutrient medium supplemented with Plant Growth Regulators.

Momordica charantia L., commonly known as bitter melon/gourd, is a slender tendril-climbing annual vine of the family Cucurbitaceae. Bitter melon grows in tropical areas, including parts of the Amazon, Asia, and the Caribbean, and is cultivated throughout South America. It is a common food of the tropics used in the treatment of many diseases and is also known for its potent hypoglycemic actions. A steroidal sapogenins known as charantin, insulin-like peptides, and alkaloids have been reported to have hypoglycemic or other actions of potential benefit in diabetes mellitus. The present chapter gives a comprehensive review of the tissue culture of Momordica charantia. There are two ways of regeneration, direct organogenesis and indirect organogenesis; both take place through the production of adventitious buds and somatic embryogenesis. The present review gives a complete in vitro regeneration protocol of M. charantia. 

Catharanthus roseus (C. roseus) is an important alkaloid-yielding medicinal and ornamental plant belonging to the family Apocynaceae. The genus Catharanthus is well studied and reported to contain biologically active terpenoid indole alkaloids (TIAs) with over 130 compounds isolated and identified. It has great medicinal importance in treating various ailments to treat diseases as diabetes, malaria, menorrhagia, Hodgkin’s disease, etc. In view of the immense importance in the pharmaceutical industry, micropropagation of C. roseus has been the best alternative for continuous source of plants and also for in vitro production of secondary metabolites. Various explants have been studied for micropropagation; however, nodal explants were the most suitable. For surface sterilization, 0.1% HgCl2 or 70% ethanol, followed by sodium hypochlorite and Bavistin (carbendazim), was optimum to control the microbial contamination. Murashige and Skoog (MS) medium was the most widely used for its success rate. 2,4-D for callus initiation and BAP, along with zeatin and activated charcoal, were reported to be promising for regeneration of plantlets. The 100% acclimatization of plantlets on transfer to field depends on the soil mixture and environmental conditions and humidity in the initial stages of transfer from in vitro cultures.

Saraca asoca (Family - Fabaceae) is well-known medicinal tree species used in codified and non-codified systems of traditional medicine. Tree parts, viz., bark, flower, leaf, root and fruit, are used to treat various disorders. A huge number of pharmaceutical products were prepared using bark as one of the major ingredients. Ashoka tree is categorized as vulnerable by the International Union for Conservation of Nature (IUCN) and endangered by the Conservation Assessment and Management Plan (CAMP) due to its overexploitation and limited distribution. Unsustainable utilization, deforestation and climate changes are the major threats to the existence of the species. Aiming at the conservation of the Ashoka tree, ample research works were performed to standardize the micropropagation techniques. The present chapter discusses the efforts made towards conservation and micropropagation studies on the Ashoka tree. 

Yam (Dioscorea spp.), a tropical monocot flowering, perennial multispecies crop, belongs to the family Dioscoreaceae. It is a valuable source of medicines and food security crops in yam-growing regions in Asia, Africa, and southern American countries. More than 600 yam species are widely cultivated in tropical and subtropical countries and used as food and medication for various human diseases. It provides big starchy tuberous roots as a source of carbohydrates, protein, antioxidants, minerals, and vitamins. It is also high in vitamin C, B6, manganese, potassium, and antioxidant compounds, which nourish and protect against oxidative cell damage in the human body. In addition, they are rich in potent plant compounds, including anthocyanins, a color-producing chemical that helps to reduce blood pressure and inflammation and protect against cancer and diabetes. Exceptionally, yam is an excellent crop for food security and human health. Micropropagation of medicinal yam is essential for the large-scale multiplication and conservation of endangered species. So far, in micropropagation of medicinal yam spp., very few studies have been conducted. These studies used axillary buds, nodal cuttings, mature, immature leaves, and shoot tips as explants for micropropagation. Several tissue culture techniques are available for micropropagation of yam, especially direct and indirect organogenesis for in-vitro propagation for large-scale generation of plantlets.

Rauvolfia serpentina (L). Benth. ex Kurz., commonly known as Sarpagandha (Indian snakewood), of the family Apocynaceae, is a medicinally important woody shrub. Since ancient times, the root of this shrub has been used for treating numerous diseases, especially hypertension, mental agitation and cardiovascular diseases. In addition to eighty different alkaloids, all well-known for their pharmaceutical properties, the plant also contains reserpine, recognized as the world's first antihypertensive drug. Thus, the demand for this plant has only grown in the pharmaceutical industry. However, overexploitation and abysmal traditional propagation methods have endangered this valuable species' natural vegetation, creating an unpleasant gap between the demand and availability. In this scenario, the in vitro micropropagation technique comes as an alternative strategy to help replenish this threatened shrub's natural vegetation loss and commercial needs. Furthermore, the beneficial features of the plant tissue culture technique by providing genetically uniformed disease-free true-to-type plant propagation within a short time, and conserving elite variety plantlets makes this technique an inevitable tool for the rapid production of economically important plants in the 21st century. Therefore, this chapter focuses on the different in vitro plant tissue culture techniques applied to regenerate R. serpentina plants. In addition, the roles of various physical and chemical factors that could affect the regeneration rate, geographical distribution, bioactive compounds and their bioactivity have also been discussed. The comprehensive data could be helpful for further studies on this valuable plant.

The genus Wrightia belongs to the Apocyanaceae family and encompasses 32 species. This genus has many pharmacological properties and is used for many of the human ailments in the traditional systems of medicine. It also has commercial importance for its timber, dye, etc. Due to its commercial importance, some of the species of this genus, like Wrightia tinctoria and W. arborea are overexploited and have become endangered. There is a need to conserve these species. One of the techniques to conserve plants and multiplication is micropropagation. In this chapter, regeneration studies that include collection, sterilization, shoot and root generation, and acclimatization of Wrightia tinctoria and W. arborea are described.

 Decalepis hamiltonii Wight & Arn. (Family: Apocynaceae) is a climber native to Southern Peninsular India, commonly called Swallow Root. The plant is used in Ayurveda, Siddha and other traditional systems of medicines as a blood purifier, appetizer, rejuvenator, wound healing agent, etc. Apart from this, various other medicinal uses and pharmacological properties created a great demand for this plant that has resulted in destructive harvesting practices in the wild. The plant is generally reproduced through seeds; however, in most of the cases, germination is an intricate process due to its poor seed viability and delayed seed production. Hence, its population has gradually declined due to over-harvesting of medicinally important tuber. International Union of Conservation of Nature (IUCN) declared all the species of Decalepis as ‘Critically Endangered Globally’. In the present chapter, complete information on traditional uses, phytoconstituents and micropropagation of ethnomedicinally important and critically endangered species D. hamiltonii is discussed. 

Tinospora cordifolia (Willd.) Hook. f. & Thomson belongs to the family Menispermaceae. The origin of the species is from Indian subcontinent to Indo-China. The plant has antipyretic, antiperiodic, anti-inflammatory, antirheumatic, spasmolytic, hypoglycaemic and hepatoprotective properties. Due to over-exploitation of this medicinally important species, attention has been given to its conservation through invitro micropropagation techniques. The present chapter emphasizes the ample research work on in-vitro regeneration and enhancement of secondary metabolites from T. cordifolia. Additional information on the importance of this genus in various systems of medicine, active components, biological activities and its sustainable utilization for the welfare of mankind is also discussed in the present chapter.

The genus Hoya (Family: Apocynaceae) has more than 500 species, comprising mainly of epiphytes and geographically distributed in South America, Southeast Asia, Indo-Malesia and Australian regions. Most of the species are cultivated for their ornamental, aromatic and showy flowers. Philippines is one of the countries with the highest diversity of Hoya species. As seed setting is very rare in most of the species, it necessitated the development of conservation strategies through ex situ conservation methods using vegetative or micropropagation techniques. Present chapter provides detailed information on the traditional uses, phytoconstituents, conservation status and micropropagation studies of the ornamental and medicinally important genus Hoya. 

Genus Kaempferia comprises about 124 species distributed in Southeast Asia and is well known for it's diverse medicinal, nutritional and industrial values. The plants of the genus are rhizomatous, perennial, and oil-yielding plants; some are also used as spices. The essential oil obtained from the plants has a considerable market value worldwide. The rhizomes of these plants were used in traditional medicine due to the presence of diverse bioactive compounds and used to treat urinary tract infections, fever, cough, hypertension, metabolic disorder, asthma, rheumatism, epilepsy, skin diseases, etc. Seed dormancy, seasonal outgrowth and seed made through crosspollination were found to be non-viable, which are the prime limitations of ex situ conservation regarding this genus. To overcome this type of problem, in vitro tissue culture is the way to get the plants available over the year without any limitations. This chapter is based mainly on exploring those bioactive compounds containing species of the genus Kaempferia, and obtaining an alternative resource of phyto-compounds for use in pharmaceuticals and conserving them through an artificial way to get them throughout the year without exploiting the area and genotypic alteration.

The present review summarises the in vitro multiple plantlet regeneration of Stemona tuberosa. MS medium fortified with 7 mg/L Kn was found to be the optimum for multiple shoot induction from axillary buds. Excision and culture of nodal segments from the in vitro shoots on medium containing 7 mg/L Kn and 4 mg/L TDZ showed a maximum number of shoot multiplication. Shoots developed were rooted best on ½ strength MS with 1 mg/L IAA. Plantlets established in pots exhibited 85% survival.

The Oxalidaceae family is known for small herbs, shrubs and small trees with economic and medicinal properties in folklore medicines. The genus Oxalis is distributed worldwide and is famous for tuberous and ornamental cultivars. The present study established reproducible in vitro protocols for mass multiplication of Oxalis corniculata L. via. micropropagation and indirect organogenesis using different explants. Murashige and Skoog (MS) medium augmented with various cytokinins, auxins and gibberellic acid and combinations with respect to the different protocols. In micropropagation, shoot tip and node explants cultured on a medium with 6-benzyl adenine (BA) 3.0 mg/L, 6-furfuryladenine (Kn) 1.0 mg/L and naphthalene acetic acid (NAA) 0.5 mg/L produced the highest average of 35.1 and 28.5 shoots after 25 days of culture, respectively. Gibberellic acid (GA3 ) treatment was satisfactory in shoot elongation, and rooting of shoots was best on indole-3-butyric acid (IBA) 3.0 mg/L than indole-3-acetic acid (IAA) and NAA. In indirect organogenesis, internode, leaf and petiole explants produced green, compact nodular calli at varying frequencies on medium fortified with auxins. The maximum frequencies of shoot regeneration and shoot numbers were observed on a medium containing BA 1.0 mg/L and IBA 0.5 mg/L. Further, the shoot elongation was achieved with BA and GA3 , and rooting was best achieved on IBA 3.0 mg/L with Kn 0.5 mg/L. All the plantlets were successfully hardened and acclimatized under the greenhouse condition with maximum survival of 95%. The current protocols established via meristem and callus mediated cultures would help in bioprospecting of this less explored medicinal plant.

Habitat destruction and over-harvesting have resulted in the gradual disappearance of many medicinally important plants from their natural habitat. At present, their number is highly reduced in the wild. To conserve the genetic stocks of such plants, in vitro propagation can be utilized successfully. One such medicinally important plant that needs to be conserved is Pseudarthria viscida (L.) Wight & Arn. It is a perennial viscid pubescent semi-erect, diffuse undershrub belonging to the family Fabaceae. It is an essential component of many famous Ayurvedic formulations like Dashamoola, Mahanarayana taila, and Dhantara taila. The root is the most important part of the plant with high medicinal value. Major chemical compounds reported to be present in the roots are 1,5 dicaffeoyl quinic acid, oleic acid, tetradecanoic acid, rutin, quercetin, gallic acid, ferulic acid, and caffeic acid. The present study focused on in vitro regeneration and mass propagation of P. viscida. Fresh young leaves, nodes, and internodal segments were used as explants. Murashige and Skoog medium (MS medium), Gamborg’s (B5 ) medium, and White’s mediums were selected for in vitro regeneration and mass propagation. Among the various media used, the MS medium gave a successful result in in vitro culture by showing a response within four weeks, and the percentage of response was also high compared to B5 and White’s medium. The leafy explant was found to be more suitable for profuse callus induction, somatic embryogenesis, and indirect organogenesis than that of internodal and nodal explants, whereas nodal explant was best for direct organogenesis in P. viscida. Of the different combinations tried, NAA (Naphthalene acetic acid) + BAP (6-Benzyl aminopurine) combinations were best for callus induction, somatic embryogenesis and indirect organogenesis. 2.5 mg/L BAP was best for shoot induction from nodal explants, whereas 2.5 mg/L NAA was best for root induction from in vitro regenerated micro shoots as explants. Well-developed plantlets were transferred to greenhouse and later to natural conditions. This study thus reports an efficient protocol for plant regeneration, and this could be vital for the multiplication and field transfer of this ethnomedicinal plant. Based on the ethnomedicinal potential, there is an urgent need for organized cultivation of this vulnerable plant for its conservation and sustainable utilization.