Micropropagation of Medicinal Plants: Volume 1 E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
This volume presents information about protocols for micropropagation of more than 40 species of medicinal plants. The contents combine knowledge about the scientific principles of micropropagation with state of the art updates in tissue culture techniques presented by plant scientists.
The readers will learn about techniques required to grow plants in challenging conditions that aim to reduce the impacts of injudicious harvesting, deforestation, climate change, pollution, urbanization and other factors that limit the ability to meet current demand. General topics such as biotization and pharmaceutical investigation are also included to guide readers about the significance of these plants in research and development for new medicines.
The book provides protocols for micropropagation of important medicinal plants like Rauvolfia serpentina, Catharanthus roseus, Withania somnifera, Tylophora indica, Bacopa monnieri, Aloe vera, Phyllanthus amarus, Allium sativum, Moringa oleifera, Operculina turpethum, Glycyrrhiza glabra, Pterocarpus marsupium, Vetiver grass, Ruta graveolens, Tinospora cordifolia, Kaempferia, Hedychium, Decalepis hamiltonii, Saraca asoca, Wrightia tinctoria, Wrightia arborea, Artemisia absinthium, Aegle marmelos, Atropa acuminata, Atropa belladonna, Alpinia species, Hedychium species, and Cissus species.
This book is a handy reference for medicinal chemists, horticulturists and pharmacists who want to learn about the growth and conservation of important medicinal herbs and plants.
Readership
Medicinal chemists, horticulturists and pharmacists.
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Veröffentlichungsjahr: 2024
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PREFACE
The cultivation of medicinal plants, especially high-value medicinal plants, is creating a new dimension in the field of agriculture. However, the cultivation of medicinal plants is not easy. It is a challenging task because of very little knowledge of seed and pollination biology, nutrient and microhabitat requirements, pest management and growing seasons. Not much effort has been made to search for elite specimens and their propagation.
In recent years, plants in their natural habitat have become rare due to large-scale destruction for medicinal uses, long intervals for vegetative, flowering and fruiting stages, short viability of seeds and low seed germination. It is, therefore, important to conserve these medicinal plants because of their commercial importance. Conventional propagation methods cannot replace the depleting population because the seeds show a low percentage of germination, and vegetative propagation methods are sometimes unsuccessful. The development of standardized micropropagation techniques for the improvement of medicinal plant species is an important issue for preserving biodiversity. Further propagation through seeds may not fulfil the commercial demand of herbals and therapeutically important medicinal species. In vitro culture can be a valuable technique for clonal mass propagation and conservation of these medicinal plants within a short period of time.
Micropropagation, however, is highly labour oriented and, thereby, commercial companies are outsourcing plant multiplication activities to low-labour cost areas. Hence, in technologically advanced countries, the great potential of micropropagation for large-scale plant multiplication can be tapped by cutting down the cost of production per plant, pursued by applying the low-cost tissue culture, adopting practices, and optimizing the use of equipment and resources to reduce the unit cost of micropropagule and plant production without compromising the quality. Furthermore, the development and rapid multiplication of new medicinal plant cultivars are required to meet the demand of the industry all year round. The existing and refined protocols for in vitro culture, as well as their direct applications in improving and developing new cultivars, regularly supply plant material year round. Moreover, in vitro long-term storage of valuable germplasm would immensely provide benefits to both the industry and academic institutes. The outcome of recent studies carried out in various research laboratories and institutions shows optimized micropropagation protocols for many medicinally-important species and well-developed in vitro techniques, such as thermotherapy and cryotherapy for virus-free production, exploitation of somaclonal variation, long-term shoot culture conservation, and plant rejuvenation.
The present book gives the protocols for micropropagation of more than 40 species of medicinal plants. This book smartly combines scientific principles with the state-of-the-art in tissue culture techniques presented by experienced authors. I wish to express my gratitude to all the authors who contributed to the review chapters and research papers. I thank them for their cooperation and erudition. I hope that this will be a sourcebook for the cultivation and improvement of medicinal plants. I request that readers give their suggestions to improve in future editions.
List of Contributors
Biotization of Medicinal Plant Cultures by Endophytes: A Promising Approach to Enrich Therapeutics
Srinivasan T.1,Rajya Lakshmi Y.V.2,Shanmukha Anand P.3,Sunitha P.3,Aruna Lakshmi K.4,*Abstract
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.
INTRODUCTION
Need for Micropropagation of Medicinal Plants
Medicinal plants have been an important source of human therapy since time immemorial. All over the world, 70-80% of people use herbal medicines [1]. Although the exact number of medicinal plants in the world is not known, about 50,000 species are used in traditional and allopathic medicines, of which ⅔ are harvested from their natural habitat [2]. The plants are harvested extensively for various purposes: (1) For veterinary purposes and the well-being of livestock (2) In folk medicine, for snake bite, and anti-ophidic activity (3) for traditional medicine, and (4) Allopathy [3, 4] (Table 1). Additionally, some of the trees, like cancer trees and guggle trees, are used for wood, package, and gum purposes, without the awareness of the medicinal properties of such trees, and the fact that they are in critically endangered condition (Table 1). An added threat to the medicinal plants is their use to treat antibiotic resistance. Antibiotic resistance is reported all over the world, caused by the misuse or repeated use of antibiotics [5]. In attempts to treat patients with multiple drug resistance (MDR), plant extracts are in practice, which is a further provocation to the medical plants.
Further pressure on the existence/sustenance of these plants is a change in climatic conditions, like urbanization, that disturbs the habitat and eventually influencing the loss of species [6]. Moreover, some of the medicinal plants are highly heterozygous, for e.g., rhubarb, or have low seed set and poor percent germination, such as long pepper and black oil plants, or some of them are male biased (5 males: 1 female) as in jojoba, or have limited planting material as seen in bael (Table 1), and lack of suitable asexual propagation techniques [7-11]. As a result of these factors, some of th trees are critically endangered, e.g., Commiphora wighiti, while others are on the verge of extinction, (endangered), e.g., species of Commiphora, Curcuma, Piper, and of Taxus (Table 1) [12].
Quick and reliable propagation techniques to rescue these plants are mandatory. True-to-type clones could be obtained through micropropagation. It is reiterated that plant tissue culture is the realistic alternative for asexual reproduction. Micropropagation can be done in 3 ways: 1. Tissue and organ culture 2. Cell cultures in bioreactors, and 3. Genetic transformation for the production of metabolites.
Micropropagation of Tissue and Organ Culture
Plant tissue culture, a century old, provides techniques for many of the medicinally important plants, including tree species, but the studies where the clones are authenticated through molecular and biochemical assays are included in the present chapter (Table 1).
Mass Cell Cultures in Bioreactors
An interesting aspect of some of these plants is the diverse variety of primary and secondary metabolites secreted by them that are consumed by human beings medicine and drugs. For secondary metabolite production, the entire plant is not regenerated, and a callus is generated from which the metabolite is extracted, which is less expensive, and research has advanced in this area [13]. The production of secondary metabolite of in vitro tissues may be as much as the mother plant, like in Jyotishmati [14], or it may be higher in the clones than in the mother plant, as in sweet flag, king of bitters, Jaborandi (Table 1).
Genetic Transformation for the Production of Metabolites
Recently, in 2021, Cortelo’s group screened 702 cancer cell lines with plant extracts obtained from 95 plant species that showed anti-cancer activity [15]. After identifying the active metabolite in the plant extract, research progressed to clone the responsible gene in the host plant-genetic transformation to have enhanced metabolite synthesis. Among different gene transfer methods, Agrobacterium mediated transformation is preferred because it is inexpensive with precise insertion of the transgene in the host DNA [16]. The Ti/Ri plasmid of A. tumefaciens/rhizogenes is a natural gene vector that produces crown galls/hairy roots in the transformed tissues. Transformation through Agrobacterium species is achieved in many plants, a few of which are Bacopa monnieri, Artemisia annua, Catharanthus roseus, Rauvolfia mircantha, and Withania somnifera [17, 18]. Bacopa monnieri is transformed with A. tumefaciens, which contains tryptophan genes from Catharanthus roseus, secreting 25 times more tryptophan than the untransformed plants [17, 19]. A. rhizogenes transformed root of Withania somnifera produced 2.7-fold more withanolide-A than the untransformed roots [20]. The Ri-transformed plant roots are more successful as the hairy roots produce secondary metabolites in higher quantities and are not transported to other tissues in the plants [21, 22].
Current Trade/economical Aspects of Plant Tissue Culture (PTC) Medicinal Plants in India and the World
During the 1990s, about 500 million plants/annum were being produced through PTC globally [23]. Western Europe has 37 PTC units and produces 212 million plants, the Netherlands with 67 units produces 62 million plantlets, while Germany produces 8 million plants with 21 functional units [24]. India has 73 commercial PTC units, on average, it produces 5-10 million plants annually [23].
According to the International Union of Conservation of Nature (IUCN) Globally 40, 468 vascular species are being extinct. In India 2,142 are red-listed, of which 8 are extinct, 432 species are threatened and 54 are near threatened condition. The biodiversity – genetic, species and habitat of India is greater than many other countries in the world, which is 8% of the world’s biodiversity and occupies 2.4% of the area in the world [12]. Western Ghats of India are a rich source of medicinal plants and the Indian Government is taking preventive measures to preserve the plants and the secondary metabolite production of the medicinal plants there [25]. Global earnings on PTC are 14 billion dollars while the estimate in India is ~ 1 billion/year [23]. Different Organizations viz., the Food and Agriculture Organization (FAO), the United Nations Industrial Development Organization (UNIDO), the World Health Organization (WHO), and the International Development Research Centre (IDRC) are coordinating to work in order to meet the needs meet the need [26].
Future Needs for Propagation
The purpose of micropropagation is to preserve elite genotypes, produce pathogen-free plants in large numbers, select cells for bioreactors liquid cultures, and use them further for genetic transformation. India is known for supplying high-quality drugs at cheap prices [27]; India aims to build a triangle of traditional medicine, modern medicine, and modern science for which medicinal plants are required in high numbers [28]. About 0.1 million secondary metabolites are discovered from nearly 0.5 million plants which could be extracted from plant extracts [29]. It has been proposed that the biologically active metabolites can be extracted from the plants and could meet the commercial need [30].
Although a large amount of money is being invested to rescue the plants holding medicinal significance, the number of threatened species is still increasing [31]. In order to cope-up with the alarming situation, large-scale micropropagation and clones with high yielding of metabolites are mandatory.
The general protocols for the micropropagation of medicinal plants, and for culture of secondary metabolites are described below.
General Protocol for Micropropagation of Medicinal Plants
Although micropropagation protocols are available in the literature [32] and in the market, there is no universal protocol that suits every plant species. They must be standardized for each plant species, respective explants, developmental stage, and physiology of the crop. Also, based on the objective, whether it is the rescue of the desired genotype or callus for secondary metabolite secretion, the protocol must be chosen/ normalized. A general protocol for micropropagation of medicinal plants is described below (Fig. 1).
Nutrition Media
Usually, the media composition includes a carbon source, macro and micronutrients, vitamins, and hormones with or without agar/phytagel; pH is adjusted between 5-6 prior to autoclaving at 1.05 kg/cm2 (15 psi) and 121°C, for 15-20 minutes. The most common media used for micropropagation are Murashige and Skoog (MS), or Gamborg’s B-5 (B-5) or Woody plant medium (WPM) [33]. The hormones are selected based on the explant and purpose of the micropropagation.
Surface Sterilization of the Explants
Among medicinally important plant species, different plant tissues (explants), viz. seeds, rhizome segments, nodes, internodes, axillary buds, shoot tips, and leaves, are usually cultured to obtain calli and plantlets [34]. The explants are surface sterilized with different reagents for varying time periods, as presented in the next section.
Culture Conditions
The cultures are incubated usually at 24- 26°C under a 16 hrs. photoperiod of fluorescent tubes (40-56mol µm−2 s−1) and are usually subcultured in 3-5 weeks.
Plantlet Formation and Acclimatization
The response of the explant to the culture conditions may follow different pathways, viz., embryogenesis or organogenesis with or without a callus phase. The plantlets are initially adapted to vermiculite for 2-4 weeks and then gradually transferred to soil. The general micropropagation techniques are presented in Fig. (1).
Assessment of Regenerants
Although micropropagation should yield pure clones, sometimes somaclonal variants appear in cultures and among plantlets [35]. Hence, every step of micropropagation is systematically analyzed through cytology and molecular methods for somaclonal variation.
Liquid Suspension Cultures for Metabolites
A group of medicinal plants and pharmaceutical crops are cultivated for plant extracts, and small and large therapeutic molecules [36]. The extracts and the therapeutic molecules are used for preparing vaccines or treating diseases. To obtain the metabolites in large quantities, liquid suspension culture is the opted method for which initially calli are induced from the explants in solid media, then are later transferred to liquid suspensions. For commercial production of therapeutic molecules, the suspension cultures are transferred to liquid phase bioreactors as obtained in Taxol [37].
Aberrant Features/characteristics of Tissue Culture Plantlets
While PTC does yield clones in large numbers in a short time in limited space, the downside of the sterile technique is stated below:
The in vitro plants are generated and maintained under highly controlled conditions of nutrition, photoperiod, temperature, and humidity. As the cultures are maintained in closed containers, the cultures are in high relative humidity (95%) due to which plantlets show a thinner layer of wax, abnormal stomata, and guard cells, discontinuous cuticle, uneven-deposition of cellulose and lignin in the stems and leaves [59, 60]. In vitro plantlets have poorly developed root systems and reduced photosynthetic activity [61]. These developmental aberrations challenge the adaptation of the in vitro plants to field conditions and result in poor hardening success [62].
Many methods of abiotic hardening have been developed like the use of: a) photoautotrophic culture systems with high light intensity and reduced or eliminated sugar in the medium b) Ventilating culture vessels [63] c) anti-transpirants [64]. However, all these methods are limited to research laboratories and have not led to commercialization. Thus, hardening remains to be the bottleneck of tissue culture. Conventional methods of acclimatization cannot ensure a high percentage of survival and alternate approaches of acclimatization need to be evolved for micropropagation to be a viable proposition.
The use of certain bioagents such as Arbuscular Mycorrhiza, rhizosphere bacteria, and fungi in acclimatizing in vitro raised plantlets is a promising innovation. We propose that microorganisms can be applied potentially at different steps to yield beneficial results as shown in Fig. (1).
WHAT ARE ENDOPHYTES?
Microbes entered the world earlier than plants but for the past 407 million years they both evolved together influencing each other in the process [65]. Some microbes have evolved to supplement or complement the plant's physiological processes and become friends with the plants, while some evolved to exploit the gaps in the plant system and have become their foes [66]. The friendly microbes present in all spheres of the plant must have become an integral part of the endosphere and are commonly known as endophytes [67]. However, at present the most acceptable definition is “any organism that at some part of its life cycle, colonizes the internal plant tissues without causing any type of harm to the host plant” [68]. Nevertheless, due to extensive studies it is understood that the endophytes colonize every part of the plant and interact with plants through mutualism, antagonism, and rarely parasitism [69].
Fig. (1)) Targeted stages of micropropagation for endophyte inoculation.Endophytes influence plant growth, development, and stress tolerance. Endophytes are highly variable not only in number but also in type based on the host plant, plant part, developmental stage, environmental factors, etc [70, 71]. However, the consequence of the plant - endophyte interaction is not uniform and ubiquitous. It also depends on the environmental factors and genotype of both plant and endophytic organisms [72]. The endophytes like pathogens enter the plant system, evade the immune response, and colonize the best-suited plant tissue. They primarily enter through openings in the roots, stomata, wounds, and hydathodes [73-75]. A few of the microbes even modify the plant cell wall for their entry and spread inside the plant. Upon entry into the tissue, the endophytes escape the plant immunity in many ways. They translocate from the endodermis to the vascular system where plant immune attack is less [76, 77]. They survive oxidative stress by producing the required antioxidant enzymes like superoxide dismutases, catalases, etc [78, 79]. The endophytes modify or suppress the molecules that act as microbe-associated molecular patterns (MAMP) weaken the plant defense reaction and colonize the host [79, 80].
The colonized local endophytes favour the host plant in multiple ways. Some of the endophytes enhance nutrient availability, and transform minerals like nitrogen, phosphorus, potassium, and iron to plant utilizable form; such that plants absorb them easily, grow well, and have high yields [81-83]. Endophytes induce or produce plant hormones like gibberellins, auxin, and ethylene and influence plant growth and development by modulating the hormones. They also induce or produce enzymes, such as Aminocyclopropane-1-carboxylate deaminase (ACCD) that promote cell division, elongation, and differentiation [84, 85]. Plants experience biotic and abiotic stresses which are mitigated by endophytes. The biotic stress is negated by quorum sensing, antibiosis, and induction of plant immunity. The impact of abiotic stress is mitigated by endophytes. They directly or indirectly induce the antioxidant mechanism as well as osmolyte production. This reduces the oxidative stress and osmotic stress-induced damage thus enhancing membrane stability. The hormones released by endophytes also improve the photosynthetic capacity, and stomatal regulation, promote cell division, stimulate the growth of the root system, and acquisition of water and nutrients [66, 86-88].
Diversity in the Endophyte Population
Endophytes generally refer to all organisms present in the endosphere of a plant, but this microbiota is not uniform; it varies among the tissues of the same plant and in the same plant over time. Endophytes have a significant role in plant health and productivity, but their individual contributions and synergistic effects with other endophytes remain unknown. Endomicrobiota is being viewed as a possible replacement for chemicals for the next green revolution and a source of unique medicinal compounds in this era of drug resistance and new diseases [68, 79]. Therefore, to understand and exploit endophytes most effectively, we must know and comprehend the diversity of these organisms.
Endophytes consist primarily of fungi and bacteria. They are found in all parts of the plants. Compared to other plant parts, endophytes are abundant in the stems of woody plants and the roots of herbs. Fungal endophytes account for around 70% of all endophytes reported to date, whereas bacteria account for approximately 30% [89]. Bacterial endophyte diversity may be substantially less thoroughly catalogued than fungal endophyte diversity due to their diminutive size, low biomass, and uncertain ecological activities. Despite the vast diversity of endophytic bacteria, the number of distinct bacteria discovered in a single plant typically ranges from 10 to 200 for culture-based studies and from 20 to 600 for non-culture-based studies [90]. The usual number of bacterial cells per gram of tissue is between 103 and 104, except for root nodules, which contain up to 107 cells per gram of tissue [91]. Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes have been discovered to be the most numerous bacterial phyla in a wide variety of plant species, with Pseudomonas, Bacillus, Pantoea, and Acinetobacteria being the most prevalent bacterial genera [92, 93].
More than a million fungal endophytes are thought to infest about 300,000 species of terrestrial plants worldwide [94, 95]. Non-culture-based studies estimate 40 to 1200 fungal endophyte species per plant, whereas culture-based investigations estimate 5 to 350 species per plant [90]. Non-balansiaceous fungi are the more prevalent of the two basic categories of endophytic fungi. Ascomycota, followed by Basidiomycota and Glomeromycota, is the predominant phylum of fungal endophytes. Acremonium, Alternaria, Cladosporium, Coniothyrium, Fusarium, Geniculosporium, Phoma, and Pleospora are all common genera [96, 90]. Some groups, like Xylariaceace, Colletotrichum, Phyllosticta, and Pestalotiopsis, are most common in the tropics, while others are common in both tropical and temperate climates (e.g., Fusarium, Phomopsis, and Phoma) [97, 90].
Numerous limitations have an influence on the diversity and density of endophytes outlined previously. Only 30% of embryophyta plant families have data on fungi, while 10.5% of plant families have data on bacteria. These investigations are based on 1,702 distinct taxa representing 254 families [98]. The studies focused almost entirely on a single species of plant at a time, and initially, just one part of the plant was examined in each study. Early research also focused mostly on isolating and identifying endophytic fungus before shifting its attention to bacteria [99-103]. Most of the current endophytic research on plants focuses on either fungus or bacteria; very few examine both. Furthermore, the endophytes reported depend on the type of medium and culture conditions used. Therefore, the variety and density of the endo microbiota in the host plant will not be accurately reflected in these studies. Hence, the actual diversity and density of endophytes responsible for plant health and the generation of bioactive chemicals are unknown. The given endophytic data should serve as the foundation for biotization investigations, around which researchers can test various permutations and combinations of the endophytes.
Isolation of Endophytes
Endophytes are diverse and widespread throughout the plant endosphere. Although methods for the identification of endophytes are advancing with time, isolating endophytes continues to rely on culture-based methods. Endophyte isolation is always challenging as ephiphytes constantly present a hurdle. This is addressed by effective surface sterilization of the explants. The effectiveness of the sterilization procedure determines whether all epiphytes are eliminated, and the growth of diverse endophytes. The selection and duration of sterilant depend upon the explant, which had to be standardized, as otherwise, it affects the yield of the endophyte. Endophyte growth is further influenced by the media composition and culture conditions.
The standard steps of a sterilization process consist of washing the plant material with running tap water, chemical sterilization with one or more chemicals, followed by rinses of sterile water. The first and final steps of every surface sterilization protocol are common, however, the second step is highly variable. Step two is dependent on the plant material, hence needs to be standardized for each plant and explant respectively.
The two-step chemical sterilization method includes ethanol wash followed by sodium hypochlorite (NaOCl), hydrogen peroxide (H2O2), or mercuric chloride (HgCl2) [104]. In the first step, 65- 85% ethanol is used [105], which being a lipid solvent and protein denaturant affects the functioning of the cell membrane. Ethanol is also a dehydrating agent, so very high concentrations for prolonged periods that cause phototoxicity are avoided [106]. At a concentration of 90% or above, ethanol is effective against fungal and bacterial spores [107, 108]. For the total removal of spores or any escapes, a second chemical is used. In the second step of chemical sterilization, sodium hypochlorite is preferred over hydrogen peroxide and mercury chloride. Microorganisms that produce catalase can breakdown H2O2 and impede the disinfection process [109, 104]. Although HgCl2 is effective, it must be used sparingly because of the risks it poses to people and the environment. NaOCl is a wide-spectrum disinfectant. It produces hypochlorous acid (HOCl) and hypochlorite ion (-OCl), which degrade proteins, amino acids, and DNA, and damage the cell completely [110]. HOCland -OCl ions can oxidize a cell, HOCl is more effective due to its high penetrating capacity and is usually used at a concentration of 0.5% to 5% [105, 104]. H2O2 is also a broad-spectrum anti-microbial agent. It contains a peroxide ion that is a powerful oxidant and causes oxidative damage to proteins, lipids, and DNA [109]. It is used at concentrations ranging from 3% to 90% for long periods of incubation to have a sporicidal effect [111-113], because of which its usage is limited. HgCl2 is a broad-spectrum disinfectant, that has been in use for a long time. Heavy metal and chloride ions contribute to the process of disinfection. Mercury atom induces cell enlargement and disruption [114]. The electronegativity of chloride ions oxidizes peptide bonds and denatures proteins. HgCl2 is used at concentrations between 0.01% and 1%, however, its use is restricted due to its toxicity [104]. For the surface sterilization of complex explants, such as seed and bark, HgCl2 is still used when alternatives are inadequate. In the last five years, Ethanol and NaOCl have been used extensively in endophyte isolation for surface sterilization (Table 2).
Based upon the endophyte to be isolated, the culture conditions are accordingly manipulated. For fungal isolates, potato dextrose agar medium is used mostly [115]. The medium is supplemented with antibacterial agents like chloram- phenicol, ampicillin, streptomycin, etc. After one week of incubation, the developed fungal cells are subcultured to obtain pure colonies. The isolates are stored in 15%–20% glycerol at -80 degrees Celsius [115, 116]. The predominant phyla of the isolated bacterial endophytes are Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes [92]. For isolation of endophytic actinobacteria, the water agar (1.5%), yeast extract agar, humic acid-vitamin agar, cellulose-proline agar, starch casein agar, starch-glycerol-nitrate agar, xylan-arginine agar, and succinate-arginine agar are used. To obtain pure isolates, the initial colonies are subcultured in ISP (International Streptomyces Project) media [117-119]. For the isolation of Proteobacteria, Firmicutes, and Bacteroidetes, Peptone-yeast agar or yeast-mannitol agar media are used. Pure cultures are generated using the same media. Luria – Bertani medium is also used for the isolation of bacteria [120, 121]. The media are supplemented, each with antifungal compounds such as nystatin and cycloheximide [117, 118]. Isolates are stored in 15-20% glycerol at -80°C [122, 123]. The isolated bacteria or fungi are identified based on morphology and biochemistry [124, 125] and are authenticated through 16S and 18S rDNA analysis [115, 116].
Endophytes from Medicinal Plants
In the natural habitat, medicinal plants have the luxury of diverse endophytes promoting their growth, development, and defense system. During the process, several bioactive compounds are generated, some of which have remarkable therapeutic significance. Micropropagation of medicinal plants by in vitro techniques reduces the diversity and quantity of bioactive compounds produced. The biotization of medicinal plants with suitable endophytes is imperative. The purpose of endophytes in the biotization is not only to prime the production of medicinally important compounds but also to enhance the response of the explant culture. Therefore, the endophytes that produce phytohormones, and secondary metabolites, and induce diverse secondary metabolite-producing pathways in host plants must be identified and exploited appropriately.
A few decades ago, the endophytes that are a source of bioactive compounds of medicinal significance were identified. The Pacific yew tree (Taxus brevifolia) is used to treat breast, lung, ovarian, and Kaposi's sarcoma cancers. The Pacific Yew endophytic fungus Taxomyces andreanae produced the anticancer compound paclitaxel (taxol) [126]. Taxol, whose production has been extensively investigated, is a diterpenoid, produced largely by diterpene synthases [127]. gibberellic acid, a tetracyclic diterpenoid, is produced by these enzymes as well [128]. It is understood that these endophytes can also produce gibberellic acid and promote the micropropagation of Taxus species. Further, many endophytic fungi of other host plant species, such as Seimatoantlerium tepuiense, Seimatoantlerium nepalense [129], Tubercularia sp. strain TF5 [130], Metarhizium anisopliae [131] etc., were also found to produce taxol. Endophyte M. anisopliae colonized maize roots and increased their salicylic acid content [132]. Low to moderate levels of salicylic acid always promoted the growth and development of plants, including the production and transport of Indole acetic acid (IAA) [133].
Trametes hirsuta and Phialocephala fortinii, endophytes of Podophyllum hexandrum and Podophyllum peltatum, respectively, produce Podophyllotoxin, which is well known for its anticancer and antiwart properties [134, 135]. Phialocephala fortinii and other species of this genus induced phytohormone production in the host [136, 137]. Vincristine, which is extracted from Catharanthus roseus, is also generated by Fusarium oxysporum, an endophyte of C. roseus [138]. The synthesis of gibberellic acid-3 (GA3) by endophytic F. oxysporum [139] increases the likelihood of boosting the proliferation of the host plant. The antioxidant, antimicrobial, anticancer, anti-asthmatic, and antihypertensive properties of Rumex gmelini Turcz are well-documented. The coculture with its endophytes, notably species of Aspergillus, Fusarium, and Ramularia, improved the synthesis of bioactive chemicals responsible for these activities [140].
Several endophytes that are documented to affect the generation of medicinally significant secondary metabolites directly or indirectly also demonstrated growth-promoting characteristics of the host. Therefore, the endophytes that triggered the synthesis of the medicinally significant bioactive compounds might be evaluated for growth promotion and be utilized in biotization studies.
IN VITRO BIOTIZATION OF MEDICINAL PLANTS
Biotization, a phenomenon analogous to vaccination, was initiated by Herman [150] where in vitro grown potatoes were bacterized with strains of Bacillus species to have enhanced transplant stress tolerance. This procedure gained popularity; potato producers use it still, as it avoids greenhouse expenses and enhances stress tolerance. Tissue culture plantlets face several biotic and abiotic challenges through micropropagation viz., malfunctioning of stomata, under-developed vasculature, reduced photosynthetic potential, and poor root system devoid of root hairs. Recently, a growing body of evidence has been available suggesting bacterial and fungal endophyte association at various stages of micropropagation imparts stress tolerance to the in vitro propagules.
Among different endophytes, the strains of Bacillussps are the most effective in promoting plant growth and development when co-cultured with crop plants. Bacillus species strain PsjN did not grow on standard plant tissue culture medium, without the plant, but forms a population of epiphytes when co-cultured with potato and/or tomato [151, 152]. The plants inoculated with bacteria exhibited improved growth, better water management, and enhanced pathogen resistance after they were transplanted into the soil [153-157].
The initial attempts of endophyte biotization focused mainly on agricultural crops like tomatoes, grapes, potato, etc., with little emphasis on medicinal crops. However, in the recent years, endophyte inoculation is extended to horticulture, forestry, and pharmaceutical crops. There have been a few reviews [158-163] sparkling the endophyte biotization of agriculture and horticulture crops but not of medicinal plants. This is the first review which is presenting the biotization of medicinal plants exclusively.
Effect of Microbial Endophytes on the Growth and Secondary Metabolite Production of In vitro Plants
Endangered medicinal plants with anti-cancerous metabolites, e.g., Gloriosa superba [164] or those with hardening problems e.g., Handroanthus were the first attempted plants for biotization. Initial attempts included fungal strains, but later bacterial endophytes had gained prominence in biotization. Among the fungal endophytes, a commercial mix of Glomus species, Piriformospora indica, and Plant Growth Promoting Hyaline Sterile Fungus (PGP-HSF) are commonly used (Table 3). Among the bacterial endophytes, Pseudomonas species is mostly used followed by Azispirullum brasiliense and Bacillus species. Recently, native endophytes isolated from soil or plants are also being used for in vitro biotization [165].
As evident in Table 3, most studies report a direct relation between biomass production and the concentration of the bioactive compounds in the host. However, a few studies suggest a negative relation between biomass and secondary metabolite production. Several studies report enhanced biomass and secondary metabolite production when inoculated with microbial consortia rather than individual inoculations [166-168]. Conversely, a mixture of inoculants had a negative effect on biomass and yield compared to the single inoculations. Pseudomonas mucidolens had a different effect on clonal lines of thyme, lavender, spearmint, and oregano which had good biomass, high phenolic and rosmarinic acid as well as other bioactive substances; and has thus been used as a tool for identification of clones [169, 170].
It is evident from these studies, that endophyte colonization specificity in the host tissues critically influences plant growth and plant secondary metabolites content. In this regard, the work of Maggini and associates [164, 171, 172] to understand the mechanisms of interaction between the host and the endobiont needs special mention. An elegant “in vitro infection model” with the medicinal plant Echinacea purpurea
