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- Herausgeber: Bentham Science Publishers
- Kategorie: Fachliteratur
- Serie: The Chemistry inside Spices & Herbs: Research and Development
- Sprache: Englisch
The Chemistry inside Spices & Herbs: Research and Development brings comprehensive information about the chemistry of spices and herbs with a focus on recent research in this field. The book is an extensive 2-part collection of 20 chapters contributed by experts in phytochemistry with the aim to give the reader deep knowledge about phytochemical constituents in herbal plants and their benefits. The contents include reviews on the biochemistry and biotechnology of spices and herbs, herbal medicines, biologically active compounds and their role in therapeutics among other topics. Chapters which highlight natural drugs and their role in different diseases and special plants of clinical significance are also included.
Part I focuses on the general aspects of spice biotechnology, structure activity relationships and the natural products that can be used to treat different diseases - such as neurological diseases, inflammation, pain and infections. This part also covers information about phenolic compounds, flavonoids and turmeric supplements.
This book is an ideal resource for scholars (in life sciences, phytomedicine and natural product chemistry) and general readers who want to understand the importance of herbs, spices and traditional medicine in pharmaceutical and clinical research.
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Seitenzahl: 510
Veröffentlichungsjahr: 2000
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FOREWORD
The book titled “The Chemistry inside Spices and Herbs: Research and Developments (Volume-1)” Edited by: Dr. Pankaj Kumar Chaurasia and Dr. Shashi Lata Bharati has an excellent collection of 10 chapters written by the experts of their subjects from countries like India, Iran, and Egypt. Each chapter of the book, attractively written by the experts, is full of research as well as academically momentous information. This book brilliantly deals with biologically valuable spices, herbs, their related chemistry, biochemistry, structure-activity relationships, biologically as well as pharmaceutically valuable active natural compounds, roles in the natural treatment of various human problems, treatment of neurobiological disorders, roles as antifungal and antibacterial agents, naturally-derived analgesics and anti-inflammatory agents, phenolic compounds, flavonoids, curcumin, turmeric, natural therapy, and so on.
In the present time of pandemic and other problems, when the whole world is searching for various types of immunity boosters to fight this virus, this volume may be helpful in this direction in order to provide in-depth information because there are different types of spices, herbs and their constituents discussed in the book which are radiantly useful in the treatment of various human problems and enhancements of immunity. In my view, after giving a thorough look at the contents, this book may be very advantageous for academicians, researchers and scientists working in the field of spices, herbs, their related chemistry, natural medicinal therapy, and so on. I am congratulating the editors of the book for producing such a useful, academically as well as a scientifically relevant book by compiling the comprehensive chapters contributed by the experts of various countries. I strongly recommend this volume for UG and PG students of life sciences, natural chemistry, biochemistry, natural medicinal studies and scientists working in aforesaid areas.
PREFACE
Pankaj Kumar ChaurasiaShashi Lata BharatiPlants are the boon of nature on the earth for us in many ways. They detoxify the environments and save the lives living on this earth. Out of several advantages of plants, their different parts and/or substances are known for their noteworthy medicinal values. Spices and herbs which are involved in our daily routine life are the treasure of good health. Spices, a routine part of the kitchen, as well as herbs of our garden, are full of medicinal virtues and benefits and can be significantly used for the treatment of various disorders and diseases of humans. Spices are actually fruits, seeds, barks, roots and other parts of the plants widely used for enhancing the taste, color and quality of the foods (https://en.wikipedia.org/wiki/Spice) and are the source of various valuable chemical constituents of pharmaceutical significances while herbs are leafy green or flowering parts of the various plants with savory or aromatic properties (https://en.wikipedia.org/wiki/Herb). They are the major source of Ayurveda and other traditional culture of treatments and also have a great potential in the modern time. Spices and culinary herbs and their various chemical constituents involved in the treatment of various problems, diseases and wounds have been beautifully covered in this book.
In the present time of the serious pandemic COVID 19 period, demands of pharmaceutically valuable spices and herbs have been surprisingly enhanced all over the world because they have a substantial and valuable position as nutraceutical which doubtlessly are due to their significant healthy, nutritious and immunity boosting properties. Actually, the main objective of the construction of this book was to collect the more significant valuable researches and information on spices and herbs, which are being widely used in our daily life either in the form of taste enhancing savory materials or quality improving materials or beautiful home decoration and so on. Collection of weighty researches on biologically active pharmaceutically interesting chemical compounds and their compositions and structure activity relationships of these compounds was the second most interesting objective of this book.
This book is full of scientific knowledge on spices, herbs, associated internal chemistry and wide biological performances. It includes biochemistry and biotechnology of spices and herbs, antimicrobial properties, analgesics and anti-inflammatory agents, cure of neurobiological disorders, phenolic compounds, flavonoids, structure activity relationship, biologically active compounds and isolation, and so on.
This volume consists of total ten chapters and each chapter has been written by the various learned experts of their field. Learned experts come from different countries like India, Iran and Egypt. This unique collection of chapters may be highly beneficial for the students of graduate and post graduate level studying in the field of life sciences, biotechnology and biochemistry, plant sciences and for researchers and scientists working research in the field of spices, herbs, compounds with biological activity, natural treatment and natural pharmacology. The book is full of updated knowledge, information and recent researches, and without any doubt, it will be very much fruitful for the readers.
Chapter 1, titled “Spices Biotechnology: Opportunities and challenges”, written by Hamid et al., provides an overview of various biotechnological solutions that increase the quality and productivity of spice plants.
Chapter 2, titled “Spices, the guards against the evil microbes: Antimicrobial properties of spices”, written by Jacob et al., highlights the effect of various spices on various micro-organisms, the various metabolites in spices that lend this ability and also reviews.
Chapter 3, titled “Spices and Herbs in the Treatment of Neurobiological Disorders”, written by Trivedi et al., deals with the role of spices and herbs for the cure of neurobiological disorders. Based on the investigations on herbal plants and neurological substrates in disease conditions, herbal medicines can be effectively used in the treatment of various neurological disorders.
Chapter 4, titled “Spices and Herbs in Bacterial and Fungal Resistance”, written by Trivedi et al., describes the use of spices and herbs against bacteria and viruses. The use of spices and herbs presents a great potential alternative or supplementary medicine to reduce side effects, progressively increasing the resistance of pathogens induced by the use of allopathic drugs.
Chapter 5, titled “Naturally Isolated Compounds from Spices and Herbs and their Medicinal Uses”, is written by Ramteke, A.M. This chapter includes a wide variety of isolated compounds such as phenolic compounds and flavanoids present in spices, which are now experimentally documented to possess antioxidant, anti-inflammatory, antimutagenic and anticarcinogenic activities. It also includes a list of spices compounds that are experimentally evidenced to control cardiovascular diseases, diabetes, cataract, cancer, etc.
Chapter 6, titled“Naturally-derived Analgesics and Anti-Inflammatory Agents”,written by Fayez et al.,covers all the nutraceuticals and phytochemicals – derived from medicinal plants– which have been reported to possess analgesic and/or anti-inflammatory effects over the period between 2018 up to June 2020.
In Chapter 7, titled “Phenolic compounds and their Biological and Pharmaceutical activities”, Kumar et al. have summarized information on the biological and pharmaceutical activities related to different classes of phenolic compounds.
Chapter 8, titled “Structure Activity Relationship of flavonoids: An update”, written by Khare et al., focuses on the majority of polyphenols present in the daily diet, which mainly exist as glycosides with different sugar units and acetylated sugars at different positions of the polyphenol skeletons.
Chapter 9, titled “Biologically active compounds and Structure-Activity Relationship” has been written by Ganatra, S.H. He has discussed all three methods in detail, along with examples. It also provides the practical procedure to use available computational tools. The final aim of this chapter is not only to provide the theoretical background of drug discovery using structure activity relationships, but also to provide practical methods.
Chapter 10, titled “Turmeric Supplementation and Its Valued Clinical Connections”, demonstrates the renowned significance of turmeric in the treatment of various health issues and its role as a food supplement concisely.
List of Contributors
Spices Biotechnology: Opportunities and Challenges
Rasmieh Hamid1,Feba Jacob2,*,Mehrnaz Entesari3,Shri Hari Prasad2,Shivaji Ajinath Lavale2Abstract
Spices have been used since ancient times as a flavoring agent as well as an important medicinal resource. Biotechnology, using strategies such as cell, organ, and tissue culture, genetic engineering, and the application of nucleic acid markers can escalate the productivity and efficiency of spices. Cell, tissue, and plant organ culture have enabled the rapid and mass reproduction of many disease-free spice plants, which are uniform genetically and qualitatively. In recent years, cell and limb suspension (stem and hair roots) have been considered for producing secondary metabolites and for studying the biosynthesis pathway of metabolites. Plant genetic engineering has helped in the genetic identification and manipulation of enzymes of the biosynthetic pathway of secondary metabolites. Gene transformation has improved the production of secondary metabolites that have yield limitations. Molecular markers are powerful tools for accurately identifying important medicinal species, examining genetic diversity, classifying hereditary reserves, and determining their genetic map irrespective of their age, physiological, and environmental conditions. Next-generation sequencing (NGS) methods like restriction-site-associated DNA sequencing (RAD-seq) have revolutionized the study of genetic diversity, and the enzymes and genes implied in the secondary metabolites biosynthetic pathways can be studded by transcriptome profiling (RNA-seq). The ground-breaking genome editing techniques like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), sequence-specific nucleases of transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases could help in customizing the plants according to the requirements. This article provides an overview of various biotechnology solutions that increase the quality and productivity of spice plants.
INTRODUCTION
Spices are mainly the aromatic parts of plants that have been dried. The Food and Drug Administration (FDA) has defined spices as: “aromatic vegetable substances in whole, crushed, or ground form, the notable characteristic of which in food is preparation as opposed to nutrition.” [1]. Flavors are regularly derived from the dried part of plant-like buds, barks (cinnamon), fruits/berries (cloves, black pepper, chili), blooms (cloves, saffron), seeds (cumin), or roots (ginger, turmeric) that contain unstable oils or fragrant scents and aromas [1, 2]. The majority of the well-known spices and herbs come from Asia, the Middle East, or Mediterranean countries and have been used since ancient times [3]. Spices and herbs have occupied, and still occupy, significant roles as seasoning specialists, food additives, and meds for quite a long time. Over the last few decades, the investigation into their medical advantages has expanded essentially; the same number of spices and flavors are considered to have properties that reduce the risk of chronic disease development. Specifically, a few of the potential wellbeing benefits of herbs and flavors include conferring security against cancer, chronic inflammation, cardiovascular illness, type 2 diabetes, neurodegenerative conditions and obesity [3-6]. Several herbs have been renowned for their anti-inflammatory, antioxidant, and anti-microbial properties [7, 8]. Additionally, the use of certain herbs and flavors will help in reducing the use of salt as the sole flavoring agent (i.e., lower sodium admissions), which has cardiovascular benefits [9]. Black pepper, turmeric, clove, vanilla, cardamom, nutmeg, ginger, cinnamon, tamarind, etc., constitute the major flavors, whereas fennel, fenugreek, coriander and cumin are imperative seed flavors. While anise, celery, lavender, oregano, saffron, sage and thyme are critical homegrown flavors. The transcriptomes of Piper player nigrum and Piper player colubrinum were analyzed to understand the host-pathogen activity in black pepper, with a focus on Phytophthora foot rot tolerance. The productivity of spices is poor, owing to the lack of high-yielding, pest and diseases resistant varieties, and also due to postharvest losses. Ordinary breeding programs were found to be time-devouring and lumbering in perpetual flavors, such as cardamom and black pepper. Dearth of sources of biotic and abiotic stress resistance within the evolved germplasm made the process even more arduous. Furthermore, crops like ginger and turmeric have no or very few seeds, rendering traditional breeding systems ineffective. Creating varieties with high yielding and disease resistance, under such circumstances, through biotechnology, is imperative for the improvement of spices. The use of biotechnological methods to achieve the above has increased dramatically in
recent years through marker-assisted breeding, development of novel varieties, and commercial propagation.
COMPARATIVE GENOMICS AND GENE TAGGING
Comparative genomics compares various genomic features like genes, regulatory sequences, DNA sequence, gene order and various genomic structural landmarks of several organisms. A crucial step in breeding is recognizing the loci of beneficial genes (high yield, quality, cost-efficiency, and pest and disease resistance). It may be a capable and swift strategy since it does not necessitate several generations of closely supervised parent strain breeding [9]. The detailed transcriptome of Piper nigrum and Piper colubrinum was conducted w.r.t host-pathogen interaction in black pepper with more focus to the Phytophthora foot rot tolerance [10]. The root transcriptome sequencing of black pepper [11] was done by the SOLiD platform and a detailed dataset of 10,338 UniGenes was found to be crucial for the molecular breeding of black pepper. The 4472 anticipated proteins appeared to have approximately 52% homology with the Arabidopsis proteome. The comparative proteome analysis of two roots revealed 615 differentially expressed proteins [12]. Hu, Hao [13] depicted the black pepper fruit transcriptome in conjunction with the piperine biosynthetic pathway and found 40,537 UniGenes included in piperine biosynthesis. The molecular mechanisms underlying foot rot susceptibility were understood by comparing the transcriptome of resistant (Piper flaviflorum) and susceptible (P. nigrum cv. Reyin-1) species. It was observed that the genes consolidated within the phenylpropanoid metabolism pathway were highly up-regulated in resistant species [10]. Karthika, Prasath [14], compared the ginger (Zingiber officinale Rosc.) and mango ginger (Curcuma amada Roxb.) transcriptomes in response to bacterial wilt infection and they observed that 105 genes were only expressed in C. amada (safe species) in reaction to contamination by Ralstonia solanacearum. These genes were linked to pathogen defence through hypersensitive, systemic acquired, and cell death responses mediated by salicylic acid (SA). Out of the 54 differentially expressed transcription factors, 32 showed upregulation in C. amada, which includes GATA, WRKY, zinc finger, MYB and leucine zipper protein domain transcription factors. The transcriptome of two samples of the elite ginger variety Suprabha obtained from two separate agro-climatic zones of Odisha was analyzed by Gaur, Das [15]. The novel transcripts coding for terpenoids related to anticancer and antimalarial in the transcriptome of Curcuma longa was reported by Annadurai, Neethiraj [16]. Comparative transcriptome (rhizome-specific) evaluation of C. longa and Curcuma aromatica associated with curcumin content provided information about the genetic basis and regulation of curcumin biogenesis [17]. Differential expression analysis identified two novel polyketide synthase genes (clpks1 and clpks2), which showed similarity to Musa acuminata polyketide synthase type 2 (MaPKS2) and M. acuminata polyketide synthase type 4 (MaPKS4) that were found to be upregulated in C. longa [17]. Babu, Jose [18] analyzed the transcriptome assembly of the turmeric variety Suvarna (CL-Suv). The transcriptome from seeds, leaves, and flowers of Coriander (Coriandrum sativum L.) was sequenced and analyzed by Tulsani, Hamid [19], 8676 unigenes were assigned to 153 KEGG pathways in this study. Among them, 291 unigenes were related to terpenes biosynthesis. Paul, Mathew [20] explored the possibility of using comparative transcriptome analysis to point out the candidate genes responsible for the black pepper foot rot field tolerance. DD-RT PCR on cDNA fragments was used to compare transcriptome profiles, and the bands that were differentially expressed were sequenced. Sequence analysis showed the participation of signal proteins and defence enzymes like Aspartyl protease, beta-glucosidase enzyme, Cytochrome P450 signal protein, Nitrous oxide reductase family maturation protein, nucleoredoxin 1-1 enzyme, Phosphatase 2C-like domain-containing protein, Premnaspirodiene oxygenase, putative disease resistance protein RGA3 and Serine/Threonine Protein kinase WAG1 in field tolerance of black pepper to foot rot. Additional insights into the molecular function of tolerance were acquired by pathway analysis. Jiang, Liao [21], analyzed the transcriptome and phytohormone profiles of ginger (Zingiber officinale Rose) in reaction to postharvest dehydration stress. Transcriptome profiling found out a total of 1415, 2726, and 6641 genes were differentially expressed after 2 h, 12 h, and 24 h of water-loss stress treatment, respectively in comparison with that during zero h of ginger rhizomes. Moreover, 518 DEGs shared comparable expression patterns throughout twenty-four h of dehydration stress. These genes are specifically enriched in plant hormone signalling, carotenoid biosynthesis, starch and sugar metabolism, phenylalanine metabolism, fatty acid elongation, and phenylpropanoid biosynthesis.
Cloning and Genes Isolation
Genes involved in biotic and abiotic stresses and agronomically critical characters were distinguished in most spice crops [22]. Pathogenesis related candidate genes may also be distinguished using sequence data from libraries, extracted, and then integrated into promising varieties utilizing transgenic techniques. A family or genus, wild relatives of crops may have a set of genes for various biotic and abiotic resistance, agronomically important characteristics, etc [23]. Since hybridization based breeding programs to mobilize genes from wild relatives are challenging, the transgenic approach to join the genes is preferable.
Genetic Transformation
Diseases, a lack of resistant varieties, and post-harvest declines are the main causes of lower spice yields [24]. Genetic transformation has great potential to overcome restrictions of conventional breeding methods and produce high yielding and disease resistant transgenic plants [25]. Plant transformation is considered as both a basic scientific method in plant biology and a practical tool for transgenic plant advancement [26].
Gene transformation is a powerful tool for increasing productivity. There are various methods for gene transformation; such as Agrobacterium-tumefaciens transformation, particle bombardment, and electroporation for gene transfer on herb and spice plants, but there are two fundamental classifications for gene delivery: biological and non-biological system [27].
NON- BIOLOGICAL GENE TRANSFORMATION SYSTEM
There are several non-biological systems, which are used for gene delivery via plant or protoplast. Non-biological systems like chemical treatment of isolated protoplasts by PEG, electroporation, lipofection, or fusion of protoplasts with liposomes, microinjection, and biolistic. In a direct gene transformation system; chemical solution including PEG, Polyethylene glycol (generally is used only PEG) is incubated with DNA fragment and protoplast. Protoplast is the most appropriate explant in this technique. Due to accessibility and simplicity, this protocol has been reported in numerous plants [28, 29]. There are some reports of using protoplast fusion mediated (PEG mediated) for production of abiotic or biotech disease tolerance or somatic hybridization in vanilla species [30], ginger [31], and coriander [32].
Lipofection mediated transformation involves liposomes (as artificial circular lipid with an aqueous interior for carrying DNA fragment), which can be stimulated via PEG to integrate into protoplasts [33]. A sudden electrical discharge for creating small pores in the plasma membrane is used in the electroporation system for the transformation of DNA to protoplast. Transformed protoplasm has the potential to regenerate transgenic plants. Electroporation is introduced as a reproducible system if a good quality protoplast is produced. In the microinjection method, DNA fragment is transferred mechanically to a specific target, which normally is the protoplast. The process is applied through a glass micro capillary-injection pipette. Using a micromanipulator is not practical for transformation in the plant due to the presence of the cell wall, however, it has been effectively used for the transformation of large animal and human cells [29, 34]. Although used rarely for gene transformation, biolistic gene transformation is an alternative non-biological method and has been referred to as an important and famous method for gene transformation to spices plants.
Biolistic Micro-Projectile Bombardment Gene Transformation
The micro projectile bombardment method (also mentioned as particle bombardment, particle gun method, particle acceleration, and biolistic) has been widely introduced as a routine, reliable, and physical gene delivery system [33]. In this method, DNA or RNA gene is coated on microinjection (which normally is tungsten or gold with the size of 1-4 m) then bombarded into callus explants. Micro-carrier size, explant target distance, and helium bombardment pressure and the constructs (circular or linear plasmid) used are factors affecting the efficiency of biolistic transformation. Among the various explants such as microspore, pollen, and shoot meristem reported as explants, embryogenic callus has a higher potential for uniform regeneration after the bombardment and has been considered as an optimum explant for biolistic gene transformation [35]. There are numerous reports of reproducible transformation protocol in capsicum [35], ginger [36], turmeric [37], cumin [38] via biolistic system. However, multi-copy integration, which causes transgenic silencing, has been reported as a major concern [39].
Biological System Agrobacterium-Mediated Gene Transformation
Agrobacterium tumefaciens mediated transformation is a natural mechanism for gene transformation in numerous plants. Even though, there are various approaches for gene transformation, the use of Agrobacterium is superior and more popular than other methods especially in dicotyledonous plants, due to more efficiency with lower cost, reproducibility, high capacity to transfer large inserts of DNA, and low copy number. This technology has been widely used for gene transformation (stable or transient) in many spices [40].
IN PLANTA GENE TRANSFORMATION
Another way to use agrobacterium in gene transformation is in planta, i.e. DNA transfer directly in the intact plants without using tissue culture methods [41]. This minimizes somaclonal variation and saves time significantly, decrease the costs and labour. The pollen tube pathway method is an in planta method that is effective only after pollination in plants. The DNA transformation process takes place by cutting the styles then using a syringe to transfer the DNA material down the pollen tube. This technique was successfully used in black pepper for improving Phytophthora capsici resistance [42]. Several genes have been transferred to spice plants via biological and non-biological methods for various purposes, which is discussed below (Fig. 1).
Fig. (1)) Important purposes of gene transfer via biological and non-biological gene transformation systems and their results in spice plants.Introducing a More Convenient Protocol
Varghese and Bhat [43], reported an efficient Agrobacterium-mediated gene transformation method in black pepper using somatic embryo explants for and GUS reporter gene. They succeeded in regenerating 9 plants per gram of embryo genic mass for the first time without using growth regulators and any genetic variation [43]. Sinojo et al 2014, also optimized somatic embryogenesis methods for Agrobacterium-mediated genetic transformation of a pathogenesis-related gene (PR5) in black pepper [44]. Compared with other solanaceous crops, pepper varieties (Capsicum annuum) are highly recalcitrant, so they have shown a very poor response toward transformation by Agrobacterium and regenerative capacity [45, 46]. In capsicum varieties, transformation frequency and shoot regeneration rate are highly genotype-dependent, also Agrobacterium-mediated transformation rate was low for cut-injured cotyledon and hypocotyl [47, 48].
A protocol for generation and gene transformation of two elite Indian cultivars of chili pepper (Capsicum annuum L.) was established through Agrobacterium tumefactions, strain LBA4404 containing pCAMB1A2301 plasmid for expression of GUS and NPT-II as reporter and marker genes respectively. Results of GUS assay, PCR, Southern blotting as well as RT-PCR analyses confirmed transformation [49].
Due to the lack of seed set in ginger, there is a high limitation of diversity in its gene pool. Moreover, all breeding programs and vegetative reproduction via rhizomes lead to the spread of soil-borne diseases [50]. However, there are many successful reports of ginger (Zingiberaceae) transformation via Agrobacterium-mediated and biolistic methods. The opening report of gene transformation of ginger was reported via biolistic methods on embryogenic callus as an explant for GUS expression [51]. Successful transformation with the biolistic method through protoplast explant of ginger was published with high GUS gene expression [45]. In Agrobacterium-mediated methods, two strains of Agrobacterium LBA 4404 including p35SGUSInt and EHA 105 with binary vector pCAMBIA1301 containing GUS reporter were used. Gene transformation stability was confirmed by PCR [25]. High transformation efficiency in Agrobacterium transformation was reported in a new quick transient transformation protocol by using LBA4404 strain containing pGFPGUSPlus when the explants incubated with Agrobacterium for 2 days as the co-cultivation stage [26]. In comparison with ginger, there are a few reports of gene transformation in turmeric. He and Gange, (2013) reported two-development transformation systems (leaf-based transient expression and callus-based stable expression) via Agrobacterium transformation. Agrobacterium strain EHA105 consisting of plasmid pBISN1, optimized for both transient and stable transformation. Transgenic plants were confirmed by PCR, Southern blot as well as GUS essay analysis [52]. There is a report of using biolistic as an alternative transformation method for Capsicum species too [53, 54].
Natural Component Gene
There are several diseases, which reduce performance in spice and herb plants and cause annual losses around the world. In this section, some of these diseases, as well as the solution proposed by genetic engineering, are mentioned.
As some of the spice plants (such as Turmeric and Ginger) have underground rhizome, they are vulnerable to accumulate pathogens and are susceptible to soil-borne diseases. Pepper varieties (Capsicum) are susceptible to numerous pathogens counting bacteria, fungi, viruses, and nematodes. So some approaches are aiming at the production of red pepper transgenic with high resistance [55].
Appropriate conventional crop improvement methods in the field of disease resistance in plants are problematic and insufficient [37]. Genetic engineering methods by identifying candidate R-Genes (resistance-Genes), cloning, and transformation, are suggested as the novel solution to obtain disease-resistant cultivar [55]. Joshi et al., 2010 isolated five NBS-LRR resistance gene candidates, via generated primers based on conserved domains of resistance genes. They suggested this NBS analogs can be a guideline for isolating more R-Gene in wild relatives turmeric for the genetic improvement of Curcuma [56]. In another report, by using molecular genetic methods, tree R-Gene was found out to be the most stable reference genes for developing Phytophthora- resistant black pepper [24, 42].
Furthermore, there are many available reports for validation, cloning, or expression of the genes related to defense mechanisms against various diseases in spices. Primary genes, expressed in black pepper via Agrobacterium-mediated transformation, were NPT II gene (neomycin phosphotransferase) and GUS gene in 1994 and 1998 respectively. There are some reports of the transformation of CP genes (as the genes resistance to CMvirus and ToMvirus) through Agrobacterium-mediated transformation to chili pepper [46, 57]. Gene of BC1 (linked with chili leaf curl Joydebpur virus) was induced in hypocotyl explants of six deferent cultivars of red pepper, by a methodical Agrobacterium-mediated transformation protocol. Transgenic lines were validated by PCR and Southern blotting analysis [58].
The primary efficient biolistic gene transfer method in turmeric was reported for the transformation of plasmid pAHC25 that included by the bar (Glufosinate) as an herbicide gene and GUS reporter. The stability of transformation was confirmed by the results of the GUS assay and PCR analysis [59].
Plant Tissue Culture
Plant tissue culture involving in vitro direct or indirect regeneration from various explants is a fundamental approach to take advantage of biotechnological applications in plants [60]. Progress in plant tissue culture has led to the development of other biotechnological methods. In the case of spice plants in vitro method has generally been used for overcoming the poor germination seed problem and improving mass propagation, producing disease-free plants and germplasm conservation.
Mass Propagation
The most crucial factors for plant mass propagation efficiency are genotypes, growth regulators, the culture medium, and the physical factors. The different parts of a plant such as leaves, terminal buds [61], bulblet [62], rhizomes [63, 64], stem and root fragments [65], have been studied as explants for spice micro-propagation. In addition, depending on the genotype, different compounds of growth regulators affect productivity [66, 67]. Numerous in vitro techniques led to established several efficient protocols for large-scale in vitro propagation in various spices. Using different dosages of cytokinins such as BA or 2ip, were reported for improving shoot regeneration in black pepper [68], ginger [65], garlic [62], turmeric [69]. An efficient protocol for micro-propagation of large cardamom was established by culturing rhizome buds as explant in MS medium containing 1 mg each of BA and IBA (tissue-cardamom3). In vitro, culture methods have been able to improve reproduction in garlic also in MS medium containing 2ip and NAA for proliferation step [62]. In Myrtle micro-propagation, modified WPM medium supplied with BA and IBA was reported as an optimum culture medium in comparison with MS and applying different concentrations of IBA or NAA were used for rooting step in another report [70]. A high rate of in vitro propagation of curcuma (almost 18 shoots) was reported by using thidiazurone as a growth regulator in MS medium [71].
Somatic Embryogenesis
Effective and developmental production of somatic embryos is a prerequisite for commercial crop production. Somatic embryogenesis is the process by which somatic embryos develop from a group of somatic cells or tissues. These embryos are similar to zygote embryos (embryos from sexual fertilization) and can be transformed into seedlings in a suitable culture medium. Plant reproduction using somatic embryogenesis from a single cell has been demonstrated in many spices and herbs. Therefore, in this case, according to the different potential in different cells for the production of natural compounds, plants with superior characteristics can be produced compared to the primary plant. Most of the reports confirmed that decreasing the concentration of growth regulators in culture medium improves somatic embryogenesis. 2,4-D is referred to as an important auxin for callus induction and somatic embryogenesis. A blend of 2,4-D and with a cytokinin, same as BA on MS medium, has a progressive effect on somatic embryogenesis and callus induction in spices. In ginger, a high number of somatic embryos, 87.7% and 93.3%, were formed by indirect and direct culturing in MS liquid medium using a combination of 2,4-D and BA via leaf sheath explants, respectively [72, 73]. Guo and Zhang reported the somatic embryogenesis of four ginger cultivars by cell suspension culture in liquid MSN medium containing 2,4-D and Kinetin [74]. The first report for direct somatic embryogenesis of turmeric with 91.1% efficiency was reported via using solid MS medium containing 2,4-D in dark condition and liquid MS medium with BA [48]. High-frequency black pepper plantlet regeneration via somatic embryogenesis was reported in several protocols [75, 76]. Application of endophytic fungi in somatic embryogenesis culture for promoting growth and hardening of in vitro cultured plants was established in Black pepper [70]. The highest somatic embryogenesis frequency (100%) was reported in Panax notoginseng in liquid MS medium contusing 2,4-D via Bioreactor cultures [77].
In-vitro Culture by Bioreactors
Automation of the micropropagation process can play a major role in overcoming the limitations of conventional laborious methods. Bioreactors are widely used for producing microbial, animal, or plant metabolism. Although applying bioreactors has been largely intended for cell suspension or hairy roots of spices and herb plants, the optimization of bioreactors for embryogenesis and tissue or organ culture has been reported in the number of studied spices [78]. The temporary Immersion (TI) system is a famous kind of bioreactor for tissue and organ culture. AKA et al., 2019, reported the optimized protocol for Myrtle micropropagation and rooting by TIB. The efficiency of Myrtle plantlet in all growth factors (Number of roots, plantlet and root length, root fresh and dry weight) in TIS was better when compared to the conventional method [79]. TI system was used for the mass improvement of the propagation of Vanilla also [80]. Three kinds of bioreactor systems were compared for micropropagation of Vanilla planifolia, TI, and RITA systems were introduced as a suitable system for commercial mass propagation and reduction of cost and labour in this spice respectively [77]. The same experiment was carried out for improving shoot and bulblet generation in garlic. However shoot propagated performance was significantly upper in the CI system, the BI system was introduced as an optimal system for bulblet formation in garlic [81].
In vitro Conservation and Cryopreservation
It is important to slow down the growth of spice shoots for the maintenance of their germplasm. In vitro conservation is one of the reliable methods for the maintenance of different vegetatively produced plant germplasm [82]. Increasing the concentration of sucrose in rhizome formation medium, using different concentrations of macroelements including EDTA and iron in MS medium and various kinds and amount of gelling agents are in vitro approaches reported for extending conservation period in spices [83]. In a successful report of in vitro turmeric conservation, low-cost medium (up to 73% cost reduction) including commercial sugar and bacteriological agar as a carbon source and gelling agent were used respectively. In vitro, conserved turmeric after 12 months does not have any significant variation in their RAPD profile when compared to the mother plants [84]. Primary in vitro conserved cardamom plantlets were achieved using ½ MS medium without growth regulators and decreasing osmotic potential in the culture medium. In a subsequent study, the efficiency of carbendazim as a fungicide on the conservation of Curcuma and ginger shoot explants was reported. The genetic stability of conserved plants was confirmed by the RAPD profile after 3 years [85]. There are several successful reports for in vitro clonal micropropagation and conservation in ginger and turmeric [86-88] also.
One of the important approaches to micropropagation is cryopreservation [89]. Cryopreservation refers to the storage and degradation of germplasm usually in liquid nitrogen at -196°C. During this time, all cell division and metabolism operations are stopped, and germplasm can be maintained safely without any genetic changes. In vitro maintenance of some spices germplasm such as wasabi [89-91], garlic [92-94], piper [95, 96], ginger [96, 97], via cryopreservation is increasingly applied. The cryopreservation technology for black pepper, cardamom, turmeric, and their germplasms using methods like vitrification, encapsulation, and encapsulation-vitrification methods is available [98-101]. Cryopreservation of Coriander (Coriandrum sativum L.) somatic embryos using air desiccation and sucrose preculture was reported by Popova, Kim [102]. González-Benito and Iriondo [103], also used LN 2 for Celery Cryopreservation.
Secondary Metabolites Production
Secondary metabolites are complex chemical organic matter that plants produce during their lifetime; however, they do not have any important role in their growth and vital activities, mainly produce against biotic and abiotic stresses or attracting pollinating insects. Mass production of these natural components on a large scale through chemical methods is mainly “difficult or impossible”. Appling tissue culture methods like cell suspension cultivation, organ culture, and polyploidy induction are suitable solutions for the rapid and mass secondary metabolites production in plants.
There are available reports for enhancing natural components in spices by micropropagation. The significance of various MS salt concentrations, as well as sucrose were evaluated on four major volatile constituents of Chenopodium ambrosioides L. in vitro condition. The results showed that all four natural compounds have changed under the influence of changing culture medium [104]. Another successful protocol for in vitro culture of Spilanthes acmella MURR via shoot tip explants was given recently [105, 106].
In most plant species, the induction of polyploidy by increasing cell size has created the ability to produce stronger vegetative organs. Growth organs are the source of a variety of commercially valuable secondary metabolites. Therefore, it is possible to induce polyploidy which can play an imperative role in improving the quantity and quality of these valuable compounds [107]. A significant rise in the production of secondary metabolism has been observed in comparison with numerous polyploidy plants with their diploid counterparts, such as Astragalus [108], Artemisia [109], Jujube [110], Lemon balm [111]. Colchicine is the most important chemical agent in chromosomal doubling, which is widely used in spice and herb plants. Colchicine inhibits the formation and polymerization of microtubules through binding to a microtubule protein, called tubulin; hence chromosomes enter the cell together at the metaphase stage, making it an active polyploid inductor [112]. In various experiments, the range of 0.01 up to 0.5% has been reported as an optimal concentration for colchicine [108].
Agrobacterium rhizogenes soil born Gram-negative bacterium is a principal agent for Hairy root disease. The infection by the bacteria culminated in production of hairy roots near the site of bacterial entry. Hairy root induction has been tried on various spices plants, hence resulted in an upturn in the production capacity of metabolites by them. Rapid growth, short duplication duration, and having more efficiency for the production of the various natural component of hairy root make them a permanent source for the secondary metabolites production. Many available reports for the usage of hairy root culture for secondary metabolites production such as; Sotolon from Trigonella foenum in airlift bioreactor [113], Sarpagin alkaloids from Rauvolfia serpentine [114, 115], α-phellandrene and apiole (as an essential oil from) in dill [116].
Protoplast Culture
Protoplast is a plant cell in which the cellulose wall has been removed. In other words, protoplasm has only a thin plasma membrane that surrounds the cell. Plasma has many applications in direct and indirect DNA transformation through electroporation and PEG mediated transformation as well as in the transient system. Protoplast culture has been reported successful in spices. Effective protocol in protoplast culture from cell suspension and leaf tissue of turmeric, cardamom, black pepper, and ginger, from the root of fennel, from mesophyll of fenugreek, from the shoot of garlic, have been elucidated [117].
Molecular Markers
Germplasm diversity is essential for a successful breeding program. Variation is significant for the increase in the genetic base since it raises the chances of discovering dynamically exceptional genes for which the alleles from the two parents are different (that is, the genetic distance) [118]. DNA markers are a powerful tool for distinguishing spice species effectively, as they are independent of age, physiological and environmental conditions. The profile obtained from the DNA fingerprint of a spice plant is the same. Also, the physical shape of the sample is not important for its evaluation and in addition to fresh tissue; it can be extracted from the dry tissue of the DNA. For species or varieties of medicinal plants that are morphologically and phytochemically similar, DNA markers are very important, as they can be used to accurately differentiate. Several kinds of nucleic acid-based markers, like RFLP, RAPD, AFLP, SNP, and SSR are used to study the genetic structure of organisms (Table 1). In last years, many studies have been performed to find out the relationship between DNA markers and quantitative and qualitative variations of active drug compounds among species and near relatives of spice plants (Table 2). In order to study geographical origins and investigation of interrelationships of Indian cardamom, the molecular level profiling of 11 species including 5 major tribes, viz., Amomum, Alpinia, Aframomum, Elettaria, and, Hedychium as well as 96 collections of cardamom germplasm using RFLP, ISSR and RAPD markers was performed by Babu, Divakaran [119]. Tamayo 2007 performed the molecular profiling of chosen cardamom genotypes in Columbia using AFLP molecular markers. The genetic diversity among the various species was appraised using various strategies (Table 2), in addition to conventional molecular markers. Next-generation sequencing (NGS) based genotyping approaches are being used of late in whole-genome sequencing and re-sequencing research programs. An enormous number of single-nucleotide polymorphisms (SNPs) identified from multiple specimens by sequencing can be used to investigate within-species polymorphism, establish haplotype maps, and conduct genome-wide association studies (GWAS). NGS has made the tedious screening of plant germplasm feasible and cost-effective [120]. Using SNP markers has been pretty powerful due to its abundance in plants, cost-effectiveness, the flexible technique, little error rate and high speed of detection [121]. GBS (genotyping by sequencing) is a modern method that uses second-generation sequencing methods to classify and represent SNPs in a smaller scale genome-wide [122]. Using restriction enzymes in GBS reduces repetitive regions and thus the genome complexity, resulting in the most rapid bioinformatics analysis for large genomes [123, 124]. Therefore, this technique, is a rapid, genome-wide, high-throughput, and cost-effective method for SNP finding [125].
This technique could aid in genotyping genomes without prior knowledge also it is useful for plant genetic diversity investigation in genome-wide spectrum [126]. Recently, GBS has been utilised in exploring the genetic heterogeneity of many crop species, such as capsicum, barley, maize, sorghum, soybean, tomato, and wheat [127-134]. Employing molecular markers, transcriptome and genome sequencing, qRT-PCR approaches can aid classical methods of breeding through the clonal selection and improving elite genotypes.
DNA Barcoding Technology
DNA barcoding is a new molecular recognition tool in which short genomic DNA fragments are used as and identifier marker in different species. Paul Hebert in 2003, firstly suggested this technique, wherein a comprehensive barcode is habituated by DNA screening; a DNA barcode database and recognition platform are settled, and the DNA data are analysed and compared by bioinformatics analysis to identify species [171, 172]. The use of DNA barcoding has helped overcome the limitations of conventional morphology-based identification methods that bank on long-term skills. In due course, instinctive identification might be possible. Barcoding DNA is a step forward and an effective alternate to classic biological characterisation methods [173, 174]. Chen et al. studied the variabilities of nuclear gene sequences as well as plastid genomes, of herbal plants and their closely related species and generated a medicinal plant DNA barcode investigation system [175, 176]. Afterwards, Wang et al. showed that the ITS2 sequences can be successfully utilized to identify various types of components of soybean pods, which can serve as a new technique for certifying clinical drug safety [177, 178]. M. Zhang, et al., used the ITS2 and psbA-trnH sequences for developing a DNA barcoding technique for the verification and uncovering of adulterants in powdered spices. The ITS2 and psbA-trnH sequences effectively distinguished sixteen types of spices and their usual adulterants. A significant degree of adulteration was observed in the adulteration detection test of 91 commercially available powdered spices. Congeneric plant, vegetal admixture, or cheaper crop-based products were the commonly detected adulterants [179]. The DNA barcoding method is thus a powerful tool for the regulation of the spice market.
CRISPR–Cas9 System for Engineering Resistance to Viruses Infecting Spices
Modern preventive methods are used to manage viral diseases. The control of viral vectors, the development of virus-free plants using different methods, and quarantine regulation are the preventive measures used. Nonetheless, these controls have limitations as the virus vectors develop resistance to pesticides [180]. The recent methods consist of pathogen-derived resistance, RNA interference-mediated resistance, and ribozyme-mediated resistance. Since the last few years, clustered, regularly interspaced, short palindromic repeats (CRISPR)–Cas 9 tools have been used for targeted silencing of viral pathogens in plants. With this technology, it is possible to simultaneously target multiple viruses at various sites and the results are quite favourable. CRISPR is widely distributed in bacterial and archaeal genomes and provides defence against invading viruses and plasmids [181]. The CRISPR locus has short repeats of prokaryotic DNA interspersed with short segments of ‘spacer DNA’ from the bacterial virus or plasmids they were previously exposed to. CRISPR spacers identify and cleave these exogenous genetic elements like RNA interference in eukaryotic organisms. This interference technique has massive potential and applications like altering the germline of humans, animals, other organisms, and plants [182]. The Cas9 protein and guide RNAs are delivered into the cell so that the genome can be specifically cut at any desired location. Thus CRISPR-Cas system can be efficiently used to develop resistance to DNA and RNA plant viruses through editing or introducing novel traits, precisely at the loci of interest, into plants [183]. It can also be used for manipulating the host genome itself to insert viral immunity. To date, there are only a few reports of using this technology in spices genome editing, Costa et al used CRISPR-Cas9 based strategy to engineer Saccharomyces cerevisiae for producing curcumin from ferulic acid [184]. Thus, this method has great potential to overcome viral and bacterial diseases in spices.
Conclusion and Future Perspectives
The availability of nutritious food to nourish the ever-growing population is crucial. Conventional breeding is inadequate to improve the growth and yield of these forgotten plants. Biotechnology based breeding methods (BBBMs) are the solution for high throughput improvement of spice plants in a rapid ways. Biotechnology can be a key device to accomplish maintainable farming and agriculture-based industry, by the progress of food creation in terms of amount, quality, and wellbeing and at the same time protecting the earth. There has been noteworthy advancement in the field of biotechnology for molecular characterization, micropropagation, and protection, and genetic resources management, management of infections, diseases and pests. Distinguishing markers connected to significant agronomic characters will help in MAS to cut short reproducing time. The utilization of recombinant DNA innovation for biotic and abiotic stress tolerance needs a lot of research before they can be adequately used. Although projects have been started in numerous research facilities for in vitro optimal metabolite creation, these methods are to be refined and scaled up for conceivable mechanical creation of the items. Due to their business potential, strengthening, and using biotechnology in spices will be significant in the coming decade. Microbial mediation through T. harzianum, T. viride, P. florescens,
