Released this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\\n\\n
We wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
IntechOpen is proud to announce that 191 of our authors have made the Clarivate™ Highly Cited Researchers List for 2020, ranking them among the top 1% most-cited.
\n\n
Throughout the years, the list has named a total of 261 IntechOpen authors as Highly Cited. Of those researchers, 69 have been featured on the list multiple times.
\n\n\n\n
Released this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\n\n
We wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
Note: Edited in March 2021
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\r\n\tNext-generation textiles represent an exciting and interesting topic within the textiles sector. They are an intersection set between life science (for example medicine, microbiology, and comfort or strain) and technical applications (textile chemistry, engineering, and testing and certification). Developments in one of these areas affect the other one; for example, the invention of superabsorbent and gel-forming materials affected the production of a new type of baby diapers. Next-generation textiles can also be considered an important part of technical textiles, being used for different purposes such as chemical and biohazard protection. They present an important aspect from an economic point of view and the necessity for their production has been increasing; for example, a huge necessity for smart medical textiles comes from the increase of the elderly population in developed countries. In the last few decades, the rapid development of command cotton fabrics also occurred. This affects all textile sectors, for example, biodegradable fibers for implantations, three-dimension spacer fabrics, and reduction of bacterial growth by using silver ion-based textiles finishing. In this and other ways, the fields concerning the next-generation textiles have been growing rapidly and are becoming a more complex area to understand.
",isbn:"978-1-80355-883-7",printIsbn:"978-1-80355-882-0",pdfIsbn:"978-1-80355-884-4",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,isSalesforceBook:!1,isNomenclature:!1,hash:"093f9e26bb829b8d414d13626aea1086",bookSignature:"Dr. Hassan Ibrahim",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/11124.jpg",keywords:"Next-Generation Textile, Intelligent Textile, Smart Textile, Technical Textile, Next-Generation Material, Medical Textile, Sustainable Textile, Nanofiber, Fabric, Smart Material, Biodegradable Fiber, Technological Innovation",numberOfDownloads:131,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"November 5th 2021",dateEndSecondStepPublish:"February 24th 2022",dateEndThirdStepPublish:"April 25th 2022",dateEndFourthStepPublish:"July 14th 2022",dateEndFifthStepPublish:"September 12th 2022",dateConfirmationOfParticipation:null,remainingDaysToSecondStep:"6 months",secondStepPassed:!0,areRegistrationsClosed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:"Dr. Hassan Ibrahim was the Egyptian National Representative of the Chemistry and Human Health Division Committee (VII) at the International Union of Pure and Applied Chemistry (IUPAC) in 2018-2019 and is currently a member of several national committees of pure and applied chemistry. 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1. Introduction
The term “molecular pharming,” blend of pharmaceutical and farming, surfaced in the literature in the 1980s to refer to the production of high-value compounds in transgenic animals. Nowadays, the expression is mainly employed to the production of recombinant pharmaceutically relevant proteins or secondary products in plants [1, 2, 3].
The roots of molecular pharming can be traced back to the mid-1980s when plants started to be genetically engineered to act as bioreactors that produced pharmaceutically relevant proteins. Barta et al. [4] demonstrated that tobacco and sunflower callus tissues were capable of expressing transcripts of a human growth hormone fusion gene. Although no protein was detected, this was the first report of plants expressing human genes and established plants as a potential production system for recombinant therapeutic proteins. Later on, the expression of a full-sized IgG in tobacco [5] was a major breakthrough since it revealed the ability of plants to produce complex functional mammalian proteins of pharmaceutical relevance. In 1990, the “authenticity” of plant-derived recombinant proteins was proved even further with the production of the first human protein (serum albumin) with confirmed native structure in tobacco and potato [6].
After several studies that demonstrated the capacity of various plant species and systems to produce recombinant pharmaceutical proteins and peptides, during the 1990s, the field of molecular pharming gained support and interest from the plant biotechnology community. The scientific attention was followed by commercial interest, with many start-up companies being created to capitalize the advantages of plants in relation to the established platforms. These advantages include being a more cost-effective, scalable, and safer means of producing pharmaceutically relevant proteins and peptides. In opposition to the fermentation-based traditional platforms that require a massive investment in bioreactors, plant-based production systems can be established with minimal investment and offer a myriad of different hosts and platforms [7]. However, the expectation that plants could easily compete for the market share of some well-established biopharmaceutical platforms, such as Chinese hamster ovary (CHO) cells, and that they could motivate the mainstream pharmaceutical industry to switch platforms was overinflated. The CHO epithelial cell lines are the most commonly used mammalian hosts for industrial production of therapeutic recombinant proteins. The technical limitations of plants, especially their lower yields compared to mammalian cell lines, allied to the colossal existing investment in fermentation infrastructures, the unfavorable public opinion on OGMs, and regulatory uncertainty, lead the mainstream pharmaceutical industry to be cautious and to a consequent stagnation of the molecular pharming field in the 2000s [8, 9]. This situation induced a change of paradigm concerning molecular pharming: the initial vision of a highly scalable and low-cost production system, while still valid, was replaced by the idea of a production system for certain niche products that are not easily manufactured by conventional systems [8, 9].
Molecular pharming embraces several platforms and technologies with different advantages and limitations, related by their use of plant tissues. Conversely to conventional biopharmaceutical production systems that are based on few selected platforms, particularly the bacterium Escherichia coli, yeasts such as Pichia pastoris, and mammalian cell lines such as Chinese hamster ovary (CHO) cells [3], pharming platforms range from plant cells or unicellular plants growing in bioreactors to whole plants growing in soil or hydroponic environments. Further, the technologies include stable integration of DNA into the nuclear genome or plastid genome and transient expression by infiltrating leaves with expression vectors based on Agrobacterium tumefaciens, plant viruses, or hybrids [3, 8]. This great diversity of molecular pharming confers adaptability and flexibility, allowing the selection of the most suitable platform for each product, but has also conduced to fragmentation. This fragmentation meant that in the early days of molecular pharming there was no driving force to establish molecular pharming as a single competitive platform. Consequently, no actions were made to match the industry requirements for high yields, standardized procedures, and good manufacturing practices (GMP) [7, 9]. More recently, efforts have been made to mimic the mainstream biopharmaceutical industry and place a focus only on a small number of platforms, namely, plant cell cultures, nuclear transgenic plants, and leafy plants transiently transformed [3, 10]. Since 2010 the attention of the biopharmaceutical industry to molecular pharming has been renewed as result of its consolidation on a small number of platforms and some target products that meet industry demands [8, 9].
In 2012, the FDA approval of the first recombinant plant-derived therapeutic for human use, Protalix Biotherapeutics’ taliglucerase alfa (Elelyso™), was an important breakthrough for molecular pharming. The enzyme taliglucerase alfa is a carrot cell-expressed human recombinant β-glucocerebrosidase and is prescribed for the treatment of Gaucher’s disease, a lysosomal storage disorder [11]. Imiglucerase, a recombinant form of glucocerebrosidase commercialized under the name Cerezyme®, was already produced in CHO cells. In this production platform, the enzyme required subsequent in vitro exposure to mannose residues in order to have biological activity, resulting in a time-consuming and expensive manufacturing process. Besides, this platform also has potential safety problems, namely, the risk of viral contamination, allergies, and other adverse reactions. In comparison, the plant-based platform is safer and less time-consuming and has reduced production costs, since the mannose units are posttranslationally added in vivo [11]. Glucocerebrosidase is a clear example of a target product in which safety, cost, and downstream processing issues were solved by switching from a traditional platform to molecular pharming. Another example that gathered mediatic exposure was ZMapp, a cocktail of three chimeric monoclonal antibodies targeting the Ebola virus surface glycoprotein produced in Nicotiana benthamiana using a hybrid transient expression system, the magnICON system. ZMapp was developed during the Ebola outbreak of 2014 by Mapp Biopharmaceutical Inc. (San Diego, USA), following initial studies on nonhuman primates [12]. ZMapp has since been used in humans under emergency compassionate protocols [13] and randomized controlled trials [14].
Following these examples of success, there has been a continuous increase in clinical trial applications and manufacturing capacity, which has also been correlated with the conception of more tangible regulations concerning plant-derived pharmaceuticals.
Although plants are still unlikely to substitute the established platforms [8], the recent promising developments in the field of molecular pharming demonstrate that glucocerebrosidase was not a lone case of success and that plant-based platforms could provide countless opportunities for the biopharmaceutical market. Plants combine the advantage of a full posttranslational modification potential with simple growth requirements and theoretically unlimited scalability in the case of field-grown whole plants. Plant-based platforms are versatile and allow the targeting of recombinant proteins and peptides produced to different organs or subcellular compartments, which provides an additional protection against proteolysis. Finally, plants are a safe host for therapeutic protein and peptide production since they do not harbor human or animal pathogens [15]. Therefore, instead of facing the red ocean of established pharmaceutical industries [16], molecular pharming is now evolving as a disruptive technology that creates its own marketplace by offering rapid drug development and production, unparalleled scalability, unique quality attributes such as tailored glycan structures, individualized therapies, and oral or topical applications of minimally processed plant tissues, thus reducing downstream costs [17].
2. Plant platforms for the production of therapeutic proteins and peptides
The continuous development of genetic engineering technologies for plants has resulted in an expansion of well-established plant-based platforms [18]. Molecular pharming encompasses platforms based on stably transformed whole-plants transgene insertion in the nuclear or plastid genome, transient expression using agroinfiltration, viral and hybrid vectors; microalgae and aquatic plants (e.g., duck-weed) stably transformed; and in vitro culture systems (e.g., cell suspensions, hairy roots, and moss protonema) [19]. Each platform has particular advantages and limitations; therefore its selection is done on a case-by-case basis, depending on economic considerations as well as on the product characteristics and intended use [20].
2.1 Platforms based on transgenic plants
Transgenic plants have been the most widely used platforms for recombinant protein production. To obtain stable transgenic lines, the gene encoding the desired protein is cloned into an expression construct, which generally includes a promoter and regulatory elements that ensure efficient RNA processing and protein synthesis [21]. This expression construct is then stably integrated into the plant nuclear genome, resulting in the stable inheritance of the transgene and expression of stable pharmaceutical proteins over generations [22]. Two major transformation strategies have been employed to insert expression constructs into the nuclear genome: Agrobacterium-mediated transformation in dicotyledonous species (dicots) and particle bombardment of DNA-coated gold or tungsten beads in monocotyledonous species (monocots) [3]. Transgenic plant lines offer several advantages as platforms for molecular pharming: they are suitable for long-term production of recombinant pharmaceutical proteins and are highly scalable, as each line can be used to produce seeds, which increase the number of plants in every generation. Ultimately, the production capacity of recombinant pharmaceutical proteins in transgenic plants is practically unlimited, as it only depends on the number of hectares available for the plant culture. The major drawbacks of transgenic plants are the long development and scale-up timescales, the unreliable production yields, and the potential spread of pharmaceutical crops in the environment and into the food chain by outcrossing and seed dispersal [3].
The development of simple transformation technologies has expanded the number of host plants available for molecular pharming. Currently, the major molecular pharming transgenic platforms are based on leafy crops, seeds, fruits, and vegetable crops. Leafy crops are benefic in terms of biomass yield and high soluble protein levels. Additionally, leaf harvesting does not need flowering and thus considerably reduces contamination through pollen or seed dispersal [23]. One disadvantage of leafy crops is that proteins are synthesized in an aqueous environment, which is more prone to protein degradation, resulting in lower production yields [24]. In fact, the mature leaves possess very large extra cytoplasmic vacuolar compartments containing various active proteolytic enzymes that are involved in the degradation of native and foreign proteins. This is particularly problematic in the case of therapeutic peptide production because short heterologous peptides have an inherent instability in plant cells [25]. In addition to the protein instability, the harvested material has limited shelf life and needs to be processed immediately after harvest.
Tobacco has been the most widely used leafy crop for molecular pharming. The major advantages of using tobacco to express pharmaceutical proteins are its high biomass yield, well-established technology for gene transfer and expression, year-round growth and harvesting, and the existence of large-scale infrastructure for processing [23]. However, the natural production of nicotine and other alkaloids in tobacco poses some safety issues in its use as a host system for heterologous protein production. Therefore, tobacco varieties with low nicotine and alkaloid levels have been produced to diminish the toxicity and overcome those safety issues. Recent studies have led to the approval of the first monoclonal antibody produced in transgenic tobacco plants, in phase I clinical trial [26]. Additionally, a 2018 publication reported the stable expression of adalimumab (a monoclonal antibody against tumor necrosis factor-alpha (TNF-α)) in tobacco plants [27]. Other leafy crops commonly used in molecular pharming include alfalfa and clover [19].
As an alternative to leafy crops, plant seeds have proven to be versatile hosts for recombinant proteins of all types, including peptides or short and long polypeptides as well as complex, noncontiguous proteins like antibodies and other immunoglobulins [28]. The expression of proteins in seeds can overcome the shortcomings of leafy crops in terms of protein stability and storage. Seeds possess specialized storage compartments, such as protein bodies and vacuoles, which provide the appropriate biochemical environment for protein accumulation, thus protecting the proteins expressed in seeds from proteolytic degradation [29]. Reports have demonstrated that antibodies expressed in seeds remain stable for at least 3 years at room temperature without detectable loss of activity [30]. Furthermore, the small size of most seeds permits to achieve a high recombinant protein concentration in a small volume, which facilitates extraction and downstream processing and reduces the costs of the overall manufacturing process [31]. One essential property of seeds is dormancy, which not only permits the stability of recombinant proteins but also allows a complete decoupling of the cycle of cultivation from the processing and purification of the protein [28]. Finally, proteins expressed in the seed do not normally interfere with vegetative plant growth, and this strategy also reduces exposure to herbivores and other nontarget organisms such as microbes in the biosphere [21]. Several crops have been studied for seed-based production, including cereals, such as maize, rice, barley, and wheat; legumes, such as pea and soybean; and oilseeds such as safflower and rapeseed. Maize has several advantages for seed-based expression of proteins; it has the highest biomass yield among food crops, and it is easy to transform, in vitro manipulate, and scale up [24]. These potentialities were explored by Prodigene Inc. for the production of the first commercially available plant-made protein, avidin (a protein with affinity for biotin used in biochemical assays). Other maize-derived protein products developed by this company include β-glucuronidase, aprotinin, laccase, and trypsin [32]. Prodigene was the first company to demonstrate the commercial benefits of plant-based platforms and was also a forerunner in the study of the economic impact of downstream processing in molecular pharming, having developed several successful approaches to recover intact and functional recombinant seeds from maize [3].
Maize has also been used to produce recombinant pharmaceutical proteins, including enzymes, vaccines, and antibodies [32, 33]. One of the most notable therapeutic proteins produced in maize is Meristem Therapeutics’ gastric lipase, an enzyme intended for the treatment of exocrine pancreatic insufficiency—a disease significantly affecting cystic fibrosis sufferers—that has completed phase II clinical trial. In addition to this enzyme, Meristem Therapeutics has developed two other maize-derived products, human lactoferrin (phase I clinical trial), whose intellectual property was later acquired by Ventria Bioscience (http://www.ventria.com/), and collagen (pre-clinical stage).
Rice is another leading platform for recombinant protein and peptide production. Similar to maize, rice is easy to transform and scale up, but unlike maize, rice is self-pollinating, which reduces the risk of horizontal gene flow. Ventria Bioscience, in its ExpressTec platform, has used rice to produce recombinant pharmaceutical proteins, including human albumin, transferrin, lactoferrin, lysozyme, and vaccines against human rabies and Lyme disease. Its lead therapeutic candidate VEN100, whose active ingredient is lactoferrin, has been shown to reduce significantly antibiotic-associated diarrhea in high-risk patients and recently completed phase II clinical trial [34]. Rice has also been widely used as host for peptide expression, especially for the production of allergen peptides (e.g., pollen and mite allergies) [35, 36]. Recent studies report that rice has the potential to offer an oral delivery system for vaccine antigens and therapeutic proteins and peptides [25, 35, 37].
Barley seeds have also been developed as commercial platforms. In comparison to other cereal crops, barley is less widely grown. However, this fact added to the self-pollinating nature of barley can be viewed as an advantage since the risk of contamination and outcrossing with non-transgenic crops is minimized. Considering this benefit, an Iceland-based company, ORF Genetics (https://orfgenetics.com/), has targeted barley grain as the expression host for several human cytokines and growth factors [19]. Other molecular pharming companies, such as Ventria Bioscience and Maltagen, have also been developing barley-based production platforms. Although barley is still recognized for its recalcitrance to transformation, over the last decade some progress has been made in the development of reliable transformation procedures [38].
The use of legume seeds, such as soybean and pea, for the production of recombinant pharmaceutical proteins, has been less explored than cereal-based platforms, with platforms based on legume seeds having yet to achieve commercial success. However, the fact that legume seeds have exceptionally high protein content (20–40%) can be exploited to achieve high yields of recombinant protein [39]. Soybean seeds have been used to express recombinant growth factors [40, 41], coagulation factors [42], and vaccine peptides [43]. Transgenic pea seeds have been previously used to produce a single-chain Fv fragment (scFV) antibody used in cancer diagnosis and therapy [44]. In another study, pea seeds were used to produce a vaccine that showed high immunogenicity and protection against rabbit hemorrhagic disease virus [45].
Safflower and rapeseed seeds are rich in oil and are, thus, referred as oilseeds. Oilseeds can provide useful recombinant pharmaceutical protein production systems. SemBioSys (http://www.sembiosys.ca/), with its oleosin-fusion platform, has been a pioneer in that field. Oleosins are the principal membrane proteins of oil bodies; oleosins confer peculiar structural properties to the oil bodies that offer simple extraction and purification procedures [46]. In the oleosin-fusion platform the recombinant protein is fused with oleosin and consequently targeted to the oil bodies. The fusion protein is then recovered through simple purification of the oil bodies and separated from oleosin by endoprotease digestion. Commercial production of hirudin in safflower by SemBioSys constituted the first report of an oilseed-derived protein [47]. The company has been focusing on safflower as its primary host ever since, with safflower-derived insulin being in phase I clinical trial [32].
Finally, fruit and vegetable crops can also be employed for molecular pharming. A major advantage of protein expression in fruit and vegetable crops is that edible organs can be consumed uncooked, unprocessed, or partially processed, making them particularly suitable for the production of recombinant subunit vaccines, nutraceuticals, and antibodies designed for topical application [29]. The oral delivery of recombinant therapeutics is one of the differentiating factor of molecular pharming in comparison to mainstream biopharmaceutical production systems, with several pharmaceutical products being produced in tomato fruits, potato tubers, and lettuce leaves for this purpose [3]. Tomato fruits are particularly useful for protein expression because the fruits are palatable as raw tissue but can also be lyophilized and stored for a long time [25]. Recently, human coagulation factor IX (hFIX) was expressed specifically in tomato fruits, constituting the first report on the expression of hFIX in plant [48]. Another study described the expression in tomato fruits of a thymosin α1 concatemer [49], an immune booster that plays an important role in the maturation, differentiation, and function of T cells. The thymosin α1 concatemer derived from transgenic tomatoes exhibited biological activity and was proven to stimulate the proliferation of mice splenic lymphocytes in vitro. Moreover, thymosin α1 specific activity was higher when produced in tomato than in Escherichia coli, demonstrating the authenticity of the plant-made product. Other examples of tomato fruit expression include F1-V [50], a candidate subunit vaccine against pneumonic and bubonic plague, and β-secretase [51], to serve as a vaccine antigen against Alzheimer’s disease.
In conclusion, platforms based on transgenic plants are a promising alternative to the conventional biopharmaceutical production platforms since they provide a stable source of pharmaceutical proteins and are also the most scalable of all molecular pharming platforms. This scalability of transgenic plants ensures the production of recombinant pharmaceutical proteins at levels previously inaccessible, namely, the commodity bulk production of monoclonal antibodies. In the current scenario of growing pharmaceutical demand, especially in developing countries, the use of transgenic plants can be game changing since they provide a highly scalable and low-cost means of producing medicines.
2.2 Platforms based on transplastomic plants
Transplastomic plants are a valuable alternative to transgenic plants for the production of recombinant pharmaceutical proteins. Transplastomic plants are obtained by the insertion of expression constructs into the plastid genome by particle bombardment. Since the Agrobacterium T-DNA (transfer DNA) complex is targeted to the nucleus, it is unsuitable for gene transfer to chloroplasts [24, 52]. Following the transformation procedure, the bombarded leaf explants are regenerated, and transplastomic plants with homoplastomic transformation (in which every chloroplast carries the transgene) are finally selected, recurring to a selection medium containing spectinomycin or in combination with streptomycin [53].
Plastid transformation can result in high yields of heterologous proteins because multiple copies of the genome are present in each plastid, and photosynthetic cells may contain hundreds or thousands of plastids [54]. As an example, the expression of a proteinaceous antibiotic in tobacco chloroplasts has achieved up to 70% of the total soluble proteins, which is the highest recombinant protein accumulation accomplished so far in plants [55]. Furthermore, chloroplasts provide a natural biocontainment of transgene flow since genes in chloroplast genomes are maternally inherited and consequently not transmitted through pollen, thereby avoiding unwanted escape into the environment. Other advantages of chloroplast engineering include the ability to express several genes as operons, and the accumulation of recombinant proteins in the chloroplast, thus reducing toxicity to the host plant [24].
Finally, transplastomic production platforms offer the possibility of oral delivery [54, 56]. In fact, it has been demonstrated that chloroplast-derived therapeutic proteins, delivered orally via plant cells, are protected from degradation in the stomach, probably due to the bioencapsulation of the therapeutic protein by the plant cell wall. They are subsequently released into the gut lumen by microbes that digest the plant cell wall, where the large mucosal intestine area offers an ideal system for oral drug delivery [57].
A shortcoming of expressing proteins via the chloroplast genome is that routine plastid engineering is still limited to tobacco, a crop that is not edible and thus unsuitable for oral delivery of therapeutic proteins. In addition, the synthesis of glycoproteins is not possible in the chloroplast system, as plastids do not carry out glycosylation [24]. Nevertheless, the expression of human somatotropin [58] in tobacco established that chloroplasts are capable of properly folding human proteins with disulfide bonds. In another study, the production of native cholera toxin B subunit [59] demonstrated the capacity of chloroplasts to fold and assemble oligomeric proteins correctly. Other therapeutic proteins expressed in tobacco chloroplasts include interferons alpha-2a and alpha-2b [60, 61] and anti-cancer therapeutic agents such as human soluble tumor necrosis factor (TNF) [62] and azurin [63]. Recently, chloroplast transformation of lettuce has also been developed [64, 65] to provide oral delivery transplastomic systems [66, 67]. Several therapeutic proteins were produced in lettuce chloroplast, namely, proinsulin [66, 67], tuberculosis vaccine antigens [68], and human thioredoxin 1 protein [69]. The chloroplast production platform has yet to achieve commercial success, though the referred developments in this field augur a promising future for therapeutic protein production in chloroplasts.
2.3 Transient expression platforms
Transient expression is a phenomenon that occurs when genes are introduced into plant tissues and are expressed for a short period without stable DNA integration into the genome [3]. Traditionally, transient expression was used to verify expression construct activity and to test recombinant protein stability. This strategy allowed the identification and elimination of initial transformation problems, and thus the prospect of regenerating the desired transgenic lines was significantly improved. Recently, there has been an emergence of transient expression for the commercial production of recombinant pharmaceutical proteins. The advantages of transient expression platforms include the ease of manipulation, speed, low cost, and high yield of proteins. In comparison to transgenic plants, transient expression permits to achieve higher recombinant protein yields because there are no position effects (suppression of transgene expression by the surrounding genomic DNA following integration) [70].
Transient expression systems utilize the beneficial properties of plant pathogens to infect plants, spread systemically, and express transgenes at high levels, causing the rapid accumulation of recombinant proteins [8]. Currently, the major transient expression platforms are based on Agrobacterium tumefaciens, plant viruses, or hybrid vectors that utilize components of both (magnICON® technology).
The agroinfiltration method involves the vacuum infiltration of a suspension of recombinant A. tumefaciens into the plant leaf tissue, with the transgenes being then expressed from the uninterrupted T-DNA [8, 71]. Using this method, milligram amounts of recombinant protein are produced within a few weeks without the need to select transgenic plants, a process that takes months to years to be completed. This system has been commercially developed in tobacco [72] and alfalfa [73] but is also applicable to other crops such as lettuce [74], potato [75], and Arabidopsis [76]. An advantage of Agrobacterium-mediated transient expression is the fact that it allows to produce in plants complex proteins assembled from subunits [70].
Another transient expression technology is based on the use of plant viruses. In this technology, the gene of interest is inserted among viral replicating elements, episomically amplified and subsequently translated in the plant cell cytosol [77]. To date, the most efficient and high-yielding platforms have been developed using RNA viruses [78]. These plant viruses include Tobacco mosaic virus (TMV), potato virus X (PVX), and Cowpea mosaic virus (CPMV) (reviewed in [8]). The advantages of virus-based production include the rapid recombinant protein expression, the systemic spread of the virus, and the fact that multimeric proteins such as antibodies can also be produced by coinfecting plants with noncompeting vectors derived from different viruses [79, 80]. Transient expression vectors based on virus have been used to express peptides and long polypeptides (at least 140 amino acids long) as fusions to the coat protein, resulting in the assembly of chimeric virus particles (CVPs) displaying multiple copies of the peptide or polypeptide on its surface [77, 81]. Transient expression based in plant viruses has been commercially adopted by the now-closed Large Scale Biology Corporation (Vacaville, USA) that used a TMV-based vector for the production of patient-specific idiotype vaccines for the treatment of B-cell non-Hodgkin’s lymphoma, which had successfully passed the phase I clinical trials [82].
Finally, the third transient expression strategy is based on hybrid systems that incorporate components of the T-DNA transfer and virus replication systems [3]. These hybrid systems use deconstructed viruses obtained by removing the coat protein (responsible for systemic movement) of the noncompeting virus strains and use Agrobacterium as the vehicle for the systemic delivery of the resulting viral vectors to the entire plant. These systems effectively address most of the major shortcomings of earlier plant-based technologies by providing the overall best combination of the following features: high expression level, high relative yield, low up- and downstream costs, very fast and low-cost R&D, and low biosafety concerns [83]. Consequently, there has been a commercial development based on several hybrid systems. One of most notable examples is the magnICON® system developed by Icon Genetics (https://www.icongenetics.com/) (formerly owned by Bayer Innovation, Dusseldorf, Germany; now a subsidiary of Nomad Bioscience, Halle, Germany), which features a deconstructed Tobacco mosaic virus (TMV) genome and A. tumefaciens as a delivery vehicle [83]. Another example is the iBioLaunch platform developed by the Fraunhofer Center for Molecular Biotechnology, which also features a deconstructed TMV genome [3]. Finally, the CPMV-HT platform is based on a deleted version of Cowpea mosaic virus RNA-2 and also allows the “hypertranslation” of recombinant proteins without virus spreading [8].
Examples of therapeutic recombinant proteins produced in these platforms have been generally reviewed in [3]. Recombinant protein production using transient expression is now being mobilized to a large scale with several companies developing scalable, automated plant-based GMP biomanufacturing facilities to efficiently produce large amounts of pharmaceuticals within weeks. Such facilities include the ones of the Fraunhofer Center for Molecular Biotechnology (Newark, DE) (https://www.fraunhofer.org/), Medicago Inc. (Quebec, Canada) (http://www.medicago.com/), Icon Genetics (Bayer; Halle, Germany) (http://www.icongenetics.com/), Texas A & M (College Station, TX), and Kentucky BioProcessing LLC (Owensboro, KY) (http://www.kbpllc.com/) [19].
In conclusion, the ability of transient plant expression systems to produce large quantities of recombinant protein, coupled to the use of current technology to increase yields, and the many promising technical solutions seems to be favorable compared with mammalian- or insect cell-based systems in quality, cost, and scale [19]. In case of emerging threats, transient platforms are advantageous since they produce large amounts of recombinant proteins rapidly (milligram quantities per plant within a few days) and can be scaled up quickly, currently providing the only reliable platform for rapid response situations [9]. During the H1N1 pandemic, the first batches of H1N1 virus-like particles (VLPs) could be produced by Medicago Inc. as soon as 3 weeks after the Centers for Disease Control and Prevention released the new influenza hemagglutinin sequence [73]. Similar lead times were reported for the H5N1 VLP vaccine [84]. Recently, the application of tobacco plant-based transient production systems, at Kentucky BioProcessing (KBP), to produce antibody lots against Ebola, was shown to significantly decrease the amount of time required for production over traditional methods, increase the quantity of antibody produced, and reduce the cost of manufacturing. Finally, at the other end of the market scale, transient expression platforms are economical for the production of pharmaceuticals for very small markets, such as orphan diseases and individualized therapies.
2.4 Callus and plant cell suspension cultures
Plant cell suspension cultures grow as individual cells or small aggregates and are usually derived from callus tissue by the disaggregation of friable callus pieces in shake bottles and are later scaled up for bioreactor-based production. Recombinant pharmaceutical protein production is achieved using transgenic explants to derive the cultures or by transforming the cells after disaggregation, usually by co-cultivation with A. tumefaciens. The co-cultivation of plant cell suspensions and recombinant A. tumefaciens has also been used for the transient expression of proteins [85]. Since these plant cell suspension cultures are grown in sterile contained environments, they provide a cGMP-compatible production environment that is more acceptable to the established pharmaceutical industry and regulatory authorities [3, 86]. These systems have added benefits of complex protein processing compared to bacteria and yeasts and increased safety compared to mammalian cell systems, which can harbor human pathogens. Another advantage of plant suspension cultures is the very low maintenance cost in comparison to other fermenter-based eukaryotic systems such as mammalian or insect cells. Moreover, the possible secretion of the target protein into the culture medium simplifies downstream processing and purification procedures [87, 88]. Nevertheless, plant cell cultures also have some limitations such as poor growth rates, somaclonal variation (particularly due to chromosomal rearrangements, common in plant cell cultures generated by calli), and gene silencing, together with the inhibition of product formation at high cell densities, formation of aggregates, cell wall growth, as well as shear-sensitivity for some species [89]. However, high levels of functional recombinant protein in plant cell suspension cultures were already obtained [87]. Besides, the previously mentioned first licensed recombinant pharmaceutical protein, Elelyso™, was produced in plant cell suspension cultures (reviewed in [88]). Tobacco has been the most popular source of suspension cells for recombinant protein production. Tobacco plants proliferate rapidly and are easy to transform, but other plant species have also been used to generate suspension cells, including rice and Arabidopsis thaliana, alfalfa, soybean, tomato, Medicago truncatula, and carrot [85, 88, 90]. Carrot suspension cells have been used by the aforementioned Protalix Biotherapeutics to produce a recombinant glucocerebrosidase. This case of commercial success shows that suspension cell cultures have potential as a viable system for large-scale protein production. Recently, carrot callus cultures, expressing epitopes from the cholesteryl ester transfer protein, were accessed for the potential of becoming an atherosclerosis oral vaccine [91].
3. Optimization of plant expression levels
The lower expression levels in comparison to the established biopharmaceutical platforms were one of the major obstacles for the commercialization of molecular pharming [9]. Therefore, numerous techniques have been developed to enhance protein expression, including codon optimization of protein sequences, to match the preferences of the host plant, targeting subcellular compartments that allow proteins to accumulate in a stable form; the use of strong, tissue-specific promoters; and the testing of different plant species and systems [25].
Protein synthesis can be increased by optimizing the components of the expression construct to maximize transcription, mRNA stability, and translation or by diminishing the impact of epigenetic phenomena that inhibit gene expression [92]. In this field, the general strategy is to use strong and constitutive promoters, such as the cauliflower mosaic virus 35S RNA promoter (CaMV 35S) and maize ubiquitin-1 promoter (ubi-1), for dicots and monocots, respectively. However, organ- and tissue-specific promoters are also being used to drive expression of the transgenes to a specific tissue or organ such as the tuber, the seed, and the fruit. Additionally, inducible promoters, whose activities are regulated by either chemical or external stimulus, may equally be used to prevent the lethality problem. Furthermore, transcription factors can also be used as boosters for the promoters to further enhance the expression level of the transgenes [53].
Protein stability can be increased by targeting proteins to cell compartments that reduce degradation. Protein targeting also affects the glycan structures added to proteins and the type of extraction and purification steps required to isolate the protein from the plant matrix. Proteins can be targeted to the secretory pathway by an N-terminal signal peptide, which is cleaved off for the release of the protein into the endoplasmic reticulum (ER). Proteins that do not require posttranslational modification, e.g., glycosylation, for their activity, can be targeted to the chloroplast using N-terminal transit peptides [93]. In addition, the target gene can be used to transform chloroplast directly, with highly enhanced protein accumulation. Moreover, posttranslational modifications of the ER lumen can also be avoided by expressing the protein as translational fusion with oleosin protein, which target the expression of the foreign protein to oil bodies of the seeds [28]. Other subcellular compartments like the protein-storing vacuoles are now being explored for recombinant protein accumulation, as it has been observed in rice seed endosperm [94].
4. Downstream processing
In the early years of molecular pharming, scientific studies were focused on demonstrating that plants could produce adequate quantities of recombinant pharmaceutical proteins and confer an oral delivery means. This led to downstream processing and the costs associated to it being basically overlooked. Downstream processing is now known to be an economically critical part of biomanufacturing processes (it can account for up to 80% of the total cost in a therapeutic protein production line) and also to be a key component of the regulatory process for evaluating the safety of pharmaceutical products [7]. The goal and the general steps for downstream processing are similar between plant and other expression systems: to recover the maximal amount of highly purified target protein with the minimal number of steps and at the lowest cost. The basic steps for downstream processes include tissue harvesting, protein extraction, purification, and formulation [22]. However, since in molecular pharming the costs of downstream processing are product-specific rather than platform-specific, the evaluation of downstream processing strategies and costs associated to it has to be done on a case-by-case basis. Nevertheless, even if unit operations have to be developed based on the properties of the product, others have to be developed based on the properties of the expression host. Plants produce process-related contaminants that require specific processing steps to ensure removal of fibers, oils, superabundant plant proteins such as RuBisCO, and potentially toxic metabolites such as the alkaloid nicotine in tobacco [8]. These secondary metabolites can be recovered from plant cells or tissues using methods such as adsorption, precipitation, and chromatography, often requiring phase partitioning and the use of mixtures of organic solvents. Several approaches have been used to facilitate downstream processing, including secretion of recombinant proteins, eliminating the plant cell disruption step; targeting of proteins into the protein bodies, oil bodies, or plastoglobules; and the use of affinity tags such as poly-histidine tags with the target protein, allowing protein purification by affinity chromatography [25]. In addition, oral delivery of whole plants or crude extracts containing the pharmaceutical relevant proteins can also be a way to simplify downstream processing and to easily distribute medicines to those in need. Furthermore, the optimization of plant’s expression level can also ease downstream processing, with higher protein concentrations conducting to higher protein volumes [7].
Finally, several purification strategies have been investigated to separate target transgenic proteins from host plant proteins, which are tailored for each individual protein based on its solubility, size, pI, charge, hydrophobicity, or affinity to specific ligands, and the parallel characteristics of plant host proteins. Chromatographic methods, such as affinity chromatography, have been the most extensively used. However, recently increasing attention is being paid to non-chromatographic methods to provide alternatives for large-scale production [22].
5. Heterologous production of bioactive angiotensin I-converting enzyme inhibitory (ACEI) peptides
In the broad range of known bioactive peptides, angiotensin I-converting enzyme inhibitory (ACEI) peptides derived from food proteins have attracted particular attention and have been studied the most comprehensively for their ability to prevent hypertension [95]. In this chapter we will further focus on the possibility to genetically engineer crop plants to produce and deliver antihypertensive ACEI peptides, therefore creating alternative sources to fight hypertension and prevent cardiovascular disease.
5.1 Cardiovascular disease and the renin-angiotensin system
Cardiovascular disease (CVD) has been recognized as the leading cause of death in developed countries. Hypertension or high blood pressure is one of the major independent risk factors for CVD [96]. States of CVD include conditions such as coronary heart disease, peripheral artery disease, and stroke. Hypertension is a condition defined by a blood pressure measurement of 140/90 mmHg or above and is thought to affect up to 30% of the worldwide adult population [95]. The kinin-nitric oxide (KNO) system and the renin-angiotensin system (RAS), Figure 1, play a crucial role in the control of hypertension by the action of angiotensin I-converting key enzyme (EC 3.4.15.1; ACE) [96, 97, 98, 99].
Figure 1.
The kinin-nitric oxide (KNO) system and the renin-angiotensin system (RAS). The left side (KNO system) shows the mechanism of the action of ACEI on ACE that cleaves bradykinin, a nonapeptide acting as vasodilatory hormone, and causes the formation of an inactive heptapeptide. In the right side (RAS system), the inhibition of ACE activity plays an important physiological role in regulation of blood pressure by inhibiting the conversion of the hormone angiotensin I to angiotensin II, a potent vasoconstrictor (figure adapted from Erdmann et al. [96]).
Several synthetic ACE inhibitors such as captopril, enalapril, and lisinopril have been prescribed for the treatment of hypertension, congestive heart failure, and diabetic neuropathy [100]. However, their consumption is associated with various side effects including cough, skin rashes, hypotension, loss of taste, angioedema, reduced renal function, and fetal abnormalities [95]. The side effects associated to synthetic ACE inhibitors and the high prevalence of hypertension have led scientists to search for natural and safer therapies. Interestingly, the study of ACEI peptides has revealed that they do not have significant effects on blood pressure in normotensive subjects, suggesting a convenient mechanism that avoids acute hypotensive effects. Based on this finding, it is hypothesized that ACEI peptides could be used in initial treatment of mildly hypertensive individuals or even as supplemental treatments [101].
5.2 Antihypertensive ACEI peptides
So far, several ACEI peptides have been identified in food proteins, mainly in milk, eggs, and plants, currently constituting the most well-known class of bioactive peptides [102, 103, 104]. These peptides are inactive within the sequence of parent proteins, but they can be released by enzymatic proteolysis in vivo or in vitro, for example, during gastrointestinal digestion or during food processing. A common feature shared by the majority of ACEI peptides is the generally short sequence, i.e., 2–12 amino acids in length. However, some larger inhibitory sequences have been identified in milk fermented with Enterococcus faecalis [105] and Lactobacillus casei Shirota [106], in koumiss [107], tuna [108], bonito [109], and rotifer [110]. Studies have also indicated that binding to ACE is strongly influenced by the substrate’s C-terminal tripeptide sequence. Hydrophobic amino acid residues with aromatic or branched side chains at each of the C-terminal tripeptide positions are common features among potent inhibitors. The presence of hydrophobic Pro residues at one or more positions in the C-terminal tripeptide region seems to positively influence a peptide’s ACE-inhibitory activity [95]. In general, the peptides showing higher activity against ACE have Tyr, Phe, Trp, or Pro at their C-terminus [95]. The peptides TQVY from rice [111], MRW from spinach [112], and YKYY from wakame [113] are some examples of this principle. Table 1 reviews some examples of ACEI activities of plant origin, whose peptides responsible for such activity may be potential sources for the heterologous production of ACEI peptides.
Examples of ACEI peptide activity from different plant origin.
Different values for the same plant product related to the ACEI peptide sequence.
The most common method to produce and identify ACEI peptides is through enzymatic hydrolysis of food proteins with gastrointestinal enzymes such as pepsin and trypsin or with commercial proteases such as Alcalase™ [127]. ACEI peptides have also been produced with Lactobacillus, Lactococcus lactis, and E. faecalis strains during milk fermentation [105, 106]. Nevertheless, there are problems associated to this type of industrial production of ACEI peptides, including the difficulty to isolate the peptide of interest from the complex mixture of compounds produced by enzymatic hydrolysis, the high cost, low recovery, and the low bioavailability. These disadvantages denote the need to develop new and alternative approaches for their production.
5.3 Heterologous production of ACEI peptides in plants
In recent years, the application of recombinant DNA technologies for the production of ACEI peptides at a large scale and low cost has gathered attention in the biotechnology community. Investigation has been focused on the development of expression methods for antihypertensive peptide production in different plant crops [128]; and here, we tried to provide some promising examples.
Thus far, the main strategies that have been adopted are as follows: the overexpression of ACEI peptide precursor proteins and the production of particular peptides as heterologous components [101], the modification of some storage proteins to produce chimeric proteins carrying ACEI peptides [101], and also the generation of multimer proteins containing tandem repeats of ACEI peptides, flanked by protease recognition sequences that allow the peptide release during gastrointestinal digestion.
5.3.1 Rice
Transgenic rice plants that accumulate novokinin (RPLKPW), a potent antihypertensive peptide designed according to the structure of ovokinin (2–7) (RADHPF), as a fusion with the rice storage protein glutelin, have been generated. The engineered peptide is expressed under the control of endosperm-specific glutelin promoters and specifically accumulates in seeds. Oral administration of either the RPLKPW-glutelin fraction or transgenic rice seeds to spontaneously hypertensive rats (SHRs)—the main model for assessing the in vivo activity of ACEI peptides (e.g., [108, 111, 122])—significantly reduced systolic blood pressures, suggesting the possible application of transgenic rice seed as a nutraceutical delivery system and particularly for administration of antihypertensive peptides [129].
Wakasa et al. [130] attempted the generation of transgenic rice seeds that would accumulate higher amounts of novokinin peptide by expressing 10 or 18 tandemly repeated novokinin sequences, with the KDEL endoplasmic reticulum retention signal at the C-terminus, and using the glutelin promoter along with its signal peptide. Although the chimeric protein was unexpectedly located in the nucleolus and the accumulation was low, a significant antihypertensive activity was detected after a single oral dose to SHRs. More importantly, this effect was observed over a relatively longer duration time, with intervals of 5 weeks between doses as low as 0.0625 g transgenic seeds per kg.
5.3.2 Soybean
Soybean [Glycine max (L.) Merr.] is an attractive option for the production of ACEI peptides given that soybean seeds contain a large amount of total protein. Therefore, there has been an effort to generate soybean lines with improved ACEI properties foreseeing the creation of novel functional foods.
Matoba et al. [128], introduced novokinin (RPLKPW) into homologous sequences of a soybean β-conglycinin α’ subunit by site-directed mutagenesis. Founded on first achievements from an E. coli expressed protein, the muted β-conglycinin α’ subunit carrying novokinin repeats were also expressed in soybean. This chimeric protein accumulated at levels of up to 0.2% of extracted protein from transgenic soybean seeds [131]. Still, the levels of expression were too low, and it was not possible to assess the in vivo effects of these soybean seeds.
Novokinin has also been expressed in transgenic soybean seeds in a fusion form along with a β-conglycinin α’ subunit. Interestingly, a reduced systolic blood pressure was observed in SHRs after administering a dose of 0.15 g kg−1 of protein extracts. A similar effect was attained following administration of a 0.25 g kg−1 dose of defatted flour. Thus, it was concluded that this chimeric protein produced in soybean possessed an antihypertensive activity [132].
Additionally, a synthetic gene of His-His-Leu (HHL), an ACEI peptide derived from a Korean soybean paste, was tandemly multimerized to a 40-mer, ligated with ubiquitin as a fusion gene (UH40), and subsequently expressed in E. coli. Following digestion with leucine aminopeptidase, the 405-Da HHL monomer was recovered by reverse-phase high-performance liquid chromatography (HPLC). MALDITOF mass spectrometry, glutamine-TOF mass spectrometry, N-terminal sequencing, and measurement of ACE-inhibiting activity confirmed that the resulting peptide was the HHL [133]. The potential use of this antihypertensive chimeric protein in soybean has yet to be assessed.
5.3.3 Tomato and tobacco
A modified version of amarantin, the main seed storage protein of Amaranthus hypochondriacus, carrying four tandem repeats of the ACEI dipeptide Val-Tyr into the acidic subunit of amarantin, was expressed in cell suspension cultures of Nicotiana tabacum L. NT1. Protein hydrolysates obtained from transgenic calli showed high levels of inhibition of the angiotensin-converting enzyme, with an IC50 value of 3.5 μg ml−1, and 10-fold lower levels than that of protein extracts of wild-type cells (IC50 of 29.0 μg ml−1) [134]. This was the first time that a chimeric protein comprising an ACEI peptide was produced in plant cell suspension cultures.
This modified version of amarantin was also expressed in the fruit of transgenic tomato plants. Protein hydrolysates from transgenic tomato fruits showed in vitro ACE inhibition, with IC50 values ranging from 0.376 to 3.241 μg ml−1; this represented an increase of up to 13-fold in the inhibitory activity when compared with the protein hydrolysates of non-transformed fruits [135]. These two results suggest the possible application of tobacco plant cell suspension cultures and transgenic tomato fruits for massive production of this engineered version of amarantin, which could be especially used as an alternative hypertension therapy [134, 135].
5.3.4 Amaranth
Although amaranth has not been genetically modified to produce ACEI peptides, the feasibility of developing a modified amarantin acidic subunit has been widely assessed [129, 134, 135, 136, 137, 138, 139]. Recently, the in vivo effect of an E. coli-modified amarantin protein, four units of Val-Tyr dipeptides (VY) in tandem, and one of Ile-Pro-Pro tripeptides (IPP) incorporated in the amarantin acidic subunit (AMC3) was evaluated in SHRs in a one-time oral administration experiment. This study showed that enzymatic hydrolysates of AMC3-containing ACEI peptide (4xVY and IPP) sequences had significant in vivo antihypertensive action [138]. The positive reports of amarantin expression in E. coli [136, 138, 139] along with the sustained expression of amarantin-modified proteins in tobacco [134] and tomato [135] prospect the successful production of ACEI peptide fusion proteins in amaranth.
5.3.5 Lettuce and Medicago truncatula
Lettuce (Lactuca sativa) is a commercially important crop belonging to the Asteraceae family. It is a diploid (2n = 18), autogamous species with a genome size of 2.7 Gb [140]. This crop is particularly suitable for oral delivery of therapeutics as its raw leaves are consumed by humans, and the time to obtain an edible product is only weeks, compared to the months needed for crops such as tomato or potato. Therefore, recently lettuce has been investigated as a production host for edible recombinant therapeutics [66, 67, 141]. Furthermore, the fact that stable transformation procedures for both nuclear [142] and plastid genomes [64], and transient expression [74], are widely available, is also an advantage. Lettuce has been used as production host for several recombinant therapeutics, virus-like particles (VLPs) and monoclonal antibodies [143], antigens [142, 144], and human therapeutic proteins [66, 69].
Medicago truncatula is a model plant from the legume family. It is a diploid (2n = 16), autogamous species, with a relatively small genome and short life cycle of 3–5 months. These characteristics enable this species to be used in molecular genetic studies and expression of foreign genes [145]. The phylogenetic distance to economically important crops is crucial in the choice of this plant by many researchers and funding agencies, since it allows comparative studies within the legume family. The methodologies for the establishment of long-term cell suspension culture are well recognized [146], and the potential of M. truncatula as expression host has also been established for the production of feed additives [20, 87], human hormones [90], and human enzymes [147].
The use of these two species in molecular pharming is at the center of a recent collaboration between the Plant Cell Biotechnology (PCB) Laboratory (ITQB UNL), the Cell Differentiation and Regeneration Laboratory (iBiMED UA), and the Institute of Plant Genetics (IPG PAS). This cooperation foresees the usage of these two species as exceptional hosts for the heterologous production and/or delivery of ACEI peptides, and a resume of this ongoing project is here schematically presented (Figure 2). This figure also provides an overview of the technologies involved in different plant platforms discussed in this chapter.
Figure 2.
Schematic representation of the technologies involved in different plant platforms for the production of therapeutically important proteins and peptides. Plastid transformation by particle bombardment can result in regeneration of transplastomic plants, revealing high-yield heterologous production, with the possibility of protein/peptide oral delivery or purification. Nuclear transformation can be accomplished by particle bombardment or by Agrobacterium-mediated transformation, resulting in the regeneration of stable transgenic plants. Finally, the technology based on transient expression, here with the example of agroinfiltration. We present Medicago truncatula and lettuce as examples: (a) M. truncatula co-culture of leaf explants with Agrobacterium, (b) and (c) plant regeneration via somatic embryogenesis according to Araújo et al. [145], (d) and (e) establishment of a cell suspension culture from callus for protein/peptide production [146, 147], (f) lettuce leaf explant co-culture with Agrobacterium, (g) and (h) plant regeneration via shoot organogenesis at PCB lab, (i) lettuce transgenic plants which can be used for oral delivery, (j) and (k) agroinfiltration of lettuce leaf explants according to Negrouk et al. [74], and (l) example of a control explant (left) and transient expression of a 35S::GUS(int) cassette in lettuce leaves (right).
6. Conclusions
Molecular pharming has been recently and extensively reviewed, and the future of this technology has gathered some optimistic expectations. A myriad of studies have already demonstrated the capacity of various plant species and systems to produce recombinant pharmaceutical proteins and peptides. This technology has already been put to the test in case of emerging threats, where transient platforms proved to be strategic for rapid production of large amounts of recombinant proteins in response to pandemic situations. However, their usefulness for the production of functional foods still falls short of expectations, as well as the attainment of its full potential in bioactive peptide production. With the improvement of known plant platforms and development of new genetic engineering techniques and their exploration, it is forthcoming an evolution in the production of heterologous bioactive peptides, to which we hope to contribute with our ACEI pharming project. The advent of genome editing techniques (with the advantage of site- specific gene insertion), like the CRISPR/Cas9 methodology, will undoubtedly increase and democratize plant transformation events and will certainly contribute to the increase of genetically modified species for molecular pharming purposes.
Acknowledgments
We acknowledge financial support from Fundação para a Ciência e Tecnologia (FCT), Portugal, through the research Grant SFRH/BPD/74784/2010 (Duque AS) and funding, through the research unit GREEN-it “Bioresources for Sustainability” (UID/Multi/04551/2013) and FCT/COMPETE/QREN/EU-FEDER (for Institute of Biomedicine iBiMED: UID/BIM/ 04501/2013). We also acknowledge the Program for Scientific Cooperation FCT/Poland 2017/2018 and Angelini Pharmaceuticals and PREMIVALOR (for Angelini University Award 2012/2013).
\n',keywords:"plant engineering, recombinant protein, bioactive peptides, angiotensin I-converting enzyme, ACEI peptides",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/65771.pdf",chapterXML:"https://mts.intechopen.com/source/xml/65771.xml",downloadPdfUrl:"/chapter/pdf-download/65771",previewPdfUrl:"/chapter/pdf-preview/65771",totalDownloads:1675,totalViews:0,totalCrossrefCites:1,totalDimensionsCites:2,totalAltmetricsMentions:3,introChapter:null,impactScore:1,impactScorePercentile:69,impactScoreQuartile:3,hasAltmetrics:1,dateSubmitted:"June 4th 2018",dateReviewed:"January 15th 2019",datePrePublished:"February 22nd 2019",datePublished:"June 10th 2020",dateFinished:"February 21st 2019",readingETA:"0",abstract:"Molecular pharming is a cost-effective, scalable, and safe system to produce high-quality and biologically active recombinant therapeutic proteins. Thus, plants are emerging alternative platform for the production of pharmaceutically relevant proteins such as vaccines, antibodies, antibody derivatives, and some serum-derived proteins. Additionally, plants have also been used to produce bioactive and immunogenic peptides. The efficacy, selectivity, specificity, and low toxicity make them particularly well-suited therapeutic agents for various indications, for instance, cardiovascular and infectious diseases, immunological disorders, and cancer. In the broad range of known bioactive peptides, angiotensin I-converting enzyme inhibitory (ACEI) peptides derived from food proteins have attracted particular attention for their ability to prevent hypertension. So far, several ACEI peptides have been identified in food proteins, mainly in milk, eggs, and plants. The industrial production of ACEI peptides is based on enzymatic proteolysis of whole food proteins, which leads to the release of small bioactive peptides with ACE-inhibitory activity. The problems associated to such procedures, namely, cost and loss of functional properties, have demonstrated the need to develop more straightforward methods to produce ACEI peptides. One viable hypothesis, discussed in this chapter, is to genetically engineer crop plants to produce and deliver antihypertensive peptides.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/65771",risUrl:"/chapter/ris/65771",book:{id:"7595",slug:"genetic-engineering-a-glimpse-of-techniques-and-applications"},signatures:"Carolina Gomes, Filipe Oliveira, Sandra Isabel Vieira and Ana Sofia Duque",authors:[{id:"165802",title:"Dr.",name:"Ana Sofia",middleName:null,surname:"Duque",fullName:"Ana Sofia Duque",slug:"ana-sofia-duque",email:"sduque@itqb.unl.pt",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:null},{id:"269041",title:"MSc.",name:"Carolina",middleName:null,surname:"Gomes",fullName:"Carolina Gomes",slug:"carolina-gomes",email:"carolinangomes@gmail.com",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:null},{id:"269043",title:"MSc.",name:"Filipe",middleName:null,surname:"Oliveira",fullName:"Filipe Oliveira",slug:"filipe-oliveira",email:"filipesnoliveira@gmail.com",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:null},{id:"269045",title:"Prof.",name:"Sandra",middleName:null,surname:"Vieira",fullName:"Sandra Vieira",slug:"sandra-vieira",email:"sivieira@ua.pt",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:{name:"University of Aveiro",institutionURL:null,country:{name:"Portugal"}}}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Plant platforms for the production of therapeutic proteins and peptides",level:"1"},{id:"sec_2_2",title:"2.1 Platforms based on transgenic plants",level:"2"},{id:"sec_3_2",title:"2.2 Platforms based on transplastomic plants",level:"2"},{id:"sec_4_2",title:"2.3 Transient expression platforms",level:"2"},{id:"sec_5_2",title:"2.4 Callus and plant cell suspension cultures",level:"2"},{id:"sec_7",title:"3. Optimization of plant expression levels",level:"1"},{id:"sec_8",title:"4. Downstream processing",level:"1"},{id:"sec_9",title:"5. Heterologous production of bioactive angiotensin I-converting enzyme inhibitory (ACEI) peptides",level:"1"},{id:"sec_9_2",title:"5.1 Cardiovascular disease and the renin-angiotensin system",level:"2"},{id:"sec_10_2",title:"5.2 Antihypertensive ACEI peptides",level:"2"},{id:"sec_11_2",title:"5.3 Heterologous production of ACEI peptides in plants",level:"2"},{id:"sec_11_3",title:"5.3.1 Rice",level:"3"},{id:"sec_12_3",title:"5.3.2 Soybean",level:"3"},{id:"sec_13_3",title:"5.3.3 Tomato and tobacco",level:"3"},{id:"sec_14_3",title:"5.3.4 Amaranth",level:"3"},{id:"sec_15_3",title:"5.3.5 Lettuce and Medicago truncatula",level:"3"},{id:"sec_18",title:"6. Conclusions",level:"1"},{id:"sec_19",title:"Acknowledgments",level:"1"}],chapterReferences:[{id:"B1",body:'Lössl AG, Clarke JL. Conference scene: Molecular pharming: Manufacturing medicines in plants. Immunotherapy. 2013;5:9-12'},{id:"B2",body:'Ma JKC, Christou P, Chikwamba R, et al. 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Comprehensive Reviews in Food Science and Food Safety. 2014;13:114-134'},{id:"B103",body:'Ibrahim HR, Ahmed AS, Miyata T. Novel angiotensin-converting enzyme inhibitory peptides from caseins and whey proteins of goat milk. Journal of Advanced Research. 2017;8:63-71'},{id:"B104",body:'Daskaya-Dikmen C, Yucetepe A, Karbancioglu-Guler F, et al. Angiotensin-I-converting enzyme (ACE)-inhibitory peptides from plants. Nutrients. 2017;9:316'},{id:"B105",body:'Quirós A, Ramos M, Muguerza B, et al. Identification of novel antihypertensive peptides in milk fermented with Enterococcus faecalis. International Dairy Journal. 2007;17:33-41'},{id:"B106",body:'Rojas-Ronquillo R, Cruz-Guerrero A, Flores-Nájera A, et al. Antithrombotic and angiotensin-converting enzyme inhibitory properties of peptides released from bovine casein by Lactobacillus casei Shirota. International Dairy Journal. 2012;26:147-154'},{id:"B107",body:'Chen Y, Wang Z, Chen X, et al. Identification of angiotensin I-converting enzyme inhibitory peptides from koumiss, a traditional fermented mare’s milk. Journal of Dairy Science. 2010;93:884-892'},{id:"B108",body:'Lee S-H, Qian Z-J, Kim S-K. A novel angiotensin I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats. Food Chemistry. 2010;118:96-102'},{id:"B109",body:'Hasan F, Kumada Y, Hashimoto N, et al. Fragmentation of angiotensin-I converting enzyme inhibitory peptides from bonito meat under intestinal digestion conditions and their characterization. Food and Bioproducts Processing. 2006;84:135-138'},{id:"B110",body:'Lee JK, Hong S, Jeon J-K, et al. Purification and characterization of angiotensin I converting enzyme inhibitory peptides from the rotifer, Brachionus rotundiformis. Bioresource Technology. 2009;100:5255-5259'},{id:"B111",body:'Li G-H, Qu M-R, Wan J-Z, et al. Antihypertensive effect of rice protein hydrolysate with in vitro angiotensin I-converting enzyme inhibitory activity in spontaneously hypertensive rats. Asia Pacific Journal of Clinical Nutrition. 2007;16:275-280'},{id:"B112",body:'Yang Y, Marczak ED, Yokoo M, et al. Isolation and antihypertensive effect of angiotensin I-converting enzyme (ACE) inhibitory peptides from spinach Rubisco. Journal of Agricultural and Food Chemistry. 2003;51:4897-4902'},{id:"B113",body:'Suetsuna K, Nakano T. Identification of an antihypertensive peptide from peptic digest of wakame (Undaria pinnatifida). The Journal of Nutritional Biochemistry. 2000;11:450-454'},{id:"B114",body:'Sheih I-C, Fang TJ, Wu T-K. Isolation and characterisation of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from the algae protein waste. Food Chemistry. 2009;115:279-284'},{id:"B115",body:'Sornwatana T, Bangphoomi K, Roytrakul S, et al. Chebulin: Terminalia chebula Retz. fruit-derived peptide with angiotensin-I-converting enzyme inhibitory activity. Biotechnology and Applied Biochemistry. 2015;62:746-753'},{id:"B116",body:'Priyanto AD, Doerksen RJ, Chang C-I, et al. Screening, discovery, and characterization of angiotensin-I converting enzyme inhibitory peptides derived from proteolytic hydrolysate of bitter melon seed proteins. Journal of Proteomics. 2015;128:424-435'},{id:"B117",body:'Li G-H, Wan J-Z, Le G-W, et al. Novel angiotensin I-converting enzyme inhibitory peptides isolated from Alcalase hydrolysate of mung bean protein. Journal of Peptide Science. 2006;12:509-514'},{id:"B118",body:'Jakubczyk A, Karaś M, Baraniak B, et al. The impact of fermentation and in vitro digestion on formation angiotensin converting enzyme (ACE) inhibitory peptides from pea proteins. Food Chemistry. 2013;141:3774-3780'},{id:"B119",body:'Pihlanto A, Akkanen S, Korhonen HJ. ACE-inhibitory and antioxidant properties of potato (Solanum tuberosum). Food Chemistry. 2008;109:104-112'},{id:"B120",body:'Marczak ED, Usui H, Fujita H, et al. New antihypertensive peptides isolated from rapeseed. Peptides. 2003;24:791-798'},{id:"B121",body:'Chen J-R, Okada T, Muramoto K, et al. Identification of angiotensin I-converting enzyme inhibitory peptides derived from the peptic digest of soybean protein. Journal of Food Biochemistry. 2002;26:543-554'},{id:"B122",body:'Kodera T, Nio N. Identification of an angiotensin I-converting enzyme inhibitory peptides from protein hydrolysates by a soybean protease and the antihypertensive effects of hydrolysates in 4 spontaneously hypertensive model rats. Journal of Food Science. 2006;71:C164-C173'},{id:"B123",body:'Gouda KGM, Gowda LR, Rao AGA, et al. Angiotensin I-converting enzyme inhibitory peptide derived from glycinin, the 11S globulin of soybean (Glycine max). Journal of Agricultural and Food Chemistry. 2006;54:4568-4573'},{id:"B124",body:'Vallabha V, Tiku PK. Antihypertensive peptides derived from soy protein by fermentation. International Journal of Peptide Research and Therapeutics. 2014;20:161-168'},{id:"B125",body:'Liu M, Du M, Zhang Y, et al. Purification and identification of an ACE inhibitory peptide from walnut protein. Journal of Agricultural and Food Chemistry. 2013;61:4097-4100'},{id:"B126",body:'Thewissen BG, Pauly A, Celus I, et al. Inhibition of angiotensin I-converting enzyme by wheat gliadin hydrolysates. Food Chemistry. 2011;127:1653-1658'},{id:"B127",body:'Pihlanto A, Makine S. Antihypertensive properties of plant protein derived peptides. In: Bioactive Food Peptides in Health and Disease. Rijeka, Croatia: InTech; 2013. DOI: 10.5772/54565'},{id:"B128",body:'Matoba N, Doyama N, Yamada Y, et al. Design and production of genetically modified soybean protein with anti-hypertensive activity by incorporating potent analogue of ovokinin. FEBS Letters. 2001;497:50-54'},{id:"B129",body:'Yang L, Tada Y, Yamamoto MP, et al. A transgenic rice seed accumulating an anti-hypertensive peptide reduces the blood pressure of spontaneously hypertensive rats. FEBS Letters. 2006;580:3315-3320'},{id:"B130",body:'Wakasa Y, Zhao H, Hirose S, et al. Antihypertensive activity of transgenic rice seed containing an 18-repeat novokinin peptide localized in the nucleolus of endosperm cells. Plant Biotechnology Journal. 2011;9:729-735'},{id:"B131",body:'Nishizawa K, Kita A, Doi C, et al. Accumulation of the bioactive peptides, novokinin, LPYPR and rubiscolin, in seeds of genetically modified soybean. Bioscience, Biotechnology, and Biochemistry. 2008;72:3301-3305'},{id:"B132",body:'Yamada Y, Nishizawa K, Yokoo M, et al. Anti-hypertensive activity of genetically modified soybean seeds accumulating novokinin. Peptides. 2008;29:331-337'},{id:"B133",body:'Jeong D-W, Shin DS, Ahn C-W, et al. Expression of antihypertensive peptide, His-His-Leu, as tandem repeats in Escherichia coli. Journal of Microbiology and Biotechnology. 2007;17:952-959'},{id:"B134",body:'Santos-Ballardo DU, Germán-Báez LJ, Cruz-Mendívil A, et al. Expression of the acidic-subunit of amarantin, carrying the antihypertensive biopeptides VY, in cell suspension cultures of Nicotiana tabacum NT1. Plant Cell, Tissue and Organ Culture. 2013;113:315-322'},{id:"B135",body:'Germán-Báez LJ, Cruz-Mendívil A, Medina-Godoy S, et al. Expression of an engineered acidic-subunit 11S globulin of amaranth carrying the antihypertensive peptides VY, in transgenic tomato fruits. Plant Cell, Tissue and Organ Culture. 2014;118:305-312'},{id:"B136",body:'Luna-Suárez S, Medina-Godoy S, Cruz-Hernández A, et al. Modification of the amaranth 11S globulin storage protein to produce an inhibitory peptide of the angiotensin I converting enzyme, and its expression in Escherichia coli. Journal of Biotechnology. 2010;148:240-247'},{id:"B137",body:'Castro-Martínez C, Luna-Suárez S, Paredes-López O. Overexpression of a modified protein from amaranth seed in Escherichia coli and effect of environmental conditions on the protein expression. Journal of Biotechnology. 2012;158:59-67'},{id:"B138",body:'Medina-Godoy S, Rodríguez-Yáñez SK, Bobadilla NA, et al. Antihypertensive activity of AMC3, an engineered 11S amaranth globulin expressed in Escherichia coli, in spontaneously hypertensive rats. Journal of Functional Foods. 2013;5:1441-1449'},{id:"B139",body:'Morales-Camacho JI, Dominguez-Dominguez J, Paredes-Lopez O. Overexpression of a modified amaranth protein in Escherichia coli with minimal media and lactose as inducer. Recent Patents on Biotechnology. 2013;7:61-70'},{id:"B140",body:'Truco MJ, Ashrafi H, Kozik A, et al. An ultra-high-density, transcript-based, genetic map of lettuce. G3: Genes, Genomes, Genetics. 2013;3:617-631'},{id:"B141",body:'Martínez-González L, Rosales-Mendoza S, Soria-Guerra RE, et al. Oral immunization with a lettuce-derived Escherichia coli heat-labile toxin B subunit induces neutralizing antibodies in mice. Plant Cell, Tissue and Organ Culture. 2011;107:441-449'},{id:"B142",body:'Liu C-W, Chen JJW, Kang C-C, et al. Transgenic lettuce (Lactuca sativa L.) expressing H1N1 influenza surface antigen (neuraminidase). Scientia Horticulturae. 2012;139:8-13'},{id:"B143",body:'Lai H, He J, Engle M, et al. Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce. Plant Biotechnology Journal. 2012;10:95-104'},{id:"B144",body:'Huy N-X, Yang M-S, Kim T-G. Expression of a cholera toxin B subunit-neutralizing epitope of the porcine epidemic diarrhea virus fusion gene in transgenic lettuce (Lactuca sativa L.). Molecular Biotechnology. 2011;48:201-209'},{id:"B145",body:'Araújo SS, Amaral Duque ASRL, Dos Santos DMMF, et al. An efficient transformation method to regenerate a high number of transgenic plants using a new embryogenic line of Medicago truncatula cv. Jemalong. Plant Cell, Tissue and Organ Culture. 2004;78:123-131'},{id:"B146",body:'Duque AS, Pires AS, Dos SDM, et al. Efficient somatic embryogenesis and plant regeneration from long-term cell suspension cultures of Medicago truncatula cv. Jemalong. In Vitro Cellular & Developmental Biology-Plant. 2006;42:270-274'},{id:"B147",body:'Pires AS, Santos RB, Nogueira AC, et al. Production of human lipocalin-type prostaglandin D synthase in the model plant Medicago truncatula. In Vitro Cellular & Developmental Biology-Plant. 2014;50:276-281'}],footnotes:[],contributors:[{corresp:null,contributorFullName:"Carolina Gomes",address:null,affiliation:'
Institute of Plant Genetics, Polish Academy of Sciences (IPG PAS), Poland
Cell Differentiation and Regeneration Laboratory, Department of Medical Sciences, Institute of Biomedicine (iBiMED), Universidade de Aveiro, Portugal
'},{corresp:"yes",contributorFullName:"Ana Sofia Duque",address:"sduque@itqb.unl.pt",affiliation:'
Plant Cell Biotechnology Laboratory, Green-it Unit, Instituto de Tecnologia Química e Biológica António Xavier (ITQB NOVA), Portugal
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1. Introduction
Plant secondary metabolites (PSM) are small organic molecules produced during plant metabolism that can function as a plant defense against herbivores, pathogens, neighboring plants, or environmental stresses [1, 2, 3]. Although proven to be incorrect, PSM [4, 5] used to be defined as (1) the part of metabolites not present in nonplant organisms or as (2) the part of plant metabolites not required for simple growth and development. These outdated PSM definitions still reflected some properties of PSM—they are widespread in the plant kingdom and are beyond the highly conserved primary metabolites, which are required in plant growth and development, such as proteins, carbohydrates, lipids, and nucleic acids. Hence, they represent plant diversity. The description of PSM often starts from the sessile property of terrestrial plants [1, 2, 6], where they cannot flee from the threat or stress from the environment and hence have to develop strategies to defend or reduce the threat or stress. PSM are their strategies.
Environmental factors, such as temperature, salinity, and water, are also called abiotic stresses [7]. The herbivores, pathogens, and neighboring plants are also called biotic stresses. Plant metabolites can be classified into primary metabolites, secondary metabolites, and plant hormones [3]. The defense function of secondary metabolites is often realized by integration with physical structures, such as cell wall, cutin, suberin, wax, and bark. According to Hartman [1], plant secondary metabolites are often lineage-specific and aid plants in interacting with the biotic and abiotic environment. For example, pine trees and mint plants often contain terpenes, peppers often contain capsaicin, and sicklepod contains anthraquinone derivatives for defense. The production of secondary metabolites can be constitutive or induced. Some plant secondary metabolites, such as anthraquinone derivatives, in sicklepod are routinely produced, and they are called constitutive secondary metabolites. The production of secondary metabolites demands a high metabolic cost on the host plant; thus, many of these compounds are not produced in large quantities until after insects have begun to feed. These secondary metabolites are called induced secondary metabolites [7].
The number of secondary metabolites reported is vast, and they have widespread applications. The most prominent application of the plant secondary metabolites is in the pharmaceutical industry, where about 25% of the drugs in use by humans are derived from medicinal plants [8]. The type and concentration(s) of the secondary molecule(s) produced by a plant are determined by the species, genotype, physiology, developmental stage, and environmental factors during its growth [2].
The application of plant secondary metabolites in agriculture is the focus of this chapter. In standard agricultural practices, the species, physiology, and development stages usually follow biological laws, and we cannot do much to change them. The genotype and environmental factors are currently where most work has been focused on in agriculture. According to Hartman [1], the functions of plant secondary metabolites could fall into three categories—(1) defense and competition involving herbivores (arthropods, vertebrates, and invertebrates), pathogens (viruses, bacteria, and fungi), and plants (allelopathy); (2) attraction and stimulation (pollination, seed dispersal, food-plant recognition, oviposition, sequestration, and symbiosis); and, (3) abiotic stresses defense. Compared to other reviews on secondary metabolites, this review chapter focuses on the agricultural applications of plant secondary metabolites, specifically categories (1) and (3).
2. Secondary metabolites as resources to reduce crop biotic stresses
2.1 Main groups of plant secondary metabolites
PSM are widely spread in the whole plant kingdom. As they are lineage-specific, the total number of PSM is much more than the number of primary metabolites [5]. PSM derive from primary metabolites using a limited number of key pathways. Their functional diversity is gained by adding diverse combination of reactive functional groups [9]. Terpenoids are the largest group of PSM and occur in all plants, including over 22,000 compounds. The simplest terpenoid is isoprene (C5H8), a volatile gas produced during photosynthesis in leaves. Terpenoids are classified into monoterpenoids consisting of two isoprene units, sesquiterpenoids (three units), diterpenoids (four units), and triterpenoids (six units), depending on how many isoprene units are in their structures [7]. Mint plants (Mentha spp.) produce large quantities of the monoterpenoids menthol and menthone stored in glandular trichomes on the epidermis [7]. Pyrethrins are monoterpenoid esters produced by chrysanthemum plants that act as insect neurotoxins (Saxona 1988). Gossypol (Gossypium hirsutum) from cotton is a diterpenoid [7]. The fresh scent of lemon and orange peel results from a class of triterpenoids called limonoids. The active ingredient of neem oil, azadirachtin, is a powerful limonoid isolated from neem trees (Azadirachta indica) [10]. Phenolics are another large group of PSM, which includes a wide variety of defense-related compounds, such as flavonoids, anthocyanins, phytoalexins, tannins, lignin, and furanocoumarins [7]. Flavonoids are one of the largest classes of phenolics. Soybean contains a large amount of isoflavone [7]. Tannins are water-soluble flavonoid polymers produced by plants and stored in vacuoles. Tannins are toxic to insects because they bind to salivary proteins and digestive enzymes, including trypsin and chymotrypsin, resulting in protein inactivation. Alkaloids are a large class of bitter-tasting nitrogenous compounds found in many vascular plants and include caffeine, cocaine, morphine, and nicotine [7]. Capsaicin and related capsaicinoids produced by members of the genus Capsicum are the active components of chili peppers and have their characteristic burning sensation in hot and spicy foods [7]. Anthraquinones are present in different plant families, such as Leguminosae, Polygonaceae, Rubiaceae, Rhamnaceae, Scrophulariaceae, Liliaceae, Verbenaceae, and Valerianaceae [11]. Anthraquinone derivatives from sicklepod (Leguminosae) have been used to repel deer from browsing soybean [12]. Chlorogenic acid (CGA) or caffeoylquinic acid (CQA) exists in all plants [13], suggesting they are among the oldest PSMs.
2.2 PSM as resources to reduce crop biotic and abiotic stresses
Crop biotic stresses come from microbial pathogens, nematodes, insects, and mammalian herbivores. Crop abiotic stresses come from drought, salinity, temperature, ultraviolet, etc. Plant secondary metabolites can help to reduce these stresses. For example, some secondary metabolites containing benzene rings can absorb ultraviolet (UV) light and release the energy in the visible light range as fluorescence to avoid crop damage from UV light.
3. Use of secondary metabolites to reduce biotic and abiotic stresses
3.1 Extraction of secondary metabolites
Secondary metabolites have a defense function in plants [1, 2]. The simplest way to utilize secondary metabolites for crop protection is to extract the secondary metabolites and apply them to crops for protection against pathogens, insects, and mammalian herbivores.
3.1.1 Secondary metabolites used as a deer repellent
Deer is the primary pest in row crop production in the US. This was first concerned in the 1960s and gradually confirmed by the agricultural community during the following 40 years [14, 15]. The annual loss of row crops in the US was estimated to be up to $4.53 billion [14]. Deer repellent is one of the primary strategies to solve crop deer damage. Among them, deer repellent with putrescent egg solids as active ingredients occurred in the 1990s and still dominates the deer repellent market today. Deer acceptance of food is dependent on the concentration of secondary metabolites present [16]. They usually avoid plants containing high concentrations of terpenes, tannins [17], and gossypol (cotton). Sicklepod (Senna obtusifolia L.) is one of the southern US’s top ten most troublesome weeds [18]. It belongs to the Leguminosae family and is famous for its high concentrations of anthraquinone derivatives [19], another group of secondary metabolites. Anthraquinone was reported as a mammalian animal repellent since the 1940s [20, 21]. To protect soybean damage from deer, deer repellents were developed using sicklepod fruits [12]. After several modifications of the extraction protocol, the sicklepod extract matched the deer repelling efficacy of Liquid Fence® Deer & Rabbit Repellent, a popular commercial deer repellent with putrescent egg solids as active ingredients. Besides the anthraquinone derivatives, some other plant secondary metabolites were used as deer repellents, such as capsaicin in pepper plants, and monoterpenoids menthol and menthone in peppermint (the active ingredients in Deer Out™, a commercial deer repellent).
3.1.2 Secondary metabolites as insecticides
One of the best examples of secondary metabolites used as an insecticide was the development of the popular insecticide bifenthrin. The pyrethrins from chrysanthemum (Chrysanthemum cinerariaefolium) flower extract were used to develop this insecticide. The safety of this product is, however, questionable. Sesbania extracts developed using a similar extraction method were applied on soybean leaves and exposed to soybean loopers in a 40 mm rearing cup for 24 hours. The looper mortality reached 60% in cups containing sesbania extract-treated soybean leaves.
3.2 Germplasm screening for secondary metabolites
3.2.1 Cotton germplasm screening for gossypol
Gossypol is a unique diterpenoid in the cotton genus Gossypium. Cotton germplasm is not as big as soybean and rice, but variations in gossypol content in cotton leaves are still significant. Low gossypol variety suffering heavy insect defoliation was observed (Dr. Saha personal communication). Unlike food crops, genetically modified cotton is not debated so critically, so Bt-based GMO method was adopted early to prevent insect defoliation. Gossypol screening is still a cultivar selection and breeding direction to defend insects and nematodes.
3.2.2 Allelopathic crop screening
Allelopathy is another term introduced to the science of plant ecology to describe the addition of chemical compounds (toxic or nontoxic) from a plant into the environment that affects the germination, growth, health, development, and population biology or behavior of another plant species [22]. Weeds are considered the most severe biotic constraint on crop production, with yield losses ranging from 45 to 95%, depending on environmental conditions and agronomic practices [23].
3.2.2.1 Rice allelopathy
Rice (Oryza sativa) is the most important grain crop cultivated in the world. More than half of the world’s population has rice as their primary food source [24]. Weed infestation is the main reason for rice yield loss. The most common weeds found in rice fields worldwide are Echinochloa species, such as Echinochloa cruss-galli and Echinochloa colona, and weedy rice species (Oryza sativa) [25]. According to Oerke [26], weed species account for more than one-third of the losses in global rice production. Therefore, using integrated pest management (IPM), including the use of allelopathic varieties, can be an important tool to control weed species and manage weed resistance to synthetic herbicides.
A diversity of allelochemical compounds, such as fatty acids, phenolic acids, indoles, steroids, and others were found to be released by different parts of the plants, in root exudates, and rice soil [27]. Yet, rice inhibits weed growth primarily by secreting momilactone B, a diterpene produced from geranylgeranyl diphosphate (GGPP) [28]. It has been shown that momilactones A and B released by allelopathic rice varieties inhibit shoot and root growth of E. crus-galli (Figure 1). Additionally, weed species growing near rice deficient in momilactone biosynthesis produced more biomass when compared to the ones growing near wild-type rice [29].
Figure 1.
Chemical structure of momilactones A and B, allelopathic molecules released by rice plants.
The rice germplasm has a large variation when testing for allelopathy. However, it was found that among the Brazilian and Asian cultivars tested, only about 3–4% showed greater allelopathic potential [30]. Similar results were found when testing allelopathic cultivars able to suppress the growth of weeds, such as E. crus-galli, Cyperus difformis, and several aquatic weeds [31]. Thus, allelopathy is still an area to be investigated since this information can be used to improve rice production.
3.2.2.2 Cotton allelopathy
Weeds are a continuous hazard to agriculture in the United States, costing farmers up to $20 billion each year [32]. Herbicide resistance in weeds influences the long-term effectiveness of weed management practices globally [33]. Pesticide residues in food and the environment, as a result, are a significant public health hazard [34]. The use of weed suppressive traits in crop types, commonly known as allelopathy, is one of the potential weed control techniques in cotton production [35]. Several studies have reported using allelopathic crop varieties in weed management, including rice, wheat, sunflower, and canola [36, 37]. However, there is limited research on the direct allelopathic effect of cotton on weeds. A few research studies have established that cotton produces allelochemicals, which can impede the growth of pigweeds in other investigations [38]. According to preliminary studies on cotton allelopathy [39], cotton root showed significant quantities of four phenolic chemicals, including p-hydroxybenzoic acid, ferulic acid, gallic acid, and vanillin. A greenhouse study was conducted using eleven cotton chromosome substitution (CS) lines for allelopathy screening against Palmer amaranth (Amaranthus palmeri) (PA) [40]. The cotton lines were tested using a modified stair-step assay. Reductions in PA height and chlorophyll concentration were measured for each cotton line. Variations in PA height among the CS lines were more prominent 21 days after establishment. CS-B22sh and T26lo were most effective in reducing Palmer amaranth height; 77 and 68% height reduction, respectively. A multivariate cluster analysis revealed that CS-B22sh and CS-T26lo were clustered in one group, suggesting similar allelopathic potential against Palmer amaranth. Allelochemicals, produced by the allelopathic cotton CS lines, are a potential bioherbicide and a possible alternative to synthetic herbicides.
3.2.2.3 Sweetpotato allelopathy
Sweetpotato [Ipomoea batatas (L.) Lam.] is a nutrition-rich food with high fiber, vitamins, and antioxidants. Weed management is a major concern for sweetpotato producers [41] as weeds result in significant crop yield loss and higher production costs [42]. Being a plant of vine nature, sweetpotato grows close to the soil surface, and hand-weeding is one of the most effective mechanical options for weed management in sweetpotato fields [43]. To maintain and promote crop productivity and reduce labor requirements, chemical herbicides have been widely applied for weed control. However, long-term and large-scale herbicide applications have increased the number of herbicide-resistant weeds, environmental issues, loss of biodiversity, and threats to ecosystem safety [44]. Allelopathy can be a possible strategy for integrated, sustainable, and ecological weed management. Allelopathic properties of sweetpotato have been demonstrated to reduce the growth and development of weeds, such as alfalfa, yellow nutsedge, Palmer amaranth, and Mikania micrantha (Figure 2) [45, 46]. Alfalfa root growth was inhibited by methanol and aqueous extracts from sweetpotato leaves, stems, and roots [47]. Aqueous extracts from sweetpotato leaves or roots reduced the biomass, root and shoot length, and inhibited the germination of Lactuca sativa [42]. Leaf leachates from sweetpotato cultivars, Sinyulmi, Sinhwangmi, Purple, and Jami demonstrated an inhibitory effect on alfalfa [47]. Palmer amaranth growth was inhibited when they were irrigated with water-containing root exudates from different sweetpotato varieties [46].
Figure 2.
Allelochemicals are released by above- and belowground parts of sweetpotato (donor) plants suppressing the surrounding receiver plants.
Some sweetpotato varieties produce several allelochemicals, such as coumarin, chlorogenic acid, caffeic acid, hydroxycinnamic and trans-cinnamic acids [48] which were weed suppressive in rice. In terms of concentration, sweetpotato leaves were found to have the highest concentration of phenolic compounds, followed by stems and roots [47]. The allelopathic effect of sweetpotato on cowpea was reported when cowpea was grown as the following crop on the same field due to the presence of leaf litters and decaying residues of sweetpotato. Allelopathic varieties with the potential to suppress weed growth may be useful for breeding cultivars designed for organic production systems.
3.2.2.4 Sorghum allelopathy
Sorghum [Sorghum bicolor (L.)] is an annual grass belonging to the family Poaceae and subfamily Panicoideae. It originated in Africa and migrated to other continents [49]. Accounting for more than 22% of the world’s sorghum production, the United States leads in the production globally [50]. Sorghum has wide-ranging utilization as food, fodder, technology, and construction [51]. The importance of sorghum is increasing globally due to its high functional value and ability to acclimatize to changing environmental conditions, especially drought [52]. Allelopathic or weed suppressive potential of sorghum has been documented in the past four decades. Several allelochemicals, such as sorgoleone and its analogs (Figure 3), phenolic acids, and their aldehyde derivatives, determine sorghum’s allelopathic potential [53]. The amount of allelochemical production depends on the plant part and age of the sorghum plant, environmental conditions, and the receiver plant. Sorgoleone, a lipophilic secondary metabolite, is the primary allelochemical produced by sorghum which consists of a quinone ring and aliphatic chain [54]. Its analogs contain aliphatic side chains or additional methoxy groups in the ring [55, 56].
Figure 3.
Structures of sorgoleone and dihydrosorgoleone (reduced analog).
Phenolic acids (Figure 4) with phytotoxic activities, such as gallic, syringic, p-hydroxybenzoic, benzoic, vanillic, p-coumaric, and benzoic acids are also produced by sorghum [57]. However, the amount of production of these compounds depends on the type of cultivar [58] and the development stage of the sorghum plant [59].
Figure 4.
Chemical structure of allelopathic phenolic compounds.
Weed suppressing potential of sorghum on several weed species has been explored by using it as a cover crop, intercrop, crop rotation, sorghum water extract, soil incorporation of sorghum residue, and allele-herbicides derived from sorghum [60, 61]. Sorghum extracts can be combined with lower herbicide doses to effectively manage the weeds and reduce the overall herbicide introduction into the environment. Sorghum residues combined with 50% of the labeled rate of trifluralin were effective in preventing yield loss in broad beans [62]. Aqueous extracts from Brassica–sunflower–sorghum reduced weed biomass of several species, such as Purple nutsedge, bermudagrass, crowfoot grass, horse purslane, field bindweed, jungle rice, and goosegrass. The extent of suppression was comparable with the full rate of atrazine or S-metolachlor with half rate of atrazine [63]. Sorghum water extract combined with a reduced rate of herbicides such as isoproturon and metsulfuron-methyl demonstrated similar weed control as the full rate of these herbicides in the wheat field [64, 65]. A combination of water extracts from sunflower, rice, and sorghum can reduce the rates by 27–67% for herbicides such as ethoxysulfuron, butachlor, and pretilachlor in rice fields [66]. The utilization of allelopathy in agriculture can be a more sustainable and cost-effective strategy for weed management.
3.2.2.5 Allelopathic cover crops
The method of using cover crops in agricultural fields has been a widespread practice among a broad range of farms. Cover crops are crops that are grown prior to harvested crops to help increase the potential of the harvested crops [67]. In agricultural systems, the practice of using cover crops is shown to improve the quality of the soil by virtue of incorporating crop residues (organic matter) [68], Using a cover crop approach can also be beneficial via enhanced hydro-availability, decrease evaporation from the soil, as well as escalate the biodiversity of the soil.
An additionally impactful use for using the cover crop method in agricultural systems is its ability to suppress weeds due to either physical biomass of the terminated cover crop essentially smothering the weedy plants, physical shading of the cover crop causing inhibition of sunlight to the weeds, as well as via the production of allelochemicals from the cover crop. Allelochemicals are the product of allelopathy, which is positive or negative impact of one plant (the allelopathic plant) on another plant. Allelochemicals can increase or decrease the nutrient availability to surrounding plants by virtue of the symbiotic microbes [69]. It is appropriately thought that the use of cover crops with allelopathic properties in an agricultural field can have positively novel effects on the growth, ability to thrive, and production yields of so-called “cash crops”.
During a study in a semiarid area of Texas, USA, during a 3-year period, cotton that was cultivated following cover crop termination showed a shorter plant height and seed and lint yields. Simultaneously, the plant density did not affect the cover crops. Benzoxaziones concentrations in the soil were 2 to 3-fold higher under the cover crop treatments than in the fallow (control) plot. Though allelopathy may not be the only factor to cause these findings, it is likely to have played a significant role [70].
During a study on non-chemical weed suppression in vegetable fields, it was shown that there was a correlation between the quantity of cover crop biomass with the level of weed suppression (Figures 5 and 6). An 8 t ha−1 or greater cover crop biomass is possibly a significant enough level to have sufficient weed suppression [71]. Although this level of weed suppression may not have everything to do with allelopathy from the cover crops, it certainly played a critical role [72].
Figure 5.
Effects of various cereal cover crops in different vegetable production systems on the dry biomass production (g m−2) of weed species at the time of cover crop termination in 2005 (gray bars) and 2006 (white bars). Vertical lines represent standard errors of the means (p < 0.05).
Figure 6.
Effects of various legume cover crops in different vegetable production systems on the dry biomass production (g m−2) of weed species at the time of cover crop termination in 2005 (gray bars) and 2006 (white bars). Vertical lines represent standard errors of the means (p < 0.05).
In a study focused on weed germination and the growth of IdaGold mustard, a seed germination bioassay technique was used. Phenol (allelochemical) concentrations were measured during this study. The total concentration of phenols in the soil was negatively correlated with the level of weed germination (Figure 7). Also, there were low concentrations of phenol in the soil that contained live microbes (<20 ng). Additionally, the germination rates were lower when compared to a nonmicrobe-containing soil with the same concentrations of phenol [73].
Figure 7.
Germination is inhibited by high concentrations of soil phenols.
Numerous studies have demonstrated the weed suppressive property of allelopathic cover crops, which is a piece of good news for farmers [74]. There is a need for more research on the possible positive growth effects of allelopathic cover crops on the cash crops’ ability to thrive.
3.3 Secondary metabolites biosynthesis regulation
While PSMs have a constitutive part, i.e., routinely produced, they are also induced. This is mainly reflected in pathogen-induced resistance (including PSM production) and herbivore-induced resistance (including PSM production) [75]. The former can be traced back to 120 years ago, while the latter be traced back to 50 years ago. Recently it was realized that both were similar in nature and were controlled by plant hormones, salicylic acid, and jasmonic acid, respectively [75].
Another group of PSMs, allelochemicals, is generally thought of as constitutive, i.e., routinely produced. Compared to PSM induced by pathogens and herbivores, allelochemical induction is a big gap in our knowledge, although the study of allelopathy can be traced back 90 years ago. A primary reason for the difference is that for pathogen/herbivore-induced PSM production, the PSM may (or may not) need activation upon induction (they do not need to be expelled out of the plant body to defend), while in allelopathy, the PSM needs to be expelled out of the plant body to be effective. Sporadic information on the induction of root exudation exists in the literature; for example, Dineli et al. [76] studied the translocation and root exudation of herbicide after foliar treatment of wheat and ryegrass using 14C-labeled diclofop-methyl and triasulfuron. The results showed the presence of untreated plants (wheat or ryegrass) in the same pot as triasulfuron-treated ryegrass or wheat induced the exudation of the herbicide 7 to 32 times more. In the case of diclofop, the induced root exudation of the herbicide was 3 to 6 times more in the presence of untreated wheat or ryegrass. The root exudated herbicides suppressed the adjacent plants, indicating a form of allelopathy. This study demonstrated that the presence of adjacent plants induces the release of allelopathic compounds. An immediate question following this case study is—could the biosynthesis of allelopathic compounds (PSM) be induced? If so, how were the signals transmitted during these processes, including the release of the compounds?
As we reviewed previously, PSMs are biopesticides widely used in agriculture. As the PSM are lineage-specific, the selection of a specific crop cultivar or cover crop is similar to selecting what kind of biopesticides to use. Similarly, understanding and application of PSM induction is the dose control of the selected biopesticides. Furthermore, in the pathogen and herbivore-induced resistance (expressed as PSM), the resistance was often called systematic acquired resistance, meaning the resistance was expressed as normal PSM for toxicity and included thickening of cell wall lignin, etc. Hence such systemic acquired resistance is more effective and lasts longer than toxic PSM increase. In this context, filling the knowledge gap of induction of allelopathic compound biosynthesis and release is similar to understanding the dose control of bioherbicide.
3.3.1 Use of plant hormones to regulate secondary metabolite biosynthesis
Generally, it has been accepted that salicylic acid (SA) and jasmonic acid (JA) or methyl jasmonate (MeJA) are recognized plant hormones specialized for defense. These defense hormones have been used to induce PSM production to defend pathogens and herbivores in agricultural studies [77]. This method has not been used in allelopathy.
3.3.2 Use of plant extracts to induce secondary metabolites production
A field study looked at the deer and insect repelling efficacy of coffee senna extract on soybean [12]. After 40 days, the soybean leaf holes were significantly lower than the control or other treatments. This was in contrast to the leaf disc assay results, where soybean loopers were exposed to both coffee senna and sesbania extracts for 24 hours. The soybean looper mortality for sesbania extract was higher than that of coffee senna. A possible explanation for the difference between the field and leaf disc results is that leaf disc experiments used detached leaves. In contrast, field experiments used living soybean plants where the coffee senna extract might have induced defense response in soybean plants. The active ingredient of coffee senna extract may be SA and JA, or a new type of defense response inducer, which is to be determined.
3.3.3 Other chemicals for crop defense activation
Besides plant hormones and plant extracts, some inorganic chemicals have also been used as crop defense activators. Juric et al. [78] reported that Ca2+ and Cu2+ increased secondary metabolites contents in lettuce. Such chemical crop activator is much less toxic for humans and their defense effects last much longer than insecticides or fungicides, hence they are more preferable to the agriculture community.
3.4 Use of transcriptomic and genomic tools to employ secondary metabolites to reduce crop stresses
During the past ten years, the transcriptome was widely used to study the gene expression of secondary metabolites [79]. While plant secondary metabolites are thought to be the readouts of plant defense activation, usually PSM quantity increase can be detected around 20 days or more after treatment (defense activation). PSM increase can be detected from several hours to 40 hours by transcriptome analysis (qPCR). Senna tora is a medicinal plant in Asia, and it is also a close relative to the weed sicklepod (Senna obtusifolia) in the US. Both sicklepod and Senna tora fruits contain high contents of anthraquinone secondary metabolites. Kang et al. [80] used differential expression analysis and showed that the expression level of genes involved in the anthraquinone biosynthetic pathway regulates differently depending on the degree of tissues and seeds development.
With improvements in sequencing technology, the sequencing cost has plunged during the past decades. Crop or cultivar genome is not far from being available. One discovery with the available genome sequences is that plants devote a significant amount of their genes to secondary metabolites, implying plant ecological functions are equivalent to its growth and development. Kang et al. [80] sequenced the genome of S. tora, and found that the CHS-L gene family expanded most notably in S. tora. This might explain in part why S. tora was rich in anthraquinones.
4. Conclusions
Compared to the estimated number of primary metabolites of 10,000, PSMs are estimated to be more than 200,000 in the plant kingdom. These PSMs function in various ecological roles, including defending pathogens, herbivores, and neighboring plants. Use of these PSM in agriculture includes (1) extraction of the PSM and applying it directly to the crop to reduce biotic stresses, (2) use of PSM in vivo/in situ by screening crop cultivars with desired PSM profiles to achieve better resistance to pests, (3) use of PSM biosynthesis regulation or plant defense activators to achieve defense readiness, (4) filling the knowledge gap on allelochemical induction, biosynthesis, and release, as it will be helpful in improving weed management practices in agriculture, and (5) employing transcriptomic and genomic tools to understand PSM biosynthesis and pathways.
Acknowledgments
The authors appreciate the funding from the Mississippi Soybean Promotion Board (MSPB) and Cotton Incorporated. This work is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, Hatch project under accession number 230100, and is a contribution of the Mississippi Agricultural and Forestry Experiment Station.
Conflict of interest
The authors declare that this work was presented in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
\n',keywords:"allelopathy, pathogen defense, herbivore defense, plant defense, cover crops, sustainable pest management, organic farming",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/82403.pdf",chapterXML:"https://mts.intechopen.com/source/xml/82403.xml",downloadPdfUrl:"/chapter/pdf-download/82403",previewPdfUrl:"/chapter/pdf-preview/82403",totalDownloads:20,totalViews:0,totalCrossrefCites:0,dateSubmitted:"March 10th 2022",dateReviewed:"March 17th 2022",datePrePublished:"June 26th 2022",datePublished:null,dateFinished:"June 26th 2022",readingETA:"0",abstract:"Plant secondary metabolites (PSM) are small molecules of organic compounds produced in plant metabolism that have various ecological functions, such as defense against pathogens, herbivores, and neighboring plants. They can also help to reduce abiotic stresses, such as drought, salinity, temperature, and UV. This chapter reviewed the ecological functions of the PSM and how people utilize these metabolites to reduce crop biotic and abiotic stresses in agriculture. Specific topics covered in this review are (1) extraction of PSM from plant parts and its application on crops; (2) screening of crop/cover crop germplasms for high PSM content and with resistance to pathogens, herbivores, and/or neighboring plants; (3) regulation of PSM biosynthesis (including plant hormones and defense activators) to increase plant readiness for defense; (4) transcriptome and genome technology improvements in the last decade leading to valuable tools to characterize differential gene expression and gene composition in a genome, and lineage-specific gene family expansion and contraction. In addition, there is a critical need to understand how the biosynthesis and release of allelochemicals occur. Filling this knowledge gap will help us to improve and encourage sustainable weed control practices in agriculture.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/82403",risUrl:"/chapter/ris/82403",signatures:"Ziming Yue, Varsha Singh, Josiane Argenta, Worlanyo Segbefia, Alyssa Miller and Te Ming Tseng",book:{id:"11331",type:"book",title:"Secondary Metabolites - Trends and Reviews",subtitle:null,fullTitle:"Secondary Metabolites - Trends and Reviews",slug:null,publishedDate:null,bookSignature:"Dr. Ramasamy Vijayakumar and Dr. Suresh Selvapuram Sudalaimuthu Raja",coverURL:"https://cdn.intechopen.com/books/images_new/11331.jpg",licenceType:"CC BY 3.0",editedByType:null,isbn:"978-1-80355-208-8",printIsbn:"978-1-80355-207-1",pdfIsbn:"978-1-80355-209-5",isAvailableForWebshopOrdering:!0,editors:[{id:"176044",title:"Dr.",name:"Ramasamy",middleName:null,surname:"Vijayakumar",slug:"ramasamy-vijayakumar",fullName:"Ramasamy Vijayakumar"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Secondary metabolites as resources to reduce crop biotic stresses",level:"1"},{id:"sec_2_2",title:"2.1 Main groups of plant secondary metabolites",level:"2"},{id:"sec_3_2",title:"2.2 PSM as resources to reduce crop biotic and abiotic stresses",level:"2"},{id:"sec_5",title:"3. Use of secondary metabolites to reduce biotic and abiotic stresses",level:"1"},{id:"sec_5_2",title:"3.1 Extraction of secondary metabolites",level:"2"},{id:"sec_5_3",title:"3.1.1 Secondary metabolites used as a deer repellent",level:"3"},{id:"sec_6_3",title:"3.1.2 Secondary metabolites as insecticides",level:"3"},{id:"sec_8_2",title:"3.2 Germplasm screening for secondary metabolites",level:"2"},{id:"sec_8_3",title:"3.2.1 Cotton germplasm screening for gossypol",level:"3"},{id:"sec_9_3",title:"3.2.2 Allelopathic crop screening",level:"3"},{id:"sec_9_4",title:"3.2.2.1 Rice allelopathy",level:"4"},{id:"sec_10_4",title:"3.2.2.2 Cotton allelopathy",level:"4"},{id:"sec_11_4",title:"3.2.2.3 Sweetpotato allelopathy",level:"4"},{id:"sec_12_4",title:"3.2.2.4 Sorghum allelopathy",level:"4"},{id:"sec_13_4",title:"3.2.2.5 Allelopathic cover crops",level:"4"},{id:"sec_16_2",title:"3.3 Secondary metabolites biosynthesis regulation",level:"2"},{id:"sec_16_3",title:"3.3.1 Use of plant hormones to regulate secondary metabolite biosynthesis",level:"3"},{id:"sec_17_3",title:"3.3.2 Use of plant extracts to induce secondary metabolites production",level:"3"},{id:"sec_18_3",title:"3.3.3 Other chemicals for crop defense activation",level:"3"},{id:"sec_20_2",title:"3.4 Use of transcriptomic and genomic tools to employ secondary metabolites to reduce crop stresses",level:"2"},{id:"sec_22",title:"4. 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In: Agrawal AA, Tuzun S, Bent E, editors. Induced Plant Defenses against Pathogens and Herbivores: Biochemistry, Ecology and Agriculture. St. Paul, MN, USA: APS Press; 2000'},{id:"B76",body:'Dinelli G, Bonetti A, Marottii M, Busi S, Catizone P. Root exudation of diclofop-methyl and triasulfuron from foliar-treated durum wheat and ryegrass. Weed Research. 2007;47:25-33'},{id:"B77",body:'Beyer SF, Bel PS, Flors V, et al. Disclosure of salicylic acid and jasmonic acid-responsive genes provides a molecular tool for deciphering stress responses in soybean. Scientific Reports. 2021;11:20600'},{id:"B78",body:'Jurić S, Stracenski SK, Król-Kilińska Ż, Žutić I, Uher SF, Đermić E, et al. The enhancement of plant secondary metabolites content in Lactuca sativa L. by encapsulated bioactive agents. Scientific Reports. 2020;10:3737. DOI: 10.1038/s41598-020-60690-3'},{id:"B79",body:'Kang S-H, Lee W-H, Lee C-M, Sim J-S, Won SY, Han S-R, et al. De novo transcriptome sequence of Senna tora provides insights into anthraquinone biosynthesis. PLoS ONE. 2020b;15(5):e0225564'},{id:"B80",body:'Kang SH, Pandey RP, Lee CM, et al. Genome-enabled discovery of anthraquinone biosynthesis in Senna tora. Nature Communications. 2020a;11:5875'}],footnotes:[],contributors:[{corresp:null,contributorFullName:"Ziming Yue",address:null,affiliation:'
Department of Crop and Soil Sciences, Mississippi State University, USA
Department of Crop and Soil Sciences, Mississippi State University, USA
'},{corresp:"yes",contributorFullName:"Te Ming Tseng",address:"t.tseng@msstate.edu",affiliation:'
Department of Crop and Soil Sciences, Mississippi State University, USA
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IntechOpen’s Academic Editors and Authors have received funding for their work through many well-known funders, including: the European Commission, Bill and Melinda Gates Foundation, Wellcome Trust, Chinese Academy of Sciences, Natural Science Foundation of China (NSFC), CGIAR Consortium of International Agricultural Research Centers, National Institute of Health (NIH), National Science Foundation (NSF), National Aeronautics and Space Administration (NASA), National Institute of Standards and Technology (NIST), German Research Foundation (DFG), Research Councils United Kingdom (RCUK), Oswaldo Cruz Foundation, Austrian Science Fund (FWF), Foundation for Science and Technology (FCT), Australian Research Council (ARC).
Open Access publication costs can often be designated directly in the grants or in specific budgets allocated for that purpose. Many of the most important funding organisations encourage, and even request, that the projects they fund are made available at no cost to the wider public. IntechOpen strives to maintain excellent relationships with these funders and ensures compliance with mandates.
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In order to help Authors identify appropriate funding agencies and institutions, we have created a list, based on extensive research on various OA resources (including ROARMAP and SHERPA/JULIET) of organizations that have funds available. Before consulting our list we encourage you to petition your own institution or organization for Open Access funds or check the specifications of your grant with your funder to ascertain if publication costs are included. Where you are in receipt of a grant you should clarify:
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Please note that this list is not a definitive one and is updated regularly. To suggest possible modifications or the inclusion of your institution/funder, please contact us at funders@intechopen.com
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Please be aware that you must be a member, or grantee, of the institutions/funders listed in order to apply for their Open Access publication funds.
Open Access publication costs can often be designated directly in the grants or in specific budgets allocated for that purpose. Many of the most important funding organisations encourage, and even request, that the projects they fund are made available at no cost to the wider public. IntechOpen strives to maintain excellent relationships with these funders and ensures compliance with mandates.
\n\n
In order to help Authors identify appropriate funding agencies and institutions, we have created a list, based on extensive research on various OA resources (including ROARMAP and SHERPA/JULIET) of organizations that have funds available. Before consulting our list we encourage you to petition your own institution or organization for Open Access funds or check the specifications of your grant with your funder to ascertain if publication costs are included. Where you are in receipt of a grant you should clarify:
\n\n
\n\t
Does your institution already have a budget for covering Open Access publication costs?
\n\t
Does your grant list Open Access publication fees as legitimate direct/indirect costs?
\n
\n\n
If you are associated with any of the institutions in our list below, you can apply to receive OA publication funds by following the instructions provided in the links. Please consult the Open Access policies or grant Terms and Conditions of any institution with which you are linked to explore ways to cover your publication costs (also accessible by clicking on the link in their title).
\n\n
Please note that this list is not a definitive one and is updated regularly. To suggest possible modifications or the inclusion of your institution/funder, please contact us at funders@intechopen.com
\n\n
Please be aware that you must be a member, or grantee, of the institutions/funders listed in order to apply for their Open Access publication funds.
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In this context, he has developed and validated new methodologies (e.g., Capillary Electrophoresis coupled to Laser-Induced Fluorescence, CE-LIF) whose application enabled him to determine both the amounts of biochemical markers (Desmosines) in urine/serum of patients affected by Chronic Obstructive Pulmonary Disease (COPD) and the activity of proteolytic enzymes (Human Neutrophil Elastase, Cathepsin G, Pseudomonas aeruginosa elastase) in sputa of these patients. More recently, Prof. Iadarola was involved in developing techniques such as two-dimensional electrophoresis coupled to liquid chromatography/mass spectrometry (2DE-LC/MS) for the proteomic analysis of biological fluids aimed at the identification of potential biomarkers of different lung diseases. He is the author of about 150 publications (According to Scopus: H-Index: 23; Total citations: 1568- According to WOS: H-Index: 20; Total Citations: 1296) of peer-reviewed international journals. 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He also obtained an MSc in Molecular and Genetic Medicine, and a Ph.D. in Clinical Immunology and Human Genetics from the University of Sheffield, UK. He also completed a short-term fellowship in Pediatric Clinical Immunology and Bone Marrow Transplantation at Newcastle General Hospital, England. Dr. Rezaei is a Full Professor of Immunology and Vice Dean of International Affairs and Research, at the School of Medicine, Tehran University of Medical Sciences, and the co-founder and head of the Research Center for Immunodeficiencies. He is also the founding president of the Universal Scientific Education and Research Network (USERN). Dr. Rezaei has directed more than 100 research projects and has designed and participated in several international collaborative projects. He is an editor, editorial assistant, or editorial board member of more than forty international journals. He has edited more than 50 international books, presented more than 500 lectures/posters in congresses/meetings, and published more than 1,100 scientific papers in international journals.",institutionString:"Tehran University of Medical Sciences",institution:{name:"Tehran University of Medical Sciences",country:{name:"Iran"}}},{id:"180733",title:"Dr.",name:"Jean",middleName:null,surname:"Engohang-Ndong",slug:"jean-engohang-ndong",fullName:"Jean Engohang-Ndong",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/180733/images/system/180733.png",biography:"Dr. Jean Engohang-Ndong was born and raised in Gabon. After obtaining his Associate Degree of Science at the University of Science and Technology of Masuku, Gabon, he continued his education in France where he obtained his BS, MS, and Ph.D. in Medical Microbiology. He worked as a post-doctoral fellow at the Public Health Research Institute (PHRI), Newark, NJ for four years before accepting a three-year faculty position at Brigham Young University-Hawaii. Dr. Engohang-Ndong is a tenured faculty member with the academic rank of Full Professor at Kent State University, Ohio, where he teaches a wide range of biological science courses and pursues his research in medical and environmental microbiology. Recently, he expanded his research interest to epidemiology and biostatistics of chronic diseases in Gabon.",institutionString:"Kent State University",institution:{name:"Kent State University",country:{name:"United States of America"}}},{id:"188773",title:"Prof.",name:"Emmanuel",middleName:null,surname:"Drouet",slug:"emmanuel-drouet",fullName:"Emmanuel Drouet",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/188773/images/system/188773.png",biography:"Emmanuel Drouet, PharmD, is a Professor of Virology at the Faculty of Pharmacy, the University Grenoble-Alpes, France. As a head scientist at the Institute of Structural Biology in Grenoble, Dr. Drouet’s research investigates persisting viruses in humans (RNA and DNA viruses) and the balance with our host immune system. He focuses on these viruses’ effects on humans (both their impact on pathology and their symbiotic relationships in humans). He has an excellent track record in the herpesvirus field, and his group is engaged in clinical research in the field of Epstein-Barr virus diseases. He is the editor of the online Encyclopedia of Environment and he coordinates the Universal Health Coverage education program for the BioHealth Computing Schools of the European Institute of Science.",institutionString:null,institution:{name:"Grenoble Alpes University",country:{name:"France"}}},{id:"131400",title:"Prof.",name:"Alfonso J.",middleName:null,surname:"Rodriguez-Morales",slug:"alfonso-j.-rodriguez-morales",fullName:"Alfonso J. Rodriguez-Morales",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/131400/images/system/131400.png",biography:"Dr. Rodriguez-Morales is an expert in tropical and emerging diseases, particularly zoonotic and vector-borne diseases (especially arboviral diseases). He is the president of the Travel Medicine Committee of the Pan-American Infectious Diseases Association (API), as well as the president of the Colombian Association of Infectious Diseases (ACIN). He is a member of the Committee on Tropical Medicine, Zoonoses, and Travel Medicine of ACIN. He is a vice-president of the Latin American Society for Travel Medicine (SLAMVI) and a Member of the Council of the International Society for Infectious Diseases (ISID). Since 2014, he has been recognized as a Senior Researcher, at the Ministry of Science of Colombia. He is a professor at the Faculty of Medicine of the Fundacion Universitaria Autonoma de las Americas, in Pereira, Risaralda, Colombia. He is an External Professor, Master in Research on Tropical Medicine and International Health, Universitat de Barcelona, Spain. He is also a professor at the Master in Clinical Epidemiology and Biostatistics, Universidad Científica del Sur, Lima, Peru. In 2021 he has been awarded the “Raul Isturiz Award” Medal of the API. Also, in 2021, he was awarded with the “Jose Felix Patiño” Asclepius Staff Medal of the Colombian Medical College, due to his scientific contributions to COVID-19 during the pandemic. He is currently the Editor in Chief of the journal Travel Medicine and Infectious Diseases. His Scopus H index is 47 (Google Scholar H index, 68).",institutionString:"Institución Universitaria Visión de las Américas, Colombia",institution:null},{id:"332819",title:"Dr.",name:"Chukwudi Michael",middleName:"Michael",surname:"Egbuche",slug:"chukwudi-michael-egbuche",fullName:"Chukwudi Michael Egbuche",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/332819/images/14624_n.jpg",biography:"I an Dr. Chukwudi Michael Egbuche. I am a Senior Lecturer in the Department of Parasitology and Entomology, Nnamdi Azikiwe University, Awka.",institutionString:null,institution:{name:"Nnamdi Azikiwe University",country:{name:"Nigeria"}}},{id:"284232",title:"Mr.",name:"Nikunj",middleName:"U",surname:"Tandel",slug:"nikunj-tandel",fullName:"Nikunj Tandel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/284232/images/8275_n.jpg",biography:'Mr. Nikunj Tandel has completed his Master\'s degree in Biotechnology from VIT University, India in the year of 2012. He is having 8 years of research experience especially in the field of malaria epidemiology, immunology, and nanoparticle-based drug delivery system against the infectious diseases, autoimmune disorders and cancer. He has worked for the NIH funded-International Center of Excellence in Malaria Research project "Center for the study of complex malaria in India (CSCMi)" in collaboration with New York University. The preliminary objectives of the study are to understand and develop the evidence-based tools and interventions for the control and prevention of malaria in different sites of the INDIA. Alongside, with the help of next-generation genomics study, the team has studied the antimalarial drug resistance in India. Further, he has extended his research in the development of Humanized mice for the study of liver-stage malaria and identification of molecular marker(s) for the Artemisinin resistance. At present, his research focuses on understanding the role of B cells in the activation of CD8+ T cells in malaria. Received the CSIR-SRF (Senior Research Fellow) award-2018, FIMSA (Federation of Immunological Societies of Asia-Oceania) Travel Bursary award to attend the IUIS-IIS-FIMSA Immunology course-2019',institutionString:"Nirma University",institution:{name:"Nirma University",country:{name:"India"}}},{id:"334383",title:"Ph.D.",name:"Simone",middleName:"Ulrich",surname:"Ulrich Picoli",slug:"simone-ulrich-picoli",fullName:"Simone Ulrich Picoli",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/334383/images/15919_n.jpg",biography:"Graduated in Pharmacy from Universidade Luterana do Brasil (1999), Master in Agricultural and Environmental Microbiology from Federal University of Rio Grande do Sul (2002), Specialization in Clinical Microbiology from Universidade de São Paulo, USP (2007) and PhD in Sciences in Gastroenterology and Hepatology (2012). She is currently an Adjunct Professor at Feevale University in Medicine and Biomedicine courses and a permanent professor of the Academic Master\\'s Degree in Virology. She has experience in the field of Microbiology, with an emphasis on Bacteriology, working mainly on the following topics: bacteriophages, bacterial resistance, clinical microbiology and food microbiology.",institutionString:null,institution:{name:"Universidade Feevale",country:{name:"Brazil"}}},{id:"229220",title:"Dr.",name:"Amjad",middleName:"Islam",surname:"Aqib",slug:"amjad-aqib",fullName:"Amjad Aqib",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229220/images/system/229220.png",biography:"Dr. Amjad Islam Aqib obtained a DVM and MSc (Hons) from University of Agriculture Faisalabad (UAF), Pakistan, and a PhD from the University of Veterinary and Animal Sciences Lahore, Pakistan. Dr. Aqib joined the Department of Clinical Medicine and Surgery at UAF for one year as an assistant professor where he developed a research laboratory designated for pathogenic bacteria. Since 2018, he has been Assistant Professor/Officer in-charge, Department of Medicine, Manager Research Operations and Development-ORIC, and President One Health Club at Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan. He has nearly 100 publications to his credit. His research interests include epidemiological patterns and molecular analysis of antimicrobial resistance and modulation and vaccine development against animal pathogens of public health concern.",institutionString:"Cholistan University of Veterinary and Animal Sciences",institution:{name:"University of Agriculture Faisalabad",country:{name:"Pakistan"}}},{id:"333753",title:"Dr.",name:"Rais",middleName:null,surname:"Ahmed",slug:"rais-ahmed",fullName:"Rais Ahmed",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/333753/images/20168_n.jpg",biography:null,institutionString:null,institution:{name:"University of Agriculture Faisalabad",country:{name:"Pakistan"}}},{id:"62900",title:"Prof.",name:"Fethi",middleName:null,surname:"Derbel",slug:"fethi-derbel",fullName:"Fethi Derbel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/62900/images/system/62900.jpeg",biography:"Professor Fethi Derbel was born in 1960 in Tunisia. He received his medical degree from the Sousse Faculty of Medicine at Sousse, University of Sousse, Tunisia. He completed his surgical residency in General Surgery at the University Hospital Farhat Hached of Sousse and was a member of the Unit of Liver Transplantation in the University of Rennes, France. He then worked in the Department of Surgery at the Sahloul University Hospital in Sousse. Professor Derbel is presently working at the Clinique les Oliviers, Sousse, Tunisia. His hospital activities are mostly concerned with laparoscopic, colorectal, pancreatic, hepatobiliary, and gastric surgery. He is also very interested in hernia surgery and performs ventral hernia repairs and inguinal hernia repairs. He has been a member of the GREPA and Tunisian Hernia Society (THS). During his residency, he managed patients suffering from diabetic foot, and he was very interested in this pathology. For this reason, he decided to coordinate a book project dealing with the diabetic foot. Professor Derbel has published many articles in journals and collaborates intensively with IntechOpen Access Publisher as an editor.",institutionString:"Clinique les Oliviers",institution:null},{id:"300144",title:"Dr.",name:"Meriem",middleName:null,surname:"Braiki",slug:"meriem-braiki",fullName:"Meriem Braiki",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/300144/images/system/300144.jpg",biography:"Dr. Meriem Braiki is a specialist in pediatric surgeon from Tunisia. She was born in 1985. She received her medical degree from the University of Medicine at Sousse, Tunisia. She achieved her surgical residency training periods in Pediatric Surgery departments at University Hospitals in Monastir, Tunis and France.\r\nShe is currently working at the Pediatric surgery department, Sidi Bouzid Hospital, Tunisia. Her hospital activities are mostly concerned with laparoscopic, parietal, urological and digestive surgery. She has published several articles in diffrent journals.",institutionString:"Sidi Bouzid Regional Hospital",institution:null},{id:"229481",title:"Dr.",name:"Erika M.",middleName:"Martins",surname:"de Carvalho",slug:"erika-m.-de-carvalho",fullName:"Erika M. de Carvalho",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229481/images/6397_n.jpg",biography:null,institutionString:null,institution:{name:"Oswaldo Cruz Foundation",country:{name:"Brazil"}}},{id:"186537",title:"Prof.",name:"Tonay",middleName:null,surname:"Inceboz",slug:"tonay-inceboz",fullName:"Tonay Inceboz",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/186537/images/system/186537.jfif",biography:"I was graduated from Ege University of Medical Faculty (Turkey) in 1988 and completed his Med. PhD degree in Medical Parasitology at the same university. I became an Associate Professor in 2008 and Professor in 2014. I am currently working as a Professor at the Department of Medical Parasitology at Dokuz Eylul University, Izmir, Turkey.\n\nI have given many lectures, presentations in different academic meetings. I have more than 60 articles in peer-reviewed journals, 18 book chapters, 1 book editorship.\n\nMy research interests are Echinococcus granulosus, Echinococcus multilocularis (diagnosis, life cycle, in vitro and in vivo cultivation), and Trichomonas vaginalis (diagnosis, PCR, and in vitro cultivation).",institutionString:"Dokuz Eylül University",institution:{name:"Dokuz Eylül University",country:{name:"Turkey"}}},{id:"71812",title:"Prof.",name:"Hanem Fathy",middleName:"Fathy",surname:"Khater",slug:"hanem-fathy-khater",fullName:"Hanem Fathy Khater",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/71812/images/1167_n.jpg",biography:"Prof. Khater is a Professor of Parasitology at Benha University, Egypt. She studied for her doctoral degree, at the Department of Entomology, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, USA. She has completed her Ph.D. degrees in Parasitology in Egypt, from where she got the award for “the best scientific Ph.D. dissertation”. She worked at the School of Biological Sciences, Bristol, England, the UK in controlling insects of medical and veterinary importance as a grant from Newton Mosharafa, the British Council. Her research is focused on searching of pesticides against mosquitoes, house flies, lice, green bottle fly, camel nasal botfly, soft and hard ticks, mites, and the diamondback moth as well as control of several parasites using safe and natural materials to avoid drug resistances and environmental contamination.",institutionString:null,institution:{name:"Banha University",country:{name:"Egypt"}}},{id:"99780",title:"Prof.",name:"Omolade",middleName:"Olayinka",surname:"Okwa",slug:"omolade-okwa",fullName:"Omolade Okwa",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/99780/images/system/99780.jpg",biography:"Omolade Olayinka Okwa is presently a Professor of Parasitology at Lagos State University, Nigeria. She has a PhD in Parasitology (1997), an MSc in Cellular Parasitology (1992), and a BSc (Hons) Zoology (1990) all from the University of Ibadan, Nigeria. She teaches parasitology at the undergraduate and postgraduate levels. She was a recipient of a Commonwealth fellowship supported by British Council tenable at the Centre for Entomology and Parasitology (CAEP), Keele University, United Kingdom between 2004 and 2005. She was awarded an Honorary Visiting Research Fellow at the same university from 2005 to 2007. \nShe has been an external examiner to the Department of Veterinary Microbiology and Parasitology, University of Ibadan, MSc programme between 2010 and 2012. She is a member of the Nigerian Society of Experimental Biology (NISEB), Parasitology and Public Health Society of Nigeria (PPSN), Science Association of Nigeria (SAN), Zoological Society of Nigeria (ZSN), and is Vice Chairperson of the Organisation of Women in Science (OWSG), LASU chapter. She served as Head of Department of Zoology and Environmental Biology, Lagos State University from 2007 to 2010 and 2014 to 2016. She is a reviewer for several local and international journals such as Unilag Journal of Science, Libyan Journal of Medicine, Journal of Medicine and Medical Sciences, and Annual Research and Review in Science. \nShe has authored 45 scientific research publications in local and international journals, 8 scientific reviews, 4 books, and 3 book chapters, which includes the books “Malaria Parasites” and “Malaria” which are IntechOpen access publications.",institutionString:"Lagos State University",institution:{name:"Lagos State University",country:{name:"Nigeria"}}},{id:"273100",title:"Dr.",name:"Vijay",middleName:null,surname:"Gayam",slug:"vijay-gayam",fullName:"Vijay Gayam",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/273100/images/system/273100.jpeg",biography:"Dr. Vijay Bhaskar Reddy Gayam is currently practicing as an internist at Interfaith Medical Center in Brooklyn, New York, USA. He is also a Clinical Assistant Professor at the SUNY Downstate University Hospital and Adjunct Professor of Medicine at the American University of Antigua. He is a holder of an M.B.B.S. degree bestowed to him by Osmania Medical College and received his M.D. at Interfaith Medical Center. His career goals thus far have heavily focused on direct patient care, medical education, and clinical research. He currently serves in two leadership capacities; Assistant Program Director of Medicine at Interfaith Medical Center and as a Councilor for the American\r\nFederation for Medical Research. As a true academician and researcher, he has more than 50 papers indexed in international peer-reviewed journals. He has also presented numerous papers in multiple national and international scientific conferences. His areas of research interest include general internal medicine, gastroenterology and hepatology. He serves as an editor, editorial board member and reviewer for multiple international journals. His research on Hepatitis C has been very successful and has led to multiple research awards, including the 'Equity in Prevention and Treatment Award” from the New York Department of Health Viral Hepatitis Symposium (2018) and the 'Presidential Poster Award” awarded to him by the American College of Gastroenterology (2018). He was also awarded 'Outstanding Clinician in General Medicine” by Venus International Foundation for his extensive research expertise and services, perform over and above the standard expected in the advancement of healthcare, patient safety and quality of care.",institutionString:"Interfaith Medical Center",institution:{name:"Interfaith Medical Center",country:{name:"United States of America"}}},{id:"93517",title:"Dr.",name:"Clement",middleName:"Adebajo",surname:"Meseko",slug:"clement-meseko",fullName:"Clement Meseko",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/93517/images/system/93517.jpg",biography:"Dr. Clement Meseko obtained DVM and PhD degree in Veterinary Medicine and Virology respectively. He has worked for over 20 years in both private and public sectors including the academia, contributing to knowledge and control of infectious disease. Through the application of epidemiological skill, classical and molecular virological skills, he investigates viruses of economic and public health importance for the mitigation of the negative impact on people, animal and the environment in the context of Onehealth. \r\nDr. Meseko’s field experience on animal and zoonotic diseases and pathogen dynamics at the human-animal interface over the years shaped his carrier in research and scientific inquiries. He has been part of the investigation of Highly Pathogenic Avian Influenza incursions in sub Saharan Africa and monitors swine Influenza (Pandemic influenza Virus) agro-ecology and potential for interspecies transmission. He has authored and reviewed a number of journal articles and book chapters.",institutionString:"National Veterinary Research Institute",institution:{name:"National Veterinary Research Institute",country:{name:"Nigeria"}}},{id:"158026",title:"Prof.",name:"Shailendra K.",middleName:null,surname:"Saxena",slug:"shailendra-k.-saxena",fullName:"Shailendra K. Saxena",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRET3QAO/Profile_Picture_2022-05-10T10:10:26.jpeg",biography:"Professor Dr. Shailendra K. Saxena is a vice dean and professor at King George's Medical University, Lucknow, India. His research interests involve understanding the molecular mechanisms of host defense during human viral infections and developing new predictive, preventive, and therapeutic strategies for them using Japanese encephalitis virus (JEV), HIV, and emerging viruses as a model via stem cell and cell culture technologies. His research work has been published in various high-impact factor journals (Science, PNAS, Nature Medicine) with a high number of citations. He has received many awards and honors in India and abroad including various Young Scientist Awards, BBSRC India Partnering Award, and Dr. JC Bose National Award of Department of Biotechnology, Min. of Science and Technology, Govt. of India. Dr. Saxena is a fellow of various international societies/academies including the Royal College of Pathologists, United Kingdom; Royal Society of Medicine, London; Royal Society of Biology, United Kingdom; Royal Society of Chemistry, London; and Academy of Translational Medicine Professionals, Austria. He was named a Global Leader in Science by The Scientist. He is also an international opinion leader/expert in vaccination for Japanese encephalitis by IPIC (UK).",institutionString:"King George's Medical University",institution:{name:"King George's Medical University",country:{name:"India"}}},{id:"94928",title:"Dr.",name:"Takuo",middleName:null,surname:"Mizukami",slug:"takuo-mizukami",fullName:"Takuo Mizukami",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/94928/images/6402_n.jpg",biography:null,institutionString:null,institution:{name:"National Institute of Infectious Diseases",country:{name:"Japan"}}},{id:"233433",title:"Dr.",name:"Yulia",middleName:null,surname:"Desheva",slug:"yulia-desheva",fullName:"Yulia Desheva",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/233433/images/system/233433.png",biography:"Dr. Yulia Desheva is a leading researcher at the Institute of Experimental Medicine, St. Petersburg, Russia. She is a professor in the Stomatology Faculty, St. Petersburg State University. She has expertise in the development and evaluation of a wide range of live mucosal vaccines against influenza and bacterial complications. 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\r\n\tBiodiversity provides food security and constitutes a gene pool for biotechnology, especially in the field of agriculture and medicine, and promotes the development of ecotourism.
\r\n
\r\n\tCurrently, biologists admit that we are witnessing the first phases of the seventh mass extinction caused by human intervention. It is estimated that the current rate of extinction is between a hundred and a thousand times faster than it was when man first appeared. The disappearance of species is caused not only by an accelerated rate of extinction, but also by a decrease in the rate of emergence of new species as human activities degrade the natural environment. The conservation of biological diversity is "a common concern of humanity" and an integral part of the development process. Its objectives are “the conservation of biological diversity, the sustainable use of its components, and the fair and equitable sharing of the benefits resulting from the use of genetic resources”.
\r\n
\r\n\tThe following are the main causes of biodiversity loss:
\r\n
\r\n\t• The destruction of natural habitats to expand urban and agricultural areas and to obtain timber, minerals and other natural resources.
\r\n
\r\n\t• The introduction of alien species into a habitat, whether intentionally or unintentionally which has an impact on the fauna and flora of the area, and as a result, they are reduced or become extinct.
\r\n
\r\n\t• Pollution from industrial and agricultural products, which devastate the fauna and flora, especially those in fresh water.
\r\n
\r\n\t• Global warming, which is seen as a threat to biological diversity, and will become increasingly important in the future.
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\r\n\tThe environment is subject to severe anthropic effects. Among them are those associated with pollution, resource extraction and overexploitation, loss of biodiversity, soil degradation, disorderly land occupation and planning, and many others. These anthropic effects could potentially be caused by any inadequate management of the environment. However, ecosystems have a resilience that makes them react to disturbances which mitigate the negative effects. It is critical to understand how ecosystems, natural and anthropized, including urban environments, respond to actions that have a negative influence and how they are managed. It is also important to establish when the limits marked by the resilience and the breaking point are achieved and when no return is possible. The main focus for the chapters is to cover the subjects such as understanding how the environment resilience works, the mechanisms involved, and how to manage them in order to improve our interactions with the environment and promote the use of adequate management practices such as those outlined in the United Nations’ Sustainable Development Goals.
\r\n\tPollution is caused by a wide variety of human activities and occurs in diverse forms, for example biological, chemical, et cetera. In recent years, significant efforts have been made to ensure that the environment is clean, that rigorous rules are implemented, and old laws are updated to reduce the risks towards humans and ecosystems. However, rapid industrialization and the need for more cultivable sources or habitable lands, for an increasing population, as well as fewer alternatives for waste disposal, make the pollution control tasks more challenging. Therefore, this topic will focus on assessing and managing environmental pollution. It will cover various subjects, including risk assessment due to the pollution of ecosystems, transport and fate of pollutants, restoration or remediation of polluted matrices, and efforts towards sustainable solutions to minimize environmental pollution.
\r\n\tWater is not only a crucial substance needed for biological life on Earth, but it is also a basic requirement for the existence and development of the human society. Owing to the importance of water to life on Earth, early researchers conducted numerous studies and analyses on the liquid form of water from the perspectives of chemistry, physics, earth science, and biology, and concluded that Earth is a "water polo". Water covers approximately 71% of Earth's surface. However, 97.2% of this water is seawater, 21.5% is icebergs and glaciers, and only 0.65% is freshwater that can be used directly by humans. As a result, the amount of water reserves available for human consumption is limited. The development, utilization, and protection of freshwater resources has become the focus of water science research for the continued improvement of human livelihoods and society.
\r\n
\r\n\tWater exists as solid, liquid, and gas within Earth’s atmosphere, lithosphere, and biosphere. Liquid water is used for a variety of purposes besides drinking, including power generation, ecology, landscaping, and shipping. Because water is involved in various environmental hydrological processes as well as numerous aspects of the economy and human society, the study of various phenomena in the hydrosphere, the laws governing their occurrence and development, the relationship between the hydrosphere and other spheres of Earth, and the relationship between water and social development, are all part of water science. Knowledge systems for water science are improving continuously. Water science has become a specialized field concerned with the identification of its physical, chemical, and biological properties. In addition, it reveals the laws of water distribution, movement, and circulation, and proposes methods and tools for water development, utilization, planning, management, and protection. Currently, the field of water science covers research related to topics such as hydrology, water resources and water environment. It also includes research on water related issues such as safety, engineering, economy, law, culture, information, and education.
",coverUrl:"https://cdn.intechopen.com/series_topics/covers/41.jpg",keywords:"Water, Water Resources, Freshwater, Hydrological Processes, Utilization, Protection"}],annualVolumeBook:{},thematicCollection:[],selectedSeries:null,selectedSubseries:null},seriesLanding:{item:{id:"7",title:"Biomedical Engineering",doi:"10.5772/intechopen.71985",issn:"2631-5343",scope:"Biomedical Engineering is one of the fastest-growing interdisciplinary branches of science and industry. The combination of electronics and computer science with biology and medicine has improved patient diagnosis, reduced rehabilitation time, and helped to facilitate a better quality of life. Nowadays, all medical imaging devices, medical instruments, or new laboratory techniques result from the cooperation of specialists in various fields. The series of Biomedical Engineering books covers such areas of knowledge as chemistry, physics, electronics, medicine, and biology. 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Dr. Koprowski has authored more than a hundred research papers with dozens in impact factor (IF) journals and has authored or co-authored six books. Additionally, he is the author of several national and international patents in the field of biomedical devices and imaging. Since 2011, he has been a reviewer of grants and projects (including EU projects) in biomedical engineering.",institutionString:null,institution:{name:"University of Silesia",institutionURL:null,country:{name:"Poland"}}},subseries:[{id:"7",title:"Bioinformatics and Medical Informatics",keywords:"Biomedical Data, Drug Discovery, Clinical Diagnostics, Decoding Human Genome, AI in Personalized Medicine, Disease-prevention Strategies, Big Data Analysis in Medicine",scope:"Bioinformatics aims to help understand the functioning of the mechanisms of living organisms through the construction and use of quantitative tools. The applications of this research cover many related fields, such as biotechnology and medicine, where, for example, Bioinformatics contributes to faster drug design, DNA analysis in forensics, and DNA sequence analysis in the field of personalized medicine. Personalized medicine is a type of medical care in which treatment is customized individually for each patient. Personalized medicine enables more effective therapy, reduces the costs of therapy and clinical trials, and also minimizes the risk of side effects. Nevertheless, advances in personalized medicine would not have been possible without bioinformatics, which can analyze the human genome and other vast amounts of biomedical data, especially in genetics. The rapid growth of information technology enabled the development of new tools to decode human genomes, large-scale studies of genetic variations and medical informatics. The considerable development of technology, including the computing power of computers, is also conducive to the development of bioinformatics, including personalized medicine. In an era of rapidly growing data volumes and ever lower costs of generating, storing and computing data, personalized medicine holds great promises. Modern computational methods used as bioinformatics tools can integrate multi-scale, multi-modal and longitudinal patient data to create even more effective and safer therapy and disease prevention methods. Main aspects of the topic are: Applying bioinformatics in drug discovery and development; Bioinformatics in clinical diagnostics (genetic variants that act as markers for a condition or a disease); Blockchain and Artificial Intelligence/Machine Learning in personalized medicine; Customize disease-prevention strategies in personalized medicine; Big data analysis in personalized medicine; Translating stratification algorithms into clinical practice of personalized medicine.",annualVolume:11403,isOpenForSubmission:!0,coverUrl:"https://cdn.intechopen.com/series_topics/covers/7.jpg",editor:{id:"351533",title:"Dr.",name:"Slawomir",middleName:null,surname:"Wilczynski",fullName:"Slawomir Wilczynski",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0033Y000035U1loQAC/Profile_Picture_1630074514792",institutionString:null,institution:{name:"Medical University of Silesia",institutionURL:null,country:{name:"Poland"}}},editorTwo:null,editorThree:null,editorialBoard:[{id:"5886",title:"Dr.",name:"Alexandros",middleName:"T.",surname:"Tzallas",fullName:"Alexandros Tzallas",profilePictureURL:"https://mts.intechopen.com/storage/users/5886/images/system/5886.png",institutionString:"University of Ioannina, Greece & Imperial College London",institution:{name:"University of Ioannina",institutionURL:null,country:{name:"Greece"}}},{id:"257388",title:"Distinguished Prof.",name:"Lulu",middleName:null,surname:"Wang",fullName:"Lulu Wang",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRX6kQAG/Profile_Picture_1630329584194",institutionString:"Shenzhen Technology University",institution:{name:"Shenzhen Technology University",institutionURL:null,country:{name:"China"}}},{id:"225387",title:"Prof.",name:"Reda R.",middleName:"R.",surname:"Gharieb",fullName:"Reda R. Gharieb",profilePictureURL:"https://mts.intechopen.com/storage/users/225387/images/system/225387.jpg",institutionString:"Assiut University",institution:{name:"Assiut University",institutionURL:null,country:{name:"Egypt"}}}]},{id:"8",title:"Bioinspired Technology and Biomechanics",keywords:"Bioinspired Systems, Biomechanics, Assistive Technology, Rehabilitation",scope:'Bioinspired technologies take advantage of understanding the actual biological system to provide solutions to problems in several areas. Recently, bioinspired systems have been successfully employing biomechanics to develop and improve assistive technology and rehabilitation devices. The research topic "Bioinspired Technology and Biomechanics" welcomes studies reporting recent advances in bioinspired technologies that contribute to individuals\' health, inclusion, and rehabilitation. 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We encourage the submission of manuscripts that provide novel and mechanistic insights that report significant advances in the fields. Topics can include but are not limited to: Biotechnology such as biotechnological products and process engineering; Biotechnologically relevant enzymes and proteins; Bioenergy and biofuels; Applied genetics and molecular biotechnology; Genomics, transcriptomics, proteomics; Applied microbial and cell physiology; Environmental biotechnology; Methods and protocols. Moreover, topics in biosensor technology, like sensors that incorporate enzymes, antibodies, nucleic acids, whole cells, tissues and organelles, and other biological or biologically inspired components will be considered, and topics exploring transducers, including those based on electrochemical and optical piezoelectric, thermal, magnetic, and micromechanical elements. 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