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This handbook presents how plant in vitro technologies can overcome current limitations in the production of important plant-derived substances. It explains the advantages of plant in vitro technologies, notably the independence from climatic and soil conditions and the ability to synthesize diverse bioactive substances under controlled conditions. Apart from making diverse metabolites, which can be used e.g. as pharmaceuticals, agrochemicals, flavors, colors, biopesticides or food additives, more easily and more efficiently available, the methods described in this handbook also offer the advantage that rare and threatened plants, which provide access to interesting and desired substances, can be better protected, when the substances are harvested from suitable plant in vitro systems. In times of increasing demand for natural plant-derived products, the described methodologies will be key to ensuring efficient and sustainable access to plant-derived products. They will also help and support in the research and investigation of plant secondary metabolites.
Despite these advantages, still only few substances are being produced at industrial scale by in vitro plant cell cultivation systems to date. This handbook therefore advertises the recent achievements and research in the field, focused on solving limitations in yield and bioprocessing conditions. Leading experts summarize the methodology, which can help overcome drawbacks like low yields of target products or problems associated with the cultivation in bioreactors. Readers will find comprehensive information on fundamentals for using different types of plants in vitro as matrix for sustainable production of valuable secondary metabolites. The handbook summarizes the core information on phytochemistry, bioreactor technology and monitoring of plant cells and tissues in bioprocesses. It also discusses selected applications and safety assessment of food and cosmetic ingredients from plant cell and tissue.

Inhaltsverzeichnis

Frontmatter

Historical Background

Frontmatter

1. History of Plant Biotechnology Development

Abstract
It is difficult to write a review on the history of plant biotechnology, especially after the excellent works of Vasil (Plant Cell Rep 27(9):1423–1440, 2008) Thorpe (Mol Biotechnol 37:169–180, 2007), and Sussex (Plant Cell 20(5):1189–1198, 2008). It is even more difficult to overview the current state of this fast-developing field. Nevertheless, in this review we will make an attempt not only to make a narrative of main stages but also to show the links between plant biotechnology and latest progress in biological science.
Plant biotechnology has its roots deep in human civilization but was established just a century ago. Starting outside the science mainstream of the time period, classical plant biotechnology slowly but steadily grew into a recognized discipline. The explosive growth of biology research at the end of the twentieth century brought plant biotechnology to the fast-track line. The field grew very rapidly and currently turned into a key tool for fundamental research and practical uses. Currently plant biotechnology has been essentially grown, and new disciplines as omics technologies as genome editing have arisen which further intensify both fundamental and practical studies in biology and make a bridge with other scientific areas as informatics, nanotechnology, and so-called digital and intelligent science. As such modern biotechnology speeds up the development of the Fourth Industrial Revolution (Schwab, The fourth industrial revolution. World Economic Forum. ISBN 1944835008, 2016).
Ivelin Pantchev, Goritsa Rakleova, Atanas Pavlov, Atanas Atanassov

2. Plant Tissue Culture Technology: Present and Future Development

Abstract
The application of plant tissue cultures in fundamental and applied studies on various biological species has been rapidly growing. The use of in vitro technology for commercial propagation of plant species and for the production of bioactive components from them has become profitable industry worldwide.
Various regeneration systems (protoplast cultures and somatic embryogenesis) and their importance for the advance of strategically significant priorities in the development of biotechnological science in agriculture, medicine, and pharmacy are treated in the present chapter.
We believe that in the future development of the in vitro technology the major priorities could be conservation of plant genetic resources; restoring the balance between research studies related to genetic transformation of plants with the aim of providing sufficient, quality and safety foods for the world population, on the one hand, and the studies aimed at determining the risk of growing and consuming them, on the other; creating transgenic plants maintaining a constant level of induced protein; and, last but not least, the use of plant resources possessing valuable biologically active substances.
Svetla Yancheva, Violeta Kondakova

Metabolic Phytochemistry

Frontmatter

3. Metabolite Profiling of In Vitro Plant Systems

Abstract
Gas chromatography-mass spectrometry is one of the base analytical platforms used in plant metabolite profiling. The remarkable recent methodological and technological developments in GC-MS profiling expand the possibilities for its application in different fields of plant science including plant biotechnology. The methods of extraction, fractionation, derivatization, and metabolite identification, associated with GC-MS metabolite profiling, along with examples demonstrating the power and applicability of GC-MS in plant in vitro studies, have been presented in this chapter.
Strahil Berkov, Liliya Georgieva, Borjana Sidjimova, Milena Nikolova

4. Microbial Transformations of Plant Secondary Metabolites

Abstract
The aim of this chapter is to present the authors’ view on the place and role of microbial transformation reactions as a perspective means of processing of plant-derived biologically active compounds into metabolites with new and/or increased activity and availability and decreased toxicity. Some microbial transformations providing information regarding metabolism in humans and mammals of plant-derived secondary metabolites applied as drugs and/or food additives are also considered.
Blaga Mutafova, Pedro Fernandes, Sava Mutafov, Strahil Berkov, Atanas Pavlov

Secondary Metabolites

Frontmatter

5. Engineering Cell and Organ Cultures from Medicinal and Aromatic Plants Toward Commercial Production of Bioactive Metabolites

Abstract
Production of secondary metabolites from in vitro cell and hairy root cultures (CHRC) of medicinal and aromatic plants (MAP) is considered a promising alternative to gathering plant material from MAP natural populations, often a reason for their overexploitation and even extinction. However, most of the valuable secondary metabolites extracted from different MAP species are present in very low amounts in the respective CHRC. Plant metabolic engineering offers an attractive opportunity to increase the content of target secondary metabolites in engineered transgenic CHRC for production at feasible levels. Moreover, applying metabolic engineering makes it possible to redirect target metabolic pathway(s) in the transgenic CHRC to produce new compounds not present in the wild plant itself. This chapter describes the strategies and experimental toolbox for plant metabolic engineering with examples from engineering secondary metabolite production in CHRC from MAP, as well as a review of these century reported studies on metabolic engineering of CHRC. The directions and prospects for CHRC metabolic engineering applications in production of valuable secondary metabolites are discussed
Krasimir Rusanov, Atanas Atanassov, Ivan Atanassov

6. Agrobacterium rhizogenes-Mediated Transformation of Plants for Improvement of Yields of Secondary Metabolites

Abstract
The transgenic hairy root culture has revolutionized the role of tissue culture of plants in the synthesis of secondary metabolites. It was shown that hairy roots in the most cases exhibit higher biosynthetic capacity for secondary metabolite production comparing to the non-transgenic roots. A big number of medicinal compounds have been produced using this approach. However, the mechanism of influence of T-DNA genes on secondary metabolite production is not completely understood. The stimulatory effect of single rol genes (rolA, rolB, rolC) on secondary metabolite production was demonstrated for a number of plant species that are widely used in pharmacology. It is interesting to note that these rol genes are present in naturally transgenic Linaria, Ipomoea, and Nicotiana plants. Many species from these genera are used as medicinal. Besides, naturally transgenic plants could be a good model for study of possible evolutionary function of rol genes in the control of secondary metabolites for plant protection.
Tatiana V. Matveeva, Sophie V. Sokornova

7. Amaryllidaceae Alkaloid Accumulation by Plant In Vitro Systems

Abstract
Over 300 alkaloids possessing a wide range of biological activities have been isolated from plants belonging to Amaryllidaceae family. Galanthamine, used for the palliative treatment of Alzheimer’s disease, is the only one commercialized. The biodisponibility of Amaryllidaceae alkaloids is low. In vitro culture offers an alternative interesting approach for the biotechnological production of these valuable alkaloids. The feeding with different exogenous factors of Leucojum aestivum, L. aestivum ‘Gravety Giant,’ and Narcissus in vitro cultures was reported, and the effects on the biosynthetic pathway of both galanthamine and lycorine were studied.
Dominique Laurain-Mattar, Agata Ptak

8. Sustainable Production of Polyphenols and Antioxidants by Plant In Vitro Cultures

Abstract
Phenolic compounds represent big group of plant secondary metabolites that influence flavor, color, and texture and can be used as food additives, nutraceuticals, and pharmaceuticals.
However, there are some limitations in obtaining sufficient amount of these bioactive compounds from plants, because they are rather seldom or occur naturally in plant tissues only at very low concentrations. Alternatively, it is possible to synthesize them chemically, but this way if oft technologically not possible or very sophisticated and economically infeasible.
Plant in vitro cultures provide an attractive route to produce high-value plant-derived products and therefore can be an alternative source of valuable phenolics.
Moreover, compounds synthesized by plant in vitro cultures are natural products and therefore can be more easily accepted by consumers as artificially synthetized substances.
The synthesis of phytochemicals by plant in vitro cultures in contrast to these in plants is not depending on environmental conditions and can be regulated through standard physical and chemical conditions in bioreactor, which helps to avoid qualitative and quantitative fluctuations in product yield.
The process of obtaining valuable phytochemicals can be represented as a multistage technology, each link of which can vary individually in dependence of specific requirements of in vitro cultures (e.g., phytohormones, nutrients, light) or properties of end product (e.g., antioxidative potential, stability).
For the establishment of high-producing and fast-growing cell lines, the parent plants should be selected (Murthy et al. Strategies for enhanced production of plant secondary metabolites from cell and organ cultures. In: Production of biomass and bioactive compounds using bioreactor technology (pp. 471–509). Springer Plus). The expression of synthetic pathways can be influenced by environmental conditions, the supply of precursors, and the application of elicitors (Schreiner, Eur J Nutr 44(2):85–94, 2005) as well as altered by special treatments like biotransformation and immobilization (Georgiev et al., Appl Microbiol Biotechnol 83:809–823, 2009). The efficiency of bioprocessing can be increased by the simplification of methods for product recovery and afterward its stabilization.
This chapter reviews the recent advances in the optimization of environmental factors for production of phenolics by plant in vitro cultures, new developments in bioprocessing of plant cell, hairy root and organ cultures, and emerging technologies on phytochemical recovery.
Iryna Smetanska

9. Production of Iridoid and Phenylethanoid Glycosides by In Vitro Systems of Plants from the Buddlejaceae, Orobanchaceae, and Scrophulariaceae Families

Abstract
The plants belonging to Buddlejaceae, Orobanchaceae, and Scrophulariaceae families are rich sources of iridoid and phenylethanoid glycosides, which are widely used as anti-inflammatory, hypoglycemic, and nourishing agents. Recent years have seen the application of various in vitro culture systems as alternative source of these metabolites. We discuss the use of callus, cell suspension cultures, shoot cultures, and the whole regenerated plants as possible approaches for production of the compounds. Additionally, methods of efficiently improving metabolite accumulation in in vitro cultures through elicitation, precursor feeding, and both Agrobacterium rhizogenes- and A. tumefaciens-mediated genetic transformations (hairy roots, transformed plants) among the plant families are also presented.
Ewelina Piątczak, Renata Grąbkowska, Halina Wysokińska

10. Taxus Cell Cultures: An Effective Biotechnological Tool to Enhance and Gain New Biosynthetic Insights into Taxane Production

Abstract
Mass phytochemical production in biotechnological platforms based on plant cell and organ cultures provides an alternative to the field cultivation of plants. The system is being successfully applied to produce plant bioactive compounds scarce in nature, including taxol, a potent chemotherapeutic agent, and its analogs. Additionally, plant cell cultures are a potent tool to shed light on the biosynthesis of phytochemicals and its control. Several studies with Taxus spp. cell cultures, focused on increasing taxane production, have gained considerable molecular understanding of how these compounds are metabolized in the target cell cultures, particularly by the application of omics tools. This chapter summarizes the state of the art in the biotechnological production of taxol and related taxanes used for the semisynthesis of a new taxane generation. Special emphasis is given to the application of the latest cutting-edge technologies that reveal the molecular changes taking place in plant cells subjected to optimized conditions for taxane biosynthesis and accumulation.
Heriberto Vidal-Limon, Raúl Sanchez-Muñoz, Abbas Khojasteh, Elisabeth Moyano, Rosa M. Cusido, Javier Palazon

11. Challenges for the Cultivation of Plant Cells on the Example of Hypericum perforatum and Taxus chinensis

Abstract
Medicinal plants are sustainable bio-factories for valuable active pharmaceutical ingredients (API). They are commonly grown in the field and their extracts have a given combination of constituents. There is some variation due to climate fluctuations and plant diseases (microbial infections), genotypic changes, soil differences, etc. Additionally, fertile agricultural areas are increasingly limited. However, these variations are undesired because they are non-controllable and can affect the batch conformity of a drug significantly. This is a challenge for producers of phyto-pharmaceuticals, and the variations in the API composition are compensated by mixing extracts from various batches to achieve the required continuous quality of an authorized drug. These drawbacks of field cultivation are overcome by well-defined bioreactor-based cultivation. Biomass growth and API production take place under variable but controllable cultivation conditions, resulting in customized extracts. Variation of the cultivation conditions leads to qualitative and/or quantitative changes in the metabolome. During bioreactor cultivation, plant cells tend to stay connected after division, which leads to the formation of aggregates. The size of shear-sensitive plant cell aggregates influenced by hydrodynamic forces resulting from mechanical agitation was often recognized as an intangible parameter, which might be responsible for general variability in plant cell culture processes. To date, however, the bioreactor approach is not often industrially implemented. This chapter provides an overview of the challenges in the cultivation of plant cell systems, briefly illustrated by (i) research on Hypericum perforatum tissue cultures into up-to-date approaches for production of hyperforin and hypericin, possibly functional at a pre-commercial level in the future, and (ii) effects of hydrodynamic mechanical forces on Taxus chinensis submerged cultures for production of paclitaxel.
Mariam Gaid, Thomas Wucherpfennig, Stephan Scholl, Ludger Beerhues, Rainer Krull

12. Mass Production of Artemisinin Using Hairy Root Cultivation of Artemisia annua in Bioreactor

Abstract
Malaria is endemic disease of the tropical countries primarily due to their specific climatic conditions. Artemisinin has been widely used for the treatment of patients of cerebral malaria in combination therapy with other antimalarial drugs such as quinine and chloroquine. It has been extracted from the leaves of the plant Artemisia annua which grows naturally in many countries except for humid tropical countries. However, yield of the drug from dry tissue has been in the range of 0.01–0.5%; with the result, a 1000 kg of dry plant leaves yield only 6 kg of artemisinin after solvent extraction and liquid chromatography-based purification protocols. In order to alleviate these problems, scientists have been exploring alternate in vitro production protocols particularly by plant cell/hairy root cultivation of A. annua to supplement the overall artemisinin availability. Hairy root cultivation could be one of the potent in vitro alternative production techniques for artemisinin as it has an inherent advantage of better biochemical stability and less doubling time than plant cell cultivation. The present report attempts to provide a comprehensive overview of mass production of artemisinin particularly in vitro production using various bioreactors and different cultivation modes.
Nivedita Patra, Ashok K. Srivastava

13. Plant In Vitro Systems as Sources of Food Ingredients and Additives

Abstract
In the recent years, people prefer to consume food with natural additives, especially those with plant origin because of the increased reports for carcinogenic and other side effects of some synthetic ones. Plant in vitro cultures have recently received great attention as an effective technology for the production of valuable secondary metabolites used as food ingredients and additives. The advantages of plant cell, tissue, and organ cultures over living plants, in terms of secondary metabolite production, are well understand: in the laboratory, growth conditions and parameters can be controlled and optimized; separation of target compounds is much easier; large-scale growth of plant cells in liquid culture in bioreactors can be achieved and ultimately commercialized. This chapter provides an overview and examples of plant in vitro systems producing food colorants, antioxidants, flavors, and sweeteners.
Radka Vrancheva, Nadezhda Petkova, Ivan Ivanov

14. Safety Assessment and Regulations for Food Ingredients Derived from Plant In Vitro Systems

Abstract
Plant cell and tissue cultures present numerous advantages for the production of food ingredients. This technology has made a great progress in the past two decades, with a few examples of products reaching the market. Safety assessment of plant in vitro products is a key issue for the development of sustainable commercial processes. Experts have proposed various strategies and methods for assessing the potential risks for the end product arising from the production processes, and throughout the years these have been implemented as guidelines and national regulations. The international approach in safety assessment of single chemically defined plant and cell culture food ingredients is based on the concept of substantial equivalence developed for other categories for novel food products. For products in the form of complex mixtures, conventional metabolism and toxicokinetic studies should be provided for toxicologically relevant constituents with known or demonstrable biological or toxicological activity on a case-by-case basis. The present chapter gives an overview of the current international regulatory basis and proposed schemes for the safety assessment of plant cell and tissue culture-derived food ingredient with some suggestions for the future needs in this field.
Angel Angelov, Velitchka Gotcheva

Bioreactor Technology and Monitoring

Frontmatter

15. Bioreactor Technology for Sustainable Production of Plant Cell-Derived Products

Abstract
The successful cultivation of plant cell and tissue cultures for the production of valuable chemical components requires the selection of an appropriate bioreactor. Selection criteria are determined based on a number of factors that are intrinsic to particular plant cell or tissue cultures and are influenced by the process objectives. Due to the specific properties of plant cell and tissue cultures, bioreactor systems may differ significantly from those used for microorganism or animal cell cultures. Furthermore, the differences from one plant culture to another can be immense; it is obvious that the optimal bioreactor system for a plant suspension cell culture is different to one for a plant tissue culture in many ways.
General considerations are presented, and based on these key points, selection criteria are used to establish a “bioreactor chooser” tool. The particular details of the most relevant bioreactor types for plant cell and tissue cultures are listed and described.
To produce valuable products, the process also needs to be scaled up to an economically justifiable size, which is usually done either by scaling up the size of the bioreactor itself or by bioreactor parallelization. Therefore, the most significant influencing factors are also discussed.
Sören Werner, Rüdiger W. Maschke, Dieter Eibl, Regine Eibl

16. Monitoring of Plant Cells and Tissues in Bioprocesses

Abstract
Plant cell and tissue cultures represent a suitable alternative as production systems for valuable plant secondary metabolites. Unlike traditional extraction from agricultural grown plants, the active ingredient production in biotechnological processes with in vitro cultures takes place in closed bioreactors under controlled conditions. This allows a year-round production with constant quality and quantity. However, the development of biotechnological processes with plant in vitro cultures is often time-consuming and requires parallelized screening systems. Furthermore, the design, optimization, and control of economic processes presuppose knowledge about the physiological state of the biological system and the kinetic parameters of biomass and product formation. To gain access to these data, suitable process-monitoring methods are required which provide information about the physiology of the process, both on a macroscopic and on the single cell level. However, due to the morphology of plant cell and tissue cultures, many methods for bioprocess monitoring that are used for mammalian and microbial cultures are not applicable. This chapter covers methods that are appropriate for monitoring of biotechnological processes with plant cell and tissue cultures: The conductivity of the growth medium is a powerful parameter to estimate the growth of complex plant cell aggregates and tissue structures. The next section describes the application of the RAMOS – a small scale cultivation system – for heterotrophic and phototrophic plant cell and tissue cultures. Flow cytometry is a tool to obtain segregated data of bioprocesses. Further, we describe a novel approach of cell immobilization for physiological studies and the design of bioprocesses, the 3D Green Bioprinting.
Juliane Steingroewer, Christiane Haas, Katja Winkler, Carolin Schott, Jost Weber, Julia Seidel, Felix Krujatz, Sibylle Kümmritz, Anja Lode, Maria Lisa Socher, Michael Gelinsky, Thomas Bley

17. Bioreactor Technology for Hairy Roots Cultivation

Abstract
Bioreactor technology is an integral requisite to the development of scale-up production process of many plant-based high-value products. The proper selection and design of the bioreactor is required to determine the optimal industrial scale bioprocess and the subsequent capital investment. A primary cause of the lack of success in commercial production of secondary compounds using hairy root culture systems is their low yield. To increase the production of hairy root-based bioactive compounds, several strategies, like elicitation, metabolic engineering, and up-scaling, etc., have been adopted. Out of these strategies, the up-scaling in bioreactor deals with the principle of large-scale metabolite production in proportion of high biomass growth. The goal of an effective bioreactor is to control, contain, and positively influence a biological reaction in an incessant way in order to get desired productivity. This chapter provides a descriptive account on up-scaling of hairy root cultures for various purposes including secondary metabolite production. This chapter also discusses the hitherto reports on up-scaling of hairy root cultures of various plant species in terms of modifications in designing of bioreactors for incessant tissue growth concomitantly with metabolite production.
Shakti Mehrotra, Sonal Mishra, Vikas Srivastava

Other Applications

Frontmatter

18. Platforms for Plant-Based Protein Production

Abstract
Plant molecular farming depends on a diversity of plant systems for production of useful recombinant proteins. These proteins include protein biopolymers, industrial proteins and enzymes, and therapeutic proteins. Plant production systems include microalgae, cells, hairy roots, moss, and whole plants with both stable and transient expression. Production processes involve a narrowing diversity of bioreactors for cell, hairy root, microalgae, and moss cultivation. For whole plants, both field and automated greenhouse cultivation methods are used with products expressed and produced either in leaves or seeds. Many successful expression systems now exist for a variety of different products with a list of increasingly successful commercialized products. This chapter provides an overview and examples of the current state of plant-based production systems for different types of recombinant proteins.
Jianfeng Xu, Melissa Towler, Pamela J. Weathers

19. Hairy Roots and Phytoremediation

Abstract
Contamination of the environment arises either from natural geological processes or due to human activities and has created an alarming situation worldwide. Biological strategies for cleaning up contaminated biosphere have gained much importance in recent years and are preferred over other conventional physical and chemical methods because these are environmentally friendly and cost-effective. Phytoremediation is an ecologically compatible approach using plants to remediate polluted environment. Currently hairy roots have emerged as a notably competent research tool for phytoremediation among the various biological systems investigated for this purpose. Infection of certain plants caused by Agrobacterium rhizogenes is expressed in the form of hairy root disease. The disease is characterized by adventitious roots with copious root hairs developing elaborately from or next to the infection site. The plant genome receives a set of genes from a segment of the large root inducing (Ri) plasmid of A. rhizogenes. Under the effect of these genes, the inherent hormonal balance of the plant is altered resulting in the development of hairy roots. In nature, plant roots are the primary organs having contact with the environmental contaminants. Thus, hairy roots have been used in phytoremediation research as physiologically they resemble the normal roots of the mother plants. Several studies demonstrate the potentiality of hairy roots in removing a vast array of both organic and inorganic pollutants from the environment. In addition, microorganisms colonizing the rhizosphere of hairy roots have also proved to improve the efficacy of hairy roots in eliminating contaminants. The purpose of this review is to summarize the applications of hairy roots in different phytoremediation strategies and provide examples and prospects of the use of hairy roots in the removal of organic and inorganic contaminants from the environment.
Anrini Majumder, Smita Ray, Sumita Jha

Backmatter

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