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Cell biotechnology and plant cell culture

The document discusses the advancements in plant cell biotechnology and the production of secondary metabolites through various methods including callus formation, protoplast culture, and tissue propagation. It outlines key concepts such as totipotency, plant hormones, organogenesis, and cell culture techniques, emphasizing their applications in generating pharmaceuticals and enhancing plant breeding. Additionally, it details the roles of secondary metabolites, their accumulation, degradation, and the importance of specific cultivation methods for achieving optimal yields and industrial applications.

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CELL BIOTECHNOLOGY PLANT CELL CULTURE Recent approaches and Development for production of secondary metabolites.
1. Biotechnology • Plant Cell Biotechnology • General terms 2. Plant Cell Culture 3. Development in Plant Cell Culture 4. Recent Approaches for High Production of secondary metabolites ( Drugs) CONTENTS: 2
The definition of biotechnology usually includes the development of methods by which biological processes may be controlled such that their rate of production enables economic industrial production (phytoproduction), or by which living material is obtained that can be utilized in industry, agriculture and forestry, as well as in gardening and breeding (plant breeding). 3 PLANT CELL BIOTECHNOLOGY: BIOTECHNOLOGY: Plant Cell Biotechnology is broadly concerned with experimental research on plants and plant like organisms (fungi and cyanobacteria).(Stafford, Morris, & Fowler, 1986)
GENERAL TERMS: 4 Cultures of plant cells taken from their natural environment and placed under controlled conditions. Callus a more or less loose association of cells without visible morphological differentiation. Cell suspension denotes cultures consisting of single cells or the smallest cellular association without differentiation, submersed in a turbulent medium. Protoplasts are naked cells of varied origin without cell walls, which are cultivated in liquid as well as on solid media Callus, Cell Suspension and Protoplast: Each cell contains every single attribute which appears in the differentiated plant. • Morphological totipotency was achieved by the regeneration of completely normal differentiated plants from callus and protoplasts • Chemical totipotency was brought by the examination of anthraquinone accumulation in the calli of different parts (root, leaf, shoot, fruit) of Morinda citrifolia . Totipotency: Sterile organs or pieces of tissue used to gain dedifferentiated cells by proliferation at sectional planes or wounded areas are called explants. These Explants are placed in specific solid culture media which, because of their phytohormonal content, encourage cell proliferation. Explant: As a rule, cytokinins and auxins are used as phytohormones. 2,4-Dichlor-phenoxyacetic acid is the most powerful, and is therefore called the dedifferentiation hormone. Phytohormones: Cytokinins Auxins 6-Benzyl-aminopurine (6-BA) Indole-3-acetic acid (IAA) 6-Furfuryl-aminopurine (kinetin) I-Naphthalene acetic acid (NAA) 2,4-Dichlorphenoxyacetic acid (2,4-D)
Callus Formation: Usually, the ratio between auxin and cytokinin concentrations determines whether a culture grows in a disorganized fashion as callus or develops shoots or roots. A particularly effective agent for callus formation is 2,4-di- chlorophenoxyacetic acid, generally characterized by its great effectiveness. Its effect is usually increased by the addition of cytokinin. Organogenesis: By reversing the conditions suitable for callus formation, the meristematic cells or groups of cells developing in the course of callus formation are stimulated to organogenesis. In many dicotyledonous callus cultures, bud formation is generally initiated by a ratio of 100/10, while callus development is favored by the inverse ratio 10/100. The relevant ratios in tobacco are different, but they do demonstrate the dependence of the effect on the donor material. However, the relative effectiveness of different auxins must be kept in mind. In some cases, culturing without one of the two hormones is sufficient to induce organogenesis. 5 AUXIN TO CYTOKININ RATIO:
6 AUXIN TO CYTOKININ RATIO:
GENERAL TERMS: 7 • Soma clonal variations can be obtained by subcultring the callus into different particular stress environment either by induced mutation using mutagens or by subculturing. • They are clones but with phenotypic and genotypic variations. Soma clonal variations: • Somatic embryogenesis is an artificial process in which a plant or embryo is derived from a single somatic cell. • Desired product can be obtained from plant by somatic embryogenesis. • Through somatic emryogenesis we get genetically uniform cell. • Bipolar in nature ( whole plant can grow) • Disease free large scale production • Synthatic seed production • E.g; Somatic culture of tobacco plant to extract nicotine. Somatic embryogenesis: • Morphogenesis (from the Greek morphê shape and genesis creation, literally "the generation of form") is the biological process that causes a cell, tissue or organism to develop its shape. • Organogenesis is the differenciation of cells into organ system. In monopolar growth either root or shoot is grown at a time. Morphogenesis:
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Cell suspension cultures are usually inoculated with the help of a callus fragment put into a liquid medium. The minimal amount needed is about 2 to 3 g/l00 ml medium. 10 PROTOPLAST CULTURE: CELL SUSPENSION CULTURE : 1. Physical Methods • Grinding with glass beads 2. Enzymatic Methods • Cellulose • Hemicellulose • Pectinase • The enzyme incubation can be carried out either successively in separate enzyme solutions or in a mixture of these enzymes for a long time (5-8 h) at low temperatures (8-25 °C), or alternatively for a short period ( < 2 h) at high
11 GENERAL CULTURE TECHNIQUES : 1. Sterlization • Sterlization of plant tissue by sodium hypochlorite, bezalkonium chloride, bromine water. • Sterlization of nutrient solution by autoclaving, tyndallization or sterile filtered. • Sterlization of glassware and tools • Air sterilization 2. Composition of media: • Inorganic components • Macronutrients • Organic components • Vitamins • Growth regulators
12 TISSUE CULTURE IS WIDELY USED IN: PLANT TISSUE CULTURE: • Plant tissue culture is a set of techniques for aseptic culture of cells, tissues, organs and their components under desired physical and chemical conditions in vitro and controlled conditions. • It explore conditions that promote cell division and genetic reprogramming in vitro conditions • It is an important tool in basic and applied sciences as well as in commercial applications. • Obtaining disease free plant. • Rapid propagation of plants those that are difficult to propagate. • Somatic hybridization. • Genetics improvement of commercial plants e.g; transgenic plants • Obtaining androgenic and gynogenic haploid plants for breeding programs.
13 HISTORY: • Gottlieb Haberlant justifiably is known as the father of plant tissue culture. He predicted that eventually a complete and functional plant could be generated from a single cell. (Totipotency) • Other studies led to the culture of isolated root tips. • The approach of using explants with meristematic cells produce the successful and indefinite culture of tomato root tips. • Only in 1985 was the sweetening agent from the flowers and leaves of the verbena plant, Lippia dulcis, which exceeds saccharose l000-fold in its sweetening power and which was used already by the Aztecs to sweeten their food, identified as a colorless sesquiterpene oil, hernandulcin. • The psychogenically active compounds from the "holy" fungi of the Indians, psilocybin and psilocin from the leaves of the genus Psilocybe, are now being utilized in modern medicine due to their structural similarity to the neurotransmitter serotonin.
14 CULTIVATION METHODS: • Once protoplasts are separated from the isolation medium and transferred to a suitable growth medium, adaptation and regeneration processes begin. Adaptation is necessary because the growth conditions differ from those in the tissue's previous environment. Stabilization: • The culture media for protoplasts are very similar to those for individual cells. However, in the former it is necessary to create conditions favoring the formation of cell walls and ensuring the stability of naked cells. Thus, the addition of polyethylene glycol (PEG) 1500 to the medium often accelerates the uniform deposition of micro fibrils. Wall Formation: • Polysaccharides such as hemicellulose synthesized for cell wall formation are initially largely excreted into the medium. Studies on the structure of newly formed 1. PROTOPLAST CULTURE:
15 CULTIVATION METHODS: Medium Components: Particular attention is paid to the effect of ion concentrations on protoplast division. Depending on the origin, the auxin/cytokinin ratio must also be specifically adapted. Highly differentiated mother cells (e.g. leaf cells) usually require a low auxin/kinetin ratio, while protoplast cultures obtained from actively growing cell cultures or meristematic tissue require high auxin/cytokinin ratios. Sensitivity: Further, a particularly great sensitivity to certain components of the medium and environmental conditions (light, shearing forces) is characteristic of protoplast cultures. For example, freshly isolated protoplasts are usually highly light-sensitive, at least during the first 4-7 days. 1. PROTOPLAST CULTURE
16 CULTIVATION METHODS: The preferred donor materials for individual cells are young leaves, calli and protoplasts. Cultures of N. tabacum and Phaseolus vulgaris, filtered off by a mesh of 0.1 to 0.3 mm, consist of 90% single cell. The three basic techniques: • nurse culture • plating technique • culture in a microchamber 2. CULTURES OFSINGLE CELLS:
17 CULTIVATION METHODS: 2. CULTURES OFSINGLE CELLS:
18 CULTIVATION METHODS:  Nurse Culture: The isolated cells to be cultured are fixed on a piece of filter paper on the surface of an actively growing nurse callus. The contact through the absorbent paper is sufficient to maintain a supply of nutrients and unknown growth factors. Once the developing microcalli have attained the size necessary for survival (200-400 µm), they are individually cultivated on a fresh medium. 2. CULTURES OFSINGLE CELLS:
19 CULTIVATION METHODS:  Plating and Feeder-Layer Technique; Culture in a Microchamber: • Conditioned Medium: During a nurse culture, unknown growth factors accumulated in a suspension culture or on the supporting filter paper are transmitted by the medium from cell to cell. In this way, the medium is enriched with these substances. Such a medium is thus called conditioned if it contains such substances excreted by any living cells which ensure survival and reproduction of cells cultivated at a suboptimal density. The effectivity of such media depends in part on the age of the nurse culture. However, nutrients depleted from the medium during preculturing must be replaced, otherwise they may be inhibitory. 2. CULTURES OFSINGLE CELLS:
20 CULTIVATION METHODS: • Plating and Feeder-Layer Techniques: Plating Technique. In this technique, cultures with a cell density less than the critical density are mixed with the medium containing agar (0.6%) at a temperature of 30-35 °C and poured into petri dishes to a depth of 1 mm. The low agar density makes it possible to follow the development of single cells through an inverse microscope. The culture may be divided into individual cubes, called agar beads. Plating success is determined by the ratio of the number of cells in the original suspension to the number of colonies developing within the following 21 days. The goal is to attain the highest possible plating efficiency (PE), the number of colonies per plated single cells, at the least possible cell density. The lower boundary has so far been around 5000/ml. 2. CULTURES OFSINGLE CELLS:
21 CULTIVATION METHODS: • Plating and Feeder-Layer Techniques: Feeder-Layer Technique: In order to add a nutrient supply and stimulate division, living cells or protoplasts unable to divide are added to the agar medium as a feeder layer. 2. CULTURES OFSINGLE CELLS:
22 CULTIVATION METHODS: • Culture in a Microchamber: In micro chambers, the problem of insufficient cell density is solved by reducing the amount of medium. Starting from the minimum concentration that ensures plating success, the amount was reduced to 0.6 µl, 0.25-0.5 µl and even to microdroplets of 10-25 nl Such mini-cultures of a single cell or protoplast may be cultivated by either hanging them from a slide held in place by the medium's surface tension (hanging drop culture), or on a slide or in mini-agar caverns (1 x 1 x 1.75 mm) open above. 2. CULTURES OFSINGLE CELLS:
23
24 CULTIVATION METHODS: From the point of view of an application-oriented industry, experiments performed on the scale of petri dishes and Erlenmeyer flasks cannot be economically utilized. Even in laboratory fermenters with a capacity of 5-501, questions of scaling-up to industrial scale are usually unanswerable. Therefore, industrial production conditions are usually simulated in so-called pilot plants at a scale up to 1000 l. The results thus obtained can be applied to larger reactors, provided the geometric and dynamic parameters remain constant in the scaling factor. However, this is possible without further difficulties only if a single parameter is rate-determining. This factor must be kept constant during scaling-up.  Determining Factors: The aspects that must be considered in culturing large volumes may be classified as process and culture conditions. The extent of scaling-up is determined by factors of physical, chemical, biochemical and biological nature. 3. MASS CULTIVATION METHOD:
25 CULTIVATION METHODS: 3. MASS CULTIVATION METHOD:
26
27 GROWTH OF CELL AND TISSUE CULTURE: Measurement methods: Changes in growth may be measured by fresh and dry weight, cell mass, cell number, mitotic index or indirectly by the conductivity of the medium. Fresh Weight: The method of fresh-weight determination neglects the various water contents of the material. Therefore, the values of callus cultures, frequently determined as total weight of callus, medium layer and petri dish, experience large variations due to evaporation via the medium's surface. More exact values are obtained by determining the weight after complete separation from the culture medium. This is possible when the material is cultured on separating layers of cellulose or nylon. Dry Weight: Quantification by means of dry weight excludes error due to varying endogenous water contents. This requires repeated drying, usally at 60°C, to the point of constant weight. Up to fresh weights of 500 mg, a linear relationship between fresh and dry weight is assumed. GROWTH PROCESS:
28 GROWTH OF CELL AND TISSUE CULTURE: Cell Mass: This may be determined by densification by centrifugation (ca. 2000 g, 5 min) of a particular percentage of the volume (ca. 4-7 ml) in graduated, conical centrifuge tubes. In order to avoid errors due to water absorption by the cells, the so-called packed cell volume (PCV) must be recorded immediately following the separation process. Cell Number: To determine the number of cells per unit volume, existing cell clumps or aggregates must be separated into isolated cells - not only in callus cultures but also in most suspension cultures. This is commonly done using chrome-trioxide alone or in combination with hypochlorous acid. Possible alternatives are EDT A and pectinase. GROWTH PROCESS:
29 GROWTH OF CELL AND TISSUE CULTURE: Conductivity: The inverse relationship between the conductivity and fresh or dry weight of the medium allows the determination of growth without taking samples (which would affect the sterility of the culture). In fully synthetic media, conductivity is determined almost exclusively by salt concentrations. As long as the pH of the medium remains above 3 (CH + < 10-3 mol/I), the concentration of hydrogen ions does not affect conductivity. Cellulose Concentration: Calcofluor-white ST (0.1 % aqueous) allows monitoring of changes in the concentrations of cell wall polymers from fJ-glycosidic bound glucose molecules such as cellulose or callose. The textile brightener specifically binds to fJ-l,4-glucans and intensely fluoresces following stimulation with shortwave blue light. In this way, even traces of these compounds may be identified GROWTH PROCESS:
PLANT CELLS AS PRODUCERS OF SECONDARY METABOLITES: 30
SECONDARY METABOLITES: 31 Secondary metabolites, also called specialised metabolites, toxins, secondary products, or natural products, are organic compounds produced by bacteria, fungi, or plants which are not directly involved in the normal growth, development, or reproduction of the organism. They were labelled secondary compounds in contradistinction to primary compounds due to: 1. an apparently limited taxonomic distribution 2. Synthesis occurring only under certain conditions 3. An apparent lack of function 4. No apparent necessity for life.
32 SOME PLANT SECONDARY METABOLITES AND THEIR APPLICATION: Metaobolites Application Species Ajmalicine Circulation Catharanthus roseus Atropine Anti-cholinergic Atropa belladonna Hyoscyamine Hyoscine Theophylline Anti-cholinergic Anti-cholinergic Hyoscyamus spp. Datura spp. Camellia sinensis Diosgenin Contraceptive Dioscorea spp. Quinine Anti-Malarial Cinchona spp Eugenol Local anesthetic Syzygium aromaticum Morphine Analgesics Papaver somniferum
33 STORAGE OF SECONDARY METABOLITES:
34 STORAGE OF SECONDARY METABOLITES: Accumulation: Turnover: Depending on the object, secondary compounds are either secreted into the surrounding medium or stored intracellularly. There they experience turnover processes with characteristic half-lives. Their degradation was first proven in cell suspension cultures. Degradation and synthesis often occur simultaneously. The extent of their accumulation is mainly determined by three cell capacities: synthetic capacity, storage capacity and the capacity to metabolize the compounds in transport and detoxification processes. Individual plant organs differ in their significance in this process.
35 PHARMACEUTICALLY ACTIVE NATURAL PRODUCT SYNTHESIS VIA PLANT CELL CULTURE TECHNOLOGY: Most secondary metabolites are often present in extremely low amounts in the plant, often less than 1% of the total carbon. This paucity can make natural harvestation impractical for bulk production, especially in the case of slow growing species. The significant engineering challenge is then to find a means by which to produce the desired natural products in a way that is both sustainable and financially feasible. Production of these products in a microbial or fungal host by transferring the biosynthetic pathway is possible.
PRODUCTION OPTIONS FOR NATURAL PRODUCTS: 36 Chemical synthesis of natural products is possible and commercially feasible, particularly for those with relatively simple chemical structures such as aspirin (derived from the natural product salicylic acid) and ephedrine. In many cases, however, the metabolite has a complex structure, which can include multiple rings and chiral centers, so that a synthetic production process becomes prohibitively costly. Many natural products used in cancer treatment, including compounds such as paclitaxel, vinblastine, and camptothecin, fall into this latter class, so an alternative method of supply is necessary.
37 PRODUCTION OPTION FOR NATURAL PRODUCTS : • Depending on the nature of the plant, extraction directly from harvested plant tissue may be an option. Especially if a plant can be cultivated en masse, this can be attractive on a commercial basis. • The anticancer drugs vincristine and vinblastine, among other medicinally valuable metabolites such as ajmalicine and serpentine, are found in the Madagascar periwinkle Catharanthus roseus. • Even though these important alkaloids, particularly vincristine and vinblastine, naturally occur at very low levels in C. roseus less than 3 g per metric tons the fast growing nature of the periwinkle makes field cultivation most practical at the present time. • However, the relative inefficiency and high cost of whole plant extraction implies that an improved method of supply would be useful for these valuable anticancer agents.
38 PRODUCTION OPTION FOR NATURAL PRODUCTS : • When natural supply is limited due to a combination of low yields and slow growth rates, in vitro cultures provide an attractive alternative. Most plant species can be cultured in vitro in either an undifferentiated or differentiated state. • As many secondary metabolites are produced by specialized cells, organ cultures such as shoots or roots can exhibit similar metabolite profile patterns compared to the native plant, whereas undifferentiated cultures often accumulate secondary metabolites to a lesser extent, and sometimes not at all.
39 PRODUCTION OPTION FOR NATURAL PRODUCTS : • The anticancer compound camptothecin, produced by the ornamental tree Camptotheca acuminata as well as Nothapodytes fetida and Ophiorrhiza pumila among other species, has been shown to accumulate in undifferentiated cultures in very low or even undetectable amounts compared to root cultures in which production levels were comparable to the intact plant. • Similarly, no artemisinin, a potent antimalarial drug, was found in cell suspension cultures of Artemisia annua, while trace amounts were detected in shoot cultures.
40 PRODUCTION OPTION FOR NATURAL PRODUCTS : • Root cultures can be transformed into hairy roots using the soil dwelling bacteria Agrobacterium rhizogenes, resulting in cultures which are genetically stable, capable of unlimited growth without additional hormones, and have an increased capacity for secondary metabolite accumulation. • Undifferentiated suspension cultures, which can be more easily scaled to levels suitable for commercial production. There are currently 14 plant cell culture processes which have been commercialized for production of secondary metabolites (including products used in applications other than pharmaceuticals such as food and cosmetics).
41 PRODUCTION OPTION FOR NATURAL PRODUCTS :
PLANT SUSPENSION CELL CULTURE TECHNOLOGY: 42 • Production of metabolites via plant cell suspension culture is renewable, environmentally friendly, and from a processing standpoint, amenable to strict control, an advantage in regards to meeting Food and Drug Administration manufacturing standards. Technology developed for other cell culture and fermentation systems (e.g., mammalian and yeast) can be readily adapted for large scale applications with plant cells, easing difficulties associated with scale-up. • A notable example of the success of plant cell culture systems, due in large part to innovative research and the application of novel technologies, is paclitaxel synthesis and supply. Paclitaxel, produced by Taxus spp., is an important anticancer agent used as a first line treatment for several types of cancer, including breast, ovarian, and non small cell lung cancer, and has also shown efficacy against AIDS-related Kaposi sacoma. Production of paclitaxel via cell culture technology has been studied since the 1980s as an alternative supply source to harvest of the slow growing yew tree, since a single dose of 300 mg requires the sacrifice of a 100 year old tree
43 PLANT SUSPENSION CELL CULTURE TECHNOLOGY :
44 PLANT SUSPENSION CELL CULTURE TECHNOLOGY : • The primary challenges impeding regular commercial application of plant cell culture technology are low and variable yields of metabolite accumulation. • Some metabolites do not accumulate in appreciable quantities in undifferentiated cells. In these cases, manipulation of genes within the biosynthetic pathway is needed to utilize plant cell cultures for bulk production.
45 HAIRY ROOT CULTURES ASASOURCE OF SECONDARY METABOLITES • The hairy root system based on inoculation with Agrobacterium rhizogenes has become popular in the two last decades as a method of producing secondary metabolites synthesized in plant roots. • The hairy root phenotype is characterized by fast hormone-independent growth, lack of geotropism, lateral branching, and genetic stability. The secondary metabolites produced by hairy roots arising from the infection of plant material by A. rhizogenes are the same as those usually synthesized in intact parent roots, with similar or higher yields. • This feature, together with genetic stability and generally rapid growth in simple media lacking phytohormones, makes them especially suitable for biochemical studies not easily undertaken with root cultures of an intact plant.
46 HAIRY ROOT CULTURES ASASOURCE OF SECONDARY METABOLITES • During the infection process, A. rhizogenes transfers a part of the DNA (transferred DNA, T-DNA) located in the root-inducing plasmid Ri to plant cells, and the genes contained in this segment are expressed in the same way as the endogenous genes of the plant cells.
47 METABOLIC ENGINEERING AND DIRECTED BIOSYNTHESIS • The engineering of biosynthetic pathways within a plant cell to enhance accumulation of a constitutively produced metabolite is an appealing strategy in which exciting progress has been made in the past decade. • A variety of tools have been employed to both identify unknown genes and characterize secondary metabolite pathway regulation, including precursor feeding, gene over expression, application of metabolic inhibitors, and mutant selection. • Additionally, elicitation, in relation to improving bulk yields in cell culture, can also be used as a powerful tool to investigate pathway regulation based on gene expression.
48 METABOLIC ENGINEERING TOOLS: • A metabolic engineering approach involves the manipulation of targets within a cell. Techniques are therefore needed both for the identification of these targets (i.e., genes, proteins, metabolites) as well as for their exploitation. • As many secondary pathways are still partially undefined, elucidating pathway genes and their control elements is an active research area. • The subsequent identification of rate influencing steps within a biosynthetic pathway can then be useful in providing targets for a rational engineering strategy.
49 METABOLIC ENGINEERING TOOLS: • Plant cell cultures, including both suspension cultures and hairy root cultures, have proven to be an extremely useful platform for metabolic studies, as a fast growing and renewable source of material. • Whole plants can also be valuable, particularly as models to study complex spatial and temporal control mechanisms associated with environmental stimuli and morphogenesis from a global metabolic perspective.
50 METABOLIC ENGINEERING TOOLS: • Several approaches have been used to identify the enzymes and their corresponding genes which catalyze biosynthetic pathway steps. • For the paclitaxel pathway, a successful approach utilized by the Croteau laboratory incorporated feeding cell free Taxus extracts with precursors to isolate and identify intermediate metabolites and enzymes. • This approach led to the identification of taxadiene synthase, which catalyzes the first committed step of the taxane pathway. • Genes were subsequently identified from a cDNA library using PCR amplification based on degenerate primers designed to recognize conserved regions from homologous enzymes in other plants whose DNA sequences were known.
51 METABOLIC ENGINEERING TOOLS: • Differential display methods (via reverse transcription and PCR) comparing mRNA transcripts between elicited and unelicited cells supplemented with a homology-based search of a cDNA library from elicited cells, as well as random sequencing of the same induced library, have also proven to be extremely effective in gene discovery (for a comprehensive review of molecular genetics in Taxus.
52 TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE YIELDS: Optimization of cultural conditions: • Number of chemical and physical factors like media components, phytohormones, pH, temperature, aeration, agitation, light affecting production of secondary metabolites affect culture productivity. • Several products were found to be accumulating in cultured cells at a higher level than those in native plants through optimization of cultural conditions. Manipulation of physical aspects and nutritional elements in a culture is perhaps the most fundamental approach for optimization of culture productivity. • For example, ginsenosides by Panax ginseng and Dixon, rosmarinic acid by Coleus bluemei, shikonin by Lithospermum, ubiquinone-10 by Nicotiana tabacum, berberin by Coptis japonica were accumulated in much higher levels in cultured cells than in the intact plants.
53 TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE YIELDS: Precursor feeding: Exogenous supply of a biosynthetic precursor to culture medium may also increase the yield of the desired product. This approach is useful when the precursors are inexpensive. The concept is based on the idea that any compound, which is an intermediate, in or at the beginning of a secondary metabolite biosynthetic route, stands a good chance of increasing the yield of the final product. Attempts to induce or increase the production of plant secondary metabolites, by supplying precursor or intermediate compounds, have been effective in many cases.
54 TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE YIELDS: Precursor feeding: For example, • amino acids have been added to cell suspension culture media for production of tropane alkaloids, indole alkaloids etc. • Addition of phenylalanine to Salvia officinalis cell suspension cultures stimulated the production of rosmarinic acid. • Addition of the same precursor resulted stimulation of taxol production in Taxus cultures. • Feeding ferulic acid to cultures of Vanilla planifolia resulted in increase in vanillin accumulation. • Furthermore, addition of leucine, led to enhancement of volatile monoterpenes in cultures of Perilla frutiscens, where as addition of geraniol to rose cell cultures led to accumulation of nerol and citronellol
55 TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE YIELDS: Elicitation: Plants produce secondary metabolites in nature as a defense mechanism against attack by pathogens. Elicitors are signals triggering the formation of secondary metabolites. Perhaps the most notable strategy for improving metabolite yields is elicitation. An elicitor can be defined as any compound that induces an upregulation of genes. Some elicitors target secondary metabolic genes, which are often associated with defense responses to perceived environmental changes.
56 TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE YIELDS: Elicitation: • Elicitors include natural hormones, nutrients, and many fungi-derived compounds. In particular, jasmonic acid and its methyl ester methyl jasmonate (MJ), are naturally occurring hormones involved in the regulation of defense genes as part of a signal transduction system. • Applied exogenously, they have been shown to induce secondary metabolic activity and promote accumulation of desired metabolites in numerous plant systems, including Taxus spp. and C. roseus. • Different elicitors may act on different segments of the biosynthetic pathway. For instance, MJ elicitation compared to salicylic acid elicitation in Taxus spp. cultures resulted in different relative increases of metabolic intermediates, suggesting that each elicitor preferentially directs flux toward, and possibly away from, different intermediate taxanes.
57 TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE YIELDS: Elicitation: While many of the specific targets of elicitors have yet to be conclusively identified, elicitation can be an extremely useful tool in conjunction with gene expression profiling for identifying rate-influencing steps in secondary biosynthetic pathways.
58 TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE YIELDS: Immobilization of plant cell cultures: • It has long been considered for increasing metabolite accumulation, as the potential of higher cell densities, continuous removal of products/inhibitors, and protection for shear-sensitive plant cells provide a number of advantages. • Immobilization can be simply achieved using a gel matrix such as alginate; however this becomes costly at a larger scale, especially when the product of interest is not secreted and must be released using sonication or treatments with an organic solvent.
59 TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE YIELDS: Immobilization of plant cell cultures: • Recently, immobilization of T. baccata cells in calcium-alginate beads was shown to produce one of the highest reported levels of paclitaxel accumulation among academic laboratories (43 mg/L). • Immobilization also has the potential to simplify product extraction and purification, as immobilized cultures of Linum usitatissimum excrete the pharmaceutically active metabolite dehydrodiconiferyl alcohol 4--D-glucoside (DCG) to a greater extent than suspension cultures.
Stafford, A., Morris, P., & Fowler, M. (1986). Plant cell biotechnology: a perspective. Enzyme and microbial technology, 8(10), 578-587. Endress, R., & Endress, R. (1994). Plant cell biotechnology: Springer. Kolewe, M. E., Gaurav, V., & Roberts, S. C. (2008). Pharmaceutically active natural product synthesis and supply via plant cell culture technology. Molecular pharmaceutics, 5(2), 243-256. Aboujaoude, E., Salame, W., & Naim, L. (2015). Telemental health: a status update. World psychiatry, 14(2), 223-230. Mulabagal, V., & Tsay, H.-S. (2004). Plant cell cultures-an alternative and efficient source for the production of biologically important secondary metabolites. Int J Appl Sci Eng, 2(1), 29-48. 60 REFERENCES:
THANK YOU Sidra sidratulmuntaha1786@gmail.com 02332013039 M.Phil Pharmacognoy 2nd Semester

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In this document
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Overview of plant cell culture focusing on recent developments for secondary metabolite production.

Defining biotechnology, plant cell biotechnology, and general terms related to cultures and totipotency.

Describes the significance of auxin-to-cytokinin ratios in callus formation and organogenesis.

Outlines somatic embryogenesis, variations, and callus formation metrics including profit margins.

Methods for propagating plant cells and the significance of physical and enzymatic techniques.

Cultural techniques including sterilization, media composition, and importance of plant tissue culture.

Highlights pioneers in plant tissue culture and notable historical advancements in the field.

Cultivation methods focusing on protoplast culture, cell division, and scaling-up techniques.

Methods to measure growth of cell and tissue culture through fresh/dry weight and conductivity.

Defines secondary metabolites, their features, applications, and storage processes in plants.

Various methods for natural product synthesis and challenges in producing pharmaceutical compounds.

Discusses metabolic engineering strategies to enhance secondary metabolite production in plants.

Describes traditional methods to enhance cell culture yields including elicitation and immobilization.

Lists references cited in presentation and provides contact details for further inquiries.

Cell biotechnology and plant cell culture

  • 1.
    CELL BIOTECHNOLOGY PLANT CELLCULTURE Recent approaches and Development for production of secondary metabolites.
  • 2.
    1. Biotechnology • PlantCell Biotechnology • General terms 2. Plant Cell Culture 3. Development in Plant Cell Culture 4. Recent Approaches for High Production of secondary metabolites ( Drugs) CONTENTS: 2
  • 3.
    The definition ofbiotechnology usually includes the development of methods by which biological processes may be controlled such that their rate of production enables economic industrial production (phytoproduction), or by which living material is obtained that can be utilized in industry, agriculture and forestry, as well as in gardening and breeding (plant breeding). 3 PLANT CELL BIOTECHNOLOGY: BIOTECHNOLOGY: Plant Cell Biotechnology is broadly concerned with experimental research on plants and plant like organisms (fungi and cyanobacteria).(Stafford, Morris, & Fowler, 1986)
  • 4.
    GENERAL TERMS: 4 Cultures ofplant cells taken from their natural environment and placed under controlled conditions. Callus a more or less loose association of cells without visible morphological differentiation. Cell suspension denotes cultures consisting of single cells or the smallest cellular association without differentiation, submersed in a turbulent medium. Protoplasts are naked cells of varied origin without cell walls, which are cultivated in liquid as well as on solid media Callus, Cell Suspension and Protoplast: Each cell contains every single attribute which appears in the differentiated plant. • Morphological totipotency was achieved by the regeneration of completely normal differentiated plants from callus and protoplasts • Chemical totipotency was brought by the examination of anthraquinone accumulation in the calli of different parts (root, leaf, shoot, fruit) of Morinda citrifolia . Totipotency: Sterile organs or pieces of tissue used to gain dedifferentiated cells by proliferation at sectional planes or wounded areas are called explants. These Explants are placed in specific solid culture media which, because of their phytohormonal content, encourage cell proliferation. Explant: As a rule, cytokinins and auxins are used as phytohormones. 2,4-Dichlor-phenoxyacetic acid is the most powerful, and is therefore called the dedifferentiation hormone. Phytohormones: Cytokinins Auxins 6-Benzyl-aminopurine (6-BA) Indole-3-acetic acid (IAA) 6-Furfuryl-aminopurine (kinetin) I-Naphthalene acetic acid (NAA) 2,4-Dichlorphenoxyacetic acid (2,4-D)
  • 5.
    Callus Formation: Usually,the ratio between auxin and cytokinin concentrations determines whether a culture grows in a disorganized fashion as callus or develops shoots or roots. A particularly effective agent for callus formation is 2,4-di- chlorophenoxyacetic acid, generally characterized by its great effectiveness. Its effect is usually increased by the addition of cytokinin. Organogenesis: By reversing the conditions suitable for callus formation, the meristematic cells or groups of cells developing in the course of callus formation are stimulated to organogenesis. In many dicotyledonous callus cultures, bud formation is generally initiated by a ratio of 100/10, while callus development is favored by the inverse ratio 10/100. The relevant ratios in tobacco are different, but they do demonstrate the dependence of the effect on the donor material. However, the relative effectiveness of different auxins must be kept in mind. In some cases, culturing without one of the two hormones is sufficient to induce organogenesis. 5 AUXIN TO CYTOKININ RATIO:
  • 6.
  • 7.
    GENERAL TERMS: 7 • Somaclonal variations can be obtained by subcultring the callus into different particular stress environment either by induced mutation using mutagens or by subculturing. • They are clones but with phenotypic and genotypic variations. Soma clonal variations: • Somatic embryogenesis is an artificial process in which a plant or embryo is derived from a single somatic cell. • Desired product can be obtained from plant by somatic embryogenesis. • Through somatic emryogenesis we get genetically uniform cell. • Bipolar in nature ( whole plant can grow) • Disease free large scale production • Synthatic seed production • E.g; Somatic culture of tobacco plant to extract nicotine. Somatic embryogenesis: • Morphogenesis (from the Greek morphê shape and genesis creation, literally "the generation of form") is the biological process that causes a cell, tissue or organism to develop its shape. • Organogenesis is the differenciation of cells into organ system. In monopolar growth either root or shoot is grown at a time. Morphogenesis:
  • 8.
  • 9.
    METHODS OF PROPAGATION: *According to a Survey 9 40-60% Average Profit Margin $175,000 Average Annual Revenue per Doctor 3% Market Growth Per Year
  • 10.
    Cell suspension culturesare usually inoculated with the help of a callus fragment put into a liquid medium. The minimal amount needed is about 2 to 3 g/l00 ml medium. 10 PROTOPLAST CULTURE: CELL SUSPENSION CULTURE : 1. Physical Methods • Grinding with glass beads 2. Enzymatic Methods • Cellulose • Hemicellulose • Pectinase • The enzyme incubation can be carried out either successively in separate enzyme solutions or in a mixture of these enzymes for a long time (5-8 h) at low temperatures (8-25 °C), or alternatively for a short period ( < 2 h) at high
  • 11.
    11 GENERAL CULTURE TECHNIQUES: 1. Sterlization • Sterlization of plant tissue by sodium hypochlorite, bezalkonium chloride, bromine water. • Sterlization of nutrient solution by autoclaving, tyndallization or sterile filtered. • Sterlization of glassware and tools • Air sterilization 2. Composition of media: • Inorganic components • Macronutrients • Organic components • Vitamins • Growth regulators
  • 12.
    12 TISSUE CULTURE ISWIDELY USED IN: PLANT TISSUE CULTURE: • Plant tissue culture is a set of techniques for aseptic culture of cells, tissues, organs and their components under desired physical and chemical conditions in vitro and controlled conditions. • It explore conditions that promote cell division and genetic reprogramming in vitro conditions • It is an important tool in basic and applied sciences as well as in commercial applications. • Obtaining disease free plant. • Rapid propagation of plants those that are difficult to propagate. • Somatic hybridization. • Genetics improvement of commercial plants e.g; transgenic plants • Obtaining androgenic and gynogenic haploid plants for breeding programs.
  • 13.
    13 HISTORY: • Gottlieb Haberlantjustifiably is known as the father of plant tissue culture. He predicted that eventually a complete and functional plant could be generated from a single cell. (Totipotency) • Other studies led to the culture of isolated root tips. • The approach of using explants with meristematic cells produce the successful and indefinite culture of tomato root tips. • Only in 1985 was the sweetening agent from the flowers and leaves of the verbena plant, Lippia dulcis, which exceeds saccharose l000-fold in its sweetening power and which was used already by the Aztecs to sweeten their food, identified as a colorless sesquiterpene oil, hernandulcin. • The psychogenically active compounds from the "holy" fungi of the Indians, psilocybin and psilocin from the leaves of the genus Psilocybe, are now being utilized in modern medicine due to their structural similarity to the neurotransmitter serotonin.
  • 14.
    14 CULTIVATION METHODS: • Onceprotoplasts are separated from the isolation medium and transferred to a suitable growth medium, adaptation and regeneration processes begin. Adaptation is necessary because the growth conditions differ from those in the tissue's previous environment. Stabilization: • The culture media for protoplasts are very similar to those for individual cells. However, in the former it is necessary to create conditions favoring the formation of cell walls and ensuring the stability of naked cells. Thus, the addition of polyethylene glycol (PEG) 1500 to the medium often accelerates the uniform deposition of micro fibrils. Wall Formation: • Polysaccharides such as hemicellulose synthesized for cell wall formation are initially largely excreted into the medium. Studies on the structure of newly formed 1. PROTOPLAST CULTURE:
  • 15.
    15 CULTIVATION METHODS: Medium Components: Particularattention is paid to the effect of ion concentrations on protoplast division. Depending on the origin, the auxin/cytokinin ratio must also be specifically adapted. Highly differentiated mother cells (e.g. leaf cells) usually require a low auxin/kinetin ratio, while protoplast cultures obtained from actively growing cell cultures or meristematic tissue require high auxin/cytokinin ratios. Sensitivity: Further, a particularly great sensitivity to certain components of the medium and environmental conditions (light, shearing forces) is characteristic of protoplast cultures. For example, freshly isolated protoplasts are usually highly light-sensitive, at least during the first 4-7 days. 1. PROTOPLAST CULTURE
  • 16.
    16 CULTIVATION METHODS: The preferreddonor materials for individual cells are young leaves, calli and protoplasts. Cultures of N. tabacum and Phaseolus vulgaris, filtered off by a mesh of 0.1 to 0.3 mm, consist of 90% single cell. The three basic techniques: • nurse culture • plating technique • culture in a microchamber 2. CULTURES OFSINGLE CELLS:
  • 17.
  • 18.
    18 CULTIVATION METHODS:  NurseCulture: The isolated cells to be cultured are fixed on a piece of filter paper on the surface of an actively growing nurse callus. The contact through the absorbent paper is sufficient to maintain a supply of nutrients and unknown growth factors. Once the developing microcalli have attained the size necessary for survival (200-400 µm), they are individually cultivated on a fresh medium. 2. CULTURES OFSINGLE CELLS:
  • 19.
    19 CULTIVATION METHODS:  Platingand Feeder-Layer Technique; Culture in a Microchamber: • Conditioned Medium: During a nurse culture, unknown growth factors accumulated in a suspension culture or on the supporting filter paper are transmitted by the medium from cell to cell. In this way, the medium is enriched with these substances. Such a medium is thus called conditioned if it contains such substances excreted by any living cells which ensure survival and reproduction of cells cultivated at a suboptimal density. The effectivity of such media depends in part on the age of the nurse culture. However, nutrients depleted from the medium during preculturing must be replaced, otherwise they may be inhibitory. 2. CULTURES OFSINGLE CELLS:
  • 20.
    20 CULTIVATION METHODS: • Platingand Feeder-Layer Techniques: Plating Technique. In this technique, cultures with a cell density less than the critical density are mixed with the medium containing agar (0.6%) at a temperature of 30-35 °C and poured into petri dishes to a depth of 1 mm. The low agar density makes it possible to follow the development of single cells through an inverse microscope. The culture may be divided into individual cubes, called agar beads. Plating success is determined by the ratio of the number of cells in the original suspension to the number of colonies developing within the following 21 days. The goal is to attain the highest possible plating efficiency (PE), the number of colonies per plated single cells, at the least possible cell density. The lower boundary has so far been around 5000/ml. 2. CULTURES OFSINGLE CELLS:
  • 21.
    21 CULTIVATION METHODS: • Platingand Feeder-Layer Techniques: Feeder-Layer Technique: In order to add a nutrient supply and stimulate division, living cells or protoplasts unable to divide are added to the agar medium as a feeder layer. 2. CULTURES OFSINGLE CELLS:
  • 22.
    22 CULTIVATION METHODS: • Culturein a Microchamber: In micro chambers, the problem of insufficient cell density is solved by reducing the amount of medium. Starting from the minimum concentration that ensures plating success, the amount was reduced to 0.6 µl, 0.25-0.5 µl and even to microdroplets of 10-25 nl Such mini-cultures of a single cell or protoplast may be cultivated by either hanging them from a slide held in place by the medium's surface tension (hanging drop culture), or on a slide or in mini-agar caverns (1 x 1 x 1.75 mm) open above. 2. CULTURES OFSINGLE CELLS:
  • 23.
  • 24.
    24 CULTIVATION METHODS: From thepoint of view of an application-oriented industry, experiments performed on the scale of petri dishes and Erlenmeyer flasks cannot be economically utilized. Even in laboratory fermenters with a capacity of 5-501, questions of scaling-up to industrial scale are usually unanswerable. Therefore, industrial production conditions are usually simulated in so-called pilot plants at a scale up to 1000 l. The results thus obtained can be applied to larger reactors, provided the geometric and dynamic parameters remain constant in the scaling factor. However, this is possible without further difficulties only if a single parameter is rate-determining. This factor must be kept constant during scaling-up.  Determining Factors: The aspects that must be considered in culturing large volumes may be classified as process and culture conditions. The extent of scaling-up is determined by factors of physical, chemical, biochemical and biological nature. 3. MASS CULTIVATION METHOD:
  • 25.
  • 26.
  • 27.
    27 GROWTH OF CELLAND TISSUE CULTURE: Measurement methods: Changes in growth may be measured by fresh and dry weight, cell mass, cell number, mitotic index or indirectly by the conductivity of the medium. Fresh Weight: The method of fresh-weight determination neglects the various water contents of the material. Therefore, the values of callus cultures, frequently determined as total weight of callus, medium layer and petri dish, experience large variations due to evaporation via the medium's surface. More exact values are obtained by determining the weight after complete separation from the culture medium. This is possible when the material is cultured on separating layers of cellulose or nylon. Dry Weight: Quantification by means of dry weight excludes error due to varying endogenous water contents. This requires repeated drying, usally at 60°C, to the point of constant weight. Up to fresh weights of 500 mg, a linear relationship between fresh and dry weight is assumed. GROWTH PROCESS:
  • 28.
    28 GROWTH OF CELLAND TISSUE CULTURE: Cell Mass: This may be determined by densification by centrifugation (ca. 2000 g, 5 min) of a particular percentage of the volume (ca. 4-7 ml) in graduated, conical centrifuge tubes. In order to avoid errors due to water absorption by the cells, the so-called packed cell volume (PCV) must be recorded immediately following the separation process. Cell Number: To determine the number of cells per unit volume, existing cell clumps or aggregates must be separated into isolated cells - not only in callus cultures but also in most suspension cultures. This is commonly done using chrome-trioxide alone or in combination with hypochlorous acid. Possible alternatives are EDT A and pectinase. GROWTH PROCESS:
  • 29.
    29 GROWTH OF CELLAND TISSUE CULTURE: Conductivity: The inverse relationship between the conductivity and fresh or dry weight of the medium allows the determination of growth without taking samples (which would affect the sterility of the culture). In fully synthetic media, conductivity is determined almost exclusively by salt concentrations. As long as the pH of the medium remains above 3 (CH + < 10-3 mol/I), the concentration of hydrogen ions does not affect conductivity. Cellulose Concentration: Calcofluor-white ST (0.1 % aqueous) allows monitoring of changes in the concentrations of cell wall polymers from fJ-glycosidic bound glucose molecules such as cellulose or callose. The textile brightener specifically binds to fJ-l,4-glucans and intensely fluoresces following stimulation with shortwave blue light. In this way, even traces of these compounds may be identified GROWTH PROCESS:
  • 30.
    PLANT CELLS ASPRODUCERS OF SECONDARY METABOLITES: 30
  • 31.
    SECONDARY METABOLITES: 31 Secondary metabolites,also called specialised metabolites, toxins, secondary products, or natural products, are organic compounds produced by bacteria, fungi, or plants which are not directly involved in the normal growth, development, or reproduction of the organism. They were labelled secondary compounds in contradistinction to primary compounds due to: 1. an apparently limited taxonomic distribution 2. Synthesis occurring only under certain conditions 3. An apparent lack of function 4. No apparent necessity for life.
  • 32.
    32 SOME PLANT SECONDARYMETABOLITES AND THEIR APPLICATION: Metaobolites Application Species Ajmalicine Circulation Catharanthus roseus Atropine Anti-cholinergic Atropa belladonna Hyoscyamine Hyoscine Theophylline Anti-cholinergic Anti-cholinergic Hyoscyamus spp. Datura spp. Camellia sinensis Diosgenin Contraceptive Dioscorea spp. Quinine Anti-Malarial Cinchona spp Eugenol Local anesthetic Syzygium aromaticum Morphine Analgesics Papaver somniferum
  • 33.
  • 34.
    34 STORAGE OF SECONDARYMETABOLITES: Accumulation: Turnover: Depending on the object, secondary compounds are either secreted into the surrounding medium or stored intracellularly. There they experience turnover processes with characteristic half-lives. Their degradation was first proven in cell suspension cultures. Degradation and synthesis often occur simultaneously. The extent of their accumulation is mainly determined by three cell capacities: synthetic capacity, storage capacity and the capacity to metabolize the compounds in transport and detoxification processes. Individual plant organs differ in their significance in this process.
  • 35.
    35 PHARMACEUTICALLY ACTIVE NATURALPRODUCT SYNTHESIS VIA PLANT CELL CULTURE TECHNOLOGY: Most secondary metabolites are often present in extremely low amounts in the plant, often less than 1% of the total carbon. This paucity can make natural harvestation impractical for bulk production, especially in the case of slow growing species. The significant engineering challenge is then to find a means by which to produce the desired natural products in a way that is both sustainable and financially feasible. Production of these products in a microbial or fungal host by transferring the biosynthetic pathway is possible.
  • 36.
    PRODUCTION OPTIONS FORNATURAL PRODUCTS: 36 Chemical synthesis of natural products is possible and commercially feasible, particularly for those with relatively simple chemical structures such as aspirin (derived from the natural product salicylic acid) and ephedrine. In many cases, however, the metabolite has a complex structure, which can include multiple rings and chiral centers, so that a synthetic production process becomes prohibitively costly. Many natural products used in cancer treatment, including compounds such as paclitaxel, vinblastine, and camptothecin, fall into this latter class, so an alternative method of supply is necessary.
  • 37.
    37 PRODUCTION OPTION FORNATURAL PRODUCTS : • Depending on the nature of the plant, extraction directly from harvested plant tissue may be an option. Especially if a plant can be cultivated en masse, this can be attractive on a commercial basis. • The anticancer drugs vincristine and vinblastine, among other medicinally valuable metabolites such as ajmalicine and serpentine, are found in the Madagascar periwinkle Catharanthus roseus. • Even though these important alkaloids, particularly vincristine and vinblastine, naturally occur at very low levels in C. roseus less than 3 g per metric tons the fast growing nature of the periwinkle makes field cultivation most practical at the present time. • However, the relative inefficiency and high cost of whole plant extraction implies that an improved method of supply would be useful for these valuable anticancer agents.
  • 38.
    38 PRODUCTION OPTION FORNATURAL PRODUCTS : • When natural supply is limited due to a combination of low yields and slow growth rates, in vitro cultures provide an attractive alternative. Most plant species can be cultured in vitro in either an undifferentiated or differentiated state. • As many secondary metabolites are produced by specialized cells, organ cultures such as shoots or roots can exhibit similar metabolite profile patterns compared to the native plant, whereas undifferentiated cultures often accumulate secondary metabolites to a lesser extent, and sometimes not at all.
  • 39.
    39 PRODUCTION OPTION FORNATURAL PRODUCTS : • The anticancer compound camptothecin, produced by the ornamental tree Camptotheca acuminata as well as Nothapodytes fetida and Ophiorrhiza pumila among other species, has been shown to accumulate in undifferentiated cultures in very low or even undetectable amounts compared to root cultures in which production levels were comparable to the intact plant. • Similarly, no artemisinin, a potent antimalarial drug, was found in cell suspension cultures of Artemisia annua, while trace amounts were detected in shoot cultures.
  • 40.
    40 PRODUCTION OPTION FORNATURAL PRODUCTS : • Root cultures can be transformed into hairy roots using the soil dwelling bacteria Agrobacterium rhizogenes, resulting in cultures which are genetically stable, capable of unlimited growth without additional hormones, and have an increased capacity for secondary metabolite accumulation. • Undifferentiated suspension cultures, which can be more easily scaled to levels suitable for commercial production. There are currently 14 plant cell culture processes which have been commercialized for production of secondary metabolites (including products used in applications other than pharmaceuticals such as food and cosmetics).
  • 41.
    41 PRODUCTION OPTION FORNATURAL PRODUCTS :
  • 42.
    PLANT SUSPENSION CELLCULTURE TECHNOLOGY: 42 • Production of metabolites via plant cell suspension culture is renewable, environmentally friendly, and from a processing standpoint, amenable to strict control, an advantage in regards to meeting Food and Drug Administration manufacturing standards. Technology developed for other cell culture and fermentation systems (e.g., mammalian and yeast) can be readily adapted for large scale applications with plant cells, easing difficulties associated with scale-up. • A notable example of the success of plant cell culture systems, due in large part to innovative research and the application of novel technologies, is paclitaxel synthesis and supply. Paclitaxel, produced by Taxus spp., is an important anticancer agent used as a first line treatment for several types of cancer, including breast, ovarian, and non small cell lung cancer, and has also shown efficacy against AIDS-related Kaposi sacoma. Production of paclitaxel via cell culture technology has been studied since the 1980s as an alternative supply source to harvest of the slow growing yew tree, since a single dose of 300 mg requires the sacrifice of a 100 year old tree
  • 43.
    43 PLANT SUSPENSION CELLCULTURE TECHNOLOGY :
  • 44.
    44 PLANT SUSPENSION CELLCULTURE TECHNOLOGY : • The primary challenges impeding regular commercial application of plant cell culture technology are low and variable yields of metabolite accumulation. • Some metabolites do not accumulate in appreciable quantities in undifferentiated cells. In these cases, manipulation of genes within the biosynthetic pathway is needed to utilize plant cell cultures for bulk production.
  • 45.
    45 HAIRY ROOT CULTURESASASOURCE OF SECONDARY METABOLITES • The hairy root system based on inoculation with Agrobacterium rhizogenes has become popular in the two last decades as a method of producing secondary metabolites synthesized in plant roots. • The hairy root phenotype is characterized by fast hormone-independent growth, lack of geotropism, lateral branching, and genetic stability. The secondary metabolites produced by hairy roots arising from the infection of plant material by A. rhizogenes are the same as those usually synthesized in intact parent roots, with similar or higher yields. • This feature, together with genetic stability and generally rapid growth in simple media lacking phytohormones, makes them especially suitable for biochemical studies not easily undertaken with root cultures of an intact plant.
  • 46.
    46 HAIRY ROOT CULTURESASASOURCE OF SECONDARY METABOLITES • During the infection process, A. rhizogenes transfers a part of the DNA (transferred DNA, T-DNA) located in the root-inducing plasmid Ri to plant cells, and the genes contained in this segment are expressed in the same way as the endogenous genes of the plant cells.
  • 47.
    47 METABOLIC ENGINEERING ANDDIRECTED BIOSYNTHESIS • The engineering of biosynthetic pathways within a plant cell to enhance accumulation of a constitutively produced metabolite is an appealing strategy in which exciting progress has been made in the past decade. • A variety of tools have been employed to both identify unknown genes and characterize secondary metabolite pathway regulation, including precursor feeding, gene over expression, application of metabolic inhibitors, and mutant selection. • Additionally, elicitation, in relation to improving bulk yields in cell culture, can also be used as a powerful tool to investigate pathway regulation based on gene expression.
  • 48.
    48 METABOLIC ENGINEERING TOOLS: •A metabolic engineering approach involves the manipulation of targets within a cell. Techniques are therefore needed both for the identification of these targets (i.e., genes, proteins, metabolites) as well as for their exploitation. • As many secondary pathways are still partially undefined, elucidating pathway genes and their control elements is an active research area. • The subsequent identification of rate influencing steps within a biosynthetic pathway can then be useful in providing targets for a rational engineering strategy.
  • 49.
    49 METABOLIC ENGINEERING TOOLS: •Plant cell cultures, including both suspension cultures and hairy root cultures, have proven to be an extremely useful platform for metabolic studies, as a fast growing and renewable source of material. • Whole plants can also be valuable, particularly as models to study complex spatial and temporal control mechanisms associated with environmental stimuli and morphogenesis from a global metabolic perspective.
  • 50.
    50 METABOLIC ENGINEERING TOOLS: •Several approaches have been used to identify the enzymes and their corresponding genes which catalyze biosynthetic pathway steps. • For the paclitaxel pathway, a successful approach utilized by the Croteau laboratory incorporated feeding cell free Taxus extracts with precursors to isolate and identify intermediate metabolites and enzymes. • This approach led to the identification of taxadiene synthase, which catalyzes the first committed step of the taxane pathway. • Genes were subsequently identified from a cDNA library using PCR amplification based on degenerate primers designed to recognize conserved regions from homologous enzymes in other plants whose DNA sequences were known.
  • 51.
    51 METABOLIC ENGINEERING TOOLS: •Differential display methods (via reverse transcription and PCR) comparing mRNA transcripts between elicited and unelicited cells supplemented with a homology-based search of a cDNA library from elicited cells, as well as random sequencing of the same induced library, have also proven to be extremely effective in gene discovery (for a comprehensive review of molecular genetics in Taxus.
  • 52.
    52 TRADITIONAL STRATEGIES TOIMPROVE CELL CULTURE YIELDS: Optimization of cultural conditions: • Number of chemical and physical factors like media components, phytohormones, pH, temperature, aeration, agitation, light affecting production of secondary metabolites affect culture productivity. • Several products were found to be accumulating in cultured cells at a higher level than those in native plants through optimization of cultural conditions. Manipulation of physical aspects and nutritional elements in a culture is perhaps the most fundamental approach for optimization of culture productivity. • For example, ginsenosides by Panax ginseng and Dixon, rosmarinic acid by Coleus bluemei, shikonin by Lithospermum, ubiquinone-10 by Nicotiana tabacum, berberin by Coptis japonica were accumulated in much higher levels in cultured cells than in the intact plants.
  • 53.
    53 TRADITIONAL STRATEGIES TOIMPROVE CELL CULTURE YIELDS: Precursor feeding: Exogenous supply of a biosynthetic precursor to culture medium may also increase the yield of the desired product. This approach is useful when the precursors are inexpensive. The concept is based on the idea that any compound, which is an intermediate, in or at the beginning of a secondary metabolite biosynthetic route, stands a good chance of increasing the yield of the final product. Attempts to induce or increase the production of plant secondary metabolites, by supplying precursor or intermediate compounds, have been effective in many cases.
  • 54.
    54 TRADITIONAL STRATEGIES TOIMPROVE CELL CULTURE YIELDS: Precursor feeding: For example, • amino acids have been added to cell suspension culture media for production of tropane alkaloids, indole alkaloids etc. • Addition of phenylalanine to Salvia officinalis cell suspension cultures stimulated the production of rosmarinic acid. • Addition of the same precursor resulted stimulation of taxol production in Taxus cultures. • Feeding ferulic acid to cultures of Vanilla planifolia resulted in increase in vanillin accumulation. • Furthermore, addition of leucine, led to enhancement of volatile monoterpenes in cultures of Perilla frutiscens, where as addition of geraniol to rose cell cultures led to accumulation of nerol and citronellol
  • 55.
    55 TRADITIONAL STRATEGIES TOIMPROVE CELL CULTURE YIELDS: Elicitation: Plants produce secondary metabolites in nature as a defense mechanism against attack by pathogens. Elicitors are signals triggering the formation of secondary metabolites. Perhaps the most notable strategy for improving metabolite yields is elicitation. An elicitor can be defined as any compound that induces an upregulation of genes. Some elicitors target secondary metabolic genes, which are often associated with defense responses to perceived environmental changes.
  • 56.
    56 TRADITIONAL STRATEGIES TOIMPROVE CELL CULTURE YIELDS: Elicitation: • Elicitors include natural hormones, nutrients, and many fungi-derived compounds. In particular, jasmonic acid and its methyl ester methyl jasmonate (MJ), are naturally occurring hormones involved in the regulation of defense genes as part of a signal transduction system. • Applied exogenously, they have been shown to induce secondary metabolic activity and promote accumulation of desired metabolites in numerous plant systems, including Taxus spp. and C. roseus. • Different elicitors may act on different segments of the biosynthetic pathway. For instance, MJ elicitation compared to salicylic acid elicitation in Taxus spp. cultures resulted in different relative increases of metabolic intermediates, suggesting that each elicitor preferentially directs flux toward, and possibly away from, different intermediate taxanes.
  • 57.
    57 TRADITIONAL STRATEGIES TOIMPROVE CELL CULTURE YIELDS: Elicitation: While many of the specific targets of elicitors have yet to be conclusively identified, elicitation can be an extremely useful tool in conjunction with gene expression profiling for identifying rate-influencing steps in secondary biosynthetic pathways.
  • 58.
    58 TRADITIONAL STRATEGIES TOIMPROVE CELL CULTURE YIELDS: Immobilization of plant cell cultures: • It has long been considered for increasing metabolite accumulation, as the potential of higher cell densities, continuous removal of products/inhibitors, and protection for shear-sensitive plant cells provide a number of advantages. • Immobilization can be simply achieved using a gel matrix such as alginate; however this becomes costly at a larger scale, especially when the product of interest is not secreted and must be released using sonication or treatments with an organic solvent.
  • 59.
    59 TRADITIONAL STRATEGIES TOIMPROVE CELL CULTURE YIELDS: Immobilization of plant cell cultures: • Recently, immobilization of T. baccata cells in calcium-alginate beads was shown to produce one of the highest reported levels of paclitaxel accumulation among academic laboratories (43 mg/L). • Immobilization also has the potential to simplify product extraction and purification, as immobilized cultures of Linum usitatissimum excrete the pharmaceutically active metabolite dehydrodiconiferyl alcohol 4--D-glucoside (DCG) to a greater extent than suspension cultures.
  • 60.
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