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Pharmacognosy practice for pharmacy students. Edited by: Dr. Noémi Tóth. Dr. Attila Hunyadi. Authors: Dr. Noémi Tóth. Dr. Attila Hunyadi. Dr. Erika Liktor-Busa. Department offering the course: Pharmacognosy Dept. Academic year/ Level: First level . A-Course Notes (lecture notes and practical notes) prepared by staff . General Introduction: The Scope of Pharmacognosy, Definitions and Basic Principles. Pharmacognosy: Is the study of medicinal products in their crude, or .
Therefore, plant tissue culture is being potentially used as an alternative for plant secondary metabolite production.
Majority of the plant secondary metabolites of interest to humankind fit into categories which categorize secondary metabolites based on their biosynthetic origin. Secondary metabolism in plants is activated only in particular stages of growth and development or during periods of stress, limitation of nutrients or attack by micro-organisms. Plants produce several bioactive compounds that are of importance in the healthcare, food, flavor and cosmetics industries.
Many pharmaceuticals are produced from the plant secondary metabolites. Currently, many natural products are produced solely from massive quantities of whole plant parts. The source plants are cultured in tropical, subtropical, geographically remote areas, which are subject to drought, disease and changing land use patterns and other environmental factors.
Secondary metabolites can be derived from primary metabolites through modifications, like methylation, hydroxylation and glycosylation. Secondary metabolites are naturally more complex than primary metabolites and are classified on the basis of chemical structure e. P containing nitrogen or not , their solubility in various solvents or the pathway by which they are synthesized Table They have been classified into terpenes composed entirely of carbon and hydrogen , phenolics composed of simple sugars, benzene rings, hydrogen and oxygen and nitrogen and or sulphur containing compounds Figure It has been observed that each plant family, genus and species produces a characteristic mix of these bioactive compounds.
All plants produce secondary metabolites, which are specific to an individual species, genus and are produced during specific environmental conditions which makes their extraction and purification difficult. As a result, commercially available secondary metabolites, for example, pharmaceuticals, flavours, fragrances and pesticides etc.
Table Classification of secondary metabolites Figure Proper selection of cell lines The heterogeneity within the cell population can be screened by selecting cell lines capable of accumulating higher level of metabolites. Manipulation of medium The constituents of culture medium, like nutrients, phytohormones and also the culture conditions, like temperature, light etc.
For e. Addition of Elicitors Elicitors are the compounds which induce the production and accumulation of secondary metabolites in plant cells.
Elicitors produced within the plant cells include cell wall derived polysaccharides, like pectin, pectic acid, cellulose etc. Product accumulation also occurs under stress conditions caused by physical or chemical agents like UV, low or high temperature, antibiotics, salts of heavy metals, high salt concentrations which are grouped under abiotic elicitors.
Addition of these elicitors to the medium in low concentration enhances the production of secondary metabolites. Addition of precursors Precursors are the compounds, whether exogenous or endogenous, that can be converted by living system into useful compounds or secondary metabolites. It has been possible to enhance the biosynthesis of specific secondary metabolites by feeding precursors to cell cultures.
For example, amino acids have been added to suspension culture media for production of tropane alkaloids, indole alkaloids.
The amount of precursors is usually lower in callus and cell cultures than in differentiated tissues. Phenylalanine acts as a precursor of rosmarinic acid; addition of phenylalanine to Salvia officinalis suspension cultures stimulated the production of rosmarinic acid and decreased the production time as well. Phenylalanine also acts as precursor of the N-benzoylphenylisoserine side chain of taxol; supplementation of Taxus cuspidata cultures with phenylalanine resulted in increased yields of taxol.
The timing of precursor addition is critical for an optimum effect. The effects of feedback inhibition must surely be considered when adding products of a metabolic pathway to cultured cells.
Permeabilisation Secondary metabolites produced in cells are often blocked in the vacuole. By manipulating the permeability of cell membrane, they can be secreted out to the media. Permeabilisation can be achieved by electric pulse, UV, pressure, sonication, heat, etc. Even charcoal can be added to medium to absorb secondary metabolites. Immobilisation Cell cultures encapsulated in agarose and calcium alginate gels or entrapped in membranes are called immobilised plant cell cultures.
Immobilization of plant cells allows better cell to cell contact and the cells are also protected from high shear stresses. These immobilized systems can effectively increase the productivity of secondary metabolites in a number of species. Elicitors can also be added to these systems to stimulate secondary metabolism.
Advantages of cell, tissue and organ cultures as sources of secondary metabolites 5. Plant cell cultures Once interesting bioactive compounds have been were identified from plant extracts, the first part of the work consisted in collecting the largest genetic pool of plant individuals that produce the corresponding bioactive substances.
However, a major characteristic of secondary compounds is that their synthesis is highly inducible, therefore, it is not certain, if a given extract is a good indicator of the plant potential for producing the compounds.
The ability of plant cell cultures to produce secondary metabolites came quite late in the history of in vitro techniques. For a long time, it was believed that undifferentiated cells, such as callus or cell suspension cultures were not able to produce secondary compounds, unlike differentiated cells or specialized organs.
Callus culture Callus is a mass of undifferentiated cells derived from plant tissues for use in biological research and biotechnology. In plant biology, callus cells are those cells that cover a plant wound. To induce callus development, plant tissues are surface sterilized and then plated onto in vitro tissue culture medium.
Different plant growth regulators, such as auxins, cytokinins, and gibberellins, are supplemented into the medium to initiate callus formation. It is well known that callus can undergo somaclonal variations, usually during several subculture cycles. This is a critical period where, due to in vitro variations, production of secondary metabolite often varies from one subculture cycle to another.
When genetic stability is reached, it is necessary to screen the different cell callus lines according to their aptitudes to provide an efficient secondary metabolite production. Hence, each callus must be assessed separately for its growth rate as well as intracellular and extracellular metabolite concentrations.
This allows an evaluation of the productivity of each cell line so that only the best ones will be taken for further studies, for example, for production of the desired compound in suspensions cultures. Cell suspension cultures Cell suspension cultures represent a good biological material for studying biosynthetic pathways. They allow the recovery of a large amount of cells from which enzymes can be easily separated.
Compared to cell growth kinetics, which is usually an exponential curve, most secondary metabolites are often produced during the stationary phase. This lack of production of compounds during the early stages can be explained by carbon allocation mainly distributed for primary metabolism when growth is very active. On the other hand, when growth stops, carbon is no longer required in large quantities for primary metabolism and secondary compounds are more actively synthesized.
However, some of the secondary plant products are known to be growth-associated with undifferentiated cells, such as betalains and carotenoids. P Plant organs are alternative to cell cultures for the production of plant secondary metabolites. Two types of organs are generally considered for this objective: A schematic representation of various organized cultures, induced under in vitro conditions, is given in Figure Shoot cultures Shoots exhibit some comparable properties to hairy roots, genetic stability and good capacities for secondary metabolite production.
They also provide the possibility of gaining a link between growth and the production of secondary compounds. Hairy root cultures Hairy roots are obtained after the successful transformation of a plant with Agrobacterium rhizogenes.
They have received considerable attention of plant biotechnologists, for the production of secondary compounds. They can be subcultured and indefinitely propagated on a synthetic medium without phytohormones and usually display interesting growth capacities owing to the profusion of lateral roots.
This growth can be assimilated to an exponential model, when the number of generations of lateral roots becomes large. During development cells undergo diverse structural and functional specialisation depending upon their position in the body.
Leaf cells bear chloroplasts and act as the site of photosynthesis. The colourless root hairs perform the function of absorbing nutrients and water from the soil and some other cells become part of the colourful petals. Normally fully differentiated cells do not revert back to a meristematic: In earlier sections of this unit you have read that the regenerative capacity is retained by all living cells of a plant.
Several horticultural plants regenerate whole plant from root, leafiand stem cuttings. Highly differentiated and mature cells such as those of pith and cortex and highly specialised cells as those of microspores and endosperm,retain full potential to give rise to full plants under suitable culture conditions.
Haberlandt was the first to test this idea experimentally. This endowment called "cellular totipotency" is unique to plants. Animal cells possibly because of their higher degree of specialisation do not exhibit totipotency.
Whole plant regeneration from cultured cells may occur in one of the two pathways: The Embryos are bipolar structures with no organic connection with the parent tissue and can germinate directly into a complete plant. On the other hand, shoots are monopolar. They need to be removed from the parent tissue and rooted to establish a plantlet. Often the same tissue can be induced to form shoots or embryos by manipulating the components of the culture conditions.
In the following sub sections we will discuss organogenesis and embryogenesis in detail. Organogenesis Organogenesis refers to the differentiation of organs such as roots, shoots or flowers. Shoot bud differentiation may occur directly from the explant or from the callus. The stimulus for organogenesis may come from the medium, from the endogenous compounds produced by the cultured tissue or substances carried over from the original explant.
Organogenesis is chemically controlled by growth regulators. Skoog while working with tobacco pith callus, observed that the addition of an auxin Indole Acetic Acid IAA enhanced formation of roots and suppressed shoot differentiation.
He further observed that adenine sulphate, Cytokinin reversed the inhibition of auxin and promoted the formation of shoots. You should know that: P 1 Organogenesis is contolled by a balance between cytokinin and auxin concentration i. A relatively high auxin: Cytokinin ration induces root formation, whereas a high cytokinin: Differential response to exogenously applied growth regulators may be due to differences in the endogenous levels of the hormones within the tissue.
Organogenesis is a complex process. Whereas in the cultured tissues of many species organogeiiesis can be demonstrated in this pattern, some plants, notably the monocots, are exceptions. Somatic Embryogenesis The process of embryo development is called embryogenesis. It is not the monopoly of the egg to form an embryo. Any cell of the female gametophyte Embryo sac or even of the sporophytic tissues around the embryo sac may give rise to an embryo. Thus we can say that 'The phenomenon of embryogenesis is not necessarily confined to the reproductive cycle".
In this subsection we will discuss -,- some examples of "embeos formed in culture", also referred to as "somatic - embryos". The first observation of somatic embryos were made m Dacus Carota.
Other plants in which the phenomenon has been studied in some detail are Ranunculus scleratus, citrus and coflea spp. In Rarrunculus scleratus somatic as well as various floral tissues, including anthers proliferated to form callus which, after limited unorganised growth differentiated several embryos. These embryos germinated in situ and a fresh crop of embryos appeared on the surface of the seedling. Guidelines for the production of secondary metabolites from plant organ cultures.
P The size of tissue culture lab and the amount and type of equipment used depend upon the nature of the work to be undertaken and the funds available. A standard tissue culture laboratory should provide facilities for: The overall design must focus on maintaining aseptic conditions. At least three separate rooms should be available one for washing up, storage and media preparation the media preparation room ; a second room, containing laminar-air-flow or clean air cabinets for dissection of plant tissues and subculturing dissection room or sterilization room ; and the third room to incubate cultures culture room.
This culture room should contain a culture observation table provided with binoculars or stereozoom microscope and an adequate light source. Additionally, a green house facility is required for hardening-off in vitro plantlets.
For a commercial set-up, a more elaborate set-up is required. Media preparation room The washing area in the media room should be provided with brushes of various sizes and shapes, a large sink, preferably lead-lined to resist acids and alkalis, and running hot and cold water. It should also have large plastic buckets to soak the labware to be washed in detergent, hot-air oven to dry washed labware and a dust-proof cupboard to store them. If the preparation of the medium and washing of the labware are done in the same room, a temporary partition can be constructed between the two areas to guard any interference in the two activities.
A continuous supply of water is essential for media preparation and washing of labware. P Figure 2. A floor plan for plant tissue culture laboratory 1. For higher or lower temperature treatments, special incubators with built-in fluorescent light can be used outside the culture room. Cultures are generally grown in diffuse light from cool, white, fluorescent tubes.
Lights can be controlled with automatic time clocks. Generally, a hour day and 8-hour nights are used. The culture room requires specially designed shelving to store cultures. Some laboratories have shelves along the walls, others have them fitted onto angle-iron frames placed in a convenient position. Shelves can be made of rigid wire mesh, wood or any building material that can be kept clean and dust-free.
Insulation between the shelf lights and the shelf above will ensure an even temperature around the cultures. While flasks, jars and petridishes can be placed directly on the shelf or trays of suitable sizes, culture tubes require some sort of support.
Metallic wire racks or polypropylene racks, each with a holding capacity of tubes, are suitable for the purpose. Dissection room or sterilization room This area should have restricted entry, which is needed to ensure the sterile conditions required for the transfer operations.
For sterile transfer operations, the laminar-air-flow cabinets are used. Temperature control is essential in this room as the heat is produced continuously from the flames of burners in the hoods. The room should be constructed in a way to minimize the dust particles and for easy cleaning. Several precautions can be taken including the removal of shoes before entering the area. The laminar horizontal flow sterile transfer cabinets are available in various sizes from many commercial sources.
They should be designed with horizontal air flow from the back to the front, and equipped with gas cocks if gas burners are to be used.
Electrical outlets are needed for use of electric sterilizers and microscopes, and if weighing is to be done in the hoods. A stainless steel working platform is most durable, easy to keep clean and to prevent the unwanted damage due to accidental fire.
P Ultraviolet light to maintain sterility inside the cabinet. UV light is a source of ozone, which can be mutagenic, therefore, utmost care is to be taken while using this. Although UV light is not necessary, a short exposure time of min to cabinet is fine sometimes. Work can be started after min of switching on the air flow, and one can work uninterrupted for long hours. The HEPA filters remove particles larger than 0. The flow of the air does not in any way hamper the use of a spirit lamp or a Bunsen burner.
Greenhouse The greenhouse facility is required to grow parent pants and to acclimatize in vitro raised plantlets. The size and facility inside the green house vary with the requirement and depends on the funds available with the laboratory.
However, minimum facilities for maintaining humidity by fogging, misting or a fan and pad system, reduced light, cooling system for summers and heating system for winters must be provided.
It would be desirable to have a potting room adjacent to this facility. Equipments and apparatus 1. The standard media pH is maintained at 5. High pressure heat is needed to sterilize media, water, labware, forceps, needles etc. A caution should be taken while opening the door of autoclave and it should be open when the pressure drops to zero. Opening the door immediately can lead to a rapid change in the temperature, resulting in breakage of glassware and steam burning of operator.
High quality microbalance are required to weigh smallest of the quantities. Additionally a top pan balance is required for less sensitive quantities. It is also used for the dry heat sterilization of clean glassware like, Petridishes, culture tubes, pipettes etc. Inverted microscope gives the clear views of cultures settled at the bottom of Petridishes. Here the sterilization of instruments is effecting by pushing them into the beads for s.
This is much safer compare to the Bunsen burner heating of instruments like, forceps, needles, scalpels etc. Preparation and handling The simplest method of preparing media is to use commercially available, dry, powdered media containing mineral elements and growth regulators. After adding sugar and other desired supplements like, plant growth regulators, make up the final volume with distilled water, adjust the pH, add agar and then autoclave the medium.
An alternative method of media preparation is to prepare a series of concentrated stock solutions which can be combined later as required. For preparing stock solutions and media, use glass-distilled or demineralized water and chemicals of high purity, analytical reagent AR grade. As its name suggests, in plant tissue culture media these components provide the elements which are required in large amounts concentrations greater than 0.
Macronutrients are usually considered to be carbon, nitrogen, phosphorous, magnesium, potassium, calcium and sulphur. It provides the elements that are required in trace amounts concentrations less than 0. These include, manganese, copper, cobalt, boron, iron, molybdenum, zinc and iodine. P iii. Iron source: It is considered the most important constituent and required for the formation of several chlorophyll precursors and is a component of ferredoxins proteins containing iron which are important oxidation: Organic supplements vitamins: Like animals, in plants too vitamins provide nutrition for healthy growth and development.
Although plants synthesize many vitamins under natural conditions and, therefore, under in vitro conditions they are supplied from outside to maintain biosynthetic capacity of plant cells in vitro. There are no firm rules as to what vitamins are essential for plant tissues and cell cultures.
The only two vitamins that are considered to be essential are myo-inositol and thiamine. Myo-inositol is considered to be vitamin B and has many diverse roles in cellular metabolism and physiology. It is also involved in the biosynthesis of vitamin C. Carbon source: This is supplied in the form of carbohydrate.
Plant cells and tissues in the culture medium are heterotrophic and are dependent on external source of carbon.
Sucrose is the preferred carbon source as it is economical, readily available, relatively stable to autoclaving and readily assimilated by plant cells.
During sterilization by autoclaving of medium, sucrose gets hydrolyzed to glucose and fructose. Plant cells in culture first utilize glucose and then fructose. Besides sucrose, other carbohydrates such as, lactose, maltose, galactose are also used in culture media but with a very limited success.
Table 3. The media elements and their functions The steps involved in preparing a medium are summarized below: Add appropriate quantities of various stock solutions, including growth regulators and other special supplements. Make up the final volume of the medium with distilled water.
Add and dissolve sucrose. After mixing well, adjust the pH of the medium in the range of 5. Add agar, stir and heat to dissolve. Alternatively, heat in the autoclave at low pressure, or in a microwave oven. If using pre-sterilized, non-autoclavable plastic culture vessels, the medium may be autoclaved in flasks or media bottles. P Allow the medium to cool to room temperature.
To make a semi-solid medium, a gelling agent is added to the liquid medium before autoclaving. Gelling agents are usually polymers that set on cooling after autoclaving.
Agar is obtained from red algae- Gelidium amansii. It is a mixture of polysaccharides. It is used as a gelling agent due to the reasons: It is obtained by purifying agar to remove the agaropectins.
This is required where high gel strength is needed, such as in single cell or protoplast cultures. It is produced by bacterium Pseudomonas elodea. It can be readily prepared in cold solution at room temperature. It sets as a clear gel which assists easy observation of cultures and their possible contamination. Unlike agar, the gel strength of gelrite is unaffected over a wide range of pH. However, few plants show hyperhydricity on gelrite due to freely available water. Plant growth regulators In addition to nutrients, four broad classes of growth regulators, such as, auxins, cytokinins, gibberellins and abscisic acid are important in tissue culture.
In contrast with animal hormones, the synthesis of a plant growth regulator is often not localized in a specific tissue but may occur in many different tissues. They may be transported and act in distant tissues and often have their action at the site of synthesis.
Another property of plant growth regulators is their lack of specificity- each of them influences a wide range of processes. The growth, differentiation, organogenesis and embryogenesis of tissues become feasible only on the addition of one or more of these classes of growth regulators to a medium.
In tissue culture, two classes of plant growth regulators, cytokinins and auxins, are of major importance. Others, in particular, gibberellins, ethylene and abscisic acid have been used occasionally. Auxins are found to influence cell elongation, cell division, induction of primary vascular tissue, adventitious root formation, callus formation and fruit growth.
The cytokinins promote cell division and axillary shoot proliferation while auxins inhibit the outgrowth of axillary buds. The auxin favours DNA duplication and cytokinins enable the separation of chromosome. Besides, cytokinin in tissue culture media, promote adventitious shoot formation in callus cultures or directly from the explants and, occasionally, inhibition of excessive root formation and are, therefore, left out from rooting media.
The ratio of plant growth regulators required for root or shoot induction varies considerably with the tissue and is directly related to the amount of growth regulators present at endogenous levels within the explants. In general, shoots are formed at high cytokinin and low auxin concentrations in the medium, roots at low cytokinin and high auxin concentrations and callus at intermediate concentrations of both plant growth regulators.
Commonly used plant growth regulators are listed in Table 4. Establishing aseptic cultures Plant tissue culture media contain sugar and so support the growth of many microorganisms bacteria and fungi. When these microorganisms reach a medium, they generally grow much faster than the cultured plant materials. Their growth and toxic metabolites will affect, and may even kill, the tissue cultures. It is, therefore, essential to maintain a completely aseptic environment inside the culture vessels.
There are several possible sources of contamination of the medium: Autoclaving media will eliminate contamination from the culture vessel or the medium. In some cases, substances such as gibberellic acid, abscisic acid ABA , urea and certain vitamins are thermolabile and break down upon autoclaving. These chemicals can be sterilized by membrane filtration using microfilters of pore size 0.
To prevent the environment of the culture room from being the source of contamination, keep the culture room as dust- free as possible and remove contaminated cultures from the area as soon as they are detected.
Ideally, the culture room should be clean, filtered air which has passed through high efficiency particulate air HEPA filters. The transfer area in most laboratories is within a laminar air-flow cabinet. A laminar air-flow cabinet has a small fan which blows air through a coarse filter to remove large dust particles and then through a fine HEPA filter to remove microbes, their spores and other particles larger than 0.
Prolonged contact with alcohol can cause skin irritation, and other health problems can result from the inhalation of fumes. Use ethanol rather than methanol, and surgical gloves when handling. P with ultraviolet light as it can permanently damage eyes and promote skin cancer. Laminar flow cabinets equipped with ultraviolet light for surface sterilization should be fitted with safety doors which can be closed when ultraviolet light is used. Plant surfaces carry a wide range of microorganisms.
The tissue must be thoroughly surface-sterilized before being placed on the nutrient medium. Discard cultures with fungal or bacterial contamination. Solutions of sodium or calcium hypochlorite are usually effective in disinfecting plant tissues. Placing tissues in a 0. Surface sterilants are toxic to plant tissues.
Choose the concentration of the sterilizing agent and the length of time to minimize tissue damage, which shows up as white, bleached areas. Take care with powdered calcium hypochlorite as it is a powerful reducing agent. If calcium hypochlorite is stored moist and the container opened later, it can explode.
Store calcium hypochlorite in a sealed container in a dry place. A summary of the six steps commonly involved in establishing and maintaining aseptic plant tissue culture follows. Collect pieces of plant material ex-plants in a screw-cap bottle.
Immerse them in a dilute solution of the disinfectant containing a wetting agent. Replace the lid and store the bottle in the laminar air flow cabinet.
Shake the bottle two or three times during the sterilization period. Remove the lid and drain carefully. Thoroughly rinse the plant material in sterilized distilled water and replace the lid.
After shaking a few minutes, discard the water. Rinse two or three times more. Transfer the material to a pre-sterilized Petri-dishes or test-tubes.
Allow to cool. Sterilize the instruments after each time they are used to handle tissue. Prepare suitable explants from the surface sterilized material using sterilized instruments scalpels, needles, forceps, etc.
Quickly remove the lid of the culture vessel, transfer the explants on to the medium, flame the neck of the vessel only if glass and replace the lid. If handling aseptic plant materials during routine subculture, omit the first two steps. Plant tissue culture techniques 1.
Introduction Plant tissue culture has become popular among horticulturists, plant breeders and industrialists because of its varied practical applications. It is also being applied to study basic aspects of plant growth and development.
The discovery of the first cytokinin kinetin is based on plant tissue culture research. P The earliest application of plant tissue culture was to rescue hybrid embryos Laibach, , , and the technique became a routine aid with plant breeders to raise rare hybrids, which normally failed due to post-zygotic sexual incompatibility.
Currently, the most popular commercial application of plant tissue culture is in clonal propagation of disease-free plants.
In vitro clonal propagation, popularly called micropropagation, offers many advantages over the conventional methods of vegetative propagation: Thus, over a million plants can be produced in a year starting from a small piece of tissue. The enhanced rate of multiplication can considerably reduce the period between the selection of plus trees and raising enough planting material for field trials. In tissue culture, propagation occurs under pathogen and pest-free conditions.
The totipotency of plant cells was predicted in by Haberlandt and the first true plant tissue culture on agar was established. Since then plant tissue culture techniques have greatly evolved. The technique has developed around the concept that a cell has the capacity and ability to develop into a whole organism irrespective of their nature of differentiation and ploidy level.
Therefore, it forms the backbone of the modern approach to crop improvement by genetic engineering. The principles involved in plant tissue culture are very simple and primarily an attempt, whereby an explant can be to some extent freed from inter-organ, inter-tissue and inter-cellular interactions and subjected to direct experimental control. Regeneration of plants from cultured cells has many other applications. Several somaclones have been processed into new cultivars.
With haploids, homozygosity can be achieved in a single step, cutting down the breeding period to almost half. This is particularly important for highly heterozygous, long-generation tree species. Pollen raised plants also provide a unique opportunity to screen gametic variation at sporophytic level. This approach has enabled selection of several gametoclones, which could be developed into new cultivars.
Even the triploid cells of endosperm are totipotent, which provides a direct and easy approach to regenerate triploid plants difficult to raise in vivo. The entire plant tissue culture techniques can be largely divided into two categories based on to establish a particular objective in the plant species: Micropropagation Growing any part of the plant explants like, cells, tissues and organs, in an artificial medium under controlled conditions aseptic conditions for obtaining large scale plant propagation is called micropropagation.
The basic concept of micropropagation is the plasticity, totipotency, differentiation, dedifferentiation and redifferentiation, which provide the better understanding of the plant cell culture and regeneration. Plants, due to their long life span, have the ability to withhold the extremes of conditions unlike animals. The plasticity allows plants to alter their metabolism, growth and development to best suit their environment.
When plant cells and tissues are cultured in vitro , they generally exhibit a very high degree of plasticity, which allows one type of tissue or organ to be initiated from another type. Hence, whole plants can be subsequently regenerated and this regenerated whole plant has the capability to express the total genetic potential of the parent plant.
This is unique feature of plant cells and is not seen in animals. Unlike animals, where differentiation is generally irreversible, in plants even highly mature and differentiated cells retain the ability to regress to a meristematic state as long as they have an intact membrane system and a viable nucleus. However, sieve tube elements and xylem elements do not divide any more where the nuclei have started to disintegrate, According to Gautheret the degree of regression a cell can undergo would depend on the cytological and physiological state of the cell.
The meristematic tissues are differentiated into simple or complex tissues called differentiation. Reversion of mature tissues into meristematic state leading to the formation of callus is called dedifferentiation. The ability of callus to develop into shoots or roots or embryoid is called redifferentiation. The inherent potentiality of a plant cell to give rise to entire plant and its capacity is often retained even after the cell has undergone final differentiation in the plant system is described as cellular totipotency.
Micropropagation vs. The latter involves a highly specialized mode of development that normally occurs only inside the seed, under the cover of several layers of parental tissues.
Consequently, the observation of developing embryos and their isolation in intact and living conditions for experimental studies have been extremely difficult. In vitro production of embryos from somatic and gametic cells has opened up the possibility of obtaining large numbers of embryos of different stages, enabling investigations on cellular, genetic and physiological control of embryogenesis induction, pattern formation, organ differentiation and maturation.
However, this technique is applicable to only limited number of species. In contrast to this, micropropagation has several advantages which are summarized here: The rapid multiplication of species difficult to multiply by conventional vegetative means.
The technique permits the production of elite clones of selected plants. The technique is independent of seasonal and geographical constraints.
It enable large numbers of plants to be brought to the market place in lesser time which results in faster return on the investment that went into the breeding work. To generate disease-free particularly virus-free parental plant stock. To raise pure breeding lines by in vitro haploid and triploid plant development in lesser time.
It can be utilized to raise new varieties and preservation of germplasm vii.
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