6 Plant Tissue Culture ‘Tissue culture is an experimental technique through which a mass of cells (callus) is produced from explants tissue. The callus produced through this process can be utilized directly to regenerate plantlets or to extract or manipulate some primary and secondary metabolites. ‘Tissue culture is the process whereby small pieces of living tissue (explants) are isolated from an organism and grown aseptically for indefinite periods on a nutrient medium. For good success in plant tissue culture, it is good to start with explants that are meristem because these cells are capable of rapid proliferaflon. The used explants include buds, root tips, nodal segments or germinating seeds and these are placed on suitable culture media where they grow into an undifferentiated mass known as Callus. Advantage of plant tissue culture over the conventional cultivation techniques: 1. Synthesis of those chemical compounds which are impossible or too difficult to synthesize chemically. Further, the compounds from tissue cultures may be easily purified because of simpler extracts and absence of significant amount of pigments. . Biogenesis of secondary metabolites can be studies. . The cultured cells could be maintained free from any microbial contamination. 4. Availability of uniform plant material at all times and manageable under regulated and reproducible condition. 5. Production of natural compounds under controlled environmental conditions, independent of soil condition and change in climate. 6. Cellular totipotency, the potency of plant cells can be revealed. 7. Immobilization of cells can be carried out which could be used for various biotransformation and biochemical reactions. HISTORICAL DEVELOPMENT OF PLANT TISSUE CULTURE: S. | Name of scientist | Year Work No. 7 | Gottlieb Haberlandt | 1902 | Develop the concept of in vitro cell culture. He was the first to culture isolated, fully differentiated cells in a nutrient medium. 2 | Kotte and Robbins | 1922 | Small excised root tips of pea and maize cultivated by Kotte. Robbin: Maintained his maize root tip | eonTextbook of Pharmacognosy and Phytochemistry cultures for a longer period by sub culturing them. White 1934, 1937 Growing root tip cultures. Initially used yeast extract in a medium containing inorganic salts and sucrose but later replaced yeast extract by three vitamins (pyridoxine, thiamine and nicotinic acid) Bonner 1937 Importance of thiamine in yeast extract. Laibach 1925, 1929 They raised zygotic embryos isolated from non- viable seeds of Linumperenne X L. austriacum to maturity on a culture medium and obtained hybrid which in natural course died out due to embryo abortion. Van Overbeek 1941 Used coconut milk (embryo sac fluid) for embryo development and callus formation in Datura, which proved a turning point in the field of embryo culture. Ball 1945 Tdentify the exact part of the shoot meristem that gives rise to a whole plant Gautheret 1934 Cultured cambium cells of some tree species (Acer pseudoplatanus and Salix caprea) on the surface of medium solidified with agar. Caplin and Steward 1948 Carrot explants that coconut milk enhanced more proliferation of callus than did auxin. 10 Skoog Skoog and Tsui 1944, 1951 ‘Adenine stimulates cell division and induces bud formation in tobacco tissue even in the presence of Indoleacetic acid. 11 ‘Skoog and Miller 1957 ‘The concept of hormonal control of organ formation from callus tissue. 12 Sanford 1948 Tnitiated studies on single cell cultures by demonstrating divisions in animal cells using conditional media. 13 Steward 1952 Designed a shaking apparatus which allowed dissociation of tissues to form cell suspension culture 14 Jones 1960 Designed a micro culture method using hanging drops of free cells in a conditioned medium. 15 Hildebrandt 1965 Substituted fresh medium enrichedPlant Tissue Culture 95 with coconut milk and 1- naphthalene acetic acid for conditioning and observed divisions in isolated tobacco hybrid cells. 16 Bergmann 1960 Developed cloned from a large number of cells. 17 Kohienbach 1966 Isolated mesophyll cells of McCleayercordata differentiated somatic embryos’ 18 Ball 1946 Successfully raised transplantable whole plants of Lupinus and Tropaeolum by culturing their shoot meristem (Tip). 19 Morel 1963 Rapid propagation of Orchids cymbidium and Odontoglossum. 20 Morel and Martin 1952, 1955 Recovered virus free Dahlia and potato plants from culture obtained by cultivating the shoot meristem of infected plants. 21 Guha and Maheswari 1966 Culture immature anthers of Daturainnoxia and were able to raise embryoids and plantlets. 22, Bourgin and Nitsch 1967 The titopotency of pollen grains by raising full haploid plants of tobacco, rice and wheat 23 San Noeum 1976 In- vitro culture of ovary isolated from Hordeumvulgare. 24 Bajaj 1976 Regenerated plant from cryopreserved plant tissues ‘ 25 Murashige 1977 Proposed artificial seed production 26 Melcher 1978 Production of somatic embryos by using protoplast fusion techniques 27 Lazer 1983 Production of cybryds by protoplast fusion technique. REQUIREMENTS FOR TISSUE CULTURE LABORATORY: Washing and storage facilities. 1, 2. 3. 4, oe . Media preparation and storage room. Transfer area for aseptic manipulations. Culture room or inoculators for maintenance of cultures under controlled conditions of temperature, light and humidity. Observation or data collection area. Washing and storage facilities: (a) Cleaning glassware : The glassware is soaked in a detergent solution for 16 hrs, then rinsed first in tap water followed by a second rinse in distilled water. If washing is done96 Textbook of Pharmacognosy and Phytochemistry-1 mechanically, in a dishwasher, the glass ware mat be treated with detergent at 70°C for 5-10 min. further cleansing involves a 5 minute rinse in hot water, 3 minutes rinse in de-ionized water, and a final hand rinse in distilled water. () Plastic lab ware: Presterilised disposable polystyrene culture container (faleon, corning) are available and used in place of glassware in order to dispense with washing. = Plastic ware may be washed with a mild non- abrasive detergent and rinse with tap water and distilled water. 2. Media preparation room: The media preparation room should be separate and away from the working laboratory. This area is to be utilized for preparation of culture media. The room should be equipped with Glassware Gulture vessels pH meter Hot plates Balance Water- bath Bunsen burner Microwave oven Autoclave Refrigerator 3. Transfer area : (a) Plastic box : This can be sterilized with an Ultraviolet (UV) light and by swabbing the floor surface with 95% ethyl alcohol when in operation. (b) Wooden hood. (©) Laminar airflow cabinet: A small motor blows air into the unit first through a coarse filter, where large dust particles are separated, and subsequently passes through a 0.3 ym HEPA filter. The air is directed either downward (vertical flow unit) or outward (Horizontal flow unit) over the working surface. The air coming out of the fine filter is ultra clean (free from fungal or bacterial contaminant) and its velocity (27 + 3 m/min) adequately prevents the micro contamination of the working area by a worked sitting in front of the cabinet. 4, Culture room : All types of plant tissues are incubated under conditions of well- controlled temperature, humidity, illumination and air circulation. Culture room should have both light and temperature programmable for 24 hrs period.Plant Tissue Culture 97 Air-conditioners and heater are used to maintain the temperature around 25 + 2°C, Lighting is adjusted in terms of quantity and photoperiod duration by using automatic clocks. Cultures are grown in diffused light although provision should also be made for maintaining cultures in higher light intensity as well as total darkness. The humidity range of 20-98%, controllable to +3% and uniform forced-air ventilation. Specially designed shelves made of glass, rigid wire mesh or wood are provided in the culture room for storing cultures. Each shelves illuminated by a separate set of fluorescent tubes. The culture vessels (flask, jars and Petri dishes) can be placed directly on the shelf or trays of suitable size whereas culture tubes require metallic racks to hold them. Characteristics of incubators and growth chambers: 1. Temperature range- 2-40°C. Temperature control, + 0.5°C. Safety high and low- temperature limits. Continuous temperature recorder. 24 hrs temperature and light programming. Adjustable fluorescent lighting up to 10,000 lux. Relative humidity range 20-98%. Relative humidity control, +3%. . Uniform forced- air distribution. 10. Capacity upto 0.7 m* of 0.5 m? shelf space. 6. Data collection area : The growth and development vf tissues cultured in vitro are generally monitored by observing cultures at regular intervals in the culture room or incubators where they have been maintained under controlled environmental conditions. Data based observations under aseptic conditions may be collected using a laminar airflow cabinet or suitable transfer area designed for the purpose, whereas for the microscopic examination of culture the usual laboratory space is used. NUTRITIONAL REQUIREMENT OF CULTURE MEDIA: Following components are required: 1. Inorganic nutrients: (a) Macronutrients: Nitrogen Phosphorous Potassium Calcium Magnesium Sulpher. Sersaaey98 Textbook of Pharmacognosy and Phytochemistry (b) Micronutrients: + Iron © Magnese e Zinc * Boron + Copper * Molybdenum . Carbon and energy source: e Sucrose © Glucose © Fructose * Others: Lactose, galactose, maltose, cellobiose. Organic supplements : (a) Vitamins : © Thiamine, nicotinic acid, pyridoxine, calcium pentothenate and myoinositol. ¢ Biotin, riboflavin, ascorbic acid, tocopherol, pantothenic acid, biotin and folic acid. (b) Amino acid * L-Glutamine, L-asparagines. e L-Glycine, L-argenine and L-cysteine. (©) Other organic supplements © Protein, coconut milk e Yeast and malt extract © Orange juice and tomato juice. (a) Activated charcoal. (e) Antibiotics: © Streptomycin e Kanamycin . Growth regulators: (a) Auxins: * Indole-3-acetic acid. 1-naphthaleneacetic acid. Indole-3-butyric acid. 2, 4-dichlorophenoxyacetic acid. Naphthoxyacetic acid. 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T). 2- methyl-4-chlorophexyacetic acid. ‘ytokinins: 6-Benzyl aminopurine or 6- benzyl adenine. 6-Dimethylaminopurine. Kinetin. Zeatin. N, N-diphenylurea. () wee ee geesePlant Tissue Culture 99 © Thidiaziron. (©) Gibberellins and Abscisic acid: GAs 5. Water ; Dematerialized water and double distilled water. 6. Solidifying agents : © Agar © Gelatin | 6. pH: Plant cells and tissues require optimum pH for growth and development in cultures. The most of culture media pH 5.0 to 6.0 before sterilization is considered optimal. The pH is adjusted by a pH meter using 0.1 M NaOH or HCl. Compositions of MS media S.No. Ingredients ‘Amount (mg/litre) ‘Stock solution 1__[ MgS0..7H:0 7400 2 | KHsPO« 3400 3__| KNOs 36000 4__| NHNOs 33000 5 CaClz.2H20. 8800 Stock solution—II 6 | HsBOs 1240 7 | MnS0s.4120 4460 8 _| ZnS0..7H20 1720 9 | NasMoO« 50 10_| CuS0:.5H20 5 11_| CoC.6H20 5 Stock solution—I1T 12 | FeSQ..7H20. 5560 13__| NAz.EDTA.2H:0 7460 ‘Stock solution-IV 14 | Inositol 20000 15_| Thiamine HCl 100 16 _| Pyridoxine HCl 100 17_| Nicotinic acid 100 18 __| Glycine 400 Preparation of the media : To prepare one litre of medium take 50 ml of stock solution-I, 5 ml each of stock solution-II-IV. Dissolve FeSO:.7H20 and NAv.EDTA.2H20 separately in 450 ml distilled water by heating and constant stirring. Mix the two solutions, adjust the pH to 5.5 and add distilled water to make up the final volume to one litre. Sterilization of media : Culture media in glass containers sealed with cotton plugs, aluminium foils or plastic closures are autoclaved at 15 psi at 121°C for 15-40 minutes from the time the medium reaches the100 ‘Textbook of Pharmacognosy and Phytochemistry—1 required temperature. Thermolabile components like carbohydrates, vitamins, growth regulators and plant extract can be conveniently sterilized by micro filtration and then added aseptically to the sterile medium. ISOLATION OF ORGANS, TISSUES AND CELLS: (a) Explants preparation: Explants is a detached portion of the plant body which is used in tissue culture to produces callus tissues, The age of explants plays a vital role in the production of callus. The desired portion is excised from the parent plant and utilized for callus induction. Seeds and grains are also considered to be good sources for preparation of explants. Seeds and grains after surface sterilization are germinated aseptically in nutrient media by placing them on double layers of pre-sterilized filter paper in Petri dishes moistened sufficiently with sterile distilled or on moist cotton plugs in Petri dishes or culture tubes. After few days, they germinate into seedlings, which are removed and surface sterilized. These can then be used as a source of explants. (b) Sterilizing plant materials (Explants): S. | Surface Sterilizing agents | Concentration Period of No. treatment Gn minutes) 1_| Sodium hypochlorite 1-3% 5-40 2 | Bromine water 12% 2-10 3_| Calcium hypochlorite 9-10% 5-30 4_| Mercuric chloride 0.1-1.0% 3-10 5_| Siler nitrate 1% 5-28 [6 _| Hydrogen peroxide 10-12 % 5-15 7 _| Benzylkonium chloride 0.01-0.1% 5:20 8_| Bthyl alcohol 70-95% 28 The aerial portion of plant such as bud, leaf and stem sections are sterilized bud submerging for 2-3 min in 70 % ethanol, followed by 2-4 rinses with sterilized distilled water. TYPE OF CULTURE: Callus culture. Protoplast culture. Shoot- tip culture. Meristem-tip culture. Flower organ culture. Fruit organ culture. Microspore and anther culture. CALLUS CULTURE Callus tissue means an unorganized proliferative mass of cells . produced from isolated plant cells, tissues or organs when grown AOwP prPlant Tissue Culture 101 aseptically on artificial nutrient medium in glass vials under controlled experimental conditions. Protocol of callus culture: 1, A fresh tap root (Carrot) is taken and washed thoroughly under running tap water to remove all surface detritus. 2. The tap root is then dipped into 5% Teepol for 10 minutes and then the root is washed. 3. The tap root is surface sterilized by immersing in 70% v/v ethanol for 60 seconds, followed by 20-25 minutes in sodium hypochlorite. 4, The root is washed four times with sterile distilled water to remove completely the hypochlorite. . The carrot is ‘then transferred to a sterilized petridish containing a filter paper. A series of transverse slice 1 mm in thickness is cut from the tap root using a sharp scalpel. 6. Each piece is transferred to another sterile petridish. Each piece contains a whitish circular ring of cambium around the pith. An area of 4 mm2 across the cambium is cut from each piece so that each small piece contains part of the phloem, cambium and xylem. Size and thickness of the explants should be uniform. Always the lid of petridish is replaced after each manipulation. 7. The cotton plug from a culture tube is removed and flamed the uppermost 20 mm of the open end. While holding the tube at an angle of 45°, an explant is transferred using forceps onto the surface of the agarified nutrient medium. 8. The closure is immediately placed on the open mouth of each tube. The foreeps are always flamed before and after use. 9. Culture tubes after inoculation are taken to the cylture room where they are placed in the racks. Cultures are inoculated in dark at 25°C. 10. After 4 weeks in culture the explants inoculated on medium with 2, 4-D will form a substantial callus. The whole callus mass is taken out aseptically on a sterile petridish and should be divided into two or three pieces. 11. Each piece of callus tissue is transferred to a tube containing fresh same medium. 12. Prolonged culture of carrot tissue produces large calluses. Significance of callus culture: 1. Callus tissue is good source of genetic or karyotypic variability, so it may be possible to regenerate a plant from callus culture. 2. Callus culture acts as a good source material for the protoplast isolation. 3. Several biochemical assays can be performed from callus culture. 4. Cell suspension culture in moving liquid medium can be initiated from callus culture. a102 Textbook of Pharmacognosy and Phytochemistry! 5. Callus culture is very useful to obtain commercially important secondary metabolites. 6. The whole plant is regenerated in large number from callus tissue through manipulation of the nutrient and hormonal constituents in the culture medium. This phenomenon is known as plant regeneration or organogenesis or morphogenesis. ANTHER AND POLLEN CULTURE Pollen grains form pollen tube and male gametes, when placed on suitable nutrient media like MS, most pollen grain follow normal development, but a few grains will form a callus instead. In a similar manner, anthers can be cultured to form embryos directly from pollen grains. These embryos can be induced to develop true haploid plants. With optimal nutrient conditions for donor plants and explants are provided, it is possible to obtain several hundred haploid plants from a single anther. For successful anther culture if is necessary to excise flower buds at first mitotic division in the uninucleatedmicrospores tetrads. Other treatments like growing on different nutrient media, cold storage, for few days to several weeks, etc. can increase the frequency of successful embryos. FLOWER ORGAN CULTURE The flowers are selected two days after pollination and subjected to surface sterilization with 5% hypochlorite solution. They are then repeatedly washed with double- distilled water. Subsequently, the surface sterilized explant is inoculated on White's medium. When cultured, such flowers produce fruits. Larger fruits are obtained on medium supplemented with hormones. MERISTEM CULTURE Cultivation of shoot apical meristem in vitro is known as meristem culture. Meristem culture involves the growth of an already existing shoot meristem and subsequent regeneration of adventitious roots from these shoots. CELL SUSPENSION CULTURE Suspension culture is a type of culture in which single cells or small aggregates of cells multiply while suspended in agitated liquid medium. It is also referred to as cell suspension culture. Protocol of cell suspension culture: 1. Take 250 ml conical flask containing autoclaved 60 ml liquid medium. 2. Transfer 3-5 pieces of pre-established callus tissue from the culture tube using the spoon headed spatula to conical flask.Plant Tissue Culture 103 3. Flame the neck of conical flask; close the mouth of the flask with a piece of aluminium foil or a cotton plug. Cover the closure with a piece of brown paper. 4, Place the flasks within the clamps of a rotary shaker moving at the 80-120 rpm (revolution per minute). 5. After 7 days, pour the contents of each flask through the sterilized sieve (pore diameter 60-90 p) and collect the filtrate in a big sterilized container. The filtrate contains only free cells and aggregates. 6. Allow the filtrate to settle for 10-15 minutes or centrifuge the filtrate at 500-1000 rpm and finally pour off the supernatant. 7, Resuspended the residue cells in a requisite volume of fresh liquid medium and dispense the cell suspension equally in several sterilized flasks. Place the flasks on shaker and allow the free cells and cell aggregates to grow. 8. at the next subculture repeat the previous steps but take only one fifth of the residual cells as the inoculum and dispense equally in flask and again place them on shaker. 9, After 3-4 subcultures, transfer 10 ml of cell suspension from each flask into new flask containing 30 ml fresh liquid medium. 10. To prepare a growth curve of the cells in suspension, transfer a definite number of the cells measured accurately by a haemocytometer to a definite olume and liquid medium and incubates on shaker. Pipette out very little aliquot of cell suspension at short intervals of time and count the cell number. Plot the cell count data of a passage on a graph paper and the curve will indicate the growth pattern of suspension culture. Type of suspension culture: 1. Batch culture: The cell material grows in a finite volume of agitated liquid medium. For instance, cell material in 20 ml or 40 ml or 60 ml liquid medium in each passage constitute a batch culture. This culture is maintained in conical flasks incubated on orbital platform shakers at the speed of 80-120 rpm. They are sub divided: (a) Slowly rotating culture: Single cells and cell aggregates are grown in a specially designed flask, the nipple flask. Each nipple flask possesses eight nipples like projections. The capacity of each flask is 250 ml. ten flasks are loaded in a circular manner on the large flat disc of a vertical shaker. When the flat disc rotates at the speed of 1-2 rpm, the cell within each nipple of the flask are alternately batched in culture medium and exposed to air. (b) Shake cultures: Single cells and cell aggregates in fixed volume of liquid medium are placed in conical flask. Conical NTextbook of Pharmacognosy and Phytochemistry flasks are mounted with help of clips on a horizontal large square plate moves by a circular motion at the speed of 60-180 rpm. (© Stirred culture: The large culture vessel is not rotated but the cell suspension inside the vessel is kept dispersed continuously by bubbling sterile air through culture medium. ‘The use of an internal magnetic stirrer is the most convenient way to agitate the culture medium safely. The magnetic stirrer revolves at 200-600 rpm. The culture vessel is a 5 to 10 litres rounded-bottom flask. (d) Spinning culture: Large volumes of cell suspension may be cultured in 10 liters bottles which are rotated in a culture spinner at 120 rpm at an angle of 45°. 2. Continuous culture system: The old liquid medium is continuously replaced by the fresh liquid medium to stabilize the physiological states of the growing cells. Normally, the liquid medium is not changed until the depletion of some nutrients in the medium and the cells are kept in the same medium for certain period. As a result, active growth phase of the cell declines the depletion of nutrient. In continuous culture system, nutrient depletion does not occur due to continuous flow of nutrient medium and the cells always remain in the steady state of active growth phase. Application of Suspension cultures: 1. Suspension cultures are idea for the production of secondary metabolites productions which are of therapeutic value. 2. It is also serves as a major source for obtaining individual isolated cells used for protoplast production. 3. It is also gives isolated cells which can be used for hydrids or hybrids production. 4. Suspension cultures are also used for the induction of mutation and genetic manipulation of plant cells. 5. Suspension cultures provide cells that are used for the immobilization of plant cells. PROTOPLAST CULTURE Protoplast is a cell without a cell wall. They contain all the normal cell organelles plus the nucleus, The cell wall of a plant cell can be decomposed and removed by the treatment of the lytic enzymes like cellulose and pectinase. Method of isolation of protoplast: 1. Mechanical method: Cutting plasmolysed tissue with sharp edged knife and releasing the protoplasts by deplasmolysis.Plant Tissue Culture 105 2. Enzymatic method: The protoplast is prepared by treating the cells with an enzyme like cellulose and pectinase which selectively digest the cell wall. Leaf | ‘Surface sterilisation Removal of epidermal tissue Cutting into small fragments ‘Small Ty of leaf tissue Immersion in hypertonic solution Plasmolyzed cells Enzyme mixture treament Incubation Removal of cell wall debris Centrifugation rotoplasts ‘Washing with sucrose and mannitol solution Pure protoplast for storage and future use Isolation of protoplast from leaf tissues by enzymatic method Protoplast fus' Following techniques are used: 1. Spontaneous fusion 2, Induced fusion 3. Electric fusion 1. Spontaneous fusion: Enzymatic degradation of cell walls is performed during isolation of protoplasts. During this process some of the protoplasts lying in close proximity may undergo fusion to produce homokaryons or homokaryocytes which contain 2- 40 nuclie. 2. Induced Fusion: It is of great utility when fusion of protoplasts from two different species or from two different sources of same species has to be achieved. Fusogen is employed for inducing fusion. Following fusogens are: (a) Sodium nitrate treatment: Isolated protoplasts are suspended in an aggregation mixture comprising of 5.5% sodium nitrate in 10% sucrose solution and incubated at 35°C to induce fusion. (b) Polyethylene glycol (PEG) treatment: When the quantity of protoplast is sufficient, protoplast suspended in 1 ml of culture106 Textbook of Pharmacognosy and Phytochemistry-1 medium is added to 1 ml of PEG solution (56%). The tube is shaken for 5 seconds and protoplasts allowed to sediment for 10 minutes. Protoplast are then washed with growth medium and observed for successful agglutination and fusion. 3. Electric fusion: Protoplast are placed in culture cell containing electrodes and a potential difference applied. This induces the protoplast to line up between the electrodes. GROWTH PROFILE OF PLANT TISSUE CULTURE: They are classifying as follows: 1, Single Cell culture 2. Callus culture 1. Single cell culture: The single cell culture exhibits various stages of growth: Stationary phase ‘Senescent phase Progressive decleration phase Linear Phase Exponential phase Lag phage —— Days of suspension culture Growth curve of single cell culture «Lag phage: The cells are exposed to a fresh medium; the cell starts or regains the ability to divide and the tissue shows slow growth. ¢ Exponential phase: This phase is characterized by rapid cell multiplication. The time of this phase varies according to the cell and its nutrient regime. Usually, it is of short duration and lasts for 3-4 generations. * Linear phase: The growth follows a linear pattern with respect to time. © Progressive declaration phase: The rate of cell division decreases with the aging of the culture. * Stationary phase: In this stage no growth of cells occurs. The rate of production of cells is equal to the rate of their death. * Senescent phase: The cells die due to unavailability of nutrients.Plant Tissue Culture 107 2. Callus culture: The following stages are: Staignary phase ———> Days of callus culture (Time) Growth curve of callus culture * Lag phase: After inoculation of the explants into the nutrient media, there is a lag time in which the cells undergo no growth; this is when the cell are trying to adjust to the new nutritional environmental conditions. * Exponential phase: The cells undergo rapid multiplication and consume nutrients from the medium leading to their depletion. « Decline phase: Depletion of the nutrient elements in the media lead to the starvation of some cells which in turn leads to the decline in the growth of callus tissue. e Stationary phase: From this stage onwards, no growth is evident. Sub-culturing of the cells is required for further growth and development. GROWTH DETERMINATION: 3 Following methods are used as: 1, Cell number: The cells in suspension culture grow by cell division and the number of cells increases. The rate of increase of cell number can be calculated simply by counting the cell number in haemocytometer under a microscope. Cell count data obtained from haemocytometer is multiplied by a factor X10° and result can be expressed in terms of cell number per unit volume of culture. 2. Packed cell volume: Definite volumes of cell suspension can be harvested from multiple replicated sets of culture. Such amount of cell suspension is transferred in a graduated conical centrifuge tube and centrifuged at 2000X g for 5 minutes. The cells will form a pellet after centrifugation. The volume of cell pellet then represents the packed cell volume. It is also called biomass volume. The packed cell volume is expressed as ml pellet ml? culture.108 ‘Textbook of Pharmacognosy and Phytochemistry 3. Cell fresh weight: the cells increase in number during growth, the liquid medium will be more turbid and as a result the optical density of the suspension culture will also be altered. The changes of optical density value can be detected by a colorimeter. 4, Nutrient Uptakes study: The growth measurement is carried out by analyzing a certain specific nutrient element in the media like glucose, sucrose, inorganic phosphates, etc, which indicates if a particular component is limiting at any stage in the culture cycle. From the date of carbohydrate analysis, the carbon conversion efficiency is calculated and the biomass subsequently measured. 5, Test for viability of cell: Cell death may occur in suspension cultures due to several factors. The cell count data will be erroneous without testing the viability. The staining method such as fluorescein diacetate is used for assessing cell viability. The fluorescein diacetate dissolved in 5 mg/ml of acetone is added to cell population at 0.01% final concentration. Dead cells fluoresce red. Evans blue also used at a final concentration of 0.01% is specific for dead cells. as soon as the stain is mixed with cell suspension, the in-viable cells _ stain blue and the viable cells remain unstained. APPLICATIONS OF PLANT TISSUE CULTURE: 1. Production of genetically variable plants. 2. Virus eradication. 3. Production of Phytoconstituents by plant tissue culture. 4. Clonal propagation 5. Study of crown gall (Tumor) by plant tissue culture. 6. Micropropagation. 7. Plant pathology and plant tissue culture. 8. Plant breeding, plant improvement and plant tissue culture. 9. Preservation of plant genetic resources or gene conservation. 10. Importance of tissue culture in Biotechnology. 11. Application of hair root culture for the production of secondary metabolites. 1. Production of genetically variable plants : In some callus culture, there is a major tendency of the callus tissue towards the numerical variation of chromosomes in the cells that occurs after a number of serial subculture may arise because of two factors. * The cells of various ploidy and genetic constitution of the initial explant may be induced to divide. ¢ Culture condition may contribute new irregularities.Plant Tissue Culture 109 Variants plants may show some useful characters such as resistance to a particular disease, herbicide resistance, stress tolerance etc. such changes are valuable for crops which are normally propagated by vegetative methods. . Virus eradication: In virus infected plants, the distribution of virus in the plants body is uneven. Apical meristem are generally either free or carry a very low concentration of viruses. The apical meristem culture is the only way to obtain a clone of virus free plant which can be multiplied vegetatively under control conditions, . Production of Phytoconstituents : The following phytoconstituents are produced by plant tissue culture as: Alkaloids: Morphine, codeine, berberine, reserpine, caffeine, Vinblastin, Vincristin, atropine, quinine, Quinidine, ete. * Glycosides: Cardiac glycoside, saponins, _ flavonoids, furocoumarin, anthraquinones (Sennosides, aloin), * Ginsenosides etc. ¢ Sugar and polysaccharides. ¢ Plant growth hormones. ¢ Steroidal hormones. « Terpenoids ° Latex. ° Oils ¢ Vitamins ¢ Tannins « Proteins and peptide PHYTOCONSTITUENTS PRODUCED BY PLANT TISSUE CULTURE Secondary Plant source Type of culture metabolites Ajmalicine Catharanthusroseus Suspension culture | Artimisinine Artemisia scoparia Suspension culture | Atropine Atropa belladonna Hairy root culture Berberine Coptis japonica Suspension culture and Cell culture Beta- carotene Rutagraveolens Callus culture Caffeine Coffee arabica Cell culture Capsaicin Capsicum annum Gell suspension culture Cardenolides Digitalis purpurea Suspension culture and Cell culture Conessine Holarrhenaantidysenterica | Callus culture Catechin, Camellia sinensis Callus culture and Suspension culture110 Textbook of Pharmacognosy and Phytochemistry Codeine Papaversomniferum Cell culture Digoxin Digitalis lanata Suspension culture Diosgenin Dioscoreacompositae Cell culture Epicatechin Camellia sinensis Callus culture Hecogenin Yucca aloifolia Callus culture Hyoscyamine Hyocyamusniger Suspension culture Lobeline Lobelia inflate Root culture Luteolin Daturapinnata Callus culture Morphine Papaversomniferum Suspension culture Nicotine Nicotianatobacum Suspension culture Papain Carica papaya Cell culture Plumbagin Plumbagozeylanica Callus culture Quercetin A. wightié Callus culture Quinine and Quinidine | Cinchona ledgeriana Root culture Reserpine Rauwolfia serpentina Suspension culture Rhein Cassia angustifolia Callus culture Solasonine Solanumxanthocarpum __| Callus culture Taxol Taxus cuspidate Cell culture ‘Tropane alkaloids Daturainnoxia Suspension culture Theobromine Coffeaarabica Suspension culture Vasicine Adhotodazeylanica Callus culture ‘Vinblastin Catharanthusroseus Cell culture Xanthotoxin Rutagraveolens Suspension culture ‘Zeaxanthin Rutagraveolens Callus culture 4. Clonal propagation: In- Vitro Clonal propagation is a type of Micropropagation. The cultured plants raised from tissue culture are derived asexually and also multiply within culture vessel by asexual means. Multiplication of genetically identical copies of a cultivar by sexual reproduction is called Clonal propagation and a plant population derived from single donor plant in tissue culture constitutes a clone. So, the variability that can arise from sexual reproduction and seed formation in a crop plant is omitted. 5. Study of crown gall (Tumor) by plant tissue culture: Plant tumor formation was induced by a bacterium, Agrobacterium tumefaciens. Sunflower Agrobacterium could induce tumor not only at the inoculated point but at a considerable distance, where secondary tumours free of bacteria are formed. Cells of these secondary tumors could be cultivated by tissue culture technique and multiplied on a medium without adding auxin and cytokinin, where as normal tissue required auxin and in some case, cytokinin. Crown gall tissues deprid of bacteria give rise to tumors by grafting. Micropropagation: The regeneration of whole plant through tissue culture is popularly called Micropropagation. This is a 6.Plant Tissue Culture i 9. 10, 1. technique where a callus mass has been initiated from a single explant taken from any living part of a donor plant and within very short time and space, a large number of plantlets can be produced from such callus tissue. Plant pathology and plant tissue culture: The virus eradication by apical meristem culture and second success of tissue culture in plant pathology is the result of its application to the problems of plant tumors, especially crown gall. Plant breeding, plant improvement and plant tissue culture: Embryo culture is useful for propagation’ of orchid, shortening the breeding cycle and overcoming seed dormancy. In meristem culture, shoot apical meristem along some surrounding tissue in vitro. It is used for Clonal propagation and recovery of virus free plants and is potentially useful in germplasm exchange and long- term storage of germplasm through freeze preservation. Anther and pollen culture has a potential application in plant breeding and plant improvement programme for the production of. haploid as well as homozygous diploid plant. Preservation of plant genetic resources or gene conservation : Modern tissue culture techniques to provide a germplasm storage procedure which uniquely combines the possibilities of disease elimination and rapid Clonal multiplication. The cell division and normal cellular reactions are totally arrested at the very low temperature of liquid nitrogen ( 196°C), which means that there should be a high level of genetic stability and that the chemical reaction responsible for cellular damage will not occur. The plant material can be stored in liquid nitrogen for desirable period. This technique could be particularly valuable for storage of any germplasm which needs to be maintained in a Clonal form. This technique is known as cryopreservation or freeze preservation of tissue or cell. Importance of tissue culture in Biotechnology: Study of biosynthesis and metabolism of steroids and cardiac glycosides. To increase the synthesis of natural compounds or new compounds by higher plant cells culture as a result of mixing or feeding transformable precursors in the culture medium. Application of hair root culture for the production of secondary metabolites : Hairy root cultures are potentially applicable to the production of all root derived secondary metabolites from dicotyledonous plants. Many of the roots synthesized compounds including Tropane alkaloids atropine and hyoscyamine, steroidal precursors such as solasodine and Catharanthus alkaloids are sufficient high value to justify the exploitation of alkaloids and hyoscyamine content of the hairyae 112 Textbook of Pharmacognosy and Phytochemistry-1 roots of Beta vulgaris, Nicotianarustica and Daturastromonium respectively. EDIBLE VACCINE Edible Vaccine involves introduction of selected desired genes into plant and then inducing these altered plants to manufacture the altered protein. HOW IT ACTS: Antigen in transgenic plant > Ingestion > Delivered by bioencapsulation > Taken up by Mcell > Pass on to the Macrophage > IgG, IgE responses > Local IgA response & Memorycells > Neutralize the attack by the real infectious agent. ADVANTAGES OF EDIBLE VACCINE: Easy to store Cost effective Heat stable Easy to administer. Acceptable to poor developing country. Fail safe . Activate both mucosal and systemic immunity. Do not required cold chain maintenance. No fear of contamination. Production of potato edible vaccine: Generating edible vaccines relies on the bacterium Agro bacterium tumefaciensto deliver into plant cells the genetic blueprints for viral or bacterial “antigens” proteins that elicit a targeted immune response in the recipient. Steps for production of edible vaccine of potatoes: 1. Leaf cut 2. Expose leaf to bacteria carrying an antigen gene and an antibiotic- resistance gene. Allow bacteria to deliver the genes into leaf cells. 3. Expose leaf to an antibiotic to kill cells that lack the new genes. Wait for surviving (gene-altered) cells to multiply and form a clump (callus). 4, Allow callus to sprout shoots and roots. 5. Put in soil. Within three months, the plantlets will grow into plants bearing antigen-laden vaccine potatoes. Advantage e Grow quickly. * Cultivate broadly. © High content Vitamin-A may boost immune response. Disadvantages © Spoils readily. WODNAMe WrPlant Tissue Culture Advantages of banana edible vaccine: « Do not need cooking. «Protein not destroyed even after cooking. « Inexpensive. * Grown widely in developing countries. Disadvantages of banana edible vaccine: « Trees take 2-3 to mature years. e Spoils rapidly after ripening 113