PLANT TISSUE CULTURE: PLANT TISSUE CULTURE PURVI H. KAKRANI, INTRODUCTION: INTRODUCTION Plant Tissue Culture (PTC) comprises of set of in vitro techniques, methods and strategies, in which a whole plant is regenerated from isolated plant cells or tissues that have been genetically transformed. This regeneration is conducted in vitro so that the environment and growth medium can be manipulated to ensure a high frequency of regeneration. Purvi Kakrani,Plant Tissue Culture 2 Plant Tissue Culture: Plant Tissue Culture Purvi Kakrani,Plant Tissue Culture 3 Why Plant Tissue Culture over Conventional Cultivation ?: Why Plant Tissue Culture over Conventional Cultivation ? Purvi Kakrani,Plant Tissue Culture 4 ADVANTAGES: ADVANTAGES Production of secondary metabolites within weeks, even for the plants that have to be cultivated for years before harvesting. The controlled culture conditions of cell culture techniques guarantee the constant quality of the drug raw material. This by-passes the undesired by-products obtained in isolating natural compounds and also the climatic factors involved in the cultivation process of the plants. Plants from different climatic regions to be cultivated at the same place. Many endangered & rare species produce unique medicinal compounds. If plant cell cultures were used to produce these valuable chemicals, then there will be no danger of rooting out a rare plant. Purvi Kakrani,Plant Tissue Culture 5 ADVANTAGES: ADVANTAGES Many medicinally important secondary metabolites produced by plants are so difficult to synthesize that it unlikely that they will ever be produced via organic synthesis. Plant cell cultures may be used for production of such complex substances . Capable to produce novel metabolites . Used for biotransformation: Biotransformation includes hydrogenation, dehydrogenation, isomerisation, hydroxylation, opening of rings, glycosylation, etc. Helpful in t racing biosynthetic pathways , using labeled precursor in the culture medium Purvi Kakrani,Plant Tissue Culture 6 ADVANTAGES: ADVANTAGES Naturally occurring plants or their metabolites cannot be patented as such. Therefore this can be done through PTC technique. Purvi Kakrani,Plant Tissue Culture 7 BASIC TERMINOLOGIES: BASIC TERMINOLOGIES PLASTICITY : Ability of the plant to adapt to the environmental conditions . This plasticity allows plants to alter their metabolism, growth and development to best suit their environment. Important aspects of this adaptation are: ability to initiate cell division from almost any tissue of the plant; to regenerate lost organs; undergo different developmental pathways in response to particular stimuli. 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. In this way, whole plants can be subsequently regenerated Purvi Kakrani,Plant Tissue Culture 8 BASIC TERMINOLOGIES: BASIC TERMINOLOGIES TOTIPOTENCY : The regeneration of whole organism depends upon the concept that all plant cells can, given the correct stimuli, express the total genetic potential of the parent plant. This maintenance of genetic potential is called‘totipotency’. EXPLANT : Small part of an organ or tissue segment, from which regeneration occurs. Purvi Kakrani,Plant Tissue Culture 9 LABORATORY REQUIREMENTS FOR PTC: LABORATORY REQUIREMENTS FOR PTC Media Room : Space where glassware & plasticware are washed, stored & used for preparation of culture media This room should be equipped with: Working benches , including a laminar flow hood ; A deep-freeze, a refrigerator, weighing balance, a hot plate-cum-magnetic stirrer, a pH meter , a vacuum pump An autoclave to sterilize media. Hot air oven for the sterilization of glassware & metallic equipments. Purvi Kakrani,Plant Tissue Culture 10 LABORATORY REQUIREMENTS FOR PTC: LABORATORY REQUIREMENTS FOR PTC Aseptic chamber/ Area for transfer of culture: Contaminant free environment necessary Requires an ordinary type small wooden hood: having a glass or plastic door, either sliding or hinged fitted with UV tube. Now-a-days laminar air flow cabinet prevalent: May be vertical or horizontal Inside the cabinet, there is arrangement for bunsen burner & a UV tube fitted on the ceiling of the cabinet Purvi Kakrani,Plant Tissue Culture 11 LABORATORY REQUIREMENTS FOR PTC: LABORATORY REQUIREMENTS FOR PTC Advantages of laminar flow hood: Flow of air does not hamper the use of bunsen burner Cabinet occupies relatively small space within the lab. Purvi Kakrani,Plant Tissue Culture 12 LABORATORY REQUIREMENTS FOR PTC: LABORATORY REQUIREMENTS FOR PTC Incubation room or Incubator : Incubate cultures in well controlled environmental conditions- temperature, humidity, diurnal illumination and air circulation For temperature: Air conditioners or room heaters- required to maintain the temperature at 25±2 ˚C Light adjusted in terms of photoperiod duration Humidity range 20- 90% controllable to ±3% Uniform forced air circulation Shelves designed in such a way that there should not be any hindrance in the light, temp & humidity maintenance Purvi Kakrani,Plant Tissue Culture 13 LABORATORY REQUIREMENTS FOR PTC: LABORATORY REQUIREMENTS FOR PTC A label having full detail about date of innoculation , name of the explant , medium & any other special info- stuck on each tray & rack to ensure identity & for maintaining the data of experiment. Also there should be shaking machine for growing suspension culture. A BOD incubator for maintaining constant temperature to facilitate the culture of callus & its subsequent maintenance. 4. Cold storage : Cold storage, i.e. 2 to 4 °C, is used for maintenance of stock block in culture for plants. This is especially useful on commercial scale. Purvi Kakrani,Plant Tissue Culture 14 LABORATORY REQUIREMENTS FOR PTC: LABORATORY REQUIREMENTS FOR PTC 5. Locker room : Employees working in the lab must first change the shoes and wear additional clean clothing such as lab coat, hair nets, etc. before entering. 6. Washing: New glassware is always washed using detergents esp. designed for the purpose to remove all traces of acid, etc. finally the glassware is rinsed with tap water & then in distilled water. Sometimes the glassware may need to be autoclaved before washing to remove agar medium & to destroy microbial contamination. Purvi Kakrani,Plant Tissue Culture 15 LABORATORY REQUIREMENTS FOR PTC: LABORATORY REQUIREMENTS FOR PTC Washing: Important requirement is provision for fresh water supply & disposal of the waste water. Space for distillation unit for the supply of distilled and double distilled water & de-ionized water. Acid & alkali resistant sink or wash basin for apparatus/ equipment washing. Working table should also be acid & alkali resistant. For the storage of dried glassware separate separate dust proof cupboards or cabinet should be provided. Purvi Kakrani,Plant Tissue Culture 16 LABORATORY REQUIREMENTS FOR PTC: LABORATORY REQUIREMENTS FOR PTC B.O.D: Biochemical oxygen demand The amount of dissolved oxygen needed by aerobic biological organisms in a body of water to break down organic material present in a given water sample at certain temperature over a specific time period. Purvi Kakrani,Plant Tissue Culture 17 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS Culture medium is defined as a liquid or gelatinous substance containing nutrients in which tissues are cultivated for scientific purposes. Culture media can be broadly classified as: Inorganic elements: Macro-nutrients Micro-nutrients Organic elements: Growth regulators Carbohydrates Vitamins Amino acids Antibiotics Miscellaneous media components. Purvi Kakrani,Plant Tissue Culture 18 MACRO- NUTRIENTS: MACRO- NUTRIENTS Purvi Kakrani,Plant Tissue Culture 19 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS Purvi Kakrani,Plant Tissue Culture 20 Sr. No. Name of the Element Added in the form Main function; deficiency & Excess 1 Nitrogen Nitrate ions; Ammonium ions Influences growth rate Essential element of molecular construction of alkaloid, protein, amino acid, hormones. In excess, it causes acidification of the medium. When deficient, it impedes growth. 2 Potassium KH 2 PO 4 Regulates osmotic potential Principal inorganic cation CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS Purvi Kakrani,Plant Tissue Culture 21 3 Phosphorus K 2 PO 4 Na 2 PO 4 Component of nucleic acid, energy transfer, component of intermediates in respiration & photosynthesis. Essential in meristematic & fast growing tissues. When in excess, there is precipitation of medium elements as insoluble phosphates. Its deficiency produces reddish purple colored organs. 4 Magnesium MgSO 4 Enzyme cofactor Central component of chlorophyll. When there is its deficiency, older leaves are yellow in color. CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS 5 Calcium CaCl2 Integral part of cell walls It gets converted into pectin, which binds to cell wall components to form a cell wall. Helps in controlling permeability & facilitates the movement of carbohydrate. Plant metabolises protein to form oxalic acid as by-product. Therefore calcium binds oxalic acid forming Calcium oxalate. Dead root & shoot tips is the sign of deficiency of calcium. 6 Sulfur SO 4 -- Component of some amino acids (methionine, cysteine) and some cofactors. Promotes root development. Responsible for deep green color of foliage. Purvi Kakrani,Plant Tissue Culture 22 MICRO- NUTRIENTS: MICRO- NUTRIENTS Purvi Kakrani,Plant Tissue Culture 23 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS Sr. No. Name of the Element Added in the form Main function 1 Manganese MnSO 4 Enzyme cofactor Essential element in chloroplast wall 2 Iron FeSO 4 Participates in energy conversion & chlorophyll synthesis Present in Fe +3 , but when needs to be utilized by plant, it gets converted to Fe +2 When the shooting buds are yellow in color, this signifies deficiency of iron. 3 Iodine KI Essential element of amino acids. 4 Copper CuSO 4 Enzyme cofactor Electron transfer reactions Purvi Kakrani,Plant Tissue Culture 24 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS 5 Cobalt CoCl 2 Component of some vitamin like Vitamin B12 6 Zinc ZnSO 4 Enzyme activator Formation of auxin chlorophyll biosynthesis 7 Molybdenum Na 2 MoO 4 Helps to convert nitrogen to ammonia & thus nitrogen fixation. Component of nitrate reductase Enzyme cofactor 8 Chlorine CaCl 2 Promotes photosynthesis 9 Boron H 3 BO 3 Promotes movement of sugar, water & hormones. Involved in nitrogen metabolism, fruiting & cell division. Purvi Kakrani,Plant Tissue Culture 25 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS N.B: Phosphorus deficiency produces reddish-purple colored organs, which signifies dominance of anthocyanin in the organ. Therefore if anthocyanin is the desired product in plant tissue culture, phosphorus should be avoided in the media. Iron deficiency causes short of chlorophyll in plants. So due to this, leaves may be fully yellow in color from starting itself or may be yellow at the veins. This property can be exploited to produce an ornamental plant variety. EDTA is usually supplied in conjunction with the iron sulfate: EDTA complexes with the iron so as to allow the slow and continuous release of iron into the medium. Iron left uncomplexed with EDTA precipitates out of the medium as ferric oxide. Purvi Kakrani,Plant Tissue Culture 26 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS N.B: Nitrogen is most commonly supplied in a mixture of nitrate ions (H + NO 3 - ) and ammonium ions (NH 4 + NO 3 - ): Nitrogen, as a source, is given in the reduced form (i.e. ammonium ions) so as to easily penetrate the macro-molecules. However, at high concentrations, ammonium ions can be toxic to the plant cell cultures. In addition, uptake of ammonium ions from the medium causes acidification of the medium. So, in order to use ammonium ions as the sole nitrogen source, the medium needs to be buffered. Now, using NO 3 - in the medium has an advantage of weakly buffering the medium, as the uptake of NH 4 + ions causes H + ions to be excreted Purvi Kakrani,Plant Tissue Culture 27 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS 2. Organic elements: Growth regulators Carbohydrates Vitamins Amino acids Antibiotics Purvi Kakrani,Plant Tissue Culture 28 GROWTH REGULATORS: GROWTH REGULATORS Purvi Kakrani,Plant Tissue Culture 29 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS Plant Growth Regulators are the organic compounds, other than nutrients which affect the morphological structure and/or physiological processes. The 5 main classes of Growth regulators are: Auxins Cytokinins Gibberellins Abscisic acid Ethylene Purvi Kakrani,Plant Tissue Culture 30 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS AUXINS : Auxins promote both cell division and cell growth. These are growth promoting substances found particularly in actively growing tissues such as apices of stem and roots. Naturally occuring auxin is IAA- Indole Acetic Acid. Many synthetic auxins are now available: Indole Butyric acid (IBA), Naphthalene Acetic Acid(NAA), 2,4- dichloro phenoxyacetic acid (2,4- D) and p- chloro phenoxy acetic acid (p- CPA). Used in concentration of 0.1 to 10 mg/ lt in PTC media. IAA is thermolabile and loses most of its activity upon autoclaving and hence sterilized by filtration. Whereas synthetic ones are thermostable and can be autoclaved. Purvi Kakrani,Plant Tissue Culture 31 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS Auxins show multiple effects like: Promotes or initiates cell division. Stimulates shoot growth. Stimulation of root growth. Controls vascular differentiation. Regulates apical dominance. Delay senescence. Promotes flowering, fruit setting and ripening. Purvi Kakrani,Plant Tissue Culture 32 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS Purvi Kakrani,Plant Tissue Culture 33 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS CYTOKININS : Antagonist to auxins . Mostly produced in the roots. Endogenous hormone- 6-furfuryl amino purine . 6- furfuryl amino purine (kinetin) is found as a degradation product of DNA or in fresh nucleic acid preparations that have been autoclaved. It remarkably stimulates the proliferation of cells. 6- benzyl aminopurine (BAP) is the 1 st synthetic analogue of kinetin, which shows similar effect. Other synthetic cytokinins like N- isopentyl aminopurine (2-iP) and zeatin . These are used for inducing morphogenesis. Used in concentration range 40 to 80 mg/ lt. Purvi Kakrani,Plant Tissue Culture 34 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS Cytokinins show effects like: Induction of morphogenesis. In higher concentrations, may induce the rapid multiplication of shoots, axillary/ adventitious buds or meristem. Inhibits rooting. Purvi Kakrani,Plant Tissue Culture 35 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS N.B.: Synthetic auxins present alone in the medium may not evoke any response unless cytokinins are added as synergistic stimulants. The same is true for cytokinins. The ratio of auxin to cytokinin determines the type of culture established or regenerated: High auxin to cytokinin ratio Root formation. High cytokinin to auxin ratio Shoot formation. An intermediate ratio Callus formation. Purvi Kakrani,Plant Tissue Culture 36 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS GIBBERELLINS : Important growth regulators produced in the leaves. Gets accumulated in the immature fruits and seeds of some plants. Large group of cyclic diterpene acids, freely soluble in water. It causes hyper-elongation of stems and stimulates flowering along with other effects such as: internode elongation, sub-apical cell division, induction of parthenocarpy . Vegetative parts also contain lower amounts of gibberellic acid, which shows wide range of activities. Autoclaving reduces the activity of gibberellic acid solution by 90%. Purvi Kakrani,Plant Tissue Culture 37 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS Purvi Kakrani,Plant Tissue Culture 38 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS ETHYLENE: Ethylene is a gaseous hormone synthesized in fungi, bacteria and in cultured cells. It intervenes: the metabolic processes; and development of higher plants. Thus exerts a strong regulating influence on plant growth. Auxins stimulate the production of ethylene. Plays important role in: Somatic embryogenesis Morphogenesis. Plant regeneration from in vitro cultures. Although the physiological significance of ethylene in tissue culture is quite obscure. Purvi Kakrani,Plant Tissue Culture 39 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS ABSCISIC ACID : Naturally occurring plant growth regulator. Causes acceleration of: Abscission of leaves. Dormancy of buds. Retardation of germination. Bud development. Low concentrations of abscisic acid may stimulate somatic embryogenesis. Stable to autoclaving. Purvi Kakrani,Plant Tissue Culture 40 CARBOHYDRATES: CARBOHYDRATES Purvi Kakrani,Plant Tissue Culture 41 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS SUCROSE: C 12 H 22 O 11. Disaccharide. Plants growing in culture cannot manufacture all the sugar they require. So a high concentration of sucrose, generally 30 g/ lt , is suggested for most media formulae. Source of sucrose: sugar from sugarcane or from sugar-beet. Occasionally, fructose or glucose are substituted for sucrose in media. D-MANNITOL: Sugar alcohol. Used as a nutrient & osmoticum , particularly when inducing plant protoplast formation and fusion. Purvi Kakrani,Plant Tissue Culture 42 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS D-SORBITOL: Sugar alcohol. It is the primary translocatable carbohydrate in some plants. Occasionally added to the media. HEXITOLS: Among hexitols , myo-inositol has been found to be important ingredient in plant tissue culture. It is considered as a growth promoter. This is a carbon source as well as has action like vitamin. Purvi Kakrani,Plant Tissue Culture 43 VITAMINS: VITAMINS Purvi Kakrani,Plant Tissue Culture 44 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS SR. NO. VITAMIN FUNCTIONS 1 B Complex : 1(a) B 4 (Adenine) Important to cells as a part of the nuclear substances (DNA & RNA). Weak cytokinin effect. Used in the form of adenine sulfate to promote shoot formation. 1(b) B 12 (Cyanocobalamine) Growth promoter. 1(c) B 3 (Niacin) Component of co-enzymes active in light energy reactions. Concentration range: 0.1 to 10 mg/lt. Purvi Kakrani,Plant Tissue Culture 45 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS 1(d) B x (PABA) Serves as antiseptic and a preservative. Important role in folic acid metabolism. 1(e) B 5 (D- Pantothenic acid) Active as a co-enzyme in fat metabolism Added as calcium salt. It should be cold-sterilized. 1(f) B 2 (Riboflavin) Active in carbohydrate metabolism. Essential to cellular respiration. 1(g) B 6 (Pyridoxine) Co-enzyme in some metabolic pathways 1(h) B 1 (Thiamine) Functions as co-enzyme to assist the Kreb’s cycle. Only 0.4mg of thiamine HCl per litre of media is required for plant tissue culture. Purvi Kakrani,Plant Tissue Culture 46 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS 2 H (Biotin) Required in media for fat, protein & carbohydrate metabolism 3 Choline An alkaloid. Has alkaline properties within B complex. Chemically related to fats, but also contains phosphorus & nitrogen. 4 M (Folic acid) Found in leaves & other plant tissues. Demonstrates co-enzyme activity. 5 Myo-inositol Sugar alcohol in B complex. In phosphate form, it is a part of various membranes, particularly those of organelles such as chloroplasts. 6 C (Ascorbic acid) Disinfectant. Anti-oxidant to prevent phenolic oxidation i.e. browning of plants. 7 E ( α - Tocopherol ) Known to promote dispersion in suspension cultures of soyabean & some other examples. Purvi Kakrani,Plant Tissue Culture 47 AMINO ACIDS: AMINO ACIDS Purvi Kakrani,Plant Tissue Culture 48 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS Amino acids are the building blocks of proteins. Some amino acids combine with nucleic acids to form nucleoproteins. Amino acids are incorporated in the media in L-forms. SR.NO AMINO ACID FUNCTION 1 Alanine Helps to increase the number of embryos in some cell cultures. 2 Arginine Helps in root initiation 3 Asparagine With proline, it assists in embryogenesis. 4 Cysteine Sulphur containing amino acid. Added as HCl salt. 5 Glutamine Contributes to somatic embryogenesis 6 Lysine When combined with proline, it enhances the quality & number of embryos 7 Serine Used in microspore (pollen) culture to grow haploid embryos. 8 Tyrosine Effective in shoot initiation. Useful as a nitrogen source. Purvi Kakrani,Plant Tissue Culture 49 ANTIBIOTICS: ANTIBIOTICS Purvi Kakrani,Plant Tissue Culture 50 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS An antibiotic is a substance produced by plants/ micro-organisms that has a toxic effect on other micro-organisms; antibiotics can retard or prevent the growth of such micro-organisms or can even kill them. These chemicals are toxic not only to contaminants, but also to cultures or explant material. So restricted use should be made for additions into the culture medium. The criteria for selection & usage: Should be water soluble. Should be made fresh Should added to the medium after autoclaving or by filter sterilization. Antibiotics commonly used: Ampicillin, Carbenicillin, Cefotaxime, Gentamcin sulfate, Polymixin B, Ribavirin, Streptomycin. Used in concentration range of 6 to 400 mg/lt. Purvi Kakrani,Plant Tissue Culture 51 MISCELLANEOUS MEDIA COMPONENTS: MISCELLANEOUS MEDIA COMPONENTS Purvi Kakrani,Plant Tissue Culture 52 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS GELLING AGENT/ SOLIDIFYING AGENT : Agar is used mainly as a gelling agent. Difco - bacto agar or inferior grade of agar can be used in 0.6 to 0.8 % for routine work. Gelatin, alginate, silica gel or acrylamide gels are sometimes used as a solid support to the media. ACTIVATED CHARCOAL : It is frequently added to rooting media to absorb root inhibiting agents. Promotes somatic embryogenesis. Added in concentration of 0.6 g/lt. NATURAL COMPLEXES : Addition of natural extract such as coconut milk, fruit juices from tomato and banana, yeast extract, malt extract & casein hydrolysate like substances promotes tissue growth. Casein hydrolysate is an undefined protein mixture that is occasionally used in media as a non-specific source of organic nitrogen. All the above complexes- their addition in the medium- make the medium undefined, since there is variation in growth promoting or inhibiting compounds in these complexes. Purvi Kakrani,Plant Tissue Culture 53 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS ANTIOXIDANTS : Anti-oxidants such as citric acid, ascorbic acid, pyrogallol, phloroglucinol & L-cysteine- are used in tissue culture to reduce excessive browning of the explants. Adsorbents like PVP and activated charcoal are also used for checking excessive browning. Purvi Kakrani,Plant Tissue Culture 54 ADDITIONAL MEDIA REQUIREMENTS: ADDITIONAL MEDIA REQUIREMENTS Purvi Kakrani,Plant Tissue Culture 55 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS LIGHT : Plays important role for inducing the culture growth. Light intensity & the period of illumination may vary from one species to another. TEMPERATURE : Temperature range from 25 to 27 °C is the most preferred for growth of callus. PHYSICAL STATE OF MEDIUM : Semi-solid & liquid media, are most commonly used for growing plant cells. CORRECT pH : Important for proper growth of the cultures. It is generally adjusted to pH range of 5.6 – 6.0 before autoclaving the culture medium. Highly acidic or alkaline ph drastically affect the uptake of nutrients in cultures. Purvi Kakrani,Plant Tissue Culture 56 CULTURE MEDIA & THEIR CONSTITUENTS: CULTURE MEDIA & THEIR CONSTITUENTS HUMIDITY : Relative humidity of up to 70 to 75% is ideal for the growth of the cultured plant tissues. Less humidity of up to 50 to 55% causes dehydration of the medium; while the higher humidity of up to 85 to 90% may promote the microbial growth on cotton plug which may contaminate the medium culture. OXYGEN : Necessary for survival of the plants. Sufficient oxygen for the medium can be provided by agitation and aeration. Relatively more oxygen is in hairy root culture medium. Purvi Kakrani,Plant Tissue Culture 57 Murashige-Skoog Medium Composition: Murashige-Skoog Medium Composition Purvi Kakrani,Plant Tissue Culture 58 0.25 NaMoO 4 • 2 H 2 O 0.83 KI 8.6 ZnSO 4 • H 2 O 16.9 MnSO 4 • H 2 O 6.2 H 3 BO 3 170 KH 2 PO 4 370 MgSO 4 • 7H 2 O 440 CaCl 2 •2H 2 O 1900 KNO 3 1650 NH 4 NO 3 mg/L Ingredients Murashige-Skoog Medium Composition (contd..): Murashige-Skoog Medium Composition (contd..) Ingredients mg/L CuSO 4 • 5H 2 O 0.025 CoCl 2 • 6H 2 O 0.025 FeSO 4 • 7H 2 O 27.8 Na 2 EDTA • 2H 2 O 37.3 myo-inositol 100.0 nicotinic acid 0.5 thiamine • HCl 0.1 pyridoxine • HCl 0.5 glycine 2.0 sucrose 30000 agar 8000 Purvi Kakrani,Plant Tissue Culture 59 STERILIZATION : STERILIZATION Purvi Kakrani,Plant Tissue Culture 60 STERILIZATION: STERILIZATION All operations in plant tissue culture is carried out under aseptic conditions, preferably under the hood of a laminar air flow cabinet. All the equipments, instruments, culture media and the plant material that are used for the purpose also need to be sterilized before use. Sterilization parameters need to be defined for the following: Equipment Transfer facilities Glassware Instruments Media Components that need to be autoclaved Components that need to be filter sterilized Plant material Purvi Kakrani,Plant Tissue Culture 61 I. STERILIZATION OF EQUIPMENT: I. STERILIZATION OF EQUIPMENT TRANSFER FACILITIES : This includes: Laminar flow cabinets Innoculation rooms Glove boxes First step is to decontaminate using a suitable disinfectant. Then in next step is by 70% alcohol. New flow cabinets should be left running overnight to clean the filters. In routine use, transfer areas should be regularly sterilised with 70% alcohol both before and after use. Transfer room and glove boxes are sterilized by exposing them to UV light for at least 15 mins. Purvi Kakrani,Plant Tissue Culture 62 I. STERILIZATION OF EQUIPMENT: I. STERILIZATION OF EQUIPMENT GLASSWARE : Autoclaved at 121 °C at a pressure of 15psi for 15min. Also dry heating method can also be applied which uses 160°C for 3hrs. But this method is usually used for protoplast media. Open glassware should be capped with a double layer of aluminium foil before sterilizing itself so that sterility is maintained after the treatment. Glassware with screw caps should always have caps loosened half a turn to avoid high pressure building up that can lead to vessel exploding. Purvi Kakrani,Plant Tissue Culture 63 I. STERILIZATION OF EQUIPMENT: I. STERILIZATION OF EQUIPMENT INSTRUMENTS : Directly flame the lower parts of the instruments before their use, e.g. scalpels, forceps, laminar air flow top. Cool before bringing it in contact with the plant tissue. Between manipulations, the instruments are stored with their working surfaces submerged in 20% ethanol in a glass vessel. Alternatively, sterilized using dry heat after first wrapping them in aluminium foil or heavy brown paper. Autoclaving is avoided since the combination of elevated temperature and steam quickly leads to corrosion. Purvi Kakrani,Plant Tissue Culture 64 II. STERILIZATION OF MEDIA: II. STERILIZATION OF MEDIA Autoclaving : Most widely used method. It uses 121 °C for 15-20 min at 15psi. After the main period of autoclaving, pressure is allowed to fall relatively slowly to avoid the media from boiling over in the flasks. Longer heating time is avoided to prevent decomposition of media. Autoclaving can result in a reduced ph, altered osmolarity, undesirable chemical modification. All these can prove detrimental to very sensitive cells in media. Filter sterilization : Useful in case of heat labile components in media. All main components of media should be homogenously mixed before filter sterilization. Usually pore size of 0.22 mcg is used in filters. Solutions for protoplast culture media should be filter sterilized. Purvi Kakrani,Plant Tissue Culture 65 III. STERILIZATION OF PLANT MATERIAL: III. STERILIZATION OF PLANT MATERIAL SR. NO Name of the Plant organ Pre-Sterilization Sterilization Post- Sterilization 1 Seed Submerged in absolute alcohol for 10sec. Wash with D.W Submerged for 20-30 min in 10% w/v Ca. hypochlorite or 1% w/v bromine Wash 3 times in sterile water & germinated on sterile damp filter paper. 2 Fruit Rinse with absolute alcohol Submerged for 10min in 2% w/v sodium hypochlorite Washed with sterile water. 3 Pieces of stem Running tap water & then with absolute alcohol Immerge for 15-30 min in 2% w/v sodium hypochlorite Wash 3 times in sterile water. 4 Strong organs Scrub and clean in running tap water 20-30 min in 2%w/v hypochlorite soln Wash 3 times in sterile water. 5 Leaves Rub surface briefly with absolute alcohol Immerse for about 1 min in 1% w/v Mercuric chloride Wash repeatedly with sterile water Purvi Kakrani,Plant Tissue Culture 66 BASIC TECHNIQUE OF PLANT TISSUE CULTURE: BASIC TECHNIQUE OF PLANT TISSUE CULTURE Purvi Kakrani,Plant Tissue Culture 67 PowerPoint Presentation: Purvi Kakrani,Plant Tissue Culture 68 Selection of part of plant from which to initiate the cuture Surface sterilization of explant Transfer of explant aseptically onto a semi-solid nutrient medium supplemented with suitable growth regulators Incubate cultures at 25-27 °C in dark or at required photoperiods. Callus of organ regeneration takes place.