HiMedia Plant Tissue Culture Manual
HiMediaPlant TissueCulture ManualHiMediaLaboratoriesTMHiMedia Laboratories Pvt. LimitedFor life is precious
Source of Life"We shall not cease from explorationAnd the end of all our exploringWill be to arrive where we startedAnd know the place for the first time."- T.S. EliotFor life is precious
YB1278 902729 010105
Quality Assurance Programmeplant tissue.Quality Assurance ProgrammeOur Quality Assurance Programme is broadly divided intothree sub categories, Raw material testingInprocess sample testingFinished product testingQuality Control of Plant tissue cultureProductsRaw Material TestingThe design of Plant Tissue Culture media requires statisticalevaluation of each ingredient and measurement of theinteraction of each ingredient with the other. Awareness ofthis is of vital importance and accordingly various qualitycontrol measures are adopted by HiMedia prior to andduring the manufacturing processes so that; desired endproducts with predictable reproducible results are obtained.Only the highest quality raw materials are used in themaking of HiMedia products. The raw materials areprocured only from approved vendors who meet all theacceptance standards. Each raw material is analysed forgrade, quality and purity as per the standards specified inUSP / EP / IP / BP / FCC / ACS. For the raw materials andchemicals whose standards are not specified, we have ourown in-house standards set by our quality assurancedepartment, Beside these standards, all the chemicals arechecked for their suitability for plant cell culture.Various raw materials incorporated in plant tissue culturemedia are :1. Inorganic Nutrients Mineral elements2. Organic Nutrients Carbohydrates Vitamins & cofactors Complex organic supplements3. Plant growth regulators4. Solidifying agents/Gelling agents1. Inorganic salts & minerals :The inorganic nutrients in plant tissue culture are mineralelements and are further classified as Macroelements andMicroelements. Most plant cells require Nitrates,Phosphates, Sulfates, Calcium, Potassium, Magnesium,Manganese, Ferrous or Ferric ions. Only analytical gradechemicals and minerals are used for the production of planttissue culture media.2. Carbohydrates :Carbohydrates are added to plant tissue culture media tosupply carbon and energy. Sucrose is the most commonlyused sugar but certain formulations also use glucose,fructose or sorbitol. Carbohydrates used as raw material aretested to ensure their identity and purity from adulterants(1,2). The metabolic and osmotic function is also assessedby preparing the complete medium along with knownstandard carbohydrates and observing the growth of the23. Vitamins :Vitamins and biochemicals are analysed as per the USP (3)and FCC, U.S.A. (2) for chemical purity and other testsspecified under the monograms. Besides the chemicalanalysis, potency is further confirmed by incorporating thevitamins in specific plant tissue culture media andmonitoring the growth characteristics.4. Complex organic supplements :Certain formulations require complex additions like bananapowder, pineapple powder or protein hydrolysates.Although these additions are known to enhance growth, it isnot known exactly which compound of these supplementsstimulates the growth process.5. Plant growth regulators :Plant growth regulators are incorporated in the plant tissueculture media to promote organogenesis. Auxin andCytokinin are the most frequently used plant hormones inplant tissue culture and gibberellins and abscisic acid areused occasionally. Plant hormones added to the media aretaken up by the tissues; their levels in the tissue arehowever transient because plant hormones are rapidlyinactivated after uptake. Morphology and growth pattern ofthe test culture is observed by incorporating the growthregulators in the tissue culture medium.6. Solidifying agents / Gelling agents :The primary purpose of a solidifying agent in plant tissueculture media is to form a solid or semisolid matrix neededto support tissue explants while allowing contact with themedium. Agar is the most commonly used solidifying agent.Besides agar, agarose, gelatine or gellan gums are alsoused. The clarity of agar gel in Plant Tissue Culture Media isan important characteristic. Ideally, the molten solutionshould be crystal clear without any haziness or deposit.Haziness in agar is due to mineral incompatibilities orpresence of debris and minute fragments which havepassed through the filters during processing. Anothercharacteristic of agar is its ability to allow diffusion ofcompounds although it locks water into a rigid gel. Thisproperty is exploited for providing required moisture andnutrients to the culture system. The agar is also analysedfor various chemical impurities before it is used for planttissue culture.Inprocess Sample TestingA sample lot is prepared from tested and approved mediaingredients and analysed in parallel with a previouslyapproved reference lot of complete medium. Afterascertaining all the specifications like colour, clarity, pH,solubility, gelling and growth response of the sample lotincluding compatibility of component ingredients, largescale lot is approved for manufacturing. Whilemanufacturing, very close attention is paid to each stage byobserving good manufacturing practice (GMP)(3). Samplesare withdrawn at intermediate stages and forwarded toquality control laboratory for appropriate evaluation as perthe test specifications. Lot in process is continuously
recorded under controlled conditions of temperature,moisture, humidity, etc. The manufacturing process iscarried out by using equipment of stainless steel or glasstaking care to avoid product contamination with toxicmetals.Finished Product TestingEach HiMedia plant tissue culture medium has definedproduct specifications. The specification includes validatedrelease criteria which stipulate the finished part inspectionnecessary for acceptability and release of each lot of finishedproduct. The final product is tested for the following criteria:PhysicochemicalparametersBiological ypHGelling [where agar/ClariGel (Gellan gum)is used]Growth promotion and study ofmorphological characteristicsof various plant tissue culturesin media.In addition, we also have unique in house criteriadeveloped specifically to zero down any lot to lot variationthat may arise due to the interplay of biological materials.Traceability & Stability StudyBeing an WHO GMP / ISO 9001:2000 / ISO 9001 :13485 /CE certified company, traceability is the most importantcriterion on which our quality systems are based. Each rawmaterial used and each production process is meticulouslydocumented. Approved final product samples and variousrecords generated during manufacturing process areretained for future reference.Stability studies are carried on representative lots todetermine the shelf-life of new products and also to verifythe efficacy of the existing product. After six months interval,representative lots are retested to verify the stability ofproducts by comparing with a reference lot.Certificate of AnalysisDehydrated plant tissue culture media are reconstituted torecommended concentrations and supplemented with theappropriate concentration of biochemicals and gellingagent depending on the cell line being used. Medium isbiologically evaluated with a callus culture (nondifferentiating) and shoot proliferating (differentiating) cellline.Callus culture Cell line Passage – One Cultural condition Temperature Humidity PhotoperiodCriteria for evaluation of cell line Increase in fresh weight of callus Lack of necrotic tissue AppearanceShoot proliferating culture Cell line Passage – One Cultural condition Temperature Humidity PhotoperiodQuality Assurance ProgrammeBesides above tests, toxicity test is carried out to ascertainthe presence of toxic and inhibitory substances by growingcallus cultures on complete medium. Morphology and freshweight is compared with a known reference lot.minimum of one plant cell line through one passage.Criteria for evaluation of cell line – Active production of shoots or plantlets Production of vigorous healthy shoots withoutmorphological aberrationsBibliography1. Ginsburg V., 1972, Methods in carbohydrate chemistry,Vol.1, Academic Press, San Diego, California. Methodsin Carbohydrate Chemistry, Vol. I, Academic Press,U.S.A.2 . Food Chemicals Codex, 2004, 5th ed., The NationalAcademic Press, Washington, D.C. Food ChemicalCodex, 5th edition, 2004.3. The United States Pharmacopoeia, 2008, USP31/NF26.The United States Pharmacopeial Convention.Rockville, MD. U.S. Pharmacopeia 28 NationalFormulary 23, 2005.Every lot released for sale is provided with a detailedCerficate of Analysis which defines the productspecifications and the actual test results. Hard copies ofCertificate of Analysis are provided on request or can bedirectly downloaded from our website(www.himedialabs.com).Quality Control of Plant TissueCulture ProductsThe growth promoting activity of all plant tissue cultureproducts viz. media, basal salt mixtures, inorganic saltsbiochemicals (i.e. vitamins, carbohydrates and plant growthregulators) and gelling agents are evaluated with a 3
Quality Assurance ProgrammeGeneral Instructions to the UsersPreliminary Precautions to beObservedThe dehydrated plant tissue culture media are highlyhygroscopic and must be stored in a cool, dry place awayfrom bright light. These media are meant for laboratory useonly.Instructions are to be read carefully given on the label. Notethe ingredients and directions of use. Also note the Usebefore date and lot number. Ensure before use that themedium has not deteriorated physically and also check thatthe Use before period is not over.Culture media have a tendency to form lumps due tofollowing conditions:iHigh humidity during storageiiContainer left open too longiiiContainer not tightly sealed every time after openingivMedium has past expiry dateReconstitution of Dehydrated MediaFor rehydration, use clean undamaged glassware anddistilled water that meets the specifications as per U.S.P./I.P.Place appropriately weighed amount of the medium in aclean dry flask, 2 - 3 times larger than the final volume ofthe prepared medium. Add part of the required amount ofdistilled water and swirl to dissolve. Now gradually add theremaining water from the side of container. For completedissolution of agar containing media, heat using hot plateor boiling water bath taking care to prevent excessiveheating or scorching of the medium.Microwave oven can also be used for heating the mediumto dissolve. After initial boiling in the microwave oven,remove the medium and gently stir. Return the medium tothe oven to heat it for regular short period intervals of time.Continue until complete dissolution of agar particles takesplace (approximately 2-3 minutes of total heating time).pH AdjustmentThe pH value of reconstituted dehydrated plant culturemedia prepared with distilled water shall produce theequivalent value as prescribed on the label at atemperature of 25 C. If old material is being used, it isrecommended to check and adjust the necessary pH priorto use as required.pH measurement and correction of the pH should becarried out at 25 C for solid as well as liquid culture mediabefore sterilization. For liquid culture media pH should bemeasured after sterilization by cooling medium to 25 C.Take an aliquot of liquid medium aseptically and measurepH. In case of solid culture media, pH should be measuredafter cooling the medium to 40 C. The pH metertemperature knob should be adjusted to 40 C. The pHshould be adjusted to the value specified and should be4corrected by adding 1N or 0.1N Hydrochloric acid orsodium hydroxide solution to a sample of known volumetaken from the reconstituted culture medium (e.g. 50 to 100ml). Finally after calculation, the required acid or alkali isadded in the remaining prepared culture medium.SterilizationDispense completely dissolved medium as desired andsterilize as per directions on the label. Generallysterilization is done at 121 C for 15 minutes in an autoclave(Steam sterilization under pressure). Temperature pressurerelation is listed in the table below.Temperature - Pressure RelationPressure ofsaturated steamTemperatureattainedP.S.I. C F5 lbs10822610 lbs11624015 lbs12125020 lbs12726025 lbs13126730 lbs134274Efficiency of autoclaving should be ascertained from time totime. At times the temperature conditions are not uniforminside the verticle autoclave at different places. This can betested by putting 2 bottles containing 2% w/v glucose in 2%w/v disodium phosphate in the autoclave at different places.Heat produces browning of solution. Bottle near steam inletwill be browner than the one away from it.It is highly recommended that the users strictly follow thedirections on the label.Heat labile enrichments or supplements are filter sterilizedand added to presterilized media. Additions are madeaseptically to liquid media cooled to room temperature andto agar media cooled to 45 - 50 C as directed on the label.Sterilization CheckIt is recommended to use calibrated and validated steamsterilizer (autoclave). Incorporating biological/chemicalindicator strips during each sterilization run to establishefficacy of autoclave should be practised.Physical measurements should be made on temperatureand pressure readings, the quality of the steam must bechecked including the steam and safety valves. In certaincountries, biological indicators are compulsory todemonstrate efficiency of sterilization cycle.Chemical indicators will demonstrate the temperaturereached or exceeded and some will indicate the time heldat the specified temperature.
Effects of OverheatingIt is suggested that all the culture media should be insolution before sterilization otherwise there are chances ofdarkening of the medium (Maillard type reaction).Pouring of Sterilized Media intoSterile ContainersSterile agar media should be poured into containers atabout 45 - 50 C to avoid the formation of condensed wateron the lids of the container. The medium should be mixedthoroughly, without bubble formation and asepticallypoured into sterile containers.Storage of Prepared MediaIf the medium is not used the same day it is prepared, itshould be stored properly in moisture proof containers toprevent drying of the medium. It is important to note thatunless the stability of the prepared medium is known itshould not be stored for longer periods. Similarly agarcontaining media should not be held at higher temperatures(40 - 50 C) for longer time as agar tends to clump.Agar plates should be stored at 2-8 C in sealed containersto prevent loss of moisture. Liquid media in test tubes orflasks should also be provided with airtight seals. Loss ofwater can result in precipitation and crystallisation of certainsubstances in the culture media.The stability of prepared plant culture media is limited and itvaries considerably. Media should not be used when morethan 2 weeks old. Media should not be stored below 0 C asthis destroys their gel structure.Before inoculation, carefully examine media containingcontainers for contamination, uneven filling or bubbles onsurface of agar, colour changes and cracks due to shrinkingand loss of volume. Discard such defective containers.Disposal of Prepared MediaPersonnel Practices : Handling of prepared media shouldbe done only by qualified personnel who have been trainedin plant tissue culture techniques. All specimens andcultures should be handled properly and should not bedisposed without autoclaving. User should ensure that anymachinery or apparatus used and by chance contaminatedmust be safely disinfected or sterilized. Cultures in reusableglass vessels (e.g. conical flasks, culture test tubes, glasspetriplates) must first be autoclaved (approximately 30minutes at 121 C) prior to disposal. All the dehydrated plant culture media are supplied in finepowder form. These products are meant for carrying outplant tissue culture related work in the laboratory asspecified under the use of individual medium and shouldnot be used for human or animal consumption directly orindirectly. Most of the powders are fluffy in nature andshould not be inhaled because inhalation may lead toirritation of the upper respiratory tract. If skin rashes occur,avoid prolonged contact with the powder and ensureexcessive dust is not produced. Any residue should bewashed off with ample cold water.To prevent the risk of inhaling fine dust, it is recommendedto use face masks while handling dehydrated media.Material Safety Data Sheets are available for individualproducts.First Aid MeasuresThe following First Aid procedures should be carried out incases of accident while handling any of the toxic orhazardous products.Inhalation : Person should be removed from the area ofexposure. Should rest and keep warm and if required seekmedical advice.Quality Assurance ProgrammeHigh temperature and prolonged heating is a commoncause of pH drift, darkening of medium, precipitation, poorgel strength and deterioration in the quality of the culturemedium.Handling of Dehydrated PlantTissue Culture MediaSkin Contact : It is suggested to remove all thecontaminated clothings immediately and wash the affectedarea thoroughly with soap and water. After completing thisprocedure, if any symptoms occur, seek medical advice.Ingestion : In case of ingestion of material, wash the mouththoroughly with plenty of water. One pint of water should bedrunk immediately after which medical advice should besought.Eye Contact : Eyes should be flushed immediately withplenty of water. If required, seek medical advice.Spillage : Following precautions must be observed ifmaterial is spilled.For Large Quantities : Wear protective overalls, gloves,eye protection and face mask. Material should be collectedin suitable container and then properly sealed. Disposal ofsuch material is to be carried out according to localregulations. Wash away the residue with plenty of water.For Small Quantities : By using protective gloves, washaway the material with large volumes of running water.5
Safety Check ListQuality Assurance ProgrammeHandling & Use6 Read the label before opening the container. Check that the product is the one required. Consider the hazards including violent reactionsbetween chemicals and use appropriate protectiveclothing and equipment. Open container carefully in well ventilated area. Take care while extracting and using hazardouschemicals and use methods which reduce the risk ofinhalation, ingestion and contact with skin, eyes andclothing. . Avoid using contaminated apparatus and instruments. Seal container tightly after use. Do not eat, drink or smoke while handling and usingchemicals. Wash hands and exposed areas thoroughly andchange contaminated clothing. Deal with spillages using precautions and methodsappropriate to the hazards. Seek medical attention immediately if affected bychemicals and use appropriate first aid until medicalattention is available.Storage Preferable storage temperature has been mentionedon the label for all items. In case of products stored at room temperature, store indry well ventilated area protected from extremes oftemperature and sources of ignition. On opening, product should be properly stored dry,after tightly capping the bottle in order to prevent lumpformation due to the hygroscopic nature of the product.Improper storage of the product may lead to lumpformation. In such cases no liability is accepted by thecompany. Secure chemicals from unauthorized use. Seggregate stock to reduce hazards. Inspect stocks from time to time and dispose offdeteriorated materials. Do not smoke where flammable chemicals are stored. Take care in handling containers with hazardousresidues. To be used by trained personnel only.
Troubleshooting For Commonly Encountered Problems WhileUsing Dehydrated Plant Tissue Culture MediaCauses Over
tissue culture media. 2. Carbohydrates : Carbohydrates are added to plant tissue culture media to supply carbon and energy. Sucrose is the most commonly used sugar but certain formulations also use glucose, fructose or sorbitol. Carbohydrates used as raw material are tested to ensure their identity and purity from adulterants (1,2).
Plant tissue culture is the growing of microbe-free plant material in an aseptic environment such as sterilized nutrient medium in a test tube and includes Plant Protoplast, Plant Cell, Plant Tissue and Plant Organ Culture. Plant tissue culture techniques have, in recent years,
Aerobic culture Anaerobic culture AFB culture Fungal culture Quantitative tissue culture (includes Aerobic culture) Deep wound culture (includes Aerobic and Anaerobic) Anaerobic Tissue Transport Media: PS59547 Tissue should sit on top of the media. Do Not Add Formalin. Tissue: (Any organ / solid tissue removed from the body i.e. kidney,
tissue culture. This resulted in the books Plant Culture Media, Vols. 1 and 2 (1987), and Plant Propagation by Tissue Culture. The latter work was first published in 1984 and then extensively revised and extended to two volumes in 1993 and 1996. The present book is based on the first volume of the 2nd edition of Plant Propagation by Tissue .
Plant Tissue Culture for the Serious Hobbyist, Teacher, Nurseryman and All Plant Lovers! Plant tissue culture involves the sterile growth of plants in containers for the purpose of mass production. ! Through the use of plant hormones and other growth regulators, small plant parts can be
Plant Tissue Culture Plant tissue culture is a technique of culturing plant cell, tissue or organ in artificial, controlled and aseptic conditions. It mainly covers micropropogation, organogenesis and somatic embryogenesis. Pest Any species, strain or biotype of plant, or pathogenic agent, injurious to plants or plant products. Procedure
Plant Tissue Culture Media 33 2.8.1. Auxins The common auxins used in plant tissue culture media include: indole-3- acetic acid (IAA), indole-3- butric acide (IBA), 2,4-dichlorophenoxy-acetic acid (2,4-D) and naphthalene- acetic acid (NAA). IAA is the only natural auxin occurring in plant tissues There are other
LifeCell Tissue Matrix LifeCell Tissue Matrix products, AlloDerm Regenerative Tissue Matrix and Strattice Reconstructive Tissue Matrix, offer a range of benefits to surgeons and their patients for soft-tissue reinforcement. LifeCell tissue matrices allow surgeons to restore many types of tissue damaged through injury, surgery or
Fjalët kyce : Administrim publik, Demokraci, Qeverisje, Burokraci, Korrupsion. 3 Abstract. Public administration, and as a result all the other institutions that are involved in the spectrum of its concept, is a field of study that are mounted on many debates. First, it is not determined whether the public administration ca be called a discipline in itself, because it is still a heated debate .
