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Presentation on
Basic & Aseptic TechniquesMedia Preparation & Sterilization
By:Aarti DwivediAarti Nagdev
Ankit Jha
Basic Steps of PTC
Introduction Principles of plant tissue culture :
Tissue culture simply directs and assistants the natural potential within the plant to put forth new growth and the multiply in highly efficiently and predictable way.
Totipotency i.e is the capacity of an individual all to regenerate in to the whole plant, the concept of totipotency (T.H. Morgan, 1901).
All the plant cells have their property since potential lies mainly in cellular differentiation. This indicates that all genes responsible for differentiation tissue or organ are able to express only under adequate culture conditions.
Introduction The three main changes or stages is the complete
development an ordered change or progress often towards a higher more complex state of a cell are Cell division Cell elongation Cell Maturation
Two kinds of plant growth are possible in vitro Organized Growth : Occurs either when organized plant parts or
organs such as the growing point apical Meristem of shoots or roots leaf initials, young flower buds and small fruit are transferred to culture (where they may continue to grow with their structure preserved ) or when these structure are formed afresh during the culture of unorganized tissues.
Unorganized Growth : Occurs when pieces of whole plant are
cultured in vitro. The tissue thus formed typically lack any recognizable structure contain only limited no of the many different kinds of specialized cells found in an intact plant.
Basic TechniquesBasic Techniques
Setting up of a tissue culture lab requires proper planning.
It is divided into 5 areasMedia preparation roomAseptic transfer areaCulture roomAnalytical roomAcclimatization room
Media Preparation RoomMedia Preparation Room
Refrigerator & freezer Water purification & storage system Glassware washing facility Continuous supply of single & double
distilled water Culture media, washing powder,
disinfectants Cabinets or shelves
Aseptic Transfer AreaAseptic Transfer Area
Laminar air flow Dissecting microscopes Dissection instruments Gas outlet Vacuum facility Sterilizer
Culture RoomCulture Room
Environmentally controlled Incubators with controlled temperature Rotary shakers Lux meter Space for cultures requiring complete
darkness
Analytical RoomAnalytical Room
Colorimeter Low speed centrifuge Inverted centrifuge Chemical reagent racks Viscosity meter Gas outlet
Acclimatization RoomAcclimatization Room
High illumination(4,000-10,000 lux) High humidity(90-100% through mist &
fog systems)
Miscellaneous ItemsMiscellaneous Items
Air conditioners Uninterrupted power supply Bunsen burners Aluminium foils Fluorescent lamps Fire fighting equipment
Media Media
No single medium supports growth of all tissues.
Some basic factorsCallus inductionOrganogenesis
Murashige-Skoog medium, White’s medium, woody plant medium
Media ComponentsMedia Components
Media ComponentsMedia Components
Media ComponentsMedia Components
MS media Macronutrients
Ammonium nitrate (NH4NO3): 1,650 mg/l
Boric acid (H3BO3): 6.2 mg/l
Calcium chloride (CaCl2 · 2H2O): 440 mg/l
Cobalt chloride (CoCl2 · 6H2O): 0.025 mg/l
Magnesium sulfate (MgSO4 · 7H2O): 370 mg/l
Cupric sulfate (CuSO4 · 5H2O): 0.025 mg/l
Potassium phosphate (KH2PO4): 170 mg/l
Ferrous sulfate (FeSO4 · 7H2O): 27.8 mg/l
Potassium nitrate (KNO3): 1,900 mg/l
Manganese sulfate (MnSO4 · 4H2O): 22.3 mg/l
Potassium iodide (KI): 0.83 mg/l Sodium molybdate (Na2MoO4 · 2H2O):
0.25 mg/l Zinc sulfate (ZnSO4·7H2O): 8.6 mg/l
Na2EDTA · 2H2O: 37.2 mg/l
•Common organic additives•i-Inositol: 100 mg/l•Niacin: 0.5 mg/l•Pyridoxine · HCl: 0.5 mg/l•Thiamine · HCl: 0.1 mg/l•IAA: 1–30 mg/l•Kinetin: 0.04–10 mg/l•Glycine (recrystallized): 2.0 g/l•Edamine (ethane-1,2-diamine): 1.0 g/l•Sucrose: 20 g/l•Agar: 10 g/l
SterilizationSterilization
Sterilization Sterilization
Physical methodsFiltration
○ Reducing microbial population in heat sensitive solutions
○ 2 types of membrane filtersGradocol membraneCellulose membrane
○ Air sterilization: HEPA filters in LAF
Sterilization Sterilization
Physical MethodsRadiation
○ UV lamps placed in ceilings or in biological safety cabinets
○ Water treatment○ Surgical area, instruments, hospitals, schools,
storage, warehouses
Chemical MethodsUsing strong disinfectants
DisinfectionDisinfection
Heat DisinfectionEating utensils & clothingRemoves all non-sporing bacteria
Chemical DisinfectantsStrong: formalinMild: ethyl alcohol, iodine, soap
Sterilization of plant tissuesSterilization of plant tissues
Plant tissuesSodium hypochlorite (NaOCl): most
common to sterilize plant tissuesCalcium hypochlorite (CaOCl): less damage
than NaOClHydrogen peroxide (H2O2): easily removed
from tissuesOther substances: bromine water, silver
nitrate, mercuric chloride
CleaningCleaning
Glassware/plasticware in 10% commercial detergent liquid.
Wash with tap water (to remove detergent).
Rinse in double distilled water, and allow to dry overnight.
Sterilization TechniquesSterilization Techniques
Refrences
A text book of Biotechnology: R.C.Dubey
Plant Tissue Culture: S.S.Purohit www.wikipedia.org