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