1. Presentation on
Basic & Aseptic Techniques
Media Preparation & Sterilization
By:
Aarti Dwivedi
Aarti Nagdev
Ankit Jha

2. Basic Steps of PTC

3. 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.

4. 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.

6. Basic Techniques
 Setting up of a tissue culture lab
requires proper planning.
 It is divided into 5 areas
Media preparation room
Aseptic transfer area
Culture room
Analytical room
Acclimatization room

7. Media 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

8. Aseptic Transfer Area
 Laminar air flow
 Dissecting microscopes
 Dissection instruments
 Gas outlet
 Vacuum facility
 Sterilizer

9. Culture Room
 Environmentally controlled
 Incubators with controlled temperature
 Rotary shakers
 Lux meter
 Space for cultures requiring complete
darkness

10. Analytical Room
 Colorimeter
 Low speed centrifuge
 Inverted centrifuge
 Chemical reagent racks
 Viscosity meter
 Gas outlet

11. Acclimatization Room
 High illumination(4,000-10,000 lux)
 High humidity(90-100% through mist &
fog systems)

12. Miscellaneous Items
 Air conditioners
 Uninterrupted power supply
 Bunsen burners
 Aluminium foils
 Fluorescent lamps
 Fire fighting equipment

14. Media
 No single medium supports growth of all
tissues.
 Some basic factors
Callus induction
Organogenesis
 Murashige-Skoog medium, White’s
medium, woody plant medium

15. Media Components

16. Media Components

17. Media Components

18. MS media
 Macronutrients •Common organic additives
 Ammonium nitrate (NH4NO3): 1,650 mg/l •i-Inositol: 100 mg/l
 Boric acid (H3BO3): 6.2 mg/l •Niacin: 0.5 mg/l
•Pyridoxine · HCl: 0.5 mg/l
 Calcium chloride (CaCl2 · 2H2O): 440 mg/l
•Thiamine · HCl: 0.1 mg/l
 Cobalt chloride (CoCl2 · 6H2O): 0.025 mg/l •IAA: 1–30 mg/l
 Magnesium sulfate (MgSO4 · 7H2O): 370 mg/l •Kinetin: 0.04–10 mg/l
 Cupric sulfate (CuSO4 · 5H2O): 0.025 mg/l •Glycine (recrystallized):
 Potassium phosphate (KH2PO4): 170 mg/l 2.0 g/l
•Edamine (ethane-1,2-
 Ferrous sulfate (FeSO4 · 7H2O): 27.8 mg/l
diamine): 1.0 g/l
 Potassium nitrate (KNO3): 1,900 mg/l
•Sucrose: 20 g/l
 Manganese sulfate (MnSO4 · 4H2O): 22.3 mg/l •Agar: 10 g/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

20. Sterilization

21. Sterilization
 Physical methods
Filtration
○ Reducing microbial population in heat
sensitive solutions
○ 2 types of membrane filters
 Gradocol membrane
 Cellulose membrane
○ Air sterilization: HEPA filters in LAF

22. Sterilization
 Physical Methods
Radiation
○ UV lamps placed in ceilings or in biological
safety cabinets
○ Water treatment
○ Surgical area, instruments, hospitals, schools,
storage, warehouses
 Chemical Methods
Using strong disinfectants

23. Disinfection
 Heat Disinfection
Eating utensils & clothing
Removes all non-sporing bacteria
 Chemical Disinfectants
Strong: formalin
Mild: ethyl alcohol, iodine, soap

25. Sterilization of plant tissues
 Plant tissues
Sodium hypochlorite (NaOCl): most
common to sterilize plant tissues
Calcium hypochlorite (CaOCl): less damage
than NaOCl
Hydrogen peroxide (H2O2): easily removed
from tissues
Other substances: bromine water, silver
nitrate, mercuric chloride

26. Cleaning
 Glassware/plasticware in 10%
commercial detergent liquid.
 Wash with tap water (to remove
detergent).
 Rinse in double distilled water, and allow
to dry overnight.

27. Sterilization Techniques

28. Refrences
 A text book of Biotechnology:
R.C.Dubey
 Plant Tissue Culture: S.S.Purohit
 www.wikipedia.org