for sem 5 GTU syllabus b.pharm

1. Presentation on
PLANT TISSUE
CULTURE
Basic & Aseptic Techniques
Media Preparation & Sterilization
By: Pooja H. Khanpara
APIP, Jamnagar

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 TechniquesBasic 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 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

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

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

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

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

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

14. MediaMedia
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 ComponentsMedia Components

16. Media ComponentsMedia Components

17. Media ComponentsMedia Components

18. 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

20. SterilizationSterilization

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

25. Sterilization of plant tissuesSterilization 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. 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.

27. Sterilization TechniquesSterilization Techniques

59. Refrences
A text book of Biotechnology: R.C.Dubey
PlantTissue Culture: S.S.Purohit
www.wikipedia.org
Pharmacognosy-III, sem -5 by Dr.A.M.Baheti and Mr. R.K.
Dumbre, pg no. 2-2 (GTU syllabus)

60. Thank you