Preparation of plant tissue culture media,types and Sterilization

Preparation of plant tissue culture media,types of Media,types of sterilization.

1. University of Agricultural Sciences, Department of Plant Biotechnology, GKVK, Bengaluru-560065 Topic : Preparation of plant tissue culture media, types and sterilization. Basic Concepts in Laboratory Techniques ( 0+1) Submitted by, SUBHAS PICHELI PALB9316 Submitted to , Dr.Shyamalamma,S. Professor Dept of Plant Biotechnology GKVK, Bengaluru

2. A growth medium or culture medium is a liquid or gel designed to support the growth of cells or small plants. Any liquid or solid preparation made specifically for the growth, storage, or transport of microorganisms or other types of cells. Robert Koch (1843-1910) could be considered the father of culture media. INTRODUCTION

3. In vitro culturing of plant tissue involves the following steps: Sterilization of glassware tools/vessels Preparation and sterilization of explant Production of callus from explants Proliferation of cultured callus Sub-culturing of callus Suspension culture GENERAL PROCEDURES INVOLVED IN PLANT TISSUE CULTURE

4. Media preparation is one of the primary and most essential steps in tissue culture. Media is prepared based on the type of tissue being cultured. Most media differentiate from each other based on the requirements of the growth of the specimen it supports. To understand the basic process of preparing media for plant tissue culture to promote. Growth and differentiation of tissues and acknowledge the basic requirements for the plant Tissue culture. To prepare culture medium based on plant species requirements. To prepare stock solution consists of macronutrient, micronutrient and organic elements. To prepare culture medium by using aseptic technique. Preparation of the tissue culture media

5. Media preparation room should have sufficient space to accommodate chemicals, lab ware, culture vessels and equipments required for weighing and mixing, hot plate, pH meter, water baths, Bunsen burners with gas supply, microwave oven, autoclave or domestic pressure cooker, refrigerator and freezer for storage of prepared media and stock solutions . MEDIA PREPARATION ROOM

6. Adding inorganic salts Adding organic compounds pH adjustment Adding gelling agents Dissolving by heating and stirring Dispensing Autoclaving Ready for culture after 24 hours Preparation of the Medium

7. This is a very crucial step for the experiment to be successful. While making the media taking individual constituents, each ingredient is separately weighed and dissolved before putting them together. After making up volume by water, pH is adjusted and then medium is autoclaved. Preferably, following four stock solutions are prepared: • Major salts (20X concentration) • Minor salts (200X concentration) • Iron (200X concentration) • Organic nutrients (200X concentration)

8. Separate stock solution for each growth regulator is prepared. Appropriate quantities are taken from stocks and mixed to constitute basal medium. Required quantity of agar, sucrose and organic supplements if needed are added separately

9. Types of Media Media is a substance of supporting and provide energy and food to the developing explant . They are of following type Solid and Liquid (Suspension culture Media) depending in their appearance. 2. Depending on their concentration as well as constituents it can varies by the type of explant and species.

10. TYPES OF MEDIA ACCORDING TO THEIR COMPOSITION Murashige and Skoog used for plant regeneration and organogenesis White`s Medium used for root and ovule culture B5 / Gamborg Medium is a type of cell suspension used for callus culture N6/ Chu for cereal anther culture Nitsch`s used for anther culture

11. Media Sterilization STERILIZATION ROOM For the sterilization of culture media, a good quality ISI mark autoclave is required and for small amount domestic pressure cookers, can also serve the purpose. For the sterilization of glassware and metallic equipments hot air oven with adjustable tray is required.

12. Culture vessel Nutrient medium Instruments used during various operation Explant Operator Environment of the tissue culture laboratory Possible sources of contamination

13. Used for glassware, metal instruments etc. dry-heating in an oven at 160-1800C for 3 hours approx. (1 hr heat up period to reach to temp and cooling period) Wrapping in aluminum foil. Cannot be used for plastic ware, however, certain plastic wares can also be heat sterilized (Instructions of the manufacturer must be read before doing this) Dry heat

14. • Autoclaving (steam under pressure) or a home pressure cooker steam pressure of 1.05kg/cm2 (temperature 121 0 C ) for 15- 45 minutes. • Time required for autoclaving varies with the volume of liquid to be sterilized • Do not close the escape valve until a steady steam comes out of autoclave • Actual time of autoclaving should be started when proper temperature is reached • Over autoclaving should be avoided • Once autoclaving is over, pressure must not lost rapidly, it should be allowed to return to atmospheric pressure slowly as rapid loss of pressure will lead to vigorous boiling of liquids inside the culture vessels • It should be opened when the pressure is zero as this might cause accidents Wet heat

15. Now a days pre-sterilized ready-to-use plastic ware is available, which can be used directly to pour medium etc. Certain components of medium like Zeatin , GA3, pantothenic acid, antibiotics etc are thermolabile and cannot be autoclaved These can be sterilized by membrane filtration and added to autoclaved medium once it has cooled down to ~ 400C.

16. filter membranes of pore size 0.45 µm or less are used Filter assemblies of different sizes are available Once the component is filter sterilized, it is collected in a sterile container which can be used immediately or dispensed in smaller amounts to be used later These filter sterilized components can be stored at 40C or -200C depending on the frequency of their usage. Filter sterilization

17. Instruments : • Sterilized by dipping in 95% ethanol followed by flaming and cooling. • Glass bead sterilizer and infra-red sterilizer are available commercially operated by electricity these instruments are safer and not a fire hazard. • Glass bead sterilizer has glass bead in a heated cavity where a temperature of nearly 2500C is maintained instruments are pushed into the cavity for 5-7s. • Infrared sterilizer also has a cavity where a temperature of nearly 7000C can be achieved by infrared wave heating. Exposure of 2-5 s is effective for sterilization of instruments like Scalpel, holder, Forceps (Small, Medium & Large).

18. Growing in nature and exposed to a variety of microbes so it is a very rich source of contaminants and needs to be surface sterilized before inoculation into the medium Variety of surface sterilizing agents are available which vary in their efficacy and toxicity Too strong treatment can kill the explants whereas too mild treatment may not yield any sterile explant So sterilizing treatment is selected on the basis of the state of explants explants are hardy and apparently contaminated a strong treatment can be given if explants are juvenile and soft, a mild treatment should be preferred In certain cases surface sterilization of the actual explant may not be required. e.g. for culturing the immature ovule the whole ovary is surface sterilized and the ovule is dissected out under aseptic conditions Generally adding few drops of surfactant in the sterilizing solution enhances its efficiency. Plant material

19. Steam sterilization/Autoclaving (121°C at 15 psi for 20-40 min) Nutrient media, culture vesels , glasswares and plastic wares Dry heat (160-180°C for 3h) Instruments (scalpel, forceps, needles etc.), glassware, pipettes, tips and other plasticwares Flame sterilization Instruments (scalpel, forceps, needles etc.), mouth of culture vessel Filter sterilization (membrane filter made of cellulose nitrate or cellulose acetate of 0.45- 0.22 μ m pore size) Thermolabile substances like growth factors, amino acids, vitamins and enzymes. Alcohol sterilization Worker’s hands, laminar flow cabinet Surface sterilization (Sodium hypochlorite, hydrogen peroxide, mercuric chloride etc) Explant . Technique Materials sterilized

20. Thank you