Tissue cultures:
*Cloning plants through tissue culture is an increasingly popular method throughout labs in both
commercial and private sectors.
Tissue culture consists of the following steps:
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Step 1. Preparation of culture medium – contains a mixture of macro and micronutrients, vitamins,
hormones, nitrogen supplements, amino acids, growth regulators and solidifying agents.
Step 2. Inoculation of explant – during this step the cultivated explant is sterilized and then carefully
transferred to a suitable container and placed on the nutrient medium
Step 3. Incubation of culture – prepared plant tissue cultures placed in a plant growth chamber or an
incubator at 25 – 30°C to support growth.
Step 4. Sub-culturing – to maintain adequate conditions for growth and development explants are
divided and transferred to new media.
Step 5. Transplantation – plants are acclimatized and then transplanted to soil
Today we will prepare a culture medium:
Equipment:
 Nitrile or latex gloves
 Erlenmeyer flask/Beaker – 1L
 Graduate cylinder – water measuring
 Bleach – for sterilizing
 Distilled water
 pH papers or probe
 Tablespoon
 Small jars/beakers - 10
 Microwave oven/ hot plate
 Rubber bands (10)
 Plastic wrap
 Sterile razor blade or utility knife
 Rubbing alcohol
 Spray bottle
 Sterile tweezers
 Small stir plate
 Pressure pot
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Ingredients for the medium:
Murashige and Skoog (MS) medium in single-use packets
Sugar
Plant Preservative Mixture (PPM)
NaOH
Acid
Agar
Plant hormones
Distilled water
Method:
1. Work on a clean and sterile area.
2. Add ultra-pure, laboratory-grade water to ~80% of the final volume to be prepared in an
Erlenmeyer flask or beaker that is at least twice the volume of medium being prepared
(800ml)
3. Add an appropriate-sized stir bar to the water and place flask or beaker on a stir plate; turn
on stir plate for moderate stirring action
4. While stirring, slowly add media components one at a time (except for gelling agent) and
allow to dissolve.
5. Add 30g sugar (use scale)
6. 4.5g MS (Micro and macro stock) (use precision scale)
7. 2ml PPM (antimicrobial) (micropipette)
8. 0.1ml hormones (micropipette)
9. 10g agar powder (use scale)
10. Carefully pour contents back into flask/beaker, add the stir bar, and place on stir plate near a
pH meter; add gelling agent (i.e. agar, gellan gum, agarose, etc.) and allow to mix while
stirring.
11. Calibrate pH meter with appropriate calibration buffers (generally pH 4 and 7 are sufficient),
then insert tip of probe into medium while stirring
12. Add appropriate acid or base solution (generally 0.1-1M hydrochloric acid or potassium
hydroxide) dropwise to medium for adjusting to desired pH*
13. Fill Erlenmeyer flask up to 1L if necessary.
14. Place flask/beaker on stir/hot plate and turn on heat to high while stirring moderately
15. Observing and wearing appropriate personal protective equipment, bring solution to a boil,
then promptly turn off heat to avoid boil over
16. Pour 10 glass containers/beakers with 100ml solution.
17. Place filled vessels in autoclave-safe containers and sterilize based on recommended
time for the volume of each vessel – use pressure cooker. (20minutes – 15Psi)
18. Wearing the proper protective equipment, carefully remove sterilized media from
autoclave and allow to cool in a clean environment.
19. Store the medium for use in the fridge.