This document provides protocols for preparing media for plant tissue culture, including constituents of the media such as inorganic salts, carbohydrates, vitamins, plant hormones, and solidifying agents. It describes that Murashige and Skoog medium is the most popular basal medium for shoot cultures. The document outlines steps for preparing the Murashige and Skoog medium, which involves measuring and mixing stock solutions of macronutrients, micronutrients, and plant growth regulators followed by adjusting the pH and autoclaving the media before use.

1. Protocols for media
preparation

2. Medium constituents
• Inorganic salt formulations
• Source of carbohydrate
• Vitamins
• Water
• Plant hormones - auxins, cytokinins, GA’s
• Solidifying agents
• Undefined supplements

3. Carbohydrates
• Plants in culture usually cannot meet their needs for fixed carbon.
Usually added as sucrose at 2-3% w/v.
• Glucose or a mixture of glucose and fructose is occasionally used.
• For large scale cultures, cheaper sources of sugars (corn syrup) may
be used.

4. Photoautotrophic culture
• Growth without a carbon source.
• Therefore need to boost photosynthesis.
• High light intensities needed (90- 150mMole/m2/s) compared to
normal (30-50).
• Usually increase CO2 (1000ppm) compared to normal 369.4ppm.
• Much reduced level of contamination and plants are easier to transfer
to the greenhouse.

5. Inorganic salt formulations
• Contain a wide range of Macro-elements (>mg/l) and microelements
• A wide range of media are readily available as spray-dried powders.
• Murashige and Skoog Medium (1965) is the most popular for shoot
cultures.
• Gamborgs B5 medium is widely used for cell suspension cultures (no
ammonium).

6. Vitamins
• A wide range of vitamins are available and may be used.
• Generally, the smaller the explant, the more exacting the vitamin
requirement.
• A vitamin cocktail is often used (Nicotinic acid, glycine, Thiamine,
pyridoxine).
• Inositol usually has to be supplied at much higher concentration
(100mg/l)

7. Plant hormones
• Auxins
• Cytokinins
• Gibberellic acids
• Ethylene
• Abscisic Acid
• “Plant Growth Regulator-like compounds”

8. ‘Plant Growth Regulator-like substances’
• Polyamines - have a vital role in embryo development.
• Jasmonic acid - involved in plant wound responses.
• Not universally acclaimed as plant hormones since they are usually
needed at high concentrations.

9. Undefined supplements
• Sources of hormones, vitamins and polyamines.
• e.g. Coconut water, sweetcorn extracts
• Not reproducible
• Do work.

10. Protocols
INTRODUCTION
• Murashige and Skoog medium (MS) is the most suitable and most
commonly used basic tissue culture medium for plant regeneration
from tissues and callus.
• This is a “high salt” medium due to its content of K and N salts.

11. Materials
• Beakers (1000, 500, and 100 ml)
• graduated cylinders (1000, and 100 ml)
• conical flasks(100 ml)
• reagent bottles (1000, 500, and 100 ml)
• micropipettes (1000 µl)
• distilled water

12. • magnetic stirrer
• Spatulas
• chemical balance
• weighing boat
• Tissue
• pH meter
• Aluminium foil
• autoclave machine

13. Chemicals
• Stock solutions of macronutrient
• Micronutrient
• Iron source
• Vitamins
• Plant growth regulators such as BAP and NAA
• Sucrose
• inositol
• Agar
• NaOH and HCl to adjust pH.

15. Method
1) Five hundred millilitres of MS medium was made. The required
volume of each stock solution were calculated and obtained into
500 ml beaker on a magnetic stirrer.
2) 15 g sucrose and 0.05 g inositol were added, and then stirred until
fully dissolved.
3) The volume was adjusted to about 500 millilitres with distilled
water.
4) 2 ml (2000 µl) of NAA was added (to get 4 mg/L NAA) then pH was
adjusted to 5.75 ±0.05 with NaOH and HCl.

16. Conti……
5) 3.1 g of agar was added and stirred for complete mixing.
6) The medium was heated up in microwave oven to dissolve the agar.
7) Medium was transferred into 500 ml reagent bottle. Bottle was
labelled before autoclaving.
8) The culture medium was autoclaved for 15-20 min at 121°C.
9) After autoclaved, medium was distributed approximately 20 ml to
each sterile disposable petri dishes in the laminar air flow and
medium are allowed to solidify followed by exposing to UV light for
20 minutes and stored for use.