Culture Media pH and Preparation Quiz

Study Notes

Preparation of Media

pH of culture media generally drops by 0.3 to 0.5 units after autoclaving and changes throughout the culture period due to oxidation and differential uptake and secretion of substances by growing tissue.
Media preparation is a crucial step for a successful experiment.
Each ingredient is separately weighed and dissolved before combining, and pH is adjusted before autoclaving.
Four stock solutions are prepared: Major salts (20X concentration), Minor salts (200X concentration), Iron (200X concentration), and Organic nutrients (200X concentration).
A separate stock solution is prepared for each growth regulator.

Selection of a Suitable Medium

A suitable medium can be devised by trying many combinations of different concentrations of major components like PGRS, salt, and sucrose.
Various combinations of low, medium, and high concentrations of minerals, auxins, cytokinin, and organic nutrients can be evaluated to get a suitable medium.

Plant Growth Regulators

Gibbrellins and abscissic acid are lesser used PGRs.
Gibbrellic acid (GA3) is used for internode elongation and meristem growth.
Abscissic acid (ABA) is used for somatic embryogenesis and for culturing woody species.

Solidifying Agents

Agar is a high molecular weight polysaccharide obtained from seaweeds and can bind water.
Agar is added to the medium in a concentration ranging from 0.5% to 1% (w/v).
Agar is preferred over other gelling agents because it is inert and does not react with media constituents or get digested by plant enzymes.
Agarose, a purified extract of agar, is used for protoplast culture.
Alternative gelling compounds like gelrite form clear gels and are easier to detect contamination.

pH

pH affects the absorption of ions and solidification of gelling agents.
Optimum pH for culture media is 5.8 before sterilization.
pH values lower than 4.5 or higher than 7.0 greatly inhibit growth and development in vitro.

Micronutrients

Manganese plays an important role in the Hill reaction of photosynthesis and is required in many enzymatic activities.
Copper plays an important role in photosynthesis and is an intermediate of the electron transport chain.
Molybdenum is essential for the conversion of Nitrate to Ammonium.
Boron is involved in different enzymatic activities.
Iron is an important enzyme co-factor and is supplied in μM quantities.

Media Formulations

White's medium is one of the earliest plant tissue culture media developed for root culture.
MS medium is widely used for many types of culture systems.
B5 medium was originally designed for cell suspension and callus cultures.
N6 medium is used for cereal anther culture.
Nitsch's medium is frequently used for anther cultures.

Synthetic and Natural Media

Synthetic media are composed of chemically defined components.
Natural media contain chemically undefined compounds (e.g., vegetable extract, fruit juice, plant extract).
Synthetic media have almost replaced natural media for tissue culture.

Expression of Concentrations

Concentrations of inorganic and organic constituents in culture media are usually expressed as mass values (mg/l or ppm or mg/l).
The International Association of Plant Physiology recommends expressing concentrations of macronutrients as mmol/l and micronutrients as μmol/l.

Sterilization Techniques

Sterilization techniques used in plant tissue culture include steam sterilization/autoclaving, dry heat, flame sterilization, filter sterilization, and alcohol sterilization.
Surface sterilization is used for explants, worker's hands, and the laminar flow cabinet.

Description

Test your knowledge on the changes in pH of culture media due to autoclaving and tissue growth, as well as the crucial steps involved in preparing culture media. Learn about weighing individual constituents, dissolving them, adjusting pH, and more.