Culture Media pH and Preparation Quiz

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.