Media for Stevia Tissue Culture 

In almost all the stevia micro-propagation experiments, Murashige and Skoog medium (MS medium) is employed. It is also most commonly used medium in plant tissue culture laboratories. Basic MS medium is to be supplemented with different plant growth application for specific type of tissue culture experiments – like callus induction, shoot induction, rhizogenesis etc.

Basal MS medium is often available in a powdered prepared form from different suppliers. For application in tissue culture, it needs to be dissolved in required quantity of water. Sometimes, specific carbon source and gelling agents are required to be added. The prepared medium is dispensed in culture vessels and is sterilized by autoclaving. After autoclaving and partial cooling, filter sterilized plant growth promoters are directly added to the medium. Then, the medium is allowed to cool and incubated for 2 – 3 days to check for contaminations. Contamination free media are then inoculated with plants explants of choice.

Composition of Basal Murashige and Skoog medium

Other major components

Carbon Source :

Sucrose (PTC grade) : Use level 2% - 5%

Gelling agent for solid and semi-solid media

Agar agar : Use level 0.8-1.0%

Water

Demineralized or double distilled water

Chemicals for pH regulation

Sodium hydroxide AR grade

Hydrochloric acid AR grade

MS Medium.jpg
Off the shelf prepared MS Basal Medium

Plant growth regulators

Auxins

It is of two types: natural which includes indole acetic acid (IAA) and synthetic which includes indole-3- butyric acid (IBA), 2,4-dichlorophenoxy-acetic acid (2,4-D) and naphthalene- acetic acid (NAA).

It is required for the cell division and cell elongation processes. Depending on its concentration, it can lead to the formation of callus (at high concentration) or root (at low concentration).

Cytokinin

The various cytokinins used in culture media are BAP (6-benzyloaminopurine), 2iP (6-dimethylaminopurine), Kinetin (N-2-furanylmethyl-1H-purine-6-amine), Zeatin (6-4-hydroxy-3-methyl-trans-2-butenylaminopurine), and TDZ (thiazuron-N-phenyl-N-1,2,3 thiadiazol-5ylurea).

It stimulates cell division which results in shoot formation and axillary shoot formation. It is also involved in the retardation of root formation. The most commonly and widely used cytokinins in the culture media are kinetin and benzyl-amino purine.

Procedure for preparation of MS Medium from individual components

For routine preparation of MS medium, five different stock solutions are first prepared. The composition of the sock solutions are provided in the table at the left. The stock solutions are either prepared in 10X concentration or 40X concentration.

Then, MS medium is prepared using those stock solutions. For preparation of 1000 ml MS medium, the stock solutions are mixed with water in the following ratio, other solid components are also added to it and a homogeneous mix is prepared.  

The pH of the medium is then adjusted to 5.7 - 5.8 with the help of 0.5 M sodium hydroxide or hydrochloric acid solution. 

Then the mix is heated in a water-bath or in a microwave oven to completely dissolve the gelling agent. The medium is then dispensed into the culture vessels. The culture vessels are then closed with appropriate caps/enclosures/lids. The medium in the culture vessels are then sterilized by autoclaving them under 1.1. kg/cm² pressure for 15 - 20 minutes.

After autoclaving, the media is taken out from the autoclave and they are allowed to cool to a temperature of approximately 45°C. The required plant growth promoters, and other thermolabile components are dissolved in appropriate solvents and filter sterilized with a syringe filter fitted with 0.2µ membrane filter. The solutions of thermlabile components are then directly added to the partially cooled MS medium with sterile pipette or droppers.  

The next step is incubating the prepared media at 28°C for 2 - 3 days. If any contamination appears in any of the media, that is discarded. The media, which are found to be free from all contaminations are used for inoculating the explants. 

Media preparation 1.jpg
Media preparation 2.jpg
Media preparation 3.jpg