PLANT TISSUE CULTURE :

• In vitro culture of plant cells, tissues as well as organs.
• Totipotency is the ability of a plant cell to perform all the functions of development which are characteristic of zygote, i.e., its ability to develop in to a complete plan.

IMPORTANT TISSUE CULTURE TECHNIQUES OF IMPORTANCE TO

PLANT PATHOLOGY:

A. Production of virus free plants through plant tissue culture:

Meristem tip culture: Cultivation of axillary or apical meristems, particularly of shoot

apical meristem, is known as meristem culture.

1. Explant:

• The explant must consist of the meristematic dome of cells together with atleast one leaf primordial.
• The infected parent plant or organ of the plant from which explant is excised is generally subjected to thermotherapy in a temperature controlled cabinet at 30 0 C to 40 0 C for six to twelve weeks to inactivate the virus.

2. Culture initiation on suitable medium:

• In general Murashige and Skoog medium has been found satisfactory for most plant species
• Culture initiation consists of surface sterilization of explants and establishing them in vitro on culture medium.
• Culture initiation often involves anti-metabolite chemicals such as ribavirin (virazole) in the tissue culture medium.

3. Shoot multiplication:

• After 2-3 weeks, the cultures are transferred to a shoot multiplication medium designed to promote axillary branching
• Higher concentration of cytokinins induces adventitious buds.
• During culture initiation and shoot multiplication phases, the cultures are generally kept at 25 0 C.

4. Rooting of shoots:

• In general, the rooting medium has low salt (1/2 or even 1⁄4 salts of MS medium) and reduced sugar levels.
• Rooting takes about 10-15 days depending on species.

5. Transfer of plantlets to soil:

• Rooted shoots are removed from the medium, agar sticking to roots is washed with tap water, and they are transplanted into plastic cups containing a suitable potting mix.
• Plants are kept in high (>90%) humidity and initially low light intensities.
• The humidity is generally decreased to the ambient level after about 7-15 days, and the light intensity is increased.
• The plants are finally exposed to greenhouse conditions

6. Indexing, clone selection and stock maintenance:

• Virus indexing is generally made by Enzyme Linked Immuno-Sorbent Assay (ELISA) or Immuno Sorbent Electron Microscopy (ISEM) for commercial multiplication.

B. Protoplast culture:

• Interspecific, intraspecific and intrageneric hybridization could be done by this technique for strain improvement of biocontrol agents to enhance the biocontrol potential for the management of pathogenic fungi.
• Isolation and self-fusion of protoplasts were achieved in Trichoderma harzianum and T. viride.

Steps in protoplast fusion:

1. Isolation of protoplasts is achieved by treating cells with a suitable mixture of cell wall degrading enzymes.

2. The pH of enzyme solution is adjusted between 4.7 and 6.0 and temperature is kept around 25-30 0 C. The osmotic concentration of enzyme mixture and of subsequent media is elevated to stabilize the protoplasts and to prevent them from bursting.

       Usually, 50-100 m mol/l CaCl 2 is added to the osmoticum as it improves plasma membrane stability.

3. The protoplasts of different strains are treated with 28-50% Poly Ethylene Glycol (fusogen) for 15-30 min followed by gradual washing of the protoplasts to remove PEG.

      The washing medium may be alkaline and contain high calcium ion concentration (50 m mol/l). Protoplast fusion occurs during washing step.

4. Selection of hybrid cells and culturing on suitable medium.