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Culture Media - The nutrient requirements of isolated protoplasts are very similar to those of cultured cells and tissues. However, since the protoplasts are devoid of a cell wall, they tend to be very efficient in uptake of nutrients from the medium. Mostly the B5 and MS media with some modifications have been found suitable. Reduction in levels of inorganic substances, especially ammonium, proves to be detrimental to protoplast survival, iron and zinc, is usually necessary.

Calcium concentrations are increased two to four times as calcium is important for protoplast membrane stability and integrity. Organic growth factors of MS and B5 likewise need to be carefully controlled in the modified media for protoplast culture and standardization for each species is imperative.

Compared to these simple media, Kao and Michayluk (1975) and Kao (1977) used rather complex media for the culture of a single protoplast of Vicia hajastana; this has proved useful for regeneration of protoplasts of many species.

Generally protoplast culture media contain 3-5% sucrose but in some systems (tobacco) a lower sugar (1.5%) content is required. Organic nitrogen in the form of CH is usually included in the medium as reduced nitrogen and NH4N03 (20 mmol 1-1) as reduced inorganic nitrogen. The vitamins used for protoplast culture are the same as used in standard tissue culture media.

Both types of growth substances (auxins and cytokinins) are used in the protoplast culture media in various combinations in order to induce cell wall formation and divisions in isolated protoplasts. 2,4-0 as the sole auxin source leads to the loss of morphogenetic potential in the protoplast derived callus. Other auxin sources are NAA or IAA.

The commonly used cytokinins are BAP, kinetin, 2-IP, or zeatin. Although the exact combination of the two types of growth hormones in the medium varies according to the species, it has been observed that protoplasts from actively growing cell cultures may find a high auxin/kinetin ratio suitable to induce divisions, while those derived from highly differentiated cells (leaf tissue) require a high kinetin/auxin ratio for regeneration.

Proper maintenance of osmolality of the culture medium is essential for stability, viability, and subsequent growth of protoplasts. During isolation and culture protoplasts require osmotic protection until they regenerate a strong wall. Inclusion of an osmoticum in both isolation and culture media prevents rupture of protoplasts.
The most widely used osmotica in a protoplast culture medium as \Well as in an enzyme mixture are sorbitol, mannitol, glucose or sucrose. Protoplasts are more stable in a slightly hypertonic solution.

For mesophyll protoplasts (cereals and pea) both sorbitol and mannitol have proven suitable as an osmotic stabilizer; to culture protoplasts of potato, sweet pea, brome grass, and cassava, sucrose has been preferred over glucose or mannitol.

In tobacco suspension culture both galactose and fructose have been used as osmotica. The osmolarity of the medium is gradually reduced by periodic addition of a few drops of fresh medium as soon as the protoplasts have regenerated walls and undergone divisions.

 
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