2.4. Protoplast viability : The isolated protoplast must have a spherical shape when observed by a light microscope, protoplast can be stained using following stain:

• Fluorescein diacetate staining method: FDA accumulates inside the plasmalemma of viable protoplasts. Live protoplasts contain esterases which cleave FDA to release fluorescein which fluoresces yellowish-green using fluorescence microscopy within 5 min. FDA dissociates from membrane after about 15 min. It is used at a concentration of 0.01% dissolved in acetone.

•  Calcofluor White staining: This staining method assures protoplast viability by detecting onset of cell wall formation. Calcofluor binds to beta linked glucosides in newly synthesized cell wall which can be observed as a fluorescent ring around the membrane. Optimum staining is achieved when 0.1 ml of protoplast is mixed with 5.0 μl of 0.1% w/v solution of CFW.

•  Protoplast viability can also be detected by monitoring oxygen uptake of cells by oxygen electrode, which shows respiration.

•  Variation of protoplast size with changing osmotic concentration also enables viability of protoplast.

 

3.  Protoplast culture

3.1. Protoplasts culture techniques

The culture requirements and the culture methods are same for both protoplasts and single cells. The main difference is the requirement of suitable osmoticum for protoplasts until they regenerate a strong wall. Isolated protoplasts are either cultured in liquid or semisolid agar or agarose media plates, sometimes the protoplast is first grown in liquid media and then transferred into the agar media plates. The following techniques have been adopted in order to maintain number of protoplast population between minimum and maximum effective densities after plating up:

i.  Liquid method : This method is preferred in earlier stages of culture as it provides (a) easier dilution and transfer, (b) the osmotic pressure of liquid media can be effectively reduced after a few days of culture (c) the cell density can be reduced or cells of special interest can be isolated easily. In Liquid medium, the protoplast suspension is plated as a thin layer in petriplates, incubated as static culture in flasks or distributed in 50-100 μl drops in petriplates and stored in a humidifier chamber.

ii.  Embedded in Agar/ Agarose : Agarose is a preferred choice in place of agar and this has improved the culture response. This method of agar culture keeps protoplast in fixed position, thus, prevents it from forming clumps. Immobilized protoplasts give rise to clones which can then be transferred to other media. In practice, the protoplasts suspended in molten (40°C) agarose medium (1.2% w/v agarose) are dispensed (4ml) into small (3.5-5cm diameter) plates and allowed to solidify. The agarose layer is then cut into 4 equal sized blocks and transferred to larger dishes (9 cm diameter) containing liquid medium of otherwise the same composition. Alternatively, protoplasts in molten agarose medium are dispensed as droplets (50-100 μl) on the bottom of petri plates and after solidification the droplets are submerged in the same liquid medium.