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The following points highlight the nine main techniques of protoplast culture. The techniques are: 1. Agar Plating Method 2. Micro-Chamber 3. Nurse Culture 4. Immobilization of Protoplast 5. Nutrient Requirement 6. Growth Regulators 7. Cultural Conditions 8. Cell Wall Regeneration 9. Regeneration.
Technique # 1. Agar Plating Method:
Protoplast can be cultured conveniently by using agar plating method. Two millilitres of protoplast suspension in culture medium is mixed with same amount of molten agar medium of the same composition in petri dish. The temperature of the medium is maintained at 4°C before mixing with protoplast suspension, which is then incubated in inverted position at 27°C. The petri dish is then sealed with paraffin to avoid desiccation.
Technique # 2. Micro-Chamber:
Construction of micro-chamber provides miniature or micro-space for initial growth and division of protoplast. Designing of micro-chamber is a simple efficient technique. One millilitre of protoplast in nutrient medium is placed in cavity space.
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Two cover slips are placed on the slide at a parting distance of 22 mm. Another cover slip is placed above, sealed completely with paraffin mineral oil and incubated under well humid conditions to avoid desiccation. Protoplasts undergo few cell divisions in micro-chamber and placed on semisolid medium.
Technique # 3. Nurse Culture:
It is a novel technique of protoplast culture in which protoplast may be obtained by certain vital nutrient factors diffused from mother callus. Initially callus is procured from mother plant and placed in the culture vessel containing shallow layer of liquid medium.
The protoplast to be cultured is placed on the filter paper disc kept above the callus tissue within the test tube. The key advantage of this method is the speedy mobilization of nutrient factors towards protoplast from nutrient medium as well as from callus.
Involvement of callus tissues as nursing effect for growth of protoplast is exemplary. Following initial growth, protoplast is then shifted to semi solid medium. It is one of the classic examples of cross feeding by nurse or feeder cells or medium previously conditioned by fast growing cells. The nurse culture has been very effective in the successive culture of protoplast of rice and wheat.
Technique # 4. Immobilization of Protoplast:
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Imprisonment of protoplast embedded in the gel matrix placed on medium was reported to be indispensable for sustainable division. Utility of gel matrix for immobilization would provide mechanical support to the protoplast. This process can enhance protoplast viability and maintains membrane integrity by inhibiting enzymatic lipid peroxidation.
In addition, diffusion of larger molecules and metabolites facilitates cell wall synthesis and finally stimulates division. This would allow combined advantage of high and low density culture and avoid aggregation. Agar or agarose are generally employed for immobilization of protoplast.
Technique # 5. Nutrient Requirement:
Composition of medium used for cell and protoplast culture shows greater degree of similarities. Composition of several media is based on the formulations of MS or B5 media. However, presence of reduced nitrogen, ammonium ion, proved to be toxic for protoplast viability in several species.
Technique # 6. Growth Regulators:
Protoplast culture requires both auxin and cytokinin in various proportions. Auxins like 2,4-D, NAA and IAA at the concentration of 0.03 to 5 mg in combination with cytokinins like kinetin, benzyl amino purine and zeatin from 0.5 to 5 mg per litre in order to facilitate growth and development.
Technique # 7. Cultural Conditions:
Protoplast requires diffused dim light because strong light inhibits protoplast division. Therefore, initial stage of protoplast culture requires complete darkness or is to be maintained at low light intensity. Temperature requirement for protoplast culture is preferred between 24 and 26°C. In addition, cold treatment or heat shock at 45°C/5 min is useful for several species. Most of the protoplasts grow well at pH 5.5 to 5.7.
Technique # 8. Cell Wall Regeneration:
Regeneration of cell wall begins immediately when protoplasts are subjected for culture. Synthesis of cell wall material and its assembly on plasmalemma begins within few hours and takes two to several days to complete the process. Newly acquired cell wall can be confirmed by plasmolysing the cell or staining with calcoflour white fluorescence of chosen concentration (0.1%).
More evidence of cell wall regeneration can be obtained by electron microscope and other techniques. Cell wall regeneration begins within few hours was evidenced when deposition of cell was material takes place. Formation of common wall may be invariable when protoplast is cultured at very high density, i.e., almost touching each other.
Aggregation of protoplast may lead to common wall formation and initiates chemical tissue. Once regeneration of cell wall completes, protoplast undergoes one or two divisions to form cell clumps or without undergoing cytokinesis. Once small colonies are produced, further growth is reduced. These are picked up and transferred on medium.
Technique # 9. Regeneration:
Regeneration of plant from protoplast has been found to be successful only in few plants, not exclusive due to its recalcitrant status, but due to lacuna in the medium conductive for differentiation (Table 10.1). Supplementing appropriate ratio between auxin and cytokinin can induce differentiation and morphogenesis. Besides, embryogenesis could occur in auxin free medium.
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In tobacco, differentiation of calli into shoots within 4 weeks in the medium is supplemented with IAA and cytokinin at 2:1 ratio. One of the major controlling factors for the induction of embryogenesis from protoplast derived callus is 2, 4-D. Further development of embryo and germination takes place in auxin free medium. Recently, effect of electrostimulation on embryo as well shoot formation has been reported in several species.
