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iii.  Feeder layer : In order to culture protoplast at low density, a feeder layer technique is adopted. A feeder layer of X-ray irradiated non-dividing but metabolically active protoplasts after washing are plated in soft agar medium. Non-irradiated protoplasts of low density are plated over this feeder layer. The protoplasts of the same species or different species can be used as a feeder layer.

 

iv.  Co-culturing : This method involves co-culture of protoplasts from two different species to promote their growth or that of the hybrid cells. Metabolically active and dividing protoplasts of two types - slow and fast growing are cultured together, the fast growing protoplast provide other species with diffusible chemicals and growth factors which helps in cell wall formation and cell division. The co-culture methods is generally used where calli arising from two types of protoplasts can be morphologically distinguished. For example, protoplasts isolated from albino plants and green plants are easily distinguishable based on color where albino protoplast will develop non green colonies.

 

Figure 12.3: protoplast culture techniques