In this article we will discuss about the stages and methods of protoplast culture.
Isolated protoplasts are cultured either in a liquid medium or semisolid agar medium in a thin layer or as small drops of nutrient medium in petridish. The medium for protoplast culture requires the same component as required for callus or suspension culture.
Increasing the calcium concentration only helps to maintain the integrity of the membrane. Generally the media require more amount of sugar. The vitamins and growth substances are used as per requirement for cell division, callus formation and then differentiation.
Plating density is another important criterion for protoplast culture, a density of 1 x 104 to 1 x 105 protoplast per ml is optimal, such high densities are helpful for earlier division of plant protoplasts whereas the density is reduced subsequently during progress of culture.
Stages of Protoplast Culture:
Protoplast culture has mainly four stages of development. The viable protoplast in culture regenerates its own wall around them and then it is prepared for cell division. Sequential cell division leads to callus formation.
From this callus by organogenetic or embryo-genetic differentiation the plantlets or embryos may develop (Fig. 20.2). But as these are all derived from a single protoplast so all regenerated are of same kind of genetic constituent.
Cell wall regeneration is the first prerequisite for cell division, after wall formation the walled cells expand and divide into two cells equally which looks like ‘8’. After the first division each daughter cell divides into two cells. Repeated division results in the formation of cell clump or cell aggregates.
All the cells derived from protoplasts do not undergo division. Several factors such as genotype of donor plant, culture medium, hormones as well as physical factors are important factors for division of protoplasts and callus formation. After callus formation these are sub-cultured at regular intervals for further differentiation by using different hormone combinations which is called plant regeneration medium (Fig. 20.3).
Methods of Protoplast Culture:
There are different methods of protoplast culture such as liquid culture, agar culture, droplet culture, co-culture, hanging droplet culture, immobilised/bead culture and feeder layer technique (Fig. 20.4A-D).
1. Liquid Culture:
This method is generally preferred in most cases during early developmental stages of protoplasts, because it allows easy dilution and transfer, protoplasts easily get divide in liquid media, osmotic pressure of the medium can be regulated and can be effectively reduced during further growth of protoplasts. The disadvantage of this method is that it does not permit the isolation of single colonies derived from one parent cell.
2. Agar Culture:
Agarose is most frequently used to solidify protoplast culture media. Protoplast suspension is taken at double density and mixed with melted agar medium at 45°C and mixed well and plated in small petridish. Here the protoplasts remain in same position and immobilised, proper plating efficiency can be obtained but the medium change can be done only after visible calli formation.
3. Droplet Culture:
Suspending protoplasts in liquid culture media are placed on petridishes in the form of droplet, the cultured protoplasts clump together at the centre of droplets. The liquid medium can be changed at regular interval.
Sometimes to induce division the newly isolated protoplast suspension is mixed with a reliable fast growing protoplast suspension and mixed protoplasts are plated. Some growth factors help to induce the proper growth and development of the isolated protoplasts.
5. Hanging Droplet Technique:
Culture of protoplasts can be done in an inverted droplet on the inner surface of the lid of petridish, a very small number of protoplasts can be cultured in this way. A thin layer of liquid medium is kept in the petridish to keep the environment inside the petridish humid.
6. Bead Culture:
The protoplasts suspension can be mixed with any kind of polymer like alginate, carrageenan, etc. and then small beads are made by dripping into the liquid medium and then cultured into liquid medium with slow shaking condition.
7. Feeder Layer:
In many cases it is desirable to reduce the plating density, then a feeder layer consisting of X-irradiated non-dividing but living protoplasts are plated in agar medium and on this layer the isolated protoplasts are plated in a thin layer of liquid medium. Here the living but non-dividing protoplasts provide necessary growth requirement for the isolated less number of protoplasts.