Isolated protoplasts can be cultured in a liquid medium , or semisolid agar medium , either in thin layers or tiny drops of nutrient medium in petridish. The medium used to cultivate protoplasts is the same as that needed for suspension or callus culture.
The increase in calcium concentration aids in maintaining the membrane’s integrity. The media generally require a greater quantity of sugar. The growth and vitamins are employed as needed to promote cell division as well as callus formation. differentiation.
The density of plating is an additional factor in the development of protoplasts. one x 10 to 1 protoplast per ml is the ideal number and such density is beneficial for early division of protoplasts from plants, whereas the density will decrease as the culture progresses.
Stages of Protoplast Culture
Protoplasts have the development of four phases. The protoplast that is viable in culture creates its own wall surrounding them and then is ready for cell division. The process of cell division in sequence results in the formation of calluses.
Through embryo-genetic or organogenetic differentiation, embryos or plants can develop. As they’re all derived from the same protoplast, they are all identical in genetic material.
Regeneration of the cell wall is the initial requirement for cell division. After the formation of walls, cells grow and split into two cells evenly which is shaped like “8”. After the initial division, each daughter cell splits into two cells. Each division is followed by an appearance of a cell clumps as well as cell aggregates.
All cells derived from protoplasts don’t undergo division. Numerous factors like the nature of the donor plant and hormones, culture medium and physical factors are crucial to the division of protoplasts as well as callus development. Following the formation of calluses, these are then sub-cultured on a regular basis to differentiate further making use of different hormone combinations. This is known as the medium for regeneration of plants.
Techniques of Protoplast Culture
There are many methods for protoplast culture , such as the liquid, Agar, droplet culture, coculture, hanging droplet cultivation, beads culture immobilised and feeder layer techniques.
1. Liquid Culture
This technique is typically preferred for the majority of cases in the early protoplasts’ development since it permits an easy transfer and dilution. protoplasts can easily divide in liquid mediums, and the and the osmotic pressure in the medium is regulated and reduced as protoplasts grow. The downside to this technique is it doesn’t allow the isolation of single colonies that originate from a single parent cell.
2. Agar Culture
Agarose is the most commonly used to solidify protoplast culture medium. Protoplast suspensions are prepared at double density, mixed with melted agar medium 45 degrees Celsius. The mixture is then well mixed and then plated into small peridish. In this case, the protoplasts remain in the their original position and remain immobilized. the proper efficiency of plating can be achieved, but the change in medium can be made only following clear calli development.
3. Droplet Culture
Suspending protoplasts in liquid culture medium can be placed onto petridishes, in the form of droplets. The cultured protoplasts form a clump in the middle of droplets. The liquid medium is changed regularly.
In order to stimulate division, The newly separated protoplast suspension is mixed an established, fast-growing protoplast suspension. The mixed protoplasts are then plated. Certain growth factors aid in stimulate the growth and development of isolated protoplasts.
5. Hanging Droplet Method
The culture of protoplasts is performed with an inverted droplet placed on the inside that is the cover of the petridish A small amount of protoplasts can be cultivated by this method. An extremely thin film of medium liquid is maintained inside the petridish to keep the surrounding environment within the petridish dry.
6. Bead Culture
The protoplasts suspension is mixed with any polymer such as alginate, carrageenan or any other. Then, small beads are formed by dripping in the medium. They are later, they are cultivated into the liquid medium using a slow shaking conditions.
7. Feeder Layer
In most cases, it is beneficial to reduce the density of plating. In this case, a feeder layer made up of non-dividing, X-irradiated protoplasts is coated in agar media and then this layer, isolated protoplasts are plated with the form of a very thin liquid medium. These living, non-dividing protoplasts fulfill the requirements for the smaller amount of protoplasts.