The first step in protoplast culture is the development of a cell wall around the membrane of the protoplast. This is followed by the cell divisions that give rise to a small colony. With suitable manipulations of nutritional and physiological conditions, the cell colonies may be grown continuously as cultures or regenerated to whole plants. Protoplasts are cultured either in semisolid agar or liquid medium. Sometimes, protoplasts are first allowed to develop cell wall in a liquid medium and then transferred to agar medium.

  • Agar culture:
    Agarose is the most frequently used agar to solidify the culture media. The concentration of the agar should be such that it forms a soft agar gel when mixed with the protoplast suspension. The plating of protoplasts is carried out by Bergmann’s cell plating technique.In agar cultures, the protoplasts remain in a fixed position, divide and form cell clones. The advantage with agar culture is that clumping of protoplasts is avoided.
  • Liquid culture:
    Liquid culture is the preferred method for protoplast cultivation for the following reasons:
    1. It is easy to dilute and transfer.
    2. Density of the cells can be manipulated as desired.
    3. For some plant species, the cells cannot divide in agar medium, therefore liquid medium is the only choice.
    4. Osmotic pressure of liquid medium can be altered as desired.

The culture media with regard to nutritional components and osmoticum are briefly described.

Nutritional components:
In general, the nutritional requirements of protoplasts are similar to those of cultured plant cells (callus and suspension cultures). Mostly, MS and B5 media with suitable modifications are used. Features of protoplast culture media are listed below:
1. The medium should be devoid of ammonium, and the quantities of iron and zinc should be less.
2. The concentration of calcium should be 2-4-times higher than used for cell cultures. This is needed for membrane stability.
3. High auxin/kinetin ratio is suitable to induce cell divisions while high kinetin/auxin ratio is required for regeneration.
4. Glucose is the preferred carbon source by protoplasts although a combination of sugars (glucose and sucrose) can be used.
5. The vitamins used for protoplast cultures are the same as used in standard tissue culture media.

Osmoticum and osmotic pressure:
Osmoticum broadly refers to the reagents/ chemicals that are added to increase the osmotic pressure of a liquid.
The isolation and culture of protoplasts require osmotic protection until they develop a strong cell wall. In fact, if the freshly isolated protoplasts are directly added to the normal culture medium, they will burst.
Thus, the addition of an osmoticum is essential for both isolation and culture media of protoplast to prevent their rupture.

The osmotica are of two types — non-ionic and ionic.

  1. Non-ionic osmotica:
    The non-ionic substances most commonly used are soluble carbohydrates such as mannitol, sorbitol, glucose, fructose, galactose, and sucrose. Mannitol, being metabolically inert, is most frequently used.
  2. Ionic osmotica:
    Potassium chloride, calcium chloride, and magnesium phosphate are the ionic substances in use to maintain osmotic pressure. When the protoplasts are transferred to a culture medium, the use of metabolically active osmotic stabilizers (e.g., glucose, sucrose) along with metabolically inert osmotic stabilizers (mannitol) is advantageous. As the growth of protoplasts and cell wall regeneration occurs, the metabolically active compounds are utilized, and this results in the reduced osmotic pressure so that proper osmolarity is maintained.

Culture Methods:
The culture techniques of protoplasts are almost the same that is used for cell culture with suitable modifications. Some important aspects are briefly given.

  1. Feeder layer technique:
    For the culture of protoplasts at low-density feeder layer technique is preferred. This method is also important for selection of specific mutant or hybrid cells on plates. The technique consists of exposing protoplast cell suspensions to X-rays (to inhibit cell division with good metabolic activity) and then plating them on agar plates.
  2. Co-culture of protoplasts:
    Protoplasts of two different plant species (one slow growing and another fast growing) can be co-cultured. This type of culture is advantageous since the growing species provide the growth factors and other chemicals which help in the generation of the cell wall and cell division. The co-culture method is generally used if the two types of protoplasts are morphologically distinct.
  3. Micro drop culture:
    Specially designed dishes namely cuprak dishes with outer and inner chambers are used for micro drop culture. The inner chamber carries several wells wherein the individual protoplasts in droplets of nutrient medium can be added. The outer chamber is filled with water to maintain humidity. This method allows the culture of fewer protoplasts for droplet of the medium.

Regeneration of Protoplasts:
Protoplast regeneration which may also be regarded as protoplast development occurs in two stages:
1. Formation of cell wall.
2. Development of callus/whole plant.

  1. Formation of cell wall:
    The process of cell wall formation in cultured protoplasts starts within a few hours after isolation that may take two to several days under suitable conditions. As the cell wall development occurs, the protoplasts lose their characteristic spherical shape. The newly developed cell wall by protoplasts can be identified by using calcofluor white fluorescent stain.The freshly formed cell wall is composed of loosely bound microfibrils which get organized to form a typical cell wall. This process of cell wall development requires a continuous supply of nutrients, particularly a readily metabolized carbon source (e.g. sucrose). Cell wall development is found to be improper in the presence of ionic osmotic stabilizers in the medium. The protoplasts with proper cell wall development undergo normal cell division. On the other hand, protoplasts with poorly regenerated cell wall show budding and fail to undergo normal mitosis.
  2. Development of Callus/whole Plant:
    As the cell wall formation around protoplasts is complete, the cells increase in size, and the first division generally occurs within 2-7 days. Subsequent divisions result in small colonies, and by the end of the third week, visible colonies (macroscopic colonies) are formed. These colonies are then transferred to an osmotic-free (mannitol or sorbitol-free) medium for further development to form callus. With induction and appropriate manipulations, the callus can undergo organogenic or embryogenic differentiation to finally form the whole plant.
    Plant regeneration can be done from the callus obtained either from protoplasts or from the culture of plant organs. There are, however, certain differences in these two calluses. The callus derived from plant organs carries preformed buds or organized structures, while the callus from protoplast culture does not have such structures.

The fragments derived from protoplasts that do not contain all the contents of plant cells are referred to as sub-protoplasts. It is possible to experimentally induce fragmentation of protoplasts to form sub-protoplasts. This can be done by application of different centrifugal forces created by discontinuous gradients during centrifugation. Exposure of protoplasts to cytochalasin B in association with centrifugation is a better approach for fragmentation of protoplasts.

There are three types of sub-protoplasts

1. Mini-protoplasts:
These are also called as karyoplasts and contain the nucleus. Mini-protoplasts can divide and are capable of regeneration into plants.
2. Cytoplasts:
These are sub-protoplasts containing the original cytoplasmic material (in part or full) but lack nucleus. Thus, cytoplasts are nuclear-free sub-protoplasts which cannot divide, but they can be used for cybridization.
3. Micro-protoplasts:
This term was suggested for sub-protoplasts that contain not all but a few chromosomes.