Protoplasts Regeneration and Isolation Plant Tissue Culture
Protoplasts Regeneration and Isolation (PTC) Protoplasts are cells that have had their cell walls removed, and possess plasma membrane and all other remaining cellular components. That is, it contains all the functional part of the cell without the barrier of the cell wall. This removal of the barrier allows the fusion of protoplast with another protoplast of different species to generate a hybrid, process known as somatic hybridization (or protoplast fusion).
A protoplast can also be fused with an enucleated (without nucleus) protoplast to generate a cybrid or cytoplast (cytoplasmic hybrids), a process known as hybridization.
A protoplast can resynthesize the cell wall, undergo repeated mitotic division, produce the daughter cells and eventually regenerate the entire plant, under suitable conditions. Hence a viable plant protoplast is totipotent.
Protocols for the regeneration of the entire plant from protoplast have been established for many plant species.
• Methods of plant protoplast isolation:
The protoplast isolation from plants can be performed by two different methods: the mechanical method and the enzymatic method. The choice of method depends on the availability and sensitivity of the cell type.
- 1. Mechanical method:
Klerceker carried out the first mechanical isolation of protoplasts in 1892 from the tissue of Stratiotes aloides L. (water soldiers or water pineapple). In this method, the cells are first plasmolyzed and then the tissue is cut. The cells which are just cut below the cell wall and the undamaged protoplast can be released either spontaneously or by agitation. This method yields low numbers of protoplasts and requires a large amount of cell source.
- 2. Enzymatic method:
Enzymes were first employed to isolate protoplasts from green plant material by Cocking (1960). He used cellulase from the fungus Myrothecium verrucaria to isolate protoplasts from root tips of Lycopersicum esculentum (Tomato). The mixture of enzymes can be used to attain optimum isolation (for example a pectinase, a cellulase, and a hemicellulase). The plant enzymes that are used can be divided into two categories namely;
– Pectinases: which dissolve the middle lamella and separating individual cells.
– Cellulase and hemicellulase: which decompose the cell wall and release the protoplast.
The protoplast isolation using enzymes can be carried out either as a two-step procedure or a one-step procedure.
- 1. Two-step procedure
This involves, as the name suggests, two different treatment in two different steps. In the first step, the macerozyme or macerate, a type of pectinase, is used to degrade the middle lamellas to release individual cells from the tissue. In the next step, the cells are treated with cellulase which dissolves the cell wall, leaving behind protoplasts. The enzyme treatment is of much shorter duration in the two-step method than in the one-step method.
2. One-step procedure
This technique is used more often than the two-step method. In this method, the tissues are mechanically loosened (e.g. by cutting strips) followed by the treatment with the mixture of enzymes, that is, pectinase and cellulase. Both the enzymes are added together in the same step for a little longer period of time.
Factors Influencing the Isolation of Protoplasts
- Plant material
- Different tissues and organs of the plant can be chosen to obtain the protoplast. The different organs that can be used for the isolation of protoplast are leaves, shoot apices, roots, coleoptiles, hypocotyls, petioles, embryos, pollen grains, calli, cell suspensions. However, the most reliable source of protoplasts are the cells of leaf mesophyll from which a high number of relatively uniform cells can be isolated. In vitro plants can yield double the amount of protoplast as compared to in vivo The type of source material, the composition of its cell wall, presence of other external layer and the cementing material in between the cells affect the isolation procedure to a large extent. The composition of the cell wall also influences the choice of enzymes.
- The composition and type of enzymes affect the isolation procedure. The density and the viability of isolated protoplasts also depend on the concentration, the period, pH and temperature of the enzyme treatment. The ratio of the enzymatic solution to the volume of the plant tissue also affects the outcome of the isolation procedure.
3. Osmotic conditions
- To avoid the osmotic shock that the protoplast can experience after the loss of the cell wall in the surrounding solution, the osmotic pressure of the solution is adjusted accordingly. The surrounding solution could be the enzymatic mixture, washing solution or the culture medium. The solution is fortified with mannitol, sorbitol, glucose or sucrose to adjust the osmotic pressure. The protoplasts are more stable in a slightly hypotonic environment rather than in isotonic solution.
Purification of Protoplasts
It is necessary to purify the protoplast and remove the isolation enzymes, undigested fragments of tissues and damaged protoplasts for the successful cultivation of protoplasts. For the purification combination of filtration, centrifugation and washing steps are employed.
The solution containing protoplasts is filtered through a metal or nylon sieve (50–100 μm) to remove larger parts of undigested tissue and cell clusters. The damaged cells and enzymes removed by repeated centrifugation (3–10 min, 75–100 × g) and resuspension in washing medium. If a lot of residues are present, 20% sucrose can be used to cover the washing medium. The protoplasts will form a ring on the upper layer of sucrose, while organelles and cell residues remain in the pellet at the bottom of the centrifugation test tube.
Viability and density of protoplasts
It is really important to check the viability and the density of the batch of protoplast isolated for the experiments following the isolation. Ideally, the sample should have high viability and sufficient density of the isolated protoplasts. To check the viability of the isolated protoplasts several procedures are used:
- Fluorescein diacetate (FDA), dyes the viable protoplasts and fluoresces under the fluorescent microscope
- Evans blue, living protoplasts do not let the pigment through the membranes
- Neutral red pigment (concentrated only in metabolically active cells)
- Observing the cytoplasmic flow as the indication of active metabolism
- Calcofluor MR2 or calcofluor white, the renewal of the cell wall is detected with the help of a fluorescent microscope.
Cultivation of Protoplasts
The important parameters for the successful cultivation of the protoplasts are the composition of the culture medium (especially the content of sugars), the temperature and the intensity of light during the incubation.
The liquid medium is usually preferred for the cultivation of the protoplasts since it makes possible to easily regulate the osmoticum in the medium, better regulation of the density of cells, easier change of the culture medium and isolation of the cells during the cultivation process.
The isolated protoplasts can be induced to regenerate, preferably, to give a whole plant. However, there are a few problems encountered during the protoplast regeneration. The protoplast could get damaged during the isolation procedure due to which their viability could significantly decrease. The damage could be reduced by shortening the duration of the treatment of the cells with degradative enzymes used during protoplast isolation. The culture conditions should be devised for some species. The regeneration conditions should be optimized for each species, this will increase the percentage of the regenerated protoplast. Each step should be carefully designed and observed to get the highest rate of regeneration from the procurement of the source to the cultivation condition and handling. Every chemicals and reagent should be of great quality and quantity should be carefully optimized