Protoplasts are plant cells that are completely naked with no cell wall however they do have plasma membranes and other components of the cell. They are functional cells, but without the presence of a barrier, cell wall. Protoplasts from various species can be merged to create a hybrid, and this is known as somatic hybridization (or protoplast fusion). Cybridization is the process of the fusion of protoplasts of normal size with nucleated (without nucleus) protoplast, resulting in the creation of a cybrid, also known as cytoplast (cytoplasmic hybrids).
The term”protoplast” was coined at the time of 1880 by Hanstein. The first protoplast was isolated was made through Klercker (1892) by using the mechanical method. The first real breakthrough in protoplast research was achieved in the year 1960 by Cocking who employed an enzyme method to aid in the elimination of cell walls.
Rakabe and his collaborators (1971) had success to create a entire tobacco plants from protoplasts. Rapid progress took place after 1980 in protoplast-to-proplast fusion, which improved the genetic material, as well as the creation for transgenic crops.
Absorption of Macromolecules, Viruses, and Organelles by Protoplasts
Protoplasts be able to absorb organelles, foreign substances and other molecules through the process of phagocytosis. This demonstrated that one cell is susceptible to infection by more than one virus particle, and that different virus can replicate within one cell. In addition, protoplasts were transformed using Ti-DNA from Agrobacterium Tumefaciens that could be used to use in genetic engineering. Absorption by plastids allows an examination of the cooperation with the nuclear as well as genomes of the plastids. Absorbed foreign genes could also make up for the absence of defects in that protoplast’s genome. The fusion between protoplasts that were inactivated from Physalis and Datura brought a nitrate reducetase activity to the mutant form of Nicotiana tabacum that lacks nitrate reductase. The transformation proved stable.
Modern methods of analysis, such as gel electrophoresisand evidence of the activity of enzymes in a gel or isoenzymes proves the effectiveness of fusion studies. Ribulose – 1,5 – bisphosphate carboxylase serves as a marker with a significant purpose since it is composed of subunits encoded by nuclear and plastidic. In addition, it provides the study of this protein for information regarding the functions of the plastids as well as the nucleus of the parent species within the product of fusion.
After protoplast techniques were established, it was the creation and later isolation of mutants vital. Auxotrophic species, i.e. strains that depend on the availability of certain nutrients, were of particular importance. Very few results were obtained One reason could be due to the fact that a significant part of angiosperm genomes is polyploid and that genetic information is present in many copies, even in haplotypes. This could easily be compensated for a deficiency gene. Additionally, plants are capable of producing the same product through different metabolic pathways. One possible solution to the issue could be through the use of monohaploid stocks (1 x instead 1 n). If it is the case with cultivars, would this suggest that the original plants must be discovered first.
What is Protoplast Culture?
The principle of protoplast cultivation is the aseptic removal of a large amount of protoplasts in good health, by eliminating their cell wall to cultivate them in an appropriate nutrient-rich medium to support their development and growth.
Protoplast is isolated by a variety of tissues in plants. A good and convenient source is mesophyll of the leaf as well as cells grown in liquid suspension cultures. Protoplast viability and yield are greatly affected by the condition of the plant and the cells.
The most important step in the isolation of protoplasts is the elimination of the cell wall, without damaging the protoplasts or the cell. Plant cells are an osmotic device. The cell wall exerts upward pressure on protoplasts in the enclosure. In the same way, the protoplast exerts pressure of equal or opposite on the wall of the cell.
Therefore, both pressures are equal. If you remove the wall of your cell taken away and the pressures balanced be altered. This means that the pressure outward of protoplast will increase and, simultaneously without a cell wall, the irresistible protoplast expansion occurs because of the massive flow of water out of an external source.
A greater pressure outward and an expansion in protoplast causes it to explode. Thus, the protoplast that is isolated is an osmotically fragile and fragile structure when it is in its infant stage.
So when the cell’s wall is being removed to isolate the protoplasts, the cells or tissue should be placed into a hypertonic liquid of sugars that are metabolically inert like mannitol in a greater concentration (13 percent) to help plasmolysis the cells away and away from its cell walls.
Mannitol, a alcoholic sugar, which is easily transferred across the plasmodesmata. It creates a stable osmotic milieu for protoplasts and wards off the normal expansion and rupture of protoplasts even after the removal of the cell wall. This is the reason the hypertonic substance is also known as osmotic stabilizer, also known as plasmolyticum or the osmolyticum.
When the cells are stabilized in this manner by plasmolysis , protoplasts are released from their cell wall that is encased by it, either mechanically or enzymeally. Mechanical isolation requires breaking the walls of every cell compartment in order to free the protoplast.
The procedure can be carried out with care on small portions of tissue under a microscope by using the micro-scalpel. However, very few protoplasts can be produced with much energy and time. The most extensive attempts to isolate mechanically involves the disintegration of tissue with a the use of a fine stainless steel-bristled brush.
This method could release more protoplasts in lesser effort, however, the yield percentage of intact protoplasts remains low. A more effective method to free the protoplasts is by digesting the cell walls that surround them using cells wall-degrading enzymes such as cellulase or hemicellulase. macerozyme, pectinase and so on. These enzymes are isolated from fungi and can be purchased on the market.
Treatment duration and concentration of enzymes are most important factors, and both should be defined for each plant tissues. Intact tissue may be treated with macerozyme or pectinase solution, which dissolves the middle layer between cells and thus separate them.
The subsequent treatment with cellulase is expected to remove the cellulosic layers of cells. This process is referred to as sequential treatment of enzymes or two step treatment instead of the mixed treatment (one step procedure) that is where both macerozyme and cellulase are combined so that the whole wall can be disintegrated in a single process.
The protoplasts that are isolated can be grown in the static liquid or in an agarified medium. The protoplast medium is composed of minerals and carbon sources, vitamins and growth hormones from plants and stabilizers of osmotic nature and, perhaps organic nitrogen sources such as coconut milk and organic acids. In the culture, protoplasts may create a new cell wall within them. When the wall is created and the protoplast is transformed into an actual cell.
The protoplast cells then start to divide into cells, and this will be followed by the creation of callus and cells. These callus also have the potential for morphogenesis as well as plant regeneration.
Application for protoplasts
- Protoplasts can be utilized to investigate membrane biology, such as the absorption of macromolecules as well as viruses.
- They are also employed in somaclonal variations.
- Protoplasts are extensively employed to transform DNA (for creating genetically altered organisms) as the cell wall can hinder the entry of DNA into the cell.
- In the case of plant cells protoplasts can be transformed into complete plants by forming a group of plant cells which then develop into a callus, followed through the regeneration of the shoots (caulogenesis) within the callus, using techniques for tissue culture in plants.
- Protoplasts can also be utilized for breeding purposes, using an approach known as protoplast fusion.
- It is used to create somatic hybrids within tissue culture. In this technique, Protoplasts of different species are stimulated to fuse using the electric fields or mixture that contains polyethylene glycol.
- In addition, protoplasts of plants that express fluorescent proteins in some cells could be utilized to perform Fluorescence Active Cell Sorting (FACS) in which only cells that fluoresce at a specific wavelength are kept.
- Utilized to identify particular cells (e.g. the guard cells that are found in leaves and pericycle cells from roots) for further research like transcriptomics.