(i) Isolation of protoplasts:
Plant cell consist of cell wall which has to be degraded if the protoplasts of the cell has to be obtained to be manipulated as required. For this purpose, the plant cell is treated with enzyme like pectinase, macerozyme, and cellulase etc., which hydrolysis the plant cell wall. The conditions are altered so that successful release of protoplast is aided. The osmotic pressure of the solution is controlled by addition of calcium chloride salts into it. This improves the plasma membrane activity. Since protoplasts are present in every plant cell it can be theoretically isolated from all the parts of plant. But most successful isolation was made possible from leaf of the plants. The leaf is surface sterilized and lower epidermis is removed, and treated with enzyme solution.
(ii) Fusion of different protoplasts:
Different protoplasts isolated are treated with different mechanisms so that they fuse together. The mechanisms followed:
High calcium high pH treatment:
Here, the different protoplasts in one solution are together treated with conditions like high calcium and high pH so that they fuse together. In some cases such extreme conditions has proved to be toxic to certain protoplasts.
Polyethlylene glycol (PEG) treatment:
This has proved to be one of the most effective methods for protoplast fusion. The cells are treated with a concentration of around 30% poly ethylene glycol which binds to plasma membrane. This is treated with calcium solution which being cationic binds to PEG. During washing, the PEG pulls out the plasmalemma leading to fusion of protoplasts in close proximity. This leads to fusion of protoplasts randomly and so is a non selective fusion process.
Electro fusion technique:
This process involves passing low voltage electric pulses in a solution of protoplasts to be fused so that they line up for fusion. The protoplast can be fused by subjecting it to brief exposure to high voltage electric current which leads to alteration of membrane so that the adjacent protoplast fuse. The protoplasts so lined up can be moved by the use of micromanipulator so that required protoplast can be fused. This is carried out in an electroporater.
Selection of hybrid cells
After a successful protoplasmic fusion experiments, a variety of structure are available like unfused protoplast and protoplast of same species fused together and protoplasts of different species fused together or hybrid cells. In order to separate the hybrid cells from other residue several techniques are followed. Mechanical isolation of fused protoplasts is done or taking advantage of natural properties exhibited by host cells so that the cells showing absence in that property indicate the hybrid cells. Another important method is by culturing all the residues and formation of calli which is then studied to identify the hybrids.
The hybrids formed are of different types like:
Symmetric hybrids: These contain the somatic chromosome of both the parental species. These are very significant as they show all the properties exhibited by parent species.
Asymmetric hybrids: These are those hybrids which preserve the genetic material of one parent organism. The chromosome content of other parent species is lost.
Cybrids: These consist of nucleus of one species and cytoplasm from both the species. They are produced by fusion of one species with another having enucleate protoplast or having inactivated nucleus or loss of chromosomes of one parent by repeated mitotic division. It can even be induced by inactivating nucleus of a protoplast prior to fusion. The cybrids produce many advantages like transfer of plasmagene of one species into the nuclear background of another species, formation of recombinants between mitochondrial or chloroplast genomes.
Thus somatic hybridization techniques help in forming wide variety of recombinants among the plasma gene of different species and plasmagenes and chloroplast genes. It also helps to form hybrid cells exhibiting chloroplast genome of one species and mitochondrial genome of another species which is not possible by ordinary means of hybridization of two plant species. These different levels of fusion and recombination helps in production of new species which has all the qualities of parent organisms or even better.