Two protoplasts can be made to fused by the application of
A. Electrofusion
B. Polyethylene glycol
C. Sodium nitrate
D. All the above

Hint: A protoplast can be defined as a cell without a cell wall or cell membrane. The membrane or walls are permeable to only selected materials. For the deliberate introduction of new products into the cell, these protective layers are removed. This can be done both mechanically and chemically.

Complete answer: The protoplast is the inner cellular mass consisting of cytoplasm as well as the organelles. The protoplasts are devoid of the cell wall or cell membrane. Protoplast formation is generally used in genetic modification experiments. These experiments mostly consist of the deliberate introduction of DNA into the host protoplasm. The cell walls or membranes exhibit a barrier to this technology and hence are dissolved or removed using certain enzymes. The protoplasts are fused to form required hybrids generally in plant biotechnology. There are various methods to fuse two protoplasts. These can be mechanical or chemical. The mechanical method can be electrofusion. Electrofusion is a technique in which an electric current is used to make the cell walls porous. This exposure to electric fields results in the fusion of parent cells. Polyethylene glycol and sodium nitrate are used in chemical protoplast fusion experiments. This is called the chemi fusion of protoplasts. Polyethylene glycol involves the treatment of protoplasts with calcium ions that shocks the protoplasts to fuse. Sodium nitrate application involves the suspension of protoplast cells in 4 ml of \[5.5%\] sodium nitrate in $10.2%$ sucrose solution. After this, the suspended protoplast pellets are kept in the water bath and the protoplasts fuse during the incubation period.
Hence, the correct answer is option D.

Note: Protoplast fusion also called somatic cell fusion provides a mean through which two parental genomes can be recombined. This is used in plants that cannot reproduce sexually. Also, protoplast fusion is helpful in studying cytoplasmic genes and their activities. The information from these fusions can be applied in plant breeding experiments.