FUSION OF PLANT PROTOPLAST

Process

Reason

ISOLATION OF PROTOPLASTS

The removal of cell wall is a prerequisite in the fusion of plant protoplasts. This can be done either mechanically or with enzymatic techniques to form a naked cell or protoplast Cell walls should be removed in order to cause rupture in the plasmodesmata connections , releasing unconnected protoplasts.

Enzymation Digestion

3 basic components are needed for the enzymatic digestion Digestive enzymes (pectinase, cellulase) Buffer at an appropriate pH (citrate-phosphate, MES-NaOH, Tris malleate, pH of 5.55.8)-used for digestion of protoplasts osmoticum used to stabilize protoplast, should be compatible with the tissue type used, (isotonic or hypotonic solution of mannitol, sorbitol or sucrose

PROTOPLAST FUSION

1. The cell membranes of adjacent protoplasts must be in close physical contact to induce fusion or spontaneous fusion 2. Formation of lipid enriched regions of the plasma membrane from the diffusion of membrane surface proteins that may involve the lipid-lipid interaction between adjacent membranes.

Cell agglutination

The use of PEG (polyethylene glycol) as agglutinator (to alter the membrane to make it susceptible to fusion) and also serves as membrane modifier. This process is necessary so that there is clumping of protoplast that may result in a mass of cell that precipitate at the bottom of the slide, in this period of tight adhesion, adjacent cells may begin to fuse The surface charge of the plasma membrane is modified because the negative protoplast surface charge must be eliminated or reduced. Normally isolated protoplasts do not fuse with each other because their surface carries negative charges around the outside of the plasma membrane and thus they have the tendency to repel each other due to their similar charges.

Membrane Modifiers

PEG Fusion
PEG is used because of its dual role as agglutinator and as membrane modifier. The IMP (intramembraneous particles) should be lessened to make the area lipid-enriched for it to undergo changes in membrane fluidity and is to make them specific sites for membrane fusion. PEGs role is to dehydrate the cell, and bind the phospholipids within the cell membranes, to form such areas. PEG is also responsible for forming tight complex of polymeric chains of PEG between the membrane bound proteins of the aggregating cells, thus necessary so that the membranes fuse together.
Melissa A. Bernardo Jessica S. Mondia MASEd: General Science