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.

Melissa A. BernardoJessica S. Mondia

MASEd: General Science

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.

3 basic components are needed for the enzymatic digestionDigestive enzymes (pectinase, cellulase)

Buffer at an appropriate pH (citrate-phosphate, MES-NaOH, Tris malleate, pH of 5.5-5.8)-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

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.

PEG Fusion

Membrane Modifiers

Enzymation Digestion

Cell agglutination

PROTOPLAST FUSION

ISOLATION OF PROTOPLASTS

ReasonProcess

FUSION OF PLANT PROTOPLAST

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. PEG’s 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

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