• The protoplast, also termed as naked plant cell refers to all the components of a plant cell excluding the cell wall.
• The term protoplast was first introduced by Hanstein in 1880 to designate the living matter enclosed by plant cell membrane.
• The isolated protoplast is unusual as the outer plasma membrane is totally exposed and is the only barrier between the external environment and the interior of living cell.
• The isolation of protoplasts from plant cells was first achieved by microsurgery on plasmolyzed cells by mechanical method (Klercker, 1892).
•  However, the yields were extremely low and this method is not useful.
• Enzymes were first used by Cocking (1960) to release protoplasts.
• He isolated the enzyme cellulase from the culture of fungus Myrothecium verrucaria to degrade the cell walls.
• Then he applied an extract of cellulase hydrolytic enzyme to isolate protoplasts from tomato root tips.
• Since then, many enzyme formulations have been employed to isolate protoplasts, and the most commonly used enzymes are now commercially available.
• The use of cell wall degrading enzymes was soon regarded as the preferred method to isolate large numbers of uniform protoplasts.
•  Under suitable conditions, in a number of plant species, these protoplasts have been successfully cultured to synthesize cell walls and now genetic manipulations have also been made.
•  Protoplasts are useful for cell fusion studies. In addition, these can also take up, foreign DNA, cell organelles, bacteria or virus particles through their naked plasma membrane.

Methods of Protoplast isolation

1. mechanical methods
2. enzymatic methods.

Mechanical method of protoplast isolation

• In this method, large and highly vacuolated cells of storage tissues such as onion bulb scales, radish root and beet root tissue could be used for isolation.
• The cells are plasmolysed in an iso- osmotic solution resulting in the evacuation of contents in the centre of cell.
• Eventually, the tissue is dissected and deplasmolyzed to release the preformed protoplasts.
• Klercker (1892) isolated protoplasts from Stratiotes aloides by this method.
•  Mechanically, protoplasts have been isolated by gently teasing the new callus tissue initiated from expanded leaves of Saintpaulia ionantha which was grown in a specific auxin environment to produce thin cell walls.
• However, this method is generally not followed because of certain disadvantages.
• It is confined to certain tissues which have large vacuolated cells.
• Yield of protoplasts is generally very low. Protoplasts from less vacuolated and highly meristematic cells do not show proper yield.
• The method is time-consuming and laborious.
• Due to the presence of substances released by damaged cells viability of protoplasts is low.
• The mechanical method is useful when there are side effects of cell wall degrading enzymes.

Enzymatic method of protoplast isolation

• Cocking in 1960 exhibited the possibility of enzymatic isolation of protoplasts from higher plants.
• He used concentrated solution of cellulase to degrade the cell walls.
• However, there was little work for the next 10 years until the commercial enzyme preparations became available.
• Takebe et al. (1968) for the first time employed commercial enzyme preparation for isolation of protoplasts and subsequently regenerated plants in 1971.
• Protoplasts are now regularly isolated by treating tissues with a mixture of cell wall degrading enzymes in solution, which contain osmotic stabilizers.
• The relative simplicity with which protoplast isolation can be achieved relies upon a variety of factors which have been discussed below:
• Physiological state of tissue and cell material:
• Protoplasts have been isolated from a variety of tissues and organs including leaves, petioles, shoot apices, roots, fruits, coleoptiles, hypocotyls, stem, embryos, microspores, callus and cell suspension cultures of a large number of plant species.
• Mesophyll tissue from fully expanded leaves of young plants or new shoots is regarded as the simple and most appropriate source of protoplasts.
• Leaf tissue is preferred as it allows the isolation of a large number of relatively uniform cells without the necessity of killing the plants.
• Moreover, the mesophyll cells are loosely arranged and enzymes have an easy access to the cell wall.
•  Leaves are taken, sterilized and lower epidermis from the excised leaves is peeled off.
• These are cut into small pieces and then further processed for isolation of protoplasts.
•  Since the physiological condition of the source tissue noticeably affects the yield and viability of isolated protoplasts, the plants or tissues must be grown under controlled conditions.
• Callus tissue and cell suspension cultures used for protoplast isolation should be in the early log phase of growth.
•  Friable tissue having low starch content generally yields better results.
• Enzymes:
• The isolation of protoplasts  depends largely on the nature and composition of enzymes used to digest the cell wall.
• There are three primary components of the cell wall which are termed as cellulose, hemicellulose and pectin substances.
• Cellulose and hemicellulose are the components of primary and secondary structure of cell wall respectively, while pectin is a component of middle lamella that connects the cells.
• Pectinase mainly degrades the middle lamella, while cellulase and hemicellulase are needed to digest the cellulosic and hemicellulosic components of the cell wall respectively.
•  Cellulase (Onozuka) R10 frequently used for wall degradation has been partially purified from the molds of Trichoderma reesei and T. viride.
• Sometimes additional hemicellulase maybe essential for recalcitrant tissues and for this Rhozyme HP 150 has been used.
• The most commonly used pectinase is macerozyme (macerase) which has been derived from the Rhizopus fungus.
• The other enzyme driselase has both cellulolytic and pecteolytic activities and has been successfully used alone for isolation of protoplasts from cultured cells.
•  Besides, there are several enzymes, e.g. helicase, colonase, cellulysin, glusulase, zymolyase, meicelase, pectolyase, etc. which are employed to treat a tissue that does not release protoplasts easily.
• Aleurone cells of barley treated with cellulase results in protoplasts with the cellulase resistant cell wall.
• These cells called sphaeroplasts which are to be treated with glusulase to digest the remaining cell wall.
• The activity of the enzyme is depends on pH and is generally indicated by the manufacturer. However, in practice the pH of enzyme solution is adjusted between 4.7 and 6.0.
• Generally the temperature of 25–30°C is sufficient for isolation of protoplasts.
• The duration of enzyme treatment is to be determined after trials. However, it may be short for 30-min duration to long duration for 20 hrs.

Types of enzymatic method of protoplast isolation

• The enzymatic isolation of protoplasts can be performed in two different ways:
• Two step or sequential method:
  • The tissue is first treated with a macerozyme or pectinase enzyme which isolates the cells by degrading the middle lamella.
  • These free cells are then treated with cellulase which releases the protoplasts.
  • In general, the cells are exposed to different enzymes for shorter periods.
• One step or simultaneous method:
  • The tissue is placed to a mixture of enzymes in a one-step reaction which consists both macerozyme and cellulase.
  • One step method is commonly used because it is less laborious.
  • During the enzyme treatment, the protoplasts isolated need to be stabilized because the mechanical barrier of cell wall which served as support has been broken.
  •  For this reason, an osmoticum is added which prevents the protoplasts from bursting.

Protoplast Isolation- mechanical and enzymatic methods

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