plant tissue culture is a diverse field including many techniques and modes for culturing of cells and tissues for research and studies.

1. PROTOPLAST CULTURE AND ITS
APPLICATIONS

2. A protoplast is a plant, bacterial or
fungal cell that had its cell wall completely or
partially removed using either mechanical or
enzymatic means.
It is described as living matter enclosed
by a plant cell membrane.

3.  Protoplast culture is the aseptic isolation & culture of protoplast in
vitro so as to obtain a viable plant.
I. ISOLATION OF PROTOPLAST:
IT CAN BE ACHIEVED BY TWO METHODS-
1. MECHANICAL
In this method, the cells are kept in suitable plasmolyticum (for
example CPW containing 13% w/v mannitol). Once the plasmolysis is
complete, while remaining in the osmoticum, the leaf lamina would be cut
with a sharp-edged knife. In this process some of the plasmolyzed cells are
cut only through the cell wall, releasing intact protoplasts.The released
protoplasts then have to be separated from damaged ones and cell debris.
Tissue damage is caused by mechanical method maximum tissue chopping with
lower protoplast yields.

4.  In this method leaf is sterilized followed by peeling of the
lower epidermis to release cells which are plasmolyzed and
then added to enzyme mixture followed by harvesting of
protoplast.
 At first, the cells are separated by the use of a maceration
enzyme – a pectin hydrolyzing enzyme(macerozyme or
Pectolyase). Once the cells are separated, they are washed in
CPW solution free of enzymes but containing plasmolyticum
by gentle centrifugation.
 Plant tissues are plasmolyzed in the presence of a mixture of
pectinases and cellulases, thus, inducing simultaneous
separation of cells and degradation of their walls to release the
protoplasts directly in a single step.

5.  Enzyme treatment results in suspension of
protoplast, undigested tissues and cellular
debris. This suspension is passed through a
metal sieve or a nylon mesh (50-100 µm) in
order to remove undigested cellular clumps.
The filtered protoplast-enzyme solution is
mixed with a suitable volume of osmoticum,
solution is centrifuged to pellet the
protoplasts, pellet of protoplast is
resuspended in osmoticum of similar
concentration as used in enzyme mixture.

6.  This can be done by two methods:-
 Feeder Layer Technique:
 In order to culture some cells, particularly at low or clonal
density, it is necessary to use a layer of less fastidious cells to
condition the medium. Often the cells are irradiated or otherwise
treated so that they will not proliferate.The protoplasts plated
into an agarified medium are exposed to X-ray dose, which
inhibites division of cells but allowed them to remain
metabolically active.After repeated washing of these inactivated
protoplasts, they are plated in agar medium and the non-irradiated

7.  Co-CultureTechnique:
 In this technique protoplasts of two different species are
cultured together for promotion of growth. On co-
culturing of two different metabolically active protoplasts,
there is a cross feeding between two types, which
enhances their sustained division at low density.This
technique is particularly suited where calli arising from
morphologically distinguishable protoplasts

9. Formation of Callus and Plantlets
Soon after the first division of the cultured protoplasts, subsequent divisions yield small cell
colonies on suitable semisolid medium. Macroscopic colonies appear within 2-3 weeks of
culture which can be then transferred to an osmoticum-free medium for callus development.
Mostly the regeneration of the plantlets has been achieved through callus mediated
organogenesis.

10. Culture medium
 The nutritional requirement of protoplast is almost similar to that of the cultured plant cells. Mostly the
MS (Murashige and Skoog) and B5 (Gamborg ) media and their modifications have been used.
 Ammonium salts have been found detrimental to protoplasts survival of many species.
 Concentration of zinc is less while the concentration of calcium is more as it enhances the membrane
stability. Osmolarity is maintained by addition of sorbitol, mannitol, glucose or sucrose and mannitol
being widely used osmoticum as it is not used by the dividing cells, thus, maintains the osmolarity of
the medium.
 Glucose is preferred carbon source. One or two amino acids are added at low concentration.
 Growth regulators are required essentially in protoplast culture generally high concentration of auxins
(NAA, 2,4-D) along with lower concentration of cytokinins (BAP, Zeatin ) is used. High light intensity
inhibits growth of protoplast hence initially protoplast is grown in dim light for few days and then
transferred to light.

11.  Protoplasts can be used to study membrane biology, including the uptake of
macromolecules and viruses .
 These are also used in somaclonal variation.
 DNA transformation (for making genetically modified organisms).
 Plant breeding, using a technique called protoplast fusion.
 Protoplasts of plants expressing fluorescent proteins in certain cells maybe
used for Fluorescence Activated Cell Sorting (FACS), where only cells
fluorescing a chosen wavelength are retained.
 Populations as Single Cellular Systems
 One of the potential properties of protoplasts that each isolated protoplast is
entirely separated from another and thus corresponds to microorganisms. In
this way, a population of protoplasts provides the best possible approach to a
single isolated cell system and can be used to study the selection of mutant cell
lines, cloning of cell populations and the synchronous infection of protoplasts
by virus.