Plant Biotechnology - Protoplast Isolation

The term protoplast was introduced by Hanstein in 1880. It refers to the cellular content excluding cell wall or can also be called as naked plant cell. It is described as living matter enclosed by a plant cell membrane. Protoplast isolation for the first time was carried out by Klercker in 1892 using mechanical method on the plasmolysed cells. The application of protoplast technology for the improvement of plants to complement conventional breeding programs.

The experiments involving protoplasts consist of three stages –

1. Protoplast isolation
2. Protoplast fusion (leading to gene uptake)
3. Development of regenerated fertile plants from the fusion product (Hybrid).

Depending upon the species and culture conditions, the protoplasts may have the potential to:

1. Regenerate a cell wall
2. Dedifferentiate to form callus
3. Divide mitotically and proliferate clonally
4. Redifferentiate into shoots, roots or embryos and produce a complete plantlet.

Since leaf tissue is a readily accessible source of genetically uniform cells, it is often desirable to use mesophyll protoplasts in somatic hybridization studies, but, leaf tissues, in general, do not yield large number of protoplasts owing to the difficulty in removing the lower epidermis.

 1.  Protoplast isolation

Protoplast isolation may be carried out by Mechanical disruption method or enzymatic method. Out of these two methods, enzymatic method is preferred as it provides better protoplast yield with low tissue damage while mechanical method causes maximum tissue chopping with lower protoplast yields. Both of these methods are described below:

i. Mechanical method

Klercker in 1892 pioneered the isolation of protoplasts by mechanical methods. In this method, the cells were 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 were cut only through the cell wall, releasing intact protoplasts while some of the protoplasts may be damaged inside many cells.

Protoplasts that were trapped in a cell and only the corner had been cut off could be encouraged to come out by reducing the osmolarity slightly to force the protoplasts swell to force their way out of the cut surface. The released protoplasts then have to be separated from damaged ones and cell debris.

ii. Enzymatic method

This method involves leaf sterilization followed by peeling of the lower epidermis to release cells which are plasmolyzed and added to enzyme mixture followed by harvest of protoplast. Either of the procedures for enzymatic isolation can be used: sequential enzymatic hydrolysis or mixed enzymatic hydrolysis.

In the sequential isolation, firstly, cells are separated by the use of a maceration enzyme – a pectin hydrolyzing enzyme such as, macerozyme or Pectolyase. Once the cells are separated, they are washed in CPW solution free of enzymes but containing plasmolyticum by gentle centrifugation (100g). The pellet is retained and resuspended in the second enzyme like, cellulases and hemicellulases, used to hydrolyse the remaining cell was component. Once the protoplasts are released, they are washed with CPW to remove the debris.

In the mixed enzymatic approach, 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.

 1.1.  Factors affecting yield and viability of protoplasts

i.  Source of material : Leaves were the most convenient source of the plant protoplasts because it allows the isolation of a large number of relatively uniform cells without killing the plants. Moreover, the mesophyll cells are loosely arranged, the enzymes have an easy access to the cell wall. The parent plant age and the conditions in which it is growing have profound effect on the yield of protoplast. The yield of protoplasts depends upon the growth rate and growth phase of the cells.

ii.  Pre-enzyme treatments : To facilitate the penetration of enzyme solution into the intercellular spaces of leaf, which is essential for effective digestion, various methods are followed. The most commonly used method is to peel the lower epidermis and float the stripped pieces of leaf on the enzyme solution in a manner that the peeled surface is in a contact with the solution. Most of the time it is not convenient to peel the epidermis, in such cases cutting the leaf or tissue into small strips (1- 2 mm wide) has been found useful.

When combined with vacuum infiltration the latter approach has proved very effective. Brushing of leaves with a soft brush or with the cutting edge of a scalpel may also improve the enzymatic action. Agitation of incubation mixture during enzyme treatment improves protoplast yield from cultured cells.

iii.  Enzyme treatment : The release of protoplast is very much dependent on the nature and concentration of the enzymes used. The two major enzymes required for the isolation of protoplast are cellulase and pectinase. The cellulase is required to digest the cellulosic cell walls and the pectinase mainly degrades the middle lamella. 

Some of the tissues may require other enzymes like, hemicellulase, driselase, macerozyme and pectolyase. The activity of enzyme is pH dependent. The pH of the enzyme solution is adjusted somewhere between 4.7 to 6.0.

2. Protoplast purification

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. The protoplast band is sucked in Pasteur pipette and is put into other centrifuge and finally suspended in culture medium at particular density.

3. Protoplast culture techniques

The culture requirements and the culture methods are same for both protoplasts and single cells. The main difference is the requirement of suitable osmoticum for protoplasts until they regenerate a strong wall. Isolated protoplasts are either cultured in liquid or semisolid agar or agarose media plates, sometimes the protoplast is first grown in liquid media and then transferred into the agar media plates. The following techniques have been adopted in order to maintain number of protoplast population between minimum and maximum effective densities after plating up:

i.  Liquid method : This method is preferred in earlier stages of culture as it provides (a) easier dilution and transfer, (b) the osmotic pressure of liquid media can be effectively reduced after a few days of culture (c) the cell density can be reduced or cells of special interest can be isolated easily.

In Liquid medium, the protoplast suspension is plated as a thin layer in petriplates, incubated as static culture in flasks or distributed in 50-100 μl drops in petriplates and stored in a humidifier chamber.

ii.  Embedded in Agar/ Agarose : Agarose is a preferred choice in place of agar and this has improved the culture response. This method of agar culture keeps protoplast in fixed position, thus, prevents it from forming clumps. Immobilized protoplasts give rise to clones which can then be transferred to other media. In practice, the protoplasts suspended in molten (40°C) agarose medium (1.2% w/v agarose) are dispensed (4ml) into small (3.5-5cm diameter) plates and allowed to solidify. The agarose layer is then cut into 4 equal sized blocks and transferred to larger dishes (9 cm diameter) containing liquid medium of otherwise the same composition.

Alternatively, protoplasts in molten agarose medium are dispensed as droplets (50-100 μl) on the bottom of petri plates and after solidification the droplets are submerged in the same liquid medium.

iii.  Feeder layer : In order to culture protoplast at low density, a feeder layer technique is adopted. A feeder layer of X-ray irradiated non-dividing but metabolically active protoplasts after washing are plated in soft agar medium. Non-irradiated protoplasts of low density are plated over this feeder layer. The protoplasts of the same species or different species can be used as a feeder layer.

iv.  Co-culturing : This method involves co-culture of protoplasts from two different species to promote their growth or that of the hybrid cells. Metabolically active and dividing protoplasts of two types - slow and fast growing are cultured together, the fast growing protoplast provide other species with diffusible chemicals and growth factors which helps in cell wall formation and cell division. For example, protoplasts isolated from albino plants and green plants are easily distinguishable based on color where albino protoplast will develop non green colonies.

 4. Protoplast development and regeneration

Protoplast starts to regenerate a cell wall within few days (2-4 days) of culture and during this process, protoplasts lose their characteristic spherical shape which has been taken as an indication of new wall regeneration. Cell wall regeneration can be confirmed by Calcofluor White staining method. There is direct relationship between wall formation and cell division.

Protoplasts which are not able to regenerate a proper wall fail to undergo normal mitosis. Protoplasts with a poorly developed wall often show budding and may enlarge several times their original volume. They may become multinucleate because karyokinesis is not accompanied by cytokinesis. Among other reasons, inadequate washing of the protoplasts prior to culture leads to these abnormalities.

And process is complete when its provided with suitable condition of light, pH and temperature newly synthesized protoplast can be visualized by staining. Once the cell wall formation is completed, cells undergo division resulting in increase size of cells.

After an interval of 3 weeks, small cell colonies appear, these colonies are transferred to an osmotic-free callus induction medium. This is followed by introduction into organogenic or embryogenic medium leading to plantlet development.