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Protoplasts (Torrey and Landgren, 1977, have defined higher plant protoplast as "cells with their walls stopped off and removed from the proximity of their neighbouring cells) represent the plant cells minus their cell wall (i.e., the naked cells) which are removed by either a mechanical or an enzymatic device. They are unusual because of being limited only by plasma membrane which is fully exposed and is the only barrier between external environment and the interior of the living cell.

Protoplasts represent the finest single cell system and offer exciting possibilities in the fields of somatic cell genetics and crop improvements. Since the isolated protoplasts grown in culture often perform better than single whole cells, they may serve as an excellent starting material for cell cloning and development of mutant lines.

Besides being useful for somatic hybridization technique, higher plant protoplasts can introduce in them foreign DNA, cell organelles, bacteria, or virus particles through their exposed plasma membrane. These unique properties of protoplasts, combined with the totipotent nature of plant cells, have opened up an entirely new area of fundamental and applied research in experimental biology and somatic cell genetics.

The use of protoplasts for the introduction of foreign germplasm, somatic hybridization, and for studies of hormone action, membrane structure and function, and cell wall synthesis are well documented for model plant systems. These aspects of protoplast applications are described in application section.

Most of the early works on protoplast technology were confined to spongy and palisade mesophyll cells obtained from mature leaves of Nicotiana and Petunia. However, at present, the protoplasts can be obtained either indirectly from in vitro cultured tissues, e.g. suspension cultures or callus tissues, or directly from the intact tissues of the plants such as pollen, fruits, tumors, leaves etc.

The mesophyll cells of leaves are most commonly used. This is because it is easy to disrupt leaves into single cells and because leaf cells have a high regeneration potential allowing mature plants to be derived from protoplsats.

(A) Isolation and Purification of Protoplasts:

Isolation of protoplasts from higher plants can be traced back to Klerker (1892), involving plasmolysing the cells and subsequent cutting of walls to release protoplasts during deplasmolysis. However, the protoplasts are isolated either by mechanical distruption or preferably by enzymatic dissolution of the cell walls.

(a) Mechanical Disruption:

The mechanical disruption of cell wall to obtain isolated protoplasts involves cutting a plasmolysed tissue with a sharp razor. This enables release of protoplasts through the cut ends of cell walls. In practice, this technique is difficult and the yield of viable protoplasts is meager.

One advantage, however, is that the complex and often deleterious effects of wall degrading enzymes on the metabolism of the protoplasts are eliminated. Protoplast isolation by this technique was first achieved almost a century ago, but this technique has limited applications because of small yield of protoplasts.

(b) Enzymatic Dissolution:

The isolation of protoplasts by enzymatic dissolution technique has been in use since 1960 when Cocking demonstrated the possibility of enzymatic isolation of a large number of protoplasts from cells of higher plants. Earlier, there was no satisfactory method of removing cell wall without injuring the cell.

But, later it was realized that fungi which grow on wood must be endowed with all the enzymes necessary to digest the plant cell wall. And indeed today most of the enzymes which are employed for protoplast isolation are extracted from fungi, while a few are isolated from intestinal juice of the Roman Snail (Helix pomatia) and from bacteria. Following table gives an idea of the fungal source of some common enzymes.

The common enzymes used in the process are pectinases, hemicellulases, cellulases. These enzymes attack the chemical constituents of the primary wall and middle lamella. Pectinases are necessary to breakup cell aggregates into individual cells (i.e., losses the cells) while hemicellulases and cellulases do break down the cell wall. However, by using enzymes one can obtain a high yield (2.5 x 106 protoplasts/g leaf tissue) of uniform protoplasts that can be cleaned of cellular debris.

There have been two approaches to the use of these enzymes, sequential procedure and mixed- enzyme procedure. Naguta and Takebe (1971) introduced sequential procedure, a two step process, to isolate protoplasts, this procedure involves treatment of sample with pectinase to loosen the cell followed by cellulase to breakdown the walls.

Simultaneously, Cocking and Power suggested a single step process, called mixed-enzymes procedure, in which they used a mixture of pectinase and cellulase enzymes to the sample. The enzyme mixture macreates the cells and simultaneously destroys their walls. Although both procedures have certain advantages and disadvantages, the mixed enzymes procedure is preferably used at present.

Technique of Enzymatic Dissolution:

The basic technique of protoplast isolation (from an excised leaf, for instance) by enzymatic process and its purification consists of following steps :

i. Surface sterilization of the leaf sample

ii. Rinsing in a suitable plasmolyticum

iii. Peeling off the lower epidermis or slicing the tissue

iv. Enzyme treatment

v. Purification of the isolated protoplasts.

Surface Sterilization of the Leaf Sample:

Mature healthy leaves are removed from the plants and they are surface sterilized using suitable sterilizant such as calcium or sodium hypochlorite solution. If the leaves are waxy or have surface appendages a few cubic centimetres of Teepol (1 % v/v) should be added as a wetting agent.

Rinsing in a Suitable Plasmolyticum:

The surface sterilized leaves are rinsed at least three times to remove all traces of hypochlorite solution. Although the leaves may be rinsed in sterile distilled water (Sterile DDH2O), it is preferable to rinse them in a culture medium adjusted to the osmolality and pH of the enzyme solution to be used: generally mannitol (13%w/v) plus agar (2% w/v) medium is used.

Peeling off the Lower Epidermis or Slicing the Tissue:

When the leaves are in the final rinsing solution, a sharp forceps is inserted below the junction of the lateral vein and midrib and the epidermis is peeled away towards the margin thus exposing the mesophyll. This facilitates enzyme-penetration. If the leaves cannot be peeled immediately, then they should be allowed to wilt before another attempt is made. If the leaves cannot be successfully peeled, then the lower epidermis is scored several times with a scalpel to aid the penetration of the enzymes.

Enzyme Treatment:

The preplasmolysed excised leaf portions are transferred to petridishes containing enzyme solution, preferably, for mixed-enzyme treatment. The enzyme solution consists of Macerozyme (0.5% w/v) plus Onozulea Cellulase (2.0% w/v) dissolved in mannitol (13% w/v) at pH 5.4. The petridishes are sealed with paraffin and wrapped with aluminium foil.-Usually the petridishes are incubated overnight (12-18 hrs) at 25°C. The leaf portions are then teared gently with forcep so that the protoplasts are released.

Purification of the Isolated Protoplasts:

The isolated protoplasts are purified by a combination of filtration, centrifugation and warming. First the enzyme solution containing isolated protoplasm is filtered through a nylon mesh (45 p pore size) to remove undigested tissue, cell clumps and cell wall debris.

Second, the filtrate is transferred to a centrifuge tube and is centrifuges for 5 minutes at 75 x g, the protoplasts form a pellet at the base of the tube while the remaining debris suspends in the supernatant. The supernatant is carefully removed with a Pasteur pipette. The pellet at the base containing protoplast is resuspended in 10 ml of Murashige Scoog (MS) medium plus mannitol (13.1% w/v) and the process is repeated three times.

The resuspension of the protoplast must be carried out carefully in order to avoid injury, for convenience, wide-mouthed pipette (10 cms) should be used. Now the isolated protoplasts are considered purified. Before the purified isolated protoplasts are transferred to suitable culture medium, it is necessary to examine them for density and viability.

After the protoplast has been examined for density and viability, they are ready for culture and are transferred to suitable culture medium.

(B) Culture Technique:

The isolated and purified protoplasts are transferred to suitable culture medium after testing their density and viability.

Method of Culture:

Protoplasts have been cultured using different methodologies, for instance, on a liquid medium employing handig drop culture technique, in microculture chambe^and in a soft agar matrix. The soft agar matrix technique is one of the better methodologies as it ensures supports for the protoplasts and permits observation of their development.

Five cubic centimetres of the purified protoplasts is added to a petridish (100 x 15 mm) and to this dish is then added 5 cm' of the complete Murasighe-Scoog (MS) medium plus 13% (w/v) mannitol, this latter solution also contains warm agar 2.0% (w/v) in the sol state at 40°C. Care is taken that the temperature does not exceed 45°C. The two aliquots are mixed by swirling the dish, and the final result is 10 cm3 of a medium containing 5 x 104 protoplasts /cm3. The culture of embedded protoplasts is incubated at 25°C in the presence of a low density white light.

Regeneration of Cell Wall:

The isolated protoplast when placed on culture medium is spherical due to the absence of rigid cell wall. Once a suitable culture medium is made available the protoplasts starts to regenerate new cell wall quickly. However, a significant role is played by the nucleus in the wall formation process.

It is considered that there is a direct relationship between wall formation and cell division, protoplasts which are not able to regenerate a proper wall, fail to undergo normal mitosis. Generally, the wall formation has been observed between the adjacent plasma membranes and the initial events of wall formation can be seen microscopically by using calcofluor White ST (0.1 % w/v). This white dye binds to wall material and exhibits fluorescence or irradication with blue light.

Formation of Plants from the Protoplast: Once the protoplast has regenerated their cell wall, they undergo cell division and form a callus. This callus 90 ft IFS Botany

can be subcultured onto plates or flasks containing a freshly prepared medium that lacks mannitol and auxin.

The medium lacking mannitol and auxin induces embryogenesis (i.e. the formation of embryoids) on the callus after about 3-4 weeks. The formation of these embryoids have been reported by Kameya and Uchimiya (1972) and Lorz et al (1979). The embryoid when provided proper care and attention, can be made to develop into seedlings and eventually result in mature plants.