Isolated Protoplast: Meaning, Technology, Culture and Other Details

Read this article to learn about the meaning, historical background, technology, culture, regeneration, fusion and contribution of isolated protoplast.

Meaning of Isolated Protoplast:

An isolated protoplast is a plant cell in which the outer wall has been removed mechanically or enzymatically.

The results of this wall removal are that the plasma membrane remains the only barrier between the cell cytoplasm contents and the external environment.

Protoplasts thus can be defined as a functional individual cell with plasma membrane as the outermost layer.

The success of isolated protoplast depends especially on the condition of the tissue and the combination of enzymes being used. Although there is no standard method available for isolation and culture of protoplasts, the procedure follows a general pattern. Cloning of protoplasts is an innovative and novel experimental approach to solve a wide range of problems concerned with the potential and possibilities of somatic hybridization by cell fusion and regeneration, uptake of macromolecules, study of the structure of plasma membrane etc.

These are the various possibilities in the crop improvement programmes. In the recent time, there has been a tremendous upsurge of interest in protoplast manipulation involving genetic engineering and pathways to modify plant genomes -in evolving new crop species with the exploitation of various tissue culture techniques.

Historical Background:

The earliest attempts of isolation of plant protoplast were made by Klerker in 1892 and by Kuster (1910) using mechanical isolation method involving the preliminary plasmolysis of the cells and subsequent dissection of the cell walls to release the protoplasts. This method was extremely slow and yielded only a few viable protoplasts. Therefore, presently mechanical isolation of protoplast is only of historical importance.

Generally the isolation of protoplasts was attempted from highly vacuolated cells of storage tissues like onion bulbs, scales, mesocarp of cucumber and beet roots etc. In the early 1960s a number of workers became interested in the synthesis and structure of the plant cell wall as a means of studying its composition and consequently initiated investigations on the activities of the fungal enzymes known to cause digestion of the cell wall.

By appropriate selection of enzyme mixtures, it became possible to liberate billions of protoplasts within a few hours. Following the enzymatic isolation technique developed by (Cocking), Takebe et al. (1968) succeeded in isolating the protoplasts of tobacco from the leaf mesophyll cells using commercially available enzymes and subsequently regenerated whole tobacco plants. Carlson (1972) achieved successful interspecific hybridization for the first time through the fusion of protoplasts of Nicotiana glauca and N. langsdorfii.

Intergeneric somatic hybridization was attempted by Melchers et al. in 1978 using tomato and potato as parental stock plants. Zeleer et al. (1978) first developed interspecific hybrid between protoplasts of normal Nicotiana sylvestris and X-ray irradiated protoplast of male sterile N. tabacum.

The fusion method was refined and further improved by Zimmerman (1981) with the development of electro-fusion technique. With the gradual refinement in the methodology of isolation, purification, fusion and culture of protoplasts, a number of Plant Biotechnologists became extremely enthusiastic about the possible utilization of these protoplasts as a tool for genetic transformation for improvement of economic crop plants. Larquin and Kado (1977) transformed plant cells with Escherichia coli plasmids (pBR 313).

A number of transgenic plants have been generated by protoplast fusion and electroporation technique (Krens et al. 1982, Fromm et al. 1985). Use of appropriate marker genes in the transformation studies enabled easier selection of transgenic plants (Paszkowsky and Saul, 1986)

Technology of Isolated Protoplast:

1. Isolation and Purification of Plant Protoplast:

The technique of protoplast isolation was originally developed in dictyledonous plants and had gradually been extended to many monocots. The basic procedure can be subdivided into a number of stages. These are physiological state of the donor plant, selection of explant, pretreatment of the explant, exposure to lytic enzyme solution and isolation, purification and culture of protoplasts. Although, each of these stages varies with the species and genotype but there are some general rules that can be applied.

2. Physiological State of the Donor Plant:

The physiological state of the donor plants for protoplast isolation has a significant effect on the yield and survival of the resulting protoplasts. In general, it is advisable to grow the plants under controlled environmental conditions and the use of systemic herbicides should be avoided.

3. Source of the Explant:

Most of the early research was conducted with the leaf mesophyll tissue obtained from mature leaves of Nicotiana and Petunia. With the gradual refinement in methodology, it has become possible to isolate healthy protoplasts from a wide range of organs and cells like hypocotyls, leaves and shoot apices of young seedlings and also from the friable callus of various plant genera (Gossypium, Pelargonium, Daucus).

Isolation of Protoplasts from Leaf Tissues Involves a Number of Basic Steps:

(a) Surface sterilization of intact leaf

(b) Removal of epidermal layer

(c) Enzyme treatment

(d) Isolation and purification etc.

Apart from the leaf mesophyll tissue, young callus tissues are also ideal materials for obtaining large quantities of protoplasts. However, older callus generally develops gaint cells with thick secondary walls, which are entirely or partially unsuitable for protoplast isolation.

Thus isolation of protoplast from callus tissues is currently being considered as most recent refinement of single cell regeneration effort which has resulted in achieving variant protoclones (protoplast derived clones) in agricultural crops.

A modified version of callus culture is the cell suspension culture, which offers excellent source material for isolating protoplasts. Protoplasts originating from cell suspension cultures have high potentiality for regeneration. Plant regeneration through protoplast culture was successfully achieved in cereals (pearl millet, barley, sorghum etc.) using protoplasts derived through cell suspension culture.

A steady supply of surface contaminant free source tissue can be obtained by culturing the excised shoot apices of dicotyledonous plants. Once the shoot has elongated and leaves expanded, the apical tip is removed to stimulate the growth of the axillary buds. Leaf and the stem tissues harvested from these axillary shoots provide a supply of surface sterile meristematic tissues.

It is difficult to regenerate plants from protoplast isolated from leaf tissue of monocots. Rice was the first cereal to be regenerated and orchard grass the first forage grass. Although a useful method for rice and subsequently for maize, the other cereals show a rapid loss of totipotency of embryogenic tissue cultures and thus require repeated initiation of tissue cultures from the source tissue.

Plasmolysis and Dissolution of the Cell wall of the Explant:

The culture media generally used for the maintenance of the tissue culture provide the basic nutrients for the culture of protoplasts. However, certain ions like ammonium ions proved to be toxic to the protoplasts. Such components could be reduced or omitted from the standard medium. Once the protoplasts have been isolated, the plasma membrane remains the only barrier between the cell contents and the external environment.

Thus the osmotic pressure of the incubating solution is particularly important. Since the protoplasts do not have a cell wall, the osmotic pressure of the external solution must be adjusted by the inclusion of non-metabolizing sugars and sugar alcohols like mannitol, sorbitol etc. One of the most common among the isolation media for protoplast is the Cultured protoplast washing (CPW) medium (Table 1).

In the last few years, a number of enzymes have become available that have wide range of wall—degrading properties and varying degrees of purity. The purity of enzymes can be a critical factor. For isolation of healthy protoplasts, leaves, stems and roots are cut into narrow strips and are incubated for one hour in the osmoticum solution containing inorganic salt mixtures such as CPW salts and 13% w/v mannitol as an osmoprotectant to prevent the cell from losing or gaining water after wall digestion.

If the source material is cultured callus tissue, the cells are broken into smaller aggregates and subsequently maintained in the osmoticum at 25°C. This preliminary treatment causes the cytoplasm to withdraw from the wall and makes wall removal less damaging.

Preplasmolysed tissues are then exposed to osmoticum, eg. CPW plus 13% mannitol containing enzymes, either as a sequential or as a mixed enzyme treatment. The incubation period may range from 2 hours to overnight. The chopped leaves of most species can be exposed to 1% cellulysin, 0.1% macerozyme R 10 and CPW 13M medium at pH 5.6 for overnight in the dark.

For cereals, the mixture contains 2% cellulysin, 0.2% macerozyme R 10, 0.5% hemicellulose, 1% potassium hextran sulphate, 11% mannitol at pH 5.8 using a TRIS malate buffer and incubated in the dark for 1-2 hours and for most cultured tissues the incubation mixture consists of 2% rhozyme HP 150, 2% meicelase, 0.03% macerozyme R 10 and CPW 13M medium at pH 5.8 incubated overnight in the dark.

The cells are then separated from this mixture by filtering through a fine mesh filter (64 µm mesh size) and centrifuged at low speed (100g for 10 min). The protoplasts are resuspended in 5 ml osmoticum without the lytic enzymes and the suspension is centrifuged as before. In the final phase of purification, the pellate is resuspended in a small volume of CPW medium and the entire suspension is slowly transferred by pipette to a dense solution (19-20% w/v sucrose solution).

Centrifugation at low speed will ultimately cause the debris to sink and the intact protoplasts to rise to the surface. This band of protoplast is then separated carefully by a pipette. At this stage the protoplasts are in a suitable stage for fusion, or may be used for the uptake of large molecules such as in plant transformation.

Culture of Isolated Protoplasts:

In order to obtain successful growth of the isolated protoplasts, it is important that a sufficiently large number of those be obtained. For tobacco, the minimum plating density (mpd) is 1 ∞ 10⁴ protoplasts ml^-1 since at this density the callus colonies arising from individual protoplasts tend to grow at an early stage of culture.

After isolation, the protoplasts still remain very fragile and need an osmoprotectant until cell walls are regenerated. The media used for the maintenance of tissue culture of the species being studied usually provide the basic requirement although the auxin and cytokinin levels require appropriate adjustment. Kao and Michayluk (1975) formulated a protoplast culture medium in which cultured protoplasts of Vicia hajastana can be grown up to callus stage. The same medium induced rapid growth of leaf mesophyll protoplasts of other crop species like pea, potato and alfalfa.

(i) Feeder Layer Technique:

One of the most popular methods for culturing the protoplasts at relatively low density is the feeder layer technique. In this method, protoplasts plated into an agarified medium (Gill et al. 1979) are exposed to X-ray dose of 2 x 10³R, which inhibited division of cells but allowed them to remain metabolically active.

After repeated washing of these inactivated protoplasts, they were plated in agar medium at a density of 2.4 x 10⁴ ml^-4 and the non-irradiated protoplasts were plated over this feeder layer at a low density (10-100 protoplasts ml^-1).

(ii) Co-Culture Technique:

In this technique protoplasts of two different species are cultured together for promotion of growth. Upon co-culturing two different metabolically active protoplasts, there is a cross feeding between two types, which, in turn, enhances their sustained division at low density.

This technique is particularly suited where calli arising from morphologically distinguishable protoplasts. For example, mechanically isolated hybrid cells co-cultured with protoplasts isolated from an albino strain will develop green colonies that are readily distinguishable from non-green colonies of albino types.

(iii) Micro Droplet Culture Technique:

This special technique is successfully employed in culturing hybrid protoplasts of Nicotiana glauca X Glycine max (Kao 1977) and Arabidopsis thaliana X Brassica campestris (Gelba and Hoffman, 1978a). For this culture, a highly specialized plate called “Cuprak dish” is used which has a small outer chamber and a large inner chamber.

The inner chamber contains numerous numbered wells, each having a capacity for 0.25-25 µ droplet of medium. Protoplasts grow in these chambers along with required volume of droplets while the outer chamber is filled with sterile water to maintain humidity inside the dish. In this technique the size of the droplets is critical for the induction of the division of the protoplasts. An increase in the droplet size generally decreases the effective plating density.

Regeneration of Protoplasts:

(i) Formation of the Cell Wall and Initiation of Division:

Regeneration of new cell wall over the plasmalemma takes place with the deposition of microfibrils and pectins immediately on culture of the protoplasts in a suitable medium. The formation of the cell wall can be clearly visualized by the use of a fluorescent dye Calcafluor White (0.1% w/v).

A direct relationship can be established between cell wall regeneration and induction of first cell division. Generally it is observed that the cultured protoplasts do not enter into first division unless the cell wall regeneration process is complete. Photosynthetic activity, oxygen uptake, size variations with osmotic changes are the parameters that usually determine the protoplast survival and their ability to divide in culture.

After the complete cell wall regeneration, protoplasts divide in presence of suitable nutrients in the culture medium. In many plant species the first division generally occurs within one week.

(ii) Formation of Callus and Plantlets:

Soon after the first division of the cultured protoplasts, subsequent divisions yield small cell colonies on suitable semisolid medium. Generally macroscopic colonies appear within 2-3 weeks of culture which can be subsequently transferred to an osmoticum-free medium for callus development.

Mostly the regeneration of the plantlets has been achieved through callus mediated organogenesis, although plant regeneration through somatic embryogenesis has been reported in Daucus carota (Dudits et al. 1976), Medicago sativa (Kao and Michyluk, 1980) and Lycopersicon peruvinum (Zapata et al. 1981).

It could be noted that regeneration of plants through protoplast culture was successfully achieved in majority of dicots and to a limited extent in monocots. Cell division was reported in protoplast cultures of a number of crop plants like wheat, sorghum, bareley, rice etc., but showed no morphogenetic potential except in rice. Plant regeneration from protoplasts taken from embryonic cell cultures of maize (Rhodes et al. 1988), sugarcane and rice (Davey and Power, 1988) was reported. So far plant regeneration from protoplasts of a wide range of species has been achieved. Some of those are listed in Table 3.

What are Somatic Hybrids and Cybrids?

One of the most useful practical applications of protoplast culture has been to develop suitable methods for facilitating somatic cell fusion and genetic engineering in higher plants. The interest in protoplast fusion technique is related to the prospect that wider crosses that are not possible by sexual means may be achieved by protoplast fusion.

For example, some plants that show physical and chemical incompatibility in normal sexual crosses may be produced by the fusion of protoplasts obtained from cultures of two different species. The fusion process involves isolation of protoplasts, fusion, plant regeneration from somatic hybrid colonies, selection of hybrid plants, confirmation of the somatic hybrid nature and, finally, screening of the regenerates for morphological variations. Fusion of protoplasts generally brings about a combination of the genetic material and involves both the nuclear and organelle genes.

The method permits gene flow from one species to another, which is not possible by sexual means. Thus a limited gene transfer is possible with wild and distant relatives and also the production of asymmetric as well as cytoplasmic hybrids is facilitated. Protoplast isolated from the same or different species can be fused under appropriate treatments, which leads to a mixing of nuclear and cytoplasmic contents.

The heterokaryons generated through fusion of protoplasts are not always genetically stable and one consequence of the fusion is that there is sometimes loss or elimination of one complete chromosome complement or one set of chloroplasts. This process of chromosomal or organelle elimination can be manipulated by irradiation treatment of one protoplast partner which will destroy the chromosomes.

Thus plantlets originated from such regenerates will, therefore, be genetically hybrids exclusively for cytoplasmic traits. Alternatively, treatment of cells of one partner with trichloroacetic acid will destoy the orgaanelle fraction, but will leave the chromosomes intact. This means that it may be possible to transfer the orgnelle fraction only into protoplasts lacking organelles in order to obtain a novel combination of cytoplasmic and nuclear components.

Fusion of Protoplasts:

In order to facilitate fusion of protoplasts, it is essential that the plasma membrane is destabilized first and then the protoplasts are brought into contact with one another. Protoplast fusion can be accomplished either by chemical or electrical methods.

(a) Chemical Method:

The simplest and most convenient method to induce fusion of protoplasts is by chemical methods of which the use of polymer polyethylene glycol (PEG) 6000 is the most suitable one. The function of the PEG is to alter the membrane characteristics so that the protoplasts become sticky and if the protoplasts are allowed to come into contact, they will adhere together and the contents will fuse. PEG has been used as a fusagenic agent in a wide range of plant species because of the reproducible high frequency of heterokaryon formation.

In this process, about 0.6 ml of PEG solution (1 g of PEG dissolved in 2 ml of 0.1 M glucose, 10 mM CaCl₂ and 0.7 mM KH₂PO₄) is added in drops to the pellate of protoplasts in a vial. Protoplasts are incubated for 40 min at room temperature. Finally, the protoplast suspension is freed from fusagenic agents by centrifugation and resuspending in culture medium.

Thus the molecular weight and the concentration of PEG, and also the time of incubation are critical in inducing successful fusion. The nature of change occurring at the membrane surface during the fusion process is not clearly known. PEG possibly acts as a bridge by which Ca⁺⁺ can bind membrane surfaces together or creates a disturbance in the surface charge during elution process.

Protoplast fusion can also be achieved by NaNO₃ treatment (Power et al. 1970). Transfer of the protoplasts in NaNO₃ solution (0.25M) followed by centrifugation causes protoplast fusion. The first successful protoplast fusion was achieved by Carlson in 1972 following this method using protoplasts of Nicotiana glauca and N. langsdorfii. Keller and Melchers developed another fusion method via high pH and Ca⁺⁺ treatment. In this process isolated protoplasts are incubated in a solution of 0.4M mannitol containing 0.05 M CaCl₂ with pH 10.5 at 37°C. Fusion occurs within 10 minutes.

(b) Electrofusion:

Protoplasts can be fused on a large scale by electrical methods using chambers in which the protoplasts are exposed to small electrical currents. The suspension of protoplasts due for fusion is placed between two electrodes. A weak AC current (400 000 Hz, 1.5 V) is passed through the electrodes, which causes the protoplasts to become positively charged on one side and negatively charged on the other.

As a result of their charge, protoplasts align themselves in groups along the line of force touching another and forming pearl chains. After a period of about 90 seconds, the AC field is replicated by a single high voltage pulse discharge of 1000 V cm^-1. This leads to the breakdown of the plasma membrane and fusion forming a continuous membrane around the pearl chains.

This is then followed by cytoplasmic fusion. Multiple fusions may occur at high concentrations of protoplasts but reducing the concentration of the protoplast suspension can prevent this. This electrofusion method is much more productive than the chemical method as up to 50% of the protoplasts can be heterokaryons.

Selection of Somatic Hybrids and Cybrids:

After fusion of protoplasts either through chemical or electrical means, there will be single un-fused parent protoplasts, fused parents of the same origin, multiple fusions as well as the desired heterokaryons. Generally 20-25% of the protoplasts may be involved in a fusion event. However, heterokaryon formation as high as 50-100% has been reported.

The Various Selection Techniques that have been Generally Employed are:

Biochemical Selection Methods:

(a) It was Carlson et al. (1972) who first demonstrated the value of a biochemically based selection procedure for somatic hybridization of two species of Nicotiana. This selection procedure was primarily based upon a prior knowledge of the nutritional requirements of mesophyll protoplasts isolated from genetically tumoured Nicotiana glauca and N. langsdotfii.

The resulting hybrids exhibit a tumourous nature. Thus the two parental populations of protoplasts are unable to grow on a medium lacking hormones. On the other hand, the somatic hybrids, however, because of the tumourous nature, produced its own auxins and cytokinins, which allow growth, division and proliferation of callus colony in an auxin free medium. Rudimentary shoots produced on such callus were grafted onto N. glauca stock in order to produce whole plants.

A comparison between some of these plants and sexually derived amphidiploids based on peroxidase isoenzyme patterns, leaf and tissue morphology and chromosome number showed that both the sexual hybrids and somatic hybrids are similar.

(b) Drug Sensitivity:

Isolated protoplasts of Petunia parodii and P. hybrida are differentially sensitive to the drug actinomycin D. The isolated protoplasts of P. hybrida develop up to macroscopic callus stage in MS medium, while those of P. parodii could only form small cell colonies.

The presence of actinomycin D in the culture medium hardly affects the regeneration potential of protoplasts of P. parodii. However, the protoplasts of P. hybrida fail to divide in presence of actinomycin D. On the contrary, heterokaryons are able to grow even in the presence of the drug and finally generate somatic hybrid plants.

(c) Auxotrophic Mutants:

Following the demonstration of interspecific somatic hybridization, Melchers and Habib (1974) have utilized two varieties of Nicotiana tabacum bearing chlorophyll defects, in order to produce interspecific hybrids. These two varieties complement in the sexually derived hybrids to form normal green colour. Protoplasts isolated from leaves of light-sensitive dihaploid parents were mixed together and fused in presence of Ca⁺⁺ ions buffered at pH 10.5. Selection was based upon the ability of the two genomes present in heterokaryon/hybrid to complement and thus to restore the normal green colour to differentiating callus and subsequently regenerated plantlets if maintained at high light intensity.

Visual Selection Method:

The selection of fusion products on the basis of visual selection has been restricted to those systems with characteristic microscopic markers such as colourless protoplasts with chloroplasts containing mesophyll protoplast (Kao et al. 1974). Patnaik labeled cell suspension protoplasts with flurescein isothiocyanate and then fused them with green mesophyll protoplasts.

When observed by fluorescence microscope, the chloroplasts fluoresce red because of the presence of chlorophyll and the isothiocyanate cells exhibit a highly characteristic green cytoplasmic fluorescence. Patnaik used micromanipulative methods to separate out green fluorescing cytoplasm containing red fluorescing chloroplasts.

Flow Cytometric Method:

The other popular but more expensive method is the use of an automated flow cytometer. In this technique the dual labelled protoplasts are projected as a stream, which is illuminated by UV. Those droplets containing the heterokaryon are identified and deflected into a separate tube.

This procedure is fully automated and can process up to 2000 protoplasts an hour. After regeneration of the isolated heterokaryon protoplasts, the heterokaryon status of the plants needs to be confirmed. This can be accomplished by morphological analysis where the plantlets should exhibit a combination of parental characters, cytological analysis, which will show whether there has been any structural or numerical chromosome aberration, and biochemical analysis, which essentially involves a comparison of the total protein and isozyme profiles of the parent and hybrid plants.

Contribution of Protoplast Fusion to Crop Breeding:

In any plant-breeding programme, one of the major objectives is the transfer of desirable genetic characters between varieties or species. It has already been described that protoplast systems are particularly very suitable for the introduction of foreign genetic materials because of their unique wall-less nature.

Originally cell hybridization was considered as a means of bringing together two different genotypes that were unable to hybridize by normal sexual means. Very wide crosses were thought possible by this means. However, in the wide crosses there was often a loss of one set of chromosomes and also organelles.

It was understood that the partners had to be closely related for the heterokaryon to be stable. An example of successful protoplast fusion involving two related species that are sexually incompatible is that of the domestic potato Solarium tuberosum with that of wild potato S. brevidens.

The intention of this cross was to introduce resistance to potato leaf roll virus and potato virus into cultivated potato from their wild counterpart. The chromosome number of potato is 2n = 48 and that of S. brevidens is 2n = 24. In order to make the two partners more compatible dihaploid plants of S. tuberosum were developed having chromosome number 2n = 24. Protoplasts of these plants were fused with those of S. brevidens.

The heterokaryons thus regenerated were found to be tetraploid and hexaploid showing chromosome numbers 48 and 72, respectively. These hybrids subsequently showed the same; levels of resistance as the S. brevidens and were largely female fertile. Another example is the introduction of male sterility.

The expression of male sterility resides in the mitochondria and chloroplast genome and somatic hybridization by protoplast fusion provides a mechanism for transferring the cytoplasm of a species with cytoplasmic male sterility to one that would benefit from this characteristic. Rice is an example where this has been successfully achieved.

In addition to these, a number of workers have successfully achieved somatic hybrids taking closely as well as distantly related species. Potrykus and Hoffmann (1973) succeeded in transplanting isolated nuclei of Petunia hybrida into isolated protoplasts of P. hybrida, Nicotiana glauca and Zea mays. Cocking and Ponjar (1969) reported that tomato fruit protoplasts take up tobacco mosaic virus (TMV) particles by pinocytosis and demonstrated the initial stages of infection in their electron microscopic studies.

In China, hybrid technology in rice is a great success. Such plants are very successful in producing hybrid seeds without emasculation. Moreover, this technology has successfully been applied to carrot, Brassica, Citrus and sugarbeet (Balasubramanian 1996). Based on detailed studies on interfamilial hybrids, it can be concluded that such hybrid cells are genetically unstable and show species-specific elimination of chromosomes belonging to one of the parents.

These hybrid lines are incapable of regeneration and morphogenesis into plants (Gleba and Hoffman 1978a). Similarly intertribal hybrids were also studied widely due to their higher genetic stability. The first intertribal cell hybrid was obtained by Gleba and Hoffmann (1978b) from callus cells of Arabidopsis thaliana crossed with mesophyll protoplasts of Brassica campestris (turnip).