Chapter 16—
The Physiology of Isolated Plant Protoplasts

16.1—
Introduction

The isolated plant protoplast is a single cell, bounded by the plasmalemma and, when first formed, containing all the normal cell components, although being separated completely from its cell wall. From a physiological viewpoint, however, the protoplast cannot be regarded simply as a cell lacking a wall, since the mechanics of isolation, in conjunction with environmental factors, undoubtedly influence its metabolism and elicit subtle ultrastructural changes. The absence of a functional cell wall may affect the permeability of the cell membrane and lead to a general leakage of solutes from the protoplast. The protoplast is also in a transient state since most protoplasts, irrespective of their immediate cultural environment, will initiate the synthesis of a new cell wall a few hours after release, and eventually revert to a single-walled cell. In spite of these considerations, and combined with a cautious extrapolation of experimental data to the presumed situation existing in the intact cell or whole plant, protoplasts provide an important biochemical tool for the biologist.

In the absence of a cell wall, the exposed plasmalemma can be examined in great detail with respect to particle uptake, permeability, possible membrane-associated functions such as disease resistance, membrane fusion, and, during cell wall synthesis, the relationship of the plasmalemma to its cell wall.

16.2—
Isolation and Culture of Protoplasts

16.2.1—
Preparation of Protoplasts

Protoplasts may be produced, under aseptic conditions, from a wide range of plant species either directly from the whole plant, or indirectly from in vitro cultured tissues. There are two basic approaches for the enzymatic isolation of protoplasts: (a) the treatment of a plant tissue with a mixture of pectinase and cellulase so as simultaneously to macerate, or separate, cells and degrade their walls (Power & Cocking, 1970); and (b) the sequential (two-step) method involving the production of isolated cells which in turn are converted into protoplasts by a cellulase treatment (Nagata & Takebe, 1970).

Since removal of the cell wall results in loss of wall pressure upon the cell, protoplasts are isolated and maintained in hypertonic plasmolytica provided by a balanced inorganic salt medium or monosaccharide sugar solution. Mannitol,

Figure 16.1
Generally applicable scheme for the isolation, washing and counting of leaf mesophyll
protoplasts. Protoplasts isolated from callus or cell suspensions are handled in the same way.

for example, is not readily transported across the plasmalemma and therefore provides a stable osmotic environment for the protoplast.

The enzymes used for the isolation of protoplasts are crude extracts of fungal origin. The pectinases are rich in polygalacturonidase activity whilst the cellulases contain hemicellulase, b -1,4-glucanase, chitinase, lipase, nucleases and pectinase.

A generally applicable scheme for protoplast production, using the mixed enzyme procedure, is shown in Fig. 16.1. In order for the enzymes to gain access to the plant tissues, as in the case of leaf palisade and mesophyll cells, the lower epidermis must be removed by peeling or partial digestion with cutinase. Certain types of leaves, particularly of the cereals, must be sliced prior to enzyme incubation, since the epidermis cannot readily be removed. Most plant tissues and organs (roots, root nodules, coleoptile, leaf epidermis, petals, germinating pollen grains, fruit placenta, tetrads and microsporocytes) will yield protoplasts after suitable adjustment of the enzyme mixture and plasmolyticum. For example, the pollen tetrad wall consists of callose and so only an enzyme rich in b -1, 3-glucanase (snail digestive juice enzyme) will liberate protoplasts.

Protoplasts are produced from calluses and cell suspensions (Fig. 16.2b) (Wallin & Eriksson, 1973) often only during the log phase in their growth cycles, since the composition of the primary cell wall varies as secondary products, such as lignin, are deposited as the culture matures, rendering it unsusceptible to complete degradation by cellulase. In general, the production of protoplasts from an untried source will always involve a consideration of enzyme purity, pectinase to cellulase ratios, protoplast yield and viability.

Following enzyme incubation, the spherical protoplasts (Fig. 16.2a, 16.2d) must be separated from cellular debris, subprotoplasts and vacuoles. Subprotoplasts are formed during early plasmolysis when the protoplast splits into two or more subunits, some of which will be enucleate and hence non-viable. Separation can readily be achieved in a variety of ways: repeated resuspension and centrifugation of protoplasts in a washing medium; flotation of protoplasts on a hypertonic sucrose solution (Fig. 16.1); passage of the incubation mixture through a nylon sieve of suitable pore size which allows only protoplasts to pass through; or by the use of a two-phase system, such as dextran-PEG (polyethylene

Figure 16.2
(a) Freshly isolated mesophyll protoplasts (40 µm diam.) in liquid medium
(Petunia hybrida).  (b) Protoplasts (60 µm diam.) released from cultured cells. The
centrally positioned nucleus is surrounded by cytoplasmic strands ( Parthenocissus
tricuspidata).  (c) Sodium nitrate induced fusion between a mesophyll (m) protoplast,
containing chloroplasts, and a colourless (c) protoplast isolated from a cell suspension.
Plastids are seen entering the cytoplasm of the colourless protoplast. (d) Electron micrograph
of a freshly isolated mesophyll protoplast (n = nucleus; ch = chloroplast; cv = central vacuole;
p = plasmalemma). (e) Uptake of  Rhizobium  (r) into vesicle within the cytoplasm of a protoplast
(p = plasmalemma). (Electron micrographs provided by Dr. M.R. Davey.)

Figure 16.3
Plating of protoplasts (whole plant or cultured cell origin) in agar solidified
nutrient medium. Whole plant regeneration from protoplasts takes approximately five months.

glycol), which is based upon a density difference between protoplasts and cells (Kanai & Edwards, 1973).

Freeze etched protoplasts, collected after washing and examined in the electron microscope or treated with fluorescent brighteners which specifically bind to cellulose, reveal the complete absence of cellulose fibres on the plasmalemma surface.

16.2.2—
Cell Wall and Whole Plant Regeneration

Protoplasts are cultured in either liquid or agar solidified nutrient media (Fig. 16.3). The culture media are very similar in composition to those required for the in vitro culture of cells, but with the addition of an osmotic stabilizer to prevent bursting. During culture, up to 90% of protoplasts isolated from highly differentiated cells undergo a rapid and immediate process of dedifferentiation whilst at the same time initiating the synthesis of a new cell wall.

Early stages of wall synthesis are preceded by extensive infoldings of the plasmalemma together with an accumulation of pectin-like substances in vesicles found in the peripheral layer of cytoplasm. These early stages of wall synthesis, detected after 18 hours, are unaffected by the presence, in the culture medium, of protein synthesis inhibitors, suggesting that synthesis of new RNA or protein is not required for wall initiation and that residual protein and endogenous hormone levels are sufficient. Structurally, the first formed envelope is amorphous and consists of pectins, but after a few days a second inner layer of cellulose fibrils is progressively laid down on the protoplast surface, eventually producing a near normal cellulose matrix after four or five days (Burgess & Fleming, 1974).

Nuclear division and cytokinesis is concomitant with cell wall formation. Occasionally cytokinesis does not occur, perhaps due to the presence of an incomplete cell wall, and gives rise to binucleate cells which may not be capable of further division.

Most protoplasts, like suspension cultured cells, have an optimum plating density as regards their division potential. At this density (usually 5 × 10⁴ – 1 × 10⁵ protoplasts/ml –¹ ), the plating efficiency, defined as a percentage of the original plated protoplasts that produce cell colonies after 28 days culture, can reach 70%. As division proceeds, the plasmolyticum level in the culture medium is reduced stepwise by the transfer of agar blocks, containing the dividing protoplasts, to the surface of a similar medium of lower osmotic pressure. Individual cell colonies are pricked out, 4–6 weeks after plating, and subcultured onto a regeneration medium. Callus masses thus produced may undergo differentiation, via organogenesis, eventually producing plantlets. Regenerating protoplasts of certain species, such as carrot, will form embryoids in liquid culture.

Whole plant regeneration from protoplasts can be achieved for an expanding list of species of distinct taxonomic groups including the Solanaceae (tobacco, Petunia ) (Frearson et al., 1973), Brassicaeeae (rape) (Kartha et al., 1974),

Papilionaceae (pea, cowpea) and Scrophulariaceae (Antirrhinum ). Whole plant regeneration is not restricted to monocotyledons or dicotyledons, haploid or diploid plants, or to protoplasts obtained directly from whole plant tissues. The majority of plants regenerated from protoplasts are normal and exhibit a high degree of fertility, but a small and variable (less than 0.1%) number of plants show morphological abnormalities owing to aneuploidy and polyploidy.

An example of this can be seen following the regeneration of plants from mesophyll protoplasts of diploid Petunia, homozygous for blue flower colour. Some regenerated plants have a tetraploid chromosome number, which is also accompanied by a flower colour change from blue to red. Since well established protoplast cultures have high plating efficiencies, and hence efficient cloning potentials, abnormal plants can arise from the expression of cell aberrations present in the tissue prior to protoplast isolation.

The nutritional and hormonal requirements of cultured protoplasts are constantly varying depending upon the stage in the regeneration process. The photosynthetic capacity and respiration rate of the protoplast is suppressed by the plasmolysing conditions. Changes in metabolic requirements during the early stages of growth could be attributed to enzyme uptake during isolation and the rigours of cell wall synthesis. These subtle changes are highlighted in the crown gall (tumour) (Scowcroft et al., 1973), where protoplasts require an exogenous growth regulator supply for the initiation of division, unlike the tumour cells which are autotrophic for auxin. Following cell wall regeneration and division, the requirement for exogenous growth regulator supply is lost, suggesting that growth regulator autonomy is, at least in part, dependent upon the presence of a functional cell wall.

16.3—
Protoplasts and Auxin Responses

Although protoplasts require both auxins and cytokinins for the initiation and maintenance of division following cell wall formation, the freshly isolated protoplast has facilitated studies of the direct and rapid physiological action of auxins upon the plasmalemma. Auxins such as IAA are known to influence cell elongation in the plant. Leaf or coleoptile protoplasts, incubated in the presence of physiological levels of IAA, respond by an increased permeability of the plasmalemma, probably to water. Protoplasts maintained in an isotonic plasmolyticum containing auxin, undergo a rapid expansion as the internal volume increases owing to extensive vacuolation, which often results in bursting (Hall & Cocking, 1974). The presence of anti-auxins, which preferentially bind to the site of action of the auxin, suppresses the bursting response indicating that these effects are the result of auxin action. As may be expected, protoplasts isolated from tumour cells which have high endogenous levels of auxins, require significantly higher levels of exogenously supplied auxin to elicit the bursting response. Vacuoles produced by disruption of protoplasts show no response to

auxins, indicating clear differences in structure and function of the tonoplast. It is difficult to relate these responses to the auxin control of growth in the plant, but the induction of membrane convolutions, seen prior to bursting, can influence the thickness of the newly synthesized wall and hence cell shape and direction of growth.

Herbicides, such as 2,4-D, are used as growth regulators at low concentrations in culture media. There appears therefore to be a balance, for some species, between controlled growth and a herbicidal effect. Protoplasts, when incubated in the presence of herbicides such as paraquat, do not respond by expansion, but collapse as a result of disruption of the plasmalemma. It may be therefore, that control of the selective action of some herbicides is found in the plasmalemma itself.

16.4—
Uptake Properties of Protoplasts

16.4.1—
Macromolecules

During isolation, and for several hours after release, protoplasts exhibit extensive pinocytic activity. This process, which may be stimulated by the plasmolysing conditions, enables macromolecules, such as ferritin and polystyrene latex particles, to enter the protoplast. Infoldings of the plasmalemma result in the formation of vesicles which contain small quantities of the surrounding medium together with any particles that may be suspended in the medium and which are less than I µm in size. Particles entering protoplasts in this way are often accumulated in the central vacuole. These basic studies have led on to an examination of uptake in relation to cell and ultimately whole plant modification, by the incorporation into protoplasts of biologically active particles. Certain microorganisms can be transformed by the incorporation into the genome of exogenously supplied DNA, which in turn can be expressed, thus modifying the cell. Comparable attempts at transforming eukaryotic cells, involving plant protoplasts, are in progress and it is clear that DNA will enter protoplasts but no evidence for the persistent expression of this input DNA currently exists at the protoplast/cell level.

16.4.2—
Chloroplasts, Nuclei and Bacteria

A further extension of the uptake phenomenon has been successfully applied to whole bacteria and plant organelles such as nuclei and chloroplasts (Bonnett & Eriksson, 1974). The precise mechanism of entry of these relatively large particles may be one of membrane fusion, in the case of nuclei and chloroplasts, rather than pinocytosis. Conditions favouring organelle uptake are very similar to those that bring about protoplast fusion. Nitrogen fixing bacteria (Rhizobium )

can enter pea leaf protoplasts during the isolation procedure (Davey & Cocking, 1972) (Fig. 16.2e) but evidence for nitrogen fixing ability of the bacterium/plant association is still awaited. Likewise transplanted nuclei may survive degradation, and following fusion with the nucleus of the recipient cell, can result in the formation of hybrid or modified cell lines. Organelles entering protoplasts as a result of uptake into vesicles may not be able to function in a strictly symbiotic manner since they are not in direct contact with the cytoplasm. However, these novel approaches to cell modification may have major implications for the plant breeder and could possibly lead to the production of more efficient economically important plant species.

16.4.3—
Virus Particles

The availability of protoplasts in large quantities has overcome many biological problems facing the investigator of host-virus relationships. In intact plants the process of infection is not synchronous and its establishment is restricted to cells with broken or damaged cell walls (Zaitlin & Beachy, 1974). Protoplasts become infected following a short incubation with intact virus or its RNA and in the presence of a polycation, such as poly-l-ornithine, which not only damages the plasmalemma but neutralizes the negative charge common to many virus particles. This allows adsorption of particles, to the plasmalemma surface, which can then enter the protoplast through damaged regions of the membrane or via pinocytic vesicles. The process of infection varies considerably depending upon the virus, but in general, newly synthesized material, possibly RNA, is detected after 2–3 days in vesicles closely associated with the endoplasmic reticulum and nucleus. Newly formed virus particles are also found within the nuclear membrane. However, it is impossible to eliminate influences of the plasmolyticum, enzyme carryover and the absence of a cell wall upon the infection process, which may therefore be dissimilar to that in whole plants. Some protoplasts resist infection with virus, and it may be possible to regenerate plants, as described previously, from such protoplasts, which are not susceptible to virus infection.

16.5—
Somatic Hybridization of Plants

Crop improvement advances with the production of new hybrids, which, in turn, are selected for the expression of desired characteristics. However, in order that plant species can preserve their genotypes there exists major incompatibility barriers. The fusion of plant protoplasts of different species offers one approach that could by-pass the sexual cycle and, provided that nuclear fusion occurs in the resultant heterokaryon, new somatic hybrids may arise following regeneration.

16.5.1—
Induced Fusion of Protoplasts

Several methods now exist for the fusion of protoplasts and all embrace the same basic concepts. It is first necessary to bring the protoplasts together so that the two plasmalemma surfaces are in contact, whilst at the same time inducing destabilized regions on the membrane. Where two such regions of adjacent protoplasts coincide, localized membrane fusion occurs, together with limited cytoplasm mixing, to produce cytoplasmic bridges. Further mixing of the cytoplasms (Fig. 16.2c) eventually results in the complete coalescence of the protoplasts which are then returned to stabilizing conditions after removal of the fusion inducing agent.

A slightly hypotonic aqueous sodium nitrate solution reduces the negative charge on the outer membrane thus allowing adhesion of protoplasts. The presence of the Na⁺ ion induces localized realignment of the lipo-protein in the membrane which in turn allows fusion to occur when two such regions come into contact. Slightly deplasmolysing conditions facilitate cytoplasmic coalescence (Power & Cocking, 1971).

PEG solutions induce tight adhesion of protoplasts (Kao et al., 1974) owing to electrostatic forces. The presence of cations, such as Ca²⁺ , in the PEG solution, enhances adhesion since the calcium ion may form a molecular bridge between the negatively polarized PEG molecule and the protein of the membrane. Water is also withdrawn from the protoplast by PEG, thus reducing the turgidity of the system and allowing closer packing of protoplasts. This dramatic disruption, and possible reversal, of the charge properties of the membrane after removal of PEG, leads to membrane fusion.

Incubation of protoplasts in solutions buffered at high pH (Keller & Melchers, 1973) and containing calcium ions also induces fusion. This may resemble the action of PEG in reversing membrane charge properties in conjunction with the establishment of molecular bridges prior to membrane fusion.

Protoplasts isolated from pollen mother cells will fuse in the absence of an inducer. In such cases the membrane structure may be quite different to the plasmalemma of somatic cells since the products of pollen mother cell development, germinating pollen grains, are ultimately involved in the fertilization process and hence membrane fusion.

16.5.2—
Culture of Fusion Products

Following fusion treatment, protoplasts are returned to the culture medium and cell wall formation proceeds. It is during the first mitotic division that nuclear fusion can occur as a result of common spindle formation, giving rise to the hybrid cell. Abortion of the nuclei, unidirectional chromosome elimination, or the inability to produce a nuclear hybrid may be expected between species that are not closely related genetically. Following regeneration into whole plants, the resulting cytoplasmic hybrids ('cybrids') may be of immense value to the

plant breeder since many plastid characteristics and resistance to certain diseases are controlled by plasmagenes.

The frequency of true hybrid cell formation is low (1 in 10⁵ ). This fact has led to the development of generally applicable cultural procedures aimed at preferentially selecting out hybrid cells. These techniques, currently receiving most attention by protoplast workers, are based upon an ability of the nuclei to complement in the hybrid cell. This involves the use of parental species that exhibit mutually exclusive biochemical features such as differential drug sensitivity (Cocking et al., 1974), light sensitivity or auxotrophic requirements.

Following selection, somatic hybrid plants have been produced between two Nicotiana species. In this particular case, the selection theory was based upon prior knowledge of the cultural requirements of the sexually produced hybrid (Carlson et al., 1972). Protoplasts of the parents, N. glauca, N. langsdorffi, did not grow in a culture medium that supported the growth of leaf protoplasts of the sexual hybrid. Following fusion with sodium nitrate, the protoplasts were plated in this medium and a few colonies were recovered which later developed into plants. Electrophoretic and karyotypic analysis confirmed that these plants were amphidiploid somatic hybrids.

In spite of this unambiguous proof that somatic hybridization is possible, it will only be when somatic hybridization is demonstrated for plant species which are truly sexually incompatible, that its potential for crop improvement will be fully realized.

Further Reading

Bajaj Y.P.S. (1974) Potentials of protoplast culture work in agriculture. Euphytica 23, 633–49. Cocking E.C. (1972) Plant cell protoplasts—isolation and development. Ann. Rev. Plant Physiol.23, 29–50.

Cocking E.C. (1974) The isolation of plant protoplasts. Methods in Enzymology (eds S. Fleischer & L. Packer), Vol. XXXI part A pp. 578–83.

Heyn R.F., Rörsch A. & Schilperoort R.A. (1974) Prospects in genetic engineering of plants. Quart. Revs. Biophys. 7, 35–73.

Kruse P.F. & Patterson M.K. (Eds.) (1973) Tissue Culture—Methods and Applications. Academic Press, London.

Nickell L.G. & Heinz D.J. (1974) Potential of cell and tissue culture techniques as aids in economic plant improvement. Genes, Enzymes and Populations (eds. A.M. Srb), pp. 109–28. Plenum Publishing Corp., New York.

Smith H.H. (1974) Model systems for somatic cell plant genetics. Bioscience24 (5), 269–76.

Street H.E. (Ed.) (1977) Plant Tissue and Cell Culture, second edition. Botanical Monographs Vol. 11. Blackwell Scientific Publications, Oxford.

Tempé J. (Ed.) (1973) Protoplastes et fusion de cellules somatiques végétales. Colloques Int. C.N.R.S., No. 212. Paris: Editions I.N.R.A.