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.