PROTOPLAST FORMATION AND FUSION

TERESA SUÁREZ AND MARGARITA OREJAS
Departamento de Genética, Facultad de Biología, Universidad de Salamanca, 37008 Salamanca, Spain

JOSÉ ARNAU AND SANTIAGO TORRES-MARTÍNEZ (storres@fcu.um.es)
Departamento de Genética, Facultad de Biología, Universidad de Murcia, Murcia, Spain

In Phycomyces. E. Cerdá-Olmedo and E. D. Lipson (eds.), 1987, p. 351-353. CSH Laboratory Press, Cold Spring Harbor, NY, USA.

Protoplast Formation

The protoplast formation method uses a commercial lytic-enzyme preparation, "Novozym 234" (Novo Industri, Bagsvaerd, Denmark), rich in chitinase, and a Streptomyces preparation, "streptozyme," rich in chitosanase. The optimal exposure time depends critically on the age and physiological conditions of the germinating cells to be treated.
Activated spores are inoculated into liquid minimal medium and incubated at 22°C with shaking (250 rpm) until the germ tubes become 10-16 µm long (slightly longer than swollen spores). This usually takes 9-14 hours, depending on the strain. Viable cells are counted by plating appropriate aliquots on agar medium. The cells are washed twice by centrifugation in 0.55 M sorbitol at 600g for 5 minutes. The pellet is resuspended in sodium phosphate buffer (10 mM, pH 6.5) containing sorbitol (0.55 M) to a final concentration of 5 x 10⁶ cells/ml. Novozym 234 and streptozyme (see below) are added at final concentrations of 1.6 mg/ml and 5.5 U/ml, respectively, and incubated at 26°C with occasional shaking, while the appearance of protoplasts is monitored with the light microscope. Good protoplast preparations usually take about 1 hour.
The protoplasts are washed twice by centrifugation in 0.55 M sorbitol at 600g for 5 minutes and counted with a hemocytometer. Total viable cells are counted by adding appropriate aliquots to 5 ml of molten regeneration medium containing 10 g/liter agar at 48°C and overlaying the mixture on solid regeneration medium with 20 g/liter agar. Regeneration medium is a suitable growth medium with 0.55 M sorbitol. Viable osmoresistant cells are counted by plating appropriate aliquots on sorbitol-free agar medium.
The regeneration rate is defined as the quotient of the number of regenerated cells (those forming colonies on regeneration medium minus the osmoresistant cells) to the original cells. Using this method, regeneration rates of up to 62% have been obtained.

Protoplast Fusion

Protoplast suspensions (10⁶ protoplasts each) from two genetically different strains are mixed and centrifuged at 600g for 5 minutes. The pellet is resuspended in 1 ml of prewarmed (33°C) fusion medium (450 g / liter polyethylene glycol, 4 kD average, and 60 mM CaCl2) and incubated for 30 minutes at 33°C with occasional gentle shaking.
The mixture is diluted in 10 ml of 0.55 M sorbitol and washed twice by centrifugation in 0.55 M sorbitol at 600g for 5 minutes. Total viable cells are counted using the overlay method described above. Viable heterokaryons are counted in the same way, using a medium where heterokaryons may be selected or screened. The proportion of heterokaryons among the viable cells using this method may be up to 3%.

Preparation of Phycomyces Cell Walls

This procedure is taken from Suárez et al. (1985). Wild-type Phycomyces cells are grown in liquid minimal medium for 3 days at 22°C, and the mycelia are collected by filtration. Ballotini number-8 glass beads (20 g) and 30 ml of distilled water are added to each 10 g of wet mycelia and homogenized three times (45 sec each) in a MSK homogenizer (Braun, Melsungen, Germany). The broken cells are washed by centrifugation in distilled water at 1000g for 8 minutes until the supernatant is clear (more than ten washings are usually needed).

Preparation of Streptozyme from Streptomyces

The procedure is modified from Price and Storck (1975). Streptomyces sp. (strain number 6) is grown overnight at 26°C with shaking in medium containing (per liter) 0.5 g of NaCl, 1 g of K₂HPO₄, 0.5 MgSO₄·7 H₂O, 10 mg of FeSO₄·5 H₂O, 5 g of D(+)-glucose, and 3 g of yeast extract (Jeniaux 1966). The overnight culture (5 ml) is inoculated into 500 ml of Jeniaux medium and incubated at 26°C with shaking until the late exponential phase (this usually takes over 24 hr).
The culture is centrifuged at 5000g for 15 minutes. The cells are then resuspended in 250 ml of induction medium (Jeniaux medium with glucose and yeast extract replaced by 10 g/liter Phycomyces cell walls), incubated at 26°C for 12 hours with shaking, and again centrifuged at 5000g for 30 minutes.
The supernatant is concentrated to one tenth of its original volume in an Amicon concentrator through a membrane with an exclusion limit of 10 kD. Alternatively, the supernatant may be brought to 95% saturation with (NH₄)2SO₄, stirred overnight at 4°C, and then centrifuged at 5000g for 60 minutes, and the pellet resuspended in a small volume of sodium phosphate buffer (20 mM at pH 6.5). In either case, the supernatant is dialyzed extensively against sodium phosphate buffer and stored at -20°C.

Assay of Chitosanase

This procedure is taken from Van Heeswijck (1984). A solution (2 mg/ml) of chitosan is prepared in 50 mM maleic acid, diluted threefold with water, and adjusted to pH 6.0 with KOH. The chitosan solution (375 µl) is warmed for 15 minutes to 30°C in an Eppendorf tube, and 125 µLl of the streptozyme to be assayed is added and incubated for 15 minutes at 30°C. The reaction is stopped by adding 100 µl of 1 M KOH. The mixture is incubated on ice for 30 minutes to allow chitosan to precipitate and centrifuged again for 10 minutes in an Eppendorf Minifuge.
The supernatant is assayed for hexosamine content using the indole method (Dische 1955): To 500 µl of the supernatant add 500 µl of a 50 g/liter solution of NaNO₂ and 500 µl of a mixture of 1 volume of acetic acid and 2 volumes of water. Shake and keep at room temperature for 10 minutes. Add 500 µl of 125 g/liter ammonium sulfamate to remove excess nitrous acid and shake repeatedly for 30 minutes. Add 2 ml of 1.4 M HCl and 200 µl of a 10 g/liter solution of indole in ethanol. Boil by immersion in a water bath for 5 minutes and then cool at room temperature. An intense orange color will appear (the occasional turbidity can be removed by adding 2 ml of ethanol and shaking). Measure absorbance at 492 nm and check against a standard curve prepared with 2-ml samples of glucosamine at final concentrations from 0 to 0.3 mM. One unit of chitosanase activity is defined as the activity required to produce 1 µmol hexosamine equivalent per minute.

REFERENCES

Dische, Z. 1955. New color reactions for the determination of sugars in polysaccharides. In Methods of biochemical analysis (ed. D. Glick), vol.2, p.313. Interscience, New York.

Jeniaux, C. 1966. Chitinases. Methods Enzymol. 8: 644.

Price, J.S. and R. Storck. 1975. Production, purification and characterization of extracellular chitosanase from Streptomyces. J. Bacteriol. 124: 1574.

Suarez, T., M. Orejas, and A.P. Eslava. 1985. Isolation, regeneration and fusion of Phycomyces blakesleeanus protoplasts. Exp. Mycol. 9: 203.

Van Heeswijck, R. 1984. The formation of protoplasts from Mucor species. Carlsberg Res. Commun. 49: 597.