The study on the isolation and culture of the protoplast from ...

Journ. Fac. Sci., Hokkaido Univ. Ser. V (Botany), 14(1): 95-114, 1987.
<strong>The</strong> <strong>study</strong> on the isolation and culture of the protoplast
from heterothallic Closterium
Takuya N AKA Y AMA, Eriko ISHII
and Hajime KAN AZA W A
<strong>The</strong> enzymatic isolation of protoplasts and their culture were studied in heterothallic
strains of Closterium peracerosum-strigosum-littorale complex. <strong>The</strong> digestion of their cell
walls proceeded from division scars distributed along the cell wall, and protoplasts were
released out from one of these scars with remaining portions of the cell wall undigested.
<strong>The</strong> yield of the protoplast varied according to cell age. When "postculture" (stationary
culture with fresh medium overnight) was carried out prior to enzyme treatment, protoplasts
were obtained in high yield from the cells of various ages. <strong>The</strong> maximum yields
of the protoplast of mt+- and mC-cells were 80-90% and 70-80%, respectively, in this <strong>study</strong>.
<strong>The</strong> cultivation of protoplasts in the presence of 100 mM CaCl, led them to initiate the
regeneration of the cell walls. Scanning electron microscopy revealed that the early stages
of the cell wall regeneration in this alga was similar to that in higher plants. Several
physiological phenomena concerning the culture of the protoplasts of this alga were also
examined.
Protoplasts have been isolated enzymatically from many kinds of plants,
cultured and regenerated into new individuals. <strong>The</strong>n they have been used
routinely as an important experimental tool in the understanding of physiology,
development and morphogenesis of plant cells. Most of these studies,
however, have been done with higher plants, and protoplasts of lower plants
have received less attention.
Recently protoplasts have been isolated from many eukaryotic algae
(reviewed by BERLINER, 1981, 1983). With regard to desmids, protoplasts
have been derived from Cosmarium (CHARDARD, 1972), Micrasterias (BERLINER
and WENC, 1976), Staurastrum and Netrium (BERLINER, 1978). For the
Closterium species, protoplasts have been isolated in C. acerosum (BERLINER,
1978) and C. peracerosum-strigosum-littorale complex (KATO et al., 1983). All
these reports described mainly protoplast obtentions, but dealt little with
cultures and regenerations of protoplast.
Closterium species are interesting materials to <strong>study</strong> some biological
problems. Several workers have studied morphologically, physiologically
and genetically about several problems using Closterium. Use of protoplast
may contribute to these studies. For example, the protoplast may be used to