The International Online Journal of Science and Mathematics Education

The Development and Validation of a Classroom Environment Scale for Filipinos

Classroom Psychosocial Environment

Antheridial Development from the Isolated Protoplast of Young Gametophyte of Tree Fern Cyathea contaminans (Hook) Copel.: A Scientific Approach
to Teaching about Spermatogenesis

Rodolfo S. Treyes, Toshiyuki Kawakami & Hideo Ikeda
Faculty of Education,
Hiroshima University,
Higashi-Hiroshima,
739-8523 Japan

AbstractIntroductionMaterials & MethodsDiscussion
ReferencesAppendix

Results

Protoplast Isolation. Immature prothallia were harvested from the cultures and used for protoplast isolation (Fig. 1A). Immature prothallia were harvested from the cultures and used for protoplast isolation after 4-hr enzyme treatment with constant shaking at 30°C. Separation of the protoplast from the enzyme solution by centrifugation at 1,000 rpm showed no harmful effect, although protoplasts are very delicate. The protoplasts were separated individually by enzymatic digestion of the cell wall. The shape of the freshly isolated protoplasts was spherical since their cell walls were already removed by enzymatic digestion. The protoplasts had an average diameter of 30~40 mm. Most of the protoplasts contained numerous compact deep green chloroplasts.

Figure 1. A, A young prothallus as the source of protoplast isolation (Bar = 0.5 mm); B, An isolated protoplast after the removal of the cell wall. The compact chloroplasts dominate the entire cytoplasm (Bar = 10 um); C, A white protoplast with fewer chloroplasts (Bar = 10 um).

Some protoplasts had less and smaller chloroplasts (average diameter = 35mm). These protoplasts were labeled white protoplasts since they were colorless. Their cytoplasm contained small-granulated structures and their nuclei appeared as translucent structures in the cytoplasm. One spherical nucleolus was observed in the nucleus (Fig. 2A). The effect of GA₇ on the culture of white protoplasts was investigated. About 50% of the isolated white protoplasts were viable after the washing treatment with 1/2 strength MS medium supplemented with 0.6 M mannitol, MES buffer and 0.25 g sucrose.

Figure 2. The events taking place during antheridial development (Bar = 10 um); A, A white protoplast showing a granulated structure—the nucleolus is clearly observed in the nucleus; B, The first unequal cell division; C, A developing antheridium.

Protoplast Culture. Three days after inoculation the shape of the protoplasts gradually changed from spherical to oblong, indicating that the cell wall had regenerated. Cell division in the white protoplast grown in 1/2 strength MS medium with GA₇ occurred 7 days after inoculation. After the first cell division, the MS medium was changed to full strength and supplemented with GA₇. The culture media was renewed every 4 days, during which time the mannitol content was gradually excluded until the isolated cells were completely adjusted to the full strength MS medium without mannitol. The effective concentration of GA₇ was determined by gradually reducing its concentration from 0.005~0 mM in each observation. Antheridial development occurred at 0.0015-mM concentration. Below this concentration, the white protoplasts cultured did not manifest any growth reaction; they remained as intact individual cells. The white protoplasts in a control (GA₇-free) culture did not undergo cell division; they remained as intact cells. No cytological change was observed.

Antheridial Development. The white protoplasts (which are the antheridial primordial cells) maintained in 1/2 strength MS medium supplemented with GA₇ underwent cell division. The first unequal cell division resulted to two daughter cells (Fig. 2B). Small green protoplasts were observed developing in the cytoplasm of the larger daughter cell. On the other hand, the cytoplasm of the smaller daughter cell contained poor granules that exhibited Brownian movement. The smaller daughter cell underwent a second cell division, producing an upper cell and lower cell (basal cell). The third cell division occurred in the upper cell where division continued resulting to the development of the primary spermatogenous cell and the first antheridial ring cell. While the antheridial ring cells continued to divide simultaneously, the primary spermatogenous cell underwent a synchronous cell division within the antheridial ring cells. The primary spermatogenous cell differentiated at a relatively early stage during the antheridial development; it underwent five successive cell divisions resulting in 32 formed spermatocytes. The development of the antheridia (Fig. 3) was completed two weeks after the inoculation of the isolated protoplast.

Figure 3. Stages of antheridial development: ai, antheridial initial; uc, upper daughter cell; sp, spermatogenous cell; cc, cap cell; rc, ring cell; s, spermatocyte.

AbstractIntroductionMaterials & MethodsDiscussion
ReferencesAppendix