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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
AbstractIntroductionResultsDiscussion
ReferencesAppendix
Materials and Methods
Prothallus Preparation. Fresh spores of Cyathea contaminans (Hook) Copel. were obtained from mature fronds of the fern growing in Central Luzon, Philippines, and stored in desiccant condition. Approximately 10 mg of stocked spores were surface sterilized by soaking in 1% sodium hypochlorite solution for 3 minutes. The spores were then washed three times with sterile distilled water. The spore suspension was centrifuged at a speed of 8,000 rpm for 3 minutes after each washing; the supernatant was discarded. After the third washing the spores were suspended in distilled water. The spore suspension was shaken to ensure the even distribution of the spores before inoculation. Approximately 1 ml of the suspension was introduced into a petri dish containing Murashige & Skoog (MS) culture medium solidified with agar; pH was adjusted to 5.8. Cultures were kept under continuous white light using fluorescent lamps with light intensity of 1,800 lux and at a temperature 25°±1°C. The biological events taking place during prothallus development were directly observed in the petri dish using an inverted microscope.
Protoplast Isolation. Three weeks after spore inoculation, immature prothallia were harvested from the cultures. The prothallia were incised at the lower central cushion (rhizoidal region)—the sites of antheridial development. The rhizoids were discarded to purify the tissues. Approximately 20 mg (fresh weight) of the cut fragments of the immature prothallia were used as the source of protoplast isolation. The tissues were cut further into smaller pieces and subjected to enzymatic digestion. The enzyme solution consisted of 2% (w/v) Cellulase Onozuka R-10, 1% (w/v) Macerozyme R-10, and 0.6 M mannitol, MES buffer; pH was adjusted to 5.8. Small petri dishes containing the tissues and the enzyme solutions were sealed with strips of laboratory parafilm and wrapped tightly with aluminum foil. The petri dishes were subjected to constant shaking (reciprocally about 100 rpm) for 4 hours at 30°C. After 4 hours of incubation, the samples were filtered through a 75-mm pore-sized stainless steel mesh to separate the protoplast from other undigested debris of the tissues. The protoplasts in suspension were transferred using a sterilized glass pipette. To remove the enzymes, the protoplast suspension was centrifuged for 5 minutes at 1,000 rpm and the supernatant was discarded. The protoplasts were washed three times with sterile washing solution, and centrifuged after each washing. The composition of the washing solution was 1/2 strength MS medium supplemented with 0.6 M mannitol, 0.05 M sucrose with a pH of 5.8 and sterilized at 115° C in an autoclave. After washing, the protoplasts were suspended in a 1/2 strength MS medium supplemented with 0.6 M mannitol, and 0.05 M sucrose without GA7. The experiment was conducted under sterile conditions.
Culture of Protoplasts. A 1/2 strength MS medium supplemented with 0.6 M mannitol, 0.25 g sucrose, 0.005~0 mM GA7, without agar, with pH adjusted to 5.8, was used for the culture of the protoplasts. The cultures were maintained in petri dishes and kept at 25°C under continuous white light (1,800 lux) using fluorescent lamps. To observe the morphological development of the protoplasts, the cultures in the petri dishes were directly examined daily using an inverted microscope. The culture media were renewed every 4 days. When the isolated cells had undergone cell wall regeneration the culture medium was changed to full-strength MS medium without mannitol.