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Spore Collection

As C-Fern sporophytes mature they produce a heteromorphic (different shaped) sequence of fronds. The first fronds are entire; the subsequent ones become more and more lobbed; the last fronds are highly dissected (branched) filiform fronds characterized by an inrolled margin or false indusium that covers developing sporangia. When the false indusium becomes brown (brown-stripe stage), the spores are sufficiently developed for harvest.

Materials:

• Scissors, razor blades, or scalpel blades
• 70% alcohol
• Collection vessels - e.g., glassine envelopes, Petri dishes, plastic tubs
• Sieves - U.S.A. Standard Testing Sieves No. 40, 60 and 100 with openings of 425, 250 and 150, respectively and collection pan
• Bunsen burner, alcohol lamp ·
• Storage vessels, clean and dry - e.g. glass jars, scintillation vials, screw cap plastic tubes
• 25 ml glass graduated cylinder
• Forceps

Procedures:

For harvesting spores from individual plants, cut the mature fertile fronds (as indicated by the brown stripe stage) and place (stuff!) them into a labeled "spore collection" containers, e.g. glassine envelope, plastic petri dish, plastic tubs, etc.

Various sizes of glassine envelopes can be obtained from photographic supply houses. The envelopes should be well sealed to prevent leakage of spores around the seams. Other collection vessels such as large plastic Petri dishes may be used. Between collections, utensils should be washed in alcohol or flame sterilized to prevent cross-contamination of genotypes. Mass collections of spores, such as from many clones of the same genotype, can be harvested and placed in large plastic tubs. Such tubs should be easily sterilized by autoclaving to prevent cross-contamination of genotypes.

Dry harvested spore material at about 30 C for about 48 hours. When material is sufficiently dry it may be stored indefinitely at room temperature, i.e. for at least several years. Refrigeration is not recommended. Small amounts of spores can be decanted directly from glassine envelopes while retaining sporophytic material. Gentle agitation can help to release additional spores. For larger collections, spores may be processed by sieving through (in order) No. 40 and No 60 sieves into a collection pan. These sieves have 425 and 250 µm openings, respectively. Small batches of dried fertile fronds are placed onto a No. 40 sieve and lightly ground with the base of a 25 ml graduated cylinder. This grinding action should release nearly all of the spores by breaking up the fertile frond material, but not completely pulverizing it. The spores should fall through the No. 40 and No. 60 sieves to the collection pan while the fertile frond material is retained. An additional sieving through a No. 100 (150 µm opening) may help to purify spores that are heavily contaminated with pulverized fertile frond material. Store dry spores at room temperature in the dark in screw cap containers.