Harvest, collect and store spores from mature fertile fronds
As C-Fern sporophytes mature they produce a heteromorphic sequence of fronds. The first fronds are entire; the subsequent ones become more and more lobed, then more and more dissected until ultimately filiform fronds are produced. Filiform fronds are characterized by a frond margin that is inrolled, referred to as a false indusium that covers the 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
Methods
HARVEST SPORES FROM MATURE SPOROPHYTES 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.
COLLECTION AND STORAGE CONTAINERS Various sizes of glassine envelopes used to be available from photographic supply houses, but now can be found from suppliers of archival storage materials. The envelopes must 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 MATERIAL Harvested spore material should be dried at about 30 C for about 48 hours. When the material is sufficiently dry it may be stored indefinitely at room temperature (cool and dry), i.e. for at least several years. Refrigeration is not recommended.
SEPARATE SPORES FROM OTHER SPOROPHYTIC MATERIAL Small amounts of spores can be decanted directly from glassine envelopes while retaining the other sporophytic material. Gentle agitation can help to release additional spores. For larger collections, spores may be processed by sequentially sieving dried sporophytic material through No. 40, and No 60 sieves into the 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. Additional sieving through a No. 100 (150 µm opening) sieve may help to further purify spores that are heavily contaminated with pulverized fertile frond material. Store dry spores in screw cap containers at room temperature in the dark.
