Freezing germplasm in liquid nitrogen
When INVAM was at the University of Florida, refrigerated cultures of some accessions
appeared to have a short storage life. As a result of these problems, Douds and
Schenck (1990) developed a procedure to store dried contents of pot cultures in
liquid nitrogen summarized in their abstract (with the most important result highlighted
Storage of spores of arbuscular mycorrhizal fungi in soil at 5°C is a common way of preserving these fungi. This method was satisfactory for Glomus intraradices but not for Gigaspora margarita, Glomus mosseae, and Acaulospora longula. Preservation of spores at -60 to -70 °C was examined. Cryoprotectants such as DMSO, glycerol, mannitol, and sucrose were ineffective using the freeze-damage sensitive species G. margarita. Incubation for 47 h in 0.75 to 1.0 M trehalose conferred a measure of freeze damage protection to the spores such that germination rates of previously frozen spores of G. margarita were one tenth to one sixth of controls. The best method of cryoprotection and cryopreservation was found to be slow-drying of pot culture soil and freezing the spores in situ . This procedure was satisfactory for the five genera of VA mycorrhizal fungi evaluated.
We found one fatal problem with this procedure in our lab, however, and that was
in manipulation of the pot culture contents to concentrate infective propagules
(hyphae, spores, roots) for storage in 2 ml cryovials. Our pot culture medium consists
of 1 part loamy soil and 2 parts sand, in which more than 80% of the sand particles
are larger than 1.0 µm in diameter. This sand could be excluded to reduce storage
volume by more than 50% using a dry-sieving procedure, but standard brass or steel
sieves have many nooks and crannies around the circumference of the wire mesh within
which hyphal fragments or small spores could lodge and remain viable even after
alcohol or chlorox soaking.
To solve this problem,
smooth stainless steel pipe (10 cm diameter with a 0.64 cm rim) was cut with
the edges trimmed so that one section could loosely fit within another. A nylon
mesh square (15×15 cm) with 1.0 µm openings (purchased from Tetko, Inc.) is sandwiched
between sections to provide a sturdy sieve plate. A sample of inoculum from a dried
and harvested pot culture is placed in the sieve and shaken on paper. With disposable
gloves, roots are separated and chopped with sterile scissors into 1-2 cm fragments.
The two steel sections are separated, washed, and immersed in a 10% chlorox solution
for 10 minutes, washed again, misted with 95% ethanol and flamed prior to reuse.
The nylon mesh section is discarded.
The portion remaining on the sieve is discarded and the portion on the paper is mixed
with root fragments and placed in cryovials (12-16 per accession). The vials are
placed in numbered positions in a case which is inserted into trays that are suspended
in liquid nitrogen. Undisturbed, tanks containing liquid nitrogen require about
4 liters of liquid nitrogen per week to remain full.
MIPs of inocula retrieved from cryopreservation give mixed results. Loss of viability
is unacceptable, so we are not using this method for routine long-term storage.
Douds, D.D., Jr. and N.C. Schenck. 1990. Cryopreservation of spores of vesicular-arbuscular mycorrhizal fungi. New Phytologist 115: 667-674.