Skip to main content

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 in bold):

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.

smooth stainless steel pipeTo 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.

Nylon mesh between two pipes  dried and harvested pot culture is placed in the sieve  Roots, sieved material and sand particles

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.

Test tubes  Test tubes labeled  Storage rack  Storage tanks

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.


References

Douds, D.D., Jr. and N.C. Schenck. 1990. Cryopreservation of spores of vesicular-arbuscular mycorrhizal fungi. New Phytologist 115: 667-674.