High throughput cryopreservation of 140,000 Physcomitrella patens mutants.
A high throughput protocol was established to preserve 140,000 mutants of a moss, Physcomitrella patens, a model plant for functional genomics studies, over liquid nitrogen. Regarding the reliable long-term storage of diverse mutant phenotypes, as well as time and cost effectiveness, each working step was optimized: 1) plant preparation, 2) freezing regime, cryogenic conditions, 3) regrowth after thawing. A prerequisite for maximum regrowth was a 1-week preculture of chopped plant material on a supplemented medium prior to freezing. Cryo vials as preculture vessels resulted in identical regrowth rates, compared to petri dishes. The cryo vial type had a significant influence on regrowth rates. A cooling rate of - 1 degrees C/min down to - 35 degrees C with a 10 min holding time before transferring plants to - 152 degrees C was the most suitable freezing regime. This protocol allows a cryopreservation of 1100 plants during a 5-day working week, practicable by one person. For more than 650 cryopreserved mutants a maximum regrowth rate of 100 % was obtained, independently of mutant phenotypes.
Schulte
J
J
Plant Biotechnology, Freiburg University, Freiburg, Germany.
Reski
R
R
D019068:Bryopsida / Q000145:classification* / Q000166:cytology / Q000235:genetics* / Q000254:growth & development; D018929:Cell Culture Techniques / Q000379:methods; D015925:Cryopreservation / Q000379:methods*; D003470:Culture Media; D016296:Mutagenesis; D009154:Mutation
D003470:Culture Media