High Throughput Cryopreservation of 140 000 Physcomitrella patens Mutants

Abstract

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 °C/min down to - 35 °C with a 10 min holding time before transferring plants to - 152 °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.

Key words

Cryopreservation - functional genomics - germplasm storage - moss - mutant collection - Physcomitrella patens (Hedw.) B.S.G.

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Section Editor: F. Salamini