Development of cryopreservation methods for cherry birch (Betula lenta L.), an endangered tree species in Canada

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Abstract
Cherry birch (Betula lenta L.) is an endangered species in Canada, with only a single natural population of 18 trees found in the Niagara region of Ontario. The tree was reportedly used for medicinal purposes by First Nations and Native American peoples. The current study describes a cryopreservation method for seeds and in vitro shoot tips of B. lenta in hopes of conserving available germplasm. Cryopreservation of mature seeds was successful, with 24% of seeds germinating in the greenhouse and 12% of seeds germinating in vitro on medium supplemented with 10 μmol·L–1 thidiazuron or 6-benzylaminopurine. Postcryopreservation regrowth of in vitro shoot tips was achieved using a droplet-vitrification protocol after preculture in 0.3 mol·L–1 sucrose for 24 h followed by osmoprotection in loading solution for 20 min and treatment with vitrification solution A3 composed of glycerol, sucrose, ethylene glycol, and dimethyl sulfoxide at 0 °C for 60 min. The highest plant regeneration (52%) was obtained after unloading (rehydration) in a medium with 0.8 mol·L–1 sucrose for 30 min. Differential scanning calorimetry analysis confirmed the absence of ice crystallization in the shoot tips during cooling and rewarming. This is the first report for successful cryopreservation of B. lenta, which will further promote initiatives for conservation of North American biocultural diversity.

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