Cryobiotechnology for maintaining genetic diversity of exceptional species: The case of Crotalaria avonensis

Valerie Pence and Megan Philpott, Cincinnati Zoo & Botanical Garden

Crotalaria avonensis is a Florida endemic found in three populations and characterized by low seed production. In the late 1990s, CREW developed protocols for tissue culture propagation from field collected shoot cuttings as well as cryopreservation methods. In order to develop a genetically representative collection for conservation, in vitro lines were established from shoots collected at all three populations from 2008-2012. Plants were produced and sent to Bok Tower Garden for further growth and for use in an outplanting by Archbold Biological Station. The resulting collection of genotypes in culture at CREW provides an example of the challenges of a genetically diverse collection of an exceptional species. C. avonensis cultures require maintenance subculturing every 2-3 months. Only a low number of replicates could be maintained for each genotype, resulting in some loss of genotypes over time. Cryopreservation offered a solution to this challenge and over the course of 16 years, a number of lines were cryopreserved. In a study of lines stored for 5.5 Ð 16 years in liquid nitrogen, there was no change in average viability of the collection in storage, although specific survival differed by genotype. A cost estimation indicated that cryopreservation could decrease the cost of maintaining the collection over 20 years by at least 1/3. Genetic analysis of the collection and the wild populations is also underway in order to determine the genetic representation of the collection.

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