Towards a standardized protocol for ex situ oak conservation using cryobiotechnologies

The Cincinnati Zoo & Botanical Garden’s Center for Conservation and Research of Endangered Wildlife (CREW) has partnered with 18 partner organizations across the United States to develop standardized and improved methods for getting oak species into tissue culture and cryopreservation as part of a three-year IMLS-funded project. At the conclusion of year one, we used 91 genotypes of eight oak species collected from Texas to Minnesota to initiate over 1,200 tubes of in vitro cultures. One of the biggest sources of loss of in vitro cultures from field collections is contamination due to fungi, bacteria, or both. While our standard sterilization protocol using a bleach solution yielded a 74% contamination rate, protocols involving an additional step of rinsing the sterilized shoots in a solution of the fungicide Benlate and antibiotics reduced the contamination rates to a minimum of 18%. We also tested the effect of adding silver thiosulfate (STS), an ethylene inhibitor, to the initiation medium. While overall the addition of STS had a positive effect on survival across species, the effects appear to be species-dependent, with some species like Quercus acerifolia and Q. hinckleyi surviving better without the addition of STS. We also tested whether initiation into tissue culture could be achieved using somatic embryogenesis of young leaves instead of shoots. While contamination rates were lower in the leaf cultures used for somatic embryogenesis than in the shoot cultures, successful embryo development appears to be at least somewhat species-dependent, with Q. georgiana in particular showing a higher success in somatic embryogenesis than in shoot initiation. Over the next two years, we will be repeating and expanding the experiments of year one using the same genotypes and species. The results of the present study should shed some light on standardized methods for ex situ oak conservation.