Cryobiotechnology for Conservation and Storage of Endangered Exceptional Hawaiian Plant Species

Megan Philpott, Valerie PenceL*, Cincinnati Zoo & Botanical Garden, United States *Speaker

Threats to global plant biodiversity compel the need for ex situ collections of species worldwide. However, the subset of species known as exceptional plants are often overlooked. These species produce few or no seeds or produce recalcitrant seeds. The Center for Conservation and Research of Endangered Wildlife (CREW) at the Cincinnati Zoo & Botanical Garden maintains a liquid nitrogen bank of exceptional plant seeds and tissues known as the CryoBioBank®. CREW has partnered with the Lyon Arboretum in Honolulu to develop cryopreservation protocols for 21 endangered exceptional Hawaiian plant species and bank them in the CryoBioBank®. To date, micropropagation protocols to produce target tissues have been tested in 13 species, and cryopreservation protocols have been tested in 7 species. As an example, in Cyrtandra gracilis, micropropagation on medium containing 2mg/L of the auxin indole-3-acetic acid and 0.1mg/L of the cytokinin 6-benzylaminopurine induces shoot organogenesis on excised leaves. These leaves were dissected into leaf bud segments and compared with shoot tips for survival after liquid nitrogen storage. Cryopreservation of shoot tips using droplet vitrification (DV) with PVS2 yielded 0% survival, while leaf bud segments yielded 20% survival. DV using PVS3 increased survival in these leaf buds to 60%. Cryopreservation of leaf bud segments immediately after bud primordia formed yielded 25% survival compared to 50% survival if the buds were left to develop into shoots before cryopreservation. Shoot tips from another species, Melicope mucronulata, have been banked in the CryoBioBank® using the DV method following an experimental survival rate of 57% after 2 days of pre- culture on a 0.3M mannitol medium instead of one day on 0.3M mannitol and one day on 0.5M mannitol medium. This project will result in the long-term protection of many endangered exceptional Hawaiian species that would otherwise be unbankable using conventional methods. (Supported by IMLS grant #MG-30-17-0055-17).

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