Role of the cotyledonary tissue in improving low and ultralow temperature tolerance of butternut (Juglanscinerea) embryonic axes

Abstract

Butternut (Juglans cinerea L.) survival is threatened in North America by the fungus Sirococcus clavigignenti-juglandacearum. To date, there is no control for this fungal disease and long-term seed storage, to ensure survival of the species, is not a viable option. Initially, low (0, –5, –10, –15, and –40°C) and ultralow (–196°C, cryopreservation) temperature tolerance of butternut embryonic axes isolated from the nuts collected from one tree was examined. Embryonic axes with approximately 3 mm of cotyledonary tissue attached to the hypocotyl area germinated after exposure to 0, –5, –10, –15, and –40°C for 4 h and to –196°C for 24 h. Percent germination after exposure to 0 and –5°C was 87 and 82%, respectively, and after –10 and –15°C was 29 and 27%, respectively. Thirty-two percent of axes germinated after –40°C, and 36% germinated after exposure to –196°C. Tolerance to –196°C was examined in the embryonic axes isolated from the nuts of 13 other trees. Significant tree-to-tree variation was found in the tolerance of the embryonic axes to low temperature. This variation corresponded to the water content of the embryonic axes; water contents of 4.8% and lower exhibited tolerance to –196°C. Reducing the water content of the embryonic axes by slow desiccation to 4.8% or less resulted in an increased tolerance to –196°C. These results suggest that low and ultralow temperature storage of embryonic axes may be a viable method for butternut ex situ conservation.