establishment of axenic seed and embryo cultures of endangered Hawaiian plant species: special review of disinfestation protocols

More than one third of the USA's federally listed endangered and threatened plants are endemic to Hawaii. Because of the accelerating rate of environmental pressures resulting from habitat degradation to loss, the Hawaiian Rare Plant Program (HRPP, Lyon Arboretum, Honolulu, Hawaii) utilizes in vitro culture and seed storage to rescue, recover, and restore Hawaii's most critically endangered native plant taxa. These in vitro methods complement existing ex situ and in situ efforts in the state. Wild-collected seeds tend to be hard to clean, and obtaining contamination-free seeds or embryos without damage or over sterilization is one of the most difficult obstacles in the successful establishment of in vitro cultures. Loss of rare and valuable propagation material occurs when seed samples succumb to contamination or mortality during the initial disinfestation stage. This study reviews pre-treatments and provides examples of three general disinfestation protocols successfully used in the HRPP to establish axenic ovule and embryo cultures of some endangered Hawaiian species. Three disinfestation protocols are described: bleach sterilization, gas sterilization, and ethanol dip and flame. Since 1998, the HRPP has been successful in establishing in vitro cultures of 135 endangered Hawaiian plant taxa representing 30 genera.