In Vitro Germination of Four Rare Endemic Hawaiian Cyanea Spp. for the Restoration of Plants Extinct in the Wild

The Hawaiian Islands are noted for a high rate of endemism and endangered species. Cyanea superba ssp. superba, C. grimesiana ssp. grimesiana, C. pinnatifida, and C. truncata are four endemic species in the Campanulaceae family that are extinct in the wild. All are targets for in vitro propagation for genetic banking and restoration efforts. Four protocols were investigated for the in vitro establishment of these species from seed, obtained in the form of mature and immature fruit or seed. Intact fruits were sterilized by the dip and flame method—dipping intact fruit in 95% ethanol then applying to flame. Seeds were sterilized by chlorine gas formed by the addition of concentrated hydrochloric acid to 85% chlorine bleach, 10% chlorine bleach with Tween® 20, or a soak in 5 ppm Plant Preservative Mixture™ biocide/fungicide (PPM) in ½ Murashige and Skoog liquid media followed by the same chlorine bleach sterilization. Immature fruit of C. superba ssp. superba were sterilized using the dip and flame method, achieving 9% germination. Mature seed germinated at 63% under bleach sterilization, compared to 41% under an application of PPM and bleach sterilization. Seeds from immature fruit of C. grimesiana ssp. grimesiana germinated at a rate of 35% using the dip and flame method, while mature seed germinated at 31% using bleach sterilization. The dip and flame method on immature fruit of C. pinnatifida yielded 37% germination. Mature seed germinated at 43% with bleach sterilization, and 47% with gas sterilization. Germination rates for C. truncata of 14% from immature fruit and 76% from mature fruit were achieved using the dip and flame method. Direct bleach sterilization of immature seed yielded 44% germination. Mature seed germinated at a rate of 97% with bleach sterilization, and 39% with the application of PPM followed by bleach sterilization. According to data from the Lyon Arboretum Seed Conservation Laboratory, under standard conditions, average germination percentages for mature seed are 34% for C. superba ssp. superba, 85% for C. pinnatifida, and 67% for C. truncata. Due to the rarity and value of these seeds, viability testing has not been done, and data for C. grimesiana ssp. grimesiana does not exist. All protocols proved effective in establishing these rare plants in vitro from seed.