In vitro establishment of Pittosporum confertiflorum Gray, an endemic plant of Hawaiʻi

Abstract

Passage of Acts 73 and 236 in 1992 and 1994 by the Hawaiʻi State Legislature, requires landscape contractors who bid on public projects to use Hawaiʻi's indigenous species in the landscaping of public buildings, facilities, and housing projects developed by the State, and has created a potentially lucrative niche market for Hawaiʻi's nurseries to create stock plantings of new, unique, and highly desirable species of native Hawaiian plants for use by the landscape industry. Pittosporum confertiflorum Gray is an endemic Hawaiian plant known as hōʻawa. This species is characterized as a dense, slow-growing, handsome broad-leaved evergreen that can be used as landscape ornamentals. However their use in local landscapes has not reached full potential due to its lengthy germination period and slow growth habit, making micropropagation suitable for its clonal multiplication. There are no studies that report any type of tissue culture protocol for any Hawaiian species of Pittosporum. Establishing Stage 1 contamination free explants without browning is a major challenge in creating a micropropagation method for P. confertiflorum. Several experiments were evaluated for control of phenolic oxidation and surface sterilization. Antioxidants citric acid (0.15 g/l) and ascorbic acid (0.10 g/L) were first used as a dip/rinse, and in later experiments, added to bleach surface sterilization solutions, sterile distilled water, and the media to reduce phenolic browning. Best practices for stock plant conditioning including preconditioning plants by rotating them from the glasshouse and into the lab, pruning and treating cuts with 0.4% BA at cuts after being brought indoors, and spraying buds with 70% ethanol the day prior to harvesting. Pretreated axillary explants were exposed to a two-step sterilization process using two different concentrations of bleach solution (10% and 5%) at different stages in the process for varying periods of time in the sonicator (Sonication 1--10, 15, or 20 m; Sonication 2--5, 10, or 15 m) (3 x 3 = 9 treatments). The effectiveness of these treatments were compared. Of 450 explants, 30% were contaminated and 42% exhibited necrosis. The best two-step sterilization treatment based on the mean was 10:5 minutes. Modifying the use of axillary bud explants to shoot tip culture and eliminating the second sonication reduced contamination to 6% and 25% necrosis. Revised sterilization methods as well as preconditioning of stock plants contributed to improved rates of contamination and necrosis.