Identification and Sterilization of Epiphytic Bacterial Flora near Hydrangea Shoot Apical Meristems

Abstract

In some plant species, bacterial contamination frequently occurs in in vitro culture. Also, in hydrangea (Hydrangea spp.), microbial contamination frequently occurs when their explants are cultured in vitro. We identified bacterial flora near hydrangea shoot apical meristems (SAMs) by analyzing their 16S ribosomal DNA (16S rDNA) sequences. Sequences of 16S rDNA fragments amplified from bacteria isolated from SAMs of 8 hydrangea cultivars were identical to those of 12 bacterial species. Because 1 to 9 bacterial species were detected only once in each cultivar, and we focused on bacteria that can colonize on Nutrient Broth medium, in this reserch we could not cover all bacterial species existing near SAMs. The appropriate chlorine concentration for sterilizing SAMs was decided using ‘Miss Hepburn’. When shoot tips with stripped SAMs were dipped in chlorine solution with 0.0005%–0.5% available chlorine concentration for 30 min, no bacterial colony was observed in the treatment with 0.05% chlorine. From this result, direct exposure of the stripped meristem to chlorine solution is a suitable method for sterilizing hydrangea shoot tip culture explants, and the bacteria causing in vitro contamination in hydrangea shoot-tip culture are not endophytic but epiphytic. A successful method for in vitro culture of hydrangea was established using 8 hydrangea cultivars in Exp. 3 and in 7 cultivars, we could gain 5 to 19 viable plants from 20 cultured explants without bacterial contamination by sterilizing shoot tips for 30 min with a sterilization solution of 0.05% available chlorine concentration after removal of all but two pairs of leaf primordia. No viable explant was gained from ‘Flambeau’, so the chlorine concentration must be reconsidered to sterilize such cultivars that have chlorine-sensitive meristems. One hundred and thirty days after culture initiation, no contamination was observed and cultured explants grew sufficiently to be transplanted out of the bottle. Surface sterilization of the SAM can be applied for many plants in which bacterial contamination poses a big problem in shoot-tip culture.