Development of novel methods for the initiation of in vitro bryophyte cultures for conservation

The value of ex situ plant collections for conservation is increasingly recognised. The Royal Botanic Gardens, Kew (RGB, Kew) has been pioneering methods for the development of such collections with the establishment of the Millennium Seed Bank and a project for the ex situ conservation of threatened UK bryophytes (mosses, liverworts, hornworts). A broad range of bryophytes are currently held in a tissue culture collection at RBG, Kew on sucrose-free 1/4 or 1/2 Murashige & Skoog or Knops minimal medium. The efficacy of the novel sterilising agent Sodium dichloroisocyanurate (NaDCC) was tested on a range of taxa, utilising a variety of bryophytic initiation material. Concentrations of 1% (w/v) for 3 min and 0.5% (w/v) for 2 min, without the addition of detergent, were found to be successful in initiating cultures from sporophytes and leafy gametophores respectively. Initiation of cultures from wild-collected sporophytes was more successful than from wild-collected gametophores. However, pre-culturing of gametophore material was found to enhance success rate of procedures. Transferring visibly clean material away from contamination post-initiation was also determined to increase protocol success rate. The ability of the biocide Plant Preservative Mixture (TM)(PPM (TM)) to control fungal and bacterial contamination in bryophyte protonemal cultures was also tested, but not found to be suitable. However, results obtained indicated that contamination may confer resistance on the moss against the phytotoxic effects of the biocide. Methods developed have wider applicability for the establishment of in vitro collections of other threatened plants.