Plant Cell,Tissueand OrganCulture 22:113-117, 1990. © 1990 Kluwer AcademicPublishers. Printedin the Netherlands. Glass beads as a solid matrix in in vitro study of the role of polyamines in cold hardiness of white clover Kenneth MacLeod & Jerzy Nowak* Department of Plant Science, Nova Scotia Agricultural College, Truro, N.S., Canada B2N 5E3 (*requests for offprints) Received 17 August 1989;accepted in revisedform 19 January 1990 Key words: cold hardiness, media exchange, polyamine inhibitors, solid matrix, Trifolium repens L. Abstract Glass beads were used as an alternative to agar in the study of the role of polyamines in cold hardiness of white clover. Plantlet growth performance, in vitro hardening and cold stress tolerance were similar on both the agar solidified medium and the liquid medium in glass beads matrix. Glass beads allowed media exchange without plant transfer and an easy monitoring of the uptake of putrescine synthesis inhibitor, ~4C di- fluoromethylornithine from the medium. The matrix is recommended in in vitro studies of whole plant physiology, screening procedures and bioassays where media exchange and/or uniform application of a selection pressure is required. There is also 60% saving on media components and the beads can be re-used after acid wash. Abbreviations: DFMA-difluoremethylarginine, DFMO-difluoromethylornithine, MS-Murashige and Skoog salts, Kcpm-thousand counts per minute Introduction Studying the role of polyamines in cold hardiness of white clover we utilized polyamine synthesis inhibitors, difluoromethylarginine (DFMA) and difluoromethylornithine (DFMO). Evidence in the literature suggested that these inhibitors can be uptaken by callus [3, 5], cell suspension [4] and protoplast cultures [7], by root fragments [12] and decapitated shoots [2]. We found in media solidified with 0.8% agar a poor uptake of ~4C-DFMO by rooted white clover plantlets. The question of whether the DFMO was strongly bound by the agar thus reducing its availability for uptake, or whether roots were unable to uptake DFMO could not be answered using agar-based medium. It was necessary therefore to use an alternative support which would allow rapid media exchange and media sampling to monitor the uptake of the in- hibitor. Neither starch [13] nor membrane raft [8] seemed amenable to our needs. This paper descri- bes utilization of glass beads as a solid matrix in in vitro experiments with rooted white clover plant- lets. Other possible applications of this solid sup- port as agar alternative are also discussed. Materials and methods Plant material, chemicals and supplies Two genotypes of white clover, Huia 5 from cv. Huia [6] and Sonja 31 from cv. Sonja previously selected in vitro for a high regenerative ability and the ability to form strong root systems at low tem- perature, respectively, were used in this experiment. MS salts [9] and other tissue culture chemicals were from Carolina Biological Supply Co. (Burlington, NC, USA). DFMA and DFMO were kindly donat- ed by Merrell Dow Research Institute (Cincinnati, OH, USA) and 5-~4C-DFMO (60mCi/mmol) and