Method In order to produce virus-free hop plants, the uppermost growth zone (= meristem) at the very tip of the shoot is first heat-treated, then cut out and prepared. The meristems obtained in this way then regenerate complete plants on special culture media. To ascertain whether the the hop plants developed from the meristems are free of viruses, the IPS 2c WG Virus Diagnostics examines the leaves for signs of the various viruses typical in hop, employing the DAS-ELISA technique (Double Antibody Sandwich Enzyme Linked Immunosorbent Assay) or an RT-PCR test (Reverse Transcriptase Polymerase Chain Reaction). As a general rule, the cheaper ELISA detection method is used when testing for hop mosaic carlavirus (HpMV) and apple mosaic ilarvirus (ApMV). The molecular technique is deployed only in testing for American hop latent carlavirus (AhpLV), hop latent virus (HpLV), hop stunt viroid (HpSVd), and hop latent viroid (HpLVD), or in cases where only very little in vitro starting material is available. Results The first step – development of the cut and prepared meristem into a small shoot – takes only a relatively short time, but the subsequent steps further shoot growth and stages of cloning onto a solid medium mean that eliminating a virus is a time-consuming process, with a timescale of up to ten months. With a view to speeding up the whole process, different parameters for culture production were examined and then optimized. Use of a fluid culture system meant that the time needed from preparation of the meristem to regeneration and cloning of the plantlet could be shortened to 3.5 -5 months, as against the 6-10 months needed with cultures on a solid medium. At the same time, the dependence on genotype of the capacity to regenerate was improved. Moreover, meristem culture produced stronger plants. Outlook Work on further optimizing the regeneration of meristems continues, the main aim being to improve the efficiency of meristem culture in eliminating pathogens. References Gatica-Arias, A. (2012): Metabolic engineering of flavonoid biosynthesis in hop (Humulus lupulus L.) for enhancing the production of pharmaceutically active secondary metabolites. University of Hohenheim, Dissertation. Kremheller, H.T., Ehrmaier, H., Gmelch, F. & Hesse, H. (1989): Production and propagation of virus-free hops in Bavaria. In: Proceedings of the International Workshop on Hop Virus Diseases 1988, Deutsche Phytomedizinische Gesellschaft, Rauischholzhausen, 131–134. Momma, T., and Takahashi, T. (1983): Cytopathology of shoot apical meristem of hop plants infected with hop stunt viroid. Phytopath. Z., 106, 272-280. Penzkofer, M. (2010): Untersuchungen zur Massenvermehrung von Phlox-Sorten in einem temporary immersion system (TIS). Fachhochschule Weihenstephan, Fakultät Gartenbau und Lebensmitteltechnologie, Diplomarbeit. Schwekendiek, A., Hanson, S.T., Crain, M. (2009): A temporary immersion system for the effective shoot regeneration of hop. Acta Hort 848, 149-156.
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