Plant-Development Cycler Project: Callus Induction and Viroid Detection in Ornamental Crops Saskia Blom1, Aylin Figiel-Lange1, Yipeng Guo1, Hulia Qimaz Izadin Khayyat1,Gerhard Artmann1, Friederike von Rundstedt2, Georg Hanka3, Rasha Bassam1, Ilya Digel1 1Institute for Bioengineering, FH Aachen University of Applied Sciences, D-52428 Jülich 2Bock Bio Science GmbH, D-28357 Bremen 3Hanka Gartenbau, D-47906 Kempen

Viroids are virus-like sub-cellular agents that belong to the smallest known pathogens and consist of a sophisticatedly folded, single-stranded RNA. Although many basic features of viroids as sub-cellular pathogens are already understood, there is still no satisfying treatment for viroid diseases. Being able to infect various plants, they cause considerable economic losses in agricultural (e.g. lemon trees) as well as in ornamental plants and crops. Recently, outbreaks of viroid diseases were detected in ornamental plants belonging to the family Solanaceae. The currently running joint project “Plant Development Cycler” is aimed on purification of horticultural crops Solanum jasminoides form viroids. In ornamental plants, the RNA molecules are usually present as latent pathogens, that is, the infection can only be proven by bio-molecular analytical methods such as real-time polymerase chain reaction (PCR). Viroid RNA represents just a tiny fraction in numerous different types of small RNA in a plant cell which demands careful RNA extraction for viroid detection. Therefore, the primary steps of our study were optimization of the necessary preparations for viroid detection by RT-PCR from Solanum jasminoides cells originating from plants and from callus. The principal preparation steps are (1) sample homogenization and (2) RNA extraction. The cells were destructed using two different methods based on the mechanical destruction. Further, four different RNA extraction kits were used with five different protocols and adapted to meet our requirements: MyBudget Plant RNA kit (Bio-Rad); Master PurePlant RNA Purification Kit (Biozym); RNeasyMini Kit (Qiagen) and E.Z.N.A Plant RNA Mini Kit (OMEGA bio-tek). For all protocols, appropriate incubation times and sample volumes were adjusted.

Fig. 2: Solanum jasminoides culture: A. Callus culture; B. Plant regeneration out of the callus can be achieved by adjustment of hormone concentration; C. Suspension culture; D. Callus tissue under 1600x magnification.

Fig. 1: Ornamental plant Solanum jasminoides is highly susceptible to viroid infections and can serve as a reservoir for viroid diseases . A. Plantation; B. Viroid infections are easily spread by a cutting machine; C. Individual plant; D. Blossom.

Fig. 4: Schematic presentation of the RNA extraction procedure and the RNA yield provided by different RNA-extraction kits

A B

D C

Fig. 3: Upper part: structure of a PSTVd viroid. Lower part: homogenization device TissueLyser and gel electrophoresis of isolated and amplified viroid RNA.

Gefördert durch: Bundesministerium für Wirtschaft und Technologie aufgrund

eines Beschlusses des Deutschen Bundestags (KF2425604MD2)