title: An improved axenic system for studying pre-infection development of the parasitic plant Orobanche ramosa.
abstract: OBJECTIVE
  Broomrapes (Orobanche spp.) are holoparasitic weeds that cause devastating losses in many economically important crops. The molecular mechanisms that control early stages of host infection in Orobanche are poorly understood, partly due to the lack of experimentally tractable in vitro systems that allow the efficient application of molecular tools. Here an improved axenic system for the analysis of pre-infection stages in O. ramosa in the absence of the host plant is described.
  
  
  METHODS
  An optimized protocol for seed disinfection, based on formaldehyde, was developed. Orobanche ramosa seeds were conditioned in Petri dishes with filter paper, stimulated by addition of the synthetic strigol analogue GR24, and the percentage of germination as well as attachment-organ formation was determined.
  
  
  RESULTS
  Treatment of O. ramosa seeds with tobacco-root exudate or with GR24 resulted in highly reproducible germination rates around 70 %. A conditioning period of 8 d was both necessary and sufficient to allow optimal germination in response to GR24. Conditioned seeds that were dehydrated for several months remained fully responsive to GR24 without the need of a new conditioning period. Treatments as short as 5 min with GR24 were sufficient to fully and irreversibly induce the seed germination response. Approximately half of the germinated seeds initiated attachment-organ development. Similar rates of attachment organ induction were also detected in the rare cases of seeds that had germinated spontaneously on water.
  
  
  CONCLUSIONS
  The results suggest that the conditioning period produces persistent changes in the seeds required for responsiveness to external stimulants. The rapid action of GR24 suggests that it may act via a receptor-mediated signalling mechanism. While germination in O. ramosa is induced by exogenous stimuli, attachment organ differentiation appears to be triggered by unknown endogenous signals. The new in vitro culture system will have useful applications for the molecular analysis of early stages of parasitic development in Orobanche.
authors.affiliation: CIFA, Alameda del Obispo, IFAPA-CICE (Junta de Andalucía) Area de Mejora y Biotecnología, Apdo. 3092, 14080 Córdoba, Spain.
authors.forename: Clara Isabel
authors.initials: CI
authors.lastname: González-Verdejo
authors.forename: Xabier
authors.initials: X
authors.lastname: Barandiaran
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Moreno
authors.forename: Jose Ignacio
authors.initials: JI
authors.lastname: Cubero
authors.forename: Antonio
authors.initials: A
authors.lastname: Di Pietro
mesh_terms: D004202:Disinfectants / Q000494:pharmacology; D018525:Germination / Q000187:drug effects; D007783:Lactones / Q000494:pharmacology; D031672:Orobanche / Q000187:drug effects / Q000254:growth & development*; D010937:Plant Growth Regulators / Q000494:pharmacology; D018517:Plant Roots / Q000187:drug effects / Q000254:growth & development; D012639:Seeds / Q000187:drug effects / Q000254:growth & development; D013997:Time Factors; D014026:Nicotiana
chemical_list: D004202:Disinfectants; C507382:GR24 compound; D007783:Lactones; D010937:Plant Growth Regulators