title: Ex vitro hairy root induction in detached peanut leaves for plant-nematode interaction studies.
abstract: BACKGROUND
  Peanut (Arachis hypogaea) production is largely affected by a variety of abiotic and biotic stresses, including the root-knot nematode (RKN)Meloidogyne arenariathat causes yield losses worldwide. Transcriptome studies of wildArachisspecies, which harbor resistance to a number of pests and diseases, disclosed several candidate genes forM. arenariaresistance. Peanut is recalcitrant to genetic transformation, so the use ofAgrobacterium rhizogenes-derived hairy roots emerged as an alternative for in-root functional characterization of these candidate genes.
  
  
  RESULTS
  The present report describes an ex vitro methodology for hairy root induction in detached leaves based on the well-known ability of peanut to produce roots spontaneously from its petiole, which can be maintained for extended periods under high-humidity conditions. Thirty days after infection with theA. rhizogenes'K599' strain, 90% of the detached leaves developed transgenic hairy roots with 5 cm of length in average, which were then inoculated withM. arenaria. For improved results, plant transformation, and nematode inoculation parameters were adjusted, such as bacterial cell density and growth stage; moist chamber conditions and nematode inoculum concentration. Using this methodology, a candidate gene for nematode resistance,AdEXLB8,was successfully overexpressed in hairy roots of the nematode-susceptible peanut cultivar 'Runner', resulting in 98% reduction in the number of galls and egg masses compared to the control, 60 days afterM. arenariainfection.
  
  
  CONCLUSIONS
  This methodology proved to be more practical and cost-effective for functional validation of peanut candidate genes than in vitro and composite plant approaches, as it requires less space, reduces analysis costs and displays high transformation efficiency. The reduction in the number of RKN galls and egg masses in peanut hairy roots overexpressingAdEXLB8corroborated the use of this strategy for functional characterization of root expressing candidate genes. This approach could be applicable not only for peanut-nematode interaction studies but also to other peanut root diseases, such as those caused by fungi and bacteria, being also potentially extended to other crop species displaying similar petiole-rooting competence.
authors.affiliation: Parque Estação Biológica, Embrapa Recursos Genéticos e Biotecnologia, CP 02372, Final W5 Norte, Brasília, DF Brazil.
authors.forename: Larissa Arrais
authors.initials: LA
authors.lastname: Guimaraes
authors.affiliation: Parque Estação Biológica, Embrapa Recursos Genéticos e Biotecnologia, CP 02372, Final W5 Norte, Brasília, DF Brazil.|Universidade de Brasília, Campus Darcy Ribeiro, Brasília, DF Brazil.
authors.forename: Bruna Medeiros
authors.initials: BM
authors.lastname: Pereira
authors.affiliation: Parque Estação Biológica, Embrapa Recursos Genéticos e Biotecnologia, CP 02372, Final W5 Norte, Brasília, DF Brazil.
authors.forename: Ana Claudia Guerra
authors.initials: ACG
authors.lastname: Araujo
authors.affiliation: Parque Estação Biológica, Embrapa Recursos Genéticos e Biotecnologia, CP 02372, Final W5 Norte, Brasília, DF Brazil.
authors.forename: Patricia Messenberg
authors.initials: PM
authors.lastname: Guimaraes
authors.affiliation: Parque Estação Biológica, Embrapa Recursos Genéticos e Biotecnologia, CP 02372, Final W5 Norte, Brasília, DF Brazil.
authors.forename: Ana Cristina Miranda
authors.initials: ACM
authors.lastname: Brasileiro
keywords: Agrobacterium rhizogenes; Arachis; Expansin like-B; Genetic transformation; Meloidogyne