Agrobacterium rhizogenes-mediated transformation of Prunus as an alternative for gene functional analysis in hairy-roots and composite plants

Abstract

Resistant rootstocks offer an alternative to pesticides for the control of soil pests. In Prunus spp., resistance loci to root-knot nematodes (RKN) have been mapped and a transformation method is needed to validate candidate genes. Our efforts have focused on the generation of transformed hairy-roots and composite plants appropriate for nematode infection assays. An efficient and reliable method using the A4R strain of Agrobacterium rhizogenes for the transformation of Prunus roots with an Egfp reporter gene is given. The rooting efficiency, depending on the genotypes, was maximal for the interspecific hybrid 253 (Myrobalan plum × almond-peach), susceptible to RKN, that was retained for subsequent studies. From the agro-inoculated cuttings, 72% produced roots, mainly at the basal section of the stem. Transformed roots were screened by microscope detection of Egfp fluorescence and molecular analyses of the integration of the transgene. The absence of residual agrobacteria in the plants was checked by the non-amplification of the chromosomal gene chvH. Egfp was expressed visually in 76% of the rooted plants. Isolated hairy roots in Petri dishes and composite plants (transformed roots and non-transformed aerial part) in soil containers were inoculated with the RKN Meloidogyne incognita. In both cases, root transformation did not affect the ability of the nematodes to develop in the root tissues. Our results showed that isolated hairy-roots can be used to validate candidate genes and the conditions in which composite plants offer a complementary system for studying the function of root genes in physiological conditions of whole plants are discussed.

TheMaGene for Complete-Spectrum Resistance toMeloidogyneSpecies inPrunusIs a TNL with a Huge Repeated C-Terminal Post-LRR Region

“…Agrobacteria were controlled by cefotaxime, but selection of transformants on kanamycin medium could not be performed because of the strong adverse effect of the antibiotics on the root growth of Prunus. Thus, roots growing autonomously and considered as hairy roots were cultured, multiplied by division at each growth time period, sampled for DNA and RNA extraction, and inoculated for evaluation of resistance to different RKN species (Bosselut et al, 2011). Fifteen independent hairy roots originating from six selected plants were infested with (1) M. incognita, (2) M. mayaguensis (= M. enterolobii), and (3) mixed populations of M. arenaria, M. javanica, and M. floridensis (5,000 J2s per individual root, two roots per transformation event and RKN species; Table II).…”

“…Independently from the hairy root transformants, sets of microplants, rooted after inoculation with agrobacteria carrying TNL1 (54 plants) or the empty vector (60 plants), were grown individually in pots as composite plants (transformed roots and untransformed aerial part) under nonsterile conditions together with control untransformed accessions (Bosselut et al, 2011). Composites microplants were infected with juveniles (J2s), and resistance was assessed after 8 weeks.…”

“…3), was transformed into Agrobacterium rhizogenes strain A4R (Tepfer, 1990) by electroporation. Microplants were inoculated by injecting the agrobacteria solution 5 to 10 mm above the base of the stem with a sterile syringe as described by Bosselut et al (2011). Cocultivation of the plants and the agrobacteria was performed at 25°C for 5 d. Independent transformation experiments were performed in order to (1) produce hairy roots in petri dishes and (2) generate composite plants (transformed hairy roots plus nontransformed aerial part; Bosselut et al, 2011).…”

Rootstock breeding in Prunus species: Ongoing efforts and new challenges

“…salicina. ), are susceptible to PPN attack worldwide (Nyczepir, 1991;Esmenjaud et al, 1996;Stalin et al, 1998;Pinochet, 2000;Rosso et al, 2004;Di Vito et al, 2005;Walters et al, 2008;Nyczepir and Thomas, 2009;Ye et al, 2009;Bosselut et al, 2011). The effects of PPN in crops are often underestimated but in general, it is accepted that on average, nematodes are annually reducing the global agricultural production by about 10% to 12% (Agrios, 2005).…”

Recent Advances in Fruit Species Transformation

“…Explant types are highly variable since it depends on the selected organogenetic process optimized for each species. Commonly, the genetic transformation protocols of fruit species employed explants such as ovules, anthers, seedlings, zygotic and somatic embryos, cotyledons, epicotyles, hypocotyles, leaf pieces, roots, meristems (Fagoaga et al, 2007;Lopez-Perez et al, 2008;Petri et al, 2008;Husaini, 2010;Malnoy et al, 2010;Bosselut et al, 2011;Petri et al, 2011;Gago et al, 2011). Typically, it is recommended that those tissues have high and active cell division to enhance the regeneration of the transgenic lines (Mante et al, 1991;Schuerman & Dandekar, 1993;Wang, 2011).…”