Ex vitro hairy root induction in detached peanut leaves for plant–nematode interaction studies

Abstract

BackgroundPeanut (Arachis hypogaea) production is largely affected by a variety of abiotic and biotic stresses, including the root-knot nematode (RKN) Meloidogyne arenaria that causes yield losses worldwide. Transcriptome studies of wild Arachis species, which harbor resistance to a number of pests and diseases, disclosed several candidate genes for M. arenaria resistance. Peanut is recalcitrant to genetic transformation, so the use of Agrobacterium rhizogenes-derived hairy roots emerged as an alternative for in-root functional characterization of these candidate genes.ResultsThe 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 the A. rhizogenes ‘K599’ strain, 90% of the detached leaves developed transgenic hairy roots with 5 cm of length in average, which were then inoculated with M. 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 after M. arenaria infection.ConclusionsThis 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 overexpressing AdEXLB8 corroborated 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.

Early responses to dehydration in contrasting wild Arachis species

“…In the present study, many genes coding for cell wall modifying proteins were activated in response to dehydration and an EXPANSIN A (EXPA) was amongst the most highly upregulated of the genes analyzed by qRT-PCR ( Fig 8 ). These results support our previous hypothesis that wild Arachis Expansins are common and crucial players in responses to multiple and simultaneous stresses, including drought, ultraviolet light exposure, and nematode infection [ 19 , 21 , 23 , 65 ]. In contrast, AQUAPORIN transcripts were downregulated in all expression analyses conducted on both species.…”

High Efficient Expression and Purification of Human Epidermal Growth Factor in Arachis Hypogaea L.

“…Therefore, the clinical application value or market demand of hEGF has become huge and booming in recent years. Hairy root is a good host for mass production of recombinant protein due to its abundant quality and extensive lateral branches [19]. Hairy root production system has received considerable interest in recent years [20,21].…”

“…Seeds of peanuts were germinated on soil and grown under greenhouse conditions (25 °C and approximately 16 h light /8 h dark photoperiod) for 30 d. Surface-sterilized leaf disks (approximately 2 × 2 cm) were inoculated with various concentrations of Agrobacterium rhizogenes R1601 and HR1601 strain (OD 600 = 0.5, 1.0, 1.5 or 2.0) for 2 min [19]. After removal of the excess strain with distilled water, leaf disks were then transferred to Murashige and Skoog medium (MS) containing cefotaxime sodium (200 mg/L) and allowed to grow in continuous light (8800 LUX) at 25 °C for about three weeks for hairy root emergence.…”

Potential of the genetically transformed root cultures of Plumbago europaea for biomass and plumbagin production

“…12 Generation of transgenic roots might be a promising approach in future owing to their specific characteristics including higher genetic and biochemical constancy, easy maintenance, fast growth, ability to synthesize a range of secondary metabolites, growth in PGRs-free media, 13,14 high lateral branching and plagiotropism. 15 As previously reported, numerous elements affect the transformation frequency in various plants such as bacterial strain, type of explant, co-cultivation media, co-cultivation period, inoculation media, inoculation temperature, type and concentration of carbohydrate source, seedling age, genotype variation and different nutrient components. [16][17][18][19] In hairy root system, some factors including medium composition, light and temperature have significant impacts on growth and production of natural products, which are frequently species dependent.…”

“…The rol genes have significant impacts on hairy roots growth and secondary metabolite synthesis by activating the special transcription factors . Generation of transgenic roots might be a promising approach in future owing to their specific characteristics including higher genetic and biochemical constancy, easy maintenance, fast growth, ability to synthesize a range of secondary metabolites, growth in PGRs‐free media, high lateral branching and plagiotropism . As previously reported, numerous elements affect the transformation frequency in various plants such as bacterial strain, type of explant, co‐cultivation media, co‐cultivation period, inoculation media, inoculation temperature, type and concentration of carbohydrate source, seedling age, genotype variation and different nutrient components .…”