A novel soybean hairy root system for gene functional validation

Abstract

Agrobacterium rhizogenes-mediated transformation has long been explored as a versatile and reliable method for gene function validation in many plant species, including soybean (Glycine max). Likewise, detached-leaf assays have been widely used for rapid and mass screening of soybean genotypes for disease resistance. The present study combines these two methods to establish an efficient and practical system to generate transgenic soybean hairy roots from detached leaves and their subsequent culture under ex vitro conditions. We demonstrated that hairy roots derived from leaves of two (tropical and temperate) soybean cultivars could be successfully infected by economically important species of root-knot nematodes (Meloidogyne incognita and M. javanica). The established detached-leaf method was further explored for functional validation of two candidate genes encoding for cell wall modifying proteins (CWMPs) to promote resistance against M. incognita through distinct biotechnological strategies: the overexpression of a wild Arachis α-expansin transgene (AdEXPA24) and the dsRNA-mediated silencing of an endogenous soybean polygalacturonase gene (GmPG). AdEXPA24 overexpression in hairy roots of RKN-susceptible soybean cultivar significantly reduced nematode infection by approximately 47%, whereas GmPG downregulation caused an average decrease of 37%. This novel system of hairy root induction from detached leaves showed to be an efficient, practical, fast, and low-cost method suitable for high throughput in root analysis of candidate genes in soybean.

The Stilbene Synthase Family in Arachis: A Genome-Wide Study and Functional Characterization in Response to Stress

“…arenaria were multiplied for three months on greenhouse-grown tomato (Solanum lycopersicum 'Santa Clara') plants and second-stage juveniles (J2) collected essentially as described by [40]. The identity of M. arenaria inoculum was confirmed by PCR analysis using a specific SCAR marker [71], as previously established [72]. GFP-positive hairy roots transformed with pPZP-AsSTS4 and pPZP-empty vectors were then challenged with approximately 1000 J2 of M. arenaria according to [38] and maintained in growth chamber conditions.…”

“…RNA purification, assessment of the RNA integrity, and cDNA synthesis were performed according to [73]. qRT-PCR reactions were conducted on a StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, USA), as described by [72]. Primer efficiency was determined by the online real-time PCR Miner tool [74], and the quantification of AsSTS4 expression was estimated using the SATqPCR website [75].…”