Development of transgenic composite Stylosanthes plants to study root growth regulated by a β-expansin gene, SgEXPB1 , under phosphorus deficiency

Key message A highly efficient transformation procedure to generate transgenic Stylosanthes roots was established. SgEXPB1 is involved in Stylosanthes root growth under phosphorus deficiency. Abstract Stylo ( Stylosanthes spp.) is an important forage legume widely applied in agricultural systems in the tropics. Due to the recalcitrance of stylo genetic transformation, functional characterization of candidate genes involved in stylo root growth is limited. This study established an efficient procedure for Agrobacterium rhizogenes -mediated transformation for generating transgenic composite plants of S. guianensis cultivar ‘Reyan No. 5’. Results showed that composite stylo plants with transgenic hairy roots were efficiently generated by A. rhizogenes strains K599 and Arqual, infecting the residual hypocotyl at 1.0 cm of length below the cotyledon leaves of 9-d-old seedlings, leading to a high transformation efficiency of > 95% based on histochemical β-glucuronidase (GUS) staining. Notably, 100% of GUS staining-positive hairy roots can be achieved per composite stylo plant. Subsequently, SgEXPB1 , a β-expansin gene up-regulated by phosphorus (P) deficiency in stylo roots, was successfully overexpressed in hairy roots. Analysis of hairy roots showed that root growth and P concentration in the transgenic composite plants were increased by SgEXPB1 overexpression under low-P treatment. Taken together, a highly efficient A. rhizogenes -mediated transformation procedure for generating composite stylo plants was established to study the function of SgEXPB1 , revealing that this gene is involved in stylo root growth during P deficiency.