Microparticle bombardment of Stylosanthes guianensis: transformation parameters and expression of a methionine-rich 2S albumin gene

Abstract

An effective protocol to generate stable transformants of the tropical forage legume Stylosanthes guianensis (Aubl.) Sw. in a selection-free system was developed. Based on transient reporter gene expression, we have obtained transformation rates of 3.47% using 30-day-old calli as target, 1300 psi helium pressure, 12.5 cm microprojectile flight distance, 10–20 mm distance between macrocarrier membrane and stopping screen and 10–20 mm gap distance between the shock wave generator and the macrocarrier. These parameters were utilized to produce transgenic S. guianensis plants expressing Be2S1 from Bertholletia excelsa that codes for a methionine-rich storage protein driven by a green-tissue specific promoter, Ats1 from Arabidopsis thaliana. Transgenic plants were identified by a PCR-based high-throughput screen in a selective agent-free system, employing pools of 20–50 regenerating shoots. The integration of the exogenous gene in the host genome was confirmed by Southern blot analysis of PCR-positive plants. The expression of the introduced gene was confirmed in leaf tissue of transgenic plants by Northern and Western blot analyses. Immunoblots of cellular fractions showed that BE2S1 expressed in Stylosanthes is mainly targeted to the vacuoles.