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 selectionfree 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 methioninerich 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.

Co‐expression of NCED and ALO improves vitamin C level and tolerance to drought and chilling in transgenic tobacco and stylo plants

“…Although Agrobacterium-mediated transformation of stylo was reported using bar gene as selective marker (Sarra et al, 1994), the transformation efficiency was low, and thus, microparticle bombardment protocol was examined later (Quecini et al, 2006). It is important to establish an effective transformation protocol in stylo for its improvements by transgenics.…”

Monitoring of cell wall modifications during callogenesis in Stylosanthes guianensis (Fabaceae) under salt stress conditions

“…Furthermore, organogenic calli have been referred to as a suitable strategy to regenerate stable stylosanthes transgenic plants (Quecini et al 2006). Nevertheless, the studies about behavior under saline conditions in stylosanthes species referred only to germination (Gonela et al 2004, Silva et al 2014) and plant growth (Lovato and Martins 1997;Gonzá-lez et al 2000;Fuentes et al 2010).…”