Direct in vitro Regeneration and Transient Gus Assay: Towards Stable Genetic Transformation in Trifolium alexandrinum L.

Abstract

The process of genetic transformation allows insertion of transgene conferring trait(s) of interest togenetically modified plants. Here, we report direct in vitro regeneration and transient gus assay as a step forward forAgrobacterium mediated genetic transformation of Berseem (Trifolium alexandrinum L.). The cotyledons from invitro grown plants of local berseem varieties “superlate Faisalabad” and “Anmol” were cultured on MS mediasupplemented with various concentrations of NAA and BAP, for direct shoot induction. Maximum regenerationwas obtained on MS medium BAP (2.0 mg/L) and NAA (0.05 mg/L). Regenerated shoots were rooted on MSmedia augmented with IBA. For transformation, in vitro grown cotyledons were co-cultured with Agrobacteriumtumefaciens strain EHA105 harboring plant transformation vector p7i-UG containing uidA gene. Histochemical GUSassay was performed for tracking transient expression of reporter gene in cotyledons. Direct shoot regeneration fromcotyledons was peculiarity of this study. Hence, establishment of efficient direct shoot regeneration combined withtransient GUS gene expression would pave the way towards efficient genetic transformation of Trifoliumalexandrinum L for valuable traits in future.