Female reproductive system of <i>Amaranthus</i> as the target for <i>Agrobacterium</i>-mediated transformation

Abstract

Agrobacterium-mediated transformation through floral dip and rapid selection process after transgenic event had become a preference as it will overcome the difficulties faced in tissue culturing procedures and lengthy time for screening transformed progenies. Therefore, in this study, three constructs, p5b5 (14,289 bp), p5d9 (15,330 bp) and p5f7 (15,380 bp) in pDRB6b vector which has hygromycin as a selectable marker gene were introduced individually into Agrobacterium tumefaciens strain (AGL1). The cell suspension was applied to Amaranthus inflorescence by drop-by-drop technique and was left to produce seeds (T 1 ). The T 1 seeds were germinated and grown to produce seedlings under non-sterile condition. Hygromycin selection on seedling cotyledon leaves results in identification of 12 putative transformants, three from p5b5, four from p5d9 and five from p5f7. All positive putative transformants that were selected at the first stage through hygromycin spraying showed positive result in leaf disk hygromycin assay and in a construct specific polymerase chain reaction-based assay. A ~750 bp amplified hygromycin gene was further verified through sequencing. Our results suggest that Amaranthus inflorescences were able to be transformed and the transformed progenies could be verified through a combination of simple and rapid methods.

“Floral-dip” transformation of Amaranthus caudatus L. and hybrids A. caudatus × A. paniculatus L.

“…Also there are two studies dedicated to the transformation through amaranth inflorescence. Umaiyal Munusamy with co-authors used the construction with selective genes (Munusamy et al, 2013), and Taipova conducted experiments with Amaranthus retroflexus, A. viridis, A. cruentus (Taipova, Kuluev, 2015).…”

“…We added 1 ml of A. tumefaciens into 50 ml of medium with 0.2 mM of acetosiringone. Next, Agrobacterium was centrifuged for 12 min, 5000 rpm and was resuspended into the medium with sucrose (50 g/l + 0.2% super wetting agent Silwet) (Munusamy et al, 2013).…”

Agrobacterium – caused transformation of cultivars Amaranthus caudatus L. and hybrids of A. caudatus L. x A. paniculatus L.

“…Also there are 2 works dedicated to the transformation through amaranth inflorescence -Umaiyal Munusamy with co-authors. They used the construction with selective genes [15]. Prof. Taipova conducted experiments with Amaranthus retroflexus, A. viridis, A. cruentus [9].…”

Achievements in Genetic Engineering of Amaranthus L. Representatives

“…There are also 3 studies devoted to amaranth transformation through inflorescences by the "floral-dip" method -Umaiyal Munusamy and co-authors. They used a vector with selective genes (Munusamy et al, 2013).…”