Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos

Abstract

ABSTRACT. Mature embryos in tissue cultures are advantageous because of their abundance and rapid germination, which reduces genomic instability problems. In this study, 2-day-old isolated mature barley embryos were infected with 2 Agrobacterium hypervirulent strains (AGL1 and EHA105), followed by a 3-day period of cocultivation in the presence of L-cystein amino acid. Chimeric expression of the b-glucuronidase gene (gusA) directed by a viral promoter of strawberry vein banding virus was observed in coleoptile epidermal cells and seminal roots in 5-day-old germinated seedlings. In addition to varying infectivity patterns in different strains, there was a higher ratio of transient b-glucuronidase expression in developing coleoptiles than in embryonic roots, indicating the high competency of shoot apical meristem cells in the mature embryo. A total of 548 explants were transformed and 156 plants developed to maturity on G418 media after 18-25 days. We detected transgenes in 74% of the screened plant leaves by polymerase chain reaction, and 49% of these Genomic integration of transgenes in mature barley embryos expressed neomycin phosphotransferase II gene following AGL1 transformation. Ten randomly selected T 0 transformants were analyzed using thermal asymmetric interlaced polymerase chain reaction and 24 fragments ranged between 200-600 base pairs were sequenced. Three of the sequences flanked with transferred-DNA showed high similarity to coding regions of the barley genome, including alpha tubulin5, homeobox 1, and mitochondrial 16S genes. We observed 70-200-base pair filler sequences only in the coding regions of barley in this study.

The distribution of cotransformed transgenes in particle bombardment-mediated transformed wheat

“…Our results showed that transgene integration occurred unevenly among the chromosomes of A, B and D genomes, with integration sites at different locations along individual chromosomes, there was only a slight tendency toward localization to distal regions. Similar to our findings, T-DNA insertion sites generated under non-selective conditions are not biased to any particular genomic region by Agrobacterium-mediated transformation (Kim and Gelvin 2007;Uçarlı et al 2015) and direct microinjection of foreign DNA is randomly integrated into chromosomes in transgenic animals (Yan et al 2013). Furthermore, transformation frequency was demonstrated to be higher for cells in the M or G2 phase compared to the S phase of the cell cycle in one study (Iida et al 1991) but in other later studies it was shown to be higher in the S and G2 (Arias et al 2006) or S and M (Villemont et al 1997) phase of the cell cycle.…”

Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava

“…Consistently, the transcript level of MePDS in MePDS -VIGS plants were 37.9%–53.1% of that in the mock, with an obvious albino phenotype and a 58%–76.5% level of chlorophyll. Compared with GV3101, Agrobacterium AGL-1 showed better effects in the VIGS system (Table 2), which might have been due to the specific hypervirulence of AGL-1 [46]. AGL-1 is derived from pTiBo542, which has a high induction of the vir gene [47], which is necessary for T-DNA transfer [48].…”