Influence of antibiotics on embryogenic tissue and Agrobacterium tumefaciens suppression in soybean genetic transformation

Abstract

The influence of different antibiotic treatments in soybean genetic transformation was evaluated. First, an assay was performed to verify how different antibiotic treatments affect soybean embryogenic tissues. The effect of carbenicillin at 500 mg L -1 was genotype-dependent. This antibiotic did not affect embryo survival of cv. IAS5, but a three-fold increase of embryo proliferation was observed for cv. Bragg, when compared to the control. On the other hand, cefotaxime at 350 and 500 mg L -1 caused death of embryogenic tissues of both cultivars. Finally, the association of cefotaxime (250 mg L -1 ) + vancomycin (250 mg L -1 ) did not affect negatively the somatic embryos of tested cultivars until 63 days of treatment. Thereafter, a second experiment was carried out to determine the efficacy of different antibiotic treatments in suppressing LBA4404 Agrobacterium tumefaciens strain in genetic transformation. On tissue culture conditions, carbenicillin at 500 and 1000 mg L -1 was not active against Agrobacterium. On the other hand, treatments with cefotaxime at 350 and 500 mg L -1 , and cefotaxime + vancomycin efficiently suppressed Agrobacterium during 49 days. Data of both experiments suggested cefotaxime + vancomycin for 49-63 days as the most appropriate treatment. This is the first work reporting the effect of antibiotics on soybean tissues. By identifying an antibiotic combination that suppressed A. tumefaciens with minimal phytotoxic effects, we are able to recommend it for improvement of soybean Agrobacterium-mediated transformation procedure.

Evaluation of novel beta-lactam antibiotics in comparison to cefotaxime on plant regeneration of Citrus sinensis L. Osb.

“…Beta-lactam antibiotics, such as carbenicillin and cefotaxime, have commonly been used to suppress and eliminate Agrobacterium, since they specifically inhibit prokaryotic cell wall synthesis and kill bacteria, with little or no detrimental effect on eukaryotic plant cells (Pollock et al 1983;Asbel and Levison 2000). Unfortunately, noticeable inhibitory effects of both antibiotics on plant cell growth, organogenesis and embryogenesis have been well documented (Nauerby et al 1997;Ogawa and Mii 2005;Wiebke et al 2006), especially in herbaceous plants. For woody fruit plants, however, little is known about the effects of beta-lactam antibiotics on in vitro growth and morphogenesis.…”

Challenges of In Vitro and In Vivo Agrobacterium-Mediated Genetic Transformation in Soybean

“…The recovered adventitious shoots grew to maturity and survived the continuous application of glufosinate-ammonium used as a selective agent for identification of transformed plants [38]. The trend observed in the study and other reports [59,63] suggest an emerging problem of antibiotic-strain relationship that specific antibiotics could be required for a specific strain of A. tumefaciens used during transformation. Aminoglycoside antibiotics are mostly used in the transformation process as selectable markers.…”

Transgenic fertile soybean plants derived from somatic embryos transformed via the combined DNA-free particle bombardment and Agrobacterium system

“…In the last years, efforts have been made to achieve an efficient Agrobacterium-mediated transformation procedure for soybean somatic embryos Finer 1997, 1998;Droste et al 2000;Wiebke et al 2006). The present work is the first report of transgenic fertile plants obtained from somatic embryogenic tissues transformed by the integrated free-DNA particle bombardment/Agrobacterium system.…”

“…Although transient expression of the gus-intron gene was successfully achieved, stable transformants were not obtained. It was further demonstrated that the soybean somatic embryos employed were too sensitive to the antibiotics required for Agrobacterium suppression after gene transfer (Wiebke et al 2006). A modification at the transformation protocol, regarding the antibiotic treatment, was suggested to maintain the viability of stable transformed cells and to allow their conversion into plants.…”