Effects of antibiotics on the elimination of Agrobacterium tumefaciens from loblolly pine (Pinus taeda) zygotic embryo explants and on transgenic plant regeneration

Abstract

Three antibiotics were evaluated for their effects on the elimination of Agrobacterium tumefaciens during the genetic transformation of loblolly pine (Pinus taeda L.) using mature zygotic embryos as targets. Agrobacterium tumefaciens strains, EHA105, GV3101, and LBA 4404, all harbouring the plasmid pCAMBIA1301, which carries the selectable marker gene, hygromycin phosphotransferase (hpt) controlled by the cauliflower mosaic virus 35S promoter and terminator, and the uidA reporter gene (GUS) driven by the cauliflower mosaic virus 35S promoter and the terminator of nopaline synthase gene, were used in this study. Exposure to 350 mg l À1 carbenicillin, claforan, and timentin respectively for up to 6 weeks did not eliminate the Agrobacterium, while antibiotics at 500 mg l À1 eradicated them from the co-cultivated zygotic embryos. All three antibiotics increased callus growth and shoot regeneration at 350 and 500 mg l À1 each, but reduced callus growth and shoot regeneration at 650 mg l À1 when compared with controls. Putative transgenic calli were selected for continued proliferation and differentiation on 4.5 mg l À1 hygromycincontaining medium. Transformed calli and transgenic plants produced on a selection medium containing 4.5 mg l À1 hygromycin were confirmed by GUS histochemical assays, by polymerase chain reaction (PCR), and by Southern blot analysis. These results are useful for future studies on optimizing genetic transformation procedures in loblolly pine.

The effect of different antibiotics on the elimination of Agrobacterium and high frequency Agrobacterium-mediated transformation of indica rice (Oryza sativa L.)

“…The inhibitory effect of cefotaxime on shooting and rooting was explained by Nauerby et al (1997). In contradiction to our investigation implying the negative effects of carbenicillin, the influence of carbenicillin on regeneration has been proved as it possesses auxin-like structural features and when broken down, displays effects similar to those of the weak auxin phenylacetic acid in the culture medium, thereby, increasing the regeneration potential of cultured explants (Nauerby et al 1997;Ling et al 1998;Tang et al 2004). Cheng et al (1998) reported that timentin can be considered to be an alternative antibiotic for those species in which the regeneration potential is negatively affected by carbenicillin and cefotaxime.…”

“…According to Matzk et al (1996) it is essential to suppress and eliminate the extra Agrobacterium present in the plant tissue after transformation to reduce the detrimental effects on growth and regeneration as well as to prevent the release of genetically modified microorganisms. The influence of different antibiotics (carbenicillin, claforan, and timentin) on the elimination of Agrobacterium and differentiation of transgenic calli and transgenic plantlet regeneration through Agrobacterium-mediated transformation in loblolly pine was well proved (Tang et al 2004). The influence of antibiotics on the growth of transgenic calli is similar to that of polyamines that improve the growth of transformed tissues (Bais et al 1999).…”

Development of Transgenic Tomato Expressing Antimicrobial Peptide Gene through Agrobacterium-Mediated Transformation

“…Van Roekel, et al (1993) reported there is a significant effect of media and Agrobacterium strains used on transformation frequency of tomato. Highfrequency of transformation also depends on the efficiency of plant in vitro regeneration and also on the elimination of bacterial cells from transformed tissues (Tang et al, 2004). In this experiment a 13% of transformation frequency was observed however frequencies have ranged from 6% in cv.…”

Growth regulators, culture media and antibiotics in the in vitro shoot regeneration from mature tissue of citrus cultivars

“…Bacterial contamination during in vitro culture of mature internodal segments from greenhouse-grown mother plants is usually responsible for the loss of explants cultured on antibiotic-free medium. However, addition of antibiotics to the culture medium requires a careful evaluation of their effects on plant regeneration, since several reports have shown that they could have positive or negative effects on in vitro morphogenesis (Costa et al, 2000;Tang et al, 2004;Mendes et al, 2009).…”