Effect of Antibiotics and Exposure to an Acidified Medium on the Elimination of Agrobacterium tumefaciens from Apple Leaf Explants and on Shoot Regeneration

Abstract

<jats:p>A range of antibiotics and short-term exposure to an acidified (pH 3.0) medium were evaluated for their effects on eliminating <jats:italic>Agrobacterium tumefaciens</jats:italic>, supervirulent strain EHA101 (pEHA101/pGT100), from leaf explants of `Royal Gala' apple (<jats:italic>Malus ×domestica</jats:italic> Borkh.) and on shoot regeneration. Exposure of leaf explants to regeneration and elongation media containing 100 μg·mL<jats:sup>-1</jats:sup> concentrations of the antibiotics carbenicillin (crb), cefotaxime (cef), and cefoxitin [=mefoxin (mef)], singly or in combination for 52 days did not eliminate <jats:italic>A. tumefaciens</jats:italic> from the explants. The percentage of regeneration on crb, cef, and mef was 97%, 11%, and 50%, respectively, compared to 67% for the controls. Short-term (1- to 18-hour) vacuum infiltration with 500 μg·mL<jats:sup>-1</jats:sup> of any of the above antibiotics did not inhibit regeneration and failed to eliminate <jats:italic>A. tumefaciens</jats:italic> from leaf explants. Cef (2000 μg·mL<jats:sup>-1</jats:sup>) did not inhibit the percentage of regeneration and was more effective than crb or mef in preventing growth of <jats:italic>A. tumefaciens</jats:italic> when vacuum infiltrated into apple leaf explants for 30 minutes. Further experiments demonstrated that the incidence of <jats:italic>A. tumefaciens</jats:italic> contamination could be reduced to 28% without negatively impacting shoot regeneration by using a 1-hour vacuum infiltration with an acidified medium, an 18-hour vacuum infiltration with cef (5000 μg·mL<jats:sup>-1</jats:sup>), and a 52-day incubation on regeneration and elongation media containing 100 μg·mL<jats:sup>-1</jats:sup> each of mef and crb. Kan resistant, GUS (β-glucuronidase) positive, putative transformants without <jats:italic>A. tumefaciens</jats:italic> were generated by adding kan (10 μg·mL<jats:sup>-1</jats:sup>) to the regeneration and elongation media.</jats:p>

Improved genetic transformation protocol for cork oak (Quercus suber L.)

“…The higher quantity of proliferating cells per explant probably increased the transformation efficiency; however, bacterial overgrowth increased as well (52.1% clusters contaminated compared with 11.6% isolated embryos), probably due to a less efficient washing after cocultivation. Thus, the elimination of bacterial contamination should be improved, applying the bactericides by means of vacuum infiltration, using more efficient bactericides or acidifying the medium for a short time, as some authors suggest (Hammerschlag et al 1997). In conclusion, clusters should be considered the explants of choice, as they present the highest transformation efficiency and are easier to collect and manipulate than isolated embryos.…”

Ethylene inhibitors and low kanamycin concentrations improve adventitious regeneration from apricot leaves

“…Timentin has been used without affecting regeneration with tobacco (Nauerby et al 1997) and tomato (Costa et al 2000). It has been demonstrated that carbenicillin breaks down to give physiologically active levels of the auxin phenylacetic acid, which could explain the positive effect that this antibiotic has on organogenesis of different species (Nakano and Mii 1993;Hammerschlag et al 1997;Yu et al 2001). This breakdown could have produced an excessive auxin concentration in apricot cells, since we have observed that increases in NAA concentration reduced and delayed regeneration from apricot leaves (data not shown).…”

Effects of β-lactam antibiotics, auxins, and cytokinins on shoot regeneration from callus cultures of two hybrid aspens, Populus tremuloides × P. tremula and P. x canescens × P. gradidentata

“…In more recent research caulogenesis was facilitated by CARB when the shoots were regenerated indirectly from callus (Suzuki et al 1998;Bhau and Wakhlu 2001;Tang et al 2004). Positive results have also been reported for direct regeneration protocols when an initial dark culture period, that would be expected to stimulate callusing prior to the onset of shoot differentiation, was included (Billings et al 1997;Hammerschlag et al 1997;Dai and Castillo 2007).…”

“…Because of their low levels of plant toxicity, b-lactams; particularly CEF, CARB, and timentin, are widely used for Agrobacterium elimination in plant transformation (Hammerschlag et al 1997;Tang et al 2004). Traditionally, cost and antibiotic potency have been among the primary criteria used to select among different b-lactams.…”