The use of amoxicillin and ticarcillin in combination with a β-lactamase inhibitor as decontaminating agents in the Agrobacterium tumefaciens-mediated transformation of Artemisia annua L

Artemisinin is a new very promising antimalarial compound produced in the areal parts of the plant Artemisia annua L. (Asteraceae). There is a great interest in the overproduction of artemisinin by means of transgenic plants. The existing Agrobacterium tumefaciens-mediated transformation procedure uses the expensive decontaminating agent vancomycin. In addition vancomycin exhibits a low activity against the Agrobacteria. This paper describes the use of the penicillin derivatives amoxicillin and ticarcillin in combination with the β-lactamase inhibitor clavulanic acid, on the market as Augmentin® and Timentin® (Smith Kline Beecham Pharma S.A., Genval, Belgium), respectively. Sensitivity studies revealed that those antibiotics still permit plant regeneration. The leaf disc transformation was performed with the C58C1 (pGV2260; pTJK136) Agrobacterium strain. Transgenic tissue was selected on 20 mg l −1 kanamycin. The integration of the foreign genes was proven by polymerase chain reactions and additional DNA sequencing. β-Glucuronidase gene expression analyses were performed. In all cases transgenic callus could be obtained, but the shoot regeneration failed. Decontamination was most efficient when high initial concentrations of Augmentin® and Timentin® (300 and 200 mg l −1, respectively) were used. Those concentrations can gradually be reduced. We may conclude that decontamination by the penicillin derived antibiotics is cheaper and more efficient than vancomycin, but is only suitable when the production of callus is desired, for example for the establishment of cell cultures.