Effects of antibiotics on contaminated callus cultures of pyrethrum

Pyrethrum [Tanacetum cinerariifolium (TREVIR.) SCHULTZ-BIP., Asteraceae] is a source of pyrethrins, natural insecticides that are widely used in agriculture and for domestic purposes. In callus cultures of pyrethrum microbial contaminants were observed routinely, therefore it was necessary to use antibiotics to maintain aseptic cultures. The antibiotics, streptomycin, cephalexin and penicillin-G were used for antibiotic pulse treatment. Their effect on callus growth and microbial elimination was investigated. Callus cultures of two different lines were incubated for 1 or 2 days in liquid medium containing different combinations and concentrations of antibiotics and then cultured for 6 weeks on solid medium without antibiotics. One day of antibiotic treatment had no effect on callus growth. After two days of treatment, however, growth was significantly inhibited. Growth suppression was genotype-dependent and high concentrations of antibiotics were found to be deleterious on callus growth. PCR analysis with eubacterial 16S rDNA primers was done for detecting bacteria, as it enables low, invisible concentrations of bacteria in callus cultures to be traced. Six weeks after antibiotic treatment no bacteria were detected in line L4 incubated for 2 days in medium containing 360 mg/l streptomycin, 160 mg/l cephalexin and 8 mg/l penicillin G. Lower concentrations of antibiotics, however, exhibited no bacteriostatic effect. These results indicate that, although the higher concentrations of antibiotics used in pulse treatment greatly reduce callus growth, they can successfully eliminate bacterial contamination from pyrethrum callus cultures.