Detection, isolation, and preliminary characterization of bacteria contaminating plant tissue cultures

In order to limit the contamination problem in plant tissue cultures experiments on selection of media suitable for detection and isolation of bacteria contaminating plant tissue explants, and preliminary characterization of isolates were made. In the first experiment aiming at detection of bacteria in plant explants four strains representing genera most often occurring at our survey of plant tissue cultures, and earlier isolated and identified (Bacillus, Methylobacterium, Pseudomonas and Xanthomonas) were streaked on five bacteriological media (NA, King B, K, R2A and 523) and on the medium used for plant culture initiation – 1 MS with milk albumin (IM). All strains grew on all media but on K and IM at the slowest rate and on 523 medium at the fastest. The IM medium proved to be useful for immediate bacteria detection at the initial stage of culture. In the second experiment, aiming at characterization of isolates on the basis of colony growth and morphology 14 strains (Agrobacterium, Bacillus, Curtobacterium, Flavobacterium, Lactobacillus, Methylobacterium – 2 strains Mycobacterium, Paenibacillus, Plantibacterium, Pseudomonas, Stenotrophomonas, Xanthomonas, and species Serratia marcescens) were streaked on five microbiological media: KB, NBY, YDC, YNA and YPGA. All strains grew on all those media but at different rates. The only exception was the strain of Lactobacillus spp., which did not grow on King B medium. This medium allowed the detection of such characteristic traits as fluorescence (Pseudomonas) and secretion of inclusions (Stenotrophomonas). The third experiment was focussed on assessment of the sensitivity of detection of specific bacteria in pure cultures and in plant tissue cultures using standard PCR and BIO-PCR techniques with genus specific primers and 2 methods of DNA isolation. Results showed that the use of Genomic Mini kit enabled an increase of the sensitivity by 100 times as compared to extraction of DNA by boiling. Moreover, the application of BIO-PCR increased sensitivity of detection from 102 to 105 times over the standard PCR. If looking for unknown cultivable bacteria more effective detection seems to be use of microbiological method enabling detection on bacteriological media single cells in the fragments of explants or in wash liquids, in which fragmented explants were shaken.

W pracy określono możliwość wykrywania obecności bakterii najczęściej spotykanych w kulturach roślinnych in vitro, należących do różnych rodzajów, przy pomocy technik mikrobiologicznej i molekularnej. Za najbardziej przydatną do izolacji uznano pożywkę 523 a także pożywkę zawierającą 1 soli mineralnych MS stosowaną do inicjacji kultur roślinnych, z dodatkiem 0,025% albuminy mlecznej. Większą czułość wykrywania bakterii przy pomocy markerowego DNA uzyskano przy zastosowaniu techniki polegającej na preinkubacji badanego materiału na pożywkach bakteriologicznych, a następnie na izolacji DNA ze wzrostu (BIO-PCR). Przy porównaniu sposobów izolacji DNA większą czułość uzyskano stosując zestaw Genomic Mini (A&A Biotechnology) niż po zastosowaniu ekstrakcji w temperaturze wrzenia.