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

Monika Kałużna,Piotr Sobiczewski,Marta Zawadzka,Elżbieta Zenkteler,Teresa Orlikowska,Artur Mikiciński

doi:10.5586/aa.2013.054

Abstract

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 – ½ 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 tis- sue 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.

Highlights

Bacterial contaminations are a serious problem in plant in vitro cultures, both in commercial plant micropropagation, by making difficult culture initiation, reducing efficiency of multiplication and rooting of shoots, as well as in research laboratories, where contamination can be the causal agent of false results in physiological experiments [1, 2, 3]
Our large survey of plant tissue cultures originating from eight laboratories in Poland resulted in obtaining 104 isolates of bacteria, which were assigned by phenotypic tests and 16S rDNA fragments sequencing to 29 genera
Sensitivity of detection in the above experiments was from 1 to 20 cells per 1 g or 1 ml of a sample. These results suggest that instead of detection specific bacterial DNA, more effective are microbiological methods, including of plating of tissue washings on bacteriological media, because in the molecular method, detectability begins at app. 15 cells whereas on bacteriological medium, it is possible to detect a single bacterium

Summary

Introduction

Bacterial contaminations are a serious problem in plant in vitro cultures, both in commercial plant micropropagation, by making difficult culture initiation, reducing efficiency of multiplication and rooting of shoots, as well as in research laboratories, where contamination can be the causal agent of false results in physiological experiments [1, 2, 3]. The diversity and abundance of genera and species of exo- and endobiotic bacteria accompanying donor plants [4, 5] is a major challenge in the sterilization of initial explants, a quick detection of bacteria in the first in vitro passages and a minimization of their adverse effect on shoot multiplication and rooting efficiency. If symptoms of bacteria colonizing plant tissues appeared within a short time, the contaminated explants should be immediately removed. Our large survey of plant tissue cultures originating from eight laboratories in Poland resulted in obtaining 104 isolates of bacteria, which were assigned by phenotypic tests and 16S rDNA fragments sequencing to 29 genera (data not published)

Results

Discussion

Conclusion