Abstract
Bacterial contamination represents a serious problem for plant tissue culture research and applications. Bacterial interference with normal plant physiology and morphology can generate misleading conclusions if the presence of bacteria is ignored. Bacterial contaminants in in vitro plant culture are typically detected by direct observation; thus, it is assumed that cultures without visible symptoms are bacteria free. Here, we demonstrate that contaminating Bacillus DNA in plant DNA solutions from asymptomatic plants can interfere with the analysis of somaclonal variation in chrysanthemum. We studied somaclonal variation in chrysanthemum using short semi-specific PCR primers based on conserved motifs in NBS–LRR disease resistance genes and in mobile elements. Instead of true somaclonal variation we found three polymorphic bands derived from contaminant bacterial DNA in plant extracts. Although the detection of asymptomatic bacteria in in vitro plant cultures is a major issue, we found that it has not been adequately addressed to date, particularly for studies on somaclonal variation. We reviewed the most commonly cited contaminant bacteria in in vitro plant culture and designed specific 16S rRNA gene-based PCR primers for the main genera causing contamination (Bacillus, Pseudomonas, Staphylococcus, Lactobacillus, Erwinia/Enterobacter and Xanthomonas). Using a panel of pure bacterial DNAs, artificial mixes of bacterial/plant DNAs, and in vitro plant cultures with and without visible contamination we demonstrated that our primers are in most instances both reliable and sensitive, and appropriate for the identification and tracking of the most frequent bacterial contaminants in plant in vitro cultures. Implications of bacterial identification to molecular analysis of somaclonal variation and plant culture decontamination are discussed.
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Acknowledgments
The authors would like to thank Dr. Emilia López Solanilla (Centre for Plant Biotechnology and Genomics, Universidad Politécnica de Madrid (UPM)–Instituto Nacional de Investigación Agraria y Alimentaria (INIA)) for providing DNA extracts from control bacteria, for carefully reviewing the manuscript and for her valuable suggestions. Additionally, we would like to thank Dra. M. Carmen Martín (Dept. Plant Biology UPM) for providing the plant tissue used to analyze somaclonal variation and also to Dr. Jaime Cubero (INIA) for supplying control Xanthomonas campestris. We are also grateful to Manuel Chimeno (Laboratorio de cultivo in vitro del Exmo. Ayuntamiento de Madrid) for providing some of the in vitro plant cultures analyzed. The author BEJB was supported by a scholarship from the Fundación Carolina (Agencia Española de Cooperación Internacional). This research work was supported by a project grant (DGI AGL2004-01929/AGR) from the CICYT (Spain), and it also benefited from research agreement (FPA040000BIO100) between UPM and the Exmo. Ayuntamiento de Madrid (Área de Gobierno de Medio Ambiente y Servicios a la Ciudad).
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Moreno-Vázquez, S., Larrañaga, N., Uberhuaga, E.C. et al. Bacterial contamination of in vitro plant cultures: confounding effects on somaclonal variation and detection of contamination in plant tissues. Plant Cell Tiss Organ Cult 119, 533–541 (2014). https://doi.org/10.1007/s11240-014-0553-x
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DOI: https://doi.org/10.1007/s11240-014-0553-x