Bacterial contamination of in vitro plant cultures: confounding effects on somaclonal variation and detection of contamination in plant tissues

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 semispecific 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.

Bacterial Contamination of Plant in vitro Cultures in Commercial Production Detected by High-Throughput Amplicon Sequencing

“…were associated with aseptically micropropagated Echinacea plantlets (Lata et al, 2006), and in vitro cultures of Astilbe (Leifert et al, 1991b). The genus Lactobacillus, highly present in GF677 and Gisela 5 cultures, has been described as in vitro contaminant (Moreno-Vázquez, 2014;Ganen et al, 2009) which could easily enter samples as inhabitant of the human skin (Leifert et al, 1991a). From plant cultures, it was detected in Pelargonium (Wojtania, 2005) and Hemerocallis (Leifert et al 1991b, Leifert et al, 1989 cultures, wherein it proved to be deleterious to last ones.…”

“…Most bacterial contaminations of in vitro plant cultures are introduced by the inefficient sterilization of explants, culture vessels, laboratory equipment or media. They occur when handling the plant material or are transmitted by mites and thrips (Moreno-Vázquez et al, 2014;Reed and Tanprasert, 1995;Van den houwe et al, 1998). The use of microbiological quality assurance (HACCP) systems, training of workers in sterile technique and use of autoclaves and laminar flowboxes have to be the first steps to avoid environmental contaminants and contamination by humans (Reed and Tanprasert, 1995).…”

“…Roughly 20 -55% of contamination losses to in vitro plant cultures arise because of bacteria (Tsoktouridis et al, 2014;Ganen et al, 2009). These bacteria may cause serious problems for in vitro gene banks, prevent the safe exchange of germplasm and may result in misleading conclusions in research studies (Moreno-Vázquez et al, 2014;Wojtania et al, 2005). Due to massive propagation, they can dominate over plants explants, limit oxygen and nutrition accessibility (Orlikowska et al, 2017).…”

“…Molecular detection based on direct DNA analysis makes these time-consuming methods unnecessary and is sensitive enough for most groups of bacteria. The 16S ribosomal RNA (rRNA) gene is the most widely used target for bacterial identification at the genus level (Moreno-Vázquez et al, 2014). Additionally, the development of new determination techniques based on DNA sequencing brought broader opportunities for studies of biological systems.…”

Identification and control of endophytic bacteria during in vitro cultures of Staphylea pinnata L.

“…The presence of bacteria in in vitro cultures is considered a negative symptom associated with the neglect of aseptic working conditions. Surface disinfection of plant material is unable to remove the latent endogenous bacteria present in tissues (Moreno-Vázquez et al, 2014). Bacterial infections can inhibit the culture development by producing phytotoxic metabolites and competing between the plant and bacteria for nutrients (Leifert and Cassells, 2001).…”