Role of Plant Tissue Cultures in International Exchange and Quarantine of Germplasm in the United States and Canada

Salih, Sarbagh; Waterworth, H. E.; Thompson, Daniel

doi:10.21273/hortsci.36.6.1015

Cited by 8 publications

(4 citation statements)

References 25 publications

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“…In fact, bacterial growth has surpassed plant growth causing detrimental effects to the host resulting in cessation of growth ( Figure 2). Latent bacteria in vitro is highly undesirable as they can interfere with research conclusions and act as potential threat to micropropagation industry (Leifert and Woodward, 1998), in vitro gene banks (Van den Houwe and Swennen, 2000) and safe exchange of germplasm (Salih et al, 2001). A range of antibiotics with different mechanisms of action were tested for their ability to suppress the growth of bacteria grown in pure culture (Table 2).…”

Section: Resultsmentioning

confidence: 99%

Identification of IAA-producing endophytic bacteria from micropropagated Echinacea plants using 16S rRNA sequencing

Lata

Silva

et al. 2006

Plant Cell Tiss Organ Cult

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The presence of latent bacteria is a serious problem in plant tissue cultures. While endophytes are generally beneficial to plants in situ, they may affect culture growth under the modified conditions in vitro. The present study was undertaken to identify and characterize endophytic bacteria associated with the medicinal plant Echinacea in tissue culture. Based on classical microbiological tests and 16S rRNA analyses, it was found that endophytic bacteria associated with aseptically micropropagated Echinacea plantlets are representatives of several genera, Acinetobacter, Bacillus, Pseudomonas, Wautersia (Ralstonia) and Stenotrophomonas. Based on TLC and HPLC analyses, we found that Pseudomonas stutzeri P3 strain produces plant hormone, auxin (indole-3-acetic acid, IAA). Antibiotic resistance was also assessed as a virulence factor. The majority of endophytic bacteria were resistant to the antibiotic kanamycin, but susceptible to chloramphenicol. Recommendations for propagating Echinacea in vitro cultures involve the addition of chloramphenicol, tetracycline, and ampicillin, antibiotics that cause no side effects on these plant species.Abbreviations: IAA -indole-3-acetic acid; MS -Murashige and Skoog medium; NA -nutrient agar; NBnutrient broth; TSA -tryptic soy agar

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Section: Resultsmentioning

confidence: 99%

Identification of IAA-producing endophytic bacteria from micropropagated Echinacea plants using 16S rRNA sequencing

Lata

Silva

et al. 2006

Plant Cell Tiss Organ Cult

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“…In the absence of an indexing, bacteria harbouring cultures would escape detection as they are not suspected as contaminated. Covert or inconspicuous bacteria in vitro, unless introduced intentionally, is highly undesirable as they can interfere with research conclusions and act as a potential threat to micropropagation industry (Leifert and Woodward 1998), in vitro gene banks (Van den Houwe and Swennen 2000) and safe exchange of germplasm (Salih et al 2001). Endophytic B. pumilus in symptomless potato tissue cultures was found to be the cause for aberrant RAPD patterns (Isenegger et al 2003).…”

Section: Discussionmentioning

confidence: 99%

Isolation of Bacillus pumilus from in vitro grapes as a long-term alcohol-surviving and rhizogenesis inducing covert endophyte

Thomas

2004

J Appl Microbiol

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P . T H O M A S . 2004.Aims: To isolate and characterize the alcohol-surviving covert bacterium associated with grape tissue culture. Methods and Results: Single colony was isolated by plating the spent rectified spirit used during grape culturing and the organism was identified as Bacillus pumilus using partial 16S rDNA sequence data. Spotting tests (1 ll) using 3-day-old broth culture having a spore content of 20-30% showed similar bacterial survival in 25-90% (v/v) aqueous ethanol for 14 days. Survival in 90% ethanol and 90% rectified spirit appeared affected thereafter with no colony growth from 1 ll samples after 4 months. Plating the samples at this stage gave similar CFU ml )1 for 25, 50 and 70% ethanol, a significant reduction in 80% ethanol and very few colonies in 90% ethanol and rectified spirit. B. pumilus-inoculated grape microcuttings showed substantial endophytic colonization of original cuttings (7AE4 · 10 6 CFU g )1 ) followed by the sprout (5AE9 · 10 5 ) and roots (2AE0 · 10 4 ). The bacterium although a poor root colonizer, induced early rooting and more roots in vitro. Inoculation at ex vitro planting resulted in significantly more roots, root weight and shoot growth. Conclusions: Bacillus pumilus could remain as a covert endophyte in grape tissue cultures and survive in aqueous ethanol for extended periods. 90% ethanol was the most effective bactericidal concentration. The bacterium showed endophytic colonization and root and shoot growth promotion. Significance and Impact of the Study: The revelation that general recommendation of 70-80% ethanol may not be the most effective bactericidal concentration for all bacteria, elucidation of the possibility of covert bacterial survival in vitro plant cultures and isolation of a potential plant growth promoting endophyte in grape.

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“…These cultures serve as the source of latent inoculum in a tissue culture laboratory. This has got vide ramifications in plant tissue culture research and commercial laboratories, for in vitro gene banks and safe exchange of in vitro germplasm (Salih et al 2001;Thomas 2004b,c;Thomas et al 2006). The bacterial isolate in this study was a distinct Gramnegative endospore former.…”

Section: Discussionmentioning

confidence: 99%

Isolation of an ethanol-tolerant endospore-forming Gram-negative Brevibacillus sp. as a covert contaminant in grape tissue cultures

Thomas

2006

J Appl Microbiol

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Aims: To characterize the alcohol-surviving bacterial isolate ARBG1 from in vitro grapes (Vitis vinifera). Methods and Results: Two bacterial strains that survived in covert form in grape cultures were isolated from the spent alcohol used for disinfecting the tools of which one (ARBG2) was characterized earlier. The present study describes characterization of the second isolate, ARBG1. Nutrient agar (NA)-derived colonies of ARBG1 displayed consistently Gram-negative staining rods (2-4 · 0AE5-0AE6 l) substantiated by KOH mucoid thread test. Older cultures (3-7 days) showed emergence of Gram-negative staining, oblong, phase-refractile cells with ellipsoidal spores. The growth and sporulation were modified by growth medium and incubation temperature with the optimum around 37°C. Identification attempts involving microscopic, biochemical, Biolog or fatty acid profiling approaches brought in mixed and inconclusive results. PCR amplification of 16S rDNA was not successful with the standard primers 27F and 1492R but with 27F and a modified primer ARBG1-RP1. The identity of the isolate was established as Brevibacillus sp. based on partial 16S rDNA sequence data from eight single colonies with Gram-positive Brevibacillus choshinensis as the closest match (99AE5%). Spotting tests on NA employing spore suspension in aqueous ethanol (0%, 25%, 50%, 60%, 70%, 80% or 90%, v/v) indicated unhindered bacterial-survival in alcohol for 1 month, and that at 2 or 4 months revealed 90% ethanol as more sporicidal than lower levels, corroborated by plating results. Grape microcuttings inoculated with ARBG1 showed substantial general colonization of shoots, roots and medium but low endophytic colonization. Conclusions: The rare type of spore-producing consistently Gram-negative bacterial isolate ARBG1 was identified as Brevibacillus sp. based on 16S rDNA sequence similarity. The alcohol-defying organism was nonpathogenic and survived in covert form in grape cultures. Aqueous 90% ethanol appeared more sporicidal than lower levels. Significance and Impact of the Study: Characterization of an unusual endospore-forming Gram-negative bacterium, observation that some bacteria may fall outside the purview of standard 16S rDNA primers, elucidation of the threats of covert bacteria in plant tissue cultures and alcohol-mediated lateral transmission of spore formers, and the revelation that 70-80% ethanol may not be the most effective bactericidal concentration for all bacteria.

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Role of Plant Tissue Cultures in International Exchange and Quarantine of Germplasm in the United States and Canada

Cited by 8 publications

References 25 publications

Identification of IAA-producing endophytic bacteria from micropropagated Echinacea plants using 16S rRNA sequencing

Identification of IAA-producing endophytic bacteria from micropropagated Echinacea plants using 16S rRNA sequencing

Isolation of Bacillus pumilus from in vitro grapes as a long-term alcohol-surviving and rhizogenesis inducing covert endophyte

Isolation of an ethanol-tolerant endospore-forming Gram-negative Brevibacillus sp. as a covert contaminant in grape tissue cultures

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