Abstract
This study establishes the widespread prevalence of fastidious or viable but non-culturable endophytic bacteria in field shoots and in unsuspicious shoot-tip cultures of papaya (Carica papaya L.) against the norm of asepsis in vitro. A total of 150 shoot-tips (approximately 10 mm) were inoculated on MS-based culture medium after surface sterilization of field-derived axillary shoots of cv. Surya during November or January (100 and 50, respectively) when 35–50% cultures showed endophytic microbial growth on culture medium. Indexing of apparently clean cultures using bacteriological media helped in detecting and removing additional 14–17% stocks with covert bacteria during the first two passages. The rest of the stocks stayed consistently index-negative during the first eight subculture cycles, but appeared positive in PCR-screening undertaken thereafter employing universal bacterial 16S rRNA gene primers indicating the association of non-cultivable bacteria. Direct sequencing of the PCR product yielded overlapping nucleotide data signifying mixed template or the presence of diverse endophytic microorganisms. This was confirmed by light microscopy of tissue sap revealing viable bacteria in considerable numbers, which were detected under phase contrast or with negative staining. Planting tissue segments or applying homogenate from these stocks on diverse bacteriological media did not induce the organisms to grow in vitro. The shoot cultures displayed variation in growth and rooting potential, the onus of such variation was solely attributable to the associated microorganisms. The findings were confirmed with additional field shoots and fresh in vitro stocks established subsequently. The observations have implications in micropropagation and all other applications involving plant cell, tissue, organ, and protoplast culture.
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Abbreviations
- BA:
-
Blood agar
- BAP:
-
6-Benzylaminopurine
- BIM:
-
Bacteriological indexing medium
- BHIA:
-
Brain heart infusion agar
- FB:
-
Fastidious bacteria
- LA:
-
Luria agar
- NA:
-
Nutrient agar
- PM:
-
Papaya medium
- TSA:
-
Trypticasein soy agar
- VA:
-
Viss et al. (1991) agar
- VBNC:
-
Viable but non-culturable
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Acknowledgments
This study was supported by a grant from the Department of Biotechnology, Government of India under the project “Identification of covert endophytic microbes in plant tissue cultures and their management and control”. This publication bears IIHR Contribution No. 83/2006.
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Communicated by P.P. Kumar.
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Thomas, P., Kumari, S., Swarna, G.K. et al. Ubiquitous presence of fastidious endophytic bacteria in field shoots and index-negative apparently clean shoot-tip cultures of papaya. Plant Cell Rep 26, 1491–1499 (2007). https://doi.org/10.1007/s00299-007-0363-2
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DOI: https://doi.org/10.1007/s00299-007-0363-2