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Plant Cell, Tissue and Organ Culture

, Volume 93, Issue 1, pp 39–54 | Cite as

Ubiquitous presence of normally non-culturable endophytic bacteria in field shoot-tips of banana and their gradual activation to quiescent cultivable form in tissue cultures

  • Pious Thomas
  • Ganiga K. Swarna
  • Prakash Patil
  • Ram D. Rawal
Original Paper

Abstract

Exploring the source of quiescent bacteria in tissue-cultured bananas (Musa sp.) we demonstrate here through a combination of bacterial 16S rDNA-based molecular technique, light microscopy and cultivation-based approaches the ubiquitous presence of endophytic bacteria in the field shoots of different genotypes (Grand Naine, Robusta, Dwarf Cavendish, Ney Poovan and exotic accessions) and their widespread prevalence in apparently clean tissue cultures. A portion of field shoot-tips (10–60%) showed cultivable endophytes, especially during rainy season, yielding 102–105 colony forming units g−1 fresh tissue in ‘Grand Naine’, which overtly expressed on tissue culture medium as well. The rest showed no colony development on diverse bacteriological media but proved PCR+ve to bacterial primers indicating the presence of normally non-culturable organisms, which was endorsed by microscopic observations. Such endophytes gradually turned cultivable rendering all visibly clean cultures as quiescent bacteria-harboring after a few (2–4) to several (8–20) passages, resulting in as much as 1.7 × 105 – 4.0 × 107 colony forming units g−1 tissue of ‘Grand Naine’ after ten passages, yielding different organisms. This study has thus exposed the ubiquitous and intense association existing between endophytes and bananas, including their quiescent survival in suspension cultures. The effect due to quiescent bacteria in micropropagated stocks could not be generalized. The observations question the fundamental principle of asepsis in plant tissue cultures and bring in new information on plant-endophtye association in vitro with implications in micropropagation, germplasm conservation, cell culture studies and molecular profiling. The possible involvement of unsuspected endophytic bacteria in tissue-culture associated phenomena like habituation and epigenetic and somaclonal variations are discussed.

Keywords

Epigenetic variation Habituation In vitro gene bank Microbial contamination Musa sp. Plant tissue culture Molecular profiling Non-culturable bacteria Somaclonal variation 

Abbreviations

BA

Blood agar base

BIM

Bacteriological indexing medium

BHIA

Brain heart infusion agar

CFU

Colony forming units

LA

Luria agar

MA

Macconky agar

NA

Nutrient agar

NCB

Non-culturable bacteria

OC

Original culture

TCM

Tissue culture medium

TSA

Trypticasein soy agar

VA

Viss et al. (1991) agar

VBNC

Viable but non-culturable

Notes

Acknowledgements

This study was supported by a grant from the Department of Biotechnology, Govt. of India under the project ‘Identification of covert endophytic microbes in plant tissue cultures and their management and control’. Contributions from D. P. Prakash, P. K. Roy and V. Bidari during their brief association with the project and laboratory assistance by N. Shivrudriah and B. Hanumanthraju are acknowledged. The supply of banana suckers by L. B. Naik, and the supply of in vitro plant material by M/s E. K. Plants (Pvt.) Ltd. Bangalore are gratefully acknowledged. Thanks to L. Anand for the critical reading of the manuscript and the suggestions. This publication bears IIHR Contribution No. 69/06.

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Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Pious Thomas
    • 1
  • Ganiga K. Swarna
    • 1
    • 2
  • Prakash Patil
    • 3
  • Ram D. Rawal
    • 4
  1. 1.Division of BiotechnologyIndian Institute of Horticultural ResearchBangaloreIndia
  2. 2.Department of BiotechnologyDr. S. G. Reddy CollegeBangaloreIndia
  3. 3.AICRP on Tropical Fruit CropsIndian Institute of Horticultural ResearchBangaloreIndia
  4. 4.Division of Plant PathologyIndian Institute of Horticultural ResearchBangaloreIndia

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