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 ThomasEmail author
  • Ganiga K. Swarna
  • Prakash Patil
  • Ram D. Rawal
Original Paper


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.


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



Blood agar base


Bacteriological indexing medium


Brain heart infusion agar


Colony forming units


Luria agar


Macconky agar


Nutrient agar


Non-culturable bacteria


Original culture


Tissue culture medium


Trypticasein soy agar


Viss et al. (1991) agar


Viable but non-culturable



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.


  1. Axelos M, Curie C, Mazzolini L, Bardet C, Lescure B (1992) A protocol for transient gene expression in Arabidopsis thaliana protoplasts isolated from cell suspension cultures. Plant Physiol Biochem 30:123–128Google Scholar
  2. Bacon CW, Glenn AE, Hinton DM (2002) Isolation, in planta detection and culture of endophytic bacteria and fungi. In: Hurst CJ, Crawford RL, McInerney MJ, Knudsen GR, Stetzenbach LD (eds) Manual of environmental microbiology, 2nd edn. ASM Press, Washington, DC, pp 543–553Google Scholar
  3. Drew RA, Smith MK, Anderson DW (1992) Field-evaluation of micropropagated bananas derived from plants containing banana bunchy-top virus. Plant Cell Tiss Org Cult 28:203–205CrossRefGoogle Scholar
  4. Habiba U, Reza S, Saha ML, Khan MR, Hadiuzzaman S (2002) Endogenous bacterial contamination during in vitro culture of banana: identification and prevention. Plant Tiss Cult 12:117–124Google Scholar
  5. Hallmann J (2001) Plant interactions with endophytic bacteria. In: Jeger MJ, Spence NJ (eds) Biotic interactions in plant-pathogen associations, CABI Publishing, Oxon, Wallingford, pp 87–119Google Scholar
  6. Hallmann J, Quadt-Hallmann A, Mahaffe WF, Kloepper JW (1997) Bacterial endophytes in agricultural crops. Can J Microbiol 43:895–914CrossRefGoogle Scholar
  7. Herman EB (2004) Recent advances in plant tissue culture viii. Microbial contaminants in plant tissue cultures: solutions and opportunities 1996–2003. Agritech Consultants, Inc., Shrub Oak, USAGoogle Scholar
  8. Horry JP, Sharrock S, Frison E (2000) Banana research and development: an international perspective. In: Singh HP, Chadha KL (eds) Banana improvement, production and utilization, Association for the Improvement in Production and Utilization of Banana, Trichy, India, pp 1–5Google Scholar
  9. Johansen DA (1940) Plant microtechnique. McGraw Hill Book Company, Inc., New YorkGoogle Scholar
  10. Kamoun R, Lepoivre P, Boxus P (1998) Evidence for the occurrence of endophytic prokaryotic contaminants in micropropagated plants of Prunus cerasus cv. ‘Montmorency’. Plant Cell Tiss Org Cult 52:57–59CrossRefGoogle Scholar
  11. Kevers C, Filaji M, Petit-Paly G, Hagège D, Rideau M, Gaspar T (1996) Habituation of plant cells does not mean insensitivity to plant growth regulators. In vitro Cell Dev Biol Plant 32:204–209CrossRefGoogle Scholar
  12. Khayat E, Duvdevani A, Lahav E, Ballesteros BA (2004) Somaclonal variation in banana (Musa acuminata cv. Grand Nanie). In: SM Jain, R Swennen (eds), Banana improvement: cellular, molecular biology, and induced mutations (
  13. Kulkarni VM, Ganapathi TR, Bapat TA, Rao PS (2004) Establishment of cell-suspension cultures in banana cv. Grand Naine and evaluation of its sensitivity to gamma-irradiation. Curr Sci 86:902–904Google Scholar
  14. Kumari S (2004) Detection and identification of endophytic bacteria associated with in vitro culture of papaya (Carica papaya L.). M.Sc. thesis. University of Agricultural Sciences, Bangalore, IndiaGoogle Scholar
  15. Leifert C, Cassells AC (2001) Microbial hazards in plant tissue and cell cultures. In vitro Cell Dev Biol Plant 37:133–138CrossRefGoogle Scholar
  16. Leifert C, Woodward S (1998) Laboratory contamination management: the requirement for microbiological quality assurance. Plant Cell Tiss Org Cult 52:83–88CrossRefGoogle Scholar
  17. Meins F Jr (1989) Habituation: heritable variation in the requirement of cultured plant cells for hormones. Annu Rev Genet 23:395–408PubMedCrossRefGoogle Scholar
  18. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  19. Ogram A, Sharma K (2002) Methods in soil microbial community analysis. In: Hurst CJ (ed) Manual of environmental microbiology, 2nd edn. ASM Press, Washington, DC, pp 554–563Google Scholar
  20. Oliveira RP, Silveira DS, Silva SO (2001) Concentraçãa de BAP ea eficieêcia de micropropagação de bananeira tetraploide (Gropo AAAB). Sci Agricola 58:73–78Google Scholar
  21. Panicker B (2005) Studies on in vitro propagation in chrysanthemum (Dendranthema grandiflora Tzvelev). Ph.D. thesis, University of Agricultural Sciences, Bangalore, IndiaGoogle Scholar
  22. Panicker B, Thomas P, Janakiram T, Venugopalan R (2007) Influence of cytokinin levels on in vitro propagation of shy suckering chrysanthemum ‘Arka Swarna’ and activation of endophytic bacteria. In vitro Cell Dev Biol Plant 43:614–622CrossRefGoogle Scholar
  23. Patil P, Singh HP (2000) In vitro multiplication of banana varieties belonging to different genome and ploidy levels. In: Singh HP, Chadha KL (eds) Banana improvement, production and utilization, Association for the Improvement in Production and Utilization of Banana, Trichy, India, pp 228–232Google Scholar
  24. Pischke MS, Huttlin EL, Hegeman AD, Sussman MR (2006) A transcriptome-based characterization of habituation in plant tissue culture. Plant Physiol 140:1255–1278PubMedCrossRefGoogle Scholar
  25. Reiter B, Sessitsch A (2006) Bacterial endophytes of the wildflower Crocus albiflorus analyzed by characterization of isolates and by a cultivation-independent approach. Can J Microbiol 52:140–149PubMedCrossRefGoogle Scholar
  26. Reiter B, Pfeifer U, Schwab H, Sessitsch A (2002) Response of endophytic bacterial communities in potato plants to infection with Erwinia carotovora subsp. atroseptica. Appl Environ Microbiol 68:2261–2268PubMedCrossRefGoogle Scholar
  27. Roels S, Escalona M, Cejas I, Noceda C, Rodriguez R, Canal MJ, Sandoval J, Debergh P (2005) Optimization of plantain (Musa AAB) micropropagation by temporary immersion system. Plant Cell Tiss Org Cult 82:57–66CrossRefGoogle Scholar
  28. Sahijram L, Soneji JR, Bollamma KT (2003) Analyzing somaclonal variation in micropropagated bananas (Musa spp.). In vitro Cell Dev Biol Plant 39:551–556CrossRefGoogle Scholar
  29. Sessitsch A, Reiter B, Pfeifer U, Wilhelm E (2002) Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes. FEMS Microbiol Ecol 39:23–32CrossRefPubMedGoogle Scholar
  30. Singh HP (2000) Perspective of banana production and utilization in India. In: Singh HP, Chadha KL (eds) Banana improvement, production and utilization, Association for the Improvement in Production and Utilization of Banana, Trichy, India, pp 20–58Google Scholar
  31. Strosse H, Van den Houwe I, Panis B (2004) Banana cell and tissue culture-a review. In: Jain SM, Swennen R (eds) Banana improvement: cellular, molecular biology, and induced mutations, (
  32. Sun J, Niu Q-W, Tarkowski P, Zheng B, Tarkowska D, Sandberg G, Chua N-H, Zuo J (2003) The Arabidopsis AtIPT8/PGA22 gene encodes an isopentyl transferase that is involved in de novo cytokinin biosynthesis. Plant Physiol 131:167–176PubMedCrossRefGoogle Scholar
  33. Thomas P (2004a) A three-step screening procedure for detection of covert and endophytic bacteria in plant tissue cultures. Curr Sci 87:67–72Google Scholar
  34. Thomas P (2004b) In vitro decline in plant cultures: detection of a legion of covert bacteria as the cause for degeneration of long-term micropropagated triploid watermelon cultures. Plant Cell Tiss Org Cult 77:173–179CrossRefGoogle Scholar
  35. Thomas P (2004c) Isolation of Bacillus pumilus from in vitro grapes as a long-term alcohol-surviving and rhizogenesis inducing covert endophyte. J Appl Microbiol 97:114–123PubMedCrossRefGoogle Scholar
  36. Thomas P (2006a) Isolation of an ethanol-tolerant endospore-forming Gram-negative Brevibacillus sp. as a covert contaminant in grape tissue cultures. J Appl Microbiol 101:764–774PubMedCrossRefGoogle Scholar
  37. Thomas P (2006b) Reemergence of covert bacteria Bacillus pumilus and Brevibacillus sp. in microbe-freed grape and watermelon stocks attributable to occasional autoclaving-defying residual spores from previous cycles. Plant Cell Tiss Org Cult 87:155–165CrossRefGoogle Scholar
  38. Thomas P (2007). Isolation and identification of five alcohol defying Bacillus spp. covertly associated with in vitro culture of seedless watermelon. Curr Sci 92:983–987Google Scholar
  39. Thomas P, Prakash GS (2004) Sanitizing long-term micropropagated grapes from covert and endophytic bacteria and preliminary field testing of plants after 8 years in vitro. In vitro Cell Dev Biol Plant 40:603–607CrossRefGoogle Scholar
  40. Thomas JE, Smith MK, Kessling AF, Hamill SD (1995) Inconsistent transmission of banana bunchy top virus in micropropagated bananas and its implications for germplasm screening. Aust J Agri Res 46:663–671CrossRefGoogle Scholar
  41. Thomas P, Prabhakara BS, Pitchaimuthu M (2006) Cleansing the long-term micropropagated triploid watermelon cultures from covert bacteria and field testing the plants for clonal fidelity and fertility during the 7–10 year period in vitro. Plant Cell Tiss Org Cult 85:317–329CrossRefGoogle Scholar
  42. Thomas P, Kumari S, Swarna GK, Gowda TKS (2007a) Papaya shoot tip associated endophytic bacteria isolated from in vitro cultures and host-endophyte interaction in vitro and in vivo. Can J Microbiol 53:380–390PubMedCrossRefGoogle Scholar
  43. Thomas P, Kumari S, Swarna GK, Prakash DP, Dinesh MR (2007b) Ubiquitous presence of fastidious endophytic bacteria in field shoots and index-negative apparently clean shoot-tip cultures of papaya. Plant Cell Rep 26:1491–1499PubMedCrossRefGoogle Scholar
  44. Thomas P, Swarna GK, Roy PK, Prakash P (2008) Identification of cultivable and originally non-culturable endophytic bacteria isolated from shoot tip cultures of banana cv. Grand Naine. Plant Cell Tiss Organ Cult. doi: 10.1007/s11240-008-9341-9 Google Scholar
  45. Van den Houwe I, Swennen R (2000) Characterization and control of bacterial contaminants in in vitro cultures of banana (Musa spp.). Acta Hort 530:69–79Google Scholar
  46. Van den Houwe I, Gun J, Swennen R (1998) Bacterial contamination in Musa shoot tip cultures. Acta Hort 490:485–492Google Scholar
  47. Viss PR, Brooks EM, Driver JA (1991) A simplified method for the control of bacterial contamination in woody plant tissue culture. In vitro Cell. Dev Biol Plant 27:42CrossRefGoogle Scholar
  48. Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703PubMedGoogle Scholar
  49. Zinder SH, Salyers AA (2005) Microbial ecology. In: Brenner DJ, Kreig NR, Staley JT (eds) Bergey’s manual of systematic bacteriology, 2nd edn, vol 2. Springer, New York, pp 101–109Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Pious Thomas
    • 1
    Email author
  • 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

Personalised recommendations