Plant Cell Reports

, Volume 11, Issue 9, pp 452–456 | Cite as

Endophytic bacteria expressing β-glucuronidase cause false positives in transformation of Dioscorea species

  • Mahmut Tör
  • Sinclair H. Mantell
  • Charles Ainsworth
Article

Summary

False positive transformants obtained during plant transformation experiments on species of the monocotyledonous genus Dioscorea (yam) are described. The false positive results were found to be due to endophytic bacteria which exist within aseptically micropropagated shoot cultures and which express β-glucuronidase (GUS). The bacteria were isolated and identified as two species of Curtobacterium. The expression of GUS in these organisms was found to be induced by a variety of glucuronide substrates. The induction of GUS activity in the bacteria can be inhibited by chloramphenicol, tetracycline, ticarcillin and sodium azide. Implications of these results for use of the gus gene in plant transformation work are discussed.

Keywords

β-Glucuronidase Dioscorea endophytic bacteria false positive transformants 

Abbreviations

DTT

Dithiothreitol

EDTA

Ethylenediamine tetra-acetic acid

ELISA

Enzyme-linked immuno-sorbent assay

GUS

β-glucuronidase

LB

Luria Bertani

MUG

4-Methylumbelliferyl-β-D-glucuronide

PNPG

p-nitrophenyl-β-D-glucuronide

PVP

Polyvinylpyrrolidone

SDS

Sodium dodecyl sulphate

TAE

Tris-acetate-EDTA buffer

X-Gluc

5-Bromo-4-chloro-3-indolyl-β-D-glucuronide

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References

  1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidmen JG, Smith JA and Struhl K (1989) Short protocols in molecular biology. A compendium of methods from current protocols in molecular biology. John Wiley and Sons, New York p61.Google Scholar
  2. Bradford MM (1976) Anal. Biochem. 72:248–254.Google Scholar
  3. Buehler HJ, Katzman PA, Doisy PP and Doisy EA (1949) Proc. Soc. Exptl. Biol. Med. 72:297–299.Google Scholar
  4. Fishman WH (1955) Adv. Enzymol. 16:361–409.Google Scholar
  5. Hu CY, Chee PP, Chesney RH, Zhou JH, Miller PD and O'Brien WT (1990) Plant Cell Reports 9:1–5.Google Scholar
  6. Jefferson RA (1987) Plant Molec. Biol. Reporter 5:387–405.Google Scholar
  7. Jefferson RA, Burgess SM and Hirsh D (1986) Proc. Natl. Acad. Sci. USA. 86:8447–8451.Google Scholar
  8. Jefferson RA, Kavanagh TA and Bevan MW (1987) EMBO J. 6:3901–3907.Google Scholar
  9. Karunairatnam MC and Levvy GA (1951) Biochem. J. 49:210–215.Google Scholar
  10. Komagata K and Suzuki KI (1986) Bergey's Manual of Systematic Bacteriology, Vol 2, P.H.A.Sneath., N.S. Mair, M.E.Sharp.and J.G.Holt, eds, pp 1313–1317.Google Scholar
  11. Leifert C, Waites WM and Nicholas JR (1989) J. Appl. Bacteriol. 67:353–361.Google Scholar
  12. Liang W (1989) M.Phil. Dissertation, University of Cambridge, England.Google Scholar
  13. Mantell SH, Haque SQ and Whitehall AP (1978) J. Hort. Science 53:95–98.Google Scholar
  14. Miller IM and Reporter M (1987) Plant, Cell and Environment 10:413–424.Google Scholar
  15. Plegt L and Bino RJ (1989) Molec. Gen. Genet. 216:321–327.Google Scholar
  16. Robinson JJ, Blin CW and Frank PF (1952) J. Bacteriol. 64:719–723Google Scholar
  17. Tör M, Ainsworth CC and Mantell SH (1990a) Abstracts VIIth Int'l. Congr. Plant Tissue and Cell Culture. (Abs# A2-138), Amsterdam, June 24–29.Google Scholar
  18. Tor M, Mantell SH and Ainsworth CC (1990b) Abstracts VIIth Int'l. Congr. Plant Tissue and Cell Culture. (Abs # A2- 139), Amsterdam, June 24–29.Google Scholar
  19. Wenzler HC, Mignery GA, Fisher LM and Park WD (1989) Plant Molec. Biol. 12:41–50Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Mahmut Tör
    • 1
  • Sinclair H. Mantell
    • 1
  • Charles Ainsworth
    • 1
  1. 1.Wye College, University of LondonWye, AshfordEngland

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