Bacterial contaminants of plant tissue culture

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Bacterial contaminants of plant tissue culture

  1. 1. Bacterial Contaminants of Plant Tissue Culture Plant Tissue Culture MBT 722 2011 Prof. Naim Iraki Amer Wazwaz 1000316
  2. 2. Importance of Controlling Contamination The medium contains many different bacterialnutrients, both original constituents of the medium and exudates from the plant cells.((Bradbury 1988When microbe(s( come in contact with plant tissue or .medium then we will have a contamination Plant ]tissues/ cells[ growing in vitroare considered to be under some stress conditions and may be predisposed to direct infection, even by bacteria not normally pathogenic to them.((Bradbury 1970
  3. 3. Contaminating Bacteria Can Be Divided intoEpiphytes CommonDisinfection can be enough Endophytes More problematic Disinfection is not enough Antibiotics are needed Pathogenic bacteria can be a contaminant
  4. 4. Contaminating Bacteria May Originatefrom ExplantsLab environment
  5. 5. Contaminating Bacteria May Originatefrom OperatorsMites and Thrips
  6. 6. Contaminating Bacteria May Originatefrom Ineffective sterilization techniquesCan contaminate cultures at any procedural step if we don‘t take strict standards
  7. 7. Procedures for producing aseptic cultures require attention toIndexing explants and cultures for contaminantsIdentifying the source of those contaminantsIdentifying and characterizing the contaminantsEliminating the contaminants withimproved cultural practices ,antibiotics orother chemicalagents
  8. 8. Indexing Cultures Serial stem slices inoculated intoliquid and agar-solidified different bacterial media 3Incubated for three weeks at30°C, detected most contaminants from more than60 aquatic ,marsh , andornamental woody plant species ((Kane. 1995Usually, a contaminant would grow on two of the three media ((Kane. 1995Contaminated cultures are sometimes rooted and transferred to the greenhouse instead of being discarded((Kane. 1995
  9. 9. Cultures and Medium Indexinga. Healthy bacteria-index-negative (left( b. Medium-indexed plateand index-positive (right( cultures of showing different bacterial typestriploid watermelon .one week post-indexing Pious Thomas , CURRENT SCIENCE, VOL. 87, NO. 1, 10 JULY2004
  10. 10. Characterization and Identification Purification by standard bacteriological methods Characterization by biochemical tests, Gram staining Identification by traditional tests which areLabor-intensive and time consumingCan be performed in any laboratory with common chemicals Through comparing with the standard strains ofBergeys Manual(Krieg and Holt 1984(
  11. 11. Modern Identification TechniquesBiolog system Detectscarbon source utilization with the reduction oftetrazolium dye inresponse to cellular respirationCan identify yeasts and fungiThrough comparing the results witha database of responses
  12. 12. Modern Identification TechniquesAnalytical Profile Index or API systemCarbon source utilization test Visual detection of the test(Leifert et at. 1989; Vemiere et al., 1993(Enzymatic oxidation/reduction interactions Allows the identification ofa limited number of Bacteria
  13. 13. Modern Identification TechniquesFatty Acid Analysis Profiles FAPUses gas chromatography to identifyover 140 separate fatty acidsFatty acid profile is matched to a libraryof 700+ bacterial species representingover 180 generaCan identify yeasts and fungi alsoMatch fatty acid methyl esters with thoseof known organisms (Buckley et al.. 1995; Chase(et al., 1992; Stead et at, 1992
  14. 14. Modern Identification Techniques16S rRNAPCR amplification/ probes for known sequence Using this system depends uponthe number and diversity ofbacteria in the databasesMany soil and plant bacteria have not been not characterized((Buckley et al. 1995For a more accurate identification the use of more than onetest isRecommended ( (Jones et al. 1993( and ( Verniere et al. 1993
  15. 15. Rate of occurrence of microbial contaminant in plant tissue cultureOdutayo et al. Afr. J. Agric. Res. Vol. 2)3(, pp. 067-072, March2007
  16. 16. The occurrence of bacteria isolates in plant tissue cultureOdutayo et al. Afr. J. Agric. Res. Vol. 2)3(, pp. 067-072, March2007
  17. 17. Antibiotic Treatments Choosing an antibiotic depends on the type of bacteriaGram negative or Gram positive Ideal antibiotics should besoluble, stable, unaffected by pH, unaffected by media,lacking side effects, broadly active, bactericidal, suitable incombination, non-resistance inducing,.inexpensive and nontoxic to human healthMany antibiotics exist that have not yet been evaluated onplants or their bacterial contaminants(Falkiner, 1990; Seckinger,.(1995
  18. 18. Antibiotic TreatmentsAntibiotics may be inactivated by environmental conditionsheat/ lightAntibiotic sensitivity is reduced in plant tissue culture media due todifferent favorable pH degreesAntibiotic concentration ] MBC [ for a particular bacteriashould be determinedPhytotoxicity varies greatly among plant species and explanttypes, so preliminary testing with plant cultures is important
  19. 19. Effects ofdifferent concentrations of antibiotics at different durations of time to ensure contamination free cultures Habiba et al. Plant Tissue Cult. 12)2( : 117-124,2002
  20. 20. Antibiotic Effects on Shoots Number and Multiplication Rate Multiplication rate of Pelargoniumshoots before (week 0(,during andafter treatment with carbenicillin or cefotaxime end of cefotaxime treatment*A. Wojtania et al.J. Fruit Ornam. end of carbenicillin treatment**Plant Res. 104 vol. 13,2005
  21. 21. Antibiotic Effects on Shoots Number and Multiplication RatePelargonium shoots after 3 weeks of the growth on the medium containing (cefotaxime (A( and carbenicillin (B A. Wojtania et al. J. Fruit Ornam. Plant Res. 104 vol. 13,2005
  22. 22. Conclusion Several steps can reduce bacterial contaminants Properly training the operatorsIndexing cultures at initiation stage/culture cycle Identifying contaminants and testing to determine the proper antibiotic
  23. 23. Thank YouQuestions

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