Re: Contamination

[jumana serhan <jumana_fuad@YAHOO.COM>]

 hello,
  i'd like to share with my experment in banana
culturing, we had tow sources for banana meristems for
initiation ( Grand Naine ) , the first one were young
plantlets that were not planted in the field yet , and
the other was larger and older and were planted in the
field .
 at the initiation ,the first one gave a very low
contamination % and a great growth rate.
 the second were worse, with a high contamination% and
weak growth rate .
 at the multiplication stage, the first one were good
in growth with a low contamination % till the third
multiplicaion , the contamination % exceeded 50% !!
 while the second one kept on the very low
contamination % , although that we multiplied them in
the same day with same techniques , and even we had a
high contamination % in the cultures that we
multiplied after the first one which were from another
generation.
  the problem is when you know that the first one were
produced by tissue culture , while the second were
not!!!!!
  so if these invisibly contaminated culture we
produce are holding a pathogenic bacteria , then we
are helping in spreading a new pathogen in our farming
land!!!!!

  any comments are welcomed
  best regards
  Jumana Serhan
--- Agricell@AOL.COM wrote:
> Latent microbial contamination of plant tissue
> cultures has been described
> since 1992, particularly by C. Leifert at the
> University of Aberdeen. The
> problem is that plant tissue culture medium is not a
> particulary good growth
> medium for many microorganisms. They can persist for
> years without clouding
> the medium or producing symptoms. The commonly used
> method for determining
> whether cultures are "clean" - holding them up to
> the light to look for
> cloudiness - is worthless, if the aim is to achieve
> an axenic culture. All
> "eyeballing"does is tell you that whatever
> contaminants are present do not
> produce visible growth in the medium that you are
> using, with the explant
> that you are growing, under the culture conditions
> that you are using. I
> wince whenever I read a paper that claims to deal
> with an axenic or clean
> culture when the author has used nothing but an
> "eyeball" test for
> contamination.
>
> The following are two recent Agricell Report
> articles on latent contamination
> that may be of interest in the contamination
> discussion:
>
> Ed Herman, Editor
> Agricell Report
>
> New Method for Detecting Latent Contaminants in
> Plant Tissues
>
> (SEPT 98, Agricell Report) It is now well
> established that microorganisms can
> survive in plant tissue cultures for many years,
> through multiple
> subcultures, with no visible indication of
> contamination. Such latent
> contaminants are often difficult or impossible to
> detect and isolate by means
> of standard bacteriological methods, but may be
> harmful to plants in vitro
> and to plants regenerated from such contaminated
> cultures. What is even more
> dangerous is that latent plant pathogens may be
> inadvertantly spread by
> micropropagated plants contaminated with latent
> pathogens.
>     At the ISHS Conference: Methods and Markers for
> Quality Assurance in
> Micropropagation, held 24-27 August 1999 in Cork,
> Ireland, University of
> London scientists G. Tsoktouridis, G. Tasiamis and
> S. Mantel described a
> PCR-based strategy and protocols that permit rapid
> detection and isolation of
> low numbers of latent bacteria in tissue
> culture-derived plants of Bilbergia.
> The technique involves amplifying a fragment of the
> 16S rDNA fragment of
> bacteria without amplifying the pure plant DNA. This
> PCR product can be used
> to classify bacteria by cloning and sequencing.
>     According to the authors, Use of any
> combinations of primers and
> subsequent sequence analysis of any amplified
> fragment allows the rapid
> detection and molecular identification of bacteria
> associated with plants.
> One combination of primers may detect eucaryotic
> bacteria . . . present as
> contaminants . . . and other combinations of primers
> would even have the
> potential to allow rapid detection of unculturable
> bacteria.
>     Tsoktouridis et al. suggest that their
> "molecular diagnostic tool" can
> yield more reliable and consistent information about
> latent contaminants than
> has hitherto been possible. They add that the
> potential benefits of the
> technique offer "an extremely attractive
> opportunity in those cases where
> high numbers of samples require rapid analysis and
> appropriate economies of
> scale."
>
> For further information:
> G. Tsoktouridis, Dept. of Agric. & Hortic., Wye
> Coll., Univ. of London, Wye,
> Ashford, Kent TN25 5AH, U.K.
>
> Persistence of Pathogens in Plant Tissue Cultures
>
> OCT 99, Agricell Report) Because autotrophic plant
> tissue culture does not
> require the presence of added sugar, it is believed
> to be less vulnerable to
> microbial contamination than heterotrophic culture.
> At the National
> University of Ireland, however, S.M. Rafferty and
> associates have found that
> human food poisoning pathogens can persist in
> autotrophic plant tissue
> cultures despite normal disinfestation procedures
> and can also persist in
> regenerated plants. Rafferty et al. inoculated
> cabbage seedlings with E.
> coli, S. marcescens and enterococci. The seedlings
> were disinfested by
> placing them in 80% ethanol for 45 seconds, 2%
> stericol for 30 minutes, then
> washed in sterile distilled water three times. After
> disinfestation, some of
> the plantlets were homogenized and the homogenate
> was plated onto an
> appropriate bacterial culture medium and incubated.
>     The authors reported at the meeting that
> bacterial isolates could be
> recovered both from direct culture of seedlings and
> from homogenates. The
> bacteria persisted during autotrophic culture,
> apparently using plant
> exudates as a carbon source, and could be repeatedly
> re-isolated through
> serial subcultures. Although some plants were
> asymptomatic, in others the
> bacteria became vitropathic (pathogenic in vitro).
> When seedlings from these
> cultures were transferred to greenhouse or field
> conditions, bacteria could
> be isolated endophytically initially and
> epiphytically for long periods of
> time, both from the plants and from the substrates
> upon which they were grown.
>     It thus appears that, despite normal
> disinfestation procedures, in vitro
> culture has the potential of spreading pathogenic
> microorganisms, even when
> the culture is performed under autotrophic
conditions.

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