Detection and control of bacterial contaminants of plant tissue cultures.

Preventing or avoiding microbial contamination of plant tissue cultures is critical to
successful micro propagation. Epiphytic and endophytic organisms can cause severe
losses to micro propagated plants at each stage of growth (Cassells. 1991; Debergh arxi
Vanderschaeghe. 1988; Leifert eot at. 1991). Bacterial contaminants are often diffiCult to
detect because they remain mostly within the plant tissue (D:ebergh and Vanderschaeghe.
1988; De Fossard and De Fossard. 1988; Viss eot al.. 1991). Contaminated plants may
have no visible symptoms. reduced multiplication and rooting rates. or may die (Leifert
et ai., 1989; 1992). Introduction of microorganisms due to poor aseptic technique or
improperly sterilized equipment can be corrected with improvements in training or
equipment handling. but eliminating internal contaminants is more problematic (Buckley et al.. 1995).

Procedures for producing aseptic cultures require attention to some or all of the
following: 1) indexing explants and cultures for
contaminants; 2) identifying the source of those contaminants; 3) identifying or characterizing the contaminants; and 4) eliminating the contaminating organism with improved cultural practices, antibiotics, or other chemical agents.

Sources and prevention of contaminants The sources of contaminated cultures usually are: difficult to determine (Le:ifert ark:i Waites. 1994). Bacteria which contaminate plant cultures may originate from explants. laboratory environments. operators. mites and thrips. or ineffective sterilization techniques- Bacteria are associated with plants as cpiphytes or endophytes (Sige:e:, 1993; Gunson and Spencer-Phillips, 1994). Explants from ficld-grown plants, diseased specimens. or from plant parts which are located closc to or bclow the soil may ~ difficult or impossible to disinfect due to both endophytic and epiphytic microbes (Leifert el al.. 1994). Contaminants of greenhouse-grown plants are mostly those associated with soil (Buckley el at.. 1995) and may originate from irrigation water (Seabrook and Farrell, 1993)-

Epiphytic bacteria may lodge in plant structures where disinfectants can not reach (Gunson and Spencer-Phillips. 1994; Leifcrt el at. 1994). Endophytic bacteria may be localized within the plant at cell junctions and the intercellular spaces of cortical parenchyma (Gunson and Spcncer-PhilJips, 1994). Contaminants found at explant initiation. present in explants from several collection dates and resistant to surface dis-infestation are likely to be endophytic (R~ el at. 1995)-

Every step of the plant tissue culture process should be considered in order to prevent contamination. These steps encompass handling of stock plants, type and handling of explants. media preparation, sub-culturing. incubation, and storage of sterile culture vessels. media. and plant cultures. Leifertand Waites (1994) suggested that stock plants used for plant tissue cultures be grown under protected conditions (greenhouses, growth chambers) to decrease the populations of epiphytic organisms. Seabrook and Farrell (1993) showed that irrigating stock planys with filtered water, rather than the standard city water, reduced bacterial contamination. Old or diluted disinfectants may lose strength and should be discarded. with only fresh mixtures used for explant dis-infestation (Leifert et al.. 1994). During sub-culturing of plant cultures. contaminants may be reduced by controlling the laboratory cleanliness and air source and by strict training of operators in aseptic technique (Leifert et aL, 1994).

Characterizing bacteria to determine the species provides important information about contamination sources. the amount of contamination from that source. and how to eliminate or prevent contaminants (Leifert et al.. 1989; 1991). Since laboratories m:e often contaminated with unique organisms, each should defme methods to prevent or treat their specific contamination problems (Leifert et al., 1991). Systems for analyzing procedures and processes may help in the design of systems to prevent or reduce microbial contamination (Leifert and Waites, 1994)

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