Bacterial Contaminants of Plant Tissue Culture

Plant Tissue CultureMBT 722

2011

Prof. Naim Iraki

Amer Wazwaz1000316

Importance of Controlling Contamination

The medium contains many different bacterial nutrients, both original constituents of the medium

and exudates from the plant cells (Bradbury 1988).

When microbe(s) come in contact with plant tissue or medium then we will have a contamination.

Plant [tissues/ cells] growing in vitro are considered to be under some stress conditions and may be predisposed to direct infection, even by bacteria

not normally pathogenic to them (Bradbury 1970).

Contaminating Bacteria Can Be Divided into

Epiphytes

Common Disinfection can be enough

EndophytesMore problematic

Disinfection is not enough

Antibiotics are needed

Pathogenic bacteria can be a contaminant

Contaminating Bacteria May Originate from

Explants

Lab environment

Contaminating Bacteria May Originate from

Operators

Mites and Thrips

Contaminating Bacteria May Originate from

Ineffective sterilization techniques

Can contaminate cultures at any procedural step if we don’t take strict standards

Procedures for producing aseptic cultures require attention to

Indexing explants and cultures for contaminants

Identifying the source of those contaminants

Identifying and characterizing the contaminants

Eliminating the contaminants with

improved cultural practices, antibiotics or other chemical agents

Indexing Cultures Serial stem slices inoculated into liquid and agar-solidified

3 different bacterial media

Incubated for three weeks at 30°C, detected most contaminants from more than 60 aquatic, marsh, and ornamental woody

plant species (Kane. 1995)

Usually, a contaminant would grow on two of the three media (Kane. 1995)

Contaminated cultures are sometimes rooted and transferred to the greenhouse instead of being discarded (Kane. 1995)

Cultures and Medium Indexing

Pious Thomas, CURRENT SCIENCE, VOL. 87, NO. 1, 10 JULY 2004

a. Healthy bacteria-index-negative (left) and index-positive (right) cultures of triploid watermelon

b. Medium-indexed plate showing different bacterial types one week post-indexing.

Characterization and Identification

Purification by standard bacteriological methods

Characterization by biochemical tests, Gram staining

Identification by traditional tests which are

Labor-intensive and time consuming

Can be performed in any laboratory with common chemicals

Through comparing with the standard strains of Bergey's Manual ) Krieg and Holt 1984(

Modern Identification Techniques

Biolog system

Detects carbon source utilization with

the reduction of tetrazolium dye in response to cellular respiration

Can identify yeasts and fungi

Through comparing the results with a database of responses

Modern Identification Techniques

Analytical Profile Index or API system

Carbon source utilization test

Visual detection of the test )Leifert et at. 1989; Vemiere et al., 1993(

Enzymatic oxidation/reduction interactions

Allows the identification of a limited number of Bacteria

Modern Identification Techniques

Fatty Acid Analysis Profiles FAP

Uses gas chromatography to identify over 140 separate fatty acids

Fatty acid profile is matched to a library of 700+ bacterial species representing over 180 genera

Can identify yeasts and fungi also

Match fatty acid methyl esters with those of known organisms (Buckley et al.. 1995; Chase et al., 1992; Stead et at, 1992)

Modern Identification Techniques

16S rRNAPCR amplification/ probes for known sequence

Using this system depends upon the number and diversity of bacteria in the databases

Many soil and plant bacteria have not been not characterized (Buckley et al. 1995)

For a more accurate identification the use of more than one test is Recommended (Jones et al. 1993) and ( Verniere et al. 1993)

Rate of occurrence of microbial contaminant in plant tissue culture

Odutayo et al. Afr. J. Agric. Res. Vol. 2(3), pp. 067-072, March 2007

The occurrence of bacteria isolates in plant tissue culture

Odutayo et al. Afr. J. Agric. Res. Vol. 2(3), pp. 067-072, March 2007

Antibiotic Treatments

Choosing an antibiotic depends on the type of bacteria Gram negative or Gram positive

Ideal antibiotics should be

soluble, stable, unaffected by pH, unaffected by media, lacking side effects, broadly active, bactericidal, suitable in combination, non-resistance inducing, inexpensive and nontoxic to human health.

Many antibiotics exist that have not yet been evaluated on plants or their bacterial contaminants (Falkiner, 1990; Seckinger, 1995).

Antibiotic Treatments

Antibiotics may be inactivated by environmental conditions heat/ light

Antibiotic sensitivity is reduced in plant tissue culture media due to different favorable pH degrees

Antibiotic concentration [ MBC ] for a particular bacteria should be determined

Phytotoxicity varies greatly among plant species and explant types, so preliminary testing with plant cultures is important

Effects of different concentrations of antibiotics at different durations of time to ensure

contamination free cultures

Habiba et al. Plant Tissue Cult. 12(2) : 117-124, 2002

Antibiotic Effects on Shoots Number and Multiplication Rate

Multiplication rate of Pelargonium shoots before (week 0),during and after treatment with carbenicillin or cefotaxime

*end of cefotaxime treatment**end of carbenicillin treatmentA. Wojtania et al. J. Fruit

Ornam. Plant Res. 104 vol. 13, 2005

Antibiotic Effects on Shoots Number and Multiplication Rate

Pelargonium 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

Conclusion

Several steps can reduce bacterial contaminants

Properly training the operators

Indexing cultures at initiation stage/culture cycle

Identifying contaminants and testing to determine the proper antibiotic

Thank You

Questions