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Controlling Contamination in
Plant Tissue Culture
Theoretically…
All (microbial) contaminants can be eliminated from
plant tissue cultures by one or more antibiotics.
In reality…
This is seldom possible.
No substitute for careful, aseptic work.
Last resort when conventional techniques fail.
Ideally:
• Isolate contaminant
• Streak it on bacterial medium plate
• Apply antibiotic discs
• Determine which antibiotic(s) are most effective
Alternate approach:
• Streak contaminant through selective media
• Determine partial characterization or identity
• Hit the library… Which antibiotics are effective?
Any phytotoxicity reported?
24 May 2004 2004 WORLD CONGRESS on In Vitro Biology
Antibiotic Effectiveness… More Reality
Negative:
• Detrimental to plastids & mitochondria
• Impair chlorophyll formation
• Inhibit amino acid incorporation
• Inadvertent development of resistant cell lines by
mutation or selection.
• Antibiotic combinations may have synergistic
phytotoxicity (i.e., 2 antibiotics show little/no toxicity
when used individually; significant toxicity when
combined).
Leifert, et al.
Plant Cell Tiss Org Cult 29:153, 1992
Positive:
• Promote growth of cell cultures
• Enhance morphogenesis while inhibiting callus
growth
• Stimulate root development in cuttings.
Not necessarily…
Stock Plant Treatment – Reduced phytotoxicity
• Hevea shoots-tips ⇒ inhibited growth w/ antibiotics
in medium.
• No inhibition if stock plant sprayed every 2 days for
2 weeks before culture.
• Contamination controlled
Enjalric, et al., Acta Hort 225: 57, 1988
Other studies have shown reduced bacterial growth
in shoot-tips & buds w/ regular antibiotic treatments
to stock pants.
24 May 2004 2004 WORLD CONGRESS on In Vitro Biology
Alternatives to Antibiotics in Medium (cont.)
Explant Treatment
• Shoot-tips sprayed before excision from stock plant
• Shoot tips/ buds dipped or soaked after excision
– e.g., Prunus shoot tips treated 1 min w/ Rifampicin before
culture gave better control than Rifampicin in medium.
• Tuber/ corm treatments
– Cyclamen tubers soaked 1-4 days before culture
produced non-contaminated cultures w/out phytotoxicity.
– Antibiotics in medium were phytotoxic.
Combining stock plant or explant treatments w/ in vitro
treatment may be necessary.
24 May 2004 2004 WORLD CONGRESS on In Vitro Biology
Antibacterial Antibiotics
Bacteristatic Bactericidal
Reversible inhibition of Lethal to Bacteria.
bacterial growth.
May have bactericidal activity Bacteristatic at low
at high concentrations. concentrations.
Includes: Includes:
• Choramphenicols • Aminoglycosides
• Tetracyclines • Cephalosporins
• Macrolides (Erythromycin) • Penicillins
• Polymyxins (Polymyxin-B)
• Rifamycins (Rifampicin)
24 May 2004 2004 WORLD CONGRESS on In Vitro Biology
The Chloramphenicols
• Solubility:
– 2.5 mg/ml in water (increased with urea).
– 50 mg/ml in ethanol.
• Solution Stability:
– 50% lost in 290 days at 20° C.
– 10% lost on heating to 115° C for 29 minutes.
– Protect from light, stable over pH 2.0-7.0.
• Incompatible with gentamicin.
• Application: [5 µg/ml for selection]
– Safe: up to 300 µg/ml stimulated corn protoplast.
Agricell Rep 9:38, 1987
– Toxic: 5-50 µg/ml Tobacco, beet, carrot, sunflower
George, Plant Prop by Tiss Cult, 1991
• Solubility:
– Free base – alcohol.
– HCl salt – water.
• Solution Stability:
– Hydrolyzes in water (tetracycline precipitates out).
– 10% lost in 24 hours at room temperature or 48 hours at 5° C.
• Incompatible with Penicillins, Polymyxin-B, and
B-complex vitamins.
• Applications:
– Safe: 25-100 µg/ml, Woody plants
Young, et al., Plant Sci Lett 34:203-209, 1984
– Toxic: Cherry, beet, carrot, tobacco
George, Plant Prop by Tiss Cult, 1993
• Water Soluble.
• Solution Stability:
– Stable at room temperature.
– Recommend 5° storage long term.
– Solutions darken without affecting potency.
• Resistance reported:
– Cross resistance with neomycin.
– Streptomycin.
• Effectiveness reduced with increasing Calcium Ions.
• Application:
– Safe: 1.5-4.5 µg/ml enhanced shoot differentiation of tobacco and
carrot calli. Owens, Plant Sci Lett 16:225-230, 1979
– Toxic: Beet, carrot, radish, Brassica spp, Tobacco
• Water Soluble.
• Solution Stability:
– 1 month at room temperature.
– A few months at 5° C.
– 18 months at -20° C.
• Resistance reported in most sensitive organisms.
• Cross-resistance with neomycin and kanamycin.
• Applications:
– 16 µg/ml (comb). Plant Cell Rept 20:22, 1993
– 25 µg/ml (comb). Ann Appl Biol 119:113, 1991
– 50-100 µg/ml (comb). J Appl Biol 119:113, 1991
– 72 µg/ml enhanced morphogenesis in tobacco and
carrot calli. Plant Sci Lett 16:225, 1979
– 1000 µg/ml (comb). Agricell Rep 20:22, 1993
– 100-200 µg/ml Syngonium & Philodendron
Acta Hort 212:87, 1987
– Toxic: Tobacco, sunflower, cherry, beet, carrot
• Applications:
– 25-100 µg/ml (comb), woody plants.
Plant Sci Lett 34:203,1984
– 50-100 µg/ml enhanced wheat regeneration.
J Plant Physiol 140:372,1992
– 100 µg/ml enhanced walnut shoot multiplication.
J Exp Bot 44:1211, 1993
– 200 µg/ml enhanced pear regeneration.
J Hort Sci 64:553, 1989
– 250 µg/ml enhanced apple regeneration.
Vitis 33:117, 1994
– 200 µg/ml increased apple shoot growth.
Plant Growth Reg 15:55, 1991
– 500 µg/ml induced Dianthus embryogenesis.
J Plant Physiol 141:721, 1993
• Water Soluble.
• Solution Stability:
– As with penicillin 24 hours at room temp.
– Maximum stability at pH 5.5-7.5.
• Incompatibility with aminoglycides
• Applications:
– 160 µg/ml reduced contamination in Piper sp
explants.
S Afr J Bot 58:500, 1992.
• Water Soluble
• Hygroscopic
• Solution Stability:
– 24 hr at room temp.
– 72 hours at 5° C.
• Activity enhanced by:
– Gentamicin, Ticarcillin, and Clavulanic acid.
• Incompatible with:
– Aminoglycosides, Tetracyclines, Amphotericin.
24 May 2004 2004 WORLD CONGRESS on In Vitro Biology
Carbenicillin (Cont.)
• Applications:
– 250 µg/ml Cattleya seedlings in vitro.
Amer J Bot 66:845, 1979.
– 500 µg/ml induced rapid callus in apple leaf explants;
inhibited regeneration.
Plant Cell Tiss Org Cult 37:257, 1994.
– Stimulated embryogenesis in Dianthus.
J Plant Physiol 141:721, 1993.
– Breakdown produces phenylacetic acid (auxin).
Plant Cell Rep 11:93, 1992.
– Toxic: Beet, carrot
• Water Soluble.
• Solution Stability:
– As with Penicillin (24 hr at room temp).
• Activity is enhanced by clavulanic acid (Timentin)
which is a B-lactamase inhibitor.
– 15-30 parts Ticarcillin: 1 part Clavulanic acid.
• Synergism reported with Aminoglycosides.
• Cross resistance between Ticarcillin and
Carbenicillin.
• Applications primarily in transformation systems
Amphotericin B
Cycloheximide
Nystatin
Griseofulvin
Petachloronitrobenzene (PCNB)
Thiabendazole
• Suspension Stability:
– 5 days at room temp.
– 1 month at 5° C.
– 2 yr at -20° C.
• Applications:
– 16 µg/ml (comb).
Plant Cell Rep 7:622,1989.
• Heat stability:
– Typically loses ~10% potency when autoclaved in
standard plant TC media (MS, B5, WPM, etc.).
– Binds to polypeptides when autoclaved so potency
reduced in protein-rich media (e.g., w/ 1 g/L Casein
Hydrolysate conc. of ~2X required).
• Recommended Concentrations:
– Seeds – 20-30+ ml/L for 8-12 hr.
– Shoot-tips (1+ cm) – 40-50 ml/L for 4-12 hr.
– Tubers – 40-50 ml/L for 12-24 hr.
– General TC media – 0.5-2 ml/L
– Callus, organogenesis, embryogenesis – 0.5-0.75 ml/L
8-Hydroxyquinoline
• Antibacterial and antifungal activity by intereferring
with microbial nucleic acid synthesis.
• Water Soluble.
• Solution Stability:
– No references.
– Stable in practical uses.
• Applications:
– Up to 1 µg/ml on Sedum callus with little toxicity.
Protoplasma 158:19, 1990.
Ribavirin (Virazole™)
Solubility: Water
Solution Stability: 24 hr at 2-6° C (prepare fresh)
Mode of action not fully understood; may be due to
competition w/ guanosine in formation of viral mRNA cap
structure or enzymes involved in production of structural
viral proteins. (www.rxmed.com)
Virus elimination in shoot-tips & buds augmented by heat
(thermo) therapy or electric shock treatment.
Some reports indicate mutation rate may be increased.
Concentration range: 20-100 µg/ml
Repeated subculture required: 7-10 for sugarcane; 6.5 months
for bamboo
Toxicity above 30 µg/ml reported.
24 May 2004 2004 WORLD CONGRESS on In Vitro Biology
Concluding Comments