Physical & Chemical Means of Controlling Contamination in Plant Tissue Culture

Dr. Gary Seckinger Dr. Kenneth C. Torres PhytoTechnology Laboratories

Like death and taxes, contamination or the threat of contamination is always with us and we need all the weapons to combat them.
L.L. Coriell National Cancer Institute Monog. 7:33, 1962

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Physical Means of Controlling Contamination Eliminate vector transmission of contaminants:

Seal Culture Vessels Parafilm, Nescofilm PVC Film (Austratec) Seal Cultures in Plastic Bags: e.g., Zip-lock Bags, Star*Pac Bags

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Chemical Control of Contamination

The Use of Antimicrobials in Plant Tissue Culture

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Antibiotics (Antimicrobials)

Bacteristatic Inhibit bacterial growth; bacteria viable but dormant. Growth resumes in antibiotic-free medium. Bactericidal Lethal to bacteria; no subsequent growth in antibiotic-free medium Antifungal (Antimycotics) Fungistatic & Fungicidal Broad-spectrum Antimicrobials Inhibit or eliminate bacteria and fungi. Antiviral Inactivate virus
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Ideally, Antibiotics in Plant Tissue Culture are:

Soluble Stable Unaffected by media pH or components Inexpensive Minimal side effects (no phytotoxicity)

Systemic in plant tissues Broad spectrum of activity Low chance of bacterial resistance Lethal to microbe Usable in combinations

Gilbert, et al., Ann Appl Biol 119:113, 1991 Falkiner, Acta Hort 225:53, 1988 & Int Assoc Plant Tiss Cult 60:137, 1990
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Theory vs. Reality

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.

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So Many Antibiotics Available Which to Use?

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?
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Antibiotic Effectiveness More Reality

Antibiotic effectiveness can be affected by: pH of medium e.g., some are more effective at alkaline pHs Ion concentration of medium high cation concentration can reduce effectiveness of some antibiotics Many antibiotics have short solution stability: e.g., the Penicillin group 24-48 hr at room temp. Phytotoxicity varies: Between plant species Between genotypes within a species Between plant parts organs vs. intact plant
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How Much Do I Use?

MIC vs. MBC MIC Minimum Inhibitory Concentration Inoculate bacteria on medium w/ increasing concentrations of antibiotic(s) Lowest concentration of antibiotic to prevent bacterial growth = MIC MBC Minimum Bactericidal Concentration Subculture from medium at & above MIC conc. Lowest antibiotic conc. in MIC test at which no bacteria grow on bacterial medium w/out antibiotic = MBC Target: Phytotoxic Concentration MBC
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=2
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If One is Good Are 2+ Better?

Most antibiotics have narrow spectrum of activity e.g., Gram +, Gram -, or Antimycotic Plant cultures may contain more than one type of contaminant may contain numerous types Therefore, Antibiotic combinations may be necessary Worse yet Some microbial species difficult to eliminate w/ one antibiotic, e.g., Hypomicrobium contamination Neither 500 g/ml Streptomycin or Carbenicillin effective. Combined 100 g/ml of both was effective.
Horsch & King, Plant Cell Tiss Oran Cult 2:21-28, 1983
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Antibiotic Effects on Growth & Development

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).
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Effect on Multiplication

Grading antibiotics based on their inhibitory effect on plant growth (reduced multiplication rate in vitro): Most Inhibitory Streptomycin Polymyxin-B Rifampicin Carbenicillin Least Inhibitory
Leifert, et al. Plant Cell Tiss Org Cult 29:153, 1992
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Antibiotic Effects on Growth & Development

Positive: Promote growth of cell cultures Enhance morphogenesis while inhibiting callus growth Stimulate root development in cuttings.

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Do I Have to Add Antibiotics to the Medium?

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.
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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.

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Antibacterial Antibiotics

Bacteristatic
Reversible inhibition of bacterial growth. May have bactericidal activity at high concentrations. Includes: Choramphenicols Tetracyclines Macrolides (Erythromycin)

Bactericidal
Lethal to Bacteria. Bacteristatic at low concentrations. Includes: Aminoglycosides Cephalosporins Penicillins Polymyxins (Polymyxin-B) Rifamycins (Rifampicin)
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The Chloramphenicols

First broad-spectrum antibiotics discovered. First isolated from Streptomyces venezuelae. Bacteristatic activity by interfering with bacterial protein synthesis. Activity spectrum similar to Tetracyclines includes Gram +, Gram -, and anaerobic bacteria.

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Chloramphenicol

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 g/ml for selection]

Safe: up to 300 g/ml stimulated corn protoplast.
Agricell Rep 9:38, 1987

Toxic: 5-50 g/ml Tobacco, beet, carrot, sunflower

George, Plant Prop by Tiss Cult, 1991

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The Tetracyclines (Quinones)

Semi-synthetic antibiotics originally derived from Streptomyces spp. Bacteristatic activity by interfering with bacterial protein synthesis. Broad-spectrum activity including Gram +, Gram -, and mycoplasmas. Includes: Tetracycline (only widely used antibiotic in this group).

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Tetracycline

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 g/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
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The Macrolides

Derived from Streptomyces spp. Bacteriostatic/bactericidal activity is dependent on dose and specific microbe. Antibiotic activity due to interference with bacterial protein biosynthesis. Effective against Gram (+) bacteria. Erythromycin only widely used antibiotic in this group.

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Erythromycin

Hygroscopic powder, mixture of Macrolide antibiotics. Solubility:

2 mg/ml in water (Merck Index). 50 mg/ml in ethanol.

Solution Stability:
5 days or longer at 5 C. Protect from light.

Resistance shown in many organisms. Antagonism & synergism with Ampicillin and Gentamicin; incompatible with Penicillin-G. Application:
32 g/ml (comb.). Toxic: Tobacco Plant Cell Rep 7:622, 1989

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The Aminoglycosides

Derived from Actinomycetales (bacterial order). Bind to 30S ribosomal subunit in bacterial cells and inhibit protein synthesis. Most active against Gram -. Gram + and anaerobic bacteria are naturally resistant. Includes:
Kanamycin Neomycins Streptomycin Gentamicin

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Kanamycin

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 g/ml enhanced shoot differentiation of tobacco and carrot calli. Owens, Plant Sci Lett 16:225-230, 1979 Toxic: Beet, carrot, radish, Brassica spp, Tobacco
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Neomycin Sulfate (Fradiomycin Sulphate)

Mixture of sulfates of Streptomyces fradiae. Water Soluble. Solution Stability:

Stable at room temperature. 5 C for long term storage, yields some color change. Maximum of two years. Protect from light.

Used in conjunction with other antibiotics (e.g., Polymyxin B) to prevent development of resistant strains. Application:
1000 g/ml shoot tip pretreatment. Toxic: Cherry, tobacco Agricell Rep 20:22, 1993

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Streptomycin Sulfate

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.

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Streptomycin Sulfate (Cont.)

Applications:
16 g/ml (comb). Plant Cell Rept 20:22, 1993 25 g/ml (comb). Ann Appl Biol 119:113, 1991 50-100 g/ml (comb). J Appl Biol 119:113, 1991 72 g/ml enhanced morphogenesis in tobacco and carrot calli. Plant Sci Lett 16:225, 1979 1000 g/ml (comb). Agricell Rep 20:22, 1993 100-200 g/ml Syngonium & Philodendron Acta Hort 212:87, 1987 Toxic: Tobacco, sunflower, cherry, beet, carrot
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Gentamicin Sulfate

Mixture of Gentamicins C1, C1A, C2, C2A, C2B. Water Soluble. Solution Stability: Stable at room temperature. Recommend 5 for long-term. Incompatible with Penicillins, Erythromycin, and Chloramphenicol. Resistance appearing in many Gram - bacteria.

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Gentamicin Sulfate (Cont.)

Resistance appearing in many Gram (-) bacteria. Application:

50 g/ml (comb). Agricell Rept 20:22, 1993. 50 g/ml no toxicity on Cattleya seedlings grown In Vitro. Amer J Bot 66:825, 1979. 250 g/ml reduced explant contamination in Piper sp. S Afr J Bot 58:500, 1992. Toxic: Datura, Cymbidium, Vanda

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The Cephalosporins

Semi-synthetic antibiotics derived from Cephalosporin C.

Natural antibiotic produced by Cephalosporium acremonium mold.

Inhibit Bacterial cell wall synthesis. Classified by generations based on antibacterial activity and introduction date. Third Generation:
Have wider spectrum and greater potency against Gram bacteria. Activity against Gram + bacterium is less than earlier generations.

Includes:
Cefotaxime
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Cefotaxime

Water Soluble. Protect from light. Solution Stability: 24 hr at room temp. 22 days at 5 C. 112 days at -10 C. Optimum pH 4.3-6.2. Activity enhanced by: Aminoglycosides (e.g., Gentamicin). Incompatible with alkaline solutions.
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Cefotaxime (Cont.)

Applications:
25-100 g/ml (comb), woody plants. Plant Sci Lett 34:203,1984 50-100 g/ml enhanced wheat regeneration. J Plant Physiol 140:372,1992 100 g/ml enhanced walnut shoot multiplication. J Exp Bot 44:1211, 1993 200 g/ml enhanced pear regeneration. J Hort Sci 64:553, 1989 250 g/ml enhanced apple regeneration. Vitis 33:117, 1994 200 g/ml increased apple shoot growth. Plant Growth Reg 15:55, 1991 500 g/ml induced Dianthus embryogenesis. J Plant Physiol 141:721, 1993
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The Penicillins

First antibiotics for therapeutic use. Bactericidal activity by inhibiting biosynthesis of bacterial cell wall. Inactivated by penicillinase-producing bacteria. Includes:
Penicillin G (Benzylpenicillin) Gram (+). Ampicillin broader spectrum than Penicillin G. Carbenicillin narrower spectrum than Ampicillin. Ticarcillin similar spectrum to Penicillin G.

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Penicillin G (Benzylpenicillin)

Off-white powder produced by Pennicillium notatum. Water Soluble. Stability:

24 hr at room temp. 7 days at 5 C. 18 months at -20 C. Optimum pH 5.5-7.5
pH above or below the accepted range accelerates hydrolysis of B-Lactam Ring.

Dilute Solutions more stable than concentrated solutions.

Incompatible with metal ions, rubber, vitamin C, Amphotericin, and Tetracycline.

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Penicillin G (Cont.)

Applications: Approximately 1650 U/mg 25-50 U/ml (comb). Ann Appl Biol 119:113, 1991. Stimulated embryogenesis in Dianthus. Agicell Rep 22:3, 1994. 10-100 g/ml Cattleya seedling in vitro. Amer J Bot 66:825, 1979. Prevent seed germination & inhibit organogenesis in several species.

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Ampicillin

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 g/ml reduced contamination in Piper sp explants. S Afr J Bot 58:500, 1992.
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Carbenicillin (-Carboxybenzylpenicillin)

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.
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Carbenicillin (Cont.)

Applications:
250 g/ml Cattleya seedlings in vitro. Amer J Bot 66:845, 1979. 500 g/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
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Ticarcillin

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
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Polymyxin-B

Bonds to bacterial membranes and alter their permeability. Bactericidal activity against Gram -. Water Soluble, Hygroscopic Powder. Solution Stability:
1 yr at 5 C.

Activity enhanced with Rifampicin. Incompatible with Ampicillin, Tetracycline, Amphotericin.

Rapidly inactivated by acids and bases.

Applications:
6-24 g/ml (comb) Woody plants Young, Plant Sci Lett 34:203, 1984.

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Rifampicin (Rifamycin, Rifampin)

Rifamycin group; interferes with bacterial nucleic acid synthesis. Spectrum: Gram +, few Gram -, Anaerobic bacteria, Mycobacteria, (some viruses at high concentrations). Solubility: DMSO [Dimethylsulfoxide] (per DBR), Slightly soluble in water and ethanol.

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Rifampicin (Continued)

Activity enhanced by: Erythromycin, Gentamicin, and Vancomycin. Incompatible with Penicillins. Applications: 6-24 g/ml (comb) Wood plants Young, Plant Sci Lett 34:203, 1984 10 g/ml (comb) Camellia Haldeman, et al., HorSci 22:306, 1987 75 g/ml (comb) Hevea Enjalric, et al., Acta Hort 225:87, 1988
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Antifungals (Antimycotics)

Amphotericin B Cycloheximide Nystatin Griseofulvin Petachloronitrobenzene (PCNB) Thiabendazole

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Amphotericin B (Amphoterrible)

Interferes with membrane permeability by binding to sterols. Soluble in DMSO.

Water-soluble preparations available (Deoxycholatesodium phosphate complex = Fungizone)

Solution Stability:
5 days at room temp. 1 month at 5 C. 1+ Yr at -20 C. Protect from light. Inactivated by low pH.
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Amphotericin B (Cont)

Incompatible with Penicillins, Gentamicin, Kanamycin, Polymyxin-B, and Streptomycin. Applications: Up to 10 g/ml on Cattleya seedlings in vitro. Amer J Bot 66:825, 1991. 62-125 g/ml (comb). Ann Appl Biol 199:113, 1991.

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Cycloheximide

Antifungal and antiviral activity. Solubility: 5 mg/ml in water (may require sonication). 50 mg/ml in ethanol. Solution Stability: 1 to 3 months at 5 C (aqueous pH 3.0-5.0) 1 to 3 years at -20 C. Autoclavable

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Nysatin (Fungicidin)

Polyene antibiotic with fungistatic and fungicidal activity by interferring with cell membrane permeability. Solubility: Soluble in DMF (dimethylformamide). Very slightly soluble in water; used as active suspension.

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Nysatin (Cont.)

Suspension Stability: 5 days at room temp. 1 month at 5 C. 2 yr at -20 C. Applications: 16 g/ml (comb).
Plant Cell Rep 7:622,1989.

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Wide-Spectrum Antimicrobials

PPM (Plant Preservative Mixture) 8-Hydroxyquinoline

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Wide-Spectrum Antimicrobials

PPM (Plant Preservative Mixture) Patented product of Plant Cell Technologies Active ingredients
5-Chloro-2-methyl-3(2H)-isothiazolone 2-Methyl-3(2H)-isothiazolone

Broad spectrum biocide (bacteria & fungi) Inhibits enzymatic activity in citric acid cycle and electron transport chain May inhibit translocation of sugars & amino acids Translocation across plant cell wall impeded
(Freshly isolated protoplasts are very sensitive to PPM)
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PPM (Cont.)

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
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Wide-Spectrum Antimicrobials

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 g/ml on Sedum callus with little toxicity. Protoplasma 158:19, 1990.
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Antiviral

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 g/ml Repeated subculture required: 7-10 for sugarcane; 6.5 months for bamboo Toxicity above 30 g/ml reported.
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Concluding Comments

Since most microbial contaminants are unknowns, and, Since microorganisms have different sensitivities to antibiotics it is difficult to specifically target microbial contaminants. Unfortunately, it is common practice to use the spray and pray technique for antibiotic application Best to check the literature first. Broad-spectrum biocides frequently have bacteristatic/ fungistatic activity continuous use may be required to control contaminants.
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References
ONeil, MJ (ed). 2001. The Merck Index 13th Edition. Merck & Co., Inc. Whitehouse Station, NJ. United States Pharmacopeia (USP XXIV). 2000. George, EF. 1993. Plant Propagation by Tissue Culture, Part 1. The Technology, 2nd Ed., Exegetics Ltd. Reynolds, JEF (ed). 1989. Martindale The Extra Pharmacopoeia. The Pharmaceutical Press. London. Dawson, RMC. 1986. Data for Biochemical Research. Clarendon Press. Oxford. TCA Manual. Procedure #15143. 1976.
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