Phy Chem Control Contamination in Ptc

Physical & Chemical Means of Controlling Contamination in

Plant Tissue Culture

Dr. Gary SeckingerDr. Kenneth C. Torres

PhytoTechnology Laboratories

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

“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. CoriellNational Cancer Institute

Monog. 7:33, 1962


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


Chemical Control of Contamination

The Use of Antimicrobials in Plant Tissue Culture


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 & FungicidalBroad-spectrum Antimicrobials – Inhibit or eliminate

bacteria and fungi.Antiviral – Inactivate virus


Ideally, Antibiotics in Plant Tissue Culture are:

Low chance of bacterial resistance

Unaffected by media pH or components

Usable in combinationsMinimal side effects(no phytotoxicity)

Lethal to microbeInexpensive

Broad spectrum of activityStableSystemic in plant tissuesSoluble

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


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.


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 effectiveAlternate approach:• Streak contaminant through selective media• Determine partial characterization or identity• Hit the library… Which antibiotics are effective?

Any phytotoxicity reported?


Antibiotic Effectiveness… More Reality

Antibiotic effectiveness can be affected by:• pH of medium – e.g., some are more effective at alkaline

pH’s• Ion concentration of medium – high cation concentration

can reduce effectiveness of some antibioticsMany 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


How Much Do I Use?

MIC vs. MBCMIC – Minimum Inhibitory Concentration• Inoculate bacteria on medium w/ increasing concentrations

of antibiotic(s)• Lowest concentration of antibiotic to prevent bacterial

growth = MICMBC – 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

= 2


If One is Good… Are 2+ Better?

Most antibiotics have narrow spectrum of activitye.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 contaminationNeither 500 µg/ml Streptomycin or Carbenicillin effective.Combined 100 µg/ml of both was effective.

Horsch & King, Plant Cell Tiss Oran Cult 2:21-28, 1983


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).


Effect on Multiplication

Grading antibiotics based on their inhibitory effect on plant growth (reduced multiplication rate in vitro):

Most InhibitoryStreptomycin ⇒ Polymyxin-B ⇒

Rifampicin ⇒ CarbenicillinLeast Inhibitory

Leifert, et al.Plant Cell Tiss Org Cult 29:153, 1992


Antibiotic Effects on Growth & Development

Positive:• Promote growth of cell cultures• Enhance morphogenesis while inhibiting callus

growth• Stimulate root development in cuttings.


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.


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.


Antibacterial Antibiotics

Bacteristatic at low concentrations.

May have bactericidal activity at high concentrations.

Includes:• Aminoglycosides• Cephalosporins• Penicillins• Polymyxins (Polymyxin-B)• Rifamycins (Rifampicin)

Includes:• Choramphenicols• Tetracyclines• Macrolides (Erythromycin)

Lethal to Bacteria.Reversible inhibition of bacterial growth.

BactericidalBacteristatic


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.


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 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, sunflowerGeorge, Plant Prop by Tiss Cult, 1991


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).


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 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


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.


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 µg/ml (comb.). Plant Cell Rep 7:622, 1989– Toxic: Tobacco


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


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 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


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 µg/ml shoot tip pretreatment. Agricell Rep 20:22, 1993– Toxic: Cherry, tobacco


Streptomycin Sulfate


– 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.


Streptomycin Sulfate (Cont.)

• 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


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.


Gentamicin Sulfate (Cont.)

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

– 50 µg/ml (comb).Agricell Rept 20:22, 1993.

– 50 µg/ml no toxicity on Cattleya seedlings grown In Vitro.Amer J Bot 66:825, 1979.

– 250 µg/ml reduced explant contamination in Piper sp.S Afr J Bot 58:500, 1992.

– Toxic: Datura, Cymbidium, Vanda


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


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.


Cefotaxime (Cont.)

• 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


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.


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.


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 µg/ml Cattleya seedling in vitro.

Amer J Bot 66:825, 1979.– Prevent seed germination & inhibit organogenesis in

several species.


Ampicillin


– 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.


Carbenicillin (α-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.


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


Ticarcillin


– 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


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 µg/ml (comb) Woody plantsYoung, Plant Sci Lett 34:203, 1984.


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.


Rifampicin (Continued)

• Activity enhanced by:– Erythromycin, Gentamicin, and Vancomycin.

• Incompatible with Penicillins.• Applications:

– 6-24 µg/ml (comb) Wood plantsYoung, Plant Sci Lett 34:203, 1984

– 10 µg/ml (comb) CamelliaHaldeman, et al., HorSci 22:306, 1987

– 75 µg/ml (comb) HeveaEnjalric, et al., Acta Hort 225:87, 1988


Antifungals (Antimycotics)

Amphotericin BCycloheximide

NystatinGriseofulvin

Petachloronitrobenzene (PCNB)Thiabendazole


Amphotericin B (“Amphoterrible”)

• Interferes with membrane permeability by binding to sterols.

• Soluble in DMSO.– Water-soluble preparations available (Deoxycholate-

sodium 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.


Amphotericin B (Cont)

• Incompatible with Penicillins, Gentamicin, Kanamycin, Polymyxin-B, and Streptomycin.

• Applications:– Up to 10 µg/ml on Cattleya seedlings in vitro.

Amer J Bot 66:825, 1991.– 62-125 µg/ml (comb).

Ann Appl Biol 199:113, 1991.


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


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.


Nysatin (Cont.)

• 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.


Wide-Spectrum Antimicrobials

PPM™ (Plant Preservative Mixture)

8-Hydroxyquinoline



PPM™ (Plant Preservative Mixture)• Patented product of Plant Cell Technologies• Active ingredients

5-Chloro-2-methyl-3(2H)-isothiazolone2-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)


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



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.


Antiviral

Ribavirin (Virazole™)Solubility: WaterSolution 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/mlRepeated subculture required: 7-10 for sugarcane; 6.5 months

for bambooToxicity above 30 µg/ml reported.


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.


References

• O’Neil, 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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Phy Chem Control Contamination in Ptc

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