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Plant Cell, Tissue and Organ Culture 50: 221–224, 1997. c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Research note

Activated charcoal does not catalyze sucrose hydrolysis in tissue culture media during autoclaving Steven R. Wann, Robert L. Veazey & Jacqueline Kaphammer Union Camp Corporation Technology Center, PO Box 3301, Princeton, NJ 08543-3301, USA Received 16 April 1996; accepted in revised form 12 September 1997

Key words: carbohydrate decomposition, media sterilization

Abstract Tissue culture media or aqueous sucrose solutions containing activated charcoal buffered to pH 5.5 and autoclaved did not undergo appreciable sucrose hydrolysis as reported. Rather, the extent of sucrose hydrolysis in media containing activated charcoal was found to be directly proportional to the hydrogen ion concentration (pH). This finding is consistent with the known mechanism of acid-catalyzed hydrolysis of acetals such as sucrose. Several types of charcoal were identified that acidified culture media to the extent that considerable acid-catalyzed sucrose hydrolysis occurred under autoclave conditions, making it appear as though activated charcoal was responsible for catalyzing sucrose hydrolysis. A simple mathematical expression was empirically derived that can be used to predict the extent of sucrose hydrolysis based on the post-autoclave pH of the media. Abbreviations: AC – activated charcoal, MES – N-morpholino-ethanesulfonic acid Activated charcoal (AC) has been used in plant tissue culture media to improve culture growth and/or promote morphogenesis in a wide variety of species. Growth-promoting effects of charcoal have been attributed to absorption of substances inhibitory to growth from the media produced either from breakdown of the media during autoclaving (Weatherhead et al., 1978) or by the cultures themselves (Fridborg et al., 1978). Additionally, alteration of medium pH to an optimum level for morphogenesis has also been reported as a beneficial effect of AC (Owen et al., 1991). Although beneficial effects of AC have been documented, it is a complex substance and the entire range of its effects on tissue culture media and the subsequent growth and morphogenesis of tissue cultures is unknown. A recent report indicated that AC catalyzed the hydrolysis of up to 90% of the sucrose in culture media to fructose and glucose (Druart and De Wulf, 1993). This report was of interest to us because fructose has been previously identified as inhibitory to the growth in vitro of some species. In papaya, shoot cultures could

be maintained for up to a year without subculture on media containing 1% fructose as the sole carbon source (Drew, 1992). The slow growth of papaya shoot cultures on fructose-containing medium enables the longterm storage of papaya germplasm at ambient temperatures, as in vitro cold storage is damaging to tissue. Slow growth of tissue cultures on fructose-containing media is presumably a result of the inhibition of glycolysis by fructose or its degradation products (Redei, 1973a, b). If AC is catalyzing the hydrolysis of sucrose in culture media to the extent that significant concentrations of fructose are produced, this could have profound effects on the growth of some species in vitro. Therefore, we set about attempting to verify and quantify the extent of sucrose hydrolysis in tissue culture media after autoclaving in the presence of AC. Sucrose solutions of various concentrations (w/v) in water, Gresshoff and Doy (1972) (GD) media, or buffer (100 mM acetate, formate or phosphate or 5 mM MES) were autoclaved at 120  C for 20 min in the presence of various types of AC (Merck Art n 2186, Sigma

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222 neutralized, Sigma acid-washed, NuChar SATM and NuChar SNTM (Westvaco Co.)). MES was employed at a much lower concentration to reflect it typical usage in plant tissue culture media. The pH of the solutions were adjusted after the addition of charcoal unless otherwise indicated. All experiments were performed at least twice and the number reported are an average of at least two determinations. After autoclaving, solutions were rapidly cooled to room temperature, the AC was removed by filtration, and the pH was determined. If sugar analysis was not performed immediately, the solutions were ‘quenched’ by neutralization (to pH = 7.0) and stored at 4  C. Sucrose does not undergo appreciable hydrolysis under these conditions. Quantitation of the sugars was done by external standard HPLC with refractive index detection. The separation was initially performed on a Biorad Aminex HPX-87C Carbohydrate Analysis column (3007.8 mm) maintained at 85  C, with an isocratic eluent of HPLC grade water at a flow rate of 0.6 ml min 1 . It was found that some buffer components interfered with the analytes using this procedure (formate coeluted with glucose for example). Analyses were subsequently performed using a Keystone Scientific Carbohydrate column (2504.6 mm) with an isocratic eluent of 3:1 acetonitrile: water at 1 ml min 1 . Samples were diluted 10-fold with water and filtered through a 0.45:m Gelman Acrodisc PVDF membrane syringe filter prior to injection (10 l). Commercial ACs used in plant tissue culture are derived from a variety of organic materials such as wood, coconut husk and peat. Therefore, it is not surprising that different ACs have different properties and produce slightly different effects when added to culture media. The effect of AC type on the pH of GD media is shown in Table 1. Some charcoals, such as those produced by Sigma, raised the pH of the media when the pH is adjusted before the AC is added, as previously shown (Owen et al., 1991). Other ACs, such as Merck Art. n 2186, and both NuCharTM types, acidify the media appreciably (by as much as 1.4 pH units or a factor of 25), especially when the pH is adjusted after the AC was added, as in the report under investigation. However, tissue culture media has some buffering capacity. Druart and De Wulf performed their experiment in aqueous sucrose solutions that have no buffering capacity. When the same ACs are added to aqueous sucrose solutions and autoclaved, the pH dropped by 2.0 units (from 5.5 to 3.5; see Table 2). Under these acidic conditions (pH = 3.5), acetals such as

sucrose undergo acid-catalyzed hydrolysis to form, at least initially, glucose and fructose. Acetals are subject to specific hydronium ion catalysis, in which the rate of hydrolysis is directly proportional to the hydrogen ion concentration (Hine, 1962). Therefore, the lower the pH, the faster the rate of acid-catalyzed sucrose hydrolysis. The low post-autoclave pH values obtained by Druart and De Wulf are within the range where acidcatalyzed hydrolysis of sucrose would be expected to occur at an appreciable rate. Verification of acid catalysis as the mechanism for sucrose hydrolysis under autoclave conditions was made by comparing the extent of sucrose hydrolysis in some of the same solutions used by Druart and De Wulf (10% sucrose with 1.0, 0.5 and 0.2% Merck Art n 2186 AC) with 10% sucrose solutions lacking AC, but buffered to the authors post-autoclave acidity. The extent of sucrose hydrolysis in solutions buffered to acid conditions but lacking AC was similar to the AC-containing solutions (see Table 2). For example, for solutions with a post-autoclave pH of 3.6, sucrose hydrolysis was 55% with 0.5% AC and 64% without AC. The slightly greater level of sucrose hydrolysis in the buffered solutions in Table 2 is probably due to the fact that they were at the post-autoclave pH from the outset, so that acid-catalyzed sucrose hydrolysis could begin immediately upon autoclaving. Solutions containing AC had to develop acidity during autoclaving, and were therefore not as acidic for as long as the buffered solutions. Furthermore, when a 10% aqueous sucrose solution containing Merck Art. n 2186 AC (0.5%) was buffered with MES to the starting pH = 5.5 so that very little acidification could occur upon autoclaving, the extent of sucrose hydrolysis was only 3% and not the 91% reported by Druart and De Wulf (see Table 2). Indeed, only 55% of the sucrose was hydrolyzed when we tried to reproduce their findings with 10% sucrose and 0.5% Merck Art n 2186 AC. The differences in the extent of sucrose hydrolysis might be due to lot-to-lot variations in the AC such that slightly different postautoclave pH values were obtained. Table 2 shows the sensitivity of sucrose hydrolysis to pH. For example, in the solutions containing 0.2 and 1.0% AC a change of 0.3 units in post-autoclave pH produced a fourfold difference in the extent of sucrose hydrolysis. The results in Table 2 demonstrate that the extent of sucrose hydrolysis in aqueous solutions can be accounted for solely on the basis of the acidity, and not on the presence of AC.

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223 Table 1. Effect of activated charcoal type (0.5%) on the pH of GD media (2% sucrose) before and after autoclaving. Charcoal type

Media pH pH adjusted to 5.8 before adding charcoal Before autoclaving After autoclaving

pH adjusted to 5.8 after adding charcoal After autoclaving

M

6.9 5.7 6.1 5.1

6.6 6.4 5.7 4.7

5.4 4.9 4.4 4.4

TM

5.3

4.5

4.4

5.8

5.5

N/A

Sigma neutralized Sigma acid-washed Merck Art. n 2186 NuChar SA (acidwashed) NuChar SN (neutralized) Control (No AC)

Table 2. The effect of post-autoclave pH (with and without activated charcoal) on sucrose hydrolysis in aqueous solution. 10% Sucrose

With AC: 1% AC (Merck) 0.5% AC 0.5% AC + 5 mm MES 0.2% AC Without AC: 100 mM Na formate 100 mm Na formate 100 mm Na formate

Pre-autoclave pH

Post-autoclave pH

Sucrose hydrolysis, %

5.5 5.5 5.5

3.5 3.6 5.3

60 55 3

5.5

3.8

14

3.6

3.4

75

3.8

3.6

64

4.2

4.0

29

In tissue culture media, acid catalyzed sucrose hydrolysis was also demonstrated over a broad range of acid concentrations (pH = 3.5-5.8) produced by autoclaving GD media containing 2% sucrose buffered in phosphate, acetate, formate, and MES. Linear regression analysis of the extent of sucrose hydrolysis as a function of hydrogen ion concentration in buffered media produced an empirical equation (Equation 1). Equation (1) can be used to predict the extent of sucrose hydrolysis in culture media after a typical autoclave cycle based on the post-autoclave pH. % Sucrose Hydrolysis

=

1:9 + 2:9  105 (10

pH ) (1 )

Under typical media preparation conditions (i.e. a post autoclave pH of 5.5) Equation (1) predicts that only about 3% of the sucrose is hydrolyzed. In most tissue culture media, with initial sucrose concentrations of 20–30 g l 1 , the concentration of fructose (and glucose) produced after autoclaving will be from 600–900 ppm. Although the original report by Druart and De Wulf incorrectly attributed to AC the ability to catalyze sucrose hydrolysis, the outcome they described remains the same. Namely, appreciable sucrose hydrolysis (albeit acid-catalyzed) can occur in culture media during autoclaving at post-autoclave pH < 5.0. Whenever media acidification occurs to this extent for any

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224 reason, due consideration should be given to the consequences of sucrose hydrolysis on the chemical and physical properties (e.g. replacement of a portion of the sucrose with fructose and glucose and an increase in media osmolarity) of the media prior to its use in tissue culture.

References Drew RA (1992) Improved techniques for the in vitro propagation and germplasm storage of papaya. HortScience 27: 1122–1224 Druart Ph & De Wulf O (1993) Activated charcoal catalyzes sucrose hydrolysis during autoclaving. Plant Cell Tiss. Org. Cult. 32: 97–99 Fridborg G, Pedersen M, Landstron L-E & Eriksson T (1978) The effect of activated charcoal on tissue culture: absorption of metabolites inhibiting morphogenesis. Physiol. Plant. 43: 104– 106

Gresshoff PM & Doy CH (1972) Development and differentiation of haploid Lycopersicon esculentum (tomato). Planta 107: 161–170 Hine J (1962) Physical Organic Chemistry (p 120). McGraw Hill Publishing Co, New York Owen HR, Wengerd D & Miller R (1991) Culture medium pH is influences by basal medium, carbohydrate source, gelling agent, activated charcoal and medium storage method. Plant Cell Reports 10: 583–586 Redei GP (1973a) Effects of degradation products of fructose on the glycolytic pathway. Z. Pflanzenphysiol. 70: 97–106 Redei GP (1973b) Effects of autoclaved fructose media on metabolites in three cruciferous plants. Z. Pflanzenphysiol. 70: 107–114 Weatherhead MA, Burdon L & Henshaw GG (1978) Some effects of activated charcoal as an additive to plant tissue culture media. Z. Pflanzenphysiol. 89: 141–147

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