Plant Cell, Tissue and Organ Culture 32: 97-99, 1993.

© 1993 Kluwer Academic Publishers. Printed in the Netherlands.

Activated charcoal catalyses sucrose hydrolysis during autoclaving

Ph. Druart ~ & O. De Wulf 2

1C.R.A., Station des Cultures Fruitibres et Marafchbres Chauss~e de Charleroi 234, 5030 Gembloux,
Belgium; 2U.E.R. de Bioindustries, Facult6 des Sciences Agronomiques, Passage des DOportOs, 5030
Gembloux, Belgium

Received 22 August 1991; accepted in revised form 10 June 1992

Key words: activated charcoal, in vitro culture, sucrose hydrolysis

Abstract

Activated charcoal frequently promotes the in vitro growth and organogenesis of plant tissues by the
absorption of compounds from the culture medium and/or from the container atmosphere.
Our experiment demonstrates that sucrose hydrolysis, which normally reaches 10% during autoclav-
ing, increases to 95% in presence of 1% of activated charcoal. This gives rise to the acidification of the
solution due to a specific reaction of the formed fructose.
Changing the available carbon source from the initial sucrose to a mixture of fructose, glucose and
sucrose, causes a osmolarity increase, a drop in the agar gelling and the formation of furfural
derivatives which are indirect consequences of the sucrose hydrolysis affecting the tissue culture media.

Introduction 1982; Misson et al. 1983; Heberle-Bors 1980;

Used in chemistry for its strong absorptive prop-
erties for both dissolved solids and gases, acti-
vated charcoal has frequently improved plant
tissue cultures (George & Sherington 1984) by
absorption of growth inhibitors, prevention of
unwanted callus growth, promotion of mor-
phogenesis, particularly embryogenesis and pro-
motion of root growth.
The advantageous effects of activated charcoal
addition includes the absorption of phenols pro-
duced by wounded tissues (Fridborg et al. 1978;
Weatherhead et al. 1978), 5-hydroxymethylfur-
fural produced by sucrose autoclaving (Weather-
head et al. 1978), agar impurities (Kolenbach &
Wernicke 1978) and ethylene from the gas phase
of the cultures (Horner et al. 1977). Activated
charcoal will also absorb medium components
such as vitamins (Weatherhead et al. 1978), cyto-
kinins and auxins (Fridborg et al. 1978), ascorbic
acid (Weatherhead et al. 1978; Takayama &
Misawa 1980; Scholl et al. 1981; Steinitz & Yahel
Johansson et al. 1982; Constantin et al. 1977).
However, activated charcoal in aqueous suc-
rose solutions and removed by filtration did not
bind the sucrose (Horner et al. 1977).
In our experiments, a sucrose solution pre-
autoclaved in the presence of activated charcoal
has been shown to improve shoot growth during
the axillary branching of some fruit-tree species
(Druart 1987). This sucrose solution also stimu-
lated somatic embryogenesis in wheat anthers
(Zhang 1988).
Materials and m e t h o d s
10% (w/v) aqueous sucrose solutions were auto-
claved at 120°C and for 20 min, in presence of
0.1, 0.2, 0.3 or 1% activated charcoal (A.C.)
(Merck Art. n°2186). The pH of the solutions
was adjusted to 5.5 prior to autoclaving. A.C.
was removed by filtration after autoclaving. Suc-
rose, glucose and fructose were quantified by
98

H.P.L.C. (Column Aminex H P X 87c, Bio-Rad 700

Laboratories). pH and osmolarity were mea- o
~00 "I"
sured with a Knight microprocessor pH meter
500
and by advanced microosmometer model 3 M.O.
E
(Advanced Instruments, Inc.) respectively. ~00 ~
IQ.
The p H of A.C. solutions was measured be- E
300
fore and after autoclaving. The pH of media L-
200 -~
containing 0.7% agar (Merck, 1613)+ the mac- 0

roelements of Lepoivre and 3% sucrose or 1.5% lOO ~E

glucose or 1.5% fructose or 1.5% glucose+ 0~ i i i
0.1 0,2 0,5
1.5% fructose was measured after autoclaving. A.C.(g/100ml)
The glucose or fructose solutions at 0.5, 1, 2 or -=- pH -e- Osmolarity
5% were analysed by UV/VIS spectrophotome-
Fig. 2. pH and osmolarity of the sucrose solution as a
ter Lambda 5 (Perkin Elmer). function of A.C. concentration.

Results

~ ~ amocl.
Sucrose hydrolysis resulting from autoclaving, Af'cr a u r a l . .
drastically increased in the presence of activated
-r
charcoal (A.C.) (Fig. 1). Less than 40% sucrose
remained after autoclaving with 0.1% A.C. and
only 3.7% with 1% A.C. These results mean
that A.C. acts as a catalyst of sucrose hydrolysis
during autoclaving. Due to the formation of a. ~ A.C.g/100nU
glucose and fructose the osmolarity and the . . . . :2 o:, o:, o:s , :o , :~
acidity of the solution increased (Fig. 2). The
Fig. 3. pH of A.C. solutions before and after autoclaving.
latter induced a lower rigidity of the gel medium.
As shown in Fig. 3 and Fig. 4 medium containing
5.3
glucose + fructose or fructose reduces the pH.
The acidification would be due to a specific 5.2

fructose reaction during autoclaving. In contrast,

autoclaving distilled water by itself or with A.C. 5,1

would tend to make it more alkaline. Z s.o

The solutions of hydrolysed sucrose were very
43
9 Ioo
8 90
8o
7 4.7
7o o SUCI~IOSE GLUCOSE FRUC'TOSE GLU.-i-FRU.
6 3 1.5 1.5 1.5 =1=
6o ~
5
o 50
4 Fig. 4. pH of autoclaved media as a function of the sugar
3 type and concentration.
2
2 2O ~
10 ! slightly coloured and induced a light browning of
1
o the culture media. Pure fructose solutions col-
0 0,1 0,2 0,5 I
oured after autoclaving whereas glucose solu-
A.C.(g/I OOml)
BB Sucrose t ~ Glucose tions did not. Spectrum-analysis (Fig. 5) con-
m Fructose 4 - Hydr. Sucrose firmed an absorbance rise between 280 and
Fig. 1. Sugar content and sucrose hydrolysis in presence of 300 nm which could correspond to the formation
A.C. of furfural derivatives.

FRtlCTOSI~ GtOCOSE
ihlere AUII©I, Autoclaved Before A I t o © l Autodlvld
I ~
300 ..... ~eO I~ " SC~
Fig. 5. Absorbance (220-420nm) of fructose and glucose
solutions as a function of autoclaving.
Conclusions
Activated charcoal stimulates sucrose hydrolysis
during autoclaving as a function of its concen-
tration. More than 95% of the sucrose auto-
claved with 1% A.C. was transformed to fruc-
tose and glucose.
The consequences to the tissue culture media
are; fructose and glucose would replace 95%
sucrose in media containing 1% A.C., the osmo-
larity would increase as a function of fructose
and glucose formation, and the pH drop, mainly
due to specific fructose reaction, would lead to
lowering the rigidity of the medium gel.
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