Plant Cell, Tissue and Organ Culture 31: 81-86, 1992.

( ~ 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Effects of autoclave-induced carbohydrate hydrolysis on, the growth of

Beta vulgar& cells in suspension

Heidi Sawyer & Ke-Cheng Hsiao*

Research and Development, Hillesh6g AB, Box 302, S-261 23 Landskrona, Sweden (*requests for
offprints)
Received 26 September 1991; accepted in revised form 31 March 1992

Key words: autoclaving, Beta vulgaris, carbohydrate hydrolysis, cell suspension, cyanide, FeNa-EDTA,
medium toxicity, oxygen consumption

Abstract

Media of de Greef & Jacobs (1979) were autoclaved either with all the nutrient components in a single
vessel (medium 1) or with the following components in separate vessels: FeNa-EDTA + CaCI 2 (medium
2), F e N a - E D T A + N a H E P O 4 (medium 3) or sucrose (medium 4). Medium 5 was prepared by
autoclaving FeNa-EDTA + NaH2PO 4 and sucrose in two separate vessels. It was found that the dry
mass yield of cell suspensions of Beta vulgaris was lowest in medium 1, followed by media 2 and 3.
There was no significant difference among media 3, 4, and 5.
The plot of dry mass yield of the cell suspensions against the rates of cyanide-initiated oxygen
consumption which indicate the extent of carbohydrate hydrolysis of the media during autoclaving,
indicated the presence of a threshold rate of about 17-20nmolml -I min -1. Dry mass yield of the
suspensions decreased rapidly when the rate exceeded this value.
For media with glucose as the source of carbohydrate, the rate of cyanide-initiated oxygen
consumption exceeded the threshold value by a factor of 1.5 to 2, depending on the volume of the
media autoclaved.

Abbreviations: F e N a - E D T A - ferric monosodium ethylenediamine-tetraacetic acid

Introduction toxic (Moye 1964; Weatherhead et al. 1978).

Sterilization by autoclaving is still widely prac-
tised, especially for relative large-scale prepara-
tions which need to be carried out routinely.
Breakdown of medium constituents or reaction
among different constituents are inevitable in
this process (van Bragt et al. 1971). A variety of
products are formed when carbohydrates are
subjected to high temperature treatment (Thean-
d e r & Nelson 1989, review). Some of these
products have been identified using fructose as a
model carbohydrate (Shaw et al. 1967, 1968).
The main component is 5-(hydroxymethyl)-2-
furaldehyde which is known to be biologically
Other toxic compounds derived from monomeric
saccharides are phenolics, a group of compounds
that are readily detected in solutions of glucose
or fructose heated in conditions similar to those
employed for the sterilization of nutrient media
(Suortti 1983).
It has been demonstrated that the growth of
unicellular entities such as protoplasts (Barbier
& Bessis, 1987), pollen (de Lange 1989) or
microorganisms (Suortti 1983; Einarsson et al.
1983) are particularly sensitive to the products of
sugar hydrolysis. At tissue or organ levels the
adverse effects of these products were also
noticed, for example, in the regeneration fre-
82
quency of callus or anther culture (Negrutiu &
Jacobs 1978; Weatherhead et al. 1978). The ex-
tent of autoclaved -induced carbohydrate break-
down can be estimated by an oxygen electrode
(Hsiao & Bornman 1991a). This method is based
on the reaction of certain degradation products
with cyanide, subsequently leading to oxygen
consumption. These reactions can also be fol-
lowed spectrophotometrically (Hsiao & Born-
man 1991b).
In our previous investigation (Hsiao & Born-
man 1991b; Schenk et al. 1991), it was demon-
strated that F e N a - E D T A , besides forming pre-
cipitates with certain micronutrients in MS
medium (Murashige & Skoog 1962), catalyses
the breakdown of sugars. It was therefore sug-
gested that the medium should be autoclaved in
two parts: F e N a - E D T A plus KH2PO 4 and the
remaining macro- and micronutrient elements
together with sugars. This procedure also has the
advantage of preventing the formation of pre-
cipitates between CaC12 and KH2PO 4. In this
paper the effects of sugar hydrolysis on the
growth of sugarbeet cells in suspension were
investigated. In addition, the utility aspect of
using the rate of cyanide-initiated oxygen con-
sumption for gauging medium toxicity was fur-
ther examined.
Materials and methods
Cultures
Cell suspensions originating from callus of Beta
vulgar& leaf discs were maintained in liquid
medium (PG0) of de G r e e f & Jacobs (1979)
supplemented with 0.44 IxM 2,4-dichloro-
phenoxyacetic acid and 0.23 IxM benzyladenine.
The suspensions, on an orbital shaker, were
maintained at 20-+ 2°C under 16-h photoperiod
with a photon flux density of 10-15 p.mol m 2 s-1
over the wavelength band 400-700 nm provided
by fluorescent tubes (Osram L36 W/77).
Preparation of culture media
P G 0 media containing 0.08 M sucrose were auto-
claved either with all the components in the
same vessels or with different components in two
to three vessels (Table 1). In one investigation
with alternative carbohydrate source, glucose
( 0 . 1 4 M ) was autoclaved separately in volumes
of 0.1 to 21 and combined with the remaining
nutrient components to give a final concentration
of 0.11 M. Autoclaving was carried out at 121°C
for 20min. Media for experiments 1 and 2
(Table 1) were of separate preparations. After
autoclaving and combining with the necessary
components, the media were dispensed 100ml
per flask (250 ml) and inoculated with 1 to 2 ml
aliquots of cell suspension. For constructing
growth curves, the cultures were initiated with
very low cell mass (approximately 0.1 mg dry
weight per ml) in order to better investigate the
time needed for the cultures to establish an
active growth.
Growth analysis
Dry mass yield was determined by measuring the
absorbance of the cell suspensions at 560nm
where the cells have the absorption minimum.
Dry mass was then calculated from the linear
equation y = 0.041 + 1.88x, where y is the dry
weight in mg ml i, x is the absorbance (Fig. 1).
Measurements were made with a Milton Roy
y = 0.041 * 1.88x
r = 0.998
/
//
/
.#
>,
8
/
0~ i i i
0 0.5 1 1.5
Absorbance (560 nm)
Fig. 1. Calibration curve for estimating dry mass yield of
suspension cultures of Beta vulgaris. Each point represents
the average of 3 dry mass measurements. Equation in the
figure is from linear regression.

Spectronic 3000 diode array spectrophotometer
(Rochester, NY, USA). For experiments 1 and 2
(Table 1) the suspensions were sampled at day
10 and day 14, respectively, after the initiation of
the cultures.
Measurements of cyanide-initiated oxygen
consumption
Protocol for polarographic measurements was
adopted from Hsiao & Bornman (1989) with the
following modifications: media were diluted 1.6
times with phosphate buffer (0.5 M, pH 8.0)
after autoclaving, and KCN (0.05 M) dissolved
in water was added to a final concentration of
0.15mM. The total assay volume was 8001xl.
Oxygen consumption was monitored at 25°C
with a Hansatech electrode (Norfolk, England).
Initial rates of the reaction were estimated from
the traces on the chart recorder.
Results
The results of different procedures of autoclav-
ing the medium on the growth of cells are pre-
sented in Table 1. The lowest dry mass yield was
Table 1. Dry mass yield of suspension cultures of Beta vulgaris and the rates of cyanide-initiated oxygen consumption of the
media autoclaved either with all nutrient components in a single or with different components in separate vessels, A, B and C.
Data are given as mean ± s . o . (n = 4).
Media
1. PG o (complete) xl*
2. A PG o (minus F e N a - E D T A x1.18
and CaCI2)
B F e N a - E D T A + CaC12 x6.67
3. A PG o (minus F e N a - E D T A x 1.18
and NaH2PO4)
B F e N a - E D T A + NaHzPO 4 x6.67
4. A PG o (minus sucrose) xl.25
B sucrose x5
5. A PG o (minus F e N a - E D T A , xl.54
NaH2PO 4 and sucrose)
B F e N a - E D T A + NaHzPO 4 x6.67
C sucrose x5
*, Multiplication factors for the concentrations due to changes in volumes.
o,, Measurements carried out at 10 and 14 days for Experiments 1 and 2, respectively.
83
from suspensions maintained in the medium
autoclaved with all the components in a single
vessel (medium 1), followed by the media in
which FeNa-EDTA+CaC1 z (medium 2) or
FeNa-EDTA + NaH2PO 4 (medium 3) was auto-
claved separately. For this three media, the dry
mass yields were inversely proportional to the
rates of cyanide-initiated oxygen consumption
(Table 1). There was no significant difference in
dry mass yields among medium 3, medium 4
(sucrose autoclaved separately) and medium 5
(sucrose and FeNa-EDTA + NaH2PO 4 auto-
claved separately), even though the rates of oxy-
gen consumption differed. The large differences
in the measurements of the rates of oxygen
consumption between experiments 1 and 2 was
due to the difference in the duration the solu-
tions were left in the autoclave after the steriliza-
tion cycle was terminated.
In a separate experiment the growth curves of
the suspension cultures maintained in media 1
and 2 was compared (Fig. 2). Cultures main-
tained in medium 2 had approximately a six-day
headstart in reaching an active growth phase.
The magnitude of dry mass yield in these results
also testified that the suspensions sampled for
dry weight determination (Table 1) were in their
Dry weight ~, mg ml- 1 Rate, nmol ml 1 m i n - 1
Exp. 1 Exp. 2 Exp. 1 Exp. 2
1.1 ± 0.3 5.4 ± 0.3 29.4±3.1 30.4±1.3
1.6 ± 0.1 7.4 ± 0.5 23.4±1.9 20.5±3.2
2.4 ± 0.3 8.1 ± 0.5 20.0±2.6 17.5±2.3
2.5 ± 0.6 8.2 ± 0.3 14.8±0.6 9.0±2.7
2.4±0.2 8.0±0.1
84

16 4 30

12
~'--~ 3 ~I + 27.5 .~O
lo •

J:
._~ 2 25 o

>,

1 22.5

0a0- ~_---I~I-----~
6 10
,
15
,
20 25 0 I I I I 20
Time, days 0.5 1 1.5 2 2.5
Volume, I
Fig. 2. Comparison of dry mass yield on cell suspensions of
Beta vulgaris grown in media autoclaved with all components Fig. 4. Effects of volumes of the glucose solutions autoclaved
in single vessel (A) or with FeNa-EDTA plus CaC12 in a on the dry mass yield (&) of cell suspensions of Beta vulgaris
separate vessel (O). and on cyanide-initiated oxygen consumption of the media
(0). Glucose (0.14 M) of different volumes were autoclaved
and combined with the remaining nutrient components to
yield media containing 0.11 M glucose. Error bars indicate
s,D. of 4 measurements.
3 10
e x p o n e n t i a l o r l i n e a r cell g r o w t h p h a s e s . T h e
p l o t o f t h e d a t a p r e s e n t e d in T a b l e 1 s h o w e d t h a t
2.5 t h e d r y m a s s yield d e c r e a s e d r a p i d l y w h e n t h e
rate of oxygen consumption exceeded about 17-
20 n m o l m l - 1 m i n 1 (Fig. 3). T h e s e results indi-
-- 2
cate the existence of a threshold value of the
~ 2 1 6 e x t e n t c a r b o h y d r a t e h y d r o l y s i s for the o p t i m a l
J~ g r o w t h o f t h e cell s u s p e n s i o n .
._~ 1.5
T h e s e n s i t i v i t y o f cells to t h e p r o d u c t s o f car-
~ 4
b o h y d r a t e h y d r o l y s i s in t e r m s of c y a n i d e -
1
i n i t i a t e d o x y g e n c o n s u m p t i o n can b e d e m o n -
s t r a t e d b y m a i n t a i n i n g cells in m e d i a in which
2
0.5 ~ o. g l u c o s e was a u t o c l a v e d s e p a r a t e l y b u t in differ-
e n t v o l u m e s . T h e d r y m a s s yield o f s u s p e n s i o n s
m a i n t a i n e d in t h e m e d i u m with glucose a u t o -
0 ' ....' ' J " ' --0 c l a v e d in 2 1 a l i q u o t with CaC12 + F e N a - E D T A
0 5 10 15 20 25 30 35
Rate, n m o l / m l / m i n in a s e p a r a t e vessel, was a p p r o x i m a t e l y 3 t i m e s
h i g h e r t h a n t h e s a m e m e d i u m a u t o c l a v e d in 0.1 1
Fig. 3. Correlation of the rate of cyanide-induced oxygen
consumption and dry mass yield of suspension cultures of a l i q u o t (Fig. 4). T h e d i f f e r e n c e in t h e r a t e s of
Beta vulgaris maintained in media autoclaved by different cyanide-induced oxygen consumption between
procedures. Numbers in the figure represent the media: t h e s e t w o v o l u m e s is o n l y a b o u t 2 0 % (Fig. 4).
with all the components autoclaved in a single vessel (med-
ium 1); with the following components autoclaved in a separ-
ate vessel- FeNa-EDTA + CaCI 2 (medium 2), FeNa-
EDTA +NaH2PO 4 (medium 3) and sucrose (medium 4); Discussion
with the following components in two separate vessels-
FeNa-EDTA + NaH,_PO4, and sucrose (medium 5). T h e r e s u l t s ( T a b l e 1) d e m o n s t r a t e d t h e a d v e r s e

effects of autoclaving all the nutrient compo-
nents in a single vessel. The advantage of auto-
claving FeNa-EDTA and CaCI 2 together but
separately from the remaining nutrient compo-
nents can be clearly seen from the growth moni-
tored over a 20-day period (Fig. 2).
Besides reducing the extent of sugar hydro-
lysis, autoclaving FeNa-EDTA separately also
prevent the precipitation of certain micronutrient
elements (Schenk et al. 1991). The reason for
autoclaving FeNa-EDTA together with
NaH2PO 4 or CaCI 2 is to prevent the formation
of precipitate between phosphate and calcium. It
turned out in our experiments that the cells of
Beta vulgaris grew better when FeNa-EDTA and
NaH2PO 4 were chosen to be autoclaved together
(Table 1). We have observed that a solution of
fructose in phosphate buffer (pH 7.0) developed
an intensive brown color after autoclaving. This
solution contains components, probably phenols,
that are autoxidizable as revealed by the sponta-
neous oxygen consumption with an oxygen elec-
trode. It is therefore a good practice to separate
FeNa-EDTA together with phosphate instead of
calcium from carbohydrates. The present results
also demonstrated that there is much to gain by
just autoclaving sucrose separately.
The changes in sucrose concentration due to
volume changes does not affect appreciably the
extent of its hydrolysis (Schenk et al. 1991).
Assuming, in addition, that precipitation is not a
factor to be considered due to the brief duration
of the trials, the variables in Table 1 can then be
plotted to reveal their relation. The results indi-
cated the presence of a threshold value at about
17-20nmolml ~min -1, above which the dry
mass yield decreased rapidly (Fig. 3).
We have observed that when glucose was used
as the carbohydrate source, cells maintained in
the medium autoclaved with all the nutrient
components in single vessel, gradually lost their
chlorophyll and ceased to grow. In media pre-
pared with glucose autoclaved separately but in
different volumes, the slight difference in the
rate of oxygen consumption resulted in large
differences in the dry mass yield of the cell
suspensions (Fig. 4). Solutions autoclaved in
smaller volumes were subjected to longer expo-
sure time to heat (Burger 1988), and the extent
of hydrolysis of carbohydrate is consequently
85
higher than similar solutions autoclaved in larger
volumes. Glucose is hydrolyzed at a rate about
5 times higher than that of sucrose (Hsiao &
Bornman 1991a). Media containing glucose auto-
claved separately from FeNa-EDTA would still
have an oxygen consumption rate exceeding the
threshold value by a factor of 1.5 to 2 (Fig. 4).
Such media would be inadequate for sustaining
optimal growth and development of cultures
which are sensitive to the products of carbohy-
drate hydrolysis.
It was suggested that the rate of cyanide-
induced oxygen consumption can be used to
indicate the toxicity of culture media (Hsiao &
Bornman 1991a). The results of present investi-
gation indicate that there is a good correlation
between the rate and medium toxicity (Fig. 4).
This method can be used, for example, to select
a convenient autoclaving procedure suitable for
particular types of tissues or cells. For the auto-
clave settings commonly used for sterilizing
growth media, i.e. 121°C and 20min, media
autoclaved in smaller aliquots are more toxic
than the same medium autoclaved in bigger
aliquots (Fig. 4). It is therefore advisable to
reduce the exposure time to high temperature or
to increase the volume of the media in order to
keep the sugar hydrolysis within a reasonable
value. However, for obtaining normal growth of
cultures of B. vulgaris maintained in a medium
containing glucose, one needs to sterilize the
medium by filtration.
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