Malus tissue cultures. I.

Sorbitol (D-glucitol) as a carbon source for

callus initiation and growth1
CALVIN
CHONGAND C. D. TAPER
Dc,partnletrt o f Horticultrrre, Facrrlty of Agricrrltrrre of McGill Urriversity,
Mncdot~aldCollege, Morrtreal, Qrre.
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Received January 10, 1972

CHOSG,C., and C. D. TAPER. 1972. Mnlrrs tissue cultures. I. Sorbitol (D-glucitol)as a carbon source for
callus initiation and growth. Can. J. Bot. 50: 1399-1404.
Callus cultures from 1-year-old twigs of the apple, Mnlrrs prm~ila,c~~ltivars McIntosh, Cortland, and
Red Delicious, and of the crabapple rootstock, Mnlus robruta No. 5, were successfully isolated and
maintained on a medium with sorbitol as the sole carbon source. Investigation with various carbon
sources, each at 3% concentration, showed that McIntosh callus grew equally well on sorbitol, sucrose,
and glucose. Cortland and Robusta cultures responded equally to sorbitol and glucose but poorly to
sucrose. The relative growths of the callus cultures on sorbitol were in the decreasing order, McIntosh,
Cortland, and Robusta. Sorbitol served as an excellent carbon source for all three cultures.

CHONG,C., et C. D. TAPER. 1972. Mnlrrs tissue cultures. I. Sorbitol (D-glucitol)as a carbon source for
callus initiation and growth. Can. J. Bot. 50: 1399-1404.
Nous avons isole avec succes des cals, formts en culture de tissus A partir de jeunes rameaux de 1 a n
de pommier, Malrrs prrnlila, variktes McIntosh, Cortland et Red Delicious et de pommetier, Malrrs
robrrsrn No. 5. Nous conservions pour utilisation substquente, ces cals sur un milieu de c ~ ~ l t u rdont
e
I'unique source d'hydrate de carbone etait le sorbitol. Nous avons observe que les cals de McIntosh
croissent aussi bien dans des milieux de culture contenant comnie source d'hydrate de carbone du
sorbitol, du sucrose ou du glucose, chacun a une concentration de 3% Les cals des varietks Cortland et
Robusta se sont dCveloppes aussi bien avcc le sorbitol qu'avec le glucose, mais peu avec le sucrose. Avec le
For personal use only.

sorbitol la variCtC Robusta avait le taux de croissance le plus rapide, suivie par la variCte Cortland, puis
McIntosh. Le sorbitol, dans les trois cas, etait une excellente source d'hydrate de carbone.

Introduction report describes the isolation and growth of

Sorbitol (D-glucitol) is an important carbo-
hydrate constituent in plants of the Rosaceae.
In the apple, sorbitol functions as a major
translocate (2, 11) and metabolite of photo-
synthesis (5) and is found in all tissues of the
plant (4, 31). Other fi~nctions of sorbitol as
intermediate metabolite (15), respiratory sub-
strate (8), and storage compound (28, 30, 31)
and its ~ossibleassociation with frost hardiness
(27) an2 various fruit (7, 8) and plant disorders
(3, 24) have been reported. This unique in-
volvement of sorbitol in the carbohydrate
metabolisn~ of the apple and related species
requires fiirther clarification.
The use of energy sources by tissue cultures
offers certain opportunities for studies of the
metabolism of such compounds that is not pos-
sible with intact plants (12). In this laboratory,
the tissue culture technique is being used as an
alternative approach for the study of sorbitol
and carbohydrate metabolism in the apple. This
'Contribution from Department of Horticulture,
Faculty of Agriculture of McGill University, Macdonald
College, Quebec, Canada.
various apple callus cultures on a nutrient
medium with sorbitol as the sole carbon source,
and, for the first time, indicates that sorbitol can
be used as an excellent energy source for higher
plant tissue culture. The relative growth of
various apple callus cultures on media with
sorbitol, sucrose, and glucose is also reported.
Materials and Methods
Origin arrd Initiatiorz of Callrrs Cultrrres
Stem callus cultures were derived from the apple,
Mahrs pumila, cultivars, McIntosh, Cortland, and Red
Delicious, and from the crabapple rootstock, Malus
robrrsta No. 5. One-year-old twigs, taken in the spring
while buds were still dormant and (or) just opening, were
surface-sterilized by soaking in 95% ethanol for 3 min
followed by flaming. The outer bark was aseptically re-
moved with a scalpel and internodal explants (0.5-1 cm)
placed on agar medium described below. Callus tissues
produced by the cambial area were excised and sub-
cultured.
Two callus c~llturesof McIntosh, MI and M2, have
been kept for routine investigations. The MI callus (Fig.
2), initiated in April 1970, under diffused light at room
temperature in the laboratory of Dr. R. K. Ibrahim, was
grown on Murashige and Skoog's medium (19) solidified
with 0.8% Difco Bacto agar and supplemented with
10% coconut milk, O.lyo casein hydrolysate, and 3%
 1400 CANADIAN JOURNAL OF BOTANY. VOL. 50, 1972

sucrose as the carbon source. Naphthalene acetic acid
and kinetin were included at 2.0 and 0.2 niglliter, re-
spectively, as recommended for apple s t c n ~callus cultures
by Messer and Lavee (18).
Preliminary investigations during the second and third
subcultures showed that coconut milk and (or) casein
hydrolysate did not increase M1 callus growth and that
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sucrose, and the agar concentration reduced to 0.55%.
All nutrient media were adjusted with NaOH to pH 5.6-
5.8 before the addition of agar, and autoclaved for 2 0
min at 15 psi, after being dispensed into individual
containers. Double-glass-distilled water was used in all
media formulations.
Tn April 1971, the second Mclntosh callus culture

3% sorbitol effectively replaced 3% sucrose as the carbon
source under various experimental conditions. Since then,
coconut milk and casein hydrolysate were excluded from
the 'standard' nutrient niedium, sorbitol substituted for
For personal use only.
(M2) and also callus cultures of Cortland (C). Red
Delicious (RD), and Robusta (R) (Fig. 1) were initiated
and grown on 'standard' sorbitol medium, under the
culture conditions described below.

ABBREVIATIONS: M I , McIntosh, culture 1; MZ, McIntosh, culture 2; C, Cortland; R; Robusta; RD, Red
Delicious; So, sorbitol; Su, sucrose; G. glucose.
FIG. 1. Five-week-old apple stem callus stock c ~ ~ l t u r originally
es initiated on sorbitol medium and main-
tained on similar niediuni; each inoculuni weighed about 150 mg. FIG. 2. TWOrepresentative replications
of 4-week-old apple callus c ~ ~ l t ~grown
~ r e s on different carbon sources (3y0); each inoculum weighed about
50 mg.
 CHONG A N D TAPER: MALUS TISSUE CULTURES 1401

Culture arrd Experitnerlral Cor~dirions
All stock and experimental cultures, except as stated
for the M1 culture, were initiated and maintained in this
laboratory under continuous cool white fluorescent
illumination (250-300 ft-c) at 27-2g°C. The M1 culture
was grown during the fifth and sixth subcult~~res under
diffused light at room temperature in the laboratory of
Dr. L. E. Powell. Stocks were subcultured at 4- to 5-
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taken only from the peripheral region of stock tissues
grown in the same container. Replications with con-
taminated bottle(s) were discarded (Table 1). At the end
of the growth period, each callus was weighed and its
dry weight determined. Analysis of variance (Table 1)
and Duncan's multiple-range test (Table 2) were per-
formed on the fresh weight values. The data are ex-
pressed in ternis of both fresh weight and percentage dry

week intervals in 125-ml Erlenmeyer flasks or in hori- weight.

zontally placed 8-oz prescription bottles, each contain-
ing 50 ml of 'standard' sorbitol niedium planted with 3
or 4 callus pieces (ca. 150 mg each).
The relative growth of McIntosh (MI), Cortiand (C).
and Robusta (R) callus tissues were coniparcd after a
4-week growth period on 'standard' rnediunz contain-
ing different carbon sources, sorbitol, sucrose, and
glucose, each at 3% concentration. In this experiment
(Tables 1, 2; Fig. 2), each individual of a treatment
consisted of one inoculum (ca. 50 mg) transplanted on
20 ml of medium per 4-oz prescription bottle. Bottles
were arranged in a split-plot design with the three callus
cultures as main treatments and the carbon sources as
subtreatments. The inocula for each replication were
TABLE 1
Analysis of variance of fresh weight (mg) in apple
callus cultures grown o n different carbon sources
For personal use only.
Results
The callus cultures of Mclntosh (M1 and M2),
Cortland (C), Robusta (R), and Red Delicious
(RD), initiated and grown under these laboratory
conditions, were markedly different in appear-
ance and growth response to the carbohydrate
regime (Figs. 1, 2; Table 2).
Descriptio~iof' Callus Cultures
The typical Mclntosh callus was relatively
rapid-growing on 'standard' sorbitol medium,
generally knobby and irregularly shaped, hard,
and green in color, although if grown in the
dark it lost its chlorophyll; if regrown in the
light it regained its typical green color. So far,
there appears to be no difference in appearance,

Source of variation D.F. Mean square F vigor, and (or) response to the carbohydrate
Callus cultures1' 2 1 457 832.0 51 .32c regime between the M1 culture (Fig. 2), which
Replicationsb 19 48647.6 1.71 has been initiated and maintained through 2
Error (a) 38 28 409.3 subcultures on the original sucrose medium
Carbon sources 2 589 776.0 36.2@ and 15 on 'standard' sorbitol medium, and the
Callus culture X
carbon source 4 101 584.0 6.251: M2 culture (Fig. l), which has been initiated
Error (b) 114 16 263.3 and maintained through 7 subcultures solely on
aInoculum weight about 50 mg; 4-week culture period. 'standard' sorbitol medium. This appears to be
bOf 24 replications plated, 4 were discarded bccausc o f contamina-
tion. true also of callus siillilarly derived from >year-
significant at P = 0.01.
NOTE: D.F. = degrees of freedom. old Mclntosl~seedlings.

TABLE 2.
Relative gromth of apple callus cultures on different carbon soixces,
each at 3% concentration

Carbon source
Callus culture Sorbitol Sucrose Glucose Mean
Fresh weight (mg)'
McIntosh (Ml) 638.5 af 586.0 a 583.1 a 602.5 A
Cortland (C) 504.9 a 257.8 b 476.2 a 413.0 B
Robusta (R) 416.5 a 136.8 b 327.1 a 293.5 C
Mean 520.0 A t 326.8 B 462.1 A
Percentage dry weight
McIntosh (Ml)
Cortland (C)
Robusta (R)
Mean
*Inoculum weight about 50 mg; 4-week culture period; average of 20 replications.
?Figures not followed by the same letter (a,b) within a row are significantly different at P = 0.05 and 0.01.
$Figures not followed by the same letter (A-C) within a row or column are significantly different at P = 0.05 and0.01.
 1402 CANADIAN JOURNAL OF
During the first three subcultures, the Cort-
land callus was similar in appearance to that of
McIntosh, although less vigorous (Fig. 1). Un-
like the McIntosh cultures which remained green
at all times, rapid loss of clllorophyll occurred
in the Cortland transfer pieces within several
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days after subculturing, although its green color
was gradually regained after 4 weeks of growth.
However, during the fourth subculture on
'standard' sorbitol medium, the ability of Cort-
land callus to form chlorophyll decreased mark-
edly, and its appearance also changed to that
of a smoother and more regularly shaped callus
(Fig. 2), which tended to be very pale green to
light yellow in color. A particular strain,
originating from this culture before the occur-
rence of the sudden loss of ability for chloro-
phyll formation, has remained typically green
under other circun~stances(Coffin, unpublished).
The Robusta callus (Figs. 1, 2) is typically of
medium vigor, compact, smooth-surfaced, gener-
ally ro~ind to oval, and lighter green than
McIntosh callus. Anthocyanin pigmentation,
For personal use only.
which generally develops during the first 10 days
after subculturing, usually persists under and
(or) around the base of the callus.
The Red Delicio~~s callus (Fig. 1) was very
slow growing, tending to be irregular, dark green,
and very hard. This culture grew at decreasing
rates through five transfers on 'standard'
sorbitol medium before failing. Growth did not
occur on medium supplemented with coconut
milk and (or) on sucrose medium.
Efect of Cnrbotl Sources
Analysis of variance (Table 1) of the callus
fresh weights showed a significant interaction
between callus cult~ireand carbon source. The
McIntosh callus grew equally well on sorbitol,
sucrose, and glucose while the Cortland and
Robusta cultures responded equally to sorbitol
and glucose but poorly to sucrose (Table 2;
Fig. 2). The relative g~owtlisof the callus cultures
on sorbitol were in the following decreasing
order: McIntosli, Cortland, and Robusta (Table
2; Figs. 1, 2). These res~iltsconfirm si~iiilardata
from preliminary experiments on the relative
growth of Robusta and McIntosh (both M 1 and
M2) c ~ ~ l t ~on
~ rthese
e s carbon sources.
Callus fresh weight was inversely related to
percentage dry weight (Table 2). Thus, the mean
percentage dry weight of each callus culture was
BOTANY. VOL. 50, 1972
in the increasing order, McIntosh, Cortland, and
Robusta. Similarly, the lower fresh weights of
Cortland and Robusta on sucrose showed cor-
respondingly greater percentage dry weights.
The ccrrelation coefficient between the fresh
weight of all subtreatments and the correspond-
ing percentage dry weights (Table 2) was highly
significant ( r = -0.928; significant at P =
0.01).
Discussion
The consistently excellent growth of the
various apple callus cultures on sorbitol and
their differential growth response on the other
carbon sources, especially the poor growth
response of both Cortland and Robusta on
sucrose, are particularly striking. Similar to
results of this study, Messer and Lavee (18)
reported consistently higher percentage dry
weight in the more vigorous of two stem callus
cultures of apple varieties. Failure to maintain
Red Delicious callus is apparently due to a
lack of proper nutrition other than the carbon
source. Factors that influence differences in the
growth and pigment formation of callus cultures
apparently are variable, complex, and not well
understood (25). Saad (25) showed that light
intensity, callus source, and media composition
are prime factors which influence the relative
growth of callus cultures of apple varieties and
their ability to develop chlorophyll and other
pigments. For instance, McIntosh leaf callus is
capable of producing abundant chlorophyll on
several kinds of media in comparison to the
production of little or no chlorophyll by Cort-
land leaf, petiole, and stem callus cultures.
Autoclaving results in the breakdown of
various carbohydrates in certain nutrient media.
Chromatography of autoclaved sorbitol medium
has indicated no breakdown of sorbitol. A
similar observation was made by Wolter and
Skoog (32) in the case of autoclaved inannitol
medium. McIntosh callus grew to the saine extent
on filter-sterilized sorbitol niedi~unas on auto-
claved sorbitol medium ; siniilar growth response
was noted 011 filter-sterilized and autoclaved
sucrose and glucose medium.
Previous tissue cultures of apple stem (18, 35,
29), cotyledon (1 3), fruit tissue (14, 25), and bud
(22) have all been grown on sucrose media.
Sucrose is generally regarded as the best carbon
source for most plant tissiie cultures, altho~ighin
 CHONG AND TAPER: MALUS TlSSUE CULTURES
certain cases glucose and other carbohydrates
are equally or more effective (10). Attempts to
use sorbitol (17, 21, 23) and other polyols as
energy sources for tissue cultures have been
mostly unsuccessf~ilalthough the triol, glycerol,
is capable of sustaining adequate growth in
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certain plant tissue cultures (10, 12). Wolter
and Skoog (32) showed that mannitol, which
occurs in significant quantities in Frnxinus
americana (33), is as effective an energy source
as sucrose for the growth of callus cultures of
this species.
Recently Nickell and Maretzki (20) found
sorbitol to sustain 25% as much growth as
sucrose in sugarcane cell suspensions. Other
species of the Gramineae, wheat (1) and oats
(6), are known to metabolize sorbitol. Evidence
from this laboratory indicates that pumpkin
cotyledon can initiate callus on sorbitol medium
(Coffin, unpublished), although sorbitol is not
able to support growth of tobacco and radish
callus cultures. Sorbitol, presumably in very
small quantities, has been reported in the tobacco
For personal use only.
plant (16). The occurrence of sorbitol (15 000
ppm in the neutral fraction) as the largest single
constituent of coconut milk (21) is particularly
noteworthv in view of its wide use as a nutrient
supplement for tissue cultures. Certain carrot
root explants are able to respond in a limited
way to sorbitol, but only in the presence of the
active fraction of coconut milk (21). The pos-
sible role of sorbitol in the nurture of embryos
has been discussed (15). Since coconut milk is
undefined. its inclusion in the nutrient media
would undoubtedly complicate studies on sor-
bit01 and carbohydrate metabolism of apple
callus cultures. Despite the traditional relative
inability of tissue cultures to use sorbitol, these
findings and the increasing evidence for sorbitol
occurrence in diverse plant species (9, 16, 21,
26), other than those of the Rosaceae, suggest
a wider metabolic significance for sorbitol in
higher plants. Further studies may reveal sorbitol
and other polyols to be effective energy sources
in the tissue cultures of many other plant species.
In other investigations various apple cotyledon
and fruit callus cultures have been initiated and
grown on 'standard' sorbitol medium. The
effect of various growth parameters and nutri-
tional requirements of Maltu tissue cultures will
be the subiect of future communications. In all
such investigations sorbitol is the standard
1403
carbon source used both for maintenance of
stock cultures and for routine experimental work.
Clearly, for Malus tissue cultures sorbitol is an
effective and excellent carbon source and is
explainable on the basis of the unique carbo-
hydrate inetabolisrn of the apple.
Acknowledgments
We are grateful to Drs. R. K. Ibrahim, De-
partment of Biological Sciences, Sir George
Williams University, Montreal, Que., and L.
E. Powell, Department of Pomology, Cornell
University, Ithaca, New York, for providing
laboratory facilities and assistance during the
developmental stages of this study. The National
Research Council of Canada is acknowledged
for financial support.
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VOL. 50, 1972
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