ISSN 10683674, Russian Agricultural Sciences, 2013, Vol. 39, No. 2, pp. 112–116. © Allerton Press, Inc., 2013.

PLANT GROWING

Effect of Sugar/Osmotica Levels on in vitro Microtuberization

of Potato (Solanum tuberosum L.)1
Alireza MotallebiAzar, Samaneh Kazemiani, and Fahimeh Yarmohamadi
Department of Horticultural Sciences, Faculty of Agriculture, Tabriz University, Iran
email: motellebiazar@gmail.com

Abstract—In order to evaluate of using microtubers for conservation of potato germplasm, the main effects
of sucrose, mannitol and PEG on percentage of microtuber initiation and formation, size and fresh weight of
microtuber were studied. PEG decreased production and weight of microtubers, whereas higher concentra
tions of sucrose promoted microtuber production, microtuber size as well as microtuber fresh weight.
Increasing of sucrose concentrations were improved efficiently in vitro microtuber production without neg
ative side effects. Addition of mannitol to all media was increased microtuberization than media containing
PEG. So, alcoholic sugars such as mannitol increased microtuberization, but after 5 weeks, microtuberiza
tion in media supplied with sucrose was more than the mannitol. Such studies might help to reduce the cost
of production of virus free potato seed from different cultivars.

Keywords: mannitol, microtuberization, PEG sucrose, Solanum tuberosum

DOI: 10.3103/S1068367413020146

1
1. INTRODUCTION
Microtubers, which are potato tubers produced in
vitro, have many properties that make them ideal
propagules for producing highquality seed potatoes.
Since microtubers are produced in vitro, they usually
stay pathogenfree if started with pathogenfree mate
rials. Compared with the other in vitro potato
propagule, microshoots, microtubers are more robust,
easier to handle, and more amenable to automatic
planting [1]. The environment and medium within the
in vitro growth vessel influences the size of the micro
tubers. Environmental factors such as photoperiod,
temperature, gaseous components and medium com
ponents [2].
One of the most important factors is the carbon
source in the medium; both its type and concentration
have profound effects on tuber growth. Plant cell, tis
sue or organ culture normally requires the incorpora
tion of a carbon source to the culture medium [3, 4].
Several tissue culture reports refer to the influence of
the carbon source on the in vitro morphogenesis of
different plant species. Among the many available car
bon sources, sucrose has been the major one [5]. In
some cases, sucrose is totally or partially replaced by
other carbon source [6, 7]. The nutritional importance
of an adequate carbon source in a culture medium is
widely known. However, the addition of medium
components, especially macronutrients and carbon
sources, represent considerable decrease in the
1 The article is published in the original.
medium osmotic potential [3]. In spite of the nutri
tional and osmotic aspects of the carbon source, dis
cussions in the literature are frequently restricted to
the nutritional aspects of the carbon source. Since the
osmotic component strongly influences plant cell, tis
sue and organ growth and morphogenesis [7], discus
sion of the results these papers are sometimes unclear
or improperly interpreted. The substitution of the cul
ture medium carbon source by osmotically active sol
utes has shown that sugars act as a carbon source and
as osmotic regulators. In this case, the most frequently
used solutes are the two alcoholic sugars mannitol and
sorbitol [3], which are permeating solutes unusually
metabolized by plants, and long chain nonpermeating
solutes, such as polyethylene glycol (PEG) [3, 6].
Mannitol was used with success in studies of the
osmotic potential of induction medium to obtain
somatic embryogenesis in sunflower [8] and spindle
tree [9]. Several authors reported the beneficial effect
of PEG supplementation on Pinus somatic embryo
maturation [6].
We reported the effect of different concentrations
of PEG, mannitol and sucrose on formation, size and
weight of microtubers in potato cv. Agria. The protocol
shall help to reduce the cost of microtuber production
for potato cv. Agria.
2. MATERIALS AND METHODS
In this experiment for study in vitro shoot multipli
cation and microtuberization, Agria cultivar (Solanum

112
 EFFECT OF SUGAR/OSMOTICA LEVELS 113

Mannitol PEG Sucrose

120

Microtuber initiation, %
100

80

60

40

20

0 0.05 0.10 0.15 0.20 0.25

Concentrations, mol L–1

Fig. 1. Percentage of microtuber initiation in different concentrations of PEG, sucrose and mannitol.

90 PEG Mannitol Sucrose

80
Microtuber formation, %

70
60
50
40
30
20
10
0
0 0.05 0.10 0.15 0.20 0.25
Concentrations, mol L–1

Fig. 2. Percentage of microtuber formation in different concentrations of PEG, sucrose and mannitol.

tuberosum L.) was used. These were made disease free
following the standard meristem culture. Diseasefree
plantlets were routinely subcultured every 4 to 5 weeks
by placing 5 single node cuttings in each culture jar.
The culture was maintained at 25 ± 2°C under 16 h
photoperiod. Each jar contained 30 mL of semisolid
MS medium (pH 5.8) supplemented with 30 g L–1
sucrose and 8 g L–1 agar.
Single node stem segments of Solanum tuberosum
L. cv. Agria were isolated from in vitro plantlets cul
tured on MS medium with 80 g L–1 sucrose containing
different concentrations of sucrose, mannitol and
PEG (0.0, 0.05, 0.11, 0.17 and 0.23 mol L–1) and 8 g L–1
agar. Cultures were maintained in dark at 18 ± 2°C, for
five weeks. Five weeks later, cultures were evaluated for
microtuberization. Records of data after 5 week’s
incubation, percentage of initiation and formation
microtubers, microtuber fresh weight (mg), microtu
ber length and diameter (mm) were done. Statistical
analysis was done by SPSS software ver. 16 and com
parison of means was carried out by Duncan’s Multi
ple Range Tests at 5% probably level.
3. RESULTS AND DISCUSSION
All of the treatments employed induced the forma
tion of microtubers, but in some treatments, did not
develop any microtubers. So, axillary buds were grown
and shoot elongated (0.5–10 cm). Analysis of variance
revealed significant differences among sucrose than
mannitol and PEG. Comparison of means for treat
ments showed that sucrose was better for microtuber
initiation and formation (85.8 and 56.4% respec
tively), microtuber size and fresh weight were
increased when nodal explants cultured in media con
taining different concentrations of sucrose.
Maximum percentage of microtuber initiation
(85.8%) was observed in sucrose treatments, but per

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114 MOTALLEBIAZAR et al.

6 PEG Mannitol Sucrose

Microtuber length, mm
5
4
3
2
1

0 0.05 0.10 0.15 0.20 0.25

Concentrations, mol L–1

Fig. 3. Mean of microtuber length in different concentrations of PEG sucrose and mannitol.

Mannitol PEG Sucrose

4.0
Microtuber diameter, mm

3.5
3.0
2.5
2.0
1.5
1.0
0.5

0 0.05 0.10 0.15 0.20 0.25

Concentrations, mol L–1

Fig. 4. Mean of microtuber diameter in different concentrations of PEG, sucrose and mannitol.

centage of microtuber initiation was decreased in
0.23 mol L–1 sucrose. Because osmotic pressure
increased in medium containing 0.23 mol L–1 sucrose.
While significantly less percentage of microtuber initi
ation was depicted by mannitol and PEG. Percentage of
microtuber initiation was decreased in media contain
ing higher concentrations of PEG (over 0.05 mol L–1).
So, PEG was decreased osmotic pressure in microtu
berization medium and created stress for nodal
explants. The microtuber initiation percentage was
increased by adding mannitol to 0.11 mol L–1 and then
decreased (Fig. 1). These observations are highly sup
ported by Gamer and Blake [10] who worked on
microtuberization in high sucrose concentrations and
obtained similar trend.
Maximum percentage of microtuber formation was
observed in high levels of sucrose (0.05–0.17 mol L–1).
Minimum percentage of microtuber formation was
found in different concentrations of PEG. The micro
tuber formation percentage was decreased by using
high concentrations of PEG in medium. The microtu
ber formation percentage was increased when explants
were cultured in media containing 0.0, 0.05 and
0.11 mol L–1 mannitol. Addition of mannitol to all
media was increased percentage of microtuber forma
tion than media containing PEG. So, alcohol sugars
such as mannitol increased microtuberization, but
after 5 weeks, microtuberization in media supplied
with sucrose was more than the mannitol (Fig. 2). The
results are in line with the findings of Kubo et al. [11]
showed that in zanthedeschia, microtuberization
decreased in higher concentrations of mannitol (high
osmotic pressure).
Length and diameter of microtuber was signifi
cantly more in media containing higher concentra
tions of sucrose. Microtuber length and diameter was
increased in 0.05 mol L–1 PEG and then decreased by
adding PEG concentrations in medium (Figs. 3 and 4).
Microtuber length and diameter was increased by add
ing mannitol from 0.0 to 0.11 mol L–1 and then micro
tuber length decreased in 0.17 and 0.23 mol L–1 man
nitol. Diameter of microtuber in media containing
mannitol was significantly less than medium that
included PEG. Its main reason may be that in media

RUSSIAN AGRICULTURAL SCIENCES Vol. 39 No. 2 2013

 EFFECT OF SUGAR/OSMOTICA LEVELS
90 PEG
80
Microtuber weight, mg
70
60
50
40
30
20
10
0 0.05
Fig. 5. Mean of microtuber fresh weight in different concentrations of PEG, sucrose and mannitol.
that included mannitol, mannitol concentration was
critical. Because length microtuber in media contain
ing mannitol was significantly more than medium that
included PEG.
Maximum Fresh weight of microtuber was given by
high levels of sucrose. Fresh weight of microtuber was
significantly decreased in 0.23 mol L–1 sucrose. Its
main reason may be that osmotic pressure increased in
media containing 0.23 mol L–1 sucrose and its increas
ing leads to slower tuberization. Fresh weight of
microtuber was decreased by adding PEG. In medium
containing mannitol, fresh weight was significantly
decreased with adding mannitol over 0.11 mol L–1
(Fig. 5).
Our findings with regard to the effect of carbon
source on microtuberization agree with those of Kubo
et al. [11], Gamer and Blake [10] who also found bet
ter microtuber production when in vitro nodal
explants were cultured in medium containing appro
priate concentration of carbon source. Also it is a well
established fact that under in vitro conditions tuber
ization in whole nodal explants is hastened in high
concentration sucrose or high osmotic pressure [10].
The desirable effect of high concentrations carbon
source on microtuberization, as obtained in the
present study, was also reported by other researcher.
Lower microtuber weight under continuous darkness
in the present study may be attributed to etiolation.
These research present results relived that the increase
in microtuberization upon supplementing the MS
medium with high sucrose level following 45 days of
culture on medium containing mannitol and PEG was
mainly due to an increase in osmotic pressure as there
was little change in microtuberization process under
different carbon source. Our results showed that the
promotion of microtuberization was more pro
nounced in sucrose treatment than mannitol confirms
RUSSIAN AGRICULTURAL SCIENCES
115
Mannitol Sucrose
0.10 0.15 0.20 0.25
Concentrations, mol L–1
the findings of Kubo et al. [11]. Also confirm the find
ings of Gamer and Blake [10] who reported that
microtubers could be induced even without the use of
growth regulators. Increasing of sucrose concentra
tions were improved efficiently in vitro microtuber
production without negative side effects. In this
research microtuberization was also faster in sucrose
treatment and was completed within 2 to 3 weeks after
culture initiation in comparison to the 4 to 5 weeks
taken by treatments with mannitol and PEG, respec
tively.
CONCLUSIONS
In conclusion, it can be said that microtuberization
can be induced in potato on medium containing dif
ferent concentrations of sucrose, mannitol and PEG.
An appropriate concentration of sucrose, mannitol or
PEG gives a wide morphogenesis response, high
microtuber weight with more percentage of microtu
ber formation. So, our results indicate the need of
developing specific protocols to maximize microtu
berization.
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RUSSIAN AGRICULTURAL SCIENCES Vol. 39 No. 2 2013