In Vitro Cellular & Developmental Biology - Plant

https://doi.org/10.1007/s11627-018-09950-6

MICROPROPAGATION

Acid pretreatment improves microtuberization of potato plantlets

Yue Teng 1 & Yan Zhang 2 & Jin Ting Guo 1 & Yu Liang Gao 3 & Kui Hua Li 1

Received: 2 January 2018 / Accepted: 3 December 2018 / Editor: Marco Buenrostro-Nava

# The Society for In Vitro Biology 2018

Abstract
To efficiently produce potato (Solanum tuberosum L.) microtubers, in vitro plantlets were pretreated with an acid solution before
culturing in Murashige and Skoog medium supplemented with 60 g L−1 sucrose. The first tubers were formed at 36 h of acid
pretreatment (AT), which was 6.2 d earlier than the untreated control. The maximum number of tubers (3.2), and highest fresh
weight per tuber (282.5 mg) were also achieved with the AT. During microtuberization, the leaf dry weight of the control was
higher than the AT from 1 to 5 d of culture. However, the dry weight of AT leaves increased rapidly after 5 d of culture and
reached the same level as the control. The tuber dry weight in the AT was significantly higher (α = 0.05) than the control during
the same culture period. Sucrose and starch content, and the enzyme activity of sucrose synthase (SS), ADP-glucose pyrophos-
phorylase (AGPase), and soluble starch synthase (SSS) in the AT and control exhibited a similar pattern as culture time
progressed. However, levels of these biochemical indexes statistically differed (α = 0.05) between the AT and control. In addition
to the sucrose content in AT, the starch content and activities of SS, AGPase, and SSS in the leaves were lower, whereas those in
tubers were higher than those in the control. The present study suggests that acid pretreatment can efficiently promote microtuber
formation and growth, which can be used for industrial production of potato seeds.

Keywords Acid stress . Acid treatment time . Microtuber . Carbohydrate metabolism

Introduction thereby restricting the development of potato virus-free tuber

Potato (Solanum tuberosum L.) microtuberization is an impor-
tant step for the production of virus-free seed potato (Hoque
2010; Aksenova et al. 2012). Microtubers are utilized for
minituber (small tubers produced from in vitro–produced
propagules) production in greenhouses or screenhouses, they
are not often planted directly in the field. As breeder seeds, the
microtubers have been widely used in potato cultivation be-
cause of their high efficiency in decontamination, longer stor-
age capability, and continuous production without seasonal
restrictions (Donnelly et al. 2003). However, a relatively low
yield of microtubers has resulted in high production costs,
Yue Teng and Yan Zhang have contributed equally to this work.
* Kui Hua Li
likuihua70@126.com
1
Agricultural College, Yanbian University, Yanji, Jilin 133002, China
2
Jilin City Academy of Agricultural Sciences, Jilin 132000, China
3
Yanbian Agricultural Sciences Academy, Longjing, Jilin 133400,
China
production (Ranalli et al. 1994). During microtuberization, the
microtuber formation and growth of potato crops are affected
by many factors such as the contents of carbohydrate and
exogenous hormones in the culture medium and the culture
temperature or light. For example, Gudeva et al. (2016) deter-
mined that the microtuber formation rate reached up to 86.6%
by increasing the sucrose concentration in Murashige and
Skoog (MS) medium (Murashige and Skoog 1962) from 30
to 90 g L−1. Piao et al. (2004) reported that the addition of 6-
benzylaminopurine to the culture medium enhanced the for-
mation of microtubers larger than 1.1 g. Gopal et al. (1998)
found that a higher yield and greater number of microtubers
were obtained with a short photoperiod (10 h) and lower tem-
peratures (day 20 ± 2 °C and night 18 ± 2 °C). In addition,
studies demonstrated that microtuberization can also be en-
hanced under stress conditions, such as cold, heat, anaerobio-
sis, and salinity (Pumisutapon and Topoonyanont 2017).
Thus, artificially setting a stress environment can be another
way to induce potato tubers.
For optimal growth and development, plants require moder-
ate environmental conditions (Zhang et al. 2005; Puijalon et al.
2007; Lipiec et al. 2013). However, a few researchers have
 TENG ET AL.
indicated that plant growth can be promoted after the plants
suffer stress conditions such as high or low temperature, light,
humidity, and acidity or alkalinity. For example, in vitro rhi-
zome growth of Alstroemeria increased significantly (α = 0.01)
after cold (0°C) and hot (38°C) pretreatments (Pumisutapon
et al. 2012). Moderate water stress promoted the elongation of
primary roots and density of root hair in Arabidopsis (Xu et al.
2013). In addition, studies showed that acid stress exerts a pos-
itive effect on potato tuber induction in a hydroponic system
(Wan et al. 1994; Barnard and Combrink 2004). Nevertheless,
these studies have not been systematically conducted to date.
In most plants, sucrose and starch are the major trans-
port and storage forms of carbohydrate, respectively
(Bánfalv et al. 1999; Miao et al. 2016). During potato
tuberization, the reducing sugar remains in dynamic equi-
librium during the metabolism of starch to sucrose, and
sucrose to starch (Ross and Davies 1992; Halford and
Paul 2003). During this process, sucrose synthase (SS),
ADP-glucose pyrophosphorylase (AGPase), and soluble
starch synthase (SSS) play important roles (Bánfalv
et al. 1996; Sweetlove et al. 1996; Arnd and Tang 1999;
Hendriks et al. 2003). Sucrose moves from the phloem
sieve elements into the storage parenchyma cells and is
converted to uridine diphosphate (UDP) glucose (UDPG)
and fructose in a UDP-dependent reaction that is cata-
lyzed by SS (Niek et al. 1997; Ruan et al. 2010). The
product of sucrose decomposition is catalyzed by
AGPase to adenosine diphosphate glucose (ADPG) to fur-
ther starch synthesis under SSS catalysis. The conversion
of sucrose to starch and starch to sucrose in plant tissues
has been studied for many years (Edwards et al. 1999;
Geigenberger et al. 2004; Baroja-Fernández et al. 2009).
However, dynamic changes of sucrose and starch and re-
lated metabolic enzymes in stress-pretreated plantlets have
not been studied to date.
To understand the effect of acid stress on potato
microtuberization, the present study pretreated in vitro
plantlets with a low acid solution for a short time before
microtuber induction and investigated tuber formation and
growth. Furthermore, the changes in the sucrose and starch
content and the activities of enzymes related to carbohy-
drate metabolism in leaves and microtubers of acid-
pretreated and unpretreated plantlets were identified to clar-
ify the mechanism of carbohydrate metabolism during po-
tato microtuberization.
Materials and Methods
Plant materials Virus-free plantlets of potato cv. Yanshu 3
were provided by the Yanbian Academy of Agricultural
Sciences, Jilin Province, China. The in vitro plantlets were
cut into 1 cm length segments (with a node) and inoculated
in a 6 × 9 cm flasks containing 20 mL MS medium supple-
mented with 20 g L−1 sucrose (Kermel Chemical reagent
Co., Ltd., Tianjin, China) and 8 g L−1 type A agar (Kermel
Chemical reagent Co., Ltd.). The pH of the culture medium
was adjusted to 5.7 by 1 M NaOH prior to autoclaving, and
the medium was autoclaved for 20 min at 121°C and
130 kPa. The flasks were maintained at 25°C with a 16 h
photoperiod under white fluorescent light (Opple Lighting
Electronic Co., Ltd., Zhongshan, China) of
30 μmol m−2 s−1 intensity. After 50 d of culture, the rooted
plantlets were used as the experimental material.
Effect of acid treatment on microtuber formation and
growth The 50-d-old whole plantlets were soaked in an acid
solution and treated for 0 (control), 12, 24, 36, 48, 60, and
72 h. The acid solution was derived from sterilized water and
adjusted to pH 4 with phosphoric acid (Kermel Chemical re-
agent Co., Ltd.). After acid treatment, the plantlets were trans-
ferred to a 6 × 9 cm flasks containing 20 mL MS medium
supplemented with 60 g L−1 sucrose and statically cultured
at 20°C in the dark. After 20 d of culture, the number of days
for microtuber formation, number of tubers, and tuber fresh
weight were scored.
Changes in sucrose and starch content and SS, AGPase,
and SSS activity The 50-d-old in vitro plantlets were
pretreated with a pH 4 acid solution for 36 h (AT), and then
cultured on MS medium supplemented with 60 g L−1 sucrose
for various days at 20°C in the dark. The plantlets not pretreated
with acid solution served as the control. The leaves and
microtubers from plantlets cultured for 1, 5, 10, 15, or 20 d
were collected, and their dry weight, sucrose and starch
content, and SS, AGPase, and SSS activity were determined.
Determination of sucrose and starch content The sucrose
content was analyzed by the resorcinol method of Wang et al.
(2015) with some modifications. Ground samples of 50 mg dry
weight were extracted two times using 4 mL of 80% (v/v)
boiling ethanol for 40 min. The supernatant and precipitate
were collected separately after centrifugation at 4000×g
(Z-36HK; Hermle Labortechnik GmbH, Wehingen, Germany)
for 15 min at 4°C. The supernatant was decolorized with 10 mg
of activated charcoal at 80°C for 30 min, adjusted to a constant
volume of 10 mL and filtered through a membrane filter
(0.22 μm). Approximately, 200 μL of 2 mM NaOH (Kermel
Chemical reagent Co., Ltd.) was added to 0.4 mL of the filtrate,
which was boiled at 100 °C for 5 min. Next, 0.8 mL of 0.1% (v/
w) resorcinol (Kermel Chemical reagent Co., Ltd.) was added,
and the reaction mixture was boiled at 80 °C for 10 min. After
cooling, the sucrose content of the reaction mixture was deter-
mined using a UV-2600 spectrophotometer (Shimadzu
Corporation, Kyoto, Japan) at 480 nm.
 POTATO MICROTUBER FORMATION IN VITRO
The total starch content was determined using the HCl
(Kermel Chemical reagent Co., Ltd.) hydrolysis-3,5-
dinitrosalicylic acid (DNS; Kermel Chemical reagent
Co., Ltd.) method as described by Lu et al. (2002) with
some modifications. The precipitate collected in the ex-
periment that measured the sucrose content was used to
determine the starch content. Approximately, 3 mL of 6 M
HCl was added to the precipitate, and the mixture was
incubated in a boiling water bath at 100°C for 40 min
and centrifuged for 5 min at 4000×g (Z-36HK; Hermle
Labortechnik GmbH) after cooling. Next, the supernatant
was collected and 200–400 μL of phenolphthalein indica-
tor (Kermel Chemical reagent Co., Ltd.) was added.
Afterwards, 0.2 mL of 6 M NaOH (Kermel Chemical
reagent Co., Ltd.) was added and diluted with distilled
water to 25 mL. Approximately, 2 mL of the mixture
was added to 1.5 mL DNS and incubated at 80 °C for
10 min. After cooling, distilled water was added to the
reaction mixture for a constant volume of 15 mL and read
using a UV-2600 spectrophotometer at 540 nm (Shimadzu
Corporation). The starch content was calculated using a
glucose standard curve.
Determination of the enzyme activity of SS, AGPase, and
SSS Fresh leaf and tuber samples (0.1 g of each) were homog-
enized separately in 1 mL of extraction buffer derived from
kits for SS, AGPase, and SSS (Nanjing Solarbio Technology
Co. Ltd., Nanjing, China) in a pre-chilled mortar and pestle.
The reagents were added to the homogenate according to the
manufacturer’s instructions. The enzymatic activity of SS was
determined at 480 nm, and the enzymatic activity of SSS and
AGPase were determined at 340 nm using a UV-2600 spec-
trophotometer (Shimadzu Corporation).
Experimental design and data analysis All data are
expressed as means ± standard error (SE) of three experi-
ments. The mean values were subjected to Duncan’s multiple
range test at α = 0.05 and Student’s t test with the statistical
package BSPSS 12.0 for Windows.^
Figure 1 . Microtuber formation
and growth of potato (Solanum
tuberosum L.) after 20 d of
culture. (A) Plantlets were
pretreated with a pH 4 acid
solution for 36 h. (B) Plantlets were
not pretreated with acid solution.
Results and Discussion
Effect of acid treatment on microtuber formation and
growth The 50-d-old plantlets were soaked in a pH 4 acid
solution at varying times and then cultured in MS medium
containing 60 g L−1 sucrose. After 20 d of culture, the leaves
of acid-pretreated plantlets showed a fresh green color.
Several microtubers were formed in the axillary buds
(Fig. 1A). However, the plantlets without the acid pretreatment
formed fewer tubers (Fig. 1B). In addition, the initial time of
tuber induction in the AT was significantly (α = 0.05) reduced
and depended on the acid pretreatment time (Table 1). The
shortest time for tuber formation was found in plantlets
pretreated with an acid solution for 36 h. The initial time of
tuber induction was approximately 6 d earlier than the control
group. In addition, tuber numbers increased in all of the plant-
lets pretreated with an acid solution for various times, except
the 12-h timepoint. A maximum number of microtubers was
found at the 36-h acid pretreatment. Tuber fresh weight was
also remarkably increased by the acid pretreatments, reaching
the highest level (282.5 mg) at 36 h of acid pretreatment,
which was 2.3-fold higher than the control group
(121.4 mg). Consequently, a method of pretreating plantlets
with an acid solution for a short time, can efficiently enhance
the microtuber production of potato.
Tolerance to abiotic stresses is crucial for sustained crop
yield (Puijalon et al. 2007). In general, plant growth and de-
velopment are restricted during strong or long-term stress, but
moderate stress can result in a positive effect. During potato
cultivation, cooling the nutrient solution temperature to 18°C
in an aeroponic system significantly (α = 0.05) promoted
plant growth and tuber yield of cultivars ‘Mystere’ and
‘Chieftain’ (Oraby et al. 2015). Microtuber multiplication
and growth were favorable when the in vitro plantlets were
submerged in 750 to 1000 mM NaCl for 1 h (Pumisutapon
and Topoonyanont 2017). In addition, the tuber formation was
increased when the nutrient solution was changed to a pH of
3.5 to 4.0 for 10 h in the hydroponic system (Wan et al. 1994).
In a previous study, microtuberization was promoted when the
 TENG ET AL.

Table 1 . Effect of time of acid

pretreatment on microtuber Times of acid Initial days of tuber Tuber numbers per Tuber fresh weight (mg per
formation and growth of potato treatment (h) induction plantlet plantlet)
(Solanum tuberosum L.) plantlets.
0 10.4 ± 0.31 a 1.2 ± 0.25 c 121.4 ± 26.14 e
12 5.8 ± 0.63 c 1.4 ± 0.31 c 179.2 ± 36.13 dc
24 4.4 ± 0.31 d 1.8 ± 0.48 b 184.3 ± 33.56 c
36 4.2 ± 0.25 d 3.2 ± 0.25 a 282.5 ± 19.12 a
48 5.4 ± 0.31 c 2.4 ± 0.31 b 223.9 ± 20.17 b
60 7.0 ± 0.31 b 1.9 ± 0.25 b 164.4 ± 29.91 d
72 7.1 ± 0.48 b 1.8 ± 0.48 b 142.8 ± 30.78 f

Data are the mean ± SE (n = 3). The different letters within the same column indicate significant difference by
Duncan’s multiple range test at α = 0.05. Tuber number and fresh weight were scored after 20 d of culture of
plantlets pretreated with acid solution (pH 4)

in vitro plantlets were pretreated with a solution of pH 3 for control, with a 29-fold increase from 5 (1.6 mg plantlet−1) to
24 h (Teng et al. 2017). However, hygrophanous leaves de- 20 d (46.4 mg plantlet−1) in the AT. During all culture days,
veloped in some plantlets (data not shown). Therefore, in the the tuber dry weight was obviously higher in the AT than in
present study, plantlets were pretreated with an acidic solution the control (Fig. 2B).
at pH 4 for different time periods and the plantlets did not During potato microtuberization, in the cytosol, SS activity
show an undesirable phenotype. Tubers formed much earlier is mainly responsible for sucrose decomposition in the
and in larger quantities after a 36-h treatment. Studies have
indicated that pH levels from 5 to 7 are suitable for potato
hydroponic cultivation (Dong et al. 2005), but the tubers can
form early and have a larger size when the plants are treated
with low pH (3 or 4) for a short time (Wan et al. 1994). In the
present study, the acid pretreatment (pH 4 for 36 h) promoted
microtuberization, and the effect on tuber formation and
growth was even better than other strategies, such as the use
of plant growth regulators. Thus, the method of pretreating
in vitro plantlets with a moderate acid solution for a short time
can be efficiently used for producing microtubers in the indus-
trial production of potato seeds.

Changes in biomass, sucrose, and starch content, and re-

lated enzyme activity in leaves and tubers The biomass of
leaves and microtubers in AT and the control during the
microtuberization process was investigated. The leaf dry
weight of AT and the control increased from day 5 to 10 and
kept a constant value at the culture time periods indicated
(Fig. 2A). Compared to the control, the dry weight of AT
was significantly (α = 0.05) lower at days 1 and 5. However,
the AT and control showed no significant difference in the
following days. During the initial culture period (1 to 5 d),
the plantlets showed a low leaf dry weight in the AT, which
was probably due to the temporary intermittent nutrient ab-
sorption by the acid stimulation. However, the leaf dry weight
increased rapidly after 5 d, possible because the stress effect
was eliminated, thereby accelerating the biomass accumula- Figure 2 . Changes in dry weight in leaves and tubers of plantlets during
tion of plantlets. In addition, microtuber formation occurred potato (Solanum tuberosum L.) microtuberization. Control indicates the
plantlets without pretreatment and AT indicates plantlets pretreated with a
earlier in the AT than in the control. Tubers formed within 5 d pH 4 acid solution for 36 h, respectively. Data are the mean ± SE (n = 3).
in the AT but at day 10 in the control. The tuber dry weight The * indicates the significant difference (α = 0.05) according to
increased by increasing the days of culture in both AT and Student’s t test.
 POTATO MICROTUBER FORMATION IN VITRO

Figure 3 . Changes in the sucrose

and starch content and enzyme
activity of sucrose synthase (SS),
ADP-glucose pyrophosphorylase
(AGPase), and soluble starch
synthase (SSS) in plantlet leaves
during potato (Solanum
tuberosum L.) microtuberization.
Control indicates plantlets
without pretreatment and AT
indicates plantlets pretreated with
a pH 4 acid solution for 36 h,
respectively. Data are the mean ±
SE (n = 3). The * indicates the
significant difference (α = 0.05)
according to Student’s t test.

carbohydrate pathway, and the degraded products from su-
crose are UDPG substrates for starch synthesis (Cao and
Tibbitts 1997; Emes et al. 2003; Koch 2004). The glucose-1-
phosphate enters the amyloplast where amylose and amylo-
pectin are synthesized by AGPase and SSS (Jeon et al. 2010).
Therefore, starch accumulation is closely related to the su-
crose content, and the activity of enzymes involved both su-
crose and starch synthesis. In the present study, to clarify the
mechanism of the carbohydrate metabolism, the changes in
carbohydrate content and metabolic enzyme activity were de-
termined. The sucrose and starch content, and the activity of
SS, AGPase, and SSS exhibited a similar pattern in the AT and
the control (Figs. 3 and 4). The sucrose content in the leaves
increased from day 1 to 5 and reached the maximum level at
5 d (Fig. 3A). This phenomenon was probably a result of
plantlets rapidly absorbing the sugar from the culture medium
with high osmotic pressure at the initial culture period and
transferring the sugar and synthesizing sucrose in the leaves
(MacGregor et al. 2008). After 5 d of culture, the sucrose
content in leaves decreased with an increase in the number
of days in culture (Fig. 3A), which was attributed to the trans-
fer of the sucrose in leaves to the tubers (Geigenberger et al.
2004). In addition, the sucrose content in AT leaves was dra-
matically (α = 0.05) higher than the level in the control from
day one to 20. This result demonstrates that moderate acid
stress can enhance sugar uptake in plantlets. The leaf SS ac-
tivity was lower in the AT than in the control at the same
culture time but did not obviously change with an increase
 TENG ET AL.

Figure 4 . Changes in the sucrose

and starch content and enzyme
activity of sucrose synthase (SS),
ADP-glucose pyrophosphorylase
(AGPase), and soluble starch
synthase (SSS) in potato
(Solanum tuberosum L.) tubers.
Control indicates plantlets
without pretreatment and AT
indicates plantlets pretreated with
a pH 4 acid solution for 36 h,
respectively. Data are the mean ±
SE (n = 3). The * indicates the
significant difference (α = 0.05)
according to Student’s t test.

in the number of culture days in both the AT and control
(Fig. 3B). Sucrose synthase is involved in sucrose degradation
(Geigenberger et al. 2004), thus its activity in leaves closely
matched the data on the sucrose content.
Moreover, the AGPase activity of leaves in the AT and
control increased with an increase in the number of culture
days. Compared with the control, AGPase activity in the AT
was significantly (α = 0.05) lower during all of the culture
days (Fig. 3C). The SSS activity of leaves in the AT and
control showed slight changes, which decreased after 1 d of
culture and remained stable in the following days. The SSS
activity was lower in the AT than in the control at 1 d
(Fig. 3D). The starch content in the AT and control increased
from 1 to 10 d and remained stable in the following days.
However, it was lower in the AT than in the control during
all of the culture days (Fig. 3E).
In microtubers, sucrose content (Fig. 4A) and SS activity
(Fig. 4B) of AT increased from 10 to 15 d and subsequently
decreased, and the levels of the control at 15 d were higher
than that at 20 d. The sucrose content and SS activity did not
obviously differ in the AT and control at 15 and 20 d. In
addition, the AGPase activity (Fig. 4C) and starch content
(Fig. 4E) of the AT and control increased with an increase in
the culture duration and peaked at 20 d. However, they were
significantly (α = 0.05) higher in the AT than in the control at
the same culture time. The SSS activity was similar to the
changes in sucrose and SS activity. The maximum values were
found at 15 d in the AT and control. Furthermore, the SSS
 POTATO MICROTUBER FORMATION IN VITRO
activity was higher in the AT than in the control at the same
culture time (Fig. 4D).
In plant tissue culture, most plant tissues require an exog-
enous source of carbohydrates because they do not efficiently
photosynthesize (Lian et al. 2014). Sucrose is the essential
carbon source in plant tissue culture and is involved not only
in metabolism-related organogenesis and respiration but also
in osmoregulation (Thorpe and Meier 1972). During potato
microtuberization, the transcription of genes involved in tuber
storage metabolism can be induced by high amounts of su-
crose contained in the culture medium (Raíces et al. 2003;
Rolland et al. 2006). Thus, sucrose is considered a specific
signal that plays an important role in tuber development
(Geigenberger et al. 1999; Koch 2004). The present study
determined the changes in the sucrose and starch content
and the activity of SS, AGPase, and SSS in the leaves and
microtubers of potato plantlets. These results can provide a
theoretical foundation to research the mechanism of
microtuber formation by acid induction in the future.
Conclusions
The acid pretreatment promoted the formation and growth of
microtubers. When the plantlets were pretreated with an acid
solution of pH 4 for 36 h, the first tubers were found 6.2 d
earlier compared to the control, and the maximum number of
tubers (3.2) and fresh weight (282.5 mg) were achieved.
During the microtuberization, the leaf dry weight was higher
in the control than in the AT during the initial culture period of
1 to 5 d. Moreover, the dry weight of AT leaves reached an
equal level with the control after 5 d of culture. In addition, the
dry weight of tubers in the AT was significantly (α = 0.05)
higher than in the control at the same culture times. The
changes in the sucrose and starch content and the enzymatic
activity of SS, AGPase, and SSS in the AT and control exhib-
ited similar patterns. However, levels of these biochemical
indexes differed between the AT and control. The present
study suggests that acid pretreatment is an efficient method
for microtuber induction and development, which can be used
for the industrial production of potato seeds.
Funding information Project 31260470 is supported by the National
Natural Science Foundation of China.
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