JIPB
Journal of Integrative
Plant Biology
Expression of auxin synthesis gene tms1 under
control of tuber-specific promoter enhances
potato tuberization in vitro
1
Signaling Systems Laboratory, Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow 127276, Russia, 2Plant
Biotechnology Laboratory, Branch of Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino
142290, Russia, 3Laboratory of Plant Physiology, Wageningen University, 6708 PB, Wageningen, the Netherlands, 4Department of Molecular
Basis of Ontogenesis, Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia. *Correspondence:
[email protected]
Abstract Phytohormones, auxins in particular, play an
important role in plant development and productivity.
Earlier data showed positive impact of exogenous auxin on
potato (Solanum tuberosum L.) tuberization. The aim of this
study was to generate potato plants with increased auxin
level predominantly in tubers. To this end, a pBinB33-tms1
vector was constructed harboring the Agrobacterium auxin
biosynthesis gene tms1 fused to tuber-specific promoter of
the class I patatin gene (B33-promoter) of potato. Among
numerous independently generated B33:tms1 lines, those
without visible differences from control were selected for
detailed studies. In the majority of transgenic lines, tms1
gene transcription was detected, mostly in tubers rather
than in shoots. Indoleacetic acid (IAA) content in tubers
and the auxin tuber-to-shoot ratio were increased in tms1expressing transformants. The organ-specific increase in
auxin synthesis in B33:tms1-transformants accelerated and
intensified the process of tuber formation, reduced the
dose of carbohydrate supply required for in vitro tuber-
INTRODUCTION
Tuberization is a morphogenetic process associated with the
vegetative reproductive development of plants. Potato
tuberization is of particular importance, because potato is
the fourth most important common food crop (after cereals),
and the global production of potato tubers exceeds 300
million tons/year (Barrell et al. 2013). In potato plants, tuber
formation occurs normally on stolons and is dependent on a
number of external factors (e.g. day length, temperature, soil
composition) that affect plant regulatory systems, especially
hormonal ones. The sensitivity to external factors depends on
potato genotype (Snyder and Ewing 1989). To date, it is
commonly accepted that potato tuber formation is controlled
by a complex cross-talk of various hormones and other
substances including miRNA (Vreugdenhil and Struik 1989;
Ewing 1995; Prat 2004; Sarkar 2008; Lucas et al. 2013;
Aksenova et al. 2014). The most convincing data concern the
role of gibberellins, which were found to promote stolon
growth but strongly suppress tuber formation. In contrast,
www.jipb.net
ization, and decreased the photoperiodic dependence of
tuber initiation. Overall, a positive correlation was
observed between tms1 expression, IAA content in tubers,
and stimulation of tuber formation. The revealed properties of B33:tms1 transformants imply an important role for
auxin in potato tuberization and offer prospects to magnify
potato productivity by a moderate organ-specific enhancement of auxin content.
Keywords: Auxin; gene expression; Solanum tuberosum; tms1;
transgenic potato; tuberization; tuber yield
Citation: Kolachevskaya OO, Alekseeva VV, Sergeeva LI, Rukavtsova
EB, Getman IA, Vreugdenhil D, Buryanov YI, Romanov GA (2014)
Expression of auxin synthesis gene tms1 under control of tuberspecific promoter enhances potato tuberization in vitro. J Integr Plant
Biol XX:XX–XX. doi: 10.1111/jipb.12314
Edited by: Lars Ostergaard, John Innes Centre, UK
Received Aug. 15, 2014; Accepted Nov. 24, 2014
Available online on Nov. 24, 2014 at www.wileyonlinelibrary.com/
journal/jipb
© 2014 Institute of Botany, Chinese Academy of Sciences
cytokinins, abscisic acid, ethylene, and jasmonic and tuberonic
acids were shown to have a stimulating effect in a number of
experiments (reviewed by Aksenova et al. 2014). With regard
to auxins, the effect of this class of plant hormones on tuber
formation was ambiguous and much less studied. In earlier
studies, a certain stimulating effect on tuberization by auxin
treatment has been revealed (Ito and Kato 1951; van Schreven
1956; Harmey et al. 1966). Later, it was found that the degree
and even the direction (stimulating or inhibiting) of the effect
of auxin on tuber formation depends on several conditions:
hormone concentration used (Kumar and Wareing 1974),
quality of light (Aksenova et al. 1994), endogenous auxin to
cytokinin ratio (Macháčková et al. 1997; Sergeeva et al. 2000),
carbohydrate supply (Aksenova et al. 2000), and potato
genotype (Romanov et al. 2000). The stimulating effect of
exogenous auxin was more pronounced when the hormone
was added to the cultivation medium at low concentrations,
and plants were cultivated under red light and at suboptimal
sucrose concentration in the medium. Measurements of
endogenous auxin content in tubers showed a positive
XXX 2015 | Volume XXXX | Issue XXXX | XXX-XX
Research Article
Oksana O. Kolachevskaya1, Valeriya V. Alekseeva2, Lidiya I. Sergeeva1,3, Elena B. Rukavtsova2, Irina A. Getman1,
Dick Vreugdenhil3, Yaroslav I. Buryanov2 and Georgy A. Romanov1,4*
2
Kolachevskaya et al.
correlation between the tuber growth rate and the content of
auxin (Marschner et al. 1984; Puzina et al. 2000). Inside the
tuber, the highest concentration of IAA was observed in the
core zone, which is responsible for the tuber growth
(Borzenkova and Borovkova 2003). According to other
reports, the level of endogenous IAA in stolons increased
prior to initiation of tubers, and then decreased, remaining
low throughout the subsequent period of tuber growth
(Obata-Sasamoto and Suzuki 1979; Koda and Okazawa 1983).
Studies at the molecular level described changes in the
expression of auxin-responsive and signaling genes during the
tuber initiation. In particular, the transition from longitudinal
to transverse cell division at swelling stolon tips was
accompanied by several fold decrease in the expression of
auxin transcription factor ARF6 (Faivre-Rampant et al. 2004).
Consistent with these data, it was shown that the expression
of a number of auxin-dependent genes in potato Solanum
tuberosum subsp. andigena decreased in the early period of
tuber initiation when stolon tips begin to swell (Hannapel
et al. 2004; Hannapel 2007). However, more detailed analysis
showed that the expression of many of the auxin-responsive
genes increased in stolons rapidly during tuber initiation
(Kloosterman et al. 2005, 2008). Among these genes were
StPIN–auxin transport genes, StYUC–auxin biosynthesis gene,
arcA-like genes, and others. Quantification of auxin content
confirmed its dramatic rise in the stolon tips prior to tuber
swelling (Roumeliotis et al. 2012). Taken together, these
results pointed to an important role of auxin during the
initiation and growth of potato tubers.
In recent decades, the research potential was significantly
increased with the advent of such an approach as genetic
engineering (Barrell et al. 2013). A great number of transgenic
plants were generated using a variety of foreign genes
(including those for hormone synthesis) or DNA constructs
inactivating endogenous genes (Csukasi et al. 2009). Comparison of such transformants with control plants allowed
elucidation of the role of specific genes and their products
in plant growth and development as well as plant responses to
environmental changes. In particular, potato transformants
were obtained expressing the Agrobacterium ipt gene for
cytokinin synthesis (Ondřej et al. 1990; Macháčková et al. 1997;
Sergeeva et al. 2000; Zubko et al. 2005) or the AtCKX2 gene for
cytokinin degradation (Raspor et al. 2012). Some experiments
with transgenic plants indicated the importance of the
cytokinin to auxin endogenous ratio for the optimal potato
tuberization (Macháčková et al. 1997; Sergeeva et al. 2000).
However, works aimed to generate potato transformants
with genes that directly affect auxin metabolism and/or
signaling were still very rare, probably due to the uncertainty
regarding the role of auxin in the tuber formation.
Constitutive expression of a bacterial gene, iaaL, encoding
the auxin-conjugating enzyme IAA-lysine synthase, led to an
increase in tuber number in transgenic plants (Fladung 1993).
Other transgenic potato plants expressed the agrobacterial
rolB gene under the control of tuber-specific patatin promoter
(B33 promoter) (Aksenova et al. 1999, 2000). It is known that
rolB gene expression in plants produces an auxin-like effect
presumably due to the increased sensitivity of cells towards
auxin (Spano et al. 1988; Maurel et al. 1991). B33:rolB
transformants differed from control plants by earlier in vitro
tuber formation and less sucrose requirement for tuberXXX 2015 | Volume XXXX | Issue XXXX | XXX-XX
ization. The average weight of individual tubers in B33:rolB
transformants significantly exceeded the weight of nontransgenic tubers (Aksenova et al. 2000; Romanov et al.
2000).
In the present study, we used another agrobacterial gene,
tms1, to generate plants with enhanced auxin biosynthesis.
This gene is known to encode tryptophan-2-monooxygenase
(iaaM), which converts L-tryptophan to indole-3-acetamide
(IAM), one of the precursors of auxin (Delker et al. 2008). The
enzyme iaaH, whose activity was found in plants (Mano
et al. 2010), uses this precursor to yield the active auxin IAA. In
order to avoid a widespread auxin accumulation which was
shown to adversely affect plant development and function
(Klee et al. 1987; Sitbon et al. 1992; our data not shown), we
used the organ-specific promoter of the class I patatin gene
instead of a constitutive promoter. The class I patatin (B33)
promoter is expressed mainly in tubers and tuberizing stolons,
although limited expression was observed in other potato
organs: leaves, stems, and roots (Rocha-Sosa et al. 1989; Park
1990; Liu et al. 1991; Deryabin et al. 2003; Romanov et al. 2007).
Our study showed that the expression of the tms1 gene under
control of a patatin promoter resulted in organ- and stagespecific increase in IAA content in potato plants. This local
increase in auxin content exerted virtually no effect on the
vegetative growth of transgenic plants but accelerated and
intensified the process of tuber formation, resulting in increase
in potato productivity, especially at suboptimal photoperiod.
RESULTS
tms1 integration into the potato genome and transgene
expression
For the purpose of our study, the vector pBinB33-tms1 was
constructed where the auxin biosynthesis gene tms1 was
fused to the tuber-specific promoter of the class I patatin gene
(B33 promoter, Figure 1), and used for potato transformation.
More than 20 independent transformants resistant to the
selective drug (kanamycin) were obtained and propagated in
vitro. To detect clones harboring the desired construct B33:
tms1, polymerase chain reaction (PCR) was performed on DNA
isolated from leaves of kanamycin-resistant plants. The
designed primers were aimed to amplify the DNA fragment
starting within the B33 promoter and ending within the tms1
sequence, thus revealing the B33-tms1 junction.
Indeed, in contrast to control (untransformed) plants, in
the majority of regenerants, DNA amplification took place, and
the amplicon size was as predicted (375 bp, Figure 2A). Among
the two dozen lines of transformed plants, those which had no
visible phenotypic differences from control plants were
selected. The expression of tms1 in the selected plants was
analyzed by reverse transcription (RT)-PCR after RNA extraction from tubers and shoots. The endogenous class I patatin
gene served as a control gene whose expression, as expected,
was confined mainly to tubers (Figure 2B). The expression
of the tms1 transgene was reliably detected in several
independent lines of transformants which were further
propagated for subsequent study. Along with potato clones,
which contained and expressed tms1, other clones were
identified which harbored the transgene but, for unknown
reasons, its transcription was not detectable (e.g. clones A4–15,
www.jipb.net
tms1 enhances potato tuberization
Figure 1. Scheme for constructing a recombinant vector
pBinB33-tms1, containing the tms1 gene under the control of
B33 patatin gene promoter
tms1, agrobacterial gene for IAA synthesis; pBS, Bluescript
KSþ phagemid cloning vector for Escherichia coli; Bc, S, R, B, H,
sites of restriction endonucleases BclI, SmaI, EcoRI, BamHI, and
HindIII, respectively; polyA, polyA DNA sequence; B33, class I
patatin gene B33 promoter; pBS-tms1, vector Bluescript KSþ
with integrated tms1; KmR, selectable kanamycin resistance
gene; pBin-B33, binary vector with a B33 patatin gene
promoter; nptII, selectable gene for kanamycin and neomycin
resistance; LB, RB, left and right T-DNA borders. Colored
arrows denote the direction of transcription.
A4–26). These “silent” transgenic clones did not differ in
morphology or productivity from wild-type plants. In some
clones, like A4–24, the tms1 expression in a certain period was
too low, causing unreliable transcript detection (Figure 2B).
Most likely, the differences in transgene expression between
transformants were caused by the genomic site of the foreign
gene integration, and/or eventual influence of the transformation process on genome functioning.
Quantitative analysis of tms1 gene expression in tubers and
shoots of the transformants
The determination of tms1 expression levels in tubers and
shoots of four independent lines of transformants was carried
out by real-time PCR (Figure 3). Transgene expression
predominated in tubers, especially during the first period
(4 weeks) which corresponded to their initiation and early
growth (Figure 3, left bars). That time, the tms1 expression
level in tubers was 10.2 fold higher in line A1–2, 89 fold in line
A4–7, and 11.6 fold in line A4–8 (8 week old plants) than in
respective shoots. In the later period, tms1 expression levels
varied depending on the line (Figure 3, middle and right bars).
Line A1–2 (Figure 3A) and A4–8 (Figure 3C) demonstrated
rather stable tms1 expression with subsequent smoothing of
www.jipb.net
3
Figure 2. Transgene presence and activity in independently
transformed potato lines
(A) Polymerase chain reaction (PCR) analysis of genomic DNA
for the presence of the B33:tms1 construct with the primers on
the B33-tms1 junction. p1, p2 designate clones of Escherichia
coli plasmid DNA containing B33-tms1 construct. Numbers
(e.g. 2, 7, 8) indicate potato lines A1–2, A4–7, A4–8, and so
forth, independently transformed with the same vector. C, M,
and W correspond to control (non-transformed) plants, DNA
molecular weight marker, and water PCR control, respectively. (B) Reverse transcription (RT)-PCR analysis of the
expression of tms1 and patatin (control) genes in potato
tubers and shoots. Plants were cultivated under long day (LD)
conditions for 4 weeks on the medium containing 5% sucrose.
cDNA was synthesized by RT on RNA template and amplified
with primers for tms1 (top row) or pat (control patatin gene)
(bottom row). Potato clones are designated by numbers as in
(A); C and M correspond to control plants and DNA molecular
weight marker, respectively. Additional symbols “t” and “s”
indicate tuber or shoot RNA source, respectively. Positions of
amplicons of expected size are marked on the right.
the differences between tubers and shoots. Line A4–7
(Figure 3B) showed high and dominant activity of tms1 in
tubers during the first 8 weeks followed by a sharp decline of
expression by the 12th week of cultivation. Unlike the others,
line A4–24 demonstrated low and equal to shoots expression
of tms1 in tubers by the 4th week, the maximum expression by
the 8th week, and then fell down (Figure 3D). For comparison,
quantitative (q)RT-PCR data on the levels of expression of the
endogenous patatin gene in tubers and shoots of control
plants were included (Figure 3D). Overall, the results show
that tms1 was preferentially expressed in tubers, but the
expression levels varied over time and between lines.
Endogenous IAA content in tubers and shoots of the
transformants
The endogenous IAA content was determined in tubers
of 4 week old plants by ultra-performance liquid chromatography/mass spectrometry (UPLC-TQ-MS/MS) method. Of the
XXX 2015 | Volume XXXX | Issue XXXX | XXX-XX
4
Kolachevskaya et al.
four transformed lines, three (A1–2, A4–7, and A4–8) showed
increased (by 3.1–4.7 fold vs control) IAA content in tubers
(Figure 4). Line A4–24, which by this time displayed the lowest
level of transgene expression (Figure 3D), had also the lowest
IAA content in tubers. The IAA content in shoots was tested in
parallel. These measurements showed a marked increase in
the tuber-to-shoot ratio of auxin content in most transgenic
lines: up to 1.1 in line A1–2, 3.7 in line A4–7, and 0.9 in line A4–8
versus 0.34 in control plants.
Effect of tms1 transgene expression on the dynamics of
tuber formation
Analysis of tuberization dynamics was carried out using in
vitro plants cultivated under different light regimes. When
plants were cultivated in the dark at low (1% and 3%) sucrose
concentrations in the medium, no significant difference
between control and transformed plants was observed
(data not shown). At the suboptimal (5%) sucrose content
in the medium, tubers appeared in A1–2 transformants a week
earlier than in control plants (Figure 5A), and tuber formation
proceeded more rapidly than in the control. At the optimal
(8%) sucrose content, the tuber formation was 1.5 fold more
extensive in transformants than in control plants, starting
from the 1st week (Figure 5B). Such differences persisted
during subsequent cultivation (up to 6 weeks) on a medium
containing 5% sucrose but leveled off at a high (8%) sucrose
content. The difference in average weight of a single tuber
between transgenic and control plants was either absent (at
8% sucrose) or statistically not significant (at 3–5% sucrose)
(Table 1A; Figure 5B, insert).
During plant cultivation under a light regime favorable for
tuberization (short day, SD) and at suboptimal (5%) sucrose
content, all investigated transgenic plants formed tubers
5–7 d earlier and more extensively than control plants
(Figure 6A), while at the optimal (8%) sucrose the tuberization
activity in most transgenic lines exceeded that of the control
up to 2 fold, starting from the 1st week (Figure 6B). By the end
of the experiment (7 weeks in culture), differences in tuber
numbers between transgenic and control plants became less
pronounced (1.25–1.6 fold). The average fresh weight of a
single tuber of transgenic plants grown on medium containing
5% or 8% sucrose typically exceeded control values: significantly (up to 2.5 fold) in lines A4–7 and A4–8, and as a trend in
line A1–2 (Table 1B).
3
Figure 3. Continued.
XXX 2015 | Volume XXXX | Issue XXXX | XXX-XX
Figure 3. Quantitative analysis of the tms1 expression in
tubers and shoots of transformed potato lines of various age
(A) line A1–2. (B) line A4–7. (C) line A4–8. (D) line A4–24. (E)
Patatin gene expression in control plants. Note that the y-axis
is logarithmic. Plants were cultivated for indicated time under
long day (LD) conditions on the medium containing 5%
sucrose. cDNA was synthesized by reverse transcription on
RNA templates. Quantitative reverse transcription polymerase chain reaction was done with primers for tms1 and potato
actin (reference gene). The bars in (A–D) represent relative
tms1 transcripts abundances at logarithm scale, filled and
hatched bars designate tubers and shoots, respectively. Data
were normalized with respect to the expression of a reference
actin gene.
www.jipb.net
tms1 enhances potato tuberization
5
Figure 4. Content of endogenous indoleacetic acid (IAA) in
different lines of B33:tms1 transformants of potato
Plants were cultivated for 4 weeks under long day (LD)
conditions on the medium containing 5% sucrose. Data show
IAA content in tubers (filled bars) and shoots (hatched bars)
versus the control plants (dashed line). The IAA content in
tubers of control plants corresponding to 21 7 ng/g dry
weight was taken as 1.
Under light conditions unfavorable for tuber formation
(long day, LD), the transformants showed the greatest
differences from the control lines. At low (3%) sucrose in
the medium, transgenic tuber induction started at 2–3 weeks
of culturing, while control plants began to form tubers only
from the 4th week (Figure 7A). Enhanced tuber formation in
most transgenic lines (except A4–24) was observed also at 5%
sucrose (Figure 7B). Particularly at 8% sucrose in the medium,
transformants developed tubers faster and more extensively
than the control, reaching 4–5 fold higher levels of tuber
formation in the first 3 weeks and retaining more than 2 fold
advantage over control to the end (7 weeks) of experiments
(Figure 7C). The average weight of a single tuber in transgenic
lines exceeded that in the control by 1.8–3.5 fold in a medium
containing 3% sucrose, by 1.8–2.7 fold in a medium with 5%
sucrose, and 1.4–2.6 fold in a medium with 8% sucrose
(Table 1C). In total, line A1–2 showed on LD marked positive
changes at all three sucrose concentrations, line A4–8 at two
concentrations (5% and 8%), while for lines A4–7 and A4–24 a
significant increase in the individual tuber weight was noted in
the single series of experiments (line A4–7 at 3% sucrose, line
A4–24 at 5% sucrose) (Table 1C). Thus, the expression of the
transgene tms1 accelerated tuber formation and magnified
tuber number under both favorable (SD) and, to a greater
degree, non-favorable (LD) light regimes. Evident correlation
was observed between the extent of tms1 expression/IAA
increase and the stimulation of tuberization.
By increasing number and weight of tubers, their average
yield (fresh mass of tubers per plant) in transformants
significantly (by 1.7–6.3 fold) exceeded that in control plants
under most growth conditions (Table 2). This effect was
particularly evident under non-favorable photoperiod (LD)
and high (5%–8%) sucrose concentration, being significant for
www.jipb.net
Figure 5. Tuberization dynamics of B33:tms1 transgenic and
control plants cultivated in the darkness
Plants were grown for indicated time on Murashige-Skoog
medium containing different sucrose concentrations: 5% (A) or
8% (B). Insertion in (B) shows 6 week old tubers from
transgenic (on the left) and control (on the right) plants.
almost all transgenic lines. A minimal deviation from control
was observed at 3% sucrose, especially under LD.
DISCUSSION
To generate potato plants with elevated level of auxin
biosynthesis, we used the tms1 gene from Agrobacterium
tumefaciens. In agrobacteria, auxin synthesis occurs in two
stages: first, IAM is derived from tryptophan, catalyzed by
iaaM; second, IAM is converted to IAA (Delker et al. 2008).
Enzymes for IAA synthesis from IAM were detected in plants
too, particularly in Arabidopsis and tobacco, and genes (AMI1)
encoding these enzymes showed homologs in genomes of
some plant species (Mano et al. 2010), including potato (SN
Lomin, pers. comm., 2014). Indeed, the tms1 gene expression
XXX 2015 | Volume XXXX | Issue XXXX | XXX-XX
6
Kolachevskaya et al.
Table 1. Average fresh weight (mg) of a single tuber from cultivated plants, depending on the cultivation conditions
A Darkness conditions
Line
Tuber weight (mean SD) of plants grown on medium with sucrose content
Control
А1–2
3%
25 8
20 6
5%
55 5
45 5
8%
65 14
65 6
33 12a
38 10a
44 7a
21 5a
30 10a
81 15a
10115a
114 10b
133 12b
89 9a
80 10a
12030a
190 8b
200 10b
67 15a
32 5a
112 20b
58 1c
18 4a
30 9a
77 4a
166 17b
88 6a
210 90b
140 9b
80 3a
210 40b
80 8a
108 20c
85 7a
B Short day conditions
Control
А1–2
А4–7
А4–8
А4–24
C Long day conditions
Control
А1–2
А4–7
А4–8
А4–24
Different superscript letters (a, b, c) indicate significant differences at P < 0.05.
in transgenic potato plants led to increased auxin level, the
allocation of extra auxin being in accordance with the
promoter specificity. This increase in IAA content corroborates the presence of AMI1-like activity in potato plants.
In a foregoing work, we obtained potato transformants
expressing the tms1 gene under the control of a constitutive
viral promoter, 35S CaMV; these plants exhibited an increase
in auxin level in all organs and demonstrated an inferior
growth and decreased tuberization ability (published in
abstract form). Evidently, excessive widespread auxin led to
deterioration of plant development. Similarly, a negative
effect of auxin overproduction on tuber formation was shown
in a recent work by Kim et al. (2013) where the AtYUC6 gene
under the control of the constitutive 35 S CaMV promoter was
used to enhance IAA synthesis in transformed potato.
Our previous experiments with local exposure of plants to
low concentrations of exogenous auxin revealed a positive
effect of this hormone on tuber initiation and growth
(Aksenova et al. 2000; Romanov et al. 2000). In such a
context, to optimize the anticipated stimulatory impact of
endogenous IAA on the tuberization, we designed a transforming vector pBinB33-tms1 providing the activation of auxin
biosynthesis at the right time and the right place, without
disturbing the plant vegetative growth. In this construct, the
tms1 gene was put under the control of the tuber-specific
class I patatin gene promoter. Among potato plants transformed with the B33:tms1 construct, some were found
that did not differ in phenotype and growth rate from
control plants, but demonstrated enhanced tuber formation
(Figures 6, 7; Tables 1B, C). This acquired property was
preserved in in vitro propagated transgenic plants for at least
3 years.
Polymerase chain reaction data with four independent
lines of transformants confirmed the tms1 transgene expression, more active in tubers than in shoots, especially in the
first period of culturing (Figures 2, 3) when the bulk of tubers
XXX 2015 | Volume XXXX | Issue XXXX | XXX-XX
was formed (Figures 5–7). Accordingly, the endogenous IAA
content in tubers of most lines under study (except line A4–24
with low tms1 expression level) was significantly higher than in
the control plants (Figure 4), thus markedly increasing tuberto-shoot IAA ratio. The enhanced expression level of B33-tms1
in non-tuber tissues (Figure 3) is presumably due to in planta
accumulation of sucrose taken up from the culture medium
because sucrose is a known activator of the patatin B33
promoter (Rocha-Sosa et al. 1989; Wenzler et al. 1989;
Naumkina et al. 2007).
The relatively high activity of the tms1 gene in tubers of line
A1–2 (Figure 3) correlated with an increased IAA content in
tubers (Figure 4) and a high and stable rate of tuber formation
in this line (Figures 5–7) under all cultivation conditions. At LD,
the A1–2 transformants developed the largest tubers by
7 weeks of cultivation (Table 1C), and at SD, displayed a similar
trend (Table 1B). Plants of lines A4–7 and A4–8 with the high IAA
content in tubers (Figure 4) exhibited extensive tuberization
dynamics as well, producing tubers of enhanced weight under a
variety of conditions: SD and 5% sucrose, SD and 8% sucrose, and
(line A4–8) LD and 5% sucrose (Table 1B, C).
Line A4–24 also expressed the tms1 gene, but by the first
weeks at relatively low level and nearly equally in tubers and
shoots (Figure 3D). The endogenous IAA content in tubers of
these plants was by that time at the control level (Figure 4),
and this line had the smallest differences from the control
plants in dynamics of tuber formation(Figures 6, 7). Only after
4–5 weeks of cultivation at the tuber-inducing sucrose content
and SD (Figure 6), A4–24 plants got ahead of the control by
tuber number and, in a unique case, average weight of a single
tuber (Table 1C). The tuberization promotion in this line
correlated with a concomitant increase in the level of tms1
expression (Figure 3D).
The transgenic plants grown in vitro seem to become less
dependent on sucrose for tuber formation. In tms1 expressing
lines at low (3%) sucrose combined with LD, tubers appeared
www.jipb.net
tms1 enhances potato tuberization
7
Figure 6. Tuberization dynamics of B33:tms1 transgenic and
control plants cultivated under short day (SD) conditions
Plants were grown for indicated time on Murashige-Skoog
medium containing different sucrose concentrations: 5%
(A) or 8% (B).
earlier and their number exceeded the control by up to 2.5 fold
(Figure 7A). Additional experiments have shown that tms1
transformants were able to produce tubers at extremely low
(1%) sucrose while the control plants were not. At high (5%–8%)
sucrose content in the medium, tuber number and weight
increased, but while in transgenic plants at LD the average
weight of a single tuber reached maximum at 5% sucrose, in
control plants the same maximum was achieved at the highest
(8%) sucrose content (Table 1B). All these data indicate that a
critical need in carbohydrate supply for tuberization is
reduced with tms1 transgene expression. Collectively, this
highlights the tight interplay between hormonal and carbohydrate signaling systems of potato in the process of tuber
formation, although the molecular mechanism of such crosstalk remains to be elucidated.
These positive changes of the tuberization may be
presumably due to strengthening tuber sink capacity caused
by extra auxin. The local enhancement of endogenous auxin
www.jipb.net
Figure 7. Tuberization dynamics of B33:tms1 transgenic and
control plants cultivated under long day (LD) conditions
Plants were grown for indicated time on Murashige-Skoog
medium containing different sucrose concentrations: 3%
(A), 5% (B), or 8% (C).
synthesis in potato stimulates tuberization in several ways: it
speeds up the onset of tuberization, reduces its dependence
on sucrose supply, and magnifies the proportion of plants
with tubers and the average weight per tuber. Together, these
changes led to a significant increase in tuber yield (Table 2). In
line A1–2 with rather high and stable transgene expression and
3 fold elevation of IAA content, the productivity was steadily
increased: tuber yield at final harvest exceeded the control by
2–6 fold under all six culturing conditions. The smallest
difference (three samples from a total of six) in tuber yield
from the control showed line A4–24 where the IAA content
was the least. Lines A4–7 and A4–8 were intermediate in the
XXX 2015 | Volume XXXX | Issue XXXX | XXX-XX
8
Kolachevskaya et al.
Table 2. Average tuber yield per one plant of potato lines under different growth conditions
Tuber yield (mg) of plants grown on medium with sucrose content:
Light regime
SD
LD
Line
3%
5%
Control
А1–2
А4–7
А4–8
А4–24
Control
А1–2
А4–7
А4–8
А4–24
3.3 1
9.5 2b
4.4 1a
3.15 1a
3.0 1a
6.1 2a
36.9 8b
27.2 5b
6.7 2a
8.4 0.9a
a
8%
26.7 4
57.3 7b
37.9 5a
63.3 9b
50.5 6b
21.6 6a
90.3 9c
43.0 8b
124.7 13d
48.2 5b
a
40.1 6a
96.0 13c
157.7 12b
164.0 18b
46.9 5a
30.0 5b
172.2 14b
72.5 12c
101.3 17c
74.4 9c
LD, long day; SD, short day. Statistical calculations were performed for each light mode separately. Different superscript letters
(a, b, c, d) indicate significant differences at P < 0.05
yield enhancement (four samples from a total of six; Table 2).
The stimulation of tuberization was manifested under both SD
and LD conditions, more evident under the latter. It should be
noted that B33 promoter functions not only in tubers but also
in stolons at an early stage of tuber formation (Wenzler et al.
1989; Liu et al. 1991), hence transgene expression in stolons
may precede and stimulate tuber initiation. Accordingly, Sun
et al. (2011) found an increased tuber initiation in soil growing
transgenic potato with rice sucrose transporter gene driven
by the same patatin B33 promoter.
Thus, one can conclude that introduction of the additional
auxin biosynthesis gene tms1 under the control of the tuberspecific B33 promoter stimulated tuber formation in transgenic potato plants. These results corroborate the important
role of auxin in the processes of potato tuber initiation and
growth. The moderate organ-specific increase in auxin level
may be suggested as a promising approach for biotechnological improvement of potato productivity.
MATERIALS AND METHODS
Plant materials
The study was performed with potato plantlets (Solanum
tuberosum L.) cv. Désirée which belongs to intermediate
maturity group (http://www.europotato.org/). Plants were
propagated by single-node stem cuttings and grown in vitro
on standard Murashige-Skoog (MS) medium solidified with
0.8% (w/v) agar and supplemented with 2% sucrose as
described previously (Aksenova et al. 2000; Romanov et al.
2000). In tuberization experiments, uniform single-node
cuttings from the middle part of the plants were used.
Cuttings were cultivated in vitro on the same medium but with
different sucrose content: 1%, 3%, 5%, or 8% (w/v). Culture
conditions were darkness or SD (8 h light, favorable
conditions) and LD (16 h light, non-favorable conditions)
photoperiods with 24/22 °C day/night temperature. The
number of plantlets with tubers was recorded every week
and the fresh weight of every tuber was determined at the
end of experiments (6–7 weeks). All experiments were done
at least in triplicate, with 15 plants per replicate. Graphs and
tables show mean values with standard deviations.
XXX 2015 | Volume XXXX | Issue XXXX | XXX-XX
Recombinant DNA techniques
Agrobacterium tumefaciens-derived tms1 gene (GenBank
accession no. NC_003065) encoding iaaM was cloned into
the plant binary vector pBin-B33 under the control of a tuberspecific class I patatin gene promoter (B33-promoter,
GenBank accession no. X14483). The vector was kindly
provided by Professor L Willmitzer (Potsdam-Golm, Germany).
pBluescriptII KSþ (pBS; Stratagene, La Jolla, CA, USA;
GenBank accession no. X52327.1) was used as an appropriate
intermediate vector. The gene cloning scheme is shown in
Figure 1. pDQ14 plasmid served as a source of tms1, containing
its sequence 2,270 bp long (Atlas 1993). DNA of the tms1 gene
was amplified by PCR using Pfu polymerase forming products
with blunt ends. At both ends of the gene BclI, a restriction
site was introduced as part of the oligonucleotide primers: 50 CGTGATCAATGTCACCTTCACC-30 and 50 -CGTGATCACTAATTTCTAGTGCGG-30 .
The PCR product of the tms1 gene was fused to the SmaIdigested pBS plasmid using T4 DNA ligase. The resulting
plasmid with the tms1 gene was designated pBS-tms1. Because
the BclI restriction enzyme is sensitive to dam DNA
methylation, the Escherichia coli strain GM272 defective in
dcm and dam methylases was transformed by the pBS-tms1
plasmid. The gene tms1 was excised from the unmethylated
pBS-tms1 DNA by BclI site, and the resulting DNA fragment
was inserted into the pBin-B33 vector treated with BamHI. The
pBin-B33 vector comprises the class I patatin gene (B33)
promoter, a polyA sequence and a polylinker with unique
restriction sites (Klee et al. 1984). Recombinant clones were
PCR-selected and gene orientation relative to the promoter
was determined by hydrolysis at SalI and HindIII internal
restriction sites. The resulting recombinant pBinB33-tms1
vector was transferred to A. tumefaciens strain CBE21 by direct
transformation. Polymerase chain reaction with the same
primers was used for the selection of recombinant clones.
Plant transformation and regeneration
Potato transformation and regeneration was performed
mainly as described (Rocha-Sosa et al. 1989) using leaf
explants treated with a suspension of A. tumefaciens
harboring the pBinB33-tms1 vector. After 2 d of incubation
in liquid MS medium with agrobacteria, the explants were
www.jipb.net
tms1 enhances potato tuberization
transferred to the callus-inducing solidified (0.8% agar) MS
medium containing antibiotics (250 mg/L cefotaxime, 50 mg/L
kanamycin) and phytohormones (5 mg/L a-naphthaleneacetic
acid (a-NAA), 0.1 mg/L N6-benzyladenine) and incubated for
7–8 d under LD at 27 °C. The explants developing calli were
transferred to shoot regeneration MS medium with 1.6%
glucose, phytohormones (2.2 mg/L zeatin riboside, 20 mg/L aNAA, 15 mg/L gibberellic acid) and antibiotics to completely
remove agrobacteria, incubated first for 14–17 d and then
were transferred repeatedly to the fresh medium of the same
composition every 10 d until shoots were formed on the calli.
These shoots were finally transferred onto MS medium with
antibiotics and then propagated by stem cuttings similarly to
starting plants. Primers to amplify agrobacterial virD2 gene
and tissue culture tests without antibiotics were used to prove
the absence of agrobacteria in the regenerants.
Analysis of transgene expression
Total RNA was extracted from 200–300 mg of fresh tissues
(tubers or two upper internodes of shoots) by means of TRIzol
method (Invitrogen, San Diego, CA, USA) and treated with
DNase I. cDNA was synthesized on the RNA template with MMuLV reverse transcriptase (Fermentas, Vilnius, Lithuania)
according to manufacturer protocol. The absence of genomic
DNA in cDNA samples was confirmed by PCR with primers on
an intron-containing fragment of the patatin gene. Qualitative
transgene expression was determined using RT-PCR with
primers: Tms_PL2, 50 -CACTAACGACAGTTGCGGTGCAA-30 and
Tms_PR2 50 -GCGACCATCGGCCAGCCTTTT-30 . DNA sequence
encoding potato class I patatin (GenBank accession no.
DQ114416) was employed as a control gene with primers:
Pat_PL, 50 -AAACAATCGACCCTTTGCTG-30 and Pat_PR 50 -TGCACACGAGTTTCTCCAAG-30 . Quantitative transgene expression
was determined using real-time qRT-PCR with primers: Tms1LP
50 -TAACGGTGTGTCTGTGAGCC-30 and Tms1RP 50 -CAGGAAATAGGTGCTGGGCA-30 . The conditions for qRT-PCR were as
follows: pre-denaturation at 95 °C for 60 s, followed by 35
cycles of denaturation at 95 °C for 50 s, annealing at 60 °C for
20 s, and a final extension step at 72 °C for 30 s. DNA sequences
encoding potato actin (GenBank accession no. X55749) and btubulin (GenBank accession no. 609267) were employed (with
similar results) as reference genes with primers (Nicot et al.
2005): ActLP 50 -GCTTCCCGATGGTCAAGTCA-30 and ActRP 50 GGATTCCAGCTGCTTCCATTC-30 ; and TubLP 50 -ATGTTCAGGCGCAAGGCTT-30 and TubRP 50 -TCTGCAACCGGGTCATTCAT-30 .
Each data of transcript content represents mean value
( SD) of three technical replicates.
IAA content determination
Determination of the endogenous content of free IAA was
accomplished by UHPLC-MS/MS using the Acquity UPLC
system coupled with the Xevo tandem quadrupole mass
spectrometer Xevo (Waters, Milford, MA, USA). The equipment for hormone measurements was kindly provided by the
Netherlands Organization for Scientific Research (Equipment
834.08.001 to HJB). Tubers and upper parts of shoots from
4 week old axenic plants were taken for assays. Finely
powdered freeze-dried plant tissue (20 mg) was extracted
twice with 1 mL of cold methanol containing 0.1 nmol/mL [13C6]
IAA as internal standard; extracts were purified by column
chromatography. The IAA amounts were determined from a
www.jipb.net
9
calibration curve with known amounts of standards, using the
software MassLynx4.1 (Waters).
Statistics
The experiments were performed in 3–5 biological replicates.
Graphics and tables show the arithmetic means with standard
deviations. Evaluation of significance of differences was
performed on the basis of Student’s criterion using t-test and
anova statistical programs.
ACKNOWLEDGEMENTS
We thank Salomé Prat for valuable advices concerning potato
transformation. The work was supported by the Russian
Science Foundation (grant no. 14-14-01095), and partly (until
July, 2014) by the Program of the Presidium of the Russian
Academy of Sciences “Molecular and Cell Biology”.
REFERENCES
Aksenova NP, Konstantinova TN, Sergeeva LI, Macháčková I,
Golyanovskaya SA (1994) Morphogenesis of potato plants in
vitro . I Effect of light quality and hormones. J Plant Growth Regul
13: 143–146
Aksenova NP, Konstantinova TN, Golyanovskaya SA, Schmülling T,
Kossmann J, Willmitzer L, Romanov GA (1999) In vitro growth and
tuber formation by transgenic potato plants harboring rolC or rolB
genes under control of the patatin promoter. Russ J Plant Physiol
46: 513–519
Aksenova NP, Konstantinova TN, Golyanovskaya SA, Kossmann J,
Willmitzer L, Romanov GA (2000) Transformed potato plants as a
model for studying the hormonal and carbohydrate regulation of
tuberization. Russ J Plant Physiol 47: 370–379
Aksenova NP, Sergeeva LI, Kolachevskaya OO, Romanov GA (2014)
Hormonal regulation of tuber formation in potato. In: Ramawat
KG, Merillon JM, eds. Bulbous Plants. Biotechnology. CRC Press,
New York, London. pp. 3–36
Аtlas RM (1993) Handbook of Microbiological Media. CRC Press Inc.,
New York
Barrell PJ, Meiyalaghan S, Jackobs JME, Conner AJ (2013) Applications
of biotechnology and genomics in potato improvement. Plant
Biotechnol 11: 907–920
Borzenkova RA, Borovkova MP (2003) Developmental patterns of
phytohormone content in the cortex and pith of potato tubers as
related to their growth and starch content. Russ J Plant Physiol
50: 119–124
Csukasi F, Merchante C, Valpuesta V (2009) Modification of plant
hormone levels and signaling as a tool in plant biotechnology.
Biotechnol J 4: 1293–1304
Delker C, Raschke A, Quint M (2008) Auxin dynamics: The dazzling
complexity of a small molecule’s message. Planta 227: 929–
941
Deryabin AN, Trunova TI, Dubinina IM, Burakhanova EA, Sabel’nikova
EP, Krylova EM, Romanov GA (2003) Chilling tolerance of potato
plants transformed with a yeast-derived invertase gene under the
control of the B33 patatin. Russ J Plant Physiol 50: 449–454
Ewing EE (1995) The role of hormones in potato (Solanum tuberosum
L.) tuberization. In: Davies PG, ed. Plant Hormones. Physiology,
Biochemistry and Molecular Biology. Kluwer, Dordrecht. pp. 698–
724
XXX 2015 | Volume XXXX | Issue XXXX | XXX-XX
10
Kolachevskaya et al.
Faivre-Rampant O, Cardle L, Marshall D, Viola R, Taylor MA (2004)
Changes in gene expression during meristem activation processes
in Solanum tuberosum with a focus on the regulation of an auxin
response factor gene. J Exp Bot 55: 613–622
Marschner H, Sattelmacher B, Bangerth F (1984) Growth rate of
potato tubers and endogenous contents of indolylacetic acid and
abscisic acid. Physiol Plant 60: 16–20
Fladung M (1993) Influence of the indoleacetic acid-lysine synthetase
gene (iaaL) of Pseudomonas syringae subsp. savastanoi on yield
attributes of potatoes. Plant Breed 111: 242–245
Maurel C, Barbier-Brygoo H, Spena A, Tempe J, Guern J (1991) Single
rol genes from the Agrobacterium rhizogenes alter some of the
cellular response to auxin in Nicotiana tabacum. Plant Physiol 97:
212–216
Hannapel DJ (2007) Signalling in induction of tuber formation. In:
Vreugdenhil D, ed. Potato Biology and Biotechnology. Elsevier,
Amsterdam. pp. 237–256
Nicot N, Hausman J-F, Hoffman L, Evers D (2005) Housekeeping gene
selection for real-time RT-PCR normalization in potato during
biotic and abiotic stress. J Exp Bot 56: 2907–2914
Hannapel DJ, Chen H, Rosin FM, Banerjee AK, Davies PJ (2004)
Molecular control of tuberization. Am J Potato Res 81: 263–274
Obata-Sasamoto H, Suzuki H (1979) Activities of enzymes relating to
starch synthesis and endogenous levels of growth regulators in
potato stolon tips during tuberization. Physiol Plant 45: 320–324
Harmey MA, Crowley MC, Clinch PEM (1966) The effect of growth
regulators on tuberization of cultured stem pieces of Solanum
tuberosum. Eur Potato J 9: 146–151
Ito H, Kato T (1951) The physiological foundation of tuber formation of
potato. Tohoku J Agric Res 1: 1–14
Kim JI, Baek D, Park HC, Chun HJ, Oh D-H, Lee MK, Cha J-Y, Kim W-Y,
Kim MC, Chung WS, Bohnert HJ, Lee SY, Bressan RA, Lee S-W, Yun
D-J (2013) Overexpression of Arabidopsis YUCCA6 in potato results
in high-auxin developmental phenotypes and enhanced resistance to water deficit. Mol Plant 6: 337–349
Klee HJ, Horsch RB, Hinchee MA, Hein MB, Hoffman NL (1987) The
effects of overproduction of two Agrobacterium tumefaciens
auxin biosynthetic genes in transgenic petunia plants. Genes Dev
1: 86–96
Klee HJ, Montoya A, Horodyski F, Lichtenstein C, Garfinkel D, Fuller S,
Flores C, Peschon J, Nester E, Gordon M (1984) Nucleotide
sequence of tms genes of the pTiA6NC octopine Ti plasmid: Two
gene products involved in plant tumorigenesis. Proc Natl Acad Sci
USA 81: 1728–1732
Kloosterman B, De Koeyer D, Griffiths R, Flinn B, Steuernagel B, Scholz
U, Sonnewald S, Sonnewald U, Bryan GJ, Prat S, Bánfalvi Z,
Hammond JP, Geigenberger P, Nielsen KL, Visser RGF, Bachem
CWB (2008) Genes driving potato tuber initiation and growth:
Identification based on transcriptional changes using the POCI
array. Funct Integr Genomics 8: 329–340
Kloosterman B, Vorst O, Hall RD, Visser RGF, Bachem CW (2005) Tuber
on a chip: Differential gene expression during potato tuber
development. Plant Biotechnol J 3: 505–519
Koda Y, Okazawa Y (1983) Characteristic changes in the levels of
endogenous plant hormones in relation to the onset of potato
tuberization. Jpn J Crop Sci 52: 592–597
Kumar D, Wareing PF (1974) Studies on tuberization of Solanum
andigena. II Growth hormones and tuberization. New Phytol 73:
833–840
Liu XY, Rocha-Sosa M, Hummel S, Willmitzer L, Frommer WB (1991) A
detailed study of the regulation and evolution of the two classes
of patatin genes in Solanum tuberosum L. Plant Mol Biol 17: 1139–
1154
Ondřej M, Macháčková I, Čatský J, Eder J, Hrouda M, Pospišilová J,
Synková H (1990) Potato transformation by T-DNA cytokinin
synthesis gene. Biol Plant 32: 401–406
Park WD (1990) Molecular approaches to tuberization in potato. In:
Vayda ME, Park WD, eds. The Molecular Biology of the Potato.
Redwood, Melksham. pp. 43–55
Prat S (2004) Hormonal and daylength control of potato tuberization.
In: Davies PJ, ed. Plant Hormones. Biosynthesis, Signal Transduction, Action! Kluwer Acadamic Publishers, Dordrecht, Boston,
London. pp. 538–560
Puzina TI, Kirillova IG, Yakushkina NI (2000) Dynamics of indole-3acetic acid in organs of potato at different stages of ontogenesis
and its role in regulation of tuber growth (in Russian). Izv Acad
Nauk Ser Biol 2: 170–177
Raspor M, Motyka V, Žižková E, Dobrev PI, Trávníčková A, ZdravkovićKorać S, Simonović A, Ninković S, Dragićević I (2012) Cytokinin
profiles of AtCKX2-overexpressing potato plants and the impact
of altered cytokinin homeostasis on tuberization in vitro. J Plant
Growth Regul 31: 460–470
Rocha-Sosa M, Sonnewald U, Frommer WB, Stratmann M, Schell J,
Willmitzer L (1989) Both developmental and metabolic signals
activate the promoter of a class I patatin gene. EMBO J 8: 23–29
Romanov GA, Aksenova NP, Konstantinova TN, Golyanovskaya SA,
Kossmann J, Willmitzer L (2000) Effect of indole-3-acetic acid and
kinetin on tuberization parameters of different cultivars and
transgenic lines of potato in vitro. Plant Growth Regul 32: 245–
251
Romanov GA, Naumkina EM, Ashapkin VV, Vanyushin BF (2007)
Methylation of GCGG sites of the patatin promoter is organspecific and inversely correlates with its activity. Dokl Biochem
Biophys 417: 327–330
Roumeliotis E, Kloosterman B, Oortwijn M, Kohlen W, Bouwmeester
HJ, Visser RGF, Bachem CWB (2012) The effects of auxin and
strigolactones on tuber initiation and stolon architecture in
potato. J Exp Bot 63: 4539–4548
Sarkar D (2008) The signal transduction pathways controlling in planta
tuberization in potato: An emerging synthesis. Plant Cell Rep 27:
1–8
Lucas WJ, Groover A, Lichtenberger R, Furuta K, Yadav S-R, Helariutta
Y, He X-H, Fukuda H, Kang J, Brady SM, Patrick JW, Sperry J,
Yoshida A, López-Millán A-F, Grusak MA, Kachroo P (2013) The
plant vascular system: Evolution, development and functions. J
Integr Plant Biol 55: 294–388
Sergeeva LI, de Bruijn SM, Koot-Gronsveld EAM, Navratil O,
Vreugdenhil D (2000) Tuber morphology and starch accumulation
are independent phenomena: Evidence from ipt-transgenic
potato lines. Physiol Plant 108: 435–443
Macháčková I, Sergeeva L, Ondřej M, Zaltsman O, Konstantinova T,
Eder J, Golyanovskaya S, Rakitin V, Aksenova N (1997) Growth
pattern, tuber formation and hormonal balance in in vitro potato
plants carrying ipt gene. Plant Growth Reg 21: 27–36
Sitbon F, Hennion S, Sundberg B, Little CHA, Olsson O, Sandberg G
(1992) Transgenic tobacco plants coexpressing the Agrobacterium tumefaciens iaaM and iaaH genes display altered growth and
indoleacetic acid metabolism. Plant Physiol 99: 1062–1069
Mano Y, Nemoto K, Suzuki M, Seki H, Fujii I, Muranaka T (2010) The
AMI1 gene family: Indole-3-acetamide hydrolase functions in auxin
biosynthesis in plants. J Exp Bot 61: 25–32
Snyder E, Ewing EE (1989) Interactive effects of temperature,
photoperiod, and cultivar on tuberization of potato cuttings.
Hortic Sci 24: 336–338
XXX 2015 | Volume XXXX | Issue XXXX | XXX-XX
www.jipb.net
tms1 enhances potato tuberization
Spano L, Mariotti D, Cardarelli M, Branca C, Constantino P (1988)
Morphogenesis and auxin sensitivity of transgenic tobacco with
different complements of Ri T-DNA. Plant Physiol 87: 479–
483
Sun A, Dai Y, Zhang X, Li C, Meng K, Xu H, Wei X, Xiao G, Ouwerkerk
PBF, Wang M, Zhu Z (2011) A transgenic study on affecting potato
tuber yield by expressing the rice sucrose transporter genes
OsSUT5Z and OsSUT2M. J Integr Plant Biol 53: 586–595
van Schreven DA (1956) On the physiology of tuber formation in
potatoes. I. Premature tuber formation. Plant Soil 8: 75–86
www.jipb.net
11
Vreugdenhil D, Struik PC (1989) An integrated view of the hormonal
regulation of tuber formation in potato (Solanum tuberosum).
Physiol Plant 75: 525–531
Wenzler HC, Mignery GA, Fisher LM, Park WD (1989) Analysis of a
chimeric class-I patatin-GUS gene in transgenic potato plants:
High-level expression in tubers and sucrose-inducible expression
in cultured leaf and stem explants. Plant Mol Biol 12: 41–50
Zubko E, Macháčková I, Malbeck J, Meyer P (2005) Modification of
cytokinin levels in potato via expression of the Petunia hybrida
Sho gene. Transgenic Res 14: 615–618
XXX 2015 | Volume XXXX | Issue XXXX | XXX-XX