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In vitro polyploidy induction: changes in morphology, podophyllotoxin

biosynthesis, and expression of the related genes in Linum album (Linaceae)

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DOI: 10.1007/s00425-017-2671-2

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In vitro polyploidy induction: changes in
morphology, podophyllotoxin biosynthesis,
and expression of the related genes in
Linum album (Linaceae)

Neda Javadian, Ghasem Karimzadeh,

Mohsen Sharifi, Ahmad Moieni &
Mehrdad Behmanesh

Planta
An International Journal of Plant
Biology

ISSN 0032-0935
Volume 245
Number 6

Planta (2017) 245:1165-1178

DOI 10.1007/s00425-017-2671-2

1 23
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 Author's personal copy
Planta (2017) 245:1165–1178
DOI 10.1007/s00425-017-2671-2

ORIGINAL ARTICLE

In vitro polyploidy induction: changes in morphology,

podophyllotoxin biosynthesis, and expression of the related genes
in Linum album (Linaceae)
Neda Javadian1 • Ghasem Karimzadeh1 • Mohsen Sharifi2 • Ahmad Moieni1 •

Mehrdad Behmanesh3

Received: 15 December 2016 / Accepted: 4 March 2017 / Published online: 14 March 2017
Ó Springer-Verlag Berlin Heidelberg 2017

Abstract genes involved in the PTOX biosynthetic pathway,

Main conclusion Induction of tetraploidy was per- including phenylalanine ammonia-lyase (PAL), cinnamoyl-
formed and podophyllotoxin production increased by Coa reductase (CCR), cinnamyl-alcohol dehydrogenase
upregulating the expression level and enzyme activity of (CAD), and pinoresinol-lariciresinol reductase (PLR). The
genes related to its biosynthesis in tetraploid compared tetraploid plants had larger leaves and stomata (length and
to diploid Linum album. width) and lower density stomata. Increasing the ploidy
level from diploid to tetraploid resulted in 1.39- and 1.23-
Linum album is a valuable medicinal plant that produces
fold enhancement of PTOX production, respectively, in the
antiviral and anticancer compounds including podophyl-
leaves and stem. The increase in PTOX content was
lotoxin (PTOX). To achieve homogeneous materials,
associated with upregulated activities of some enzymes
in vitro diploid clones were established, and their nodal
studied related to its biosynthetic pathway and the
segments were exposed to different concentrations and
expression of the corresponding genes. The expression of
durations of colchicine. This resulted in successful tetra-
the PAL gene and PLR enzymatic activity had the most
ploidy induction, confirmed by flow cytometry, and is
positive correlation with the ploidy level in both leaf and
being reported for the first time. The highest efficiency of
stem tissues. Our results verified that autotetraploid
tetraploid induction (22%) was achieved after 72 h expo-
induction is a useful breeding method, remarkably
sure to 2.5-mM colchicine treatment. The stable tetraploids
increasing the PTOX content in the leaves and stem of L.
were produced after being subcultured three times, and
album.
their ploidy stability was confirmed after each subculture.
The effects of autopolyploidy were measured on the mor-
Keywords Linum album  Podophyllotoxin  Tetraploidy 
phological and phytochemical characteristics, as well as
Gene expression  Flow cytometry
enzyme activity and the expression levels of some key

Electronic supplementary material The online version of this Introduction

article (doi:10.1007/s00425-017-2671-2) contains supplementary
material, which is available to authorized users.
& Ghasem Karimzadeh
karimzadeh_g@modares.ac.ir
1
Department of Plant Breeding, Faculty of Agriculture,
Tarbiat Modares University, P. O. Box 14115-336, Tehran,
Iran
2
Department of Plant Biology, Faculty of Biological Sciences,
Tarbiat Modares University, Tehran, Iran
3
Department of Genetics, Faculty of Biological Sciences,
Tarbiat Modares University, Tehran, Iran
Polyploidy has long been considered a prominent process
in the evolution of plant species, and it plays a key role in
plant species diversity (Mishra et al. 2010). Polyploidy can
arise via duplication of a whole genome (autopolyploid) or
by combining two or more genomes from different species
(allopolyploid) (Lee and Chen 2001). Polyploids often
exhibit valuable new phenotypic traits compared with their
diploid progenitors (Głowacka et al. 2010). However, the
underlying mechanisms for the appearance of these new
phenotypes are poorly understood in polyploid plants. They

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1166
probably associate with alterations in gene expression
through increased variation in dosage-regulated gene
expression, altered regulatory interactions, and rapid
genetic and epigenetic changes (Osborn et al. 2003). Linum
album is a herbaceous plant endemic to Iran, a cross-pol-
linated, diploid (2n = 2x = 26), perennial of the Linaceae
family, having a podophyllotoxin (PTOX) metabolite
(Mohagheghzadeh et al. 2006). This substance is a phar-
maceutically natural product belonging to the lignan
chemical group. It is used as a precursor for the synthesis of
antitumor drugs like (Etoposide, Teniposide, and Etopo-
phos) (Gordaliza et al. 2004; Ahmadian Chashmi et al.
2012), but its industrial production is not economically
feasible. Therefore, various strategies have been used to
increase the production of PTOX in this plant (Yousefzadi
et al. 2010b). Autopolyploidy can lead to changes in
cytological, biochemical, physiological, and developmental
characteristics in many species (Lavania 2005; Majdi et al.
2010; Abdoli et al. 2013; Tavan et al. 2015). Natural
polyploidy does not exist in all plant genera, so it has been
artificially induced in some plants over the past decades
(Dhooghe et al. 2011). Synthetic autopolyploidy can be
induced in plant cells by several antimitotic agents such as
colchicine (Planchais et al. 2000). In many studies, col-
chicine, a poisonous alkaloid extracted from autumn crocus
(Colchicum autumnale), is used to produce autopolyploids
in plants (Thao et al. 2003; Majdi et al. 2010; Ye et al.
2010; Abdoli et al. 2013; Tavan et al. 2015).
A literature survey revealed that artificial autopoly-
ploidy induction can potentially increase the production of
some secondary metabolites in medicinal plants when these
compounds are in the vegetative organs, such as diosgenin,
artemisinin, parthenolide, and scopolamine in Dioscorea
zingiberensis (Heping et al. 2008), Artemisia annua (De
Jesus-Gonzalez and Weathers 2003), Tanacetum parthe-
nium (Majdi et al. 2010), and Hyoscyamus muticus (De-
hghan et al. 2012) plants, respectively. Hence, the
induction of tetraploidy can be used as a method for
improving the production of secondary metabolites in
medicinal plants (Lin et al. 2011). Research on the induc-
tion of tetraploidy in medicinal plants has mainly focused
on the production of plants with a higher secondary
metabolite content, and few studies have been performed in
connection with the assessment of gene expression chan-
ges, along with a secondary metabolite biosynthetic path-
way, in newly synthesized autotetraploid plants. Analysis
of gene expression changes in relation to secondary
metabolite biosynthesis pathways in some synthetic
autopolyploids of medical plants showed that transcript
levels of a number of genes increased, owing to the change
in the ploidy level. (Mishra et al. 2010; Lin et al. 2011).
However, transcriptome analyses in synthetic autopoly-
ploids of Isatis indigotica, Solanum phureja, and Paspalum
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Planta (2017) 245:1165–1178
notatum showed that only few genes display significant
expression changes compared with those of their diploid
counterparts (Martelotto et al. 2005; Lu et al. 2006; Stupar
et al. 2007).
Several plants such as L. album that are cross-pollinated
tend to exhibit significant genetic variation, both among
individuals within a population and between populations
(Millar and Libby 1989). Hence, the existence of genetic
variation among individuals can affect on their response to
ploidy levels and other characteristics. One of the best
ways to achieve homogeneous plant materials with no
genetic variation is the production of in vitro clones
through tissue culture methods. Hence, in the present study,
in vitro clones of L. album were produced via tissue cul-
ture. The aim of this research was to establish an efficient
protocol for in vitro tetraploid induction in L. album using
colchicine treatments to assess the different morphological
and phytochemical characteristics of tetraploid and diploid
plants. Furthermore, with the alteration of the ploidy level,
the relationships between expression changes and the
enzyme activity of some key genes involved in the
biosynthesis of lignans were analyzed.
Materials and methods
Plant material and in vitro propagation
Shrubs of L. album Kotschy ex Boiss were collected from
natural habitats in Taleghan city (36°290 N, 50°350 E and
altitude of 1870 m), Iran. This plant was recognized by
comparison with a voucher specimen from the herbarium
of the Faculty of Biological Sciences, Tarbiat Modares
University, Tehran, Iran. Stems from the shrubs were
separately excised and washed in running water for 5 min.
After removing the leaves, the stems were surface sterilized
by immersion in 70% (v/v) ethanol for 30 s, and 1% (v/v)
sodium hypochlorite for 10 min, and finally rinsed three
times with sterile distilled water. The stems of each shrub
were cut into segments with at least two axillary buds and
were placed on shoot proliferation MS culture medium
(Murashige and Skoog 1962) supplemented with
0.4 mg l-1 Kin, 30 g l-1 sucrose, and 1% (w/v) agar, and
incubated at 25 °C with a 16-h photoperiod under 16/8-h
light/dark. The regenerated shoots of each shrub were
subcultured on the same medium every 6 weeks to produce
clones of L. album. Eventually, six in vitro diploid clones
of L. album were produced.
Extraction and analysis of PTOX in different clones
To select a clone containing the highest amount of
PTOX, clones were assessed for their PTOX content.
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Planta (2017) 245:1165–1178
Hence, dried and powdered leaves and stems, 0.5 g per
clone, were extracted by sonication in methanol (80%
v/v) for 1 h. For the partition of compounds in the
extract, dichloromethane and water (1:1 v/v) were added
and centrifuged for 10 min at 70009g. The under frac-
tions containing dichloromethane and PTOX were col-
lected, dried, and dissolved in 500 ll of methanol and
then injected into high-performance liquid chromatog-
raphy (HPLC) (Yousefzadi et al. 2010a). Three biologi-
cal replicates were used for each sample. The
quantification of PTOX in the samples was determined,
using HPLC as described previously by Yousefzadi et al.
(2010a). PTOX was monitored by UV absorption at
290 nm and identified by comparison of the retention
time and UV spectral peaks of the sample with the
authentic standard of PTOX.
In vitro induction of polyploidy in nodal segments
Regenerated shoots from axillary buds of the clones
containing the highest content of PTOX were selected for
tetraploid induction. The 4–7-mm-long nodal segments
were obtained from 60-day-old shoots. They were placed
in liquid shoot regeneration medium supplemented with
filter-sterilized colchicine (Sigma-Aldrich, USA) at con-
centrations of 0, 1.25, 2.5, and 5 mM, and incubated at
24 ± 2 °C on an orbital shaker (100 rpm) in darkness for
24, 48, 72, and 96 h. A total of 30 nodal segments were
used per treatment in three replications. Liquid shoot
generation medium without colchicine was used as a
control. After these treatments, the nodal segments were
washed three times with sterile distilled water and then
cultured on shoot regeneration medium. After 60 days,
the survival rate of nodal segments, the number of
regenerated shoots, and the tetraploid induction rate were
recorded.
Flow cytometric (FCM) analysis
DNA-ploidy levels of in vitro shoots (approximately
5 cm in height) obtained from colchicine treatments
were analyzed according to the method described by
Loureiro et al. (2007). These in vitro shoots were
regenerated from axillary buds from the clone containing
the highest content of PTOX. Glycine max cv. Polanka
(2n = 2x = 22, 2C = 2.5 pg) was used as the internal
reference standard plant (Doležel et al. 1994). Approxi-
mately, 1 cm2 leaf tissue of L. album and one cm2 of
internal standard were chopped with a sharp razor blade
in one ml Woody Buffer Plant (WBP; Loureiro et al.
2007). The suspension of isolated nuclei was filtered
through a Partec (Partec, Münster, Germany) 30 lm
nylon mesh filters to remove cell debris. The nuclear
1167
suspension of each sample was treated with 50 lg ml-1
RNase (Sigma-Aldrich Corporation, MO, USA) to pre-
vent staining of double-stranded RNA and stained with
50 lg ml-1 propidium iodide (PI, Fluka). Samples were
then incubated on ice and analyzed by BD FACSCan-
toTM flow cytometer (BD Biosciences, Bedford, MA,
USA), using the BD FACSDivaTM Software. More than
5,000 stained nuclei were analyzed per sample. Data
were first transferred to Flowing Software version 2.5.0,
followed by Partec FloMax Software version 2.4e (Par-
tec, Münster, Germany). The absolute DNA content of L.
album was calculated based on the G1 peak means
(Doležel and Bratoš 2005; Mahdavi and Karimzadeh
2010; Karimzadeh et al. 2010, 2011) as follows:
Sample 2C DNA ðpgÞ
¼ ðSample G1 peak mean=Standard G1 peak meanÞ
 Standard 2C DNA ðpgÞ:
The mean coefficients of variation (CV%) of FCM
measurements of either L. album sample or reference
standard plants were less than 5%. The confirmed FCM-
based tetraploid plants were designed as the zero vegetative
generation of the first-generated tetraploid plants (T1V0).
Subsequently, T1V0 were subcultured three times on shoot
proliferation medium to produce T1V1, T1V2, and T1V3
plants. In all generations, plants were again re-analyzed by
FCM to re-confirm the stability of their tetraploidy status.
Only regenerated tetraploids that maintained their ploidy
level were considered for subculturing for the production
of the next generation. The control diploid plants were
simultaneously subcultured with tetraploid plants in dif-
ferent vegetative generations. The following analysis was
conducted on plants that kept their tetraploidy from gen-
eration T1V0 to generation T1V3.
Morphological analysis
Length, width, and density of stomata were determined in
the lower epidermis of the leaves by the impression
method reported by Khazaei et al. (2009). The stomatal
density and size of each leaf were photographed. Their
density was counted at 209 magnification, and their
length and width were measured at 1009, using a DP12
digital camera interfaced to a BX50 Olympus microscope
(Olympus Optical Co., Ltd., Tokyo, Japan) and
MicroMeasure 3.3 software. The length and the width of
the leaves were also measured by a digital caliper. These
characteristics were measured, using 30 in vitro-derived
leaves collected from five of the 60-day-old T1V3 tetra-
ploid and diploid plants. In addition, the diameter of the
basal stem was measured from 30 in vitro-regenerated
plants of each ploidy level.
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1168
PTOX measurement
The leaves and the stems of in vitro T1V0 and T1V3 tetra-
ploid and those of diploid plants (60 days old) were sepa-
rately dried at room temperature. The content of PTOX was
determined according to the method described in the section
‘‘Extraction and analysis of PTOX in different clones’’.
Total phenolic and flavonoid measurement
Total phenolic and flavonoid contents were determined in
the leaves and the stems of T1V3 tetraploid and in those of
diploid plants, according to the method described by Akkol
et al. (2008). These compounds were extracted by 80% (v/
v) methanol. Total soluble phenolics were estimated from
the calibration curve of gallic acid standard solutions by the
Folin Ciocalteu method. Total flavonoid contents were
calculated from the calibration curve and prepared, using
rutin, by the aluminum chloride method. Total phenolic
and flavonoid contents were expressed as mg of gallic acid
and rutin equivalent per 1 g of dry weight, respectively.
Lignin measurement
Lignin analysis was carried out on the leaves and stem of
T1V3 tetraploid and in those of diploid plants, by an
improved acetyl bromide procedure (Iiyama and Wallis
1988). The lignin content was also determined from
absorbance at 280 nm and a specific absorption coefficient
value of 20 g-1 l-1 cm.
PAL and CAD assay
For determination of PAL and CAD enzyme activities,
extracts of leaf and stem tissues (100 mg) of T1V3 tetra-
ploid and diploid plants (60 days old) were prepared as
described by Garden (2003). All steps were carried out in an
ice bath (at 4 °C). The extracts were centrifuged at (20 min,
13,000g) at 4 °C, and the supernatants were collected for
enzyme assay. Protein concentrations were determined
according to Bradford (1976), with bovine serum albumin
(BSA) as a standard. PAL and CAD activities were mea-
sured spectrophotometrically by monitoring the formation of
trans-cinnamic acid at 290 nm and conifer aldehyde at
390 nm, respectively, as described by Wang et al. (2006).
PLR assay
The protein extract was prepared by the method of Garden
(2003) and then concentrated using an Amicon Ultra cen-
trifugal filter device with cutoff 30 kDa (Millipore). The
protein concentration was determined according to Brad-
ford (1976). The concentrated protein was used for the
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Planta (2017) 245:1165–1178
determination of PLR activity according to the method of
Hemmati (2007). The assay mixtures (500 ll) consisted of
400 lg protein extract, 10 mM (?)-pinoresinol, 25 mM
NADPH, and 0.1 M potassium phosphate buffer (pH 7.1).
The reaction mixture containing protein and buffer was
pre-incubated for 15 min at 30 °C, and then NADPH was
added to initiate enzyme reaction. After overnight incu-
bation, the reaction was stopped and extracted with
(3 9 500 ll in total) ethyl acetate. The combined ethyl
acetate phases were dried under vacuum. The residue was
dissolved in 100-ll methanol and subjected to HPLC
analysis according to the method reported by Yousefzadi
et al. (2010a). PLR activity was assayed by monitoring the
formation of (?)-lariciresinol. (?)-lariciresinol was moni-
tored by UV absorption at 290 nm and identified by
comparison of the retention time and UV spectral peaks of
the sample with the authentic standard of lariciresinol.
Total RNA extraction and gene expression analyses
The three genes of PAL, CCR, and CAD, based on their
involvement in the phenylpropanoid production pathway
and the PLR as a main key gene in lignan production, were
selected to elucidate the expression level of genes by
quantitative polymerase chain reaction (PCR). Total RNA
of leaf and stem tissues from 60-day-old T1V3 tetraploid
and diploid plants was extracted, using a RibospinTM plant
kit (GeneAll, Korea). The extracted RNA was treated with
DNase I (Sigma, USA) at 37 °C for 30 min to remove any
genomic DNA contamination, according to the manufac-
turer’s instruction. The quality and quantity of RNA were
assessed using agarose gel electrophoresis and Nanodrop
(Thermo Scientific, Germany) spectrophotometer analyses,
respectively. Two micrograms of total RNA from each
sample was reverse transcribed by an anchored oligo (dT18)
primer and RevertAid RT (Thermo Scientific) to synthesize
the first-strand cDNA, according to the manufacturer’s
protocol. Specific primers used for real-time quantitative
PCR (qPCR) were from the partial cDNA sequences
(Supplementary Table S1) (Yousefzadi et al. 2012). The
qPCR reactions were performed with an ABI PRISM 7500
sequence detection system (Applied Biosystems, USA) and
using the SYBR Green PCR Master Mix (Takara, Japan).
PCR amplification was carried out with an initial phase at
95 °C for 15 min, followed by 40 cycles of 15 s at 95 °C
and 20 s at 58 °C. L. album Actin gene expression was
selected as an internal control for data normalization. To
gain a standard curve for each gene transcript, a serially
diluted cDNA was used. The relative expression of each
gene was analyzed by the 2DDCT method, as described by
Livak and Schmittgen (2001). The expression analyses
were performed with three biological replicates for each
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Planta (2017) 245:1165–1178
sample and two technical replicates for each biological
sample.
Statistical analysis
The effects of different concentrations and the durations of
colchicine on survival rate, tetraploidy induction, tetraploid
induction efficiency, and regenerated shoots were statisti-
cally analyzed, using a factorial experiment on the basis of
completely randomized design (CRD), with three replica-
tions (10 explants per replication). The means were com-
pared using the Least Significant Difference (LSD) test at
the different significance levels of 0.05, 0.01, and 0.001,
respectively, by Minitab Release 16 (Minitab Inc., State
College, PA, USA; Ryan and Joiner 2001) and SAS
Release 9.0. (SAS Institute Inc 2002) softwares. Tetra-
ploidy induction efficiency was calculated as follows
(Bouvier et al. 1994; Majdi et al. 2010):
Induction efficiency ¼ %Seedling survival
 %Tetraploidy induction:
Differences between mean values between diploid and
tetraploid plants for other analyses were evaluated using
the Student t test. All data were examined for normality
and homogeneity of variances prior to analyses.
Results
Selection of the highest PTOX clone
To ensure that the observed morphological, phytochemical,
and molecular changes between synthetic autotetraploid
and diploid were not caused by genetic variation in L.
album, in vitro clones of this plant were provided via tissue
culture and then used for the different measurements. The
content of PTOX in clones was measured to select the
clone containing the highest content of PTOX to induce
tetraploidy. Analysis of variance (ANOVA) showed that
PTOX content between clones had significant differences
(P \ 0.01, Supplementary Table S2). The fifth clones,
which were identified to have the highest content of PTOX
(Fig. 1), were selected for tetraploidy induction.
Survival rate and regenerated shoots of colchicine-
treated nodal segment
The effects of different concentration and duration of col-
chicine treatments on survival rate and shoot number were
examined 60 days after the exposure of the treatments.
ANOVA showed that the interaction of concentra-
tion 9 duration of colchicine significantly affected the
survival rate and the number of shoots (P \ 0.05 and
1169
50
Podophyllotoxin content (µg g FW)
40
a
-1
30
ab
b
20
b
b
b
10
0
1 2 3 4 5 6
Clone number
Fig. 1 Podophyllotoxin content in in vitro-derived shoots of different
2x clones of L. album. Bars represent means (n = 3) ± SE. Different
letters are significantly (P \ 0.01) different according to LSD test
P \ 0.001, respectively; Supplementary Table S3). After
colchicine treatments, some nodal segments turned brown
and died. The survival rate gradually decreased in all the
concentrations of colchicine, along with increased duration
of treatment. The highest and the lowest survival rates
(83.33 and 26.66%) resulted from treating nodal segments
with 1.25 mM for 24 h and 5-mM colchicine for 96 h,
respectively. In 5-mM colchicine concentration, the highest
decrease in the rate of survival (63.33–26.67%) was
observed from 48 to 96-h duration of treatment, and this
was significantly different from the other treatments
(Table 1). Shoots produced from nodal segments were also
influenced by the different colchicine treatments. Higher
concentrations and longer durations decreased the number
of shoots produced. The number of shoots produced from
colchicine-treated nodal segments ranged from 3.5 to 1.41.
The lowest number of shoots produced was obtained from
treatment by 5 mM for 96 h colchicine. In this concen-
tration of colchicine, no significant difference was detected
between 96- and 48- or 72-h treatment durations (Table 1).
Tetraploidy determination
Flow cytometric analysis of colchicine-treated and control
in vitro-regenerated plants was carried out to identify their
ploidy levels. The FCM histograms indicated that the ratio
of reference standard plant and L. album peak positions in
the tetraploid plants was approximately twice that of the
diploid plants. Furthermore, tetraploids, mixoploid plants
(2x ? 4x), were also produced from in vitro colchicine
treatments of nodal segments. In the FCM, the peak of a
control diploid (2x) was adjusted to appear at the 70–75
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1170 Planta (2017) 245:1165–1178

Table 1 Mean comparison of different concentrations and durations of colchicine on tetraploidy induction characteristics in L. album
Colchicine concentration Treatment duration Survival rate Tetraploidy rate Tetraploid induction efficiency Shoot production
(mM) (h) (%)a (%)b (%)c (no.)d

0 (control)e 24 96.67a ± 3.33 0.00f ± 0.00 0.00d ± 0.00 4.30a ± 0.10

a f d
48 96.67 ± 3.33 0.00 ± 0.00 0.00 ± 0.00 3.96ab ± 0.03
a f d
72 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 4.01a ± 0.08
ab f d
96 93.33 ± 3.33 0.00 ± 0.00 0.00 ± 0.00 3.95ab ± 0.10
bc f d
1.25 24 83.33 ± 3.33 0.00 ± 0.00 0.00 ± 0.00 3.50b ± 0.11
bc ef cd
48 80.00 ± 5.77 6.67 ± 3.33 5.33 ± 2.73 2.91c ± 0.10
72 76.67cde ± 6.67 13.33cde ± 3.33 10.67bc ± 3.67 2.59cd ± 0.02
96 66.67efg ± 3.33 10.00def ± 0.00 6.67cd ± 0.33 2.55cd ± 0.15
2.5 24 76.67cde ± 3.33 10.00def ± 5.70 7.33cd ± 4.06 2.95c ± 0.04
def bc ab
48 70.00 ± 5.77 23.33 ± 3.33 16.67 ± 3.71 1.99ef ± 0.08
fg a a
72 60.00 ± 5.77 36.67 ± 3.33 22.00 ± 3.66 1.61fg ± 0.06
96 56.67gh ± 3.33 20.00bcd ± 5.70 11.00bc ± 2.65 1.57fg ± 0.03
efg cde bcd
5 24 66.67 ± 3.33 13.33 ± 3.33 9.00 ± 2.52 2.26de ± 0.08
fg ab ab
48 63.33 ± 6.67 26.67 ± 3.33 16.67 ± 2.19 1.76efg ± 0.08
h bcde bcd
72 46.67 ± 3.33 16.67 ± 3.33 8.00 ± 2.00 1.50g ± 0.08
i cde cd
96 26.67 ± 6.67 13.33 ± 3.33 4.00 ± 0.67 1.41g ± 0.04
LSD LSD5% = 13.14 LSD1% = 12.07 LSD1% = 9.01 LSD0.1% = 0.47
Each value represents the mean ± SE of three replicates. Means in the same column followed by the same letter are not significantly different at
given LSD value
a
The percentage of survived nodal segments compared to total of the treated nodal segments
b
The percentage of shoots that converted to tetraploid
c
Calculated by formula: survival rate (%) 9 tetraploidy rate (%)
d
Mean number of the regenerated shoots of the treated nodal segments
e
Nodal segments cultured in liquid shoot culture medium without colchicine

channel (Fig. 2a). The peak of tetraploid plants (4x) was
expected at channel 140–150 (Fig. 2b), and peaks of
mixoploid plants were expected at both channels (Fig. 2c).
The standard peak was adjusted to appear at about channel
40 (Fig. 2a, b). The mean 2C DNA contents of diploids and
tetraploids were determined as 4.34 ± 0.08 and
8.36 ± 0.12 pg, respectively. The mean CV% of each
DNA peak of either sample or reference standard plants
was less than 5% in all histograms. Since the mixoploid
plants consisted of different numbers of both diploid and
tetraploid nuclei, they were not considered for further
analyses. ANOVA showed that the interaction of concen-
tration 9 duration of colchicine had a significant impact on
tetraploidy induction (P \ 0.01, Supplementary Table S3)
and tetraploidy induction efficiency percentage (P \ 0.01,
Supplementary Table S3). The highest percentage of tet-
raploidy induction was 36.67% when nodal segments were
treated with 2.5 mM colchicine for 72 h. The result of this
treatment showed no significant difference with 26.66%
tetraploidy induction, which was produced from 5-mM
colchicine for 48 h. In all concentrations of colchicine,
prolonging the duration of treatment from 72 to 96 h
resulted in decreased tetraploidy induction. The efficiency
of tetraploid induction is a suitable parameter to determine
the best treatment for the induction of tetraploidy, because
it considers both survival and tetraploid rate production
(Lehrer et al. 2008; Majdi et al. 2010). Hence, the 2.5-mM
colchicine for 72 h appeared to be the most effective
treatment in tetraploid induction efficiency (22%), which
resulted from a 60% survival rate and 36.67% tetraploid
induction. However, no significant difference was detected
between this concentration for 48 h with 5 mM colchicine
concentration for 48 h. In concentrations 1.25 and 2.5 mM,
the tetraploidy induction percentage and tetraploidy
induction efficiency increased with increasing duration
from 24 to 72 h.
Morphological changes
Remarkable differences in the morphological characteris-
tics of in vitro-derived leave and stems were identified
between diploid and tetraploid plants in the T1V3 gener-
ation under the same growing condition. Table 2 shows
morphological differences between diploid and tetraploid
plantlets. Specifically, tetraploid plants showed signifi-
cantly 1.8-fold thicker stem, 1.8-fold wider and longer

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Planta (2017) 245:1165–1178
Fig. 2 Flow cytometric histograms of nuclei isolated from in vitro-
derived leaves of Linum album 2x (a), 4x (b), and mixoploid
(c) plants. The left peaks refer to G1 phases of the cell cycle of
reference standard plant (Glycine max cv. Polanka, 2C
DNA = 2.50 pg) and the right peaks refer to the G1 of L. album
samples. The mean coefficient of variations (CV%) of the peaks was
less than 5%. a. u. = arbitrary units
leaves, 1.4-fold wider, and 1.6-fold longer stomata on the
lower leaf surface but 48% less stomatal density than in
diploid leaves (Figs. 3, 4).
Effect of tetraploidy on lignin, total phenolic,
and flavonoid
The results indicated that the contents of lignin, total
phenolic and flavonoid in the stems, and leaves of
1171
tetraploid plants increased significantly (P \ 0.05) com-
pared to those of diploid plants. The leaves and stems of
tetraploid plants had more lignin content (1.37- and 1.18-
fold increase), total phenolics (1.8- and 2.1-fold increase),
and flavonoids (1.55- and 1.53- fold increase) than diploid
plants (Fig. 5).
Effect of tetraploidy on PTOX
PTOX content was higher (P \ 0.05; Supplementary
Fig. S1) in the leaves than in the stems in both the diploid
and tetraploid plants and in both T1V0 and T1V3 genera-
tions. The leaves of the tetraploid plants contained 1.66-
fold more PTOX in T1V0 and 1.33-fold in T1V3 compared
to those of the diploid plants in the same vegetative gen-
erations. On the other hand, tetraploid stems contained a
higher amount of PTOX in both T1V0 and T1V3 (1.48-
and 1.33-fold increase, respectively, Fig. 6) compared to
those in the diploid stems in the same vegetative genera-
tions. In tetraploids and diploids, no remarkable changes
were identified in the content of PTOX in T1V3 in com-
parison with that in T1V0 (Supplementary Fig. S2).
PAL, CAD, and PLR enzyme activity
Our results showed that PAL and CAD enzyme activities
were much higher than PLR activity in different tissues of
tetraploid and diploid plants. PAL activity in the leaves and
stems of tetraploids showed significant (P \ 0.05) 1.64-
and 1.47-fold increases, respectively, compared with those
of diploids. CAD activity was significantly enhanced in
tetraploid leaves compared to that in diploids. No PLR
activity was recognized in total protein extraction of
diploid leaves and stems, but its activity in the tetraploid
leaves and stems was increased 2.51- and 1.68-fold
(lM lariciresinol mg-1 protein h-1), respectively (Fig. 7).
Gene expression
Quantitative PCR was performed to study the influence of
increasing the ploidy level on the expression of several
genes involved in the PTOX biosynthetic pathway in
in vitro-derived leaves and stems of L. album. The results
showed that the transcription levels of PAL, CCR, and CAD
genes were significantly increased in the leaves and stems
of tetraploids compared to those of diploids, but the
expression of the PLR gene was significantly increased
only in the leaves of tetraploids compared to diploids. A
higher expression of PAL, CAD, and PLR genes was
detected in the leaves than in the stems of tetraploids,
whereas the CCR expression was conversely higher in the
stems than in the leaves of tetraploids. The PAL gene
showed a higher level of transcription than CCR, CAD, and
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1172 Planta (2017) 245:1165–1178

Table 2 Comparison of morphological characteristics between 2x and 4x Linum album

Ploidy level Stem diameter (mm) Leaf Stomata
Width (mm) Length (mm) Width (lm) Length (lm) Density (mm2)

2x 0.82b ± 0.21 1.64b ± 0.04 4.52b ± 0.10 29.35b ± 0.87 39.94b ± 0.80 47.67a ± 0.95
a a a a a
4x 1.49 ± 0.22 2.88 ± 0.08 8.01 ± 0.15 41.17 ± 1.36 64.76 ± 1.32 24.76b ± 0.56
Data are means (n = 32) ± SE. Values in each vertical column, followed by different letters, are significantly (P \ 0.01) different according to
two-sample Student’s t test

increased ratio of the expression level of their related

genes, whereas the reverse occurred in PLR. In fact, tet-
raploidy induction had more effect on enzymatic activity
than on the expression level of both PAL and CAD genes,
while in contrast, it had more effect on the expression level
of PLR gene than on its enzymatic activity (data not
shown).

Discussion

The present study is the first report of the successful

Fig. 3 In vitro-derived shoots of 2x (a) and 4x (b) of Linum album.
Bar = 1 cm
PLR genes in the leaves and stems of tetraploids than in
those of diploids (Fig. 8). Comparing the ratio of the
expression level of PAL, CAD, and PLR in tetraploids to
those in diploids showed that the increased ratio of enzy-
matic activity of CAD and PLR was more than the
induction of tetraploidy in L. album by treating diploid
nodal segments of in vitro clones with colchicine. Indeed,
the in vitro clones of L. album were established to achieve
uniform plant materials, because L. album is cross-polli-
nated, having genetic variation among individual plants
(Millar and Libby 1989). In vitro treatment of nodal seg-
ments with colchicine was successfully applied to the
induction of polyploidy in different plant species, including
Zingiber officinale (Adaniya and Shirai 2001), Pyrus
pyrifolia (Kadota and Niimi 2002), Punica granatum (Shao
et al. 2003), Alocasia (Thao et al. 2003), Zizyphus jujuba
Mill. (Gu et al. 2005), and Thymus persicus (Tavan et al.
2015). The effect of colchicine on the survival rate and the
number of shoots produced in the nodal segments depended

Fig. 4 Stomatal size and density in lower leaf epidermis of 2x (a) and 4x (b) Linum album plants

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Planta (2017) 245:1165–1178 1173

Fig. 5 Histograms showing the a b

contents of total phenols (a),

Total phenols content (mg g-1 DW Galic acid)

25 16
flavonoids (b), and lignins (c), a 2x a 2x
in in vitro-derived leaf and stem 4x

Flavonoids content (mg g-1 DW Rutin)

of 2x and 4x Linum album. Bars 4x
20
represent means (n = 3) ± SE.
12
Different letters are significantly
(P \ 0.05) different according
to Student’s t test 15 b
b
8
a
10
a

b 4 b
5

0 0
Leaf Stem Leaf Stem

c
20 2x
4x
a
b
Lignin (% Cell wall dry weight)
15

10 a

b
5

0
Leaf Stem

a b
0.8 0.8
2x 2x
Podophyllotoxin content (mg g-1 DW)

4x 4x

0.6 0.6

a
a
0.4 0.4
b b

0.2 a 0.2
a
b b

0 0
Leaf Stem Leaf Stem

Fig. 6 Podophyllotoxin content in 2x and 4x L. album in in vitro- represent means (n = 3) ± SE. Different letters are significantly
derived leaf and stem of zero vegetative generation (T1V0) plant (P \ 0.05) different according to Student’s t test
(a) and of the third vegetative generation (T1V3) plant (b). Bars

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1174 Planta (2017) 245:1165–1178

Fig. 7 Activities of PAL (a), a

CAD (b), and PLR (c) enzymes
b

CAD activity µM Conifer aldehyde mg-1 protein min-1

in in vitro-derived leaf and stem

PAL activity µM Cinnamic acid mg-1 protein min-1

25 55
of 2x and 4x Linum album. Bars a 2x a 2x
represent means (n = 3) ± SE.
4x 4x
Different letters are significantly
(P \ 0.05) different according
to Student’s t test 20 45
b

15 b 35

a a
10 25
a
b

5 15
Leaf Stem Leaf Stem

c
PLR activity µM Lariciresinol mg-1 protein min-1 4
2x
4x

3
a

2
a

b b
0
Leaf Stem

on the concentration and the duration of treatments. In and Karimzadeh 2010; Karimzadeh et al. 2010, 2011). In
general, these two parameters reduced with the higher the present study, the highest percentage of tetraploid
concentration and longer duration of exposure to colchicine induction efficiency was detected as 22% after treating of
treatment. Similar results have been reported in other the in vitro nodal segment with 2.5-mM colchicine for
plants, including Alocasia micholitziana (Thao et al. 2003), 72 h. In the high concentration of colchicine (5 mM),
two species of Miscanthus (sinensis and 9 giganteus) lower treatment duration (48 h) was the most effective
(Głowacka et al. 2010), and Jatropha curcas (De Oliveira treatment for tetraploid induction efficiency (16.67%), but
et al. 2013). The mortality and reduction in the shoot longer duration caused reduction in the percentage of
production of colchicine-treated nodal segment can be induction efficiency. Such an interaction between the
associated with chemical damage by colchicine, which concentration and duration of colchicine treatments was
inhibited growth, and the penetration of colchicine into the also reported in other studies (Aina et al. 2012; De Oliveira
apical cell layers, which affected the normal division of the et al. 2013). The highest efficiency of tetraploid induction
cells (Thao et al. 2003; Ascough et al. 2008). in L. album was 22%, which compared favorably with
The ploidy level in the shoot regeneration of nodal those reported for Smallanthus sonchifolius (2.5%;
segments treated with colchicine was determined by flow Viehmannová et al. 2009), Populus pseudo-simonii
cytometry. It is a commonly used method for ploidy (14.1%; Cai and Kang 2011), and Phlox subulata (18%;
screening, because it is convenient, fast, and reliable Zhang et al. 2008), using in vitro colchicine treatment for
(Doležel and Bratoš 2005; Doležel et al. 2007; Mahdavi chromosome doubling. In other words, the tetraploid

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Planta (2017) 245:1165–1178 1175

Fig. 8 Expression of PAL (a), a b

CCR (b), CAD (c), and PLR 5 3
2x 2x
(d) genes in in vitro-derived leaf 4x
and stem of 2x and 4x Linum a 4x

CCR gene expression (fold)

album. Bars represent means 4

PAL gene expression (fold)

(n = 3) ± SE. Different letters
are significantly (P \ 0.05)
a
2
different according to Student’s a
t test. Relative expression of
3 a
genes calculated using qRT-
PCR based on the Ct values. The b
obtained Ct value for each 2 b
sample was normalized using 1
the housekeeping gene Actin b b
1

0 0
Leaf Stem Leaf Stem

c 3 d
2x 3 2x
4x 4x
CAD gene expression (fold)

PLR gene expression (fold)

a
2 a 2
a a

b b a
b
1 1

0 0
Leaf Stem Leaf Stem

induction efficiency in the present study in L. album was
8.8-, 1.6-, and 1.2-fold higher, respectively, than those in
the latter-mentioned reports. Some of the tetraploid plants
obtained by in vitro induction were stable after being
maintained in vitro for three cycles of subculture. Hence, in
stable tetraploids of L. album, stem diameter and the size of
leaves and stomatal cells increased, but the density of
stomata decreased. A correlation between ploidy level and
morphological characteristics has been reported previously
in numerous plant species such as P. subulata (Zhang et al.
2008), T. parthenium (Majdi et al. 2010), Ocimum basili-
cum and Dracocephalum moldavica (Omidbaigi et al.
2010a, b), Gerbera jamesonii Bolus cv. Sciella (Gantait
et al. 2011), Lychnis senno (Nonaka et al. 2011), Ullucus
tuberosus (Viehmannová et al. 2012), Echinacea purpurea
(Abdoli et al. 2013), and T. persicus (Tavan et al. 2015).
In plants, the major secondary metabolites are produced
in the phenylpropanoid pathway such as lignans, flavo-
noids, and lignins (Sreelakshmi and Sharma 2008). In the
present study, findings indicate that the content of PTOX
was higher in the leaves than in the stems, regardless of
ploidy levels. Mohagheghzadeh et al. (2006) reported that
PTOX content and related lignans varied in different tis-
sues of diploid L. album, which is in agreement with our
results. In the present study, the in vitro-derived leaves and
stems of tetraploid plants had higher PTOX content in
comparison to those of their diploid counterpart in either
T1V0 or T1V3 generations. The tetraploidy induction also
resulted in increased production of flavonoids and lignin.
Our results were in agreement with several previously
published papers that reported positive correlations
between ploidy levels and some amount of secondary
metabolites in medicinal artificially induced tetraploid
plants such as Salvia miltiorrhiza (Gao et al. 1996), A.
annua (Wallaart et al. 1999), Scutellaria baicalensis (Gao
et al. 2002), Papaver somniferum (Mishra et al. 2010), T.
parthenium (Majdi et al. 2010), and T. persicus (Tavan
et al. 2015). Hence, because of the interesting effects of

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1176
polyploidy (conspicuous changes in morphology and sec-
ondary metabolism), artificial polyploidy has been induced
in numerous plants (Thao et al. 2003; Omidbaigi et al.
2010a, b; Majdi et al. 2010; Ye et al. 2010; Abdoli et al.
2013; Dhooghe et al. 2011; Kaensaksiri et al. 2011; Tavan
et al. 2015).
The activities of some enzymes related to PTOX
biosynthetic pathway and the transcript levels of the cor-
responding genes in two organs (leaf and stem) were sur-
veyed to understand the molecular mechanism underlying
the increase of PTOX content in autotetraploid L. album.
The expression level and enzyme activity of these genes
increased in two tetraploid organs compared to those of the
diploid. Similar findings have been reported in synthetic P.
somniferum and A. annua autotetraploid, in which the
expression of some known genes related to morphinanes
and artimisinin biosynthesis increased in tetraploids com-
pared to diploids (Mishra et al. 2010; Lin et al. 2011). Our
present results indicated that duplicated genome dosage
can enhance the enzymatic activity and transcript levels of
the studied genes in PTOX biosynthetic pathway and
consequently the content of the PTOX in organs. The
results of the latter study verified that expression of some
genes is linearly correlated with ploidy level and can be
dramatically changed because of ploidy alteration. How-
ever, little data are still available elucidating the conse-
quences of autopolyploidization on gene expression, but
these alterations appear to be controlled by genetic and
epigenetic mechanisms (Lavania et al. 2012).
Conclusion
We have successfully induced in vitro tetraploidy in nodal
segments of L. album for the first time, using colchicine
treatment and also achieved stable tetraploid plants. The
highest tetraploid induction efficiency (22%) was achieved
after 72-h exposure to 2.5-mM colchicine treatment. The
tetraploids showed remarkable differences in their mor-
phological characteristics compared to diploids, such as
thicker stems, greater length and width of stomata, and
reduced stomatal density in the leaves. Hence, the doubling
of ploidy caused 1.66- and 1.48-fold increased accumula-
tion of PTOX in the leaves and in the stem, respectively.
This enhancement in PTOX content was correlated with
increased activities of some key enzymes and expression of
the corresponding genes in the PTOX biosynthetic path-
way. The tetraploidy induction had more effect on the
activities of enzyme PLR and the transcript level of the
PAL gene. As far as we know, further research will be
necessary to determine which mechanisms caused the
changes of gene expression due to increased ploidy level
123
Planta (2017) 245:1165–1178
and also to evaluate the influence of autotetraploidy on the
transcriptome and metabolome of L. album.
Author contribution statement NJ carried out all experi-
ments, analyzed data, and wrote the paper. GK and MSh
conceived and designed the experiments. GK analyzed the
data, directed through all steps of the experiments, and
participated in writing of the paper. AM conducted the
tissue culture experimental steps. MB contributed in the
design and analysis of gene expression experiment. All
authors read and approved the final manuscript.
Acknowledgements Authors gratefully acknowledge the support
provided for this survey by the Tarbiat Modares University, Tehran,
Iran. NJ thanks Dr. Najmeh Ahmadian Chashmi, Department of
Biology, University of Mazandaran, Babolsar, Iran, for her help and
advice throughout this research. We gratefully acknowledge Dr. Paul
Kron, Department of Integrative Biology, University of Guelph,
Ontario, Canada, for reviewing the manuscript and for his valuable
comments.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflicts of
interest.
References
Abdoli M, Moieni A, Naghdi Badi H (2013) Morphological,
physiological, cytological and phytochemical studies in diploid
and colchicine-induced tetraploid plants of Echinacea purpurea
(L.). Acta Physiol Plant 35:2075–2083
Adaniya S, Shirai D (2001) In vitro induction of tetraploid ginger
(Zingiber officinale Roscoe) and its pollen fertility and ger-
minability. Sci Hortic 88:277–287
Ahmadian Chashmi N, Sharifi M, Yousefzadi M, Behmanesh M,
Rezadoost H, Cardillo A, Palazon J (2012) Analysis of
6-methoxy podophyllotoxin and podophyllotoxin in hairy root
cultures of Linum album Kotschy ex Boiss. Med Chem Res
22:745–752
Aina O, Quesenberry K, Gallo M (2012) In vitro induction of
tetraploids in Arachis paraguariensis. Plant Cell Tissue Organ
Cult 111:231–238
Akkol EK, Göger F, Kosar M, Baser HC (2008) Phenolic composition
and biological activities of Salvia halophila and Salvia virgata
from Turkey. Food Chem 108:942–949
Ascough GD, Van Staden J, Erwin JE (2008) Effectiveness of
colchicine and oryzalin at inducing polyploidy in Watsonia
lepida NE Brown. HortScience 43:2248–2251
Bouvier L, Pillon FR, Lespinasse Y (1994) Oryzalin as an efficient
agent for chromosome doubling of haploid apple shoots in vitro.
Plant Breed 113:343–346
Bradford MM (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal Biochem 72:248–254
Cai X, Kang XY (2011) In vitro tetraploid induction from leaf
explants of Populus pseudo-simonii Kitag. Plant Cell Rep
30:1771–1778
 Author's personal copy
Planta (2017) 245:1165–1178
De Jesus-Gonzalez L, Weathers PJ (2003) Tetraploid Artemisia annua
hairy roots produce more artemisinin than diploids. Plant Cell
Rep 21:809–813
De Oliveira SC, Nunes ACP, Carvalho CR, Clarindo WR (2013)
In vitro polyploidization from shoot tips of Jatropha curcas L.: a
biodiesel plant. Plant Growth Regul 69:79–86
Dehghan E, Häkkinen ST, Oksman-Caldentey KM, Ahmadi FS
(2012) Production of tropane alkaloids in diploid and tetraploid
plants and in vitro hairy root cultures of Egyptian henbane
(Hyoscyamus muticus L.). Plant Cell Tissue Organ Cult
110:35–44
Dhooghe E, Van Laere K, Eeckhaut T, Leus L, Van Huylenbroeck L
(2011) Mitotic chromosome doubling of plant tissues in vitro.
Plant Cell Tissue Organ Cult 104:359–373
Doležel J, Bratoš J (2005) Plant DNA flow cytometry and estimation
of nuclear genome size. Ann Bot 95:99–110
Doležel J, Doleželová M, Novák FJ (1994) Flow cytometric
estimation of nuclear DNA amount in diploid bananas (Musa
acuminata and M. balbisiana). Biol Plant 36:351–357
Doležel J, Greilhuber J, Suda J (2007) Estimation of nuclear DNA
content in plants using flow cytometry. Nat Protoc 2:2233–2244
Gantait S, Mandal N, Bhattacharyya S, Das PK (2011) Induction and
identification of tetraploids using in vitro colchicine treatment of
Gerbera jamesonii Bolus cv. Sciella. Plant Cell Tissue Organ
Cult 106:485–493
Gao SL, Zhu DN, Cai ZH, Xu DR (1996) Autotetraploid plants from
colchicine-treated bud culture of Salvia miltiorrhiza Bge. Plant
Cell Tissue Organ Cult 47:73–77
Gao SL, Chen BJ, Zhu DN (2002) In vitro production and
identification of autotetraploids of Scutellaria baicalensis. Plant
Cell Tissue Organ Cult 70:289–293
Garden HJ (2003) Biotechnological production of podophyllotoxin by
Linum album suspension cultures. Ph.D. Dissertation, University
of Heinrich-Heine, Düsseldorf, Germany
Głowacka K, Je_zowski S, Kaczmarek Z (2010) In vitro induction of
polyploidy by colchicine treatment of shoots and preliminary
characterization of induced polyploids in two Miscanthus
species. Ind Crops Prod 32:88–96
Gordaliza M, Garcı́a PA, Corral MD, Castro MA, Gómez-Zurita MA
(2004) Podophyllotoxin: distribution, sources, applications and
new cytotoxic derivatives. Toxicon 44:441–459
Gu XF, Yang AF, Meng H, Zhang JR (2005) In vitro induction of
tetraploid plants from diploid Zizyphus jujuba Mill. cv. Zhanhua.
Plant Cell Rep 24:671–676
Hemmati S (2007) Biosynthesis of lignans in plant species of the
section Linum: pinoresinol-lariciresinol reductase and justicidin
B 7-hydroxylase. Ph.D. Dissertation, University of Heinrich-
Heine, Düsseldorf, Germany, 135 p
Heping H, Shanlin G, Lanlan C, Xiaoke J (2008) In vitro induction
and identification of autotetraploids of Dioscorea zingiberensis.
In Vitro Cell Dev Biol Plant 44:448–455
Iiyama K, Wallis AFA (1988) An improved acetyl bromide procedure
for determining lignin in woods and wood pulps. Wood Sci
Technol 22:271–280
Kadota M, Niimi Y (2002) In vitro induction of tetraploid plants from
a diploid Japanese pear cultivar (Pyrus pyrifolia N. cv. Hosui).
Plant Cell Rep 21:282–286
Kaensaksiri T, Soontornchainaksaeng P, Soonthornchareonnon N,
Prathanturarug S (2011) In vitro induction of polyploidy in
Centella asiatica (L.) Urban. Plant Cell Tissue Organ Cult
107:187–194
Karimzadeh G, Mousavi SH, Jafarkhani-Kermani M, Jalali-Javaran M
(2010) Karyological and nuclear DNA variation in Iranian
endemic muskmelon (Cucumis melo var. Inodorus). Cytologia
75:451–461
1177
Karimzadeh G, Danesh-Gilevaei M, Aghaalikhani M (2011) Kary-
otypic and nuclear DNA variations in Lathyrus sativus
(Fabaceae). Caryologia 64:42–54
Khazaei H, Mohammady S, Zaharieva M, Monneveux P (2009)
Carbon isotope discrimination and water use efficiency in
Iranian diploid, tetraploid and hexaploid wheats grown under
well-watered conditions. Genet Resour Crop Evol 56:105–114
Lavania UC (2005) Genomic and ploidy manipulation for enhanced
production of phyto-pharmaceuticals. Plant Genet Resour
3:170–177
Lavania UC, Srivastava S, Lavania S, Basu S, Misra NK, Mukai Y
(2012) Autopolyploidy differentially influences body size in
plants, but facilitates enhanced accumulation of secondary
metabolites, causing increased cytosine methylation. Plant J
71:539–549
Lee HS, Chen ZJ (2001) Protein-coding genes are epigenetically
regulated in Arabidopsis polyploids. Proc Natl Acad Sci USA
98:6753–6758
Lehrer JM, Mark HB, Jessica D (2008) Lubell induction of tetraploidy
in meristematically active seeds of Japanese barberry (Berberis
thunbergii var. Atropurpurea) through exposure to colchicine
and oryzalin. Sci Hort 119:67–71
Lin X, Zhou Y, Zhang J, Lu X, Zhang F, Shen Q, Wu Sh, Chen Y,
Wang T, Tang K (2011) Enhancement of artemisinin content in
tetraploid Artemisia annua plants by modulating the expression
of genes in artemisinin biosynthetic pathway. Biotechnol Appl
Biochem 58:50–57
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression
data using real-time quantitative PCR and the 2-DDCT method.
Methods 25:402–408
Loureiro J, Rodriguez E, Doležel J, Santos C (2007) Two new nuclear
isolation buffers for plant DNA flow cytometry: a test with 37
species. Ann Bot 100:875–888
Lu BB, Pan XZ, Zhang L, Huang BB, Sun L, Li B, Yi B, Zheng SQ,
Yu XJ, Ding R, Chen W (2006) A genome-wide comparison of
genes responsive to autopolyploidy in Isatis indigotica using
Arabidopsis thaliana affymetrix genechips. Plant Mol Biol Rep
24:197–204
Mahdavi S, Karimzadeh G (2010) Karyological and nuclear DNA
content variation in some Iranian endemic Thymus species
(Lamiaceae). J Agr Sci Tech 12:447–458
Majdi M, Karimzadeh G, Malboobi MA, Omidbaigi R, Mirzaghaderi
G (2010) Induction of tetraploidy to feverfew (Tanacetum
parthenium Schulz-Bip.): morphological, physiological, cyto-
logical, and phytochemical changes. HortScience 45:16–21
Martelotto LG, Ortiz JPA, Stein J, Espinoza F, Quarin CL, Pessino
SC (2005) A comprehensive analysis of gene expression
alterations in a newly synthesized Paspalum notatum autote-
traploid. Plant Sci 169:211–220
Millar CI, Libby WJ (1989) Restoration: Disneyland or a native
ecosystem? A question of genetics. Fremontia 17:3–10
Mishra BK, Pathak S, Sharma A, Trivedi PK, Shukla S (2010)
Modulated gene expression in newly synthesized auto-tetraploid
of Papaver somniferum L. S Afr J Bot 76:447–452
Mohagheghzadeh A, Hemmati Sh, Alfermann AW (2006) Quantifi-
cation of aryltetralin lignans in Linum album organs and in vitro
cultures. Iran J Pharm Sci 2:47–56
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Nonaka T, Oka E, Asano M, Kuwayama S, Tasaki H, Han DS, Godo
T, Nakano M (2011) Chromosome doubling of Lychnis spp. by
in vitro spindle toxin treatment of nodal segments. Sci Hortic
129:832–839
Omidbaigi R, Mirzaee M, Hassani ME, Sedghi Moghadam M (2010a)
Induction and identification of polyploidy in basil (Ocimum
123
 Author's personal copy
1178
basilicum L.) medicinal plant by colchicine treatment. Int J Plant
Prod 4:87–98
Omidbaigi R, Yavari S, Esmaeil Hassani M, Yavari S (2010b)
Induction of autotetraploidy in dragonhead (Dracocephalum
moldavica L.) by colchicine treatment. J. Fruit Ornam Plant Res
18:23–35
Osborn TC, Pires JC, Birchler JA, Auger DL, Chen ZJ, Lee HS,
Comai L, Madlung A, Doerge RW, Colot V, Martienssen RA
(2003) Understanding mechanisms of novel gene expression in
polyploids. Trends Genet 19:141–147
Planchais S, Glab N, Inzé D, Bergounioux C (2000) Chemical
inhibitors: a tool for plant cell cycle studies. FEBS Lett
476:78–83
Ryan B, Joiner BL (2001) Minitab handbook, 4th edn. Duxbury Press,
California
SAS Institute Inc (2002) The SAS System for Windows, Release 9.0.
Statistical Analysis System Institute, Cary, NC
Shao J, Chen C, Deng X (2003) In vitro induction of tetraploid in
pomegranate (Punica granatum). Plant Cell Tissue Organ Cult
75:241–246
Sreelakshmi Y, Sharma R (2008) Differential regulation of pheny-
lalanine ammonia lyase activity and protein level by light in
tomato seedlings. Plant Physiol Biochem 46:444–451
Stupar RM, Bhaskar PB, Yandell BS, Rensink WA, Hart AL, Ouyang
S, Veilleux RE, Busse JS, Erhardt RJ, Buell CR, Jiang J (2007)
Phenotypic and transcriptomic changes associated with potato
autopolyploidization. Genetics 176:2055–2067
Tavan M, Mirjalili MH, Karimzadeh G (2015) In vitro polyploidy
induction: changes in morphological, anatomical and phyto-
chemical characteristics of Thymus persicus (Lamiaceae). Plant
Cell Tissue Organ Cult 22:573–583
Thao N, Ureshino K, Miyajima I, Ozaki Y, Okubo H (2003) Induction
of tetraploids in ornamental Alocasia through colchicine and
oryzalin treatments. Plant Cell Tissue Organ Cult 72:19–25
123
View publication stats
Planta (2017) 245:1165–1178
Viehmannová I, Cusimamani EF, Bechyne M, Vyvadilová M, Greplová
M (2009) In vitro induction of polyploidy in yacon (Smallanthus
sonchifolius). Plant Cell Tissue Organ Cult 97:21–25
Viehmannová I, Trávnı́čková M, Špatenková E, Černá M, Trávnı́ček
P (2012) Induced polyploidization and its influence on yield,
morphological, and qualitative characteristics of microtubers in
Ullucus tuberosus. Plant Cell Tissue Organ Cult 109:83–90
Wallaart TE, Pras NK, Quax WJ (1999) Seasonal variations of
artemisinin and its biosynthetic precursors in tetraploid Artemi-
sia annua plants compared with the diploid wild-type. Planta
Med 65:723–728
Wang JW, Zheng LP, Wu JY, Tan RX (2006) Involvement of nitric
oxide in oxidative burst, phenylalanine ammonialyase activation
and taxol production induced by low-energy ultrasound in Taxus
yunnanensis cell suspension cultures. Nitric Oxide Biol Chem
15:351–358
Ye YM, Tong J, Shi XP, Yuan W, Li GR (2010) Morphological and
cytological studies of diploid and colchicine-induced tetraploid
lines of crape myrtle (Lagerstroemia indica L.). Sci Hortic
124:95–101
Yousefzadi M, Sharifi M, Ahmadian Chashmi N, Behmanesh M,
Ghasempour A (2010a) Optimization of podophyllotoxin extrac-
tion method from Linum album cell cultures. Pharm Biol
12:1421–1425
Yousefzadi M, Sharifi M, Behmanesh M, Moyano E, Bonfill M,
Cusido RM, Palazon J (2010b) Podophyllotoxin: current
approaches to its biotechnological production and future chal-
lenges. Eng Life Sci 10:281–292
Yousefzadi M, Sharifi M, Behmanesh M, Ghasempour A, Moyano E,
Palazon J (2012) The effect of light on gene expression and
podophyllotoxin biosynthesis in Linum album cell culture. Plant
Physiol Biochem 56:41–46
Zhang Zh, Dai H, Xiao M, Liu X (2008) In vitro induction of
tetraploids in Phlox subulata L. Euphytica 159:59–65