Plant Science 334 (2023) 111776

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Plant Science
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Polyploidy as a strategy to increase taxane production in yew cell cultures:

Obtaining and characterizing a Taxus baccata tetraploid cell line
Ainoa Escrich a, Diego Hidalgo b, Mercedes Bonfill b, Javier Palazon b, Raul Sanchez-Mun
! oz c, *,
a, **
Elisabeth Moyano
a
Department of Medicine and Life Sciences, Universitat Pompeu Fabra, Barcelona, Spain
b
Department of Biology, Healthcare and the Environment, Faculty of Pharmacy and Food Science, Universitat de Barcelona, Barcelona, Spain
c
Laboratory of Functional Plant Biology, Department of Biology, Ghent University, Ghent, Belgium

A R T I C L E I N F O A B S T R A C T

Keywords: Novel approaches to optimize the production of plant specialized metabolites are crucial to reach maximum
Cell cultures productivity of plant biofactories. Plant polyploidization frequently enhances protein synthesis and thereby in-
Colchicine creases the biosynthesis of specialized metabolites. Paclitaxel is a valuable anticancer agent scarcely produced in
Paclitaxel
nature. Therefore, plant biofactories represent a sustainable alternative source of this compound and related
Polyploidy
taxanes. With the aim of improving the productivity of Taxus spp. cell cultures, we induced polyploidy in vitro by
Taxane productivity
Taxus spp treating immature embryos of Taxus baccata with colchicine. To obtain the polyploid cell lines, calli were
induced from T. baccata plantlets previously treated with colchicine and ploidy levels were accurately identified
using flow cytometry. In terms of cell morphology, tetraploid cells were about 3-fold bigger than the diploid cells.
The expression of taxane pathway genes was higher in the tetraploid cell line compared to the diploid cells.
Moreover, taxane production was 6.2-fold higher and the production peak was achieved 8 days earlier than in the
diploid cell line, indicating a higher productivity. The obtained tetraploid cell line proved to be highly pro-
ductive, constituting a step forward towards the development of a bio-sustainable production system for this
chemotherapeutic drug.

1. Introduction Mukherjee, 2021; Madani et al., 2021; Majdi et al., 2014; Zahedi et al.,
2014).
Polyploid plants typically exhibit enlarged parts, such as bigger Chromosome doubling occurs spontaneously in some plant species
flowers or fruits, and greater resistance to environmental stressors but requires time. However, artificial systems have been developed to
compared to their diploid counterparts (Kaensaksiri et al., 2011; Liu induce polyploidy in plant species in in vivo, ex vitro and in vitro condi-
et al., 2011). It has also been demonstrated that the production of tions. The in vitro systems are the most widely used due to the shorter
specialized metabolites is affected by polyploidization (Gantait and times required for polyploidy induction and their faster propagation in a
Mukherjee, 2021; Salma et al., 2017). Polyploidy or whole-genome limited space, with sterile and controllable environmental conditions
duplication is a condition in which a normally diploid cell acquires (Eng and Ho, 2019). Moreover, with adventitious organ regeneration
one or more additional sets of chromosomes, which alters the meta- techniques, in vitro polyploidy induction has become the method of
bolism of the plant. Polyploid plants usually present better qualities choice for several plant species because it increases the mutagenesis
compared to diploid plants. Enhanced cellular activity increases the efficiency and decreases the occurrence of chimeras in comparison to in
vigor, size and productivity of the plant, as the increase in cell size and vivo methods (R”ego et al., 2011).
nuclear activity alters gene expression and enzyme biosynthesis (Eng The key step in the polyploidization process is the choice of suitable
and Ho, 2019; Gantait et al., 2011; Moghbel et al., 2015). This alteration polyploidy inducers. To date, more than 60 studies have applied anti-
in metabolic activity ultimately leads to a higher production of alka- mitotic agents for polyploidization using different plant tissues and or-
loids, terpenes and polyphenols, among other compounds (Gantait and gans and quantified the effects on specialized metabolite production

* Correspondence to: Laboratory of Functional Plant Biology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium.
** Correspondence to: Department of Medicine and Life Sciences, Universitat Pompeu Fabra, C/ Doctor Aiguader 88, 08003 Barcelona, Spain.
E-mail addresses: [email protected] (R. Sanchez-Mu! noz), [email protected] (E. Moyano).

https://doi.org/10.1016/j.plantsci.2023.111776
Received 1 March 2023; Received in revised form 24 May 2023; Accepted 14 June 2023
Available online 19 June 2023
0168-9452/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
A. Escrich et al. Plant Science 334 (2023) 111776

(Gantait and Mukherjee, 2021). During polyploidization induction in in for the first time by treating immature embryos with colchicine and in
vitro conditions, several parameters can be manipulated to obtain the this way to increase the taxane yield. A combination of three concen-
highest percentage of polyploids (Eng and Ho, 2019). The effectiveness trations of colchicine and three exposure times were applied to assess
of the antimitotic compound depends greatly on the applied concen- the best conditions to obtain polyploid T. baccata cell lines. Therefore,
tration, the duration of treatment, the induction medium, the usage of the present work evaluates the effectiveness of colchicine in inducing
shakers, the type of explant, and supplementation with DMSO (or other polyploidy in T. baccata. In the polyploid cell cultures, both the pro-
permeabilizers) to facilitate the penetration of the compound (Ascough duction of taxanes and the expression of PTX biosynthetic genes were
et al., 2008). assessed to determine the effect of polyploidization on the specialized
Among the different antimitotic agents, colchicine has proved to be metabolism of T. baccata. Overall, an in-depth characterization of
the most efficient and reliable in producing polyploids. It is a naturally morphology, taxane production, and gene expression was performed to
produced alkaloid extracted from meadow saffron, which belongs to the determine the effects of polyploidy in T. baccata cell lines.
Liliaceae family (Swarbrick, 1986). Its mechanism of action consists of
binding to tubulin heterodimers, causing the failure of microtubule as- 2. Materials and methods
sembly during mitosis at the metaphase stage (Borisy and Taylor, 1967).
This prevents chromosome separation, and therefore, by the end of the 2.1. Plant material
cycle, two sets of chromosomes will remain in a single nucleus and a
polyploid cell will be formed. Seeds containing immature embryos were collected from T. baccata
Concentration and treatment duration are inter-dependent and trees. They were sterilised by washing with sterile distilled H2O, 70%
considered the most crucial factors in the use of colchicine for the in- ethanol for 30 s, rinsed 2 times with sterile distilled H2O, 2% mercury
duction of polyploidization. In general, colchicine is applied at con- chloride (HgCl2) for 30 min, rinsed with sterile distilled H2O, 30%
centrations ranging from 0.01% (v/w) to 0.1% (v/w) for a time of 24–72 bleach with 10 drops of Tween-20 for 30 min in an ultrasonic bath, and
h. Low doses are usually unsuccessful whereas high doses can be lethal finally, rinsed 2 times with sterile distilled H2O. Sterilized seeds, isolated
(Widoretno, 2016). embryos and the new plantlets were cultured on Gupta and Durzan
Colchicine has been widely used to induce polyploidy in medicinal medium (G&D) (Gupta and Durzan, 1985) supplemented with 3% su-
plants, altering the production of specialized metabolites. An increment crose and 5 g/L of phytoagar. Isolated embryos from sterilized seeds
in the specialized metabolite levels of Centella asia#tica (Kaensaksiri et al., were cultured in darkness for six weeks, and then maintained in an 8–16
2011; Thong-On et al., 2014), Datura stramonium (Al-Taweel et al., h light-dark cycle at 25ºC. T. baccata plantlets were cultured in the same
2019), Echinacea purpurea (Abdoli et al., 2013; Yang et al., 2009), Panax temperature and photoperiod conditions.
ginseng (Kim et al., 2004), Papaver somniferum (Mishra et al., 2010),
Pogostemon cablin (Widoretno, 2016; Yan et al., 2016), Salvia miltiorrhiza 2.2. Colchicine treatment
(Gao et al., 1996) and Scutallaria baicalensis (Gao et al., 2002), among
other species, has been reported. However, polyploidization does not Embryos were treated with different concentrations of colchicine
always benefit the production of specialized metabolites. Several studies (0.01%, 0.05% and 0.1%) for three different exposure times (24 h, 48 h
have reported polyploid plants that are less productive or similar to and 72 h) in G&D liquid media. 2% (v/v) dimethyl sulfoxide (DMSO)
diploid plants, such as Solanum bulbocastarum (Caruso et al., 2013) and was added to the treatments as a permeabilizer. Sterile distilled H2O was
Miscanthus → ginganteus (Ghimire et al., 2016). added to the medium instead of DMSO for the negative controls. The
Given the growing interest in increasing the supply of plant-derived incubation with colchicine was carried out using an orbital shaker at
bioactive compounds to treat human diseases, the induction of poly- 115 rpm. Once the exposure time was over, embryos were cultured in
ploidy plants is a potentially useful strategy to achieve enhanced pro- solid fresh G&D media without colchicine in darkness at 25ºC.
duction. The well-known anticancer drug paclitaxel (PTX) is produced in
the inner bark of Taxus spp. trees, but in scarce quantities, so its current 2.3. Callus induction and cell suspension cultures
market demand cannot be met solely by extraction from natural sources.
Therefore, the use of plant cell cultures of Taxus spp. is an attractive For callus induction, B5 medium (Gamborg et al., 1968) was sup-
alternative for PTX production (Vidal-Limon et al., 2018). Different plemented with 3% sucrose, 4 mg/L of 2,4-dichlorophenoxyacetic acid,
biotechnological approaches have been used to increase the in vitro 1 mg/L of kinetin and 0.5 mg/L of gibberellic acid, pH 5.8. For callus
production of PTX and related taxanes, both empirical (Cusido et al., maintenance, B5 medium was supplemented with 0.5% sucrose, 0.5%
2014; Ramirez-Estrada et al., 2015; Escrich et al., 2021) and rational fructose, 2 mg/L of 1-naphthaleneacetic acid, 0.1 mg/L of 6-benzyla-
(Escrich et al., 2022; Sanchez-Mun ! oz et al., 2018). In this context, pol- mino purine and 0.5 mg/L of gibberellic acid, pH 5. 8. All calli were
yploidization has potential as an effective strategy to overcome the maintained at 25ºC in darkness and were subcultured biweekly. Sus-
natural scarcity of taxanes by improving the growth, productivity, and pension cell cultures were generated and two-stage cultures were
yield of Taxus spp. in vitro cell cultures. established as described by Cusido et al. (2002), as this system has
In vitro cultures of Taxus spp. can be established using different plant proved effective for the stimulation of taxane production.
tissues and organs (Chee, 1995; Ewald, 2007; Xie and Guo, 1999). For
polyploidy induction, it has been described that shoot apical meristems 2.4. Morphological analysis
and seeds can be used as starting material to obtain polyploid plants
(Salma et al., 2017), but young meristems have the highest efficiency of A magnifier colour camera (Optronics, Goleta, CA) mounted on a
polyploidization (Gantait and Mukherjee, 2021). Nevertheless, Leica MZFLIII stereomicroscope (Leica Microsystems, Bannockburn, IL)
depending on the explant type and plant species, cell permeability can was used to acquire pictures of embryos. For calli, images were taken by
be improved to facilitate the penetration of the mutagenic agents into Leica THUNDER DMi8 microscope (Leica Microsystems, Bannockburn,
cells. The addition of dimethyl sulfoxide (DMSO) in the process of IL) using LAS X 3.0.1 software. ImageJ software (Wayne Rasband, Na-
polyploidy induction increases the efficiency of the mutagenic agent tional Institutes of Health-NIH, Maryland, USA) was used to measure cell
(Chen and Gao, 2007; Grouh et al., 2011; Yan et al., 2016). However, the size and set the scale bar.
study of cell viability is recommended when DMSO is added to the media
due to its toxicity, which could compromise cell viability and influence 2.5. Flow cytometric analysis
plant growth (Eng and Ho, 2019).
This study aimed to obtain polyploid in vitro T. baccata cell cultures Nuclei were extracted from calli using the CyStain™ PI Oxprotect kit

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A. Escrich et al. Plant Science 334 (2023) 111776

(Sysmex, Gorlitz, Germany). 200 mg of callus was incubated in 500 μL of Regarding the effect of colchicine on embryo germination, the sur-
staining buffer for 1 min. Samples were filtered using disposable filters vival rate was measured for eight weeks after the treatment, and non-
CellTrics™ 50 µm (Sysmex, Gorlitz, Germany) and 1.5 mL of staining germinated and brown embryos were considered dead or aborted. The
buffer was added to the filtrate. Then, the filtrate containing the released three treatments of colchicine applied to the immature embryos showed
nuclei was stained with 2.5 μL of 4′,6-diamidino-2-phenylindole (DAPI) significant differences in terms of viability (Fig. 1A). 0.1% colchicine
(1 mg/mL). All extraction steps were carried out on ice to maintain caused the maximum mortality rate, resulting in 72% of embryos being
nuclei integrity. Samples were analysed by flow cytometry using a non-viable, followed by the 0.05% treatment, which caused 68% mor-
Gallios flow cytometer (Beckman Coulter, Brea, CA, EEUU.). The DAPI tality. On the other hand, the germination rate of the control treatment
stain was excited at 405 nm and the emission was collected from 430 to was 70%, followed by the 0.01% colchicine treatment with a germina-
470 nm. The figures from the flow cytometer analysis were obtained tion rate of 63% (Fig. 1A).
using FlowJo v10 (Ashland OR 97520). The colchicine concentration also affected the development of the
embryo. In the early stages of embryo development aerial parts and
2.6. Taxane determination roots are produced in a coordinated process. However, it was observed
that the treated embryos often did not develop roots. In some cases, they
Taxanes were extracted from the culture media and lyophilized cells only developed aerial parts and had a lower survival rate compared to
were harvested at 0, 8, 16 and 24 days as described by Cusido # et al. the diploid cells (Fig. 1). The negative control conditions produced the
(1999) and quantified by UPLC (Bonfill et al., 2003) with a Water highest rate of well-developed embryos (close to 80%), followed by the
Acquity Ultra Performance LC system (Waters, Milford, MA, USA). 0.01% colchicine treatment (50%) (Fig. 1B). In contrast, the 0.05% and
Target taxanes, 10-deacetylbaccatin III (DABIII), baccatin III (BACIII), 0.1% colchicine treatments resulted in semi-developed embryos (80%
10-deacetyltaxol (DAT), cephalomannine (CEPH) and paclitaxel (PTX), and almost 90%, respectively), which had aerial parts but not roots
were provided by Abcam (Cambridge, UK). (Fig. 1B). Apart from the alterations in development, early-stage growth
was also slower in the treated embryos than in the control samples. For
2.7. Quantitative real-time PCR (RT-qPCR) that reason, 0.01% colchicine seemed the best concentration for embryo
growth and development (Fig. 1).
For gene expression analysis, total RNA was isolated from 100 mg of A morphological study was then performed based on the develop-
lyophilized cells using the Real Plant RNA Kit (REAL, Valencia, Spain) mental differences between the treated and untreated embryos. Fig. 2
following the manufacturer’s instructions. cDNA was prepared from 1 µg shows the structural differences between the control embryos (Fig. 2A)
of RNA with SuperScript IV reverse transcriptase (Invitrogen, CA, USA) and those of the different colchicine treatments (Fig. 2B-D). Whereas
and an RT-qPCR assay was performed with SYBR Green (Biorad, Her- control embryos had correctly developed roots with numerous absor-
cules, CA, USA) using gene-specific primers designed with Primer3 (v bent hairs (Fig. 2A), the embryos treated with 0.01% colchicine had
0.4.0; Table S1), in a 384-well platform system (LightCycler ® 480 In-
strument, Roche, USA). The expression of genes encoding geranylger-
anyl diphosphate synthase (GGPPS), taxadiene synthase (TXS), 10-
deacetylbaccatin III-10β-O-acetyltransferase (DBAT), baccatin III 13-O-
(3-amino-3-phenylpropanoyl) transferase (BAPT) and 3′-N-debenzoyl-
2′-deoxytaxol-N-benzoyltransferase (DBTNBT) was quantified. TBC41
was used as a housekeeping gene for expression normalization. For each
gene, relative expression levels were normalized relative to time
0 (reference value ↑ 1). Samples for determining taxane biosynthetic
gene expression were taken at 0, 4, 8, 12, 24, 48 and 72 h.

2.8. Statistical analysis

For the statistical analyses, Shapiro’s and Levene’s tests were used to
test the normality and variance of each dataset. A two-factor ANOVA
was used as a parametric test and the Krustal-Wallis rank sum test fol-
lowed by Dunn’s Multiple Comparison test were used as non-parametric
tests. A p-value of ω 0.05(*), ω 0.01(**) and ω 0.001(***) were assumed
for significant differences. All the analyses were performed in R.

3. Results

3.1. Effect of colchicine on embryo germination and development

T. baccata embryos were treated with colchicine and DMSO was used
as a permeabilizer in all samples. Consequently, the effects of DMSO
were determined in terms of embryo development. The embryos were
cultured for 24, 48 and 72 h with 2% DMSO or H2O (control conditions).
Of the 200 embryos treated with DMSO or H2O, 70% and 71%,
respectively, had a normal development (Fig. S1). Therefore, 2% (v/v)
Fig. 1. A) Germination (green) and mortality (grey) rate of embryos. B) Early
DMSO did not affect the germination and development of embryos
steps of embryo development at different colchicine concentrations indicating
during the three exposure times (24, 48 and 72 h). the presence of only an aerial part (grey) or both root and aerial parts (orange).
The embryos treated with colchicine displayed a lagged germination, Untreated embryos (control conditions; 0% colchicine) and the different
suggesting the colchicine concentration was a more important variable colchicine concentrations (0.01%, 0.05% and 0.1%) are depicted in the x-axis.
than the exposure time (data not shown). Consequently, we focused our A p-value of ω 0.05 (*), ω 0.005 (**), and ω 0.001 (***) was used for statisti-
attention on the different colchicine concentrations. cally significant differences.

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A. Escrich et al. Plant Science 334 (2023) 111776

Fig. 2. Early stage of embryo development. Embryos treated with different concentrations of colchicine: A) 0% (negative control), B) 0.01%, C) 0.05%, D) 0.1%.
Scale bar ↑ 1 mm.

roots with scarce and burned absorbent hairs (Fig. 2B). Moreover, em- 3.3. Ploidy determination
bryos treated with 0.05% and 0.1% colchicine did not develop roots,
presenting only the radicle (Fig. 2C and D, respectively). Calli obtained from T. baccata plantlets were analyzed by flow
From the obtained embryos, the ones exhibiting notable phenotypic cytometry to reveal the ploidy levels of each cell line. The callus induced
differences and proper growth, all of them treated with 0.05% or 0.1% from the untreated embryo (0% colchicine) was used as the negative
colchicine, were selected for culture (Fig. 2). As shown in Fig. 3, the control (diploid).
putative polypoid T. baccata plants presented an abnormal growth, In the flow cytometric histograms, the peak of the diploid T. baccata
whereas control plants (diploid) had a slim, well-defined stem and clear callus (2x) was set at channel 222 K (Fig. 4 A). Therefore, the peak of
gravitropic growth (Fig. 3A). The putative polyploid plant obtained with tetraploid (4x) nuclei was expected at channel 444 K, and a peak cor-
0.1% colchicine presented a stem that was not completely developed, responding to 4x appeared at 455 K (Fig. 4B). The mix of diploid (2x)
and its growth was not phototropic (Fig. 3C). Moreover, it had smaller and tetraploid (4x) is represented in Fig. 4 C. This mix was performed to
leaves and shorter internodal distances than the diploid plant. The corroborate the polyploidy determination, where the tetraploid peak
control plants had a deep green coloration in contrast with the had approximately twice the fluorescence intensity in proportion to the
yellowish-green of putative polyploid plants obtained with 0.05% and square root of ploidy of the diploid peak (Fig. 4 C). Moreover, the sta-
0.1% colchicine (Fig. 3 A-C). The morphology associated with 0.05% tistics (mean, median and mode) between the diploid T. baccata cell line
colchicine treatment differed from the normal development of the and the induced tetraploid cell line were proportional and the difference
control plant in that the treated plants had two shoots and greater leaf between each measurement was approximately 2-fold (Fig. 4 F).
length and nodal distances, although the number of leaves was the same Regarding the morphology produced by 0.1% colchicine, a peak at
(Fig. 3B). 299 K was obtained in the cytometry analysis (Fig. 4D), which did not
correspond to a 4x peak. To determine if this was due to analytical ef-
3.2. Callus induction fects, such as aggregations of DAPI, a mixture of that sample and the
diploid control was analyzed. Two different peaks were obtained
Plantlets (control and treated with colchicine) were used to induce (Fig. 4E), one at channel 222 K belonging to 2x, and another one at
callus lines (Fig. 3D-F) to obtain polyploid cell cultures for the taxane channel 299 K belonging to an incomplete polyploidization.
production studies. The induced calli from the control plantlet (un- The statistics regarding the unusual peak found in the IP cell line
treated with colchicine) had brown cells forming aggregates (Fig. 3D). In showed that the difference in mode is approximately 1.4-fold compared
contrast, the putative polyploid calli derived from the 0.05% colchicine- to the control (Fig. 4F). Therefore, it could not be considered either a
treated plantlets were characterized by dark brown tissue and large tetraploid or a diploid, and it was described as an incomplete or inter-
aggregates, whereas the calli derived from the 0.1% colchicine-treated mediate ploidy.
plantlets had light-brown friable tissue (Fig. 3E and F, respectively).

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Fig. 3. Candidate plantlet polyploids: A) Control 2x (Diploid), B) Morphology associated with 0.05% colchicine C) Morphology associated with 0.1% colchicine.
Calli induced from A, B and C plantlets used as explants: D) Control 2x callus, E) Calli from plantlets treated with 0.05% colchicine and F) Calli from plantlets treated
with 0.1% colchicine.

3.4. Cell size determination intracellular taxane content of approximately 2.5 mg/L was observed in
the 2x cell line (Fig. 6B). Although no significant differences were found
Two types of cells were observed in the diploid line of T. baccata, in the intracellular production of taxanes between the three cell lines,
either elongated or rounded. The same cell morphologies were found in the taxane content in the tetraploid cell line was 2.5-fold lower than in
the cell cultures obtained from colchicine-treated embryos. In Fig. 5, it the diploid line at the end of the experiment.
can be observed that the diploid (2x) cell culture was a mixture of Regarding the total taxane content (Fig. 6C), the maximum pro-
elongated and rounded cells (Fig. 5A). The cell culture with intermediate duction in the diploid cell culture was 13.7 mg/L and reached at day 16.
ploidy (treated with 0.1% colchicine) looked like the diploid one On the other hand, the taxane production levels remained very low in
(Fig. 5B). Finally, the tetraploid cell line (treated with 0.05% colchicine) the IP cell line throughout the experiment, oscillating between 1.8 and
also presented a mixture of elongated and rounded cells (Fig. 5C and D). 2.5 mg/L. Finally, the highest taxane production was achieved by the
However, in the line with incomplete ploidy (IP), cell size was similar tetraploid cell (4x) line at day 8, when it was 6.2-fold (21.64 mg/L)
to the control and did not show significant differences (Table 1). higher than in the diploid cell line at this time, indicating a higher
Meanwhile, in the tetraploid (4x) cell culture, the cell size was 3.33- and productivity and greater taxane yield.
2.99-fold higher for the elongated and rounded cells, respectively, The profile of the taxanes produced was similar between the three
compared to the diploid cells (Table 1). cell lines, with DAT produced in higher quantities than PTX. The taxane
composition of the diploid cell line was more uniform compared to the
tetraploid line, as all intermediates were converted into DAT. Regarding
3.5. Effect of ploidy on taxane production taxane excretion, the studied taxanes mainly accumulated extracellu-
larly rather than intracellularly.
The total taxane content of the three cell lines, diploid (2x), IP and
tetraploid (4x) was determined by UPLC as the sum of DABIII, BACIII,
DAT, CEPH and PTX at different time-points (0, 8, 16 and 24 days). 3.6. Taxane gene expression
Fig. 6 shows the extracellular, intracellular and total taxane content.
The extracellular taxane content oscillated between 0.7 mg/L and The transcription profile of the taxane biosynthetic genes in the cell
19 mg/L (Fig. 6A). The highest content was obtained by the tetraploid lines was assessed by RT-qPCR (Fig. 7). The relative expression of target
cell line (4x) at day 8, which was 10.8-fold higher compared to the genes belonging to early (GGPPS and TXS), intermediate (DBAT) and
diploid cell line on the same day, indicating the tetraploid cell line had a late steps (BAPT and DBTNBT) of the taxane biosynthetic pathway was
higher productivity (Fig. 6A). On the other hand, the maximum analyzed. The results showed that all the target genes were

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A. Escrich et al. Plant Science 334 (2023) 111776

Fig. 4. Flow cytometric histograms of DAPI-stained nuclei from Taxus baccata calli maintained in in vitro conditions. A) Control, diploid (2x). B) Treated with 0.05%
colchicine, tetraploid (4x). C) Mixture of control and treated with 0.05% colchicine (2x ↓ 4x). D) Treated with 0.1% colchicine, unusual peak, incomplete ploidy (IP).
E) Mixture of control and treated with 0.1% colchicine. X-axis represents arbitrary units of fluorescence. Y-axis represents the number of nuclei analyzed. F) Mean,
median and mode for the three samples (diploid (2x) cell line, IP cell line and tetraploid (4x) cell line) were used as statistics for the flow cytometric analysis.

overexpressed in the tetraploid line compared to the diploid line. The which were 2.84- and 2.66-fold higher than the diploid line, respectively
basal transcription levels of the tetraploid cell line (4x) compared to the (Fig. 7). The expression of TXS in the IP cell line was 1.45- and 1.77-fold
control (2x) were between 1.84- and 12.83-fold higher (Fig. 7). higher at 4 and 24 h compared to the diploid line (Fig. 7).
Regarding the transcript levels of the early step genes, maximum Regarding the intermediate steps, DBAT transcript levels in the
GGPPS expression was observed in the 4x cell line at 48 h, being 1.76- tetraploid line were higher than in the 2x and IP cell lines, the relative
and 4.7-fold higher than the diploid and IP cell lines, respectively. For expression value at 4 h being 9.1-fold higher than in the control.
TXS, the tetraploid line showed two peaks of expression, at 12 and 72 h, The genes involved in the last steps, BAPT and DBTNBT, are known

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Fig. 5. Morphological comparison between: A) diploid cells (2x), B) incomplete ploidy cells (IP), C) rounded tetraploid (4x) cells and D) elongated tetraploid (4x)
cells. Scale bar ↑ 100 µm.

T. baccata embryos were used due to the lack of efficient protocols for
Table 1
plant regeneration and organogenesis of Taxus spp. Therefore, T. baccata
Mean cell size (μm) of the different Taxus baccata cell lines: diploid (2x),
embryos treated under different conditions were cultured to obtain
incomplete ploidy (IP), and tetraploid (4x).
plantlets, which were transferred to an induction medium to obtain
Sample Elongated cells (μm) Rounded cells (μm) polyploid calli. Finally, polyploid cell cultures of Taxus spp. were
2x 170.62 ↔ 45.78 105.33 ↔ 18.48 generated from callus tissue and, after their morphological character-
Incomplete ploidy (IP) 160.33 ↔ 20.24 112.33 ↔ 13.34 ization, their taxane content and the expression of several genes
4x 569.22 ↔ 95.46*** 315.70 ↔ 83.32***
involved in taxane biosynthesis were determined.
Statistical analysis was performed comparing 2x (control) samples with the IP The effect of colchicine on the embryo survival rate and germination
and 4x cell lines (p-value: * ω 0.05; ** ω 0.01; *** ω 0.001). capacity depended mainly on the concentration rather than the exposure
time. As the colchicine concentration increased, the rate of live and well-
to be most regulated genes of the pathway. Compared to the diploid line, developed embryos decreased. The untreated embryos (control) mostly
where the transcripts of BAPT and DBTNBT genes were almost unde- developed normally, and only 20% did not develop roots, attributed to
tectable, the transcriptional peaks in the tetraploid cell line were 7.17- potential injuries in the manual process of embryo isolation. On the
fold higher at 4 h for BAPT and 53.2-fold higher at 48 h for DBTNBT, other hand, the rooting of treated embryos occurred later than in the
showing the highest increase of all the studied genes (Fig. 7). untreated ones. This delay in rooting when using colchicine treatments
To sum up, the tetraploid cell line showed an enhanced expression of has also been reported by other authors (Gantait et al., 2011; Tavan
all the genes studied, which correlates with its high taxane production et al., 2015). In addition, in the treatments with 0.05% and 0.1%
rates. On the other hand, the IP line presented a non-homogenous colchicine, 80% and 90% of the embryos, respectively, did not develop
expression profile that clearly differs from the 2x profile. The expres- roots. The embryos that only had aerial parts and radicles were
sion rates of GGPPS, DBAT and DBTNBT genes were lower in the IP than non-viable, since root development is necessary for nutrient and water
the diploid cell line. In contrast, the expression values for the TXS and uptake from the culture medium, as well as for the proper physiological
BAPT genes in the IP line were similar to those of the tetraploid cell line regulation of the growing process (Jones and Dolan, 2012). Neverthe-
(Fig. 7). less, due to the inhibitory effect of colchicine on cell division (Ascough
et al., 2008) we can expect an increase in mortality and abnormal
4. Discussion growth associated with chemical damage by the mutagenic agent.
In mature stages, polyploidy plantlets were yellowish green, which
To the best of our knowledge, this is the first report of the successful could be due to worthless stems failing to transport water and nutrients
induction of tetraploidy in Taxus spp. by treating diploid embryos with to the aerial parts (Pate and Dieter Jeschke, 1995). Furthermore, the
colchicine in in vitro conditions. Although polyploidy is relatively lack of apical dominance and changes in tropism could be correlated
common in angiosperm trees, and perhaps all angiosperms have expe- with the worthless polyploidy stem development (Ahuja, 2005). More-
rienced polyploidy during their evolutionary history (Wendel, 2000), it over, although gymnosperm seeds, such as those of Taxus spp., only
is rather infrequent among gymnosperms (Adams and Wendel, 2005; develop one shoot, two shoots developed simultaneously in the tetra-
Ahuja, 2005). ploid plants. These observations could be related to the growth abnor-
It has been demonstrated that the in vitro treatment of nodal seg- malities of the two plant morphologies selected, the morphology of IP
ments with colchicine successfully induces polyploidy (Iannicelli et al., showing more growth abnormalities than tetraploid plantlets (4x).
2016; Inthima and Sujipuli, 2019; Javadian et al., 2017). In this study, Furthermore, the growth aberrations in the early stages of plantlet

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A. Escrich et al. Plant Science 334 (2023) 111776

Fig. 6. Taxane production of the Taxus baccata cell cultures: diploid (2x), incomplete ploidy (IP) and tetraploid (4x). A) Extracellular taxane content. B) Intracellular
taxane content. C) Total taxane content. A p-value of ω 0.05 (*), ω 0.005 (**), and ω 0.001 (***) was used for significant differences. 10-Deacetylbaccatin III
(DABIII), baccatin III (BACIII), 10-Deacetyltaxol (DAT), cephalomannine (CEPH) and paclitaxel (PTX).

growth could be explained by the fact that polyploidy is rare in gym- fluorescence peak of the tetraploid cell line was twice as intense as that
nosperms, and induced polyploids in conifers are prone to exhibit of the diploid, representing a double quantity of DNA. However, in 0.1%
growth abnormalities (Ahuja, 2005). colchicine-treated samples, an intermediate peak between the diploid
Due to the limited embryo germination and seedling development, and tetraploid peaks was observed (Fig. 4D). This peak, however, is
calli were induced from plantlets to provide enough plant material for unlikely to match triploid nuclei because it is not equidistant from the
the study. The calli corresponding to the 0.1% colchicine treatment (IP) diploid and tetraploid peaks, appearing to be the result of an incomplete
had a similar appearance to those of the diploid control, whereas those ploidy. In a study with Watsonia lepida, an unusual peak was also found
of the 0.05% colchicine treatment (4x) were dark brown and had large in the flow cytometric analysis of seedlings treated with colchicine
aggregations of cells. This callus phenotype has been correlated with (Ascough et al., 2008). Colchicine may have caused a disruption in cell
high taxane production rates (Patil et al., 2013; Sanchez-Mun ! oz et al., division in some cells, so that DNA replication was not fully accom-
2018). The ploidy level of the callus was determined by flow cytometry, plished, leading to an incomplete doubling of the chromosomes and an
a commonly used method for ploidy determination because it is fast and intermediate polyploid cell culture. Although uncommon, incomplete
reliable (Eng and Ho, 2019; Gantait and Mukherjee, 2021). The nuclei doubling and aneuploid and triploid production can occur when

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A. Escrich et al. Plant Science 334 (2023) 111776

baicalin was studied in cell cultures of Scutellaria baicalensis (Gao et al.,

2002).
In the present study, not only was the production of taxanes in the
tetraploid cell line of T. baccata significantly increased, being 10.8-fold
higher compared to the control, but the peak of production was obtained
earlier, at day 8, thus demonstrating an improved productivity and
taxane yield. The taxane production of 21.64 mg/L achieved in the
tetraploid cell line is significantly higher than previously reported yields
in diploid cell lines of T. baccata. For example, Perez-Matas et al., (2022)
obtained a maximum taxane production of 5 mg/L at day 12 of culture,
and Sanchez-Mun ! oz et al. (2018) reported total taxane contents of
2.48 mg/L in an old T. baccata cell line with low productivity and
5.36 mg/L in a high-producing line after 24 days of culture. Considering
these data, the results obtained in the present study with the polyploid
T. baccata cell line are highly encouraging, as the treatment not only
improved yields, but also shortened the time required to reach optimal
production.
Another biotechnological approach to taxane production is based
mainly on elicitation. For instance, the taxane yield of T. media cell
cultures was 140 mg/L after elicitation with methyl jasmonate and cy-
clodextrins (Sabater-Jara et al., 2014), and 85 mg/L after treatment with
coronatine and calix[8]arenes (Escrich et al., 2021). In both cases, the
taxane yield was greater than in the polyploid cell line (21.64 mg/L),
but the high economic cost of these treatments hinders their industrial
application.
Moreover, the taxane content inside the cells of the tetraploid line
was 2.5-fold lower compared to the diploid line, indicating that the
polyploid line had a higher capacity to excrete taxanes to the medium.
The facilitation of taxane excretion from cell cultures constitutes
another advantage of a polyploidy system for the biotechnological
production of taxanes. Therefore, future studies could focus on the use of
elicitors such as coronatine or methyl jasmonate, alone or in combina-
tion, with permeabilizers such as cyclodextrins or calix[8]arenes to
optimize the production of PTX in the tetraploid cell line, following the
approach we have previously used in Taxus spp. diploid cell lines
(Escrich et al., 2021; Onrubia et al., 2013; Ramirez-Estrada et al., 2015).
Nevertheless, due to the physiological and morphological differences
between the diploid and tetraploid cell lines, the elicitation conditions
will need to be careful optimized.
Fig. 7. Transcript profile of taxane pathway genes in the three cell lines: In turn, the intermediate ploidy cell line was less productive than the
diploid (2x), incomplete ploidy (IP) and tetraploid (4x). Colors in the expression diploid control in the period of time studied. Lavania et al. (2012)
profile represent each gene of the taxane pathway, with dark colors for high demonstrated that cytosine methylation in genomic regions in tandem
relative expression and light colours for low relative expression. Geranylgeranyl with ploidy upliftment diminished biometabolite production in Cym-
diphosphate synthase (GGPPS): turquoise blue; taxadiene synthase (TXS): or- bopogon spp., and it has also been demonstrated that cytosine methyl-
ange; 10-deacetylbaccatin III-10β-O-acetyltransferase (DBAT): purple; baccatin ation modulates the production of taxanes (Escrich et al., 2022;
III 13-O-(3-amino-3-phenylpropanoyl) transferase (BAPT): dark blue, and 3′-N- Sanchez-Mun ! oz et al., 2018). Therefore, the reduction in taxane pro-
debenzoyl-2′-deoxytaxol-N-benzoyltransferase (DBTNBT): green. Color scale duction of the intermediate ploidy cell line could be due to the stress
ranges represent the relative expression of each gene.
generated by an incomplete ploidy linked to epigenetic events. On the
other hand, the study carried out by Manz et al. (2022) links the cell
attempting to induce tetraploid plants, as reported in previous studies shape with its productive capacity, concluding that rounded cells are
(Zlesak et al., 2005). more productive than elongated ones. In our study, the majority of cells
Besides the alterations in the polyploid development, the cell size of in the IP cell line were elongated, which could be another reason for its
the IP line was very similar to the control line. In contrast, the tetraploid low production profile. In contrast, the tetraploid line had more rounded
cell size was significantly higher than the diploid cell line. Similar al- than elongated cells, in line with the high productivity of our polyploid
terations in cell size have been reported by other authors (Kondorosi cell line.
et al., 2000; Robinson et al., 2018; Zhang et al., 2019). Regarding Chromosome doubling often results in a higher expression of genes in
specialized metabolite production, it has been demonstrated that poly- comparison with diploid cells (Madani et al., 2021; Majdi et al., 2014).
ploids usually produce more metabolites than diploids (Salma et al., Based on the transcription profile analysis, our results support that the
2017). Higher levels of phytopharmaceutical compounds have been whole-genome duplication in the tetraploid cell line caused an increase
reported in tetraploid plants, such as a 50% increase in the morphine in the expression levels of the target taxane-related genes compared to
content of Papaver somniferum (Mishra et al., 2010) and an 11% incre- the diploid control. The genes involved in the last steps of the taxane
ment of triterpenes of Centella asiatica (Kaensaksiri et al., 2011), as well pathway, BAPT and DBTNBT, are the most regulated ones (Cusido # et al.,
as an increased production of anticancer compounds such as podo- 2002; Exposito et al., 2010; Onrubia et al., 2013; Sabater-Jara et al.,
phyllotoxin in Linum album or beta-asarone in Acorus calamus (Madani 2014). Their transcription levels in the tetraploid cell line were 7.17-fold
et al., 2021). However, to date few studies have examined the effects of and 53.2-fold higher compared to the diploid line, increasing the flux of
ploidy in cell cultures; for example, production of the antioxidant metabolites to the production of final taxanes. This enhanced expression

9
A. Escrich et al. Plant Science 334 (2023) 111776

might be correlated with the increase in transcription factors and M.R. Ahuja, Polyploidy in gymnosperms: revisited, Silvae Genet. (2005), https://doi.
org/10.1515/sg-2005-0010.
cis-elements. Last but not least, the transcription levels of all the target
S. Al-Taweel, H. Al-Amrani, T. Al-Rawi, Induction and flow cytometry, GC-MS
genes tested increased, representing an up-regulation of the taxane identification of tetraploids through colchicine treatments in Datura stramonium L,
pathway. Plant Arch. 19 (2019) 241–250.
G.D. Ascough, J. Van Staden, J.E. Erwin, Effectiveness of colchicine and oryzalin at
inducing polyploidy in Watsonia lepida N.E. Brown, HortScience 43 (2008)
5. Conclusion 2248–2251, https://doi.org/10.21273/hortsci.43.7.2248.
M. Bonfill, J. Palaz#on, R.M. Cusid# o, S. Joly, C. Morales, M.T. PiΌol, Influence of elicitors
In conclusion, in this study an in vitro tetraploid cell line derived from on taxane production and 3-hydroxy-3-methylglutaryl coenzyme A reductase
activity in Taxus media cells, Plant Physiol. Biochem. 41 (2003) 91–96, https://doi.
immature embryos of T. baccata was successfully induced for the first org/10.1016/S0981-9428(02)00013-X.
time. 0.05% colchicine proved to be the most efficient concentration for G.G. Borisy, E.W. Taylor, The mechanism of action of colchicine. Colchicine binding to
the induction of polyploidy in Taxus spp. The polyploid line showed a sea urchin eggs and the mitotic apparatus, J. Cell Biol. 34 (1967) 535–548, https://
doi.org/10.1083/jcb.34.2.535.
significant increase in cell size, the elongated and rounded cells being I. Caruso, F.D. Piaz, N. Malafronte, N. De Tommasi, R. Aversano, C.W. Zottele, M.
3.33- and 2.99-fold larger, respectively, than in the diploid cell line. The T. Scarano, D. Carputo, Impact of ploidy change on secondary metabolites and
maximum total taxane production of the tetraploid cell line was 6.2-fold photochemical efficiency in Solanum bulbocastanum, Nat. Prod. Commun. 8 (2013)
1387–1392, https://doi.org/10.1177/1934578→1300801011.
higher and occurred 8 days earlier compared to the diploid line, showing P.P. Chee, Organogenesis in Taxus brevifolia tissue cultures, Plant Cell Rep. 14 (1995)
an improved productivity and taxane yield. In addition, the extracellular 560–565, https://doi.org/10.1007/BF00231938.
taxane content in this new tetraploid cell line was 10.8-fold higher L.L. Chen, S.L. Gao, In vitro tetraploid induction and generation of tetraploids from
mixoploids in Astragalus membranaceus, Sci. Hortic. (Amst. ) 112 (2007) 339–344,
compared to the diploid line. These results were accompanied by an https://doi.org/10.1016/j.scienta.2006.12.045.
increase in the transcription levels of all the taxane-related genes stud- R.M. Cusido, M. Onrubia, A.B. Sabater-Jara, E. Moyano, M. Bonfill, A. Goossens,
ied, particularly the BAPT and DBTNBT genes, which are active in the M. Angeles Pedre! no, J. Palazon, A rational approach to improving the
biotechnological production of taxanes in plant cell cultures of Taxus spp,
last steps of the pathway. Based on these results, polyploid cell lines of
Biotechnol. Adv. (2014), https://doi.org/10.1016/j.biotechadv.2014.03.002.
T. baccata represent another step forward in the development of optimal R.M. Cusid# o, J. Palaz#
on, M. Bonfill, A. Navia-Osorio, C. Morales, M.T. Pi! nol, Improved
Taxus spp. biofactories for the production of PTX and related taxanes. paclitaxel and baccatin III production in suspension cultures of Taxus media,
Further research should focus on determining the effect of elicitors on Biotechnol. Prog. 18 (2002) 418–423, https://doi.org/10.1021/bp0101583.
R.M. Cusid# o, J. Palaz#
on, A. Navia-Osorio, A. Mallol, M. Bonfill, C. Morales, M.T. Pi! nol,
the tetraploid cells. To sum up, this promising new biotechnological Production of Taxol® and baccatin III by a selected Taxus baccata callus line and its
strategy for taxane production offers the possibility of achieving higher derived cell suspension culture, Plant Sci. 146 (1999) 101–107, https://doi.org/
yields than currently available methods. 10.1016/S0168-9452(99)00093-X.
W.H. Eng, W.S. Ho, Polyploidization using colchicine in horticultural plants: a review,
Sci. Hortic. (Amst. ) (2019), https://doi.org/10.1016/j.scienta.2018.11.010.
Funding A. Escrich, L. Almagro, E. Moyano, R.M. Cusido, M. Bonfill, B. Hosseini, J. Palazon,
Improved biotechnological production of paclitaxel in Taxus media cell cultures by
the combined action of coronatine and calix[8]arenes, Plant Physiol. Biochem. 163
This work was supported by the Spanish Ministry of Science and (2021) 68–75, https://doi.org/10.1016/j.plaphy.2021.03.047.
Innovation, with project number PID2020–113438RB-I00/AEI/ A. Escrich, R.M. Cusido, M. Bonfill, J. Palazon, R. Sanchez-Mu! noz, E. Moyano, The
10.13039/501100011033. Ainoa Escrich was supported by a fellowship Epigenetic Regulation in Plant Specialized Metabolism: DNA Methylation Limits
Paclitaxel in vitro Biotechnological Production, Front. Plant Sci. 13 (2022) 2385,
from the University Pompeu Fabra. https://doi.org/10.3389/FPLS.2022.899444.
D. Ewald, Micropropagation of yew (Taxus baccata L.), in: Protocols for
Micropropagation of Woody Trees and Fruits, Springer, Netherlands, 2007,
Declaration of Competing Interest pp. 117–123, https://doi.org/10.1007/978-1-4020-6352-7_11.
O. Exposito, K. Syklowska-Baranek, E. Moyano, M. Onrubia, M. Bonfill, J. Palazon, R.
The authors declare that they have no known competing financial M. Cusido, Metabolic responses of Taxus media transformed cell cultures to the
addition of methyl jasmonate, Biotechnol. Prog. 26 (2010) 1145–1153, https://doi.
interests or personal relationships that could have appeared to influence
org/10.1002/btpr.424.
the work reported in this paper. O.L. Gamborg, R.A. Miller, K. Ojima, Nutrient requirements of suspension cultures of
soybean root cells, Exp. Cell Res. 50 (1968) 151–158, https://doi.org/10.1016/
Data Availability 0014-4827(68)90403-5.
S. Gantait, N. Mandal, S. Bhattacharyya, P.K. Das, Induction and identification of
tetraploids using in vitro colchicine treatment of Gerbera jamesonii Bolus cv. Sciella,
No data was used for the research described in the article. Plant Cell. Tissue Organ Cult. 106 (2011) 485–493, https://doi.org/10.1007/
s11240-011-9947-1.
S. Gantait, E. Mukherjee, Induced autopolyploidy—a promising approach for enhanced
Acknowledgments biosynthesis of plant secondary metabolites: an insight, J. Genet. Eng. Biotechnol.
(2021), https://doi.org/10.1186/s43141-020-00109-8.
JP and EM conceived the project and designed the research plan. AE S.L. Gao, B.J. Chen, D.N. Zhu, In vitro production and identification of autotetraploids of
Scutellaria baicalensis, Plant Cell. Tissue Organ Cult. 70 (2002) 289–293, https://
performed the cell cultures. AE and DH performed data analysis. AE, R S- doi.org/10.1023/A:1016577002039.
M and EM interpreted the data and wrote the draft of the manuscript. S.L. Gao, D.N. Zhu, Z.H. Cai, D.R. Xu, Autotetraploid plants from colchicine-treated bud
MB and JP supervised and supplemented the writing. All authors culture of Salvia miltiorrhiza Bge, Plant Cell. Tissue Organ Cult. 47 (1996) 73–77,
https://doi.org/10.1007/BF02318968.
reviewed and approved the final version for publication. JP acquired the B.K. Ghimire, E.S. Seong, T.X. Nguyen, J.H. Yoo, C.Y. Yu, S.H. Kim, I.M. Chung,
funds for the project. Assessment of morphological and phytochemical attributes in triploid and hexaploid
plants of the bioenergy crop Miscanthus → giganteus, Ind. Crops Prod. 89 (2016)
231–243, https://doi.org/10.1016/j.indcrop.2016.04.051.
Appendix A. Supporting information M.S.H. Grouh, H. Meftahizade, N. Lotfi, V. Rahimi, B. Baniasadi, Doubling the
chromosome number of salvia hains using colchicine: Evaluation of morphological
Supplementary data associated with this article can be found in the traits of recovered plants, J. Med. Plant Res. 5 (2011) 4892–4898, https://doi.org/
10.5897/JMPR.9000459.
online version at doi:10.1016/j.plantsci.2023.111776. P.K. Gupta, D.J. Durzan, Shoot multiplication from mature trees of Douglas-fir
(Pseudotsuga menziesii) and sugar pine (Pinus lambertiana), Plant Cell Rep. 4
References (1985) 177–179, https://doi.org/10.1007/BF00269282.
J. Iannicelli, M.A. Elechosa, M.A. Ju# arez, A. Martínez, V. Bugallo, A.L. Bandoni, A.
S. Escand# on, C.M. van Baren, Effect of polyploidization in the production of essential
M. Abdoli, A. Moieni, H. Naghdi Badi, Morphological, physiological, cytological and
oils in Lippia integrifolia, Ind. Crops Prod. 81 (2016) 20–29, https://doi.org/
phytochemical studies in diploid and colchicine-induced tetraploid plants of
10.1016/j.indcrop.2015.11.053.
Echinacea purpurea (L.), Acta Physiol. Plant. 35 (2013) 2075–2083, https://doi.org/
P. Inthima, K. Sujipuli, Improvement of growth and bacoside production in Bacopa
10.1007/s11738-013-1242-9.
monnieri through induced autotetraploidy with colchicine, PeerJ (2019) 2019,
K.L. Adams, J.F. Wendel, Polyploidy and genome evolution in plants, Curr. Opin. Plant
https://doi.org/10.7717/peerj.7966.
Biol. (2005), https://doi.org/10.1016/j.pbi.2005.01.001.

10
A. Escrich et al. Plant Science 334 (2023) 111776

N. Javadian, G. Karimzadeh, M. Sharifi, A. Moieni, M. Behmanesh, In vitro polyploidy M.M. R”ego, E.R. R”ego, C.H. Bruckner, F.L. Finger, W.C. Otoni, In vitro induction of
induction: changes in morphology, podophyllotoxin biosynthesis, and expression of autotetraploids from diploid yellow passion fruit mediated by colchicine and
the related genes in Linum album (Linaceae), Planta 245 (2017) 1165–1178, https:// oryzalin, Plant Cell. Tissue Organ Cult. 107 (2011) 451–459, https://doi.org/
doi.org/10.1007/s00425-017-2671-2. 10.1007/S11240-011-9995-6.
V.A.S. Jones, L. Dolan, The evolution of root hairs and rhizoids, Ann. Bot. 110 (2012) D.O. Robinson, J.E. Coate, A. Singh, L. Hong, M. Bush, J.J. Doyle, A.H.K. Roeder, Ploidy
205–212, https://doi.org/10.1093/AOB/MCS136. and size at multiple scales in the arabidopsis sepal, Plant Cell 30 (2018) 2308–2329,
T. Kaensaksiri, P. Soontornchainaksaeng, N. Soonthornchareonnon, S. Prathanturarug, In https://doi.org/10.1105/tpc.18.00344.
vitro induction of polyploidy in Centella asiatica (L.) Urban, Plant Cell. Tissue Organ A.B. Sabater-Jara, M. Onrubia, E. Moyano, M. Bonfill, J. Palaz# on, M.A. Pedre! no, R.
Cult. 107 (2011) 187–194, https://doi.org/10.1007/S11240-011-9969-8/FIGURES/ M. Cusid# o, Synergistic effect of cyclodextrins and methyl jasmonate on taxane
5. production in Taxus x media cell cultures, Plant Biotechnol. J. 12 (2014) 1075–1084,
Y.S. Kim, E.J. Hahn, H.N. Murthy, K.Y. Paek, Effect of polyploidy induction on biomass https://doi.org/10.1111/pbi.12214.
and ginsenoside accumulations in adventitious roots of ginseng, J. Plant Biol. 47 U. Salma, S. Kundu, N. Mandal, Artificial polyploidy in medicinal plants: Advancement
(2004) 356–360, https://doi.org/10.1007/BF03030551. in the last two decades and impending prospects, J. Crop Sci. Biotechnol. 20 (2017)
E. Kondorosi, F. Roudier, E. Gendreau, Plant cell-size control: Growing by ploidy? Curr. 9–19, https://doi.org/10.1007/s12892-016-0080-1.
Opin. Plant Biol. (2000) https://doi.org/10.1016/S1369-5266(00)00118-7. R. Sanchez-Mu! noz, M. Bonfill, R.M. Cusid# o, J. Palaz#
on, E. Moyano, Advances in the
U.C. Lavania, S. Srivastava, S. Lavania, S. Basu, N.K. Misra, Y. Mukai, Autopolyploidy Regulation of In Vitro Paclitaxel Production: Methylation of a Y-Patch Promoter
differentially influences body size in plants, but facilitates enhanced accumulation of Region Alters BAPT Gene Expression in Taxus Cell Cultures, Plant Cell Physiol. 59
secondary metabolites, causing increased cytosine methylation, Plant J. 71 (2012) (2018) 2255–2267, https://doi.org/10.1093/pcp/pcy149.
539–549, https://doi.org/10.1111/j.1365-313X.2012.05006.x. J. Swarbrick. Clarke’s isolation and identification of drugs, 2nd ed..,, Pharmaceutical
S. Liu, S. Chen, Y. Chen, Z. Guan, D. Yin, F. Chen, In vitro induced tetraploid of Press,, London, 1986.
Dendranthema nankingense (Nakai) Tzvel. shows an improved level of abiotic stress M. Tavan, M.H. Mirjalili, G. Karimzadeh, In vitro polyploidy induction: changes in
tolerance, Sci. Hortic. (Amst. ) 3 (2011) 411–419, https://doi.org/10.1016/J. morphological, anatomical and phytochemical characteristics of Thymus persicus
SCIENTA.2010.10.012. (Lamiaceae), Plant Cell. Tissue Organ Cult. 122 (2015) 573–583, https://doi.org/
H. Madani, A. Escrich, B. Hosseini, R. Sanchez-Mu! noz, A. Khojasteh, J. Palazon, Effect of 10.1007/s11240-015-0789-0.
polyploidy induction on natural metabolite production in medicinal plants, W. Thong-On, P. Arimatsu, S. Pitiporn, N. Soonthornchareonnon, S. Prathanturarug,
Biomolecules 11 (2021) 899, https://doi.org/10.3390/biom11060899. Field evaluation of in vitro-induced tetraploid and diploid Centella asiatica (L.)
M. Majdi, G. Karimzadeh, M.A. Malboobi, Spatial and developmental expression of key Urban, J. Nat. Med. 68 (2014) 267–273, https://doi.org/10.1007/s11418-013-0761-
genes of terpene biosynthesis in Tanacetum parthenium, Biol. Plant. 58 (2014) 4.
379–384, https://doi.org/10.1007/s10535-014-0398-5. H. Vidal-Limon, R. Sanchez-Mu! noz, A. Khojasteh, E. Moyano, R.M. Cusido, J. Palazon,
C. Manz, M.L. Raorane, J. Maisch, P. Nick, Switching cell fate by the actin–auxin Taxus Cell Cultures: An Effective Biotechnological Tool to Enhance and Gain New
oscillator in Taxus: cellular aspects of plant cell fermentation, Plant Cell Rep. 41 Biosynthetic Insights into Taxane Production, in: Reference Series in
(2022) 2363–2378, https://doi.org/10.1007/S00299-022-02928-0/FIGURES/6. Phytochemistry, Springer Science and Business Media B.V., 2018, pp. 295–316,
B.K. Mishra, S. Pathak, A. Sharma, P.K. Trivedi, S. Shukla, Modulated gene expression in https://doi.org/10.1007/978-3-319-54600-1_5.
newly synthesized auto-tetraploid of Papaver somniferum L, South Afr. J. Bot. 76 J.F. Wendel, Genome evolution in polyploids, Plant Mol. Evol. (2000) 225–249, https://
(2010) 447–452, https://doi.org/10.1016/j.sajb.2010.02.090. doi.org/10.1007/978-94-011-4221-2_12.
N. Moghbel, M.K. Borujeni, F. Bernard, Colchicine effect on the DNA content and stomata W. Widoretno, In vitro induction and characterization of tetraploid Patchouli
size of Glycyrrhiza glabra var.glandulifera and Carthamus tinctorius L, Cult. Vitr. J. (Pogostemon cablin Benth.) plant, Plant Cell. Tissue Organ Cult. 125 (2016)
Genet. Eng. Biotechnol. 13 (2015) 1–6, https://doi.org/10.1016/j.jgeb.2015.02.002. 261–267, https://doi.org/10.1007/s11240-016-0946-0.
M. Onrubia, E. Moyano, M. Bonfill, R.M. Cusid# o, A. Goossens, J. Palaz#on, Coronatine, a D. Xie, Z. Guo, Zygotic embryo culture of taxus chinesis var. mairei and plant
more powerful elicitor for inducing taxane biosynthesis in Taxus media cell cultures regeneration through organogenesis, Isr. J. Plant Sci. 47 (1999) 287–289, https://
than methyl jasmonate, J. Plant Physiol. 170 (2013) 211–219, https://doi.org/ doi.org/10.1080/07929978.1999.10676786.
10.1016/j.jplph.2012.09.004. H.J. Yan, Y. Xiong, H.Y. Zhang, M.L. He, In vitro induction and morphological
Pate, J.S., Dieter Jeschke, W., 1995. Role of Stems in Transport, Storage, and Circulation characteristics of octoploid plants in Pogostemon cablin, Breed. Sci. 66 (2016)
of Ions and Metabolites by the Whole Plant, in: Plant Stems. Academic Press, pp. 169–174, https://doi.org/10.1270/jsbbs.66.169.
177–17. https://doi.org/10.1016/b978–012276460-8/50010–2. Y.S. Yang, D. Nilanthi, X.L. Chen, F.C. Zhao, H. Wu, Induction of tetraploids from petiole
R.A. Patil, M.E. Kolewe, S.C. Roberts, Cellular aggregation is a key parameter associated explants through colchicine treatments in Echinacea purpurea L, J. Biomed.
with long term variability in paclitaxel accumulation in Taxus suspension cultures, Biotechnol. (2009) 2009, https://doi.org/10.1155/2009/343485.
Plant Cell. Tissue Organ Cult. 112 (2013) 303–310, https://doi.org/10.1007/ A.A. Zahedi, B. Hosseini, M. Fattahi, E. Dehghan, H. Parastar, H. Madani,
S11240-012-0237-3. Overproduction of valuable methoxylated flavones in induced tetraploid plants of
E. Perez-Matas, A. Hanano, E. Moyano, M. Bonfill, R.M. Cusido, J. Palazon, Insights into Dracocephalum kotschyi Boiss, Bot. Stud. 55 (2014) 1–10, https://doi.org/10.1186/
the control of taxane metabolism: Molecular, cellular, and metabolic changes 1999-3110-55-22.
induced by elicitation in Taxus baccata cell suspensions, Front. Plant Sci. 13 (2022) Y. Zhang, B. Wang, S. Qi, M. Dong, Z. Wang, Y. Li, S. Chen, B. Li, J. Zhang, Ploidy and
2698, https://doi.org/10.3389/fpls.2022.942433. hybridity effects on leaf size, cell size and related genes expression in triploids,
K. Ramirez-Estrada, L. Osuna, E. Moyano, M. Bonfill, N. Tapia, R.M. Cusido, J. Palazon, diploids and their parents in Populus, Planta 249 (2019) 635–646, https://doi.org/
Changes in gene transcription and taxane production in elicited cell cultures of Taxus 10.1007/s00425-018-3029-0.
→ media and Taxus globosa, Phytochemistry 117 (2015) 174–184, https://doi.org/ D.C. Zlesak, C.A. Thill, N.O. Anderson, Trifluralin-mediated polyploidization of Rosa
10.1016/j.phytochem.2015.06.013. chinensis minima (Sims) Voss seedlings, Euphytica 141 (2005) 281–290, https://doi.
org/10.1007/s10681-005-7512-x.

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