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In vitro morphogenetic responses from obligatory apomictic Taraxacum belorussicum Val. N. Tikhom seedlings explants

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Taraxacum belorussicum Val. N. Tikhom, a poorly known and obligatory apomictic species, is an attractive plant material for studying the embryological, genetic and molecular mechanisms of apomixis. This work aims to obtain an efficient protocol for Taraxacum belorussicum regeneration. Four types of explants (cotyledons, hypocotyls, meristems and roots) that were taken from 2-weeks-old seedlings were used for in vitro cultures, and a fast and efficient protocol of T. belorussicum regeneration was obtained. Various ½ MS-based media containing IAA (5.71 µM), TDZ (4.54 µM) and PSK (100 nM) were chosen to assess the morphogenetic abilities of selected T. belorussicum explants. Studies on the role of PSK were done in three independent experiments, where the most significant factors were always light and darkness. All explants produced callus by the third day of culture and adventitious shoots after 7 days, although in an asynchronous indirect manner, and with different intensities for all explant types. The most preferred medium culture for hypocotyl, cotyledon and meristem explants was ½ MS + TDZ, and ½ MS + IAA + TDZ + PSK for roots which were the only explant sensitive to PSK. A short darkness pretreatment (8 days) in PSK medium was found suitable to enhance organogenesis. Secondary organogenesis was observed for regenerated plants on meristem explants from the ½ MS + IAA + TDZ + PSK medium. A weak somatic embryogenesis was observed for hypocotyl and cotyledon explants from ½ MS + IAA + TDZ and ½ MS + IAA + TDZ + PSK media. Histological and scanning electron microscope images (SEM) of T. belorussicum confirmed indirect organogenesis and somatic embryogenesis. Plant material treated with aniline blue solution revealed the presence of callose in the cell walls of cotyledon and hypocotyl explants. The presence of extracellular matrix (ECM) and heterogenic structure of callus was also verified by scanning electron microscopy and light microscopy, confirming the high morphogenetic ability of T. belorussicum.
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Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
https://doi.org/10.1007/s11240-019-01694-4
ORIGINAL ARTICLE
In vitro morphogenetic responses fromobligatory apomictic
Taraxacum belorussicum Val. N. Tikhom seedlings explants
AdriannaGałuszka1· MaciejGustab2· MonikaTuleja2
Received: 31 March 2019 / Accepted: 5 September 2019 / Published online: 13 September 2019
© The Author(s) 2019
Abstract
Taraxacum belorussicum Val. N. Tikhom, a poorly known and obligatory apomictic species, is an attractive plant material
for studying the embryological, genetic and molecular mechanisms of apomixis. This work aims to obtain an efficient pro-
tocol for Taraxacum belorussicum regeneration. Four types of explants (cotyledons, hypocotyls, meristems and roots) that
were taken from 2-weeks-old seedlings were used for invitro cultures, and a fast and efficient protocol of T. belorussicum
regeneration was obtained. Various ½ MS-based media containing IAA (5.71µM), TDZ (4.54µM) and PSK (100nM) were
chosen to assess the morphogenetic abilities of selected T. belorussicum explants. Studies on the role of PSK were done in
three independent experiments, where the most significant factors were always light and darkness. All explants produced
callus by the third day of culture and adventitious shoots after 7days, although in an asynchronous indirect manner, and with
different intensities for all explant types. The most preferred medium culture for hypocotyl, cotyledon and meristem explants
was ½ MS + TDZ, and ½ MS + IAA + TDZ + PSK for roots which were the only explant sensitive to PSK. A short darkness
pretreatment (8days) in PSK medium was found suitable to enhance organogenesis. Secondary organogenesis was observed
for regenerated plants on meristem explants from the ½ MS + IAA + TDZ + PSK medium. A weak somatic embryogenesis
was observed for hypocotyl and cotyledon explants from ½ MS + IAA + TDZ and ½ MS + IAA + TDZ + PSK media. Histo-
logical and scanning electron microscope images (SEM) of T. belorussicum confirmed indirect organogenesis and somatic
embryogenesis. Plant material treated with aniline blue solution revealed the presence of callose in the cell walls of cotyledon
and hypocotyl explants. The presence of extracellular matrix (ECM) and heterogenic structure of callus was also verified by
scanning electron microscopy and light microscopy, confirming the high morphogenetic ability of T. belorussicum.
Key message
An efficient regeneration protocol for Taraxacum belorussicum, the obligatory apomict, was obtained. Application of PSK
revealed the significance of light and darkness as a key factor in the morphogenetic response. A short darkness pretreatment
(8 days) in PSK medium was found suitable to enhance organogenesis. Histological and scanning electron microscope images
confirmed indirect organogenesis and somatic embryogenesis presence. The protocol can enhance the knowledge about this
species in studying the embryological, genetic and molecular mechanisms of apomixis.
Communicated by Sergio J. Ochatt.
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s1124 0-019-01694 -4) contains
supplementary material, which is available to authorized users.
* Monika Tuleja
monika.tuleja@uj.edu.pl
1 Department ofClinical Immunology, Jagiellonian University
Medical College, Cracow, Poland
2 Department ofPlant Cytology andEmbryology, Institute
ofBotany, Jagiellonian University, Gronostajowa Street 9,
30-387, Cracow, Poland
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506 Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
1 3
Keywords Taraxacum belorussicum· Apomixis· Phytosulfokine· Somatic embryogenesis· Organogenesis
Abbreviations
BAP 6-Benzyl amino purine
ECM Extracellular matrix
IAA Indole-3-acetic acid
BA Indole-3-butyric acid
MS Murashige & Skoog
NAA Naphthalene acetic acid
PAS Periodic acid schiff
PGRs Plant growth regulators
PSK Phytosulfokine
SE Somatic embryogenesis
SEM Scanning electron microscope
TDZ Thidiazuron
Introduction
Taraxacum belorussicum Val. N. Tikhom or dandelion,
belongs to the genus Taraxacum (Asteraceae) (Tikhomirov
2003). This genus includes many species which were used
for the analysis of embryological, genetic and molecular
aspects of apomictic mechanisms (Janas etal. 2016). In gen-
eral, dandelions have also been a part of the natural medicine
tool kit because of their medical qualities as diuretic, chol-
eric, anti-inflammatory, anti-oxidative, anti-carcinogenic
and melliferous. T. belorussicum Val. is also a raw material
for the cosmetic industry (Hu and Kitts 2003; Jamshieed
etal. 2010). Based on their specific features and heavily
urbanized areas of growth (Erofeeva 2014) some Taraxacum
species can play a role as temperate climate bio-indicators
(Jamshieed etal. 2010). T. belorussicum is a Palustria trip-
loid species (Marciniuk etal. 2010) and comes from the
area of Minsk in Belarus where it was described for the
first time by Tikhomirov (Tikhomirov 2003). It is an obliga-
tory apomict that can produce embryos in a parthenogenetic
manner, and can thus be used for the comparative study of
parthenogenesis and somatic embryogenesis.
Apomixis is a type of plant reproduction during which
the embryo is formed without full meiosis and syngamia, or
omitting either of these two processes (Noyes 2007). Taraxa-
cum is characterized by the occurrence of diplospory apo-
mixis (Koltunow 1993; Hand and Koltunow 2014), where
a meiotic prophase is observed, but chromosomes do not
create bivalents or metaphase tiles. Instead, a restitution
nucleus is formed which leads to generation of unreduced
embryo sac cells after successive mitotic divisions (Hand
and Koltunow 2014; Rodkiewicz etal. 1996). Despite this,
some apomictic species often maintain the ability to sexu-
ally reproduce, and certain seed parts have a zygotic origin
(Rodkiewicz etal. 1996).
One of the common morphogenetic pathways leading to
invitro plant regeneration is organogenesis (Huang etal.
2014), which can be indirect via callus and direct by the
creation of adventitious shoots from explant tissue (Woźny
and Przybył 2004). A second possible path of invitro mor-
phogenesis is somatic embryogenesis, with somatic embryo
induction from a somatic cell without fertilization (Williams
and Maheswaran 1986). Somatic embryos can be formed
directly from the initial explant (Kuo etal. 2005) or indi-
rectly from callus cells (Jasrai etal. 2003). Both morphoge-
netic processes are under the control of several factors, one
of which is the explant’s origin (Ebida and Hu 1993), while
others are the growth factors, the auxins, cytokinins and
polysaccharides, amino acids, vitamins and proteins (Nic-
Can etal. 2015) to which it is exposed. The ratio between
concentration of auxins and cytokinins has a crucial influ-
ence on invitro morphogenesis (Isah 2015). Phytosulfokine
(PSK), which was isolated for the first time from the Aspar-
agus officinalis mesophyll (Matsubayashi and Sakagami
1996), being a peptide hormone, plays a role of regulatory
molecule in the growth and development of plant cells, espe-
cially in somatic embryogenesis induction (Lorbiecke etal.
2005; Igasaki etal. 2003; Kobayashi etal. 1999; Maćkowska
etal. 2014; Ochatt etal. 2018).
Plant cell predisposition to undergo one of the two devel-
opmental pathways invitro can be predicted using callose
identification, as callose deposited in the cell walls is a
marker of somatic embryogenesis. In addition, the pres-
ence of cuticle and the loss of plasmodesmata connections
are attributed to this process (Fiuk and Rybczyński 2006).
Callose may be also deposited in the place of damage as
protection against microorganism infection (Ellinger and
Voigt 2014).
According to the latest data, the presence of the Extracel-
lular Matrix (ECM) in invitro culture is a specific feature
of morphogenesis or germinal tissues that is associated with
ECM signaling functions. In some cases, ECM may perform
a protective function or is seen as a specific feature of cal-
lus surfaces formed under invitro conditions (Popielarska-
Konieczna etal. 2013). The formation of ECM may be the
result of stress factors or exposure to chemical agents that
protect callus cells (Bevitori etal. 2013) and can create a
pathway for receiving and transducing signals that determine
the recognition and fate of cells, and the morphogenesis of
plant.
Apomixis is a phenomenon in which embryos arise by
bypassing the process of fertilization, as also observed in
embryo formation from somatic tissue cultured invitro. To
what extent this similarity has its justification at the molec-
ular or physiological level so far is not known and is an
interesting research challenge, for which use of apomictic
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507Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
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Taraxacum species seems to be the right choice. Therefore,
tracing the morphogenetic abilities with the hope of obtain-
ing an efficient somatic embryogenesis of this species is the
first and necessary step for later comparative studies of these
two developmental processes. Based on the growing evi-
dence suggesting that PSK has impact on acquisition of SE
and morphogenesis, while there are no reports concerning
these processes in T. belorussicum, we have attempted to
identify problems related to the development of an efficient
regeneration system for selected types of explants in invitro
conditions that included histological and SEM analysis.
Materials andmethods
Plant material
All experiments were performed with seeds of apomict
T. belorussicum Val. N. Tikhom kindly received from
Professor Jolanta Marciniuk (Institute of Biology, Uni-
versity of Natural Sciences and Humanities at Siedlce
in Poland). All seeds were sterilized by immersion in
70% ethanol (Sigma, St. Louis, MO, USA) for 30s and
then in 2% solution of sodium hypochlorite (Sigma) for
15min. Next, they were rinsed three times in sterile water
and placed in Petri dishes (Corning Incorporated, Corn-
ing, NY) containing ½ MS (Murashige and Skoog 1962)
medium supplemented with 30g/l sucrose and 8g/l agar.
In vitro plant culture conditions
All used media were based on MS medium (Murashige
& Skoog Medium, Duchefa Biochemie B.V., Haarlem,
The Netherlands). Seeds were cultured onto 20ml of
½ MS medium culture under a 16-h light photoper-
iod (cool white fluorescent light with an intensity of
39μM × m−2 × s−1) at 23 ± 2°C for 2weeks. Afterwards,
four types of explants: cotyledon (Fig. S1b), meristem
(Fig. S1c), hypocotyl (Fig. S1d) and root (Fig. S1e) were
obtained from the 2-week-old seedlings (Fig. S1a) and
cultured on several media according to different experi-
mental systems with 25 explants per Petri dish, in three
repetitions. As a control, ½ MS medium was used.
Organogenesis model regenerated plants with clearly
formed leaves were isolated from explants after 40days
of culture and transferred into test-tubes (Equimed, Cra-
cow, Poland) containing 50ml ½ MS, supplemented with
4.92μM IBA, under conditions as above for 90days with-
out subculturing. Then, regenerated well rooted plantlets
were transferred into pots filled with experimental soil
media, covered by transparent plastic boxes and kept in
the same culture conditions for the next 14days. Within
this time, all plantlets acclimatized successfully.
SE/PSK model/short darkness explants were cultured
in ½ MS medium supplemented with 5.71μM IAA,
4.54μM TDZ and 100nM PSK (phytosulfokine-α, Pep-
taNova GmbH, Germany) at 23 ± 2°C for 8days in the
dark (short darkness), then explants were transferred to
the light regime above for 60days, with two subcultures
(Fig. S2). Next, the regenerated plants were test tube cul-
tured (Equimed, Cracow, Poland) in 50ml ½ MS, supple-
mented with 4.92μM IBA under the photoperiodic light
and temperature conditions as above.
SE/PSK model/long darkness Explants were cultured
in ½ MS medium, supplemented with 5.71μM IAA,
4.54μM TDZ and 100nM PSK at 23 ± 2°C for 55days
in the dark (long darkness), and were then transferred
to the light for 35days without subculturing (Fig. S3).
Six types of media were used in this experimental model
(P1—½ MS − control, P2—½ MS + IAA + TDZ, P3—½
MS + IAA + TDZ + PSK, P4—½ MS + PSK, P5—½
MS + TDZ, P6—½ MS + TDZ + PSK). Next, the regener-
ated plants were test tube cultured in 50ml ½ MS, supple-
mented with 4.92μM IBA under the same photoperiodic
light conditions as above at 23 ± 2°C.
Histological analysis ofinvitro morphogenesis
Samples of each kind of explant after 50days of culture
were fixed in a mixture of 5% glutaraldehyde in phosphate
buffer, pH 7.2 (Sigma) in 5°C overnight, washed four times
in 0.1M phosphate buffer (pH 7.2), then dehydrated in an
ethanol series (10%, 30%, 50%, 70%, 96%; 15min each) and
kept overnight in absolute ethanol. The fixed tissue samples
were subsequently embedded in Technovit 7100 (Heraeus
Kulzer, South Bend, Indiana, USA). Next, histology blocks
of plant material were made using a solution of Technovit B
under vacuum for 24h. Plant material histology blocks slices
(7µm of thickness) were obtained by using a rotational
microtome—Microm HM 355 S I Microtome (MICROM
International GmbH, Walldorf, Germany).
Histological samples were stained using 0.1% toluidine
blue (Sigma), 0.05% aniline blue (Sigma), 0.5% naphthol
blue black (Sigma) and PAS—Periodic Acid Schiff (Sigma).
Next, sample slides were closed by histological medium
(Entellan; Merck Millipore, USA). To detect cell structures
such as the cytoplasm or nuclear cell, the slides were first
warmed, then stained with 0.1% toluidine blue for 30s and
afterwards, washed in distilled water. The callose fluores-
cence was observed in UV light (370nm) after staining with
0.1% toluidine blue for 30s and being incubated with 0.05%
aniline blue for 10min.
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508 Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
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Scanning electron microscope (SEM)
For examination utilizing the scanning electron microscope,
plant material of each type of explant was collected at the
50th day of culture and pre-fixed in 5% buffered glutaral-
dehyde (0.1M phosphate buffer, pH 7.2) for 3h at room
temperature. After dehydration in a graded ethanol series,
samples were dried at the critical point of CO2 by use of a
Quorum Technologies E3000 (Quorum Technologies Ltd.,
Lewes, UK). Afterwards, samples were coated with fine-
grained gold using automated sputter coater Jeol JFC—1100
E (JEOL, Peabody, MA, USA) for imaging via scanning
electron microscope Jeol JSM 5410 (JEOL).
Observation anddocumentation ofresults
Images of invitro cultures were made utilizing a Canon
PC 1089 (Canon, Uxbridge, Middlesex, United Kingdom)
connected to a binocular loupe Stemi SV 11 (Carl Zeiss
Microscopy, Jena, Germany) at 7-day intervals. Symptoms
of somatic embryogenesis were observed on selected types
of explants, while organogenesis was scored as an average
amount of shoots per explant for all types of explants, after
20 and 45days of culture for SE/PSK model/short darkness
and after 30 and 65days of culture for SE/PSK model/long
darkness. Regenerated plants and ex vitro culture evolution
were documented by Canon PC 1089 (Canon, Uxbridge,
Middlesex, United Kingdom). Histology samples were ana-
lyzed using a phase contrast microscope Nikon Eclipse E400
(Nikon Corporation, Tokyo, Japan) with Olympus 10x/0.25
(Olympus Corporation, Tokyo, Japan) and Olympus
40x/0.55 (Olympus Corporation) objective lenses. Samples
stained with aniline blue were analyzed with an EX330-380
(Nikon Corporation) excitation filter. Images were processed
by EOS Utility version 3 software (Canon).
Statistics
Statistical analyses were performed by One-way ANOVA
test with Tukey test for multiple comparisons and test t
with using the PRISM GraphPad 6.01 software (GraphPad
Software Inc., San Diego, California, USA). Results were
expressed as mean ± SEM per one explant for all types of
explants. Statistical significance of differences between
groups were considered at p values: *p < 0.01, **p < 0.01,
***p < 0.001, ****p < 0.0001.
Results
Induction anddevelopment ofcallus
All types of explants (meristem, hypocotyl, cotyledon, root),
after mechanical damage, produced callus at different effi-
ciency rates after 3days of culture in all experimental models
(Fig.1). Observation of root explants (Fig.1a) showed that
callus formation begins initially at the ends of explants, in
the place of scalpel cutting (Fig.1b). Next, the entire explant
swells (Fig.1c) and callus formation subsequently covers
the whole or the most part of the explant surface (Fig.1d).
Indeed, 100% of the explants started overgrowing with callus
after 8days of culture. At this time all explants showed two
callus types, non-morphogenetic (Fig.1d) and morphoge-
netic (Fig.1e). Morphogenetic callus was identified by the
formation of adventitious shoots (Fig.1f)—a trait that is not
characteristic for non-morphogenetic callus. Observation of
hypocotyl and root explants provided similar information
about the callus formation and development, albeit with some
important differences. Initially, the callus had a compact
structure and was visible on one part of the explants (Fig.1c).
The callus color concentrated around various shades of green,
while white and yellow colors were also noticed.
During the culture of selected T. belorussicum explants,
the most advanced formation and development of callus
was observed on root (Fig.1 I) and hypocotyl (Fig.1 II)
explants. Meristem explants also produced callus, but in a
moderate amount, while callus was formed with the slowest
and the least efficiency on cotyledon explants. Callusing was
most intensive in the experimental model with a complete
medium: ½ MS + IAA + TDZ + PSK (Fig.1). Explants cul-
tured on the control medium did not produce callus (data not
shown). Results obtained from other experimental models
(½ MS + IAA + TDZ and ½ MS + TDZ + PSK) revealed a
varied intensity of callusing as a phase preceding the induc-
tion of adventitious shoots, observed on hypocotyl and root
explants (Fig.1). The weakest development of callus was
noted on cotyledon explants. The callus color was from light
to dark green, as well as white, yellow and brown. Meris-
tem, cotyledon and hypocotyl explants showed the greenest
shades, thus the largest amount of chlorophyll in callus cells,
while shades of brown were visible on root explants.
Morphogenetic response ofselected explants
depending onplant growth regulators treatment
The intensity of morphogenetic response (organogenesis)
of selected types of explants was expressed as the average
number of adventitious shoots produced during invitro cul-
ture. The anecdotal presence of somatic embryogenesis did
not allow for its proper quantification.
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509Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
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Organogenesis
In the organogenesis model, the first morphogenetic
response occurred after 7days of culture on ½ MS medium
with TDZ and IAA, where analyses revealed callus for-
mation on all types of explants, but meristem and root
explants degenerated over time. Furthermore, the presence
of adventitious shoots was evident only on hypocotyl and
meristem explant surfaces (TableS1). Hence, the regenera-
tive efficiency invitro was calculated only for these explants
(Fig.2a), with an average of regenerated plants of 8.48 ± 1.7
and 17.8 ± 4.94 for cotyledon and hypocotyl explants,
respectively, whereby hypocotyl explants were selected for
subsequent studies. Noteworthy, 140 out of 251 regenerated
Fig. 1 Two paths of callus
development, indirect organo-
genesis and somatic embryo-
genesis (arrow) of T. belorus-
sicum in invitro culture: I the
development of callus from the
root explant, II the develop-
ment of callus from hypocotyl
explant and the first symptoms
of somatic embryogenesis
(marked as an oval structures);
a—explant on the first day of
culture, b—beginning of callus
formation on the cut edges of
explant, c—swelling callus and
visible changes in the explant
(after 14days of culture), d—
morphogenetic structures after a
few days of culture, e—explant
overgrown with developing
callus (60th day of root explant
culture), f—adventitious shoots
on root explant on the 80th day
of culture and hypocotyl after
28days (indirect organogene-
sis). Photos came from SE/PSK
model/short darkness; explants
were cultured on ½ MS medium
supplemented with 5.71μM
IAA, 4.54μM TDZ and 100nM
PSK. Scale 1mm
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510 Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
1 3
plants obtained from hypocotyl explants were successfully
rooted and ultimately able to function in ex-vitro conditions.
In SE/PSK model/short darkness, hypocotyl and meris-
tem explants revealed better morphogenetic response than
did cotyledon and root explants after 20 and 45days of cul-
ture on ½ MS medium supplemented with IAA, TDZ and
PSK, with a short time under dark conditions (8days). The
presence of adventitious shoots was observed on the surface
of all type of explants and thus their regenerative efficiency
was calculated. Analyses also showed that organogenesis
efficiency in terms of average amount of regenerated plants
was highest on meristem and hypocotyl explants, while that
of root and cotyledon explants was the lowest (Fig.2b). In
addition, indirect organogenesis proceeded with different
efficiency on the majority of all types of explants. However,
all the regenerated plants obtained were successfully rooted
after 28days on ½ MS medium supplemented with IBA.
In SE/PSK model/long darkness, meristem and coty-
ledon explants revealed better morphogenetic response
than did hypocotyl and root explants after 30 and 65days
of culture under an extended period of time in dark con-
ditions (55days) (Fig.2c). In this model, ½ MS medium
supplemented with TDZ turned out to be more efficient
than medium with PSK added, for cotyledon, hypocotyl
and meristem explants, while ½ MS medium supplemented
with IAA, TDZ and PSK showed the highest efficiency for
root explants. Conversely, root explants cultured on ½ MS
medium with TDZ and PSK and cotyledon explants cultured
on ½ MS medium with PSK died out after 103days of cul-
ture (Figs.3 and 4). I was also observed that indirect organo-
genesis proceeded with diverse efficiency on the majority
Fig. 2 Organogenesis efficiency
of T. belorussicum explants. a
hypocotyl and cotyledon after
40days of culture on ½ MS
with 5.71μM IAA and 4.54μM
TDZ. b hypocotyl, meristem,
cotyledon and root cultured
on ½ MS with 5.71μM IAA,
4.54μM TDZ and 100nM PSK
during 8days without access
to light and after the transfer
of the explants to light for the
subsequent 60days. Number
of adventitious shoots per one
explant were assessed twice
after 20days (T1) and 45days
(T2) of culture. c hypoco-
tyl, meristem, cotyledon and
root cultured on: P1—½ MS
(control), P2—½ MS + PSK,
P3—½ MS + TDZ, P4—½
MS + IAA + TDZ, P5—½
MS + TDZ + PSK, P6—½
MS + IAA + TDZ + PSK dur-
ing 55days without access to
light and after transferring the
explants to light for the sub-
sequent 35days. The number
of adventitious shoots per one
explant were assessed twice
after 30days (T1) and 65days
(T2) of culture. Data are pre-
sented as mean ± SEM per one
explant for all types of explants.
Statistical significance of dif-
ferences between groups were
considered at p values: *p < 0.1,
**p < 0.01, ***p < 0.001,
****p < 0.0001 (a test t, b and
c one-way ANOVA test). The
figures show the results from 25
independent explants per one
Petri dish in three repetitions
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511Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
1 3
of explants, while secondary organogenesis (adventitious
shoots formation on the regenerated plants) was notice-
able on regenerated plants from meristem explants cultured
on ½ MS medium supplemented with IAA, TDZ and PSK
(Fig.4n). All regenerated plants obtained were successfully
rooted after 28days on ½ MS medium supplemented with
IBA.
Somatic embryogenesis
In SE/PSK model/short darkness, symptoms of somatic
embryogenesis were observed on hypocotyl and cotyledon
explants (Fig.1c II). Somatic embryos were formed directly
from explant tissue cultured on all type of ½ MS media
supplemented with PSK. The highest efficiency of somatic
embryogenesis, in the form of small, light-green globular
structures, was observed on ½ MS medium supplemented
with TDZ, IAA and PSK with a short time period under
darkness (8days). Formation of somatic embryos was not
observed in the organogenesis model and SE/PSK model/
long darkness on all types of explants and in SE/PSK model/
short darkness on meristem and root explants.
Role ofphytosulfokine (PSK) intheprocess
oforganogenesis
The applied conditions in SE/PSK model/short darkness
were designed to enhance PSK activity by pre-incubation
of explant cultures in the dark for 8days. Results obtained
clearly indicate that the addition of PSK to the medium
increased the intensity and efficiency of the organogenesis
process. However, the explants were in a poor condition: part
of them began to quickly degenerate and, under these condi-
tions, most explants were overgrown with callus (especially
the root explants). To improve this, explants were subjected
to light after 8days of incubation in dark. During culture
under light conditions, a significant improvement was evi-
dent, the color of explants changing from brown yellow to
bright and dark green.
The combination of PSK with auxins and cytokinins
enhanced the process of indirect organogenesis and had
Fig. 3 The development of T. belorussicum explants (a, e, i, m
cotyledon; b, f, j, n meristem; c, g, k, o hypocotyl; d, h, l, p root)
cultured on: P1—½ MS (control), P2—½ MS + PSK, P4—½
MS + IAA + TDZ, P6—½ MS + IAA + TDZ + PSK in 23 ± 2°C after
7days of culture (SE/PSK model/short darkness). Scale: 1mm
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512 Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
1 3
a positive influence on somatic embryogenesis induction.
Results obtained from SE/PSK model/long darkness with
an extended period of explant culture in the dark (55days),
indicated that selected types of explants had undergone a
varied intensity of morphogenesis process depending on the
medium used (Fig.2c). For cotyledon, hypocotyl and mer-
istem explants, the use of a medium without PSK was much
more favorable. However, root explants developed better on
a medium with PSK. It should be noted that the used media
differed not only in the presence or absence of PSK, but also
in the auxin and cytokinins content. Still, the interaction
between PSK, auxins and cytokinins did not significantly
affect the development of explants. In the case of hypoc-
otyl, meristem and root explants, the use of PSK without
Fig. 4 The development of T. belorussicum explants (a, e, i, m, q,
u cotyledon; b, f, j, n, r, v meristem; c, g, k, o, s, w hypocotyl; d,
h, l, p, t root) cultured on: P1—½ MS (control), P2—½ MS + PSK,
P3—½ MS + IAA + TDZ, P4—½ MS + IAA + TDZ + PSK, P5—½
MS + TDZ, P6—½ MS + TDZ + PSK in 23 ± 2 °C after 28 days of
culture (SE/PSK model/long darkness). Scale: 1mm
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513Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
1 3
Fig. 5 Cross-section of T. belorussicum explants from SE/PSK
model/long darkness: cotyledon (a, b), meristem (d, e), hypoco-
tyl (g, h) and root (j, k) cultured on ½ MS medium supplemented
with 5.71 μM IAA, 4.54 μM TDZ and 100 nM PSK in 23 ± 2 °C
after 55days of culture: a explant with xylogenic centers, b somatic
embryo from the cotyledon explant, d presence of ECM between
cells of the meristem explant, e xylogenic centers (asterisk) in mer-
istem explant, g morphogenesis on the edges of the explant, h ECM
between hypocotyl cells with fragment of regenerated plant, j adven-
titious shoots as deformed relicts in the root explants, k cross-section
through the explant tissue in advanced culture and leaf of regenerated
plant, and cross-section of T. belorussicum explants from the organo-
genesis model: cotyledon (c), meristem (f), hypocotyl (i) and root
(l) cultured on ½ MS medium supplemented with 5.71μM IAA and
4.54μM TDZ in 23 ± 2 °C after 47days of culture: c leaf of regen-
erated plant with fragment of the vascular bundle, stomata (asterisk)
and starch grains in the mesophyll cells, f young stage of regenerating
leaf (asterisk) with vascular bundle inside and the xylogenic center
(asterisk), i numerous starch grains in callus cells and the xylogenic
center at the young stage of its development (asterisk), l varied shape
of callus cells, presence of ECM (asterisk) and xylogenic centers
(asterisk) at the early stage of formation. PAS reaction. Scale: 100μm
(ag, j, k), 25μm (h, i and l)
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514 Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
1 3
other agents, slightly stimulated the organogenesis process.
However, in the case of cotyledon explants, the use of PSK
without auxins and cytokinins did not bring a desired effect.
The applied light conditions in SE/PSK model/long
darkness were also designed to enhance PSK activity. After
55days, explants showed a poor condition, most of them
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515Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
1 3
began to die. So, all surviving cultures were transferred to
the light for the subsequent 35days. However, the effect of
PSK on the formation of callus was noticed while under
dark conditions. Observation of explants in the light showed
a significant improvement in their condition as they turned
from brown yellow to green (due to the restoration of chlo-
rophyll). PSK had a positive effect on somatic embryogen-
esis (visible on cotyledon explants), but deformed somatic
embryos (which were not able to convert) were observed.
Such tissues did not produce regenerated plants and the cul-
ture was terminated at this stage.
Histological evaluation ofselected explants
Explants from the organogenesis model and SE/PSK model/
long darkness were selected for histological analysis.
In the organogenesis model, histological analysis con-
firmed the presence of two types of callus tissue. This dif-
fered in the shape and size of cells. The first consisted of
loosely distributed cells with various shapes and intercel-
lular spaces (Fig.5l). The second type was distinguished
by a compact structure with meristematic centers and
adventitious shoots were formed (Fig.6e). These centers
were mainly present in the subsurface layers, hence con-
firming that organogenesis proceeded indirectly via callus
tissue. Cells in the meristematic centers were character-
ized by a dense cytoplasm, clearly visible nuclei and thin
cell walls. In addition, to the adventitious shoots, somatic
embryo-like structures were observed on the hypocotyl
explants. Moreover, in some explants, different stages of
regeneration process were observed at the same time of
culture (Fig.6d, e, k). Analyses also documented non-syn-
chronized development of regenerated plants leaves from
the meristem explants (Fig.6l). Still, explants maintained
tissue integrity—made evident by the presence of anticli-
nal cell divisions in the epidermis of meristem explants
(Fig.6j). However, the majority of callus cells were
highly vacuolated, in some cases, nuclei with well-visible
nucleoli could be observed, as well as various cytokinesis
phases. Xylogenic centers were also commonly present
among the explants and callus cells (Figs.5f, i, l, 6a).
Vascular tissue was observed in the adventitious shoots at
advanced stages of regeneration (Figs.5f, 6d) and in the
root explants as deformed relicts (Fig.5j). PAS staining
showed, in all types of explants, the presence of starch
granules accumulating around the meristematic centers,
near the cell walls, mainly in the callus cells (Fig.5i, l).
These granules were also quite noticeable in the stomates
and the mesophyll tissue of regenerated leaves, as well as
in the vascular tissue. This suggests a properly function-
ing photosynthetic system has developed (Fig.5c). Their
protein content was identified through naphthol blue black
staining. The aforementioned granules were highly con-
centrated in the cells of meristematic centers, at various
stages of development (Fig.6d–f, j–l). Both PAS and tolui-
dine blue staining revealed the presence of an extracellular
matrix (ECM) with fibrous, reticular or mucoid structures
filling the spaces between the callus cells (Fig.6a). In
contrast to the successful application of these two methods
of staining, the ECM was not visible under naphthol blue/
black conditions, which makes evident its polysaccharide
and non-protein character.
In the SE/PSK model/long darkness, histological analy-
sis of the cotyledon explants confirmed indirect organogen-
esis and the induction of indirect somatic embryogenesis
(Fig.5a, b). Results revealed structures similar to somatic
embryos formed from explant tissues overgrown with endog-
enous callus (Fig.6b). These structures were deformed at
the later stage of development. The PAS staining showed
clearly visible vascular bundles in the embryo cotyledon
explant and the absence of starch in explant and embryo
tissues (Fig.5b). Secondary xylogenic centers were seen
within the mesophyll, the explant epidermis was noted to
be continual (Fig.5a) and lobed nuclei cells were noted in
explant tissues and regenerated plants. Naphthol blue/black
staining showed a concentration of the dye in the cells of the
developing regenerated plant, in the top region of explant.
This effect may indicate formation of the apical meristem.
Double staining with PAS and naphthol blue/black revealed
the presence of secondary meristematic centers next to
Fig. 6 Cross-section of T. belorussicum explants cultured on ½ MS
medium supplemented with 5.71 μM IAA and 4.54 μM TDZ from
the organogenesis model: meristem (a, e, j), cotyledon (d), hypoco-
tyl (f, k) and root (l) after 47days of culture, and cross-section of T.
belorussicum explants cultured on ½ MS medium supplemented with
5.71μM IAA, 4.54 μM TDZ and 100 nM PSK: cotyledon (b) and
meristem (c) after 20days of culture from SE/PSK model/short dark-
ness, meristem (G), hypocotyl (H, M) and root (I, N, O) after 55days
of culture from SE/PSK model/long darkness. b fibrous structure
of ECM (asterisk) between callus cells and xylogenic centers, b, c
organogenesis on the 20th day of culture at one of the poles, d regen-
erated plant (asterisk) growing from cotyledon explant and secondary
meristematic center, e regenerated plant (asterisk) growing from the
sub-surface layer of the meristem explant with meristematic center
and one leaf, as well as an xylogenic center on the right, f regener-
ated plant from hypocotyl explant, meristematic center (asterisk), two
growing leaves and arch of the vascular tissue, g cytological differ-
entiation within the meristem, h secondary meristematic and xylo-
genic centers, i compact structure of explant with the varied shape of
cells, j young adventitious shoot, content of protein and the rest of
explant epidermis, k regenerated plant, apical meristem, two growing
leaves and vascular bundles, l different shapes of regenerated young
leaves, m enlargement of the fragment from which adventitious shoot
grow, meristematic and xylogenic centers (asterisks), vascular bun-
dles, n ECM between callus cells, o morphogenesis from pericycle.
ac Staining by 0.1% toluidine blue. Scale: 100μm (ac); di Protein
localization by staining with 0.5% naphthol blue black. Scale: 100μm
(dh), 500 μm (i); jo PAS reaction for the presence of starch in
explants and staining with 0.5% naphthol blue/black to determine the
protein localization. Scale: 25μm (n), 100μm (ko), 500μm (j, l)
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516 Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
1 3
the formed adventitious shoot on the explant during callus
formation. Based on the continuity of the epidermis, the
presence of endogenous callus was indicated. Moreover,
well-formed long vessels were observed in the place where
secondary meristematic centers were formed, while well-
visible nuclei and dense cytoplasm were reported in small
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517Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
1 3
cells surrounding vessels. This effect indicates the meris-
tematic nature of these cells. Such aforementioned changes
observed on the cross-sections are mostly noticeable in the
cotyledon’s mesophyll. Still, despite many alterations, the
tissue maintained compact nature and cells adhered closely
to each other.
Histological analysis of the meristem explant showed the
presence of secondary meristematic centers, but the continu-
ity of tissues was not maintained. Many intercellular spaces
were ECM enriched (Fig.5d). However, continuity of epi-
dermis was not maintained, and an exogenous callus was
observed next to endogenous callus (Fig.5e). Differences
in size and shape of cells, the presence of many cytokinesis
at various stages of advancement and the formation of xylo-
genic centers were observed (Fig.6g). Cells stained with
PAS and naphthol blue/black showed that these were viable.
This was confirmed by the presence of proteins and the accu-
mulation of starched in these cells, as well as their abun-
dant cytoplasm. The appearance of shoots from the meris-
tematic tissue and the presence of the first younger leaves
were observed (Fig.6c), while double staining with PAS
and naphthol blue/black revealed the structure of the tissue
surrounding the meristematic tissue. Herein, circular abun-
dant meristematic and xylogenic centers, dense cytoplasm
and large dividing nuclei were noted in the meristematic
tissue—indicating high mitotic activity. The differentiation
of callus parenchyma cells into meristematic tissues was also
seen. This is a natural development.
Histologic analysis of the hypocotyl explant revealed
changes in the axial roller and parenchyma of the primary
cortex cells (Fig.5g and h). Endogenous callus with large
spaces between cells was also present within the primary
cortex (Fig.6h). Double staining with PAS and naphthol
blue/black showed lack of epidermis continuity, abundant
endogenous and exogenous callus. Moreover, meristematic
and xylogenic centers had formed from subepidermal cells
of callusing explant and were located inside and on the
edge of the callus. The formation of adventitious shoots and
somatic embryos on the explant was also noted in the mer-
istematic centers formed in the callus (Fig.6m). In addition,
the callus was heterogenous, with different size and irregu-
lar shapes of cells. At the base of the forming adventitious
shoots, new young shoots were seen to have sprouted—indi-
cating the unsynchronized nature of indirect organogenesis.
Histological analysis of the root explant revealed a disor-
der in its anatomical structure, as well as large intercellular
spaces, lack of epidermis continuity and presence of vascular
bundles. The structure of parenchyma of the primary cortex
was of compact nature. Changes in the explant’s construc-
tion, the presence of meristematic centers, cell aggregates
and formation of callus were observed at the later stage of
explant development (Fig.5k). Furthermore, well-developed
vessels arranged in a concentric way and the explant being
overgrown with callus were evident. The cross-section
through the root explant also showed cytological changes
characterized by different size and shapes of cells (Fig.6i).
Double staining with PAS and naphthol blue/black revealed
the presence of ECM between the callus cells (Fig.6n),
while formation of adventitious shoots and the connection
of explant’s vascular bundle with the regenerated plant’s
vascular bundle were observed (Fig.6o).
The presence ofcallose inexplant cells
Analysis of histological samples revealed a weak presence of
callose only in tissues of cotyledon and hypocotyl explants,
but not in cells of root and meristem explants. In the hypoco-
tyl explant, callose fluorescence was noted in the cell wall
of peripheral cells of callus (Fig.7a) and in the cell wall of
the few parenchyma of the primary cortex cells (Fig.7b).
However, the signal of callose was uneven—this being asso-
ciated with progressive deposition of callose in the cell wall.
Analysis ofexplant organogenesis using scanning
electron microscope (SEM)
Analysis of cotyledon’s explants surface indicated the pres-
ence of adventitious shoots, showed the lack of continuity
of epidermis (Fig.7d) and revealed a variety of callus cell
shapes formed in some parts of the explant (Fig.7e, l). On
the surface of the adventitious shoot growing out from the
meristem explant, clearly visible stomata and various callus
cells were noticeable. Furthermore, the extracellular matrix
with a mantle structure was seen where adventitious shoots
were forming (Fig.7f, g, i). Observation also indicated the
presence of cotyledons on the surface of the explant at an
advanced stage of development, with cells visible on its
Fig. 7 Cross-section of T. belorussicum explants: hypocotyl (ac,
jk), cotyledon (df), meristem (gi) and root (mo) cultured on ½
MS medium supplemented with 5.71 μM IAA, 4.54 μM TDZ and
100nM PSK in 23 ± 2 °C after 20 days (ac) and 55days (do) of
culture from SE/PSK model/short darkness: a peripheral cells with
callose in the cell wall (arrow), b the presence of callose in the cell
wall (arrows), c control (from the light microscope), and from SE/
PSK model/long darkness: d organogenesis, e callus cells, g regen-
erating plantlets from endogenic callus, h organogenesis at various
stages of development, j extensive amounts of ECM covering cells
of explant, k adventitious shoots and trichomes, m indirect organo-
genesis, n fibrous structure of ECM, and cross-section of T. belorus-
sicum explants: cotyledon (f), meristem (i), hypocotyl (l) and root
(o) cultured on ½ MS medium supplemented with 5.71μM IAA and
4.54μM TDZ in 23 ± 2 °C after 45days of culture from organogen-
esis model: f the lobe structure of ECM, i dense network of ECM, l
abundant callus coated with the ECM, o regenerated plantlet grow-
ing from callus tissue of explant. ac Staining with 0.1% toluidine
blue and 0.05% aniline blue to detect the callose. Scale: 100μm. do
Scanning electron microscope. Scale: 13μm (i), 30μm (f), 180 μm
(j, l, n), 200μm (k), 300μm (e, g), 400μm (o), 600μm (d), 1000μm
(h, m)
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518 Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
1 3
surface (Fig.7h). Moreover, organogenesis and ECM forma-
tion on callus surface were noted on the hypocotyl explant
(Fig.7j), while single, hairline and single-celled structures
were visible on the regenerated plants and explants, as were
shoots at various stages of development. Apical meristem
structures and single fibers at the youngest stage of devel-
opment were observed (Fig.7k). What is more, follicular
callus, fibers, well visible fibrous of ECM and regenerated
plants at various stages of development were observed on
the root explant (Fig.7m, n). In addition, analysis confirmed
the occurrence of indirect organogenesis via callus (Fig.7o).
Discussion
Existing world literature devoted to representatives of the
genus Taraxacum is rather poor and mostly focused on their
therapeutic properties (Ahmad etal. 2000; Chunga etal.
2018), developmental and embryological processes (Janas
etal. 2016), and their application in invitro culture condi-
tions (Ermayanti and Martin 2011; Tuleja etal. 2014a).
T. belorussicum is an interesting obligatory apomictic
species (Rodkiewicz etal. 1996) and was the object of rare
embryological analysis (Małecka 1965, 1982; Janas etal.
2016). Preliminary studies of invitro plant cultures (Tuleja
etal. 2014a) revealed direct organogenesis without forming
callus, as well as slight somatic embryogenesis on explants
from immature parthenogenetic embryos. However, the
obtained results did not confirm the occurrence of direct
organogenesis. The same effect was also observed in other
species e.g. T. officinale Weber (Chen etal. 2005). One of
the possible reasons for such result can be the difference in
explant origin along with their developmental stage (Kumar
and Chandra 2010). The aforementioned experiments were
conducted on explants derived from 2-week-old seedlings.
It seems that the age of T. belorussicum explants plays a
crucial role in SE induction and this developmental pathway
is involved mostly in younger plant material (Tuleja etal.
2014a). Even the application of PSK, the peptide responsible
of somatic embryogenesis induction (Igasaki etal. 2003),
did not break this recalcitrance. However, slight signals of
SE activity were detected in 2-week-old seedlings explants,
and this was additionally confirmed by the callose presence,
treated as a SE prerequisite (Dubois etal. 1989; Tao etal.
2012), as well as the first symptom of embryogenic cell
potential (Fiuk and Rybczyński 2006).
The present study showed significant differences in suit-
ability between the applied T. belorussicum explants. In
the organogenesis model (under light condition), the best
morphogenetic response was noted in hypocotyl explants,
whereas, hypocotyl and meristem explants had greater
response in the SE/PSK model/short darkness. The hypoc-
otyl is employed as a promising type of explant in many
studies for the induction of efficient organogenesis and
somatic embryogenesis (Jach and Przywara 2000). On the
other hand, cotyledon and meristem explants in the SE/PSK
model/long darkness revealed better morphogenetic response
than did hypocotyl and root explants after 30 and 65days of
culture under an extended period of time in dark conditions.
These differences suggest that all types of explants, despite
their common origin from one plant, could give various
morphogenetic responses depending on the combination of
growth conditions and the plant regulators used.
The high morphogenetic ability of all types of T. belorus-
sicum explants was additionally confirmed by the notice-
able presence of secondary organogenesis on the meristem-
regenerated plants. However, it is worth noting that under
the applied invitro conditions, the time of culture for each
type of explant was not a factor limiting their viability and
morphogenetic potential. This indicated the high regenera-
tive potential of this species and the suitability of this plant
for research in tissue culture field experiments.
A characteristic feature of all types of T. belorussicum
explants employed in the applied experimental models was
the relatively short time of response to the used invitro con-
ditions. This effect is desirable for invitro culture of that
species. It has already been demonstrated in earlier stud-
ies on T. belorussicum (Tuleja etal. 2014a) and is not a
common feature among plants exposed to such conditions,
e.g. Cephalotus follicularis reacts scarcely after 47days of
invitro culture (Tuleja etal. 2014b).
The invitro plant regenerative capacity is influenced
by genetic properties and by several environmental con-
ditions e.g. the nutrient compositions and the exposure to
light. Generally, the light impact on regeneration process
appears to be highly context dependent (Ikeuchi etal. 2016),
light can trigger organ regeneration (Saitou etal. 1992) or
inhibit root or shoot regeneration in some plants (Bellini
etal. 2014). The incubation in dark conditions increased the
organogenetic capacity of T. belorussicum hypocotyls and
especially of the cotyledons. A similar effect of light was
reported in Arabidopsis cotyledons (Nemeth etal. 2013).
Enhancement of invitro plant regeneration capacity can
be achieved by the use of appropriate media supplemented
with PGRs. The combinations of IAA and TDZ used in these
experiments were sufficient to induce organogenesis. A simi-
lar combination of the same PGRs for Cichorium intybus
L. also resulted in a high level of morphogenesis (Yucesan
etal. 2007). Still, data shows that the use of ½ MS without
growth regulators is sufficient for rooting Plantago asiatica
(Makowczyńska and Andrzejewska-Golec 2003). However,
this approach does not work in T. belorussicum. Therefore,
the use of ½ MS medium supplemented with IBA to achieve
the effective rooting of T. belorussicum regenerated plants
turned out necessary. This fact was confirmed by studies on
Taraxacum officinale Weber (Jamshieed etal. 2010).
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519Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
1 3
The addition of PSK increased the efficiency of T.
belorussicum organogenetic potential, as also reported from
Pisum sativum and Vicia faba explants (Ochatt etal. 2018).
Data showed that the use of PSK induced callus in Oryza
sativa and Beta vulgaris protoplasts (Grzebelus etal. 2012),
as well as cell proliferation in maize, rice, carrots or aspara-
gus, and stimulated cell division in these plants (Yang etal.
2000). The obtained results from SE/PSK model/short dark-
ness are associated with data on the positive impact of PSK
on the morphogenesis process. In the case of T. belorus-
sicum, the positive effect of PSK on the organogenesis pro-
cess was not confirmed in SE/PSK model/long darkness.
Therefore, the effect of PSK on T. belorussicum explants
seems to be more favorable when the incubation time in the
dark is reduced to a few days. This feature is perhaps char-
acteristic for T. belorussicum and may be correlated with the
preferences of this plant towards light conditions. Thus, the
longer incubation time in the dark weakens the condition and
morphogenetic potential of explants. PSK, used alone, stim-
ulated mainly meristem, hypocotyl and root explants. In con-
trast, the use of PSK with auxins and cytokinins enhanced
the morphogenesis process, mainly indirect organogenesis,
in SE/PSK model/short darkness. This fact was confirmed
by data showing that the expression of biological activity
of PSK correlates with the pathway of PSK signaling via
auxins and cytokinins (Matsubayashi and Sakagami 1996;
Matsubayashi etal. 1999). It was observed as the formation
of adventitious roots on cucumber hypocotyls (Yamakawa
etal. 1998a) and adventitious buds in Antirrhinum majus
(Yang etal. 1999). However, this dependency did not apply
to SE/PSK model/long darkness and did not enhance mor-
phogenesis of T. belorussicum.
Observation of explants showed the formation of callus
after the addition of PSK. This enhanced the development of
callus and significantly weakened the condition of explants.
Thus, PSK which promoted the synthesis of chlorophyll in
cucumber cotyledons (Yamakawa etal. 1998b), could have
allowed the explants to overcome the unfavorable condi-
tions due to the lack of light. In addition, PSK is noted to
affect cell viability (Yamakawa etal. 1999) and to reduce
the occurrence of albino individuals, which confirms its
participation in chlorophyll synthesis through increasing
photosynthesis efficiency and assimilating production (Asif
etal. 2014).
Incubation with PSK in darkness affects the induction
of somatic embryogenesis on hypocotyl and cotyledon
explants. These results confirm the role of PSK in increas-
ing the intensity of somatic embryogenesis of Cryptomeria
japonica (Igasaki etal. 2003), Daucus carota (Kobayashi
etal. 1999; Maćkowska etal. 2014) and pea (Ochatt etal.
2018). However, it seems that the induced somatic embryos
of T. belorussicum become deformed after longer culture
times, probably as a result of callus development and do
not generate plantlets. In the explants, the use of different
exposure periods to light and dark conditions affects the
variable level of PSK which, in turn, influences morphogen-
esis at initial stages of cell de-differentiation, proliferation
and re-differentiation. Thus, the activity of PSK was similar
to the activity of plant growth and development regulators
(Matsubayashi etal. 2004). Moreover, the manner of PSK
function in T. belorussicum cultures probably did not differ
from the function presented in our data, subject to taking
into account the short incubation time in the dark.
Induction of callus formation can occur from any initial
explant. For this purpose, appropriate amounts of growth
regulators should be applied (Sugiyama 2000). Previous
studies showed that the use of a combination of IAA and
TDZ for cotyledon explants of Pelargonium rapaceum
has a positive effect on callus induction and proliferation
(Sukhumpinij etal. 2010). This fact was confirmed in the
present study, wherein callus formation occurred more or
less intensively depending on the combination of IAA, TDZ
and PSK.
Histological analysis confirmed the indirect course
of organogenesis and showed an increased occurrence of
starch and proteins in cells of explants near the conducting
elements, the effect of which indicates the energetic role
of these substances, especially starch, in organogenesis
(Karim etal. 2006; Pei-Lang etal. 2006). The presence of
starch in the leaves of regenerated plants also revealed their
photosynthetic activity, especially in SE/PSK model/short
darkness. The most significant accumulation of proteins was
observed in the vicinity of secondary meristematic centers,
which demonstrates the high metabolic activity of these
cells (Fisher 1968). In this work, the histological analysis
of meristem explants revealed the occurrence of two types of
organogenesis: one is based on the native development and
can be assigned to the meristematic cells of the apical mer-
istem, while the second is released through the reprogram-
ming of the differentiated somatic cells located in the tissue
surrounding the apical meristem. Both processes rely on the
phenomenon of cellular plasticity, which can be defined as
the ability to re-specify cell fate (Ikeuchi etal. 2016).
The presence of ECM singled out by histological ana-
lyzes and SEM on the surface of the hypocotyls and roots
of T. belorussicum may be the result of a stress response
to applied invitro culture conditions. According to cur-
rent data, ECM can be formed in response to stress fac-
tors such as tissue culture conditions or the chemical agents
used to protect callus cells (Bevitori etal. 2013). It seems
that the extracellular matrix (ECM) was formed to create a
path for the receiving and transmitting signals that deter-
mine the recognition of cells and morphogenesis process.
Until recently, ECM was considered as a marker of somatic
embryogenesis in Cocos nucifera, Zea mays, Drosera sp.
and Papaver sp. (Namasivayam 2007), because the cells
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520 Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:505–522
1 3
acquired embryogenic competence in the presence of ECM.
Currently, data suggest that the presence of ECM in invitro
culture is a specific feature of morphogenesis (Popielarska-
Konieczna etal. 2013) and of morphogenetic callus formed
in invitro conditions. Of note, it may also have a protective
function.
The plants obtained in invitro cultures were in very good
condition, thus they easily acclimated to the ground. The
valuable result of these experiments is obtaining somatic
embryos and enabling their comparative analysis with apom-
ictic embryos.
Conclusions
Analyzes of selected Taraxacum belorussicum explants
revealed that this plant showed morphogenetic abilities
directed towards indirect organogenesis. Results have shown
that the indirect organogenesis was not synchronized. Still,
the invitro culture conditions, through the application of
IAA, TDZ and PSK factors, were sufficient for developing
an efficient innovative regenerative system for each type
of explants. Taking into consideration results of the study,
hypocotyl explants seem to be the most promising in induc-
ing somatic embryogenesis. In addition, observation of the
culture effects revealed that callus formation is characteris-
tic and separate for the particular type of T. belorussicum
explant. Moreover, the time of T. belorussicum explant cul-
ture was not a limiting factor for their viability and mor-
phogenetic potential. This indicates the high suitability of
this species for use in invitro experiments. Results from
this study conducted on selected types of T. belorussicum
explants also confirmed the role of PSK in the process of
morphogenesis (organogenesis and somatic embryogenesis).
Herein, the short incubation of T. belorussicum explants in
PSK-enhanced culture, under short conditions of darkness,
is most beneficial for organogenesis efficiency.
Acknowledgements The authors are grateful to Professor Jolanta
Marciniuk (Institute of Biology, University of Natural Sciences and
Humanities in Siedlce) for plant material (seeds) taken from the apom-
ict Taraxacum belorussicum Val. N. Tikhom. SEM images were made
in the Laboratory of Scanning Electron Microscopy for Biological and
Geological Sciences, Institute of Zoology, Jagiellonian University.
Author contributions The idea of the experiments, data analyzing
and writing the final version of the manuscript—MT; performing the
experiments, data analyzing and writing the draft of the manuscript:
AG, MG, invaluable discussions: AG, MG, MT. The authors declare
that they have no conflict of interest.
Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution 4.0 International License (http://creat
iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appro-
priate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made.
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