Modeling of asymmetric division of somatic cell in protoplasts culture of higher plants

Abstract

The key result of the work is the selection of factors for the cultivation of protoplasts of higher plants in vitro, which allowed induction of asymmetrical cell division during the first cell cycle phase. Gibberellin has been proved to be one of the main cofactors of asymmetric division of plant cells. The objects of research were plants of the following cultivars aseptically grown in hormone-free MS medium: tobacco (Nicotiana tabacum L.), SR-1 line; Arabidopsis thaliana var. columbia (L.) Heynh; potato (Solanum tuberosum L.), Zarevo cultivar; cultivated white head cabbage (Brassica oleraceae var. capitata L.) of the following varieties: Kharkivska zymnia, Ukrainska osin, Yaroslavna, Lika, Lesya, Bilosnizhka, Dithmarscher Früher, Iyunskarannya; rape (Brassica napus L.) of Shpat cultivar; winter radish (Raphanus sativus L.) of Odessa-5 cultivar. In experiments with mesophilic and hypocotyl protoplasts of the above-mentioned plant species it has been proved that short-term osmotic stress within 16–18 hours being combined with subsequent introduction of high doses of gibberellin GK3 (1 mg/L) into the modified liquid nutrient media TM and SW led to the occurrence of pronounced morphological traits of cytodifferentiation already at the initial stages of the development of mitotically active cells in a number of higher plants. Meanwhile, in all analyzed species, there was observed the division of the initial genetically homogeneous population of mitotically active cells into two types of asymmetric division: by the type of division of the mother cell into smaller daughter cells and by the type of the first asymmetric division of the zygotic embryo in planta. In this case, the first type of asymmetric division occurred during unusual cytomorphism of the mother cells: a pronounced heart-shaped form even before the first division, which is inherent in the morphology of somatic plant embryo in vitro at the heart-shaped stage. A particular study of the effect of osmotic stress influencing protoplasts of various cultivars of white cabbage, isolated from hypocotyls of 7–9 day etiolated seedlings, revealed quite a typical consistent pattern: the acquisition and maintenance of the axis of symmetry in growing microcolonies occurred without extra exogenous gibberellin (GK3), which was added to the nutrient medium earlier. While analyzing the effect of growth regulators on the formation of microcolonies with traits of structural organization, the conclusion was made regarding the commonality of the revealed morphogenetic reactions of cells within the culture of protoplasts of higher plants in vitro with similar reactions studied earlier on other plants, both in vitro and in planta. Modeling of asymmetric cell division in protoplast culture in vitro has become possible by carrying out a balanced selection of growth regulators as well as their coordinated application through time along with changes in osmotic pressure of a nutrient medium.

Full text

Regul. Mech. Biosyst., 2020, 11(2) 93
Regulatory Mechanisms
in Biosystems
ISSN 2519-8521 (Print)
ISSN 2520-2588 (Online)
Regul. Mech. Biosyst.,
2020, 11(2), –
doi: 10.15421/022039
Modeling of asymmetric division of somatic cell
in protoplasts culture of higher plants
S. I. Kondratenko*, T. P. Pasternak**, O. P. Samovol*, O. M. Mogilna*, O. V. Sergienko*
*Institute of Vegetables and Melon Growing of National Academy of Agricultural Sciences of Ukraine, Kharkiv region, Uktaine
**Albert-Ludwigs-University Freiburg, Freiburg, Germany
Article info
Received 16.04.2020
Received in revised form
10.05.2020
Accepted 11.05.2020
Institute of Vegetables
and Melon Growing
of National Academy of
Agricultural Sciences
of Ukraine, Institutskaya st., 1,
Selektsiyne village,
Kharkiv region,
62478, Ukraine.
Tel: +38-057-748-91-91.
E-mail:
ovoch.iob@gmail.com
Albert-Ludwigs-University.
Schänzle st., 1, Freiburg,
D-79104, Germany.
Tel: +49-157-861-83-538.
E-mail:
taras.p.pasternak@gmail.com
Kondratenko, S. I., Pasternak, T. P., Samovol, O. P., Mogilna, O. M., & Sergienko, O. V. (2020). Modeling of asymmetric divi-
sion of somatic cell in protoplasts culture of higher plants. Regulatory Mechanisms in Biosystems, 11(2), –. doi:10.15421/022039
The key result of the work is the selection of factors for the cultivation of protoplasts of higher plants in vitro, which allowed in-
duction of asymmetrical cell division during the first cell cycle phase. Gibberellin has been proved to be one of the main cofactors of
asymmetric division of plant cells. The objects of research were plants of the following cultivars aseptically grown in hormone-free
MS medium: tobacco (Nicotiana tabacum L.), SR-1 line; Arabidopsis thaliana var. columbia (L.) Heynh; potato (Solanum tubero-
sum L.), Zarevo cultivar; cultivated white head cabbage (Brassica oleraceae var. capitata L.) of the following varieties: Kharkivska
zymnia, Ukrainska osin, Yaroslavna, Lika, Lesya, Bilosnizhka, Dithmarscher Früher, Iyunskarannya; rape (Brassica napus L.) of
Shpat cultivar; winter radish (Raphanus sativus L.) of Odessa-5 cultivar. In experiments with mesophilic and hypocotyl protoplasts
of the above-mentioned plant species it has been proved that short-term osmotic stress within 16–18 hours being combined with
subsequent introduction of high doses of gibberellin GK3 (1 mg/L) into the modified liquid nutrient media TM and SW led to the
occurrence of pronounced morphological traits of cytodifferentiation already at the initial stages of the development of mitotically
active cells in a number of higher plants. Meanwhile, in all analyzed species, there was observed the division of the initial genetically
homogeneous population of mitotically active cells into two types of asymmetric division: by the type of division of the mother cell
into smaller daughter cells and by the type of the first asymmetric division of the zygotic embryo in planta. In this case, the first type
of asymmetric division occurred during unusual cytomorphism of the mother cells: a pronounced heart-shaped form even before the
first division, which is inherent in the morphology of somatic plant embryo in vitro at the heart-shaped stage. A particular study of
the effect of osmotic stress influencing protoplasts of various cultivars of white cabbage, isolated from hypocotyls of 7–9 day etio-
lated seedlings, revealed quite a typical consistent pattern: the acquisition and maintenance of the axis of symmetry in growing mi-
crocolonies occurred without extra exogenous gibberellin (GK3), which was added to the nutrient medium earlier. While analyzing
the effect of growth regulators on the formation of microcolonies with traits of structural organization, the conclusion was made
regarding the commonality of the revealed morphogenetic reactions of cells within the culture of protoplasts of higher plants in vitro
with similar reactions studied earlier on other plants, both in vitro and in planta. Modeling of asymmetric cell division in protoplast
culture in vitro has become possible by carrying out a balanced selection of growth regulators as well as their coordinated application
through time along with changes in osmotic pressure of a nutrient medium.
Keywords: plant cell; osmotic stress; first asymmetric division; gibberellic acid (GA3); cell microcolony.
Introduction
One of the main problems in the framework of plant morphogenesis
is the elucidation of the nature of the processes causing polarization and
further differentiation of cells (Medvedev, 1996). Such processes include
non-equivalent or asymmetric division of the mother cell, which leads to
the occurrence of daughter cells of different functions, thereby the primary
formation and further maintenance of the axial symmetry of multicellular
structures of the plant body.
Bipolar growth is an essential trait of life activity of most plants. Es-
sentially, the plant stem increases upwards, while the root downwards,
deepening into the soil. Bipolar growth is primarily due to an increase in a
number of cells through reproduction (proliferation) in special cellular
structures: apical meristem located at the very top of the stem and at the
root tip. In nature, plants are represented by a great variety of life forms,
which are widely different in their growth features (Flindt, 1992): the
number of cells in the cell chains from the tip of the shoot to the root tip,
the ratio of shoot-to-root size, growth rate as well.
It is determined that two hormones – auxin and cytokinin – are neces-
sary for the separation of plant cells (Dante et al., 2014). It is essential that
these phytohormones are synthesized mainly at the plant poles: auxin is
being synthesized at the crown of the stem and cytokinin – at the root
apex. Through transport channels, the auxin in plants moves downward,
while the cytokinins move towards them (Polevoj, 1982; Polevoj & Sala-
matova, 1991). The role of hormonal counterflow within the organization
of growth processes is still not fully clarified. So far, it has not been possi-
ble to experimentally identify the auxin-cytokinin counterflow circuit or
study its functions as a plant growth inductor. To solve this problem, it is
considered quite effective to apply different kinds of simulation. In par-
ticular, the authors of one of the published works proposed a mathematical
model in which the bihormonal contour controls the proliferative growth
of a plant-type cell structure (Sukhoverov & Romanov, 2009).
In plants, the patterns of cell division in combination with growth and
differentiation determine the hierarchical organization of plant tissues and
organs. Cells can reproduce using symmetrical divisions, while asymmet-
rical ones are mainly related to the initiation of new cell types, neoplasms
and development patterns (Rasmussen et al., 2011; Smolarkiewicz &
Dhonukshehe, 2013). In asymmetric division, the formation of the clea-
vage plane is often associated with cell polarity. The common paradigm is
that the position of the cleavage plane will be determined by such an

Regul. Mech. Biosyst., 2020, 11(2)
94
asymmetrically distributed determinant that will lead daughter cells to dif-
ferent directions of development. An alternative view is that daughter cells
will initially have the same determinant, however, they will be exposed to
different positional signals that will cause them to develop in various man-
ners (Horvitz & Herskowitz, 1992). Therefore, elucidating the principles
that govern symmetric and asymmetric divisions provides for better un-
derstanding the cellular bases of plant development as well as plant mor-
phogenesis in general. The early embryogenesis of Arabidopsis thaliana
has been deemed to be an attractive model for studying how the positions
and orientation of cleavage planes are being selected. During the first ge-
nerations of cells, exclusive embryo geometry is indeed organized from a
single initial cell through a stereotype sequence of invariant oriented cell
divisions (Mansfield & Briarty, 1991; Capron et al., 2009). Thus, the
boundaries of the future direction of cell division have been established
and mapped using numerous genetic and cytological reverse analyses, and
these properties have been successfully used to identify the origin of deve-
lopmental defects in mutant patterns (Laux et al., 2004; Palovaara et al.,
2016). The influence of cell shape on the orientation and choice of clea-
vage planes in animal and plant cells has attracted much attention (Minc &
Piel, 2012) with special emphasis on the common rules of cleavage de-
fined as early as the late XIX century (Errera, 1888). According to the
Errera rule (Errera, 1888), plant cells will behave as soap bubbles, so that
symmetrical separations will adhere to the principle of minimum surface
area. Besson & Dumais (2011) recently rearranged the rule in a stochastic
version, according to which the choice of the cleavage plane between the
various alternatives depending on the distribution of probabilities is asso-
ciated with the plane area. It is generally accepted that the principle of
minimizing surface area, according to the Errera rule, will a priori be a
mechanism for separating plant cells in the absence of internal or external
traits (Roeder, 2012). However, the vast majority of studies supporting this
view have focused on symmetrical divisions in tissues that have been
assimilated in 2D systems (e.g., tissues with a constant cell layer and abso-
lutely anticlinical divisions). The question of whether geometric rules are
acceptable of describing a 3D division of plant cells deserves to be asked.
Recently, Yoshida et al. (2014) reported, however, that they were unable
to identify the geometric rule, which underlies the sequence of 3D cell di-
vision models in the Arabidopsis thaliana zygotic embryos.
Other authors (Moukhtar et al., 2019) questioned the existence and
nature of the rules governing cell division during early zygote embryos’
stage. Using automatic image analysis of 3D embryos, they quantitatively
determined cell shapes and models of cell division. It was found out that
the distance between the equator of a cell and the mother cell’s centroid
was an invariant trait of the generations’ course and spatial boundaries.
Being based on the developed computer cell division model, the authors
(Moukhtar et al., 2019) investigated the space of possible division planes
under geometric constraints in real three-dimensional cell forms. On the
basis of the proposed model, a new rule for predicting the position and
orientation of division planes by the geometry of the mother cell was
established, which was true for both symmetric and asymmetric cell divi-
sion. The results supported the key role of the geometric feedback loop
between the cell shape and the position within the cleavage plane in the
self-organization of the early embryo.
The above-mentioned instances of asymmetric cell division, as well
as variants of mathematical modeling of this type of division were consid-
ered on plant objects with pre-genetically determined development pro-
gram at the in planta level, i.e. the modeling concerned those cases when
the course of cytodifferential cell division were under genetic and regula-
tory control of a plant body.
In this regard, the culture of isolated protoplasts of higher plants in vit-
ro can serve as an alternative model system for identifying and studying
the cofactors of cytodifferential division and growth of plant cells as well.
The feature of this model system is that it allows one to investigate the
cofactor role of growth regulators as well as elements of osmotic and
trophic regulation of cell division of isolated somatic plant cells in vitro
(Butenko, 1981; Kuchuk, 2017).
In experiments with tobacco culture it was shown (Nawaga et al.,
1987) that the identification of endogenous gibberellin is closely correlated
with the onset of morphogenesis. The results of embryological studies on
all plant species concerned also demonstrate the direct involvement of
endogenous gibberellins in the formation of zygotic embryos in planta,
beginning with the heart-shaped development stage (Trigiano et al., 1987).
However, in a number of already prepared studies, the physiological effect
of the phytohormone was evaluated on multicellular structures, confir-
ming a failure to fully manifest its functions. A rather convenient object
for study is the culture of isolated protoplasts, where a mitotically active
somatic cell may be a potential morphogenetic unit. Therefore, this cell
type is an ideal experimental object for simulating different types of mor-
phogenesis in culture in vitro. The purpose of our research was to deter-
mine the informative possibilities of the culture of protoplasts of higher
plants in vitro as a model system capable of reproducing asymmetric
division of somatic plant cells only due to the action of exogenous factors
of growth and development controlled by a researcher.
Materials and methods
Six plant species from the Brassicaceae and Solanaceae families were
chosen as research objects, which in the culture of protoplasts during gra-
dual application of nutrient media TMmod1 (k), TMmod2 (k) and
TMmod3 (k) and SWmod1 (k), SWmod2 (k) and SWmod3 (k) (Table 1)
initiated the first symmetrical divisions of the cells, from which the unor-
ganized growing colonies were then formed. The following tube stock
plants were aseptically grown in vitro on hormone-free agarized MS me-
dium (Murashige & Skoog, 1962): SR-1 line of tobacco (Nicotiana ta-
bacum L.) (Maliga et al., 1973); wild species of Arabidopsis (A. thaliana
var. colymbia (L.) Heynh.); Zarevo variety of potato (Solanum tuberosum
L.); whitehead cabbage (Brassica oleraceae var. capitata L.) varieties:
Kharkivska zymova, Ukrainska osin, Yaroslavna, Lika, Lesia, Bilosnizh-
ka, Dithmarscher Früher and Iuinskarannia; Shpat variety of rape (Bras-
sica napus L.); Odeska-5 variety of winter radish (Raphanus sativus L.).
Protoplasts of leaf mesophyll were extracted from microclonal propa-
gated juvenile plants, with well-developed leaf area. Hypocotyl protoplasts
were excreted from ethylated seedlings for 7–9 days of cultivation. Leaf or
hypocotyl tissues were cut into strips 1 mm wide and incubated under
darkroom conditions at 28 ºС for 16–18 hours in an enzymatic solution
containing 0.5% Macerozyme (“Calbiochem”, USA), 0.5% Onozuka R-
10 (“Jacult Biochemicals”, Japan), 0.5 М sucrose and 5 mM CaCl2
(pH 5.6). The isolated protoplasts were separated from residual tissue
through metal filters with a pore diameter of 64 µm. The filtrate was trans-
ferred to centrifuge tubes (10 mL) and then centrifuged at 700 rpm for
7 min. The ring of floated protoplasts was selected by a Pasteur pipette and
washed at least twice in a W-5 medium (Medgyesy et al., 1980), precipita-
ting them each time by centrifugation for 5 minutes at 700 rpm. The number
of protoplasts in the medium was calculated using the Goriaiev chamber.
In our studies, the addition of exogenous gibberellin (GK3) to the
composition of nutrient medium growth regulators proved to be effective,
starting from the first mitotic cell division in protoplast culture and at
growth of microcolonies formed after the 5th and 6th mitosis. After tissue
fermentation and isolation procedure (Sidorov et al., 1985), protoplasts of
various plant varieties within the experimental variant of cultivation were
transferred to the appropriate nutrient media SWmod1 (d) and Tmod1 (d)
(Table 1) and cultivated on diffuse light at 22–24 ºC for 16–18 hours
under stress (hypotonic) conditions (0.36 M glucose) with a population of
104–105 cells/mL. Then the level of osmotic agents was brought to 0.5 M
(modifications of SWmod2 (d) and Tmod2 (d), respectively). The increa-
se in osmotic concentration was followed by simultaneous injection of
exogenous gibberellin (1 mg/L GK3) into nutrient medium. Further, the
level of osmotic agents was brought up to 0.5 M (modifications) during
the first 48 hours of cultivation, more than 80% of both mesophilic and
hypocotyl protoplasts of the studied plant cultivars were fully restored to
the cellular membrane. The morphological criterion for such a condition
in cultured protoplasts was a change in their spherical shape to elongated
or oval, which is typical for suspension-cultured cells (Fig. 1). The identi-
fication of regenerating cell membranes in protoplasts was carried out by
colouring them with 0.01% calcofluor (Calcofluor white, MR-2, “Serva”)
on 0.1 М phosphate buffer, pH 7.2 during 5–10 minutes following the
Herth method (Herth & Schnepf, 1980). For this purpose, microdrops of
cell suspensions of 10–20 µL were used, which were selected from the
nutrient media. Having being washed with a buffer, protoplasts were exa-

Regul. Mech. Biosyst., 2020, 11(2) 95
mined with the help of a fluorescent microscope ML-2, the luminescence
was caused by the blue light of a mercury lamp and a light blue-green
glass filter. Calculation of the percentage of protoplasts from regenerated
membranes was carried out for 24 hours and 48 hours of cultivation.
Table 1
The composition of liquid nutrient media (mg/L) for cultivation of protoplasts of higher plants in the experimental (e) and control (c) variants
The content in nutrient medium, mg/L
Components * ТМmod1
(e)
ТМmod1
(c)
ТМmod2
(e)
ТМmod2
(c)
ТМmod3
(e)
ТМmod3
(c)
SWmod1
(e)
SWmod1
(c)
SWmod2
(e)
SWmod2
(c)
SWmod3
(e)
SWmod3
(c)
Macronutrients:
NH4Cl
NH4NO3
KNO3
CaCl2x2H2O
MgSO4x7H2O
KH2PO4
0.0
200.0
1500.0
440.0
370.0
170.0
0.0
200.0
1500.0
440.0
370.0
170.0
0.0
200.0
1500.0
440.0
370.0
170.0
0.0
200.0
1500.0
440.0
370.0
170.0
0.0
200.0
1500.0
440.0
370.0
170.0
0,0
200.0
1500.0
440.0
370.0
170.0
133.0
0.0
1900.0
440.0
370.0
170.0
133.0
0.0
1900.0
440.0
370.0
170.0
133.0
0.0
1900.0
440.0
370.0
170.0
133.0
0.0
1900.0
440.0
370.0
170.0
133.0
0.0
1900.0
440.0
370.0
170.0
133.0
0.0
1900.0
440.0
370.0
170.0
Fe-helate:
Na2EDTA
FeSO4x7H2O
37.3
27.8
37.3
27.8
37.3
27.8
37.3
27.8
37.3
27.8
37.3
27.8
37.3
27.8
37.3
27.8
37.3
27.8
37.3
27.8
37.3
27.8
37.3
27.8
Micronutrients:
H3BO3
MnSO4x5H2O
ZnSO4x7H2O
NaMoO4x2H2O
KJ
CoSO4x6H2O
CuSO4x5H2O
3.0
13.2
2.0
0.25
0.75
0.025
0.025
3.0
13.2
2.0
0.25
0.75
0.025
0.025
3.0
13.2
2.0
0.25
0.75
0.025
0.025
3.0
13.2
2.0
0.25
0.75
0.025
0.025
3.0
13.2
2.0
0.25
0.75
0.025
0.025
3.0
13.2
2.0
0.25
0.75
0.025
0.025
3.0
13.2
2.0
0.25
0.75
0.025
0.025
3.0
13.2
2.0
0.25
0.75
0.025
0.025
3.0
13.2
2.0
0.25
0.75
0.025
0.025
3.0
13.2
2.0
0.25
0.75
0.025
0.025
3.0
13.2
2.0
0.25
0.75
0.025
0.025
3.0
13.2
2.0
0.25
0.75
0.025
0.025
Vitamins and other biologically
active components:
Mesoinositol
В1
В6
РР
Casein hydrolyzate
100.0
10.0
1.0
1.0
150.0
100.0
10.0
1.0
1.0
150.0
100.0
10.0
1.0
1.0
150.0
100.0
10.0
1.0
1.0
150.0
100.0
10.0
1.0
1.0
150.0
100.0
10.0
1.0
1.0
150.0
100.0
10.0
1.0
1.0
150.0
100.0
10.0
1.0
1.0
150.0
100.0
10.0
1.0
1.0
150.0
100.0
10.0
1.0
1.0
150.0
100.0
10.0
1.0
1.0
150.0
100.0
10.0
1.0
1.0
150.0
Growth regulators:
BAP (6-benzylaminopurine)
NAA (α-naphthylacetic acid)
2,4-D (2,4-dichlorophenoxyacetic acid
GA3 (gibberellic acid)
0.5
1.0
0.2
0.0
0.5
1.0
0.2
0.0
0.5
0.1
0.2
1.0
0.5
0.1
0.2
0.0
1.0
0.1
0.2
1.0
1.0
0.1
0.2
0.0
0.5
2.0
0.2
0.0
0.5
2.0
0.2
0.0
0.5
0.1
0.2
1.0
0.5
0.1
0.2
0.0
1.0
0.1
0.2
1.0
1.0
0.1
0.2
0.0
Carbohydrate sources (osmotic agents):
Glucose
(molar concentration)
Xylose
64870.0
(0.36М)
125.0
90100.0
(0.5М)
125.0
90100.0
(0.5М)
125.0
90100.0
(0.5М)
125.0
72000.0
(0.4М)
125.0
72000.0
(0.4М)
125.0
64870.0
(0.36М)
125.0
90100.0
(0.5М)
125.0
90100.0
(0.5М)
125.0
90100.0
(0.5М)
125.0
72000.0
(0.4М)
125.0
72000.0
(0.4М)
125.0
The level of acidity of nutrient media: pH 5.8 5.8 5.8 5.8 5.8 5.8 5.8 5.8 5.8 5.8 5.8 5.8
Note: * – experimental nutrient media for white head cabbage, rapeseed and radish protoplasts – TMmod1(e) for cultivation of protoplasts under conditions of hypotonic stress
(0.36 M glucose), TMmod2(e) for the cultivation of protoplasts after the first division (at the optimum for the initiation of mitotic activity of the osmotic content 0.5 M glucose)
and simultaneous removal of osmotic stress, TMmod3(e) –for culturing cells at a later stage in the formation of micro colonies; similarly intended nutrient media for protoplasts
of tobacco, potatoes and Arabidopsis – SWmod1(e), SWmod2(e), SWmod3(e).
Within 60–72 hours after about 50% of cells started the first cell divi-
sion, the suspensions were diluted with liquid media TMmod3 (d) and
SWmod3 (d) with a low auxin content (0.1 mg/L NAA) and a high one of
gibberellin (1 mg/L GK3) in order to reach the final density of 102–103
cells/mL. Phenological observations of the state of culture were carried out
on a daily basis, while the number of formed microcolonies from one
mitotic active cell and their morphological analysis for 15 days of cultiva-
tion were counted. The registration of the state of cell as well as microco-
lony development in vitro cultures of mesophilic and hypocotyl proto-
plasts of various plant varieties was carried out within two months – a
sufficient period at which in the applied experimental option of cell culti-
vation the traits of structural organization of microcolonies and their com-
plete transition to unorganized growth were completely lost. During the
first 20 days, cell culture was grown in the darkroom conditions, then
transferred to diffuse light to gradually grow the existing callus clones to
an optimum size of 3–4 mm in diameter, which is necessary for their
migration to agarized nutrient media. Photo fixing of various stages of
growth and division of floating cells in nutrient media of protoplasts and
plant cells after the cellular membrane and microcolonies repair process
was carried out by photographing them on black and white film with the
use of a Zenith camera from the photonhead to an inverted microscope of
the Olympus CKX53. The mitotic activity of cells in protoplast culture
was assessed by fixation of callus and cell aggregates in a mixture of
ethanol – acetic acid – chloroform (6 : 1 : 3). The material was dyed in 1%
aceto-orcein and cells at different stages of mitosis were counted under a
microscope on squash material. The mitotic index was determined in 5-
fold repetition when analyzing 5–7 thousand cells for each fixation point.
The above cytological parameter was determined using the following
formula:
100%

∑ of cells at mitosis stage
Mitotic index = ∑ of cells in total (1)
The analysis of the experimental data was carried out using the
ANOVA method. The differences between the values in the different va-
riants of experience in the cultivation of somatic cells of plants were deter-
mined using the Tukey criterion, where the differences were considered
reliable at P < 0.05 (with consideration of the Bonferroni correction). The
numerical data in the tables are presented as x ± SD (n = 5).
Results
When using the control variant of cultivation of mesophilic proto-
plasts – a combination of initial isosmotic conditions (0.5 M glucose) and
high doses of auxin (1 mg/L NAA for Brassicaceae plants and 2 mg/L for
Solanaceae), we observed, primarily, the initiation of symmetrical division
of daughter cells and further formation of mini-clones in the majority of
cases (Fig. 1). Application of low osmotic pressure of the nutrient medium
during the first 16–18 hours of cultivation led to changes in the morpho-
logy of the protoplasts even before full reparation of the cell wall. Namely,
under such conditions of cultivation, physiological reaction to osmotic
stress (OS) represented excessive vacuolization of protoplasts with forma-
tion of large vacuole, which polarized cell cytoplasm (Fig. 2a). Subsequ-
ently, a similar cytomorphosis persisted until the beginning of the mitotic

Regul. Mech. Biosyst., 2020, 11(2)
96
activity of the cells and led to the initiation of the first asymmetric divisi-
ons with the appearance of daughter cells with clearly visible cytoplasmic
optical density – transparent with excessive vacuolization and dense, satu-
rated with cytoplasmic elements (Fig. 2b). Previously, when conducting
methodological studies in order to select optimal concentrations of the
auxin regulator NAA, we observed the phenomenon of vacuolization in
the culture of higher plant protoplasts at the regulator’s doses exceeding
2 mg/L and the initial isosmotic conditions. However, the prolonged expo-
sure to the mentioned dose of auxin only increased the growth of cells by
stretching, neutralizing their mitotic activity for too long. Thus, the stimu-
lation of asymmetrical first cell division can be explained by the synergis-
tic effect of high doses of auxin and the initial low, compared to the con-
trol variant, osmotic pressure of the nutrient medium. In the application of
OS on the protoplasts of potatoes, tobacco and Aarabidopsis, the first
divisions with unequal distribution of cytoplasmic elements in formed
daughter cells were observed. A characteristic feature of the protoplasts of
rapeseed, cabbage and radish after the OS stage was the stimulation of
mitotic active cell division by the type of cleavage into smaller daughter
cells without determination of cytoplasmic elements. After removal of the
initial OS protoplasts of all plant species formed disorganized growing cell
clones morphologically identical to the minicalli of the control variant of
the experiment without any traits of cytodifferentiation (Fig. 3). A prolon-
ged use of hypotonic influence of the nutrient medium (for a day or more)
led to the final loss of mitotic activity and death of cell populations.
a b
c d
Fig. 1. Typical cell growth in the culture of protoplasts of the studied plant species in vitro under the control variant of cultivation:
a – recently isolated mesophilic protoplasts of rapeseed Shpat (Brassica napus L.), sown in a liquid nutrient medium TMmod1 (k) (1 mg/L NAA;
0.2 mg/L 2.4-D, 0.5 mg/L BAP, 0.5 M glucose); b – cell wall repair process within the first 48 hours of cultivation of hypocotyl protoplasts of white
cabbage Kharkivska zymnia (Brassica oleraceae var. capitata L.); c – the first symmetrical cell division of the mother cell (hypocotyl protoplasts
of white cabbage Lika cultivar (Brassica oleraceae var. capitata L.)); d – unorganized microcolony growth (highlighted with an arrow)
on the nutrient media SWmod3(k) in the culture of mesophilic tobacco protoplasts in vitro of the SR-1 line (Nicotiana tabacum L.)
a b
Fig. 2. Cell response to OS in the experimental variant of mesophilic protoplasts cultivation of higher plants in vitro: a – excessive cellular vacuolization
and polarization of cytoplasmic elements of mitotic active cells after the stages of cell wall repair process (Nicotiana tabacum L); b – the first asymmetrical
cell division (highlighted with an arrow) with unequal cytoplasmatic elements’ distribution (Arabidopsis thaliana var. colymbia (L.) Heynh.)

Regul. Mech. Biosyst., 2020, 11(2) 97
Another phenotypic demonstration was observed if the introduction
of gibberellin GK3 (1 mg/L) into liquid media was followed by simulta-
neous osmotic stress relief and reduction of NAA auxin concentration to
0.1 mg/L after the first cell division. In all the analyzed plant species under
this variant of cultivation the division of the original population of mitoti-
cally active cells into two types of unequal division was observed (by the
type of the first asymmetric division of the zygotic embryo in planta as
well as by the type of cleavage of the mother cell into smaller daughter
cells). In the experimental variant of cultivation in the selected species of
higher plants the level of manifestation of the first type of unequal division
was within 3.1–54.1%, while the second type was within 15.0–40.6%.
The symmetrical divisions were 8.5–64.9% (data of the diagram in Figure 4).
A characteristic feature of the first type of division was an unequal
distribution of cytoplasmic elements between daughter cells, among
which one cell was of a large size with optically dense cytoplasm, while
the second cell was of a smaller size and quite vacuolated with an optically
light cytoplasm. Subsequently, there was a development of two types of
microcolonies, which were formed after asymmetric cell division. One of
them had a clearly expressed part consisting of compact small cells in the
form of globules and long vacuolated cells, which were divided along the
axis (Fig. 5). The second one represented a cone-shaped microcolony,
which, as it grew, acquired forms morphologically identical to the heart-
shaped zygotic embryo in planta (Fig. 6). Thus, in this case there was an
analogy between the first asymmetric division of somatic cells in meso-
philic protoplasts culture in vitro and the first one, determined during the
germinal axis, direction of zygote separation in planta, after which apical
and basal cells were formed (Tvorogova & Lutova, 2018). The second
feature of the development of microcolonies with primary traits of cytodif-
ferentiation was the determination of the symmetry axis of the second
order, which was formed as a result of bilateral division of cells that had
optically dense cytoplasm after the first asymmetric division (Fig. 7).
The above-mentioned trajectory of microcolonies development seemed to
be typical for tobacco and Arabidopsis protoplasts.
Fig. 3. Unorganized microcolony cell growth in the control variant
derived from mesophilic protoplasts (Nicotiana tabacum L.)
Fig. 4. Percentage ratio of different directions of division of mitotic active cells in both control and research variants in mesophilic protoplasts culture
of 6 Brassicaceae and Solanaceae plant species: a – the first asymmetric zygotic embryo in planta division type; b – division of the mother cell cleavage
type into smaller daughter cells; c – symmetrical divisions; the numerical data in the diagram are presented as x ± SD (n = 5)
a b
Fig. 5. Polar morphology of microcolonies in the experimental variant of the cultivated cells derived from mesophilic protoplasts:
a – potato of Zarevo cultivar (Solanum tuberosum L); b – wild form of Arabidopsis (A. thaliana var. columbia (L.) Heynh.)

Regul. Mech. Biosyst., 2020, 11(2)
98
The developmental pathway inherent mainly in rapeseed, radish, cab-
bage, and potato cells in the presence of gibberellin was to initiate cell divi-
sion, in which the mitotically active cells were fragmented into smaller
daughter cells. In this case, protoplasts, after the cell wall was repaired,
were morphologically similar to somatic embryos in the heart-shaped
stage of development (Fig. 8). Such cytomorphosis was observed even
before the first division and only in the presence of exogenous gibberellin.
During the first and subsequent mitosis, daughter cells continued to preser-
ve the initial morphology of the mother cell, however, their cell division
had no clear orientation throughout the initial axis of symmetry. Neverthe-
less, within the process of further development, such microcolonies held
for a long time the heart-shaped form inherited from the mother cell
(Fig. 8 and 9). Except for mitotic active cells, which formed microcolonies
with morphological traits of structural organization in all plant species
under study, there were cells that divided in an isopolar way, forming
microcolonies without traits of structural organization after OS exposure
and subsequent injection of exogenous gibberellin into nutrient media
(Fig. 3). Consequently, in experiments with the culture of mesophilic pro-
toplasts of higher plants in vitro it was shown that osmotic stress followed
by the subsequent introduction of high doses of gibberellin GK3 into the
nutrient medium led to the appearance of morphological traits of cytodif-
ferentiation already at the initial stages of development of somatic cell
microcolonies in a number of higher plants.
Fig. 6. Heart-shaped microcolony with morphological traits
of structural organization (Nicotiana tabacum L.)
Fig. 7. Bilateral symmetry of microcolonies with morphological traits
of structural organization (Arabidopsis thaliana (L.) Heynh.)
Fig. 8. Structural form of microcolonies of cells in which the mother cell
has taken the morphological form of a somatic embryoid at the heart-
shaped stage of development even before the first division begins
(Brassica napus L.)
Fig. 9. Structural form of microcolonies of cells in which the mother cell
has taken the morphological form of a somatic embryoid at the heart-
shaped stage of development even before the first division begins
(Brassica oleracea var. capitata L.)
A separate study of the effect of OS on hypocotyl protoplasts of
different varieties of white cabbage isolated from tissues of 7–9 day etiola-
ted seedlings revealed one characteristic feature: the determination of cyto-
plasmic elements in this type of cells and the subsequent development of
differential morphology of microcolonies occurred without additional
injection of exogenous gibberellin (GK3) into the nutrient medium
TMmod2 (k). That is, the hypocotyl protoplasts, having undergone a cell
membrane repair process, morphologically resembled somatic embryos at
the heart-shaped stage of development in the absence of exogenous gibbe-
rellin (cytomorphosis similar to the one presented in Figure 9 for white
cabbage cells derived from mesophilic protoplasts). This fact, as well as
preliminary data obtained on mesophilic protoplast culture, gave us
grounds to assume that plant gibberellin has the same regulatory effect as
GK3. It has been established that during the period of germination of zy-
gote embryo, the endogenous content of different forms of gibberellin in
different plant species increases by several orders of magnitude (Blume
et al., 2012), which probably significantly affected the phenotypic reacti-
ons of hypoc6otyl protoplasts in terms of the action of osmosis. In the next
series of experiments for this type of cells, the evaluation of asymmetrical
division was conducted from the standpoint of phytohormonal regulation

Regul. Mech. Biosyst., 2020, 11(2) 99
of proliferation and cell differentiation. Figure 10 shows the diagram re-
flecting the percentage ratio of different types of division of mitotic active
cells in the culture of hypocotyl protoplasts of 8 varieties of white cabbage
after the application of control and trial cultivation, similar to that of
mesophilic protoplasts of higher plants (Fig. 4).
Evidence suggests that these cultures, in the framework of the experi-
mental variant, were dominated by maternal cells’ being divided smaller
daughter cells. The greatest number of them was registered in early-ripe-
ning cultivars: Dithmarscher Früher and Iyunskayarannyaya at 71.5–
79.0% (Fig. 10). In other cultivars, this figure ranged between 25.3–
67.9%. Division by type of cleavage occurred in those cells, which after
OS and subsequent administration of exogenous gibberellin (GK3) acqui-
red initial morphology with traits of structural organization (heart-shaped
one). In late ripening varieties of white cabbage, the highest percentage of
such cells (more than 50%) was observed in Kharkivska zymnia, Lika,
Lesya and Bilosnizhka cultivars (Fig. 10).
a
b
Fig. 10. The percentage ratio of different types of division of mitotic active cells in both control and research variants in culture of hypocotyl lprotoplasts
in vitro of white cabbage cultivars (Brassica oleraceae var. capitata L.): a – the first asymmetric zygotic embryo in planta division type; b – division of
the mother cell cleavage type into smaller daughter cells; c – symmetrical divisions; the numerical data in the diagrams are presented as x ± SD (n = 5)
It is common practice when at the stage of their cultivation up to the
nutrient media TMmod3(d) is applied, they continued to preserve the
differential morphology. To support mitosis, the culture was transferred to
the optimal growth medium for microcolonies TMmod3 (d) with modi-
fied content of growth regulators (1 mg/L BAP, 1 mg/L GK3, 0.2 mg/L
2.4-D and 0.1 mg/L NAA), glucose (0.4 M) as well. In the process of
further cultivation, microcolony growth was followed by a gradual loss of
differential morphology and a transition to unorganized growth. More-
over, the further presence of gibberellin (1 mg/L GK3) in the growth
regulators of TMmod3 (d) nutrient medium had a negative impact on cell
development, increasing their transition to growth by extending while
reducing mitotic activity. This affected the change in the qualitative com-
position of cell populations, in which not meristematic, but vacuolated
parenchymal cells began to predominate in an amount up to 65–70% of
the total population (Fig. 12). The dynamics of mitotic activity of cells,
which took place during the 2-month period of cell cultivation with the use
of different options of osmotic and hormonal regulation of plant cell cultu-
re growth, derived from hypocotyl protoplasts of white cabbage, is reflec-
ted in the graph in Figure 13.
The obtained results show that the value of mitotic index reliably dif-
fers in the two experimental variants of cell cultivation with the presence
and absence of GK3 in the composition of growth regulators of TMmod2
(e) nutrient medium. Moreover, the value of this statistical indicator was
higher in the options of cultivation, where gibberellic acid was not used.
Cell proliferation in the presence of the auxin regulator 2,4-D in the con-
trol variant of cultivation affected the maximum number of peaks of mito-
tic activity of cells. Altogether, the obtained results indicate that changes in
osmotic and hormone regulation had a peculiar effect on growth processes
in cultivated plant cells.

Regul. Mech. Biosyst., 2020, 11(2)
100
a b c d
Fig. 11. Typical microcolony development with morphological traits of structural organization in hypocotyl protoplasts’ in vitro culture of white cabbage
Kharkivska zymnia, Ukrainska osin, Yaroslavna, Lika, Lesia, Bilosnizhka, Dithmarscher Früher and Iyunskarannia (Brassica oleraceae var. capitata L.):
a – the first cell division (indicated by an arrow), which initiates the onset of division of a more vacuolated mother cell into smaller daughter cells;
b – growth of microcolonies of cells after their transfer to the nutrient medium ТМmod3(d); c, d – gradual transition to unorganized microcolony
growth with primary traits of structural organization on the TMmod3(d) nutrient medium
Decrease in mitotic cell activity in the framework of variants with the
use of gibberellic acid correlates well with the beginning of cell growth by
extension and inhibition of cell division. By the number of mitotic active
cells, the research variant of cultivation with application of hypotonic
stress only took an intermediate place between the control and experimen-
tal variants with application of a high dose of gibberellin in the
TMmod3(e) nutrient medium. This intermediate state was preserved up to
about 20 days of cultivation, and the mitotic index value was almost equal
to the control one (Fig. 13).
Discussion
Characterizing the effect of growth regulators of nutrient media on the
development of microcolonies of cells, we should note the generality of
our results obtained in earlier studies of regulatory action of phytohormo-
nes at the in planta level. Cells in higher plants are known to lack mobility
due to the presence of cell walls, so plant morphogenesis is largely depen-
dent on regulated cell division in a strictly defined direction (Medvedev,
1996). Accurate determination of the cell division area is extremely im-
portant for many forming processes, with the structural components of cell
cytoskeleton playing an important role in this process, among which mic-
rotubes (MT) perform the most important function (Medvedev, 1996).
The first indication that the cell begins to divide in this direction appears
immediately after interphase, as the MTs reorient in the process of prepa-
ring for mitosis. There is evidence that processing with auxin leads to tran-
sverse (relative to the long axis) orientation of MT in cells of higher plants
(Blume et al., 2012). It was found out that the impact of the auxin on the
orientation of MT is after the preliminary processing of the GK3 (Blume
et al., 2012). Gibberellins, like auxin, induce transverse (relatively long
axis) location of MT cells (Blume et al., 2012). Short-term cell extension
under the influence of gibberellin is not followed by MT retargeting. GK3
pre-treatment leads to MT reorientation if the gibberellin combines with
auxin. Data from other studies show that the orientation of MT also de-
pends on the cytokinins. It has been determined that action of kinetin on
plant cells can change the location of these structural components of cyto-
skeleton from transverse to longitudinal (Blume et al., 2012). Thus, under
the influence of phytohormones there is a dynamic modification of corti-
cal MT system in mitotic active cells in planta. In the presence of all the
above-mentioned phytohormone analogs in the nutrient medium for the
cultivation of white cabbage protoplasts, we observed similar phenomena
in the growth of cell clones. However, the induction of differential cell
division required a balanced selection of growth regulators as well as their
coordinated application in time together with the regulation of osmotic
pressure in the nutrient medium.
Fig. 12. Transition to cell growth by stretching (indicated by arrows)
in microcolony in the TMmod3(d) nutrient medium (culture of hypocotyl
protoplasts in vitro of white cabbage of Kharkivska zymnia cultivar)
Fig. 13. Features of the mitotic cell activity identified at different variants of cultivation of hypocotyl protoplasts (averaged data for 8 varieties
of white head cabbage): experiment 1 – cultivation mode with osmotic stress and subsequent introduction of gibberellin GK3 into nutrient medium;
experiment 2 – osmotic stress-only cultivation mode; the numerical data in the graph are presented as x ± SD (n = 5)

Regul. Mech. Biosyst., 2020, 11(2) 101
Assessing the contribution of osmotic stress to cytodifferentiation pro-
cesses, it should be noted that previously various types of stress agents
were widely used in vitro culture for embryogenesis induction. For exam-
ple, cold and osmotic stresses are often used for androgenesis induction in
the cultivation of isolated microspores (Ochattet al., 2009; Islam & Tuteja,
2012). The addition of abscisic acid (AA) to nutrients as one of the stress
agents has been applied to induce cytodifferentiation in carrot globular
embryos (Jameel & Abdulaziz, 2012). In our case, one of the possible
mechanisms of osmotic stress influence is the emerging of a powerful dif-
fuse gradient, which led to the intake of excess water and growth regula-
tors (primarily auxin) into the cells. Forceful hydration of both membrane
and cytoplasmic proteins could also contribute to the “unblocking” of the
functional activity of a number of enzyme systems as well as to the mobi-
lization of the genome of cells as a whole (Komaki & Sugimoto, 2012).
It is worth noting that when using a system of isolated protoplasts, the he-
terogeneity of the original cell population is determined by the absence of
synchronization in the phases of the cell cycle. Thus, within the frame-
work of our researches we observed a different ratio of cell population
being developed by cytodifferentiation. The morphology of the microco-
lonies obtained in the result of our study was common with the early
stages of development of protoplasts of both Gentiana kurroo (Niedziela
& Rybczyński, 2007) and G. straminea (Shi et al., 2016), which develo-
ped through direct somatic embryogenesis in specially selected plant lines
in the deficiency of exogenous gibberellin. Related morphology has been
observed, also in the initial division of the zygote in planta (Zhao & Sun,
2015) as well as polarization (formation by root-like projection) in the
brown algae (Bogaert et al., 2013). As already proven, in the lower algae,
such polarization is due to the polar transport of auxin. In terms of our
research, the morphological diversity of microcolonies according to the
differential types may be explained by the accelerated intermutation of
various gibberellins by cell enzyme systems (Hedden & Sponsel, 2015),
while the determinacy of symmetry axes – by the effect of GK3 on the
orientation of cytoplasm microtubes of mitotic active cells in a certain
direction, e.g. along the axis of growing microcolonies (Wenzel et al.,
2000). It is worth highlighting that gibberellin GK3 is rarely used in plant
culture in vitro, while more often at the last stage of regeneration (Edwin
et al., 2007) and is almost out of use in cultivated media at early stages of
protoplast culture development. Assessing the possible role of gibberellin
in cytodifferentiation processes, it should be noted that plants have predo-
minantly the same type of auxin (IAA) and cytokinin (zeatine), but more
than 130 different forms of gibberellin, most of which exhibit their activity
specifically and only at specific stages of plant development (Binenbaum
et al., 2018). Besides, gibberellin synthesis level in planta increases signifi-
cantly in the process of de novo formation of all organs of higher plants
and, especially, in the development of the zygotic embryo, which is quite
convincingly demonstrated in embryological studies on rape (Hays et al.,
2001). Nevertheless, studies of the phytohormone content in the unorgani-
zed growth of tobacco callus revealed a complete absence of endogenous
gibberellins (Nawaga et al., 1987). Our results show that gibberellins di-
rectly participate in regulation of somatic cell differential division. Based
on the above, there is every ground to assume that of all phytohormones, it
is the gibberellin that is the hormone determinant of morphogenetic pro-
cesses in plants. To verify the current hypothesis, we require more detailed
studies and clarifications of the regulatory functions of this phytohormone
in plants. Our morphological evaluation of microcolonies that underwent the
OS stage provides additional evidence in favour of the hypothesis, since
even the presence of only one compound from the class of gibberellins
(GK3) in the hormonal composition of the nutrient medium led to the for-
mation of microcolonies with initial morphological traits of structural organi-
zation. The generality of reaction on presence of gibberellin in mesophilic
protoplasts of the species under study gives the grounds to assume that the
phenomenon described by us represents the general physiological regularity
inherent in higher plants as a whole. Summing up the effect of GK3 gibbe-
rellin at the early stages of development of hypocotyl protoplasts of diffe-
rent varieties of white cabbage genotypes, it ought to be remarked that the
effect of this phytohormone is more negative than positive. According to
Butenko (1981), when growing an explant plant tissue in the dark-room
conditions with the use of gibberellic acid, it is possible to suppress the
function of auxin oxidase, which may adversely affect the mitotic activity
of cells due to the growth of endogenous auxin. Since under conditions of
optimal proliferation of cells, their cultivation during the first 20 days was car-
ried out in the dark-room conditions, the above-mentioned negative impact
of this phytohormone on the mitotic activity of cells could naturally occur.
The generalization of the factors that influenced the asymmetric divi-
sion of cells derived from mesophilic and hypocotyl protoplasts of higher
plants is presented in Table 2. The table shows in more detail the main
types of asymmetric cell division of the studied plant species, depending
on the time-coordinated factors of the osmotic and hormonal regulation of
the division process. At the stage of the sixth mitosis in dividing cells, mic-
rocolonies switched to unorganized growth, while losing morphological
traits of structural organization. In our opinion, the reason for such a transi-
tion to the unorganized growth of microcolonies is the incorrect choice
and balance of growth regulators in the composition of nutrient media
TMmod3 (e) and SWmod3 (e). The possibility of implementing a program
of somatic embryogenesis in protoplast culture in vitro for the studied plant
species requires additional study of the factors of cell differentiation.
As is known, isolated protoplast is the most disintegrated structure of
the plant body. Therefore, this cell type is an ideal experimental object for
studying the regularities of the “cell-plant” development cycle when simu-
lating morphogenetic processes in vitro culture. Currently, significant pro-
gress has been made in studying plant protoplasts, but the issue of their
cultivation and regeneration of plants on their basis is still of fundamental
importance. This is mainly due to the fact that the protoplast is a compo-
nent of a complex system of development, and in addition, generally, it is
subject to key stress factors when cultivated. It is known that different
types of stress can influence the regulation of genes through their expressi-
on or derepression. According to Cocking (1972), the protoplast is a da-
maged, with a missing cellular shell, isolated plant cell, which is under
osmotic stress. The principal physiological feature of protoplasts is the
ability, under certain conditions of cultivation, to regenerate the cellular
cover and begin to divide, forming cell colonies or even plants (Davey
et al., 2005). Having undergone cellular repair process, plant cells are for-
med in the same way as in a conventional suspension culture (Sidorov
et al., 1985), therefore the conditions of their cultivation are subject to
change. It is required as follows: reduction of osmotically active concen-
tration in substances, gradual change of hormonal balance being domina-
ted by суtokinins. Existing experimental practice requires an empirical
selection of all three cultivation regulatory factors (Sidorov et al., 1985),
thus a certain number of protoplast nutrient media options have been de-
veloped that have been applied to a limited number of genotypes or plant
cultivars. Violation of any of the above-mentioned elements of the regula-
tion of cultural growth as well as development leads, commonly, to cell
death. A special role in maintaining cell growth and differentiation in pro-
toplast culture belongs to the regulators auxin, among which we should
mention the special role of 2,4-D as an activator of reprogramming the
genome of special somatic cells, after which they become totipotent and
achieve their development potential, just as it occurs in egg cells after
fertilization (Peterson et al., 2016).
In our study, the occurrence of the regulator 2,4-D was obligatory as
in its absence cultivated cells of plants did not restore or completely lost
their mitotic activity. It has been established that the regulator belongs to
the class of phytohormonal herbicides, a quite toxic substance, which ne-
gatively affects the ontogenesis of plants. This has been proven in numero-
us studies on many plant species (Song, 2014). In particular, the toxicity of
this substance is manifested in chromosomal disorders and the growth of
meristematic cells in apical zones (Song, 2014). 2,4-D is poorly disposed
of by plants. It provides grounds to assume that while implementing the
program of plant morphogenesis in vitro, the regulator may disrupt rege-
neration processes in the cultivated objects due to the herbicidal effect, espe-
cially in the determination of stem organogenesis in dicotyledonous plants.
Thus, the question of the final determination of the principles of os-
motic, trophic and hormonal regulation of morphogenesis in vitro of hig-
her plant protoplasts requires further study. The identified common consis-
tent pattern of the development of somatic cells an early stage of the
growth of protoplast culture of higher plants in vitro served as a basis for
the authors’ developed method of the effective plant regeneration in proto-
plast in vitro culture of white cabbage through organogenesis. The results
of these studies are planned to be presented by us in the next article.

Regul. Mech. Biosyst., 2020, 11(2)
102
Table 2
Exogenous factors of plant cell cultivation before and after cell wall repair that influenced the asymmetric cell division
in the culture of mesophilic and hypocotyl protoplasts of six plant species of the Brassicaceae and Solanaceae families
Cultivation variants for mesophilic and hypocotyl protoplasts
control variant experimental variant
Stage of cell
development growth regulators and
glucose osmotic content the phenotypic response of cells growth regulators and
glucose osmotic content the phenotypic response of cells
nucleus
cytoplasm
vacuole
Cell wall repair in
protoplasts, the
beginning of growth
processes in cells
1–2 mg/L NAA,
0.5 mg/L BAP,
0.2 mg/L 2,4-D,
0.5 М glucose lack of vacuolization, uniform distribution
of cytoplasmic cells elements
1–2 mg/L NAA,
0.5 mg/L BAP,
0.2 mg/L 2,4-D,
0.36 М glucose
(during 16–18 hours and
followed 0.5 M glucose) unequal separation of cytoplasmic elements of
cells due to the growth of vacuoles
I mitosis
1–2 mg/L NAA,
0.5 mg/L BAP,
0.2 mg/L 2,4-D,
0.5 М glucose
symmetrical first division
1–2 mg/L NAA,
0.5 mg/L BAP,
0.2 mg/L 2,4-D,
0.5 М glucose
asymmetrical first division
0.1 mg/L NAA,
0.5 mg/L BAP,
0.2 mg/L 2,4-D,
0.5 М glucose
disorganized growth of microcolonies
1 mg/L GA3,
0.1 mg/L NAA,
0.5 mg/L BAP,
0.2 mg/L 2,4-D,
0.5 М glucose
microcolony growth with traits of structural
organization:
1) formation of cell division directions by type
of zygotic embryo development in planta;
II-VI mitosis
0.1 mg/L NAA,
0.5 mg/L BAP,
0.2 mg/L 2,4-D,
0.5 М glucose
disorganized growth of microcolonies
1 mg/L GA3,
0.1 mg/L NAA,
0.5 mg/L BAP,
0.2 mg/L 2,4-D,
0.5 М glucose
2) division of the mother cell into smaller
daughter cells.
Conclusion
The key result of the research carried out is the determination of the
development process for the cultivation of protoplast of higher plants that
provide for an indication of the process of asymmetric cell division in vit-
ro. Exogenous gibberellin has been proved to be one of the initiators of
such a division type. As it has been shown above, this class of phytohor-
mones significantly influences the initiation of morphogenetic processes in
higher plants, therefore, in our experiments, the action of gibberellic acid
(GK3) has been studied in more detail at the initial stage of cultivation of
mesophilic protoplasts of six species of plant belonging to the Brassica-
ceae and Solanaceae families.
In experiments with mesophilic protoplasts it was shown that short-
term osmotic stress within 16–18 hours in combination with the subsequ-
ent administration of high doses of gibberellin GK3 into the nutrient me-
dium led to the appearance of morphological traits of cytodifferentiation
already at the initial stages of cell microcolonies in a number of higher
plants. At the same time in all analyzed plant species there was observed
the division of the original population of mitotic active cells into two types
of asymmetric division: by the type of cleavage of the mother cell into
smaller daughter cells and by the type of the first asymmetric division of
the zygotic embryo in planta. A special study of the effect of osmotic
stress on hypocotyl protoplasts of various cultivars of white cabbage, iso-
lated from hypocotyl 7–9 day etiolated seedlings, revealed a rather charac-
teristic feature: the determination of cytoplasmic elements in this type of
cells and the subsequent development of differential morphology of mic-
rocolonies occurred without additional injection of exogenous gibberellin
(GK3) into the nutrient medium. This fact, as well as the data obtained on
mesophilic protoplasts, gave us grounds to assume that phytogenic gibbe-
rellin, localized in juvenile etiolated seedlings, which were the source of
protoplasts, had a regulatory effect similar to that of GK3.
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