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Article
Multiple Plant Regeneration from Embryogenic Calli of
Paulownia tomentosa (Thunb.) Steud
Aigul Amirova 1,2, *,† , Symbat Dossymbetova 2 , Yeldana Rysbayeva 2 , Bakdaulet Usenbekov 1 , Arman Tolegen 1
and Alibek Ydyrys 1,3,†
1 Faculty of Biology and Biotechnology, Al-Farabi Kazakh National University, al-Farabi Av. 71, Almaty 050040,
Kazakhstan; bakdaulet7@yandex.ru (B.U.); tolegenarman7@gmail.com (A.T.); ydyrys.alibek@gmail.com (A.Y.)
2 Department of Food Technology, Almaty Technological University, Tole be 100, Almaty 050008, Kazakhstan;
symbat_89@list.ru (S.D.); eldana-90@mail.ru (Y.R.)
3 Biomedical Research Centre, Al-Farabi Kazakh National University, al-Farabi Av. 71,
Almaty 050040, Kazakhstan
* Correspondence: aikoamir@mail.ru
† These authors contributed equally to this work.
Abstract: The aim of this paper was to study the effect of plant growth regulators on callus induction
and in vitro morphogenesis using various explants of Paulownia tomentosa to develop an efficient
plant regeneration protocol. Different plant organ sections (leaves, apical shoot tips, petals, nodes,
and internodes) were cultured as explants to identify the best in vitro explants responsive to callus
induction and plant regeneration. Explants were cultivated on MS media supplemented with
different concentrations of plant growth regulators (TDZ (Thidiazuron), BAP (6-Benzylaminopurine),
kinetin, and NAA (1-Naphthaleneacetic acid). It was discovered that the addition of TDZ and NAA
stimulated the induction of somatic embryogenesis. It was discovered that the MS medium with
the combination of plant growth regulators BAP (35.5 µM) and NAA (5.4 µM) with the addition of
Citation: Amirova, A.;
30.0 g/L maltose, 500.0 mg/L casein hydrolysate, and 250.0 mg/L L-proline was optimal for callus
Dossymbetova, S.; Rysbayeva, Y.; induction and multiple plant regeneration. The study of the regenerative capacity of various explants
Usenbekov, B.; Tolegen, A.; Ydyrys, of Paulownia tomentosa in vitro showed that plant regeneration depends on the type of explant, and
A. Multiple Plant Regeneration from occurs in both ways, indirectly, through the formation of callus tissues and directly on the explant,
Embryogenic Calli of Paulownia without callus formation. As a result of this study, the efficient reproducible protocol of embryogenic
tomentosa (Thunb.) Steud. Plants 2022, callus formation and multiple shoot induction in vitro of Paulownia tomentosa was developed. This
11, 1020. https://doi.org/10.3390/ system provides a clear increase in the frequency of plant regeneration from 36.3 ± 3.4% to 38.6 ± 2.3%
plants11081020 per embryogenic callus from leaves and apical shoot tips, respectively.
Academic Editors: Milan S. Stankovic
and Sharyn Perry Keywords: Paulownia tomentosa; plant growth regulators; in vitro; somatic embryogenesis; plant
regeneration
Received: 28 December 2021
Accepted: 5 April 2022
Published: 8 April 2022
economic, social, and environmental problems. One of the effective measures to prevent
desertification, land degradation, and to strengthen soil structure is to increase the planting
of trees and forests. The use of biotechnological methods of microclonal propagation
and plant regeneration in vitro increased plant production. Currently, fast growing trees
Paulownia tomentosa are widely used to protect the environment, reduce soil erosion, and
improve soil fertility [1,4,5]. Many wild plants have sufficient reserves of raw materials in
nature, but the rapid reduction of forest areas due to anthropogenic pressure and unplanned
development and the excessive use of cultivated plants contribute not only to a decrease in
their number, but also to the extinction of a number of species in nature [6], as well as to
a decrease in the number of species in biodiversity [7]. Therefore, an alternative way to
obtain more raw materials is to grow medicinal plants in botanic gardens or agricultural
fields [8,9], as well as using the methods of in vitro plant tissue culture. Currently, many
scientists are engaged in the cultivation of medicinal plants in various ways [10–12], for
example, growing and storing them in the system of organic farming without the use
of fertilizers and agricultural drugs. Alternatively, growing medicinal plants using new
biogums is used in agriculture [13].
Traditionally, Paulownia propagates by seed or root cuttings [14,15]. The main dis-
advantages of vegetative propagation methods are the genetic diversity of the resulting
planting material and the duration of the juvenile period. Seeds germinate slowly or quickly
lose germination. Propagation by root cuttings is delayed due to the damage and disease
caused by pathogenic microorganisms and pests [16].
The use of modern biotechnological methods of in vitro plant propagation can over-
come these disadvantages and increase the production of plants much better than the
traditional method of propagation by seeds and cuttings. Plant cell and tissue culture
techniques, such as microclonal propagation, organogenesis, and somatic embryogenesis
are mainly used in plant propagation Paulownia ‘Shan Tong’ (P. fortunei ∗ P. tomentosa)
in vitro [17]. Organogenesis and somatic embryogenesis are two major morphogenetic
pathways in tissue culture in vitro. Organogenesis is the formation of organs (shoots and
roots) in plant tissue culture. Somatic embryogenesis is a process in which the plant somatic
cells, under the influence of external factors, suddenly begin to behave like a zygote and
pass through various stages of embryogenesis with the formation of bipolar structures that
resemble zygotic embryos [18–20]. Somatic embryogenesis is the process of the formation of
many somatic embryos of single or multicellular origin and is used for the mass production
of plants.
One of the ways to develop reproducible plant cell and tissue technologies is to study
the cytophysiological features of the regulation of morphogenesis in vitro by changing the
composition of nutrient media [21]. The use of biotechnological methods of in vitro plant
propagation significantly expands the possibility of mass production of somatic embryos
and regenerated plants. Most of the developed protocols of plant regeneration of Paulownia
are mainly associated with the use of nodal explants. Thus, the study of regenerative
capacity of various types of explants of Paulownia (segments of leaves, petioles, internodes,
and nodes) showed that the nodal explants have a high regenerative capacity compared to
other types of explants [22,23].
Mostly, the nutrient medium of Murashige and Skoog [24] is used for the propagation
of different genotypes of Paulownia in vitro [17,25,26]. Furthermore, studying the effects of
various plant growth regulators (TDZ, BAP, IAA) on the frequency of plant regeneration,
different authors obtained data that are slightly different from each other. Some authors
reported that the TDZ was more effective when compared to BAP for the regenerated plant
induction of Paulownia ‘Shan Tong’ (P. fortunei ∗ P. tomentosa) [17,27,28]. It was observed
that the optimal ratio of auxin to cytokinin should be 1:10, which will contribute to the
regeneration of shoots. In this ratio, the highest frequency of adventitious shoots formation
was discovered [17].
Ipekci (2001) [22] and Litwinczuk and Bochnia (2012) [29] reported that the MS
medium supplemented with 4.44 mg/L BAP and 0.54 µM NAA was found to be opti-
Plants 2022, 11, 1020 3 of 14
mal for the regeneration of multiple shoots from the shoot tip of Paulownia in tissue culture
in vitro. The high efficiency of BAP over the other cytokinin for the formation of multiple
shoots of Paulownia was reported by Taha (2008) [30].
Another author showed that the addition of 13.9 µM kinetin and 2.68 µM NAA in the
culture medium was most effective for the regeneration of shoots of Paulownia tomentosa
among the various plant growth regulators (BAP, kinetin, zeatin, and NAA). The leaf
segments produced the largest number of shoots per explant (12 ± 0.4) in this medium
when two types of explants (apical shoot and leaf segments) were used. The addition of
10% of coconut water (CW) to the abovementioned medium increased the number of shoots
(up to 18) per culture. In addition, they showed that the rooting of shoots occurred well
on half-strengthened MS medium ( 12 MS) supplemented with 10.7 µM NAA within 12 to
15 days [26].
In this regard, the aim of this paper is to study the effect of various phytohormones
on callus induction and plant regeneration processes in vitro, to develop an effective repro-
ducible protocol of multiple plant regeneration of Paulownia tomentosa.
2. Results
2.1. Testing of the Various Nutrient Media for Callus Induction and Plant Regeneration
Testing of the various nutrient media for callus induction and plant regeneration
capacities of Paulownia tomentosa used MS1-MS6 nutrient media supplemented with 7.0 g/L
agar, 30.0 g/L maltose, 1000.0 mg/L casein hydrolysate, and 250.0 mg/L L-proline. It
is known that the number of apical shoot tips and nodes is limited on plants and it was
decided that their use on all tested media is not profitable. Paulownia has a lot of large leaves
and there were no restrictions in plant material for the isolation of explants. Therefore,
only leaf fragments were selected as explants to study the induction of calli on various
media (Table 1) and to select the optimal nutrient medium for obtaining the tissue culture
of Paulownia tomentosa.
Table 1. The influence of media composition on callus induction and plant regeneration from leaves
of Paulownia tomentosa.
Culture Medium Concentration of Plant Growth Regulators Callus Induction, % Plant Regeneration, %
MS1 22.2 µM BAP + 5.4 µM NAA 87.6 ± 2.6 44.4 ± 0.8
MS2 35.5 µM BAP + 5.4 µM NAA 95.5 ± 3.9 95.2 ± 1.9
MS3 4.5 µM TDZ + 2.8 µM IAA 82.4 ± 4.3 23.9 ± 2.5
MS4 13.6 µM TDZ + 2.8 µM IAA 80.2 ± 3.7 12.5 ± 1.8
MS5 13.9 µM kinetin + 2.8 µM IAA 0 0
MS6 23.2 µM kinetin + 2.8 µM IAA 0 0
Studying the influence of different plant growth regulators on callus induction re-
vealed that the MS2 medium is optimal (95.5 ± 3.9%) among the used nutrient media
(Table 1).
was not detected on the two MS media with the addition of kinetin and NAA (MS5–MS6),
but the initiation of roots directly on explants was observed (Figure 1F).
A high percentage of callus tissues formation was observed in all types of explants.
The frequency of callus induction from leaves is greater than others (Table 2). It was found
that the leaves are the best explants for callus induction.
Table 2. The callus induction and plant regeneration capacities of calli from different types of explants
of Paulownia tomentosa on MS2 medium.
The morphological study of calli sorts tissues into two types: dense white globular
and white–greenish embryogenic tissues. It is observed that the induction of white dense
globular calli begins primarily in the area of the cutting of the explants. During cultivation,
white dense globular calli were transformed into white–greenish embryogenic tissues
(Figure 1B). It was noticed that after the first subculture calli began growing fast and all
explants produced callus tissues.
It was discovered that the most optimal nutrient media for the induction of white–
greenish embryogenic calli from leaf explants was MS with a combination of TDZ and
NAA. Thus, TDZ stimulates the induction of somatic embryogenesis: the formation of
white–greenish embryogenic callus tissues after several subculturing (3–4 passages) and
spontaneous regenerated plants (Figure 2A–D) were observed. Figure 2B clearly shows the
production of regenerated seedlings from embryogenic calli (EC). Embryogenic calli are
characterized by the smallest needle-like embryogenic structures, which further develop
and differentiate into somatic embryoids (SE) and give rise to whole plants.
Thus, the addition of TDZ to the nutrient medium stimulates the induction of embryo-
genic and regenerable callus tissues. It was found that increasing the concentration of TDZ
in the medium leads to a decrease in the regenerative potential of calli. Callus induction on
the two MS media supplemented with kinetin and NAA was not detected, however, the
initiation of roots directly on explants was observed.
(A) (B)
(C) (D)
(E) (F)
Figure 1. Induction of callus
Figure tissues from
1. Induction different
of callus explants
tissues of Paulownia
from different tomentosa
explants on MS2tomentosa
of Paulownia media: on MS2 media:
morphology of white callus tissues from leaves (A,B), internodes (C), nodes (D), and petals (E);
morphology of white callus tissues from leaves (A,B), internodes (C), nodes (D), and petals (E); root
root formation directly (organogenesis) from the explants of leaf on MS media with 13.9 µ M kinetin
formation directly (organogenesis) from the explants of leaf on MS media with 13.9 µM kinetin +
+ 2.8 µ M IAA (F).
2.8 µM IAA (F).
A high percentage of callus tissues formation was observed in all types of explants.
The frequency of callus induction from leaves is greater than others (Table 2). It was
found that the leaves are the best explants for callus induction.
Table 2. The callus induction and plant regeneration capacities of calli from different types of
explants of Paulownia tomentosa on MS2 medium.
(A) (B)
(C) (D)
Figure 2. Induction of 2.callus
Figure tissuesof(A)
Induction and tissues
callus plant regeneration
(A) and plantfrom embryogenic
regeneration fromcallus from
embryogenic callus from
fragments of leaf on MS media with 4.5 µ M TDZ + 2.8 µ M IAA (B–D). Symbols in (B): SE—somatic
fragments of leaf on MS media with 4.5 µM TDZ + 2.8 µM IAA (B–D). Symbols in (B): SE—somatic
embryos, EC—embryogenic calli with small needle-like embryogenic structures at early stages of
embryos, EC—embryogenic calli with small needle-like embryogenic structures at early stages of SE
SE (micro photo).
(micro photo).
Thus, the addition of TDZ to the nutrient medium stimulates the induction of
embryogenic and regenerable callus tissues. It was found that increasing the
concentration of TDZ in the medium leads to a decrease in the regenerative potential of
calli. Callus induction on the two MS media supplemented with kinetin and NAA was
not detected, however, the initiation of roots directly on explants was observed.
(A) (B)
(C) (D)
Figure 3. Indirect and direct
Figure plant and
3. Indirect regeneration from
direct plant different types
regeneration fromof explantstypes
different on MS2 medium:on MS2 medium:
of explants
internodes and internodes
petals indirectly, through the callus formation (A) and directly, without calli calli formation,
and petals indirectly, through the callus formation (A) and directly, without
formation, on nodal explants (B); plant regeneration from leaves (C) and apical shoot tips (D)
on nodal explants (B); plant regeneration from leaves (C) and apical shoot tips (D) through the
through the callus formation.
callus formation.
During the second
Duringand further and
the second subcultures (up to 2 (up
further subcultures years)
to 2the multiple
years) plant plant regenera-
the multiple
regeneration from callus tissues occurs on the different explants of Paulownia tomentosa.
tion from callus tissues occurs on the different explants of Paulownia tomentosa.
Table 3. Relationship between the number of passages and plant regeneration in embryogenic calli
lines of Paulownia tomentosa.
Therefore, the highest regeneration capacity of callus tissues was observed on MC2
medium containing 35.5 µM BAP and 5.4 µM NAA. This medium was selected as optimal
for plant regeneration from the tested nutrient media. It was concluded that the leaves and
apical shoot tips are the best explants for somatic embryogenesis and plant regeneration. Of
course, we also successfully used other types of explants. The number of regenerated plants
increased with the time of subcultures of calli and plant regeneration capacity, continuing
for up to 2 years.
(A) (B)
(C) (D)
Figure 4. Rooting of regenerated plants on ½ MS media with 5.4 µ M NAA and transfer of rooted
Figure 4. Rooting of regenerated plants on 12 MS media with 5.4 µM NAA and transfer of rooted
plants to soil: well rooted shoots in vitro (A,B), after rooting plants transferred to the soil (C,D).
plants to soil: well rooted shoots in vitro (A,B), after rooting plants transferred to the soil (C,D).
Increasing3. the concentration of IAA and NAA up to 11.4 µ M and 10.7 µ M,
Discussion
respectively, prolonged the rooting of shoots. The appearance of roots was observed after
The use of biotechnological propagation methods allows one to increase the yield
10–15 days in media
of plants with a high concentration
in comparison with the of auxins. Itmethod
traditional was determined that a high In this article,
of seed propagation.
concentration of auxins promotes the rapid rooting of shoots.
experiments are devoted to the development of methods to increaseThe successful and rapidthe yield of the
rooting of shoots was demonstrated on media ½ MS with 5.4 µ M NAA
regeneration of plants of Paulownia tomentosa in tissue culture in vitro. or 5.7 µ M IAA.
Thus, the media ½In MSthewith 5.4 literature,
available µ M NAAit is orknown
5.7 µ M thatIAA acceleratednutrient
the well-known the rooting
medium of Murashige and
regenerated plants
Skoog of[24]
Paulownia tomentosa
is used for (Figure
the in vitro tissue4A–D).
culture and plant regeneration of Paulownia tomentosa,
An average (90%) of adaptation of the regenerated
and among the plant growth regulators, TDZ, plants
BAP,wasandachieved
IAA arewhen
used. they
were transferred from in vitro to glasses with water. The well acclimatized
As a result of our experiments, it was discovered that the addition plants were of TDZ to the
transferred to pots with soil, peat, and perlite in a ratio of 1:1:1 (Figure 4D).
nutrient medium stimulates the induction of somatic embryogenesis. However, However, the increasing
transportation the
of acclimatized plants to the soil was the most critical and difficult
concentration of TDZ leads to decreasing the regenerative potential of calli. Thestage,
at which not all plant regenerants
induction survived.
of callus tissues We achieved
on media an average
supplemented withsurvival rateNAA
kinetin and (98%)was not shown,
in vivo of regenerated plants. Then,
but an increase of rootsplants were
directly ontransferred
the explants towas
the greenhouse.
observed.
Among the tested six media with various plant growth regulators, media with a
3. Discussion combination of 35.5 µM BAP and 5.4 µM NAA turned out to be the most optimal for callus
The use ofinduction, somatic embryogenesis,
biotechnological propagation and plant regeneration.
methods allows one to increase the
yield of plants in comparison with the traditional method of seed propagation. In this
article, experiments are devoted to the development of methods to increase the yield
of the regeneration of plants of Paulownia tomentosa in tissue culture in vitro.
In the available literature, it is known that the well-known nutrient medium
Murashige and Skoog [24] is used for the in vitro tissue culture and plant
regeneration of Paulownia tomentosa, and among the plant growth regulators, TDZ,
Plants 2022, 11, 1020 10 of 14
Studying the regenerative capacities of different explants (leaves, apical shoot tips,
petioles, nodes, and internodes) showed two pathways of plant regeneration in vitro: direct
and indirect. Direct plant regeneration on explants is rare; mostly an indirect pathway
of plant regeneration through callus formation was found in the in vitro tissue culture
of Paulownia tomentosa. The earlier, direct, and indirect plant regeneration from leaf and
internode explants in Paulownia elongata was reported by Ipekci and Gozukirmizi [31,32].
Most of the developed protocols of plant regeneration of Paulownia are mainly associated
with the use of nodal explants. Thus, the study of the regenerative capacity of various types
of explants of Paulownia (segments of leaves, petioles, internodes, and nodes) indicates
that the nodal explants show a high regenerative capacity compared to other types of
explants [22,23].
Ipekci [22] and Litwinczuk and Bochnia [29] reported that the MS medium supple-
mented with 4.4 µM BAP and 0.54 µM NAA was found optimal for the regeneration
of multiple shoots from the shoot tip of Paulownia tomentosa in tissue culture in vitro. A
high efficiency of BAP over the other cytokinin for the formation of multiple shoots of
Paulownia was reported by Taha [30]. Pozoga et al. presented a high potential of in vitro
micropropagation of Paulownia tomentosa ∗ Paulownia fortunei hybrid by inducing organo-
genesis from nodular explants on 12 MS media with various concentrations of BAP [33].
Rahman et al. studied the propagation of the Paulownia tomentosa tree in vitro from apical
shoot and lateral bud explants on MS media containing various plant growth regulators:
BAP, NAA, and kinetin. The authors achieved a high percentage of plant induction (84%)
on MS medium with 11.1 µM BAP + 2.68 µM NAA, where the plant yield was 7.4 shoots per
explant [34]. Roy (2015) [26] showed that the addition of 13.9 µM kinetin and 2.68 µM NAA
in the culture medium was effective for the regeneration of shoots of Paulownia tomentosa
(Thunb.) Steud. among the various plant growth regulators (BAP, kinetin, zeatin and
NAA). The leaf segments produced the largest number of shoots per explant (12 ± 0.4)
in this medium when two types of explants were used (apical shoot and leaf segments).
The addition of 10% of coconut water (CW) to the abovementioned medium increased
the number of shoots (up to 18) per culture. In addition, they showed that the rooting of
shoots was good on half strengthened MS medium ( 12 MS) supplemented with 10.7 µM
NAA within 12 to 15 days [26]. As can be seen, many researchers of the in vitro propagation
of Paulownia frequently used the MS medium, and based on this, the authors of this paper
selected this medium for their experiments. In this experiment, authors used plant growth
regulators BAP and NAA, which stimulated the induction of somatic embryogenesis and
plant regeneration.
The tissue culture system described here provides a highly efficient and reproducible
protocol of the embryogenic callus induction and multiple shoot regeneration of Paulownia
tomentosa in vitro. The selection of EC lines with high embryogenic and plant regeneration
capacities from leaf and apical shoot explants accelerated and increased the production
of plants Paulownia tomentosa in vitro. Thus, the system developed by us provides a clear
increase in the frequency of plant regeneration from 36.3 ± 3.4 to 38.6 ± 2.3 per callus
(EC) originating from leaves and apical shoot tips, respectively, and a significantly higher
percentage of calli showing the regeneration potential relative to levels reported in the
literature on the tissue culture of Paulownia tomentosa. These lines of EC tissue retain the
ability to regenerate plants for a long period. Moreover, the developed protocol for multiple
regenerations of plants from embryogenic calli can be applied to genetic manipulations of
Paulownia tomentosa. Moreover, there is an attempt to obtain a transgenic woody plant of
Paulownia tomentosa resistant to bacterial infections [35].
4.4. Testing of the Various Nutrient Media for Callus Induction and Plant Regeneration
The study was carried out in 2 stages. In the first experiment, different types of
nutrient media (MC1–MC6) were tested using one type of explants in order to select
the best medium for callus induction and plant regeneration. Only leaf fragments were
cultured on various nutrient media (MS1–MS6). The callus induction frequency was
determined by the proportion of explants number that formed callus tissues. The frequency
of regeneration capacities was represented by the number of regenerated plants on the
variant of the experiments.
Plants 2022, 11, 1020 12 of 14
5. Conclusions
In conclusion, an efficient and reproducible protocol of the in vitro propagation of
Paulownia tomentosa has been developed. High embryogenic callus formation and multiple
shoot regenerations in vitro of Paulownia tomentosa were observed. We recommend this
reproducible technology of somatic embryogenesis and plant regeneration to use in the
propagation of valuable woody plants, to study the molecular and cellular mechanisms
of somatic embryogenesis and plant regeneration in vitro, and to use it in the genetic
transformation of Paulownia tomentosa.
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