1
The Effects of Different Cytokinin Types and Their Concentration
on in Vitro Growth of Apricot (Prunus armeniaca L.) Shoots
Metka ŠIŠKO*, Tina TERNJAK, Silva GROBELNIK MLAKAR
University of Maribor, Faculty of Agriculture and Life Sciences, Pivola 10, 2311 Hoče, Slovenia
ABSTRACT
The objective of this study was to assess the effects of different cytokinin types [(6-benzylaminopurine (BAP), 
thidiazuron (TDZ) and meta-topolin (mT)] and their concentration on in vitro growth of apricot shoots. Sufficiently 
developed shoots were transferred to different multiplication media, which differed regarding the presence of cytokinin 
and its concentration. The subcultivation period lasted nine weeks. Analysis of covariance (ANCOVA) was performed to 
investigate the influence of plant growth regulators and their concentration on the final shoot weight of obtained tissue 
per regenerating explant. The results showed that all three cytokinins had a positive effect on shoot growth. There was a 
significant difference between used concentrations in the case of TDZ and BAP, while in the case of mT, the concentration 
did not significantly affect shoots’ regeneration. In our study, the most significant increase in shoots weight was obtained 
on medium supplemented with 1.0 mg/L BAP.
Key words: Prunus armeniaca, micropropagation, in vitro propagation, BAP, TDZ, mT
Agricultura 19: No 1: 1-6(2022)
https://doi.org/10.18690/agricultura.19.1.1-6.2022
*Correspondence to:
E-mail: metka.sisko@uni-mb.si                                               
INTRODUCTION
Apricot (Prunus armeniaca L.) is one of the most important 
stone fruit crops and has been cultivated for centuries as a 
fruit crop and ornamental plant in many countries. Usually, it 
is propagated by vegetative cuttings that may be infected with 
pathogens. One of the most important and frequently present 
pathogens is the Plum pox virus (PPV), the agent responsible 
for the Sharka disease. The natural host range of this virus is 
restricted to Prunus spp. (stone fruits and ornamental trees). 
Efforts to eliminate pathogens from infected accessions are 
an important part of germplasm quarantine and exchange 
programs (Cheong Eun & An, 2015). In vitro tissue culture 
with or without thermotherapy and/or chemotherapy is an 
efficient method to obtain pathogen-free plants. Therefore, 
the optimal protocol for in vitro propagation of apricot must 
be developed. Wang et al. (2013) show that optimal protocol 
depends greatly on genotype, so the media's optimisation 
needs to be done for different germplasms.
Less information is known about apricot micropropagation 
compared to other stone fruit plants. Some research suggests 
a very low ability to form axillary shoots in apricots in vitro 
culture (Kataeva & Kramarenko, 1989; Skirvin et al., 1986) 
or an almost complete absence of axillary shoot formation 
(Snir, 1984).
In the micropropagation of apricots, the first stage 
(disinfection of buds and their induction) can be problematic. 
The success of induction itself, of course, depends to a large 
extent on the cultivar, the age of the tree from which we 
take the buds, and the environment in which the tree grows 
(Cheong Eun & An, 2015; Wang et al., 2013).
The type and concentration of PGR (plant growth 
regulators) used in micropropagation vary widely, depending 
on the species that are being studied, and they have to be 
optimized for each genotype. Cytokinins are an essential 
class of growth regulators that, along with auxins, control 
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The effects of different cytokinin types and their concentration on in vitro growth of apricot (Prunus armeniaca L.) shoots
numerous physiological and plant development processes. 
The choice of cytokinin is one of the most critical factors in 
developing of protocols for the in vitro culture of plants (San 
José et al., 2021).
BAP (6-benzylaminopurine) is one of the most frequently 
used cytokinin due to its efficiency and relatively low cost. 
However, using this cytokinin in micropropagation procedures 
can produce physiological disorders such as hyperhydricity 
and apical necrosis. The discovery of a new group of aromatic 
cytokinins, the topolins, has led to new possibilities for their 
use in the micropropagation of numerous species (Zaytseva 
et al., 2021). In the last few years, the use of meta-topolin 
(mT) and its derivatives has increased rapidly, especially in 
initiating new cultures, optimizing protocols, and inhibiting 
the negative features of some other cytokinins (San José et 
al., 2021). Meta-topoline (mT) is a cytokinin that can be used 
as an alternative to the cytokinin benzyladenine (BA); after 
the acclimatization process, the plants develop roots better 
structured and more abundant than those grown in the 
medium with an added BA (Werbrouck et al., 1996). Gentile 
et al. (2014) demonstrated that meta-topolin positively 
affects shoot growth and quality of shoots and reduces 
the proportion of plants with hyperhydricity symptoms. 
Thidiazuron (TDZ) is an aromatic natural cytokinin and is 
one of the most active cytokinin-like substances for woody 
plant tissue culture. It enables efficient micropropagation of 
many woody species. Low plant regulator concentrations (<1 
µM) can induce more significant axillary proliferation than 
many other cytokinins. A concentration higher than 1 µM, 
can stimulate the formation of callus, adventitious shoots or 
somatic embryos (Huetteman & Preece, 1993). 
This study evaluates the effects of three cytokinins (BAP, 
TDZ and mT) and their concentration on in vitro growth of 
a local apricot genotype named Pišečka marelica.
MATERIAL AND METHODS
Plant material 
The experiment was carried out in the Plant Tissue Culture 
Laboratory at Faculty of Agriculture and Life Sciences 
(FKBV), University of Maribor. Shoots were collected on 
March 22, 2017, from an adult apricot tree in the Slovenian 
plant gene bank of stone fruit trees at Pohorski dvor, FKBV, 
Hoče, NE Slovenia and transferred to the laboratory. The 
accession included in this study was a local cultivar named 
Pišečka marelica having the accession number 6339. Buds 
were used as explants and sterilised as described by Šenveter 
(2018). For this study, previously sterilised and developed 
shoots were used.
Shoot proliferation
Developed shoots approximately 1–1.5 cm long (Figure 1) 
were placed on the medium, which contained the following 
substances: Woody plant medium (WPM) (Lloyd & McCown, 
1980) mineral salts including vitamins (Duchefa, Haarlem, 
The Netherlands) supplemented with 13 g/L sucrose, 11 g/L 
D-sorbitol, 0.1 mg/L biotin, 0.01 g/L folic acid, 0.1 mg/L 
riboflavin and solidified with 6.0 g/L agar (Plant Agar, 
Duchefa, Haarlem, The Netherlands). The pH was adjusted 
to 5.7 before adding gelling agent and autoclaving. Seven 
different media were prepared according to plant growth 
regulators: one without plant regulators (control) and six 
containing 0.01 mg/L 1-naphthaleneacetic acid (NAA) and 
three different cytokinins: 6-benzylaminopurine (BAP), 
thidiazuron (TDZ), and meta-topolin (mT) each at two 
concentrations; 0.5 mg/L and 1.0 mg/L. We set 30 shoots on 
each of seven media, each shoot in its own “baby jar”. Each 
shoot was weighted before planting, and its initial weight 
was recorded. The shoots were moved to a freshly prepared 
medium every three weeks during the experiment. Cultures 
were maintained in a growth chamber at 23 °C with a 15 
hours long photoperiod (15,000 Lux). After nine weeks 
of cultivation, the fresh weight of newly formed shoots 
(final weight) was assessed. Shoots were also observed for 
hyperhydricity.
Statistical analysis
The experiment was conducted as a completely random 
design, with 30 explants per treatment. A univariate 
(control) and two–way between–groups univariate analysis 
of covariance (ANCOVA) was performed to investigate the 
influence of plant growth regulator and its concentration 
Figure 1: Regeneration of shoots of apricot (P. armeniaca, Pišečka marelica): shoot developed after disinfection and induction of bud 
(a); shoots used as an explants in experiment (b); shoots obtained on medium supplemented with meta-topolin (c)
a b c
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The effects of different cytokinin types and their concentration on in vitro growth of apricot (Prunus armeniaca L.) shoots
on the final shoot weight measurements. The initial 
shoot weight was entered as a covariate, and its effect was 
Table 1: F statistics of ANCOVA on the effects of PGR, the 
used concentration, and their interaction on shoot 
regeneration of apricot explants expressed by final 
fresh shoot weight (g) 
Factor Shoot final weight (g)
Covariate (initial 
shoot weight) 7.934**
PGR 24.340***
Concentration (C) 8.122**
PGR × C 4.666*
Meana ± SEM
PGR
BAP 1.92 ± 0.153 a
TDZ 1.97 ± 0.178 a
mT 0.55 ± 0.161 b
Concentration
0.5 mg/L 1.22 ± 0.125 b
1.0 mg/L 1.74 ± 0.125 a
*, **, *** corresponding to P < 0.05, 0.01 and 0.001, respectively 
a Covariates appearing in the model are evaluated at the 0.0412
a–b Different letters indicate significant differences among treatments 
(Pairwise comparison, Bonferroni adjustment)
Figure 2: Effect of various concentration of BAP, TDZ and mT on shoot proliferation of P. armeniaca, Pišečka marelica explants 
* Estimated means (ANCOVA)
a–b Different letters (± SEM) indicate significant differences among treatments (Pairwise comparison, Bonferroni adjustment) 
controlled in the analysis. In order to run the analysis under 
optimal conditions, we attempted to detect any deviation 
from ANCOVA assumptions that might distort the tests of 
statistical significance (Field, 2013). Statistical significance 
was indicated by a P-value < 0.05. Results are presented as 
estimated means ± standard error of the mean (SEM). A 
Bonferroni adjustment setting was applied to assess the 
between-subject effect analysis. All analyses were carried out 
using the IBM Statistics software (Version 25).
RESULTS AND DISCUSSION
It is well known that plant growth regulators (PGR) 
greatly influence shoot regeneration. TDZ and BAP are the 
most used cytokinins in Prunus regeneration studies, while 
kinetin, isopentenyl adenine (2iP), and mT have been used 
less (Ruzic & Vujović, 2008). Results about the effectiveness 
of PGR on regeneration and shoot development of Prunus 
are contradictory. Often TDZ has been reported to be more 
effective than BAP (Bhagwat & David Lane, 2004; Canli & 
Tian, 2008; Espinosa et al., 2006; Hammatt & Grant, 1998; 
Matt & Jehle, 2005; Pérez-Tornero & Burgos, 2000), but in 
other reports (Ruzic & Vujović, 2008; Tang et al., 2002) BAP 
has been found more effective than TDZ. These findings 
are in accordance with our study, where BAP shows the 
highest regeneration and gained the highest final weight of 
shoots. Different species and genotypes used in conducted 
experiments can explain contradictory results.
4
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A., Nota, P., & Caboni, E. (2014). Effect of meta-Topolin on 
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(pp.421-427). International Plant Propagators' Society.
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from leaves and internode sections of sweet cherry 
cultivars (Prunus avium L.). Plant Cell Rep, 24(8), 468-
476. https://doi.org/10.1007/s00299-005-0964-6 
Nas, M. N., Bolek, Y., & Sevgin, N. (2010). The effects of 12. 
explant and cytokinin type on regeneration of Prunus 
microcarpa. Scientia Horticulturae, 126(2), 88-94. https://
doi.org/https://doi.org/10.1016/j.scienta.2010.06.012 
Pérez-Tornero, O., & Burgos, L. (2000). Different media 13. 
requirements for micropropagation of apricot cultivars. 
Plant Cell, Tissue and Organ Culture, 63(2), 133. https://
doi.org/10.1023/A:1006430718024 
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types and their concentration on in vitro multiplication of 
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HORTSCI 
San José, M. C., Cernadas, M. J., & Janeiro, L. V. (2021). 15. 
Optimization of Micropropagation Protocols in Some 
Woody Plants Using Meta-topolin. In N. Ahmad & M. 
Strnad (Eds.), Meta-topolin: A Growth Regulator for Plant 
Biotechnology and Agriculture (pp. 221-240). Springer 
Singapore. https://doi.org/10.1007/978-981-15-9046-
7_16 
Skirvin, R. M., Chu, M. C., Mann, M. L., Young, H., 16. 
The effects of different cytokinin types and their concentration on in vitro growth of apricot (Prunus armeniaca L.) shoots
The ANCOVA results demonstrated that the PGR effect is 
the main factor influencing the shoot regeneration of apricot 
explants (Table 1). On average, BAP and TDZ resulted 
in statistically higher final shoot weight (0.92 and 0.97 g, 
respectively) than mT. The two-way interaction between 
observed factors was also detected (Table 1) and shown in 
Figure 2. The most effective in shoot regeneration was BAP 
(2.54 g) and TDZ (2.10 g) in higher – 1.0 mg/L concentration, 
and the least effective cytokinin was mT regardless of 
concentration (0.53 and 0.57 g). Interestingly, we noticed that 
the final shoot weight was for 13.7% higher using 1.0 mg/L of 
TDZ. Results are contrary to those of Ning et al. (2007), who 
reported that TDZ at lower concentrations promotes and at 
higher concentrations usually inhibits shoot regeneration.
Our data show that exogenous PGR is essential for shoot 
regeneration of apricot explants. Regeneration of apricot 
shoots cultured on plant growth regulators-free medium 
(control) was much lower (0.21 g) than explants cultured 
on a medium containing BAP, TDZ or mT (Figure 2). Nas 
et al. (2010) report that regeneration rations of P. microcarpa 
cotyledons explants cultured on PGR-free medium (control) 
were lower than explants cultured on a medium containing 
BAP, TDZ or mT, which is in accordance with our results.
Even meta-topolin was the least effective cytokinin in 
our study, the developed shoots were healthier (Figure 1), 
and the number of hyperhydric explants was reduced (no 
hyperhidricity observed) in comparison with BAP (35.3% 
hypehydricity) and TDZ (60% hyperhidricity) (data not 
shown). These data agree with Gentile et al. (2014), who 
obtained a better control of hypehidricity by using mT in 
Prunus rootstocks than BAP and TDZ.
CONCLUSION 
To our knowledge, this is the first report of cytokinin 
effect on shoot proliferation of apricot genotype named 
Pišečka marelica. The results presented could be used for 
micropropagation protocol for apricot multiplication in vitro. 
In our study, the best regeneration was obtained on medium 
supplemented with BAP in concentration of 1.0 mg/L in 
combination with 0.01 mg/L NAA.
ACKNOWLEDGMENTS
We would like to express our gratitude to Tamara Hribernik 
for assistance with the experiments.
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& Debergh, P. C. (1996). Meta-topolin, an alternative to 
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Vpliv različnih tipov citokininov in njihova koncentracija na in vitro 
rast poganjkov marelice (Prunus armeniaca L.)
IZVLEČEK
Cilj te raziskave je bil oceniti vpliv različnih tipov citokininov [(6-benzilaminopurin (BAP), tidiazuron (TDZ) in meta-
topolin (mT)] in njihove koncentracije na in vitro rast poganjkov marelice. Dobro razvite poganjke smo prenesli na različna 
razmnoževalna gojišča, ki so se razlikovala glede na dodan citokinin in njegovo koncentracijo. Poskus je trajal devet tednov. 
Vpliv dodanih rastlinskih rastnih regulatorjev  in njihove koncentracije na končno maso pridobljenih poganjkov smo preverili 
z analizo kovariance (ANCOVA). Rezultati so pokazali, da imajo vsi trije v raziskavi uporabljeni citokinini pozitiven učinek na 
rast poganjkov. Pri TDZ in BAP je bila statistično značilna razlika pri različnih koncentracijah, medtem ko pri mT koncentracija 
ni statistično značilno vplivala na rast poganjkov. Glede na naše rezultate, smo največji prirast mase dobili na gojišču z dodanim 
BAP v koncentraciji 1,0 mg/L.
Ključne besede: Prunus armeniaca, mikropropagacija, razmnoževanje in vitro, BAP, TDZ, mT
The effects of different cytokinin types and their concentration on in vitro growth of apricot (Prunus armeniaca L.) shoots