Cytokinins are involved in plant cell proliferation leading to plant growth and morphogenesis. The size, activity and maintenance of the shoot apical meristem (SAM) are defined by a balanced rate of mitotic cell divisions and functional cell differentiation that are controlled by cytokinins. In order to investigate the effect of exogenous cytokinin on SAM, morpho-anatomical changes in the shoot apices of tobacco treated with benzyladenine (BA) were compared to those of untreated control plants.Projekat ministarstva br. 17301

Budimir, Snežana

Janošević, Dušica

Stojičić, Dragana

Uzelac, Branka

Digital Repository of Archived Publications - Institute for Biological Research Sinisa Stankovic (RADaR)

Arch. Biol. Sci., Belgrade, 64 (2), 511-516, 2012 DOI:10.2298/ABS1202511U
511
EFFECT OF CYTOKININS ON SHOOT APICAL MERISTEM IN NICOTIANA TABACUM
BRANKA UZELAC1, DUŠICA JANOŠEVIĆ2, DRAGANA STOJIČIĆ3 and SNEŽANA BUDIMIR1
1 Institute for Biological Research “Siniša Stanković”, University of Belgrade, 11060 Belgrade, Serbia 
2 Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia 
3 Faculty of Science, University of Niš, 18000 Niš, Serbia
Abstract - Cytokinins are involved in plant cell proliferation leading to plant growth and morphogenesis. The size, activity 
and maintenance of the shoot apical meristem (SAM) are defined by a balanced rate of mitotic cell divisions and functional 
cell differentiation that are controlled by cytokinins. In order to investigate the effect of exogenous cytokinin on SAM, 
morpho-anatomical changes in the shoot apices of tobacco treated with benzyladenine (BA) were compared to those of 
untreated control plants. 
Key words: Benzyladenine, histology, shoot apical meristem, tobacco
INTRODUCTION
Cytokinins are an important regulatory factor of 
plant morphogenesis that influences numerous de-
velopmental programs, including the formation and 
activity of the shoot apical meristem (SAM). SAMs 
are composed of embryonic-type cells, which pro-
liferate both to maintain the meristem and to gen-
erate tissues and organs such as leaves and flowers 
throughout the life of the plant. Within the SAM, 
these cells are organized into distinct multicellular 
domains, allowing the meristem to act as a coordi-
nated whole. 
Cells of the typical angiosperm meristem are 
organized into zones and layers, characterized by 
specific cytological features and gene expression pat-
terns. The zonation model supposes the existence of 
a central zone of stem cells (CZ), surrounded by a 
peripheral zone (PZ) where the organs are initiated. 
The CZ and PZ are subtended by a rib meristem 
(RM), which generates the internal parts of the stem. 
Superimposed on this zonation is an organization 
into tunica and corpus. The surface layer, or tunica, 
is formed and maintained by the strict anticlinal ori-
entation of division planes during cell proliferation. 
In dicots, the tunica is composed of two layers, called 
L1 and L2, which surround an inner group of cells 
called corpus, which has no clearly defined stratifica-
tion (Traas and Doonan, 2001).
Meristems provide the developmental flexibility 
that allows plants to modulate their development with 
respect to the prevailing conditions because they are 
able to alter their activity in response to both internal 
and external cues (Traas and Doonan, 2001).
The role of cytokinins in regulating shoot mer-
istem establishment and maintenance is supported 
by their ability to induce the formation of organized 
shoot meristems from disorganized growing callus 
tissue (Skoog and Miller, 1957). Regulation of the cell 
cycle and the number of cycles that cells undergo in 
the meristems and organ primordia, are the primary 
512 BRANKA UZELAC ET AL.
regulatory targets of cytokinins. Both locally synthe-
sized and imported cytokinins not only control the 
cell proliferation rate (i.e. stimulate division activity 
in the SAM), but are also involved in regulating the 
transition from undifferentiated stem cells to dif-
ferentiated primordia. This dual function of cytoki-
nins in the control of SAM proliferation is achieved 
via the KNOX family homeobox genes (Chuck et 
al., 1996; Rupp et al., 1999), suggesting a model in 
which cytokinins and KNOX genes positively regu-
late each other to maintain the indeterminate state 
of the SAM. 
In the present study of cytokinin action in plant 
morphogenesis, the effect of exogenously applied 
benzyladenine on tobacco shoot apical meristem 
structure and cytophysiological zonation was histo-
logically analyzed.
MATERIALS AND METHODS
Plant material and culture conditions
Seeds of Nicotiana tabacum cv. Wisconsin 38 were 
kindly provided by the Botanical Garden of Ni-
jmegen. The tobacco seedlings were germinated and 
grown for 6 weeks on half-strength MS (Murashige 
and Skoog, 1962) medium, supplemented with 1% 
(w/v) sucrose and solidified with 0.6% (w/v) agar. 
The pH of the medium was adjusted to 5.8 prior to 
autoclaving at 114°C for 25 min. The shoot apices 
with 1-2 leaves were then transferred to MS solid 
medium supplemented with 3% sucrose containing 
benzyladenine (BA) at 1, 3, 10, 30 and 100 μM, and 
cultured for an additional 3 weeks prior to examina-
tion. The same medium, but without BA, was used 
as the control. All cultures were maintained at 25 
± 2°C under a 16-h photoperiod. A photosynthetic 
photon flux density of 45 μmol m-2 s-1 was provided 
by white fluorescent tubes (Tesla, Pančevo, Serbia, 
65 W, 4500 K). 
Histological analysis
Twenty explants, each cultured on the control and 
BA-containing medium, were sampled. For light 
microscopy, the apical segments of the main axes 
devoid of expanded leaves were used. Samples were 
fixed in FAA (formalin/acetic acid/ethanol 10:5:85), 
dehydrated in a graded ethanol series and embedded 
in paraffin at 58˚C. Sections 5 µm thick were stained 
with hematoxylin and photographed using a Leitz 
DMRB photomicroscope (Leica Germany).
Statistical analysis
Five replicates, each consisting of two explants, were 
used per treatment, and the experiment was repeated 
twice. Of a total 20 samples for each treatment, 10 
were used for histological analysis. The dimensions 
of SAMs in the median longitudinal sections were 
measured using UTHSCSH Image Tool program ver-
sion 2.0 (University of Texas Health Science Center, 
San Antonio, Texas, USA). The data were subjected 
to one-way analysis of variance and significant differ-
ences between means from each treatment were de-
termined using Fisher’s Least Significant Difference 
(LSD) test at the 95.0% confidence level. 
RESULTS
Effect of cytokinins on shoot morphology
Morphological changes induced by increased cyto-
kinin concentration included reduced apical domi-
nance, fasciation and abnormal adventitious shoot 
morphology. Axillary bud meristems were present 
in both control and BA-treated shoot tips. However, 
in the control explants, the primary shoot elongated 
and the leaves grew vigorously, while none of the ax-
illary shoots developed (Fig. 1a). Benzyladenine in 
higher concentrations inhibited the growth of the 
main axis, simultaneously accelerating whole plant 
senescence (Fig. 1b-d).
Numerous adventitious shoots were induced 
from the callus tissue formed at the basis of the BA-
treated shoot cuttings (Fig. 1b-d). Callus growth as 
a function of treatment was not recorded, but visual 
observation indicated that more calli were produced 
at intermediate BA concentrations (3-30 μM). In 
explants cultured on 1 and 3 μM BA, the adventi-
CyTOKININ EFFECT ON TOBACCO SAM 513
tious shoots were tiny leafy structures, the majority 
of which were simple with radial symmetry, and the 
leaves were ovate with an entire leaf margin (Fig. 1b). 
The numerous adventitious shoots that developed on 
higher BA concentrations (10-100 μM) were short 
with shortened internodes (Fig. 1c, d). Differentiated 
leaves were small and orbicular with thick, flattened 
leaf blades. Ectopic meristems frequently emerged 
along the midribs and at the periphery of the leaves 
that were often hyperhydrated and swollen (Fig. 1c). 
At BA concentrations ranging from 10 to 100 μM, 
short, broad and frequently bifurcated cowl-shaped 
leaves were observed (Fig. 1d).  
Histology
The structure of shoot apices was examined under a 
light microscope after three weeks of cultivation on 
BA-supplemented media and compared to that of the 
control. 
Fig. 1. Morphology of Nicotiana tabacum shoot apices cultured with various levels of benzyladenine (BA) for 3 weeks. 
(a) Control plants with elongated stems and well-developed roots. 
(b) In explants cultured on 3 μM BA, numerous adventitious shoots emerged from the callus tissue formed in the basis of the main axis. 
Adventitious shoots were tiny leafy structures, with ovate leaves. 
(c) Bushy appearance of explants cultured at 10 μM BA, with well-developed axillary shoots and supernumerary adventitious shoots 
with shortened internodes. Note their intensely green leaves are frequently fasciated, with swollen midrib and petiole.
(d) At 100 μM BA leaves of the primary explant are noticeably more yellowish, with the swollen main axis and the callus tissue formed 
in its basis. Note the very short adventitious shoots that are often displaying a cowl-shaped apical leaf (arrowhead). 
514 BRANKA UZELAC ET AL.
In the longitudinal sections taken through the 
meristems of the control explants after three weeks 
of culture, two first leaf primordia were observed on 
the flanks of the shoot apical meristem (Fig. 2a). The 
meristems of the control plants consisted of a shal-
low dome of densely stained meristematic cells with 
large nuclei and lacking large vacuole. The meristem 
measured around 200 μm across the base, and had 
between 5 and 7 layers of avacuolate cells at its high-
est point (Table 1, Fig. 2a). The control plants dis-
played a normal organization and a regular arrange-
ment into layers (L1, L2 and L3), exhibiting the usual 
planes of cell division (Fig. 2a).
In the BA-treated explants, the shoot apical mer-
istem was a rather shallow dome with a typical organ-
ization into tunica and corpus. However, compared 
to control SAM, it had an increased diameter and its 
surface was flatter and enlarged (Table 1, Figs. 2b-d). 
The meristems of explants cultured on 3-30 μM BA 
were significantly broader across the base, measur-
ing maximum diameter (about 150% of the control) 
at 10 μM BA. With a further increase of of BA con-
centration, its effect on meristem diameter gradually 
decreased (Table 1). Compared to the control mer-
istems, the regular arrangement of densely stained 
meristematic cells into distinct layers appeared to be 
somewhat less organized at BA concentrations of 10 
µM and higher. Enlarged peripheral zones contained 
a few more vacuolated cells at the meristem flanks, in 
both L2 and L3, especially at 10 and 30 µM BA (Fig. 
2c). At 100 µM BA, not so many vacuolated cells are 
present in the meristem, while the corpus was some-
what modified, having four to five layers of flat cells 
resulting apparently from coordinated periclinal 
divisions so that the corpus occasionally displayed 
stratified appearance (Fig. 2d).
The number of cells in the uppermost L1 tunica 
layer was determined in all examined sections (Ta-
ble 1). A significant increase in cell number in the L1 
layer of the SAM was observed in explants cultured 
on media containing BA at 3, 10 and 30 μM after 3 
weeks, reaching a maximum at 10 μM BA where an 
increase of nearly 50% of the control was recorded 
(Table 1, Fig. 2c). 
However, regardless of the cytokinin treatment, 
a similar SAM structure and cytophysiological zona-
tion were observed for both control and BA-treated 
plants.
DISCUSSION
In the control plants, neither axillary nor adventitious 
buds developed, whereas the BA-treated explants had 
Fig. 2. Histology of Nicotiana tabacum vegetative shoot apical 
meristem (SAM) cultured with various levels of benzyladenine 
(BA) for 3 weeks.
(a) Median longitudinal section of control SAM displaying regu-
lar organization into tunica and corpus; Bar = 50 μm.
(b) Median longitudinal section of SAM in explant treated with 
3 μM BA; Bar = 100 μm.
(c) Median longitudinal section of SAM in explant treated with 
10 μM BA; Bar = 100 μm.
(d) Median longitudinal section of SAM in explant treated with 
100 μM BA, displaying layers of flat cells as a result of coordinat-
ed periclinal divisions in the corpus (arrowhead); Bar =100 μm.
CyTOKININ EFFECT ON TOBACCO SAM 515
a bushy appearance, confirming the ability of BA to 
overcome the apical dominance of shoots. 
An oversupply of cytokinin can account for 
stunting and/or fasciation of shoots, albinism and 
hyperhydricity, altered metabolism and anatomical 
and ultrastructural abnormalities. Ramage and Wil-
liams (2004) observed that elevated exogenous cyto-
kinin levels lead to the initiation of abnormal shoots 
and that these cytokinin-induced abnormalities in 
shoot morphology are associated with an increase 
in the expression of a knotted1-type homeobox gene 
(TobH1) within the meristematic tissue. Herath et 
al. (2005) showed that in BA-treated plants of Hibis-
cus cannabinus, the primary shoot apical meristem 
of the BA-treated shoot tip was reprogrammed to 
induce several meristematic loci, which was never 
observed in the primary shoot apical meristems of 
tobacco shoot tips, regardless of the concentration of 
the BA used.
A significant increase in SAM diameter was ob-
served in the tobacco explants cultured on medium 
containing 3-30 μM BA. Further increase in BA con-
centration no longer had a stimulatory effect on cell 
division in the SAMs. As Huyluoglu et al. (2008) 
have shown for barley root tips, BA at 1 and 10 μM 
increased the mitotic indices, whereas 100 μM BA 
was shown to decrease division frequency compared 
to lower BA concentrations, exerting an inhibitory 
effect.
The BA-treated SAMs of tobacco became en-
larged due to the enlargement of both the periph-
eral and central zones. The pattern of increase in 
cell number closely followed the pattern of diameter 
changes for each BA concentration tested, suggesting 
that the increase in SAM diameter was most likely 
due to an increase in the number of its constituent 
cells. Unlike the quantitative parameters, the typical 
regular structure and organization of the SAM were 
not altered.
Cytokinin is implicated in positively regulat-
ing meristem size and rapid leaf initiation. Trans-
genic tobacco plants expressing high levels of two 
cytokinin oxidase genes from Arabidopsis (CKX1 
and CKX2), hence exhibiting reduced levels of en-
dogenous cytokinin, developed stunted shoots with 
smaller apical meristems (Werner et al., 2001). In 
addition, Werner et al. (2003) showed that the over-
expression of the CKX1 gene in transgenic Arabi-
dopsis plants significantly reduced the endogenous 
cytokinin content and resulted in a strong reduction 
of the diameter and height of the meristem, which 
was attributable mainly to fewer meristematic cells, 
but also to the significantly decreased size of meris-
tematic cells. The reduced size of the SAM, together 
with the retarded leaf formation and reduced cell 
production in the leaves, provides evidence that cy-
tokinins are required as a positive regulator of cell 
division activity in the shoot. Interestingly, the di-
ameter of the shoot apical meristem in cytokinin-
Table 1. Effect of BA concentration on diameter and number of cells in L1 layer in shoot apical meristem of tobacco
Diameter Cell number in L1
BA (μM) (μm) % of control % of control
0 202.24 ± 10.50a 100 26.6 ± 1.9a 100
1 222.55 ± 12.49ab 110 31.4 ± 1.3ab 118
3 252.69 ± 1.85bc 125 32.8 ± 0.7b 123
10 310.85 ± 14.85d 154 39.2 ± 2.2c 147
30 295.99 ± 28.22cd 146 33.4 ± 3.1b 125
100 230.31 ± 13.87ab 114 27.8 ± 1.3ab 104
1) The values are means ± standard error, n=10. 
2)  Means within a column followed by different letters are significantly different from each other at 5% level by Fisher’s least significant 
difference (LSD) test.
516 BRANKA UZELAC ET AL.
overproducing ipt transgenics of Arabidopsis thal-
iana, displaying higher steady state mRNA levels of 
the SAM-specifying homeobox genes KNAT1 and 
STM, was not significantly different from that of the 
wild type (Rupp et al., 1999). 
Another link between cytokinins and meris-
tem size comes from a mutant in Arabidopsis AMP1 
gene. Nogué et al. (2000) have shown that the amp1 
mutant has a meristem that is both larger and taller 
than the wild type, with an increased number of 
cells in the L1 layer compared to the wild type, and 
that this is associated with a higher endogenous cy-
tokinin level in the meristem of the amp1 mutant. 
This is in accordance with the findings of our experi-
ment, where the observed changes in tobacco SAM 
dimensions upon exogenous addition of cytokinin 
confirm the ability of cytokinin to increase mitotic 
activity in the SAM. 
Acknowledgment - This research was supported by the Minis-
try of Education and Science of Serbia, Grant N° 173015.
REFERENCES
Chuck, G., Lincoln, C. and S. Hake (1996). KNAT1 induces lobed 
leaves with ectopic meristems when overexpressed in Ara-
bidopsis. Plant Cell 8, 1277-1289. 
Herath S.P., Suzuki, T. and K. Hattori (2005). Light and scanning 
electron microscopic analysis of benzyl adenine induced 
multiple shoot regeneration in kenaf (Hibiscus cannabinus 
L.). Breeding Science 55, 75-80.
Huyluoglu, Z., Unal, M. and N. Palavan-Unsal (2008). Cytologi-
cal evidences of the role of Meta-topolin and Benzylad-
enin in barley root tips. Adv. Mol. Biol. 1, 31-37.
Murashige, T. and F. Skoog (1962). A revised medium for rapid 
growth and bioassays with tobacco tissue cultures. Physiol. 
Plant. 15, 473-497. 
Nogué, F., Grandjean, O., Craig, S., Dennis, E. and A. Chaudhury 
(2000). Higher levels of cell proliferation rate and cyclin 
CycD3 expression in the Arabidopsis amp1 mutant. Plant 
Growth Regul. 32, 275-283.
Ramage, C.M. and R.R. Williams (2004). Cytokinin-induced ab-
normal shoot organogenesis is associated with elevated 
Knotted1-type homeobox gene expression in tobacco. 
Plant Cell Rep. 22, 919-924.
Rupp, H.M., Frank, M., Werner, T., Strnad, M. and T. Schmülling 
(1999). Increased steady state mRNA levels of the STM 
and KNAT1 homeobox genes in cytokinin overproducing 
Arabidopsis thaliana indicate a role for cytokinins in the 
shoot apical meristem. Plant J. 18, 557-563.
Skoog, F. and C.O. Miller (1957). Chemical regulation of growth 
and organ formation in plant tissues cultured in vitro. 
Symp. Soc. Exp. Biol. 11, 118-131.
Traas, J. and J.H. Doonan (2001). Cellular basis of shoot apical 
meristem development. Int. Rev. Cytol. 208, 161-205.
Werner, T., Motyka, V., Strnad, M. and T. Schmülling (2001). Reg-
ulation of plant growth by cytokinin. Proc. Natl. Acad. Sci. 
USA 98, 10487–10492.
Werner, T., Motyka, V., Laucou, V., Smets, R., Van Onckelen, H. 
and T. Schmülling (2003). Cytokinin-Deficient Transgenic 
Arabidopsis Plants Show Multiple Developmental Altera-
tions Indicating Opposite Functions of Cytokinins in the 
Regulation of Shoot and Root Meristem Activity. Plant 
Cell 15, 2532-2550. 


Effect of cytokinins on shoot apical meristem in Nicotiana tabacum

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Effect of cytokinins on shoot apical meristem in Nicotiana tabacum

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