Plant Cell, Tissue and Organ Culture (2005) 82: 141150 Springer 2005

DOI 10.1007/s11240-004-8153-9

A comparative histological study between normal and fasciated shoots

of Prunus avium generated in vitro

Peter Kitinl, Ivan Iliev2,*, Apostolos Scaltsoyiannes3, Christos Nellas4, Athanassios

Rubos4 & Ryo Funada5,6
1
Institute of Wood Technology, Akita Prefectural University, Noshiro 016-0876, Japan; 2University of
Forestry, 10 Kliment Ohridski Boulevard, Soa 1756, Bulgaria; 3Department of Forestry and Natural
Environment, Aristotle University, 54 006 Thessaloniki, Greece; 4Tehnological Education Institute, 54 100
Thessaloniki, Greece; 5Laboratory of Wood Biology, Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan; 6Faculty of Agriculture, Tokyo University of Agriculture and Technology,
Fuchu-Tokyo 183-8509, Japan (*request for oprints; Fax: +359-2-8622830; E-mail: iviliev@ltu.bg)

Received 3 November 2003; accepted in revised form 17 December 2004

Key words: confocal microscopy, cytokinin, fasciation, histology of stem, Prunus avium, tissue culture, wild
cherry

Abstract

A combination of Murashige and Skoogs medium and N6-benzyladenine (BA) at various concentrations
(0, 0.1, 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 mg l)1) was supplied to shoot tips from root cuttings of a 50-year-
old wild-cherry tree (Prunus avium). The concentration of BA in the growing medium was a determining
factor with respect to the number of proliferated shoots per explant in vitro. Normal and fasciated shoots
were generated when BA was present at 0.5, 0.75, 1.0 and 1.25 mg l)1 in the medium and the mean numbers
of normal shoots per explant were 3.63, 5.37, 8.93 and 7.30 respectively, and those of the fasciated shoots
per explant were 0.03, 0.1, 0.47 and 0.4 respectively. Anatomical analysis by confocal microscopy of
sections of paran-embedded specimens revealed that the cell structure and organization of the cortex and
vascular cylinder in the fasciated shoots was similar to that in normal shoots. However, the cross-sectional
area of stem of the fasciations was apparently greater than that of the normal shoots. In particular, the
volume of vascular tissues, of pith and of some individual parenchyma cells in the cortex and pith was
apparently greater in fasciated shoots than in normal shoots. Increases in cytokinesis and morphogenetic
activity, such as the development of callus-like regions and the formation of adventitious shoots, were
observed in the cortex and pith throughout the fasciations. The fasciated shoots had numerous buds and
initiating new shoots at their apices while normal shoots had a single dominant axial bud.

Abbreviations: IBA indole-3-butyric acid; GA3 gibberellic acid; BA N6-benzyladenine

Introduction et al., 2001). Inheritable fasciation has been

Many tree species, shrubs, cacti and herbs develop
fasciated stems (White, 1948; Mertens and Bur-
dick, 1954; Karagiozova and Meshineva, 1977;
Boke and Ross, 1978, Srivastava and Glock, 1987;
Werner, 1988, Varga et al., 1988; Papafotiou
observed in a number of experimental systems and
the CLAVATA1 (Leyser and Furner, 1992; Clark
et al., 1993) and CLAVATA3 (Fletcher et al.,
1999) genes have been shown to be associated with
fasciation of stems of Arabidopsis. Studies of the
bacterium Rhodococcus fascians showed that
142

transfer of a gene from the bacterium to host cells
induced fasciation (Crespi et al., 1992). Once the
bacterial gene had been transferred to a host plant,
the propensity for fasciation was transferred to
other plants as cuttings or via grafts from gene-
infected plants.
There have been various attempts to explain
the origin of fasciation in vivo and in vitro but this
phenomenon is still not clearly understood. It has
been suggested that fasciations might be the result
of the growth of a single apical meristem (Nestler,
1894; Lebedeva, 1963) and, alternatively, that
fasciations are due to the adhesion of several sites
of growth (Zielinski, 1945; Vitkovskii, 1959; Ka-
ragiozova and Meshineva, 1977) or to a hormonal
imbalance within plants (Boke and Ross, 1978;
Nilsson et al., 1996).
The fasciation of shoots of Prunus avium L. in
vitro has not previously been reported, although
many experiments related to the propagation of
this species in vitro have been performed (Feutch
and Dausend, 1976; De March et al., 1993;
Hammatt and Grant, 1993, 1997, 1999). It has
been reported that exogenously applied cytokinins
induce the fasciation in some species (Iliev, 1996;
Varga et al., 1988; Papafotiou et al., 2001). In a
preliminary experiment, we have obtained normal
and fasciated shoots of in vitro propagated Prunus
avium when a cytokinin, N6-benzyladenine (BA),
was applied to the growing medium. Therefore,
this study was designed to investigate the eect of
exogenous BA in the shoot induction in vitro of
Prunus avium, and to compare the anatomical
structure of fasciated shoots with that of normal
shoots.
Materials and methods
(0, 0.1, 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 mg l)1) for
in vitro induction of adventitious shoots. The pH
of all media was adjusted to 5.6 before autoclaving
at 1.2 atm and temperature 115120 C for
20 min. The shoot cultures were grown in a culti-
vation chamber at 25 1 C with 16 h of cool
white uorescent light at photon ux density
80 lmol s)1 m)2, daily.
Ten apical shoots were used for the experiments
with each medium and the results were averaged
for three replications of each experiment. One-
Way ANOVA and a post hoc LSD test, SPSS 12.0
for Windows, were used for comparison of means.
Anatomical study
Fasciated and normal adventitious shoots were
harvested and xed in FAA solution that con-
tained 96% ethyl alcohol, glacial acetic acid, 40%
formalin and distilled water (10:1:2:7, v/v). Then
the shoots were transferred to an automatic tissue
processor (Leica Histokinette 2000; Leica Micro-
systems, Wetzlar, Germany), in which the shoots
were processed consecutively as follows: dehydra-
tion in an ethanol series, acetone, xylol and
embedding in paran wax. The paran wax
blocks were sectioned with a rotary microtome
and sections stained with Harris hematoxylin and
eosin followed by dehydration and mounting in
Table 1. Eects of the concentration of N6-benzyladenin (BA)
on the formation in vitro of normal and fasciated adventitious
shoots on apical shoot explants of Prunus avium
Concentrations Mean number of Mean number of
of BA (mg l)1) normal fascinated
adventitious shoots adventitious
per explant shoots per explant

M SE M SE
Plant material and induction of adventitious shoots
0.1 0a 0a
Root cuttings from a 50-year-old specimen of 0.25 0a 0a
Prunus avium L. were planted in a mixture of peat 0.5 3.63 0.18 b 0.03 0.03a
and perlite (1:1, v/v) for ex vitro induction of 0.75 5.37 0.15 c 0.10 0.05a
adventitious shoots and cultivated in a green- 1.0 8.93 0.15 d 0.47 0.03b
house. The resultant shoots were collected 40 days 1.25 7.30 0.26 e 0.40 0.06b
later, when they had reached 1525 mm in length. 1.5 0a 0a
The shoots were cultivated on Murashige and *Values represent the mean of 3 independent experiments each
Skoog (1962) medium that contained 0.5 mg l)1 used 10 shoots.
indole-3-butyric acid (IBA), 0.1 mg l)1 gibberellic Means in the columns followed by the same letter are not
acid (GA3) and BA at various concentrations signicantly dierent at p = 0.05 with LSD test.
 143

Figure 1. Longitudinal section through the apex of a normally developing shoot of Prunus avium cultured in vitro. (a) General view
under the confocal microscope. An arrow points to the axial bud. Basal parts of petioles (pet) of developing leaves are visible on both
sides of the axial bud (bar 100 lm). (b) Confocal image of part of the section in (a) showing part of the cortex with the epidermis
(ep), cortical parenchyma (cp) and developing vascular tissues (vt). The large arrow points to xylem cells and arrowheads point to
phloem cells. The small arrow points to part of a trichome. (bar 25 lm).
144

Entelan (Merck, Germany). Radial and cross sec- ser scanning microscope (LSM-310; Carl Zeiss,
tions were examined with either a light microscope Oberkochen, Germany). For confocal microscopy,
(BH-2; Olympus, Tokyo, Japan) or a confocal la- transmitted visible light or excitation by incident

Figure 2. Transverse section through the apical region of a normally developing shoot. (a) Single optical section, obtained by confocal
microscopy, showing the general cross-sectional view of the apex. The large arrow points to a vascular bundle that contains xylem,
phloem and cambium; small arrows point to developing vascular tissues that include phloem and cambial cells (bar 250 lm). (b)
Magnied view of the region of the vascular bundle indicated by the large arrow in (a) showing cortical parenchyma (cp), epidermis
(ep) and developing vascular tissues. The large arrow points to xylem cells, the small arrows point to cambial cells, and arrowheads
point to phloem elements. (c) Transmitted light image of the same section as in (b) with arrows as in (b). (d) The same section as in (b)
and (c), viewed under polarized light, with arrows as in (b). Strong birefringence of secondary walls of xylem cells (large arrow) and of
trichomes in the epidermal layer (ep) of the cortex is evident. Weak birefringence of the walls of the cortical parenchyma cells can also
be seen but no birefringence of the walls of cambial and phloem cells (see corresponding arrows and arrowheads in b and c),
(bar 50 lm).
 145

light from an argon-ion laser (wavelength, phenomenon has been observed during the in vitro
488 nm) with a long-pass lter (LP; 590 nm) were propagation of Betula pendula and ex vitro in
used as described earlier (Kitin et al., 2000, 2002). Spiraea and Salix craca (Iliev and Kitin, personal
communication). Further research would be re-
quired to understand this interesting aspect of
Results and discussion development of normal shoots from fasciated
shoots.
The apical shoots died on cytokinin-free MS A longitudinal section of a normally developing
medium within one week of subculture. They explant is shown in Figure 1. One axial bud and
survived for two weeks on MS medium plus developing leaves can be seen at the shoot apex
0.1 mg l)1 BA but did not form adventitious (Figure 1a). Vascular tissues and well-dierenti-
shoots (Table 1). Larger numbers of adventitious ated cortical tissues were visible below the axial
shoots were formed on explants in the presence of
1.0 mg l)1 BA (Table 1), conrming previous re-
ports that cytokinins in the medium are important
for the formation of adventitious shoots and that
the concentrations of cytokinins determine the
frequency of induction of shoots (Cornu and
Chaix, 1981; Cornu et al. 1981; Scaltsoyiannes
et al., 1998; Mandegaran et al., 1999). Further
increase of the concentration of BA up to
1.5 mg l)1 led to abundant formation of callus but
no adventitious shoots were formed.
There have been many reports on the propa-
gation of P. avium L. in vitro on MS medium plus
BA but there have been no reports on the forma-
tion of fasciated shoots of P. avium L. (Feutch and
Dausend, 1976; Cornu and Chaix, 1981; Cornu
et al., 1981; Hammatt and Grant, 1993; Hammatt
et al., 1998; Mandegaran and Roberts, 1998).
However, we observed the simultaneous forma-
tions of normal and fasciated shoots in the grow-
ing medium (Table 1). Nilsson et al. (1996)
reported that fasciated tissues in hybrid aspen
contained elevated levels of free cytokinins. How-
ever, we cannot say whether the fasciation in our
experiment was due to the direct eect of BA
concentrations in the growing medium or because
the rate of multiplication was higher, the fre-
quency of fasciated shoots formation has also
increased.
The fasciated shoots were characterized by
lateral growth and the formation of attened
stems, at the top of which there were densely ar-
Figure 3. Anatomical features of the middle part of the stem of
ranged lanceolate leaves. The larger diameter of
a normally developing shoot. (a) Single optical section, ob-
the attened stems ranged from 5 to 12 mm and tained by confocal microscopy, showing a general cross-sec-
the smaller diameter was approximately 2 mm. tional view of the middle portion of the stem. Vascular bundles
From two to ve lateral shoots, without visible are evident on the entire circumference of the pith. Arrows
point to xylem cells (bar 250 lm). (b) Magnied view of the
signs of fasciation, formed on some fasciated re- region at the arrows in (a) showing part of the cortex, the
generants. We also noted the branching of normal developing vascular cylinder with phloem cells (arrowheads)
shoots that had emerged from fasciations. Similar and xylem cells (arrows), and pith. (bar 25 lm).
146

bud (Figure 1b). Intitially, the development of the
vascular cylinder was asymmetric around the pith
of the stem (Figure 2). Only one or two well-de-
ned vascular bundles containing xylem and
phloem could be seen at the early stage of shoot
development (large arrow on Figure 2a). The rest
of the vascular tissue consisted of cambial cells and
adjacent small groups of phloem cells (small ar-
rows and arrowheads in Figure 2a2d). The cortex
was composed of cortical parenchyma cells and an
epidermal layer, which consisted of small isodia-
metric epidermal cells and trichomes. The cortical
parenchyma cells were approximately cylindrical,
appearing rounded on transverse sections and
rectangular in longitudinal sections (Figures 1 and
2).
Initially, the purpose of Harris hematoxylin
and eosin staining was to facilitate observation
of the cytological events (cell divisions and
protoplasmic changes) in the histological sections
under the conventional light microscope. In
addition, this staining protocol proved to be
useful for observation with the confocal
microscope. The cell walls of all types of cell
uoresced under the argon-ion laser after stain-
ing and, thus, cell structure was clearly visible
under the confocal microscope. The cell walls of
the phloem cells emitted brighter uorescence

Figure 4. Anatomical features of the bottom part of the stem of a normally developing shoot. (a) Single optical section, obtained by
confocal microscopy, showing a general cross-sectional view of the bottom portion of the stem. The well-developed xylem cylinder
(arrows) is clearly visible. The section passed through the base of a lateral shoot (ls) (bar 100 lm). (b) Magnied view of the region at
the arrows in (a) showing cortical parenchyma (cp), xylem (large arrows), cambium (small arrows) and pith (upper right of the image)
(bar 25 lm). (c) Longitudinal section showing xylem and adjacent tissues. Cortical parenchyma cells (cp), phloem cells (arrow-
heads), xylem (arrows) and pith parenchyma (right side of the image) are visible. (d) The same section as in (c) under polarized light,
with arrows as in (c). Strong birefringence is evident in the cell walls of the xylem cells, while weaker birefringence is evident in the
peripheral layers of the pith parenchyma cells (bar 50 lm).
 147

than other cell walls (arrowheads in Figure 2b).
The same phenomenon has been observed in
histological preparations of B. pendula after
Harris hematoxylin and eosin staining (Iliev
et al., 2003) but has not been observed after
safranin staining (Kitin et al., 2003). The
brighter uorescence of the phloem cell walls
might have been related to their chemical com-
position and it was not related to advanced
development of secondary walls since such cells
did not exhibit birefringence under polarized
light (Figure 2b2d).
The vascular tissue in normal shoots was well
developed around the pith in the middle portion of
the stem (Figure 3a), consisting of vascular bun-
dles that contained xylem (arrows in Figure 3b)
and phloem (arrowheads on Figure 3b), with
cambial cells between them. In the middle portion
of the stem there was well-dierentiated epidermis
with cuticle, as well as cortical parenchyma cells

Figure 5. Confocal microscopy images of a longitudinal, slightly oblique section through the apical region of a fasciated shoot of
Prunus avium generated in vitro. (a) General view; the upper side of the shoot corresponds to the left of the image. Numerous
longitudinally cut leaf blades are evident in the eld of view above the shoot. Arrows point to new shoots that are developing from
adventitious buds (bar 250 lm). (b) Enlarged view of a region of the section further below the image in (a). A new adventitious bud
(ab) is evident, as are developing vascular tissues, namely, xylem cells (arrow) and phloem cells (arrowheads) (bar 100 lm). (c) The
same section as in (b), viewed under polarized light, with arrows as in (b). The birefringence of xylem cells is indicated by an arrow. The
longitudinal section passes through the cortex and the outer side of vascular bundles. Therefore, only small portions of xylem cells are
visible (bar 100 lm).
148

Figure 6. Single optical section, obtained by confocal microscopy, showing the cross-section of the middle portion of a fasciated shoot
of Prunus avium that developed in vitro. The image in Figure 6 is at the same magnication as that in Figure 3a. Note the increased
cross-sectional area of the fasciated stem compared to that of the normal stem in Figure 3a. Some of the parenchyma cells of the cortex
and the pith appear abnormally large and many such cells have broken, possibly during the preparation of sections, as a consequence
of their thin walls. The vascular tissues (vt) form an almost complete cylinder around the pith (pth) (bar 250 lm).

with slightly thicker walls than those of the same
tissue in the upper portion of the stem (Figures 2b,
3b). A complete vascular cylinder with secondary
xylem that consisted of four to ve layers of cells
was evident at the base of the stem (Figure 4a, b).
The secondary walls of xylem cells were well
developed and exhibited strong birefringence un-
der polarized light. In addition, the development
of cell walls of the cells in the peripheral layers of
the pith was advanced, as indicated by their bire-
fringence (Figure 4c, d). At the base of the stem,
adventitious lateral shoots were formed (Fig-
ure 4a), were the tissues of the cortex appeared
poorly dierentiated (Figure 4a, b). The formation
of adventitious shoots and callus-like regions in
the cortex and pith suggests that meristematic
activity and morphogenesis were active at the base
of normally developing shoots.
The volume of fasciated shoots was consider-
ably larger than that of normal shoots (Figure 1a,
3, 6). The fasciated shoots had numerous buds and
new shoots in the apical region, instead of the
single axial bud found in normal shoots. The fas-
ciated shoots had no distinct apex and their apical
region was broad and at.
In the middle parts of fasciated stems
(Figure 6), the development of the vascular cylin-
der was more advanced than that of the normal
shoot shown in Figure 3. In normal stems, vas-
cular tissues were organized in bundles, while in
fasciated stems they formed almost complete cyl-
inders with several more layers of cambial cells
and xylem cells than were found in normal stems
(Figure 3, 6). Some of the parenchyma cells of the
cortex and pith were enlarged and there were re-
gions with a callus-like appearance in the fasciated
stems. In this regard, the stage of dierentiation of
the middle portion of the stems of fasciated shoots
resembled that of the bottom part of normal
shoots (Figure 4, 6). The diameters of fasciated
shoots were several times greater than those of the
normal shoots. Increases in rates of cell growth
and cell division were indicated not only by the
increased volumes of the stem tissues but also by
the formation of adventitious shoots and the
presence of callus-like regions throughout each
entire fasciated stem.
The appearance of fasciated plants has been
observed under natural conditions in the case
of Pinus silvestris (Velkov et al., 1987); Lilium
martagon, Celosia cristata and Euonimus japonica
149
(Karagiozova and Meshineva, 1977), and Syringa
yosikaea (Vitkovskii, 1959). Such plants have also
been induced by mutagens in Tagetes (Drjagina
et al., 1981). The appearance of fasciation in birch
in vitro might, in some cases, be due to p-uor-
ophenylalanine (FPA) because no fasciation was
observed in the absence of FPA (Srivastava and
Glock, 1987). Exogenously applied cytokinins in-
duced fasciation in Kalanchoe blossfeldiana (Varga
et al., 1988), B. pendula (Iliev, 1996) and Mam-
millaria elongata (Papafotiou et al., 2001). Our
observations indicated that the cytokinin BA when
applied in the growing medium increases the
multiplication rate of P. avium in vitro, and can
also induce fasciated shoots, but for understanding
the exact mechanism of its action we need further
research.
It has been suggested that a genetic mecha-
nism might operate through a hormonal imbal-
ance that is restricted to the meristem and its
immediate vicinity in fasciated stems (Boke and
Ross, 1978). However, the simultaneous regener-
ation of fasciated and normal shoots, as well as the
branching of normal shoots on fasciated shoots,
remains dicult to interpret.
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