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SI : TISSUE CULTURE
Origin, morphology, and anatomy of fasciationin plants cultured in vivo and in vitro
Ivan Iliev • Peter Kitin
Received: 28 May 2010 / Accepted: 20 October 2010 / Published online: 31 October 2010
� Springer Science+Business Media B.V. 2010
Abstract Fasciation (or cristation) is a variation in the
morphology of plants, characterized by the development of
various widened and flattened organs. According to origin,
fasciations are classified as physiological or genetic but
comparatively little is known on their epigenetic or genetic
nature at the molecular level. Physiological fasciations are
caused by natural environmental factors or artificial treat-
ments including exogenously applied growth regulators.
CLAVATA genes (CLV1, CLV2, and CLV3) have been
shown to be the main genetic factors associated with fas-
ciation. Despite the great variety of fasciation-induction
factors, fasciations have similar features of development
during the first few weeks, i.e., increased mitotic activity
and size of the apical meristem and an altered arrangement
of cells in the meristematic zones, often leading to an
increased number of organs and changes in the plastochron.
The enhanced activity of apical meristem and cambium
results in a significantly increased circumference of the
stem and enlarged proportions of pith and cortical paren-
chyma, associated with a delayed differentiation of the
vascular tissues. An elliptical or irregular shape of the cross
section of a fasciated organ corresponds to a similar shape
of the vascular cylinder. Later stages of the ontogenic
development of fasciations are species-specific, may
depend on the origin of fasciation, and in some cases may
lead to deviations from the normal structure of the epi-
dermis, shape of leaves, as well as altered development of
axillary buds. Studying the causes and patterns of devel-
opment of fasciations could provide a better understanding
of the growth processes in the vegetative apex. Further
anatomical and physiological research should focus on the
structure and activity of meristems of fasciated shoots, as
well as on their transcriptome analysis, in order to better
understand the pattern of fasciation development.
Keywords CLAVATA genes � Fasciation � Meristem �Plant growth regulators
Introduction
Fasciation (or cristation) is a variation that may occur in
the morphology of plant organs and typically involves
broadening of the shoot apical meristem, flattening of the
stem and changes in leaf arrangement. The term fasciation
comes from the Latin fascis meaning a bundle. The phe-
nomenon of fasciation is wide-spread in the plant kingdom.
During the nineteenth century there was a more pro-
nounced interest to study abnormal organ forms. The
subject was known as teratology (the science of wonders or
monsters) (reviewed in Heslop-Harrison 1952; Bos 1957;
Binggeli 1990). The scientific knowledge on fasciation was
reviewed by White (1948), Gorter (1965), Meyer (1966),
Meyerowitz et al. (1989), Binggeli (1990), and Clark et al.
(1993). Many authors, have interpreted fasciation as an
excrescence or fusion of organs due to deviations from
normal meristematic processes and to crowding of buds,
I. Iliev (&)
University of Forestry, 10 Kliment Ohridski blvd.,
1756 Sofia, Bulgaria
e-mail: [email protected]
P. Kitin
Laboratory for Wood Biology and Xylarium,
Royal Museum for Central Africa,
Leuvense steenweg 13, 3080 Tervuren, Belgium
Present Address:P. Kitin
Department of Wood Science and Engineering,
Oregon State University, Corvallis, OR 97331, USA
123
Plant Growth Regul (2011) 63:115–129
DOI 10.1007/s10725-010-9540-3
while others proposed that the ‘‘true fasciations’’ were a
transformation of a single growing point into a line (for a
review and discussion, see Clark et al. 1993).
Fasciations have been reported to occur naturally in
trees, shrubs, flowers and cacti in at least 107 plant families
and are very common in the Rosaceae, Ranunculaceae,
Liliaceae, Euphorbiaceae, Crassulaceae, Leguminosae,
Onagraceae, Compositae and Cactaceae (White 1948;
Binggeli 1990). Fasciations are especially prevalent in
species with indeterminate growth patterns of vegetative
organs and inflorescences (Binggeli 1990). They are less
common in woody plants than in herbaceous species but
occur in lianas, in many broad-leaved species and less
frequently in conifers. Among conifers, fasciation has been
reported in spruce and pines (Kienholz 1932).
Fasciated mutants are evaluated in plant breeding pro-
grams for their ornamental characteristics and are widely
cultivated by commercial growers (Krusmann 1995; Van
Gelderen and Van Hoey Smith 1997; Dirr 1998). Some
fasciations are perceived as real living sculptures and are
sought after by collectors. They are very attractive when
potted hence have ornamental values (Vallicelli 2010).
Plants with such abnormal growth are referred to as ‘cris-
tata’ following the name of the species Celosia cristata. In
Celosia cristata, the band-like shape of the inflorescence
determines the species name and made this species
attractive for floriculture. Moreover, the fasciation pheno-
type has been targeted in breeding programs for commer-
cially important species, such as some tomato cultivars,
where the significant increase in locule number and fruit
size was due to fasciation (Gorter 1965; Tanskley 2004). In
recent years, the interest on research in the fasciation
phenotype has been invigorated due to increased knowl-
edge of the plant genome and genes that control meristem
development as well as plant form (Fletcher 2002;
Tanskley 2004; Fambrini et al. 2006; Sinjushin and
Gostimsky 2006). Fasciated plant mutants have also been
used as experimental systems for analysis of the meristem
structure and function (Williams and Fletcher 2005).
In this review, we aim to summarize the classical
knowledge and recent research on the morphology and
development of fasciation in plants. Emphasis is given to
discussion on the development and anatomy of fasciation
phenotype induced in vitro since such plants represent
suitable systems for studying the regulatory mechanisms of
plant growth.
Altered growth and morphology of fasciated shoots
Fasciation is typically characterized by the development of
a flattened organ or plant part, most commonly a stem
(Fig. 1a, b) or an inflorescence (Fig. 1c). White (1948) and
Gorter (1965) described linear, circular and radiate types of
the fasciated shoots. In linear fasciation, the stem is flat-
tened and the shoot apical meristem (SAM) is enlarged and
flattened as a ribbon (Ecole 1970). As a result the shoots
have a bilateral symmetry instead of a central one.
CLAVATA1 mutant Arabidopsis plants have enlarged
apical vegetative and floral meristems, leading to fascia-
tion, altered phylotaxis, and extra floral organs and whorls
(Clark et al. 1993). Similarly, fasciation in pea is charac-
terized by abnormal enlargement of the stem apical meri-
stem leading to distortions in shoot structure (Sinjushin and
Fig. 1 Fasciated stems and inflorescences observed under natural
conditions: a fasciated stem of Spiraea 9 vanhouttei. Formation of
normal, new shoots on the fasciated stem, b fasciated stems of Salix
udensis ‘Sekka’ (S. sachalinensis F.Schmidt), c fasciated inflores-
cence of Trachicarpus fortunei
116 Plant Growth Regul (2011) 63:115–129
123
Gostimsky 2006, 2008). In the epicotyl of a fasciated pea
phenotype, the number of vascular bundles was higher than
in the wild type; as a result the SAM assumed a ring-like
shape. A detailed anatomical analysis showed that circular
type of fasciated shoot is formed as a result of the fusion
of several meristematic growth cones (Sinjushin and
Gostimsky 2006, 2008). Furthermore, the same authors
reported the presence of a significant number of underde-
veloped leaves preserved in the upper part of the shoot and
racemes with unopened flowers were located in their axils.
Several upper internodes usually remained shortened,
which resulted in a peculiar shape of the fasciated plants.
The leaf arrangement in any plant is species-specific and
its expression is not violated during fasciation. However,
the number of leaves in the node appears to depend not
only on the size of the zone of suppression of a primor-
dium, but also on the number of bundles of the leaf trace.
This is correlated with the further direction of primary leaf
primordium specialization (Guyomarc’h et al. 2004;
Szczesny et al. 2009). In secondary primordia, i.e. in those
formed as a result of cleavage of the primary primordium,
the zone of suppression is absent (Sinjushin and Gostimsky
2006). In buckwheat, fasciation causes a decrease of
growth and viability as a whole (Sakharov 1986). Also, it
was reported that fasciated plants exhibit heterosis in a
hybrid population F2 in pea (Loennig 1980, 1981) but had
low seed yield in sunflower (Jambhulkar 2002).
While there are numerous reports on the appearance of
fasciated plants in natural environmental conditions, rela-
tively few studies describe in vitro formation of fasciations.
Enlarged SAMs were observed in tissue culture in the
presence of high cytokinin concentrations (Brossard 1976;
Iliev and Tomita 2003; Iliev et al. 2003, 2011; Kitin et al.
2005). After cytokinin treatments, calli surrounded with
continuous meristematic bands that formed enlarged SAMs
were observed. These SAMs later divided dichotomously
into two normal SAMs (Chriqui 2008). The circular fas-
ciations are much rarer, characterized by a ring-shaped
growing point and produce a hollow shoot. Such appear-
ances were observed in some plants following treatments
with inhibitors of auxin transport (Ecole 1971), or in the
pin1 mutant which has altered auxin efflux (Vernoux et al.
2000). Inhibition of auxin transport can also result in fused
leaf organs (Ecole 1972) similar to what occurs in the cuc
mutants, which are altered in organ separation (Chriqui
2008). In the radiate fasciations, the SAM and the stem
have a stellate shape in transverse section (Chriqui 2008).
Fasciated shoots of Betula pendula induced in vitro
(Fig. 2a), as well as Prunus avium, and Fraxinus excelsior
formed flattened stems with densely arranged lanceolate
leaves that were dramatically increased in size (Iliev et al.
2003, 2011; Kitin et al. 2005; Mitras et al. 2009). The fas-
ciated shoots of the in vitro explants of these woody species
were not only distinct in shape from the normal shoots but
had also significantly larger dimensions (10–12 mm diam-
eters in flattened stems versus 2 mm in normal rounded
stems). Similarly, the fasciated stems of Helianthus annuus
plants were flattened and characterized by a shortened
plastochron and an altered phyllotaxis pattern (Fambrini
et al. 2006). In another in vitro experiment, the flattened
stems of Spartium junceum ranged from 4 to 9 mm in width
but the leaf arrangement and flower production were not
affected (Reboredo 1994). In some cases, the growth of
fasciated stem of genetically transformed hybrid Populus
tremula 9 Populus tremuloides, clone T89 resulted in the
stem being spiral and bifurcated (Nilsson et al. 1996).
An interesting observation was that a single fasciated
shoot could produce one to five new in vitro shoots without
visible signs of fasciation (Balotis and Papafotiou 2003;
Iliev et al. 2003, 2011; Kitin et al. 2005). The branching of
normal shoots that had emerged from fasciations has also
been noted to occur naturally in Spiraea 9 vanhouttei
(Fig. 1a), and Salix udensis ‘Sekka’ (Fig. 1c) (Iliev and
Kitin, unpublished results). Probably related to such phe-
nomenon is the observation that larger, fasciated floral
primordia give rise to more organs, and not bigger organs
(Clark et al. 1993). Also, Sinjushin and Gostimsky (2006)
noted that individual growth cones of the enlarged fasci-
ated meristem can function autonomously to a significant
degree and preserve their capacity of being autonomous
leading to defasciation. Most fasciations that appear in
vitro were found to be epigenetic (Varga et al. 1988;
Stimart and Harbage 1989; Jemmali et al. 1994). Fasciated
Cymbidium kanran rhizomes however were stable during
in vitro propagation (Fukai et al. 2000). Furthermore, it
was reported that both normal and fasciated rhizomes of
Cymbidium kanran exhibited geotropism, which resulted in
drooping branches of the normal rhizome and a crest shape
of the fasciated shoots (Fukai et al. 2000). Fasciated rhi-
zomes of Cymbidium kanran and Glycine max produced
scaly leaves associated with a linear apical meristem (Fukai
et al. 2000; Tang and Scorupska 1997).
Effect of various factors on the induction
of fasciations in vivo
The appearance of fasciated stems, shoots, and flower
stalks has been observed under natural conditions in Lilium
martagon, Celosia cristata and Euonimus japonicus
(Karagiozova and Meshineva 1977), Syringa yosikaea
(Vitkovskii 1959), Glycine max (Tang and Scorupska
1997), Arabidopsis (Medford et al. 1992), Spartium
junceum (Reboredo 1994; Reboredo and Silvares 2007).
Many other species are listed in White (1948). However,
the origin of fasciation is unknown in many of the naturally
Plant Growth Regul (2011) 63:115–129 117
123
growing or vegetatively propagated plants. There have
been various attempts to explain the origin of fasciation
in vivo and it could be classified as physiological or
genetic (White 1948; Gorter 1965; Karagiozova and
Meshineva 1977; Bairathi and Nathawat 1978; Driss-Ecole
1981; Behera and Patnaik 1982; Rance et al. 1982;
Albertsen et al. 1983; Gottschalk and Wolff 1983; LaMotte
et al. 1988; Werner 1988; Binggeli 1990; Nadjimov et al.
1999; Kitin et al. 2005).
Physiological fasciation
Physiological fasciation can be caused by a variety of
natural and artificial factors. Natural environmental factors
Fig. 2 Six-week-old silver
birch explants in vitro:
a fasciated shoot obtained after
application of 10 mg l-1 zeatin.
Subsequently a part of these
regenerants formed from 2 to 5
lateral shoots without visual
signs of fasciation; b, c cross-
sections that were cut at the
bases of a normal (b) and
fasciated (c) shoots and viewed
at the same magnification by
confocal laser scanning
microscopy (CLSM). The
normal stem is circular
isodiamteric in cross-section
with well-differentiated pith,
vascular cylinder, and cortex.
c The section shows the base
of a fasciated shoot that was
similar to the one shown in
a. The fasciated stems are
flattened with a delayed
development of the vascular
cylinder. The diameters of the
flattened stems varied from 4 to
12 mm. Staining with
Hematoxylin-Eosin and
observation by CLSM
(excitation 543 nm, emission
BP 515–565 and LP 590)
(adopted with permission, from
Iliev et al. 2003). Scalebars = 250 lm
118 Plant Growth Regul (2011) 63:115–129
123
include; attack by insect species (Peyritsch 1888; Molliard
1900; Knox 1908; Hus 1908; White 1948), mechanical
pressure and/or tension during growth in some species such
as asparagus (Binggeli 1990) and liana species (Rajput
2010), time and density of sowing; earlier sowing appears
to produce larger numbers of fasciated plants while higher
planting density decreases the percentage of fasciated
plants (Binggeli 1990), temperature fluctuation; low tem-
perature followed by high temperature caused fasciation
in Hyacinthus (Went 1944; Binggeli 1990), mineral
deficiency; zinc deficiency is known to cause fasciation
(Rance et al. 1982) and biotic stress caused by fungal and
nematode infections; the bacterium Rhodococcus fascians
was associated with fasciation (Thimann and Sachs 1966;
Crespi et al. 1992, 1994; Stange et al. 1996). Studies of
R. fascians showed that transfer of a gene from the bacte-
rium to the host cell induced fasciation. Once the bacterial
gene is transferred to a host plant, the propensity for fas-
ciation was transferred to other plants as cuttings or grafts
from the gene-infected plants (Crespi et al. 1992). It has
also been shown that stem fasciation of Lilium henryi
(Stumm-Tegethoff and Linskens 1985) and strawberries
(Steiner 1931) is associated with the presence of nematodes.
Fasciation is also caused by artificially applied factors.
Decapitation and defoliation; amputation of the main stem
of seedlings just above the cotyledons (White 1948); dur-
ing spring frost (Klers 1903–1906); crushing the young
stems of Viola tricolor (Blaringhem 1903) and cutting the
root tips of Vicia faba (Hus 1908), wounding of growing
points (Riddle 1903) as well as heavy pruning in deciduous
trees (Blaringhem 1907) induced fasciation. Enhanced
nutrition, including high rates of manuring, increases the
occurrence of fasciation (Binggeli 1990). Similarly, when
plants with indeterminate inflorescences were kept under
drought conditions prior to flowering and then subjected to
heavy watering and high nutrient levels, they produced
numerous fasciations (Hus 1908). Ionizing radiation and
chemical agents also caused fasciation in stems and inflo-
rescences (Johnson 1926; Irvine 1940; Behera and Patnaik
1981; Drjagina et al. 1981; Gottschalk and Wolff 1983;
Jambhulkar 2002; Soroka and Lyakh 2009; Abe et al.
2009). Application of some plant growth regulators causes
fasciation. For instance TIBA (2,3,5-triodobenzoic acid)
induces ring fasciation and other abnormalities such as
distortions and fusion of organs (Astie 1963). Similarly,
dry and germinated buckwheat seeds soaked in 0.1% IAA
solution produced altered phyllotaxy and fasciated bran-
ches (Yamasaki 1940). Fasciation may also be induced by
increasing or decreasing of the photoperiod (Astie 1963).
Furthermore, hard winters (Hus 1908), unfavorable
growth conditions abruptly succeeded by very favorable
conditions, variations in soil fraction (Jones 1935), heavy
manure or very rich soil (Hus 1908; De Vries 1909–1910;
White 1916; King 1918), grafting (Cobhold 1931) have
been suggested or regarded as causes of fasciation. It
appears that often fasciations are caused by conditions
which abruptly accelerate growth after the growth has been
slowed or stopped by some environmental or other factors.
However, in many of the above mentioned publications
there are not enough experimental evidences that confirm
the effect of a particular environmental factor or treatment
that caused fasciation. The information about the frequency
of appearance of fasciations is also very limited. Further-
more, many reports on fasciated plants contain no infor-
mation as to what may have induced the fasciation. For this
reason more research is clearly needed for determination of
the relative importance of the above-listed agents.
Genetic fasciation
The fasciated form of Pisum sativum L. (earlier called
P. umbellatum; Synonyms: mummy pea, crown pea, pois
turk, pois coronne, see Marx and Hagedorn 1962) was one
of the original seven Mendelian pairs of characters
(Mendel 1866). It is genetically determined in many spe-
cies (De Vries 1894; Knights 1993; Barotti et al. 1995;
Nadjimov et al. 1999; Karakaya et al. 2002). The gene
responsible for the development of fasciation was desig-
nated FASCIATA (FA) (White 1917). A hypothesis on the
monogenic nature of fasciation was proposed and the trait
was later characterized in a recent study as mono-factorial
with an incomplete penetrance and varying expressivity
(Sinjushin and Gostimsky 2008). In addition, the gene
FA2 was causing fasciation in the recessive stage and a
hypothesis of two polymeric genes was postulated
(Swiecicki 2001; Swiecicki and Gawlowska 2004).
In Mendel’s original experiment, all hybrids of F1
generation were non-fasciated, while in F2 the fasciation
and normal phenotypic classes were observed at 3:1 ratio.
However, since the gene that conditions fasciation exhibits
incomplete penetrance, the character may assume multiple
degrees of expression and the inheritance of fasciation
could also be non-Mendelian (Mertens and Burdick 1954;
Marx and Hagedorn 1962; Albertsen et al. 1983; Sinjushin
and Gostimsky 2008).
Lamprecht (1952) observed deviations from the expec-
ted ratio of genetic inheritance of fasciation and suggested
the existence of a gene FAS which could be polymeric to
the FA (for discussion, see Sinjushin and Gostimsky 2008).
A hypothesis for the existence of modifying genes influ-
encing expression of fasciation has been proposed to
explain the observed deviations from the predicted ratio
(Marx and Hagedorn 1962). Later, the studies of interac-
tion of mutants fa and fas revealed that genes FA and FAS
control consequential stages of apical meristem special-
ization in Pisum sativum (Sinjushin and Gostimsky 2008).
Plant Growth Regul (2011) 63:115–129 119
123
Srinivasan et al. (2008) studied the relationship between
spontaneous and induced mutant genes controlling stem
fasciation in chickpea (Cicer arietinum L.). Their hybrid-
ization experiments indicated the presence of a common
gene (designated fas1) for stem fasciation in the sponta-
neous chickpea mutants, whereas the gene for stem fasci-
ation in the induced mutant (designated fas2) was not
allelic to the common gene for stem fasciation.
Phytoplasmas belonging to the aster yellows group were
identified in Lilium sp. with flattened stems (Poncarova-
Vorackova et al. 1998; Bertaccini et al. 2005). Abe et al.
(2009) found that atbrca2 mutant plants, which are
hypersensitive to genotoxic stresses, displayed fasciation
and abnormal phyllotaxy phenotypes with low incidence,
and that the ratio of plants exhibiting these phenotypes was
significantly increased by c-irradiation. Laufs et al. (1998a)
and later Guyomarc’h et al. (2004) reported that MGO
mutation in Arabidopsis results in a delayed differentiation
of meristematic cells into lateral organ primordia which
leads to fasciation. A recent study indicated that MGO1
functions together with WUS in stem cell maintenance at
all stages of shoot and floral meristems and that MGO1
affects gene expression together with chromatin remodel-
ing pathways and may stabilize epigenetic states (Graf
et al. 2010).
It was suggested that fasciations might be the result of
the growing of a single apical meristem (Nestler 1894;
Shavrov 1961; Lebedeva 1963) and alternatively, that
fasciations are due to the adhesion of several sites of
growth (Zielinski 1945; Vitkovskii 1959; Karagiozova and
Meshineva 1977; Sinjushin and Gostimsky 2006) or hor-
monal imbalance within plants (Boke and Ross 1978;
Nilsson et al. 1996). The post-embryonic growth of plants
depends on the regulation of structure and size of the apical
meristems (Carles and Fletcher 2003). The shoot apical
meristem has three important roles: initiating tissues that
form organs, receiving and producing signals for regulation
of growth and development, and perpetuating itself as a
region of growth (Steeves and Sussex 1989; Kaplan and
Cooke 1997; Baurle and Laux 2003; Castellano and
Sablowski 2005; Bhalla and Singh 2006). The SAM of
dicotyledonous plants consists of three functionally distinct
zones: a peripheral zone (PZ) of rapidly dividing cells; a
central zone (CZ) of slowly dividing cells and the rib
meristem zone (RZ), which lies underneath the CZ. The
SAM’s CZ has the essential role of meristematic cell
maintenance and recovery, while the PZ produces new
lateral organs with predetermined spacing (phyllotaxis) and
regular timing of initiation (plastochron), and the RZ
initiates the pith and vascular tissues of the stem (reviewed
in Steeves and Sussex 1989). Superimposed on these zones
are three layers of cells that have separate symplasmic
domains: L1 (epidermal), L2 (sub-epidermal) comprising
tunica, and L3 (corpus) (Rinne and Van der Shoot 1998).
Anatomical studies on the temporal and spatial distribution
of cell divisions in SAMs could help the interpretation of
the functions of the genes controlling the apical meristem
(Laufs et al. 1998a; Szczesny et al. 2009). Based on cal-
culations of cell size and mitotic index, Laufs et al. (1998b)
differentiated two distinct zones in the apices of Arabid-
opsis clv3-1 and mgo mutants, a central and a peripheral
zone. The establishment and maintenance of the central
and peripheral zones and layers are essential for proper
SAM function. An increase in SAM size often results in
loss of typical arrangement of organ primordia, and ribbon-
like flattening (fasciation) (White 1948; Sharma and
Fletcher 2002; Traas and Vernoux 2002; Fambrini et al.
2006; Sinjushin and Gostimsky 2006). In another scenario,
if the indeterminate fate of the meristematic state of stem
cells is not properly maintained, the development of new
lateral primordia is suppressed (Laux et al. 1996; Long
et al. 1996). Laufs et al. (1998a) reported two recessive
mutations in Arabidopsis, MGOUN1 and MGOUN2 which
cause a reduction in the number of organ primordia, larger
meristems and fasciation of the inflorescence stem. These
authors described a form of fasciation which is radically
different from that described for CLAVATA. Instead of one
enlarged central zone of the meristem, mgo1 and 2 showed
an enlarged periphery and a continuous fragmentation of
the shoot apex into multiple meristems, which leads to the
formation of many extra branches. Furthermore, it was
demonstrated that MGO and CLV genes are involved in
different events, e.g. CLV3 gene is necessary for the tran-
sition of cells from the central to the peripheral zone,
whereas, mgo2 is impaired in the production of primordia,
hence the increased size of the mgo2 meristem could be
due to an accumulation of cells at the periphery (Laufs
et al. 1998b).
An extracellular signaling pathway in SAM mainte-
nance depends on the activities of CLAVATA genes (CLV1,
CLV2 or CLV3) which have been identified in Arabidopsis
thaliana (Leyser and Furner 1992; Williams et al. 1997;
Fletcher et al. 1999; Clark 2001), tomato (Mertens and
Burdick 1954), tobacco (Poething and Sussex 1985), and
maize (Taguchi-Shiobara et al. 2001). Plants with muta-
tions in any of the three loci show a progressive increase in
meristem size beginning in the embryo and continuing
throughout life, indicating a loss of cell division restriction
(Clark et al. 1993, 1997). This phenotype results in clavata
mutants. Their name is derived from the Latin word clav-
atus meaning club-like.
Shoot apical meristem enlargement can be caused by the
presence of either more or larger cells than normal. Anal-
ysis of clv or rolC mutant plants revealed their SAMs
contain a larger quantity of cells than wild-type SAMs
(Clark et al. 1993, 1995; Nilsson et al. 1996; Kayes and
120 Plant Growth Regul (2011) 63:115–129
123
Clark 1998). Because CLV genes do not affect cell size,
they must instead control either the rate of cell division in
the SAM central zone or the rate at which cells exit in the
central zone. The mitotic index of stem cells in the central
zone is actually slightly lower, not higher, in clv3 inflo-
rescence apices than in the wild type (Laufs et al. 1998b).
Thus it appears that CLV gene activity does not limit cell
division rates in the center of the SAM but controls stem
cell accumulation by regulating the rate at which cells in
the central zone make the transition from the meristem into
organ primordia (Fletcher 2002).
Recent experimental evidence provides new insight into
the spatial and temporal signaling pathways in the SAM. It
was found that CLV3 encodes a small secreted peptide
expressed in outer cell layers (Fletcher et al. 1999) which
binds to the leucine-rich repeat repressor kinase CLV1 and
its putative dimerization partner CLV2, which are expres-
sed in the inner cell layers (Clark et al. 1997; Stone et al.
1998; Lenhard and Laux 2003).
Another key element of the CLV signaling pathway is a
WUSCHEL (WUS) gene product found to be expressed
near the boundary of the CZ and RZ in shoot and floral
meristems (Meyer et al. 1998). The SAM and floral mer-
istems of wus mutants prematurely terminate activity after
the formation of a few organs, indicating that WUS is
necessary to promote stem cell activity and ensure con-
tinuous development (Laux et al. 1996). Conversely, CLV3
represses the expression of WUS and clv3 mutants are
prone to expansion of the SAM and development of fas-
ciation (Schoof et al. 2000).
The regular production of leaf primordia that is reflected
in stable phylotaxis and plastochron, is another primary
function of the SAM. The phyllotaxis and some times leaf
size are altered in clv, rolC and fas A. thaliana mutants as
well as in other mutants that show increased SAM size
(Nilsson et al. 1996; Itoh et al. 1998, 2000; Laufs et al.
1998a, b; Running et al. 1998; Bonneta et al. 2000; Giulini
et al. 2004; Green et al. 2005). Although the genes
involved in fasciation are also supposed to play roles in leaf
initiation, it is generally considered that the initiation pat-
tern of leaves is closely associated with the size and shape
of the SAM (Fleming 2005; Reinhardt et al. 2005). Leaves
are not generated randomly, but rather in a consistent
pattern over space and time, producing the regular phyl-
lotaxis of the plant. Plant hormones have been associated
with this process. In particular, auxin appears to be a
central player in leaf and flower formation and is a com-
ponent of phyllotactic patterning (Reinhardt et al. 2000,
2005; Vernoux et al. 2000; Jonsson et al. 2006; Smith et al.
2006).
The fasciated phenotype is expressed not only during
primary but also during secondary growth (Kitin et al.
2005). This is particularly evident in fasciated plants
growing in natural conditions. Although much progress has
been made towards understanding the genetic control of
secondary growth (Spicer and Groover 2010), virtually
nothing is known yet about the genetic mechanisms that
control cambial development in fasciated plants. The genes
commonly associated with fasciated SAM mutants appear
not to be expressed in normally developing cambium of
trees. Research on cambium development in fasciated
plants is important for understanding the genetic control of
cambium development and the opportunities it may pro-
vide for the manipulation of secondary growth of plants for
biomass production.
Effect of growth regulators on the induction
of fasciations in vitro
Fasciated plants propagated in vitro can be good models for
studying the causes and development of fasciation because
of the high level of control of the plant material and con-
ditions of growth they provide. However, to date the
available literature on in vitro cultivated fasciated plants is
still limited.
Several studies found that exogenously applied cytoki-
nins induce fasciation in Betula pendula (Iliev 1996; Iliev
et al. 2003, 2011), Kalanchoe blossfeldiana (Varga et al.
1988), Prunus avium (Kitin et al. 2005), Fraxinus excelsior
(Mitras et al. 2009), Mammillaria elongata (Papafotiou
et al. 2001), and Pisum sativum (Thimann and Sachs 1966),
Kniphofia leucocephala (McCartan and Van Staden 2003).
During the in vitro propagation of cristated Euphorbia
pugniformis most of the tip explants gave one cristate
shoot, while very few reversed to a normal shoot and the
number of cristate shoots increased with the BAP con-
centration (Balotis and Papafotiou 2003). Reduction of the
nitrogen nutrient concentration in the Murashige and
Skoog (1962) medium to one-fourth affected cristate form
stability. The in vitro behavior of cristated Euphorbia
pugniformis resembles that of cristated Mammilaria
elongata (Papafotiou et al. 2001) as far as explant type and
plant growth regulators effect on cristate shoot regenera-
tion are concerned. However, M. elongata cristated shoots
were quite stable at normal MS nitrogen concentration
(Papafotiou et al. 2001) as opposed to E. pugniformis.
It was shown that the type of the cytokinin is an
important factor for the induction of fasciated shoots. A
study of the reaction of different cultivars and varieties of
Betula pendula in vitro showed that appearance of fasci-
ated shoots was observed on media containing zeatin, only,
but their formation was not found on media containing BA
(Iliev et al. 2011). Our observations indicated that BA
when applied in the growing medium increases the multi-
plication rate of Prunus avium and also induces fasciation
Plant Growth Regul (2011) 63:115–129 121
123
(Kitin et al. 2005). Formation of fasciated shoots was
observed when BA was used but TDZ had no influence on
the formation of fasciated shoots in Fraxinus excelsior
(Mitras et al. 2009). Higher levels of zeatin and TDZ
resulted in higher frequency of shoot conversion in normal
rhizomes of Cymbidium kanran, but no shoot conversion
was observed in fasciated rhizomes (Fukai et al. 2000).
These cytokinins stimulated branching in normal rhizomes
but had no effect in fasciated rhizomes. The fasciated
rhizomes exposed to TDZ produced many scaly leaves
with a shorter plastochron, while the explants exposed to
zeatin produced small amounts of new fasciated rhizomes
with slow growth. It was suggested that the loss of shoot
conversion ability in the fasciated rhizomes of Cymbidium
kanran might be due to genetic changes or to the size of the
large linear apical meristems which requires different
phytohormonal conditions from normal rhizome require-
ments (Fukai et al. 2000). According to Ueda and Torikata
(1969) a short plastochron was the early signal of shoot
conversion in Cymbidium goeringii.
Cytokinin concentration is another key factor for the
induction of fasciated shoots in some tree species. For
example lower BA concentration (0.44 lM) was needed
for shoot formation in cristate form of M. elongata whereas
higher BA concentrations induced normal shoots The same
study showed that Murashige and Skoog (1962) medium
supplemented with 1.07 lM NAA or 0.54 lM NAA and
0.44 lM BA induced 100% of inflated cristate shoots
in shoot-tip explants. The medium supplemented with
0.89 lM BA also promoted 100% cristate formation, but
induced 50% hyperhydricity (Papafotiou et al. 2001).
In Betula pendula cultivars and varieties, the formation
of fasciated shoots was observed only when 5, 10 and
15 mg l-1 zeatin was used. There was no statistical dif-
ference in the percent of fasciated shoots formed on media
containing 5 and 10 mg l-1 zeatin but the fasciated shoots
decreased with an increase of the zeatin concentration
(Iliev et al. 2003). The appearance of fasciation in silver
birch in vitro might, in some cases, be due to p-fluoroph-
elanine (FPA) because no fasciation was observed in the
absence of FPA (Srivastava and Glock 1987). On media
free of plant growth regulators and media with the lowest
concentration, no fasciated shoots were found in Betula
pendula cultivars (0.2 mg l-1 zeatin), Prunus avium
(0.1 and 0.25 mg l-1 BAP), and Fraxinus excelsior (3.0
mg l-1 BA) (Kitin et al. 2005; Mitras et al. 2009; Iliev
et al. 2011). In contrast, hypocotyl, epicotyl, and cotyledon
explants from fasciated plants of Helianthus annuus were
able to sustain auxin-autonomous growth whereas wild-
type explants died on medium lacking plant growth regu-
lators (Fambrini et al. 2006). In this respect, it is worth
noting that in vitro cultured explants of fasciated mutants
of A. thaliana (Mordhorst et al. 1998) and Mammilaria
elongata (Papafotiou et al. 2001) showed a different
growth behavior than wild type.
The publications reporting the effect of growth regula-
tors on the frequency of fasciated plant development in
tissue culture are very limited. Appearance of fasciated
shoots in Fraxinus excelsior was less frequent (only few
fasciated shoots were observed) and was occasionally
observed when the medium contained 4.0 mg l-1 BA
(Mitras et al. 2009). The number of the fasciated shoots in
Prunus avium increased to the average of 0.5 ± 0.0 per
explant with an increase of BA to 1.0 mg l-1, but higher
concentrations had an inhibiting effect (Kitin et al. 2005).
It was shown that the percentage of fasciated shoots
formed in vitro was statistically different between different
cultivars and varieties of Betula pendula. Their spontane-
ous appearance ranged from 0.2% (var. Typica) to 2.0%
(‘Fastigiata’), but was not observed in ‘Dalecarlica’ (Iliev
et al. 2011). The rate of regenerated plants with fasciation
reached 15% from all genetically transformed plants of the
hybrid Populus tremula 9 P. tremuloides clone T89
(Nilsson et al. 1996). Therefore, it can be concluded that
the influence of the type and concentration of the plant
growth regulators is species specific and the frequency of
fasciation depended on genotype.
Fasciated tissues in hybrid aspen had a high level of free
cytokinins (Nilsson et al. 1996). It has also been shown that
plants of Helianthus annuus with fasciated stems (STF) had
higher endogenous free IAA levels; this however, did
not affect auxin sensitivity (Fambrini et al. 2006). The
observed phenotype and the higher levels of auxin detected
suggest that the STF gene is necessary for the proper
initiation of primordia and for the establishment of a
phyllotactic pattern through control of both shoot apical
meristem arrangement and hormonal homeostasis (Fambrini
et al. 2006). Furthermore, the increased hormonal levels
point to a possible hormonal control mechanism of the
development of fasciated SAM phenotypes. A possible
genetic mechanism which operates through a hormonal
imbalance restricted to the meristem and its immediate
vicinity in fasciated stems has been suggested (Boke and
Ross 1978). However, the simultaneous regeneration of
fasciated and normal shoots, as well as the branching of
normal shoots on fasciated shoots, remains difficult to
interpret and requires further research on the hormonal
levels in different parts of the plants (Kitin et al. 2005).
Experiments with season and position of explant exci-
sion showed no effect on the propagation of Mammillaria
elongata cristate form (Papafotiou et al. 2001). It was
reported that 100% of shoot-tip explants and 50–70% of
the explants below the shoot-tip of the branch responded
forming either one inflated cristate shoot, or one normal
shoot. Inflated cristate or normal shoots developed directly
on the explants, without callus intervention.
122 Plant Growth Regul (2011) 63:115–129
123
Anatomical differences between normal and fasciated
in vitro induced shoots
There are striking macroscopic differences in size and
shape between normal and fasciated in vitro induced
shoots. However, only few anatomical features of the
developing shoot clearly related to fasciation have been
identified so far (Table 1). The most obvious sign of fas-
ciation is a change of the shape of cross stem sections from
circular to elliptical or irregular (Figs. 2, 3). In normal
shoots of most plants, the vascular bundles and stem vas-
cular tissues are typically arranged in a concentric ring
around the iso-diametric pith. In contrast, fasciated stems
have bilateral symmetry or elliptical arrangement of vas-
cular bundles and the stem cross-sections are flattened or
irregular in shape (Driss-Ecole 1981; Nilsson et al. 1996;
Iliev et al. 2003; Kitin et al. 2005; Mitras et al. 2009).
Elliptical or irregular cross sections are a common feature
of the developing fasciated shoots in plants cultivated in
vivo as well (LaMotte et al. 1988; Tang and Knap 1998;
Sinjushin and Gostimsky 2006, 2008).
An increased size of SAM and structural changes of the
central and peripheral zones of the shoot apex are impor-
tant features of fasciated shoots. These features result in
altered development and abnormal morphology of the stem
and lateral organs. Fasciation phenotypes of in vivo prop-
agated plants are early manifested by meristem enlarge-
ment (Clark et al. 1993, Tang and Scorupska 1997; Laufs
et al. 1998a, b; Tang and Knap 1998; Kitin et al. 2005).
Through histological analysis, it was demonstrated that the
fasciated stems of Helianthus annuus were also associated
with an abnormal enlargement of nuclei in both CZ and PZ
of the apex, as well as a disorganized distribution of cells in
the L2 layer of the CZ (Fambrini et al. 2006). During the
later stages of fasciated stem development, a significant
increase in the proportion and total volume of the
Table 1 Anatomical differences between normal and fasciated stems induced in vitro
Normal Fasciation Growth regulator or
mutation
Species Reference
Normal SAM Enlarged SAM size and changed cell
number in the central zone of SAM
CLV; rolC; Corolla
Fasciation (CF);
Arabidopsis
BRCA2; zeatin;
BA; IAA
Silver birch, wild
cherry, common
ash, hybrid aspen,
sunflower
Nilsson et al. (1996), Laufs et al.
(1998a, b), Iliev et al. (2003), Kitin
et al. (2005), Fambrini et al. (2006),
Abe et al. (2009), Mitras et al.
(2009)
Normal SAM Enlargement of nuclei in central and
peripheral zones of apical meristem
Free IAA, STF
Gene
Sunflower Fambrini et al. (2006)
Normal SAM Apices of fasciated SAMs contain
more and smaller cells; increased
size of palisade cells in leaves
RolC Gene;
increased levels of
free cytokinins
Hybrid aspen Nilsson et al. (1996)
Normal SAM Enlargement of peripheral zones of
SAM; reduced mitotic index;
fragmentation of the shoot apex into
multiple meristems, which leads to
the formation of extra branches
MGOUN1,
MGOUN2, Mgo3
Arabidopsis Laufs et al. (1998a, b), Guyomarc’h
et al. (2004)
Circular and
isodiametric
stem, vascular
cylinder, pith
Elliptical or flattened stem, vascular
cylinder and pith; an enlarged
perimeter of vascular ring or an
increased number of vascular
bundles.
Zeatin, BA, IAA,
rolC Gene
Soybean, silver birch,
wild cherry,
common ash, hybrid
aspen, Cymbidiumkanran
Driss-Ecole (1981), Nilsson et al.
(1996), Fukai et al. (2000), Iliev
et al. (2003), Kitin et al. (2005),
Mitras et al. (2009)
Well developed
cortex and
vascular tissues
after 6 weeks
proliferation
Delayed differentiation of xylem and
cortical fibers after 6 weeks
proliferation.
Zeatin, BA, IAA,
Corolla Fasciation
(CF) mutant
Silver birch, wild
cherry, common
ash,
Iliev et al. (2003), Kitin et al. (2005),
Mitras et al. (2009)
Typically no
callus-like tissue
Occurrence of procambial cells at the
edge of pith or infrequent
occurrence of xylem cells after six
weeks proliferation; Occurrence of
callus-like tissue in the cortex
Zeatin, BA Silver birch, wild
cherry, common ash
Iliev et al. (2003), Kitin et al. (2005),
Mitras et al. (2009)
An increased volume of pith and bark,
and increased sizes of pith
parenchyma cells and cortical
parenchyma cells
Zeatin, BA, IAA,
rolC Gene
Silver birch, wild
cherry, common
ash, hybrid aspen
Nilsson et al. (1996), Iliev et al.
(2003), Kitin et al. (2005), Mitras
et al. (2009)
Plant Growth Regul (2011) 63:115–129 123
123
parenchymatic tissues as well as a several-fold increase in
the circumference relative to normal shoots of the same age
has been observed (Nilsson et al. 1996; Iliev et al. 2003;
Kitin et al. 2005). Typically, the expression of the fascia-
tion phenotype in vitro leads to an increase in the volume of
pith and cortex parenchyma. The xylem and phloem fibers
of fasciated stems are also less developed in comparison
to those of normal stems of the same age (Figs. 2, 3).
The increased proportions and volumes of pith and cortex in
fasciated stems were not solely the result of an increased
mitotic activity and larger cell numbers but also were due to
an enhanced cell enlargement as evidenced by the larger
dimensions of parenchyma cells (Nilsson et al. 1996; Kitin
et al. 2005). In contrast, 30–40% decrease in size of indi-
vidual pit cells of field-grown fasciated soybean was
reported by LaMotte et al. (1988). In plantlets of silver
birch, ash and wild cherry, a decrease in the size of
parenchymatic cells was associated with an increase in
mitotic activity and formation of callus-like tissue on the
peripheral layers of the pith (Iliev et al. 2003; Kitin et al.
2005; Mitras et al. 2009). As discussed earlier, clv mutant
SAMs are larger because they contain many more cells than
wild-type SAMs and the CLV genes appear to regulate the
rate at which cells in the central zone make the transition
from the meristematic to differentiating cell lines. While
the cell size of individual meristematic cells in clv mutants
or other in vitro propagated plants may not be affected, an
increase in the size of cells of the derivative tissues, in some
cases, is clearly related to fasciation.
Most of the research on in vitro-induced fasciation
phenotype deals with the vegetative or inflorescence apices
and little investigation has been done on the secondary
structure. Iliev et al. (2003) analyzed the histology of six-
week-old in vitro plantlets of silver birch. They found that
the concentric ring of xylem in normal stems consisted of
5–10 layers of well-developed cells, while the differentia-
tion of the vascular tissues in fasciated stems was delayed
and they formed a thin layer of 1–3 cells with little or no
signs of secondary wall development (Figs. 2, 3). More-
over, delayed xylem development and groups of undiffer-
entiated elongated cells (possibly cambium precursors)
occurred adjacent to the pith. Regions of callus-like cells
were also observed in the pith and cortex (Iliev et al. 2003;
Kitin et al. 2005; Mitras et al. 2009). Prosenchymatic
cambium-like cells were evident in longitudinal sections
and it was suggested that the increased cross-sectional area
of fasciated shoots in comparison to normal shoots was, at
least in part, the result of an increased cambial activity and
secondary growth. It has to be noted, however, that while
cambium, phloem and xylem were well-differentiated in
normal shoots, wide regions of undifferentiated parenchy-
matic cells, that might be derived from the enlarged SAM,
were predominant in the fasciated shoots (Figs. 2, 3).
Hence, it yet needs to be clarified whether the intensive
volume growth of few-week-old fasciated plantlets is of a
primarily origin (from SAM) or represents a secondary
growth by cambium.
In tissue cultures of three woody species (birch, ash, and
wild cherry), the well-differentiated tissues in older parts of
the plants were similar in both normal and fasciated shoots
and had the typical species-specific cellular and histologi-
cal structure of cortex and epidermis. Images of seedlings
of the fasciated soybean (CF) mutant also show well-
differentiated vascular tissues and bark comparable to
those in normal plants (Tang and Knap 1998). According to
Iliev et al. (2003), the secondary walls of xylem cells in
both normal and fasciated silver birch shoots had the same
types of pith and helical sculpturing. Moreover, the epi-
dermal cells, stomata and trichomes were well-differenti-
ated and identical in structure in both normal and fasciated
Fig. 3 Development of the vascular cylinder in normal and fasciated
six-week-old silver birch explants: a an enlarged view by CLSM of
the same normal explant as in Fig. 2b showing well-developed xylem
and cortex. b An enlarged view by polarized-light microscopy of
the fasciated stem in Fig. 2c showing an area of vascular tissue.
Birefringence occurs in the xylem cell walls in which helical
thickenings are seen (arrow). Pith is at the left side of the image and
cortex is at the right. Note the very early stage of development of the
vascular tissues in comparison to those in the normal stem in a. Scalebars = 50 lm. co cortex, p pith, x xylem
124 Plant Growth Regul (2011) 63:115–129
123
shoots. In general, the parenchyma cells in the pith and the
cortex in fasciated shoots had rounded shapes in cross
sections, similar to those in the normal shoots (Iliev et al.
2003; Kitin et al. 2005). However, as discussed earlier the
pith and cortical parenchyma cells in fasciated stems were
enlarged in comparison to those in normal stems.
Fukai et al. (2000) showed that epidermal cells, stomata
and rhizoids in fasciated in vitro cultured rhizomes of the
orchid Cymbidium kanran were elliptical in shape and were
arranged in a regular pattern towards the base of the rhi-
zome. On the other hand, round-shaped epidermal cells,
stomata and rhizoids were randomly orientated on the
normal rhizome. Normal rhizomes had blunt shoot tips
producing scaly leaves with a longer plastochron and nor-
mal rhizomes often branched in the middle. Axillary buds
swelled and developed into lateral rhizomes. In contrast,
axillary buds of fasciated rhizomes were inactive and
branching was rare (Fukai et al. 2000). According to the
same authors, the lack of lateral branching in fasciated
rhizomes was due to strong apical dominance produced by
the large apical meristem. A complete inhibition of axillary
buds in fasciated mutants of soybean (CF) was also reported
by Tang and Knap (1998). Furthermore, the formation of
flattened stems in the fasciated soybean plants coincided
with alterations in the phyllotaxy and plastochron.
Patterns of development of fasciations in vitro
The patterns of growth of fasciations are particularly
diverse in wild plants in natural conditions and may include
fusions of several points of growth or adjacent shoots that
grow in a parallel direction (Zielinski 1945; Vitkovskii
1959; Karagiozova and Meshineva 1977). In contrast, in
vitro originated fasciated shoots always had a single apical
meristem, although there might be differences in the
structure of the meristem layers as in the case of the
CLAVATA and MGO mutants (Laufs et al. 1998a, b). In
some instances, SAMs of the fasciation phenotype may
have several apical domes (Kitin et al. 2005; Fambrini
et al. 2006), which is somewhat similar to the ring-like type
of meristem formed by the fusion of multiple growth cones
as described by Sinjushin and Gostimsky (2006). In the
conditions of in vitro propagation, the fasciated shoots
undergo similar steps of development in the first few weeks
of growth irrespective of species or fasciation-induction
factor(s). The common pattern of fasciation development
of in vitro-induced shoots was always characterized with
an enlarged SAM with a larger number of cells, delayed
xylem differentiation, and an enlarged size of individual
parenchyma cells in the later stages of development of the
organs. The application of cytokinins, such as BA or zeatin,
is known to stimulate the meristematic activity which at a
certain concentration can cause fasciations (Iliev et al.
2003; Kitin et al. 2005). Moreover, anatomical observa-
tions of the SAMs of fasciated plants suggest that the
fasciation phenotype may be triggered by changes in the
arrangements of the cells in CZ or PZ of the apical meri-
stem (Tang and Knap 1998; Laufs et al. 1998a, b; Fambrini
et al. 2006). As discussed earlier, fasciation phenotype
development was found to be associated not only with
genetic modifications but also with shifted levels of phy-
tohormones at the enlarged SAMs (Nilsson et al. 1996;
Tang and Knap 1998; Fambrini et al. 2006).
The fasciation feature of in vitro cultured plants is most
commonly expressed in the shoots (also rhizomes or
inflorescences). Because visual signs of fasciations are
rarely found in roots or leaves, comparative anatomical
studies of normal and fasciated in vitro cultured plants
have been executed mainly on stems and relatively little
studies have addressed the anatomical structure of leaves or
roots. Guyomarc’h et al. (2004) reported that mgo3 muta-
tion in Arabidopsis affected shoot, leaf and root morpho-
genesis. As discussed earlier, the development of fasciated
stems is usually associated with larger numbers but smaller
in size leaves (Nilsson et al. 1996; Fukai et al. 2000).
Histological analysis of of rolC Arabidopsis plants sur-
prisingly showed that the smaller in overall dimensions
leaves were considerably thicker and with larger palisade
parenchyma cells than those of the wild plants (Nilsson
et al. 1996). Cell size and numbers were not affected in
mgo3 mutants despite occasional irregular cell arrangement
(Guyomarc’h et al. 2004). As development of the mgo3
Arabidopsis plants proceeded, deregulation of the phyllo-
taxis and plastochron were more noticeable leading to
the development of a wide range of rosette phenotypes
(Guyomarc’h et al. 2004).
Fasciations may appear expressed in all stems or only in
part of the stems of an individual plant as is the case when
normal stems proliferate from fasciated stems (Iliev et al.
2003; Kitin et al. 2005). Whereas in many cases fasciation
is expressed only in a restricted portion of a single plant, it
has been shown that fasciations can be propagated from
tissue to tissue through grafting, bacterial infection or
phytoplasmas (Crespi et al. 1992; Poncarova-Vorackova
et al. 1998; Bertaccini et al. 2005). It is apparent that fur-
ther anatomical investigations as well as physiological and
genetic studies on the development and functionality of
stem, roots, and leaves are needed to improve our under-
standing of how fasciations develop.
Conclusions
Fasciated individuals arise through various environmental
causes, and they do not transmit this altered state to their
Plant Growth Regul (2011) 63:115–129 125
123
progeny. In some cases fasciation arises as a mutation, the
progeny of which inherit the changed phenotype.
Many intriguing features of the development of fascia-
tions still have no satisfactory explanations, such as for
example, the different frequencies of induced fasciated
shoots at similar growth conditions, as well as the
branching of normal shoots on fasciated shoots remain
difficult to interpret and require further studies at the hor-
monal and genotype levels.
Cytokinins, particularly zeatin and BA, can induce fas-
ciation in different species. Similar to naturally occurring
fasciations, the in vitro induction of fasciations is associ-
ated with an increased meristem size and enhanced growth
of plant stems. However, while in vivo fasciations can be
caused by fusion of several apical meristematic regions or
fusion of adjacent stems or flowers, the in vitro induced
fasciation of stems is a direct result from an abnormally
enlarged SAM and changes in the developmental control of
meristematic cells. Such single SAM, however, may have
multiple apical domes and, subsequently, points of growth.
Whereas it is still an open question why the increased
activity of SAM may result in fasciation, recent evidence
suggests that the development of fasciations may be pre-
ceded by an imbalanced distribution of cells in the CZ and
PZ of SAM associated with abnormal proliferation of
meristematic cells.
Fasciated plants were shown to have increased content
of free auxin at the apical meristem regions and a possible
hormonal misbalance in comparison to normal plants that
may result in changes of the epidermal structure, leaves,
plastochron, and an inhibition of axillary buds.
Many studies have demonstrated differences in the
expression strength of different plant growth regulators and
genes related to fasciation. However, it is not yet clear
whether different growth regulators have any specificity
regarding the pattern of fasciation development. The ana-
tomical analysis to date shows that any of the fasciation-
induction agents initially causes an increased meristem size
that later leads to similar phenotypic effects of shoot
fasciation.
Despite dramatically increased biomass (at least in the
early stages of development) and shifted stem differentia-
tion and morphology, there is no clear indication for
pathogenic abnormality at the tissue and cellular levels in
fasciated shoots. The anatomical structure of phloem and
xylem cells appears similar in normal and fasciated stems.
This distinguishes the fasciations from pathogen-caused
occurrences. To our knowledge, however, no studies to
date have addressed the physiological performance of
vascular tissues in fasciated versus normal plants.
The most apparent morphological difference between
normal and fasciated stems is in the shape of the vascular
cylinder and the pattern of development of vascular tissues.
The differentiation of xylem of fasciated stems is delayed
compared to that of normal stems and this delay of dif-
ferentiation is associated with the occurrence of mitoti-
cally active cambium or callus-like regions in the stem.
Limited evidence suggests that cambial growth may have
contributed to an increased biomass of six-week-old in
vitro fasciated regenerants. Further research should focus
on the structure and activity of meristems of in vitro
induced shoots, as well as on their transcriptome analysis,
in order to better understand the pattern of fasciation
development.
Acknowledgments We thank Prof. Aposolos Scaltsoyiannes
(Aristotle University, Thessaloniki, Greece), Prof. Athanassios Rubos
and Mr. Christos Nellas (Technological Education Institute, Thessa-
loniki, Greece) for providing laboratory conditions and technical
assistance for the accomplishment of this investigation. We also thank
Dr. Geert-Jan De Klerk (Wageningen UR Plant Breeding, The
Netherlands) and Prof. Johannes Van Staden (University of KwaZulu-
Natal, South Africa), for reading the manuscript and providing many
helpful comments.
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