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: ivilievltu@yahoo.com

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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