Plant Growth Regul (2011) 63:115129 DOI 10.

1007/s10725-010-9540-3

SI : TISSUE CULTURE

Origin, morphology, and anatomy of fasciation in 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 attened organs. According to origin, fasciations are classied 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 articial treatments including exogenously applied growth regulators. CLAVATA genes (CLV1, CLV2, and CLV3) have been shown to be the main genetic factors associated with fasciation. Despite the great variety of fasciation-induction factors, fasciations have similar features of development during the rst 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 signicantly increased circumference of the stem and enlarged proportions of pith and cortical parenchyma, 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
I. Iliev (&) University of Forestry, 10 Kliment Ohridski blvd., 1756 Soa, 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

of the vascular cylinder. Later stages of the ontogenic development of fasciations are species-specic, may depend on the origin of fasciation, and in some cases may lead to deviations from the normal structure of the epidermis, shape of leaves, as well as altered development of axillary buds. Studying the causes and patterns of development 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, attening of the stem and changes in leaf arrangement. The term fasciation comes from the Latin fascis meaning a bundle. The phenomenon of fasciation is wide-spread in the plant kingdom. During the nineteenth century there was a more pronounced 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 scientic 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,

123

116

Plant Growth Regul (2011) 63:115129

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, owers 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 inorescences (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 programs 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 cristata following the name of the species Celosia cristata. In Celosia cristata, the band-like shape of the inorescence determines the species name and made this species attractive for oriculture. Moreover, the fasciation phenotype has been targeted in breeding programs for commercially important species, such as some tomato cultivars, where the signicant 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 knowledge 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 attened organ or plant part, most commonly a stem (Fig. 1a, b) or an inorescence (Fig. 1c). White (1948) and Gorter (1965) described linear, circular and radiate types of the fasciated shoots. In linear fasciation, the stem is attened and the shoot apical meristem (SAM) is enlarged and attened 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 oral meristems, leading to fasciation, altered phylotaxis, and extra oral organs and whorls (Clark et al. 1993). Similarly, fasciation in pea is characterized by abnormal enlargement of the stem apical meristem leading to distortions in shoot structure (Sinjushin and

Fig. 1 Fasciated stems and inorescences 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 inorescence of Trachicarpus fortunei

123

Plant Growth Regul (2011) 63:115129

117

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 signicant number of underdeveloped leaves preserved in the upper part of the shoot and racemes with unopened owers 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-specic 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 primordium, but also on the number of bundles of the leaf trace. This is correlated with the further direction of primary leaf primordium specialization (Guyomarch 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 sunower (Jambhulkar 2002). While there are numerous reports on the appearance of fasciated plants in natural environmental conditions, relatively 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 fasciations are much rarer, characterized by a ring-shaped growing point and produce a hollow shoot. Such appearances were observed in some plants following treatments with inhibitors of auxin transport (Ecole 1971), or in the pin1 mutant which has altered auxin efux (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 attened 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 fasciated shoots of the in vitro explants of these woody species

were not only distinct in shape from the normal shoots but had also signicantly larger dimensions (1012 mm diameters in attened stems versus 2 mm in normal rounded stems). Similarly, the fasciated stems of Helianthus annuus plants were attened and characterized by a shortened plastochron and an altered phyllotaxis pattern (Fambrini et al. 2006). In another in vitro experiment, the attened stems of Spartium junceum ranged from 4 to 9 mm in width but the leaf arrangement and ower 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 ve 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 phenomenon is the observation that larger, fasciated oral 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 fasciated meristem can function autonomously to a signicant 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 rhizomes 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 ower 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

123

118 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 crosssections that were cut at the bases of a normal (b) and fasciated (c) shoots and viewed at the same magnication 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 attened with a delayed development of the vascular cylinder. The diameters of the attened stems varied from 4 to 12 mm. Staining with Hematoxylin-Eosin and observation by CLSM (excitation 543 nm, emission BP 515565 and LP 590) (adopted with permission, from Iliev et al. 2003). Scale bars = 250 lm

Plant Growth Regul (2011) 63:115129

growing or vegetatively propagated plants. There have been various attempts to explain the origin of fasciation in vivo and it could be classied 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 articial factors. Natural environmental factors

123

Plant Growth Regul (2011) 63:115129

119

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 uctuation; low temperature followed by high temperature caused fasciation in Hyacinthus (Went 1944; Binggeli 1990), mineral deciency; zinc deciency 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 bacterium to the host cell induced fasciation. Once the bacterial gene is transferred to a host plant, the propensity for fasciation 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 articially applied factors. Decapitation and defoliation; amputation of the main stem of seedlings just above the cotyledons (White 1948); during spring frost (Klers 19031906); 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 inorescences were kept under drought conditions prior to owering 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 inorescences (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 branches (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 19091910;

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 conrm 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. Furthermore, many reports on fasciated plants contain no information 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 species (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 designated 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 Mendels 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 expected 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 inuencing expression of fasciation has been proposed to explain the observed deviations from the predicted ratio (Marx and Hagedorn 1962). Later, the studies of interaction of mutants fa and fas revealed that genes FA and FAS control consequential stages of apical meristem specialization in Pisum sativum (Sinjushin and Gostimsky 2008).

123

120

Plant Growth Regul (2011) 63:115129

Srinivasan et al. (2008) studied the relationship between spontaneous and induced mutant genes controlling stem fasciation in chickpea (Cicer arietinum L.). Their hybridization experiments indicated the presence of a common gene (designated fas1) for stem fasciation in the spontaneous chickpea mutants, whereas the gene for stem fasciation in the induced mutant (designated fas2) was not allelic to the common gene for stem fasciation. Phytoplasmas belonging to the aster yellows group were identied in Lilium sp. with attened 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 signicantly increased by c-irradiation. Laufs et al. (1998a) and later Guyomarch 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 oral meristems and that MGO1 affects gene expression together with chromatin remodeling 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 hormonal 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 SAMs 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 Arabidopsis 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 ribbonlike attening (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 inorescence 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 transition 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 maintenance depends on the activities of CLAVATA genes (CLV1, CLV2 or CLV3) which have been identied 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 mutations 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 clavatus meaning club-like. Shoot apical meristem enlargement can be caused by the presence of either more or larger cells than normal. Analysis 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

123

Plant Growth Regul (2011) 63:115129

121

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 inorescence 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 expressed 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 oral meristems (Meyer et al. 1998). The SAM and oral meristems 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 continuous 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 fasciation (Schoof et al. 2000). The regular production of leaf primordia that is reected 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 pattern 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 phyllotaxis of the plant. Plant hormones have been associated with this process. In particular, auxin appears to be a central player in leaf and ower formation and is a component 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 provide 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 conditions 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 cytokinins 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), Kniphoa 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 concentration (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 regeneration 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 fasciated 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 multiplication rate of Prunus avium and also induces fasciation

123

122

Plant Growth Regul (2011) 63:115129

(Kitin et al. 2005). Formation of fasciated shoots was observed when BA was used but TDZ had no inuence 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 requirements (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 inated 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 difference 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-uorophelanine (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 wildtype explants died on medium lacking plant growth regulators (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 regulators 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 spontaneous 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 inuence of the type and concentration of the plant growth regulators is species specic 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 difcult 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 excision 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 5070% of the explants below the shoot-tip of the branch responded forming either one inated cristate shoot, or one normal shoot. Inated cristate or normal shoots developed directly on the explants, without callus intervention.

123

Plant Growth Regul (2011) 63:115129

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 identied so far (Table 1). The most obvious sign of fasciation 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 vascular tissues are typically arranged in a concentric ring around the iso-diametric pith. In contrast, fasciated stems have bilateral symmetry or elliptical arrangement of vascular bundles and the stem cross-sections are attened 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 important features of fasciated shoots. These features result in altered development and abnormal morphology of the stem and lateral organs. Fasciation phenotypes of in vivo propagated plants are early manifested by meristem enlargement (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 signicant increase in the proportion and total volume of the

Table 1 Anatomical differences between normal and fasciated stems induced in vitro Normal Normal SAM Fasciation Growth regulator or Species mutation Reference 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) Fambrini et al. (2006) Nilsson et al. (1996)

Enlarged SAM size and changed cell CLV; rolC; Corolla Silver birch, wild number in the central zone of SAM Fasciation (CF); cherry, common Arabidopsis ash, hybrid aspen, BRCA2; zeatin; sunower BA; IAA Enlargement of nuclei in central and Free IAA, STF peripheral zones of apical meristem Gene Apices of fasciated SAMs contain more and smaller cells; increased size of palisade cells in leaves Sunower

Normal SAM Normal SAM

RolC Gene; Hybrid aspen increased levels of free cytokinins Arabidopsis

Normal SAM

Enlargement of peripheral zones of MGOUN1, SAM; reduced mitotic index; MGOUN2, Mgo3 fragmentation of the shoot apex into multiple meristems, which leads to the formation of extra branches Elliptical or attened stem, vascular cylinder and pith; an enlarged perimeter of vascular ring or an increased number of vascular bundles. Zeatin, BA, IAA, rolC Gene

Laufs et al. (1998a, b), Guyomarch et al. (2004)

Circular and isodiametric stem, vascular cylinder, pith Well developed cortex and vascular tissues after 6 weeks proliferation

Soybean, silver birch, Driss-Ecole (1981), Nilsson et al. wild cherry, (1996), Fukai et al. (2000), Iliev common ash, hybrid et al. (2003), Kitin et al. (2005), Mitras et al. (2009) aspen, Cymbidium kanran Iliev et al. (2003), Kitin et al. (2005), Mitras et al. (2009)

Delayed differentiation of xylem and Zeatin, BA, IAA, Silver birch, wild cortical bers after 6 weeks Corolla Fasciation cherry, common proliferation. (CF) mutant ash,

Typically no Occurrence of procambial cells at the Zeatin, BA callus-like tissue edge of pith or infrequent occurrence of xylem cells after six weeks proliferation; Occurrence of callus-like tissue in the cortex An increased volume of pith and bark, Zeatin, BA, IAA, and increased sizes of pith rolC Gene parenchyma cells and cortical parenchyma cells

Silver birch, wild Iliev et al. (2003), Kitin et al. (2005), cherry, common ash Mitras et al. (2009)

Silver birch, wild cherry, common ash, hybrid aspen

Nilsson et al. (1996), Iliev et al. (2003), Kitin et al. (2005), Mitras et al. (2009)

123

124

Plant Growth Regul (2011) 63:115129

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. Scale bars = 50 lm. co cortex, p pith, x xylem

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 fasciation phenotype in vitro leads to an increase in the volume of pith and cortex parenchyma. The xylem and phloem bers 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, 3040% decrease in size of individual pit cells of eld-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 inorescence apices and little investigation has been done on the secondary structure. Iliev et al. (2003) analyzed the histology of sixweek-old in vitro plantlets of silver birch. They found that the concentric ring of xylem in normal stems consisted of

510 layers of well-developed cells, while the differentiation of the vascular tissues in fasciated stems was delayed and they formed a thin layer of 13 cells with little or no signs of secondary wall development (Figs. 2, 3). Moreover, delayed xylem development and groups of undifferentiated 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 parenchymatic cells, that might be derived from the enlarged SAM, were predominant in the fasciated shoots (Figs. 2, 3). Hence, it yet needs to be claried 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-specic cellular and histological structure of cortex and epidermis. Images of seedlings of the fasciated soybean (CF) mutant also show welldifferentiated 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 epidermal cells, stomata and trichomes were well-differentiated and identical in structure in both normal and fasciated

123

Plant Growth Regul (2011) 63:115129

125

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 rhizome. 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 normal 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 attened 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 rst 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 observations 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 meristem (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 modications but also with shifted levels of phytohormones 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 inorescences). 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. Guyomarch et al. (2004) reported that mgo3 mutation in Arabidopsis affected shoot, leaf and root morphogenesis. 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 surprisingly 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 (Guyomarch et al. 2004). As development of the mgo3 Arabidopsis plants proceeded, deregulation of the phyllotaxis and plastochron were more noticeable leading to the development of a wide range of rosette phenotypes (Guyomarch 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 further anatomical investigations as well as physiological and genetic studies on the development and functionality of stem, roots, and leaves are needed to improve our understanding of how fasciations develop.

Conclusions Fasciated individuals arise through various environmental causes, and they do not transmit this altered state to their

123

126

Plant Growth Regul (2011) 63:115129

progeny. In some cases fasciation arises as a mutation, the progeny of which inherit the changed phenotype. Many intriguing features of the development of fasciations 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 difcult to interpret and require further studies at the hormonal and genotype levels. Cytokinins, particularly zeatin and BA, can induce fasciation in different species. Similar to naturally occurring fasciations, the in vitro induction of fasciations is associated 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 owers, 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 preceded 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 specicity regarding the pattern of fasciation development. The anatomical analysis to date shows that any of the fasciationinduction 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 differentiation 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 differentiation is associated with the occurrence of mitotically 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, Thessaloniki, 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 KwaZuluNatal, South Africa), for reading the manuscript and providing many helpful comments.

References
Abe K, Osakabe K, Ishikawa Y, Tagiri A, Yamanouchi H, Takyuu T, Yoshioka T, Ito T, Kobayashi M, Shinozaki K, Ichikawa H, Toki S (2009) Inefcient double-strand DNA break repair is associated with increased fasciation in Arabidopsis BRCA2 mutants. J Exp Bot 60:27512761 Albertsen MC, Curry TM, Palmer RG, Lamotte CE (1983) Genetics and comparative growth morphology of fasciation in soybeans (Glycine max (L.) Merr). Bot Gaz 144:263275 Astie M (1963) Teratologie spontanee et experimentale. Ann Sci Nat Ser 12(3):619844 Bairathi MK, Nathawat GS (1978) Morphology and anatomy of fasciated plants of Sannhemp (Crotalaria juncea L.). Flora 167:147157 Balotis G, Papafotiou M (2003) Micropropagation and stability of Euphorbia pugniformis cristate form. Acta Hortic 616:471474 Barotti S, Fambrini M, Pugliesi C, Lemzi A (1995) Genetic variability in plants regenerated from in vitro cultures of sunower (Helianthus annuus L.). Plant Breed 114:275276 Baurle I, Laux T (2003) Apical meristems: the plants fountain of youth. Bioessays 25:961970 Behera NC, Patnaik SN (1981) Induced fasciation in Celosia argentea L. Curr Sci 50:287288 Behera NC, Patnaik SN (1982) Histology of mutants in Amaranthus hypochondriacus. Indian J Genet Plant Breed 42:510 Bertaccini A, Franova J, Botti S, Tabanelli D (2005) Molecular characterization of phytoplasmas in lilies with fasciation in the Czech Republic. FEMS Microbiol Lett 249:7985 Bhalla PL, Singh MB (2006) Molecular control of stem cell maintenance in shoot apical meristem. Plant Cell Rep 25: 249256 Binggeli P (1990) Occurrence and causes of fasciation. Cecidology 5:5762 Blaringhem L (1903) Anomalies hereditaries provoquees per la traumatismes. Compt Rend Acad Sci, Paris 140:375 Blaringhem L (1907) Mutation et traumatismes. Alcan, Paris Boke NH, Ross RG (1978) Fasciation and dichotomous branching in Echinocereus (Cactaceae). Am J Bot 65:522530

123

Plant Growth Regul (2011) 63:115129 Bonneta D, Bayliss P, Sun S, Sage T, McCourt P (2000) Farnesylation is involved in meristem organization in Arabidopsis. Planta 211:182190 Bos L (1957) Plant teratology and plant pathology. Eur J Plant Pathol 63:222231 Brossard D (1976) Inuence de la concentration en kinetine sur ` lobtention et le degree de plodie des bourgeons neoformes a partir de la moelle de tabac cultivee in vitro. Z Panzenphysiol 78:323333 Carles CC, Fletcher JC (2003) Shoot apical meristem maintenance: the art of a dynamic balance. Trends Plant Sci 8:394401 Castellano MM, Sablowski R (2005) Intercellular signaling in the transition from stem cells to organogenesis in meristems. Curr Opin Plant Biol 8:2631 Chriqui D (2008) Developmental biology. In: George EF, Hall MA, De Klerk G-J (eds) Plant propagation by tissue culture, vol 1, 3rd edn. The background 283334 Clark SE (2001) Cell signaling at the shoot meristem. Nat Rev Mol Cell Biol 2:276284 Clark SE, Running MP, Meyerowitz EM (1993) CLAVATA1, a regulator of meristem and ower development in Arabidopsis. Development 119:397418 Clark SE, Running MP, Meyerowitz EM (1995) CLAVATA3 is a specic regulator of shoot and oral meristem development affecting the same processes as CLAVATA1. Development 121:20572067 Clark SE, Williams RW, Mayerowitz EM (1997) The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and oral meristem size in Arabidopsis. Cell 89:575585 Cobhold A (1931) The grafting of cacti. Gard Chron 90:108 Crespi M, Messens E, Caplan AB, van Montagu M, Desomer J (1992) Fasciation induction by the phytopathogen Rhodococcus fascians depends upon a lenear plasmid encoding a cytokinin synthase gene. EMBO J 11:795804 Crespi M, Vereecke D, Temmerman W, van Montagu M, Desomer J (1994) The fas operation of Rhodococus fascians encodes new genes required for efcient fasciation of host plants. J Bacteriol 176:24922501 De Vries H (1894) Over de erfelijkheid der fasciatien. Bot Jaarb 6:72118 De Vries H (19091910) The mutation theory, vol 1 and 2. Open Court, Chicago Dirr M (1998) Manual of woody landscape plants: their identication, ornamental characteristics, culture, propagation and uses, 5th edn. Stipes Publishing LLC, Champaign Driss-Ecole D (1981) Fasciation from excised shoot apices in Celosia cristata (Amaranthaceae) cultivated in vitro. Can J Bot 59: 13671373 Drjagina IV, Murin AV, Liskov VN (1981) Experimental mutagenesis with garden plants. In: Konotop AI (ed), Kishinev, Shtinitsa (in Russian) ` Ecole D (1970) Premieres observations sur la fasciation chez le ` Celosia cristata L. (Amarantacees). C R Acad Sci Paris 270: 477480 ` Ecole D (1971) Edication de pedoncules sous laction de lacide ` triidobenzoque chez deux Solanacees: Lycopersicum esculentum et Lycopersicum pimpinellifolium. C R Acad Sci Paris 273:634637 ` ` Ecole D (1972) Response a lapplication dacide 2, 3, 5 tri` idobenzoque (TIBA) chez deux Crassulacees: Bryophyllum daigremontianum Berger et Bryophyllum tubiorum Harv. C R Acad Sci Paris 275:557560 Fambrini M, Bonsignori E, Rapparini F, Cionini G, Michelotti V, Bertini D, Baraldi R, Pugliesi C (2006) stem fasciated, a Recessive mutation in sunower (Helianthus annuus), alters plant morphology and auxin level. Ann Bot 98:715730

127 Fleming AJ (2005) Formation of primordial and phyllotaxy. Curr Oper Plant Biol 8:5358 Fletcher JC (2002) Shoot and oral meristem maintenance in Arabidopsis. Annu Rev Plant Biol 53:4566 Fletcher J, Brand U, Running M, Simon R, Meyerowitz E (1999) Signaling of cell fate decisions by CLAVATA 3 in Arabidopsis shoots meristems. Science 283:19111914 Fukai S, Hasegava A, Goi M (2000) Morphological characteristics and shoot regeneration potential of the fasciated rhizome of Cymbidium kanran Makino. Plant Biotechnol 17:259262 Giulini A, Wang J, Jackson D (2004) Control of phylotaxy by the citokinin-inducible response regulator homologue ABPHYL1. Nature 430:10311034 Gorter CJ (1965) Origin of fasciation. In: Rhuland W (ed) Encyclopedia of plant physiology, vol 15(2). Springer, New York, pp 330351 Gottschalk W, Wolff G (1983) The alteration of the shoot system by means of mutations. In: Frankel R, Gall GAE, Grossman M, Linskens HF, Riley R (eds) Monographs on theoretical and applied genetics. Induced mutation in plant breeding, vol 7. Springer, Berlin, pp 4364 Graf P, Dolzblasz A, Wurschum T, Lenhard M, Pfreundt U, Laux T (2010) MGOUN1 encodes an Arabidopsis type IB DNA topoisomerase required in stem cell regulation and to maintain developmentally regulated gene silencing. Plant Cell 22: 716728 Green KA, Prigge MJ, Katzman RB, Clark SE (2005) CORONA, a member of the class III homeodomain leucine zipper gene family in Arabidopsis, regulates stem cell specication and organogenesis. Plant Cell 17:691704 Guyomarch S, Vernoux T, Traas J, Zhou D-X, Delarue M (2004) MGOUN3, an Arabidopsis gene with TetratricoPeptide-Repeatrelated motifs, regulates meristem cellular organization. J Exp Bot 55:673684 Heslop-Harrison J (1952) A reconsideration of plant teratology. Phyton 4:1934 Hus H (1908) Fasciations of known causation. Am Nat 42:8197 Iliev I (1996) In vitro propagation of Betula pendula Roth Youngii. In: Iliev I, Zhelev P, Aleksandrov P (eds) Propagation of ornamental plants, Ministry of the Education, Science and Technology Publishing House, Soa, pp 4454 Iliev I, Tomita M (2003) Micropropagation of Betula pendula Roth Fastigiata by adventitious shoot regeneration from leaf callus. Propag Ornam Plants 3:2026 Iliev I, Rubos A, Scaltsoyiannes A, Nellas C, Kitin P (2003) Anatomical study of in vitro obtained fascinated shoots from Betula pendula. Roth Acta Hortic 616:481484 Iliev I, Scaltsoyiannes A, Tsaktsira M, Gajdosova A (2011) Micropropagation of Betula pendula Roth cultivars by adventitious shoot induction from leaf callus. Acta Hortic (in press) Irvine VC (1940) X-radiation and growth substances as affecting growth primordial tissues. Proc Soc Exp Biol Med 43:453455 Itoh J-I, Hasegawa A, Kitano H, Nagato Y (1998) A recessive heterochronic mutation, plastochron 1, shortens the plastochron and elongates the vegetative phase in rice. Plant Cell 10: 15111521 Itoh J-I, Kitano H, Matsuoka M, Nagato Y (2000) SHOOT ORGANIZATION genes regulate shoot apical meristem organization and the pattern of leaf primordium initiation in rice. Plant Cell 12:21612174 Jambhulkar SJ (2002) Growth and morphology and inheritance of fasciation mutation in sunower. J Genet Breed 56:327330 Jemmali A, Boxus P, Dekegel D, van Heule G (1994) Occurrence of spontaneous shoot regeneration on leaf stipules in relation to hyperowering response in micropropagated strawberry plantlets. In vitro Cell Dev Biol-Plant 30P:163166

123

128 Johnson EL (1926) Effects of X-ray upon growth, development and oxidizing enzymes of Helianthus annus. Bot Gaz 82:373402 Jones DF (1935) The similarity between fasciation in plants and tumors in plants and their genetic basis. Science 81:7576 Jonsson H, Heisler MG, Shapiro BE, Meyerowitz EM, Mjolsness E (2006) An auxin-driven polarized transport model for phyllotaxis. Proc Natl Acad Sci USA 103:16331638 Kaplan DR, Cooke TJ (1997) Fundamental concepts in the embryogenesis of dicotyledons: a morphological interpretation of embryo mutants. Plant Cell 9:19031919 Karagiozova M, Meshineva V (1977) Histological characteristic of band-shaped fasciations. Ann J Soa Univ 68:1130 (in Bulgarian) Karakaya HC, Tang YH, Cregan PB, Knap HT (2002) Molecular mapping of the fasciation mutation in soybean, Glycine max (Legominosae). Am J Bot 89:559565 Kayes JM, Clark SE (1998) CLAVATA2, a regulator of meristem and organ development in Arabidopsis. Development 125:38433851 Kienholz R (1932) Fasciation in red pine. Bot Gaz 94:404410 King HG (1918) Fasciated vegetable marrow. Gard Chron 64:147 Kitin P, Iliev I, Scaltsoyiannes A, Nellas C, Rubos A, Funada R (2005) A comparative histological study between normal and fasciated shoots of Prunus avium generated in vitro. Plant Cell Tissue Organ Cult 82:141150 Klers G (19031906) Uber kunstliche metamorphosen. Abb Naturf Ges Halle 25:134 Knights EJ (1993) Fasciation in chickpeagenetics and evaluation. Euphytica 9:163166 Knox AD (1908) The induction, development and heritability of fasciation. Publ Carnegie Inst (Wash) 98:321 Krusmann G (1995) Manual of cultivated conifers. H-D Warda, Timber Press, Portland, Oregon LaMotte CE, Curry TM, Palmer RG, Albertsen MC (1988) Developmental anatomy and morphology of fasciation in the soybean (Glycine max). Bot Gaz 149:398407 Lamprecht H (1952) Polymere gene und chromosomen-struktur bei Pisum. Agri Hortique Genet 10:158167 Laufs P, Dockx J, Kronenberger J, Traas J (1998a) MGOUN1 and MGOUN2: two genes required for primordium initiation at the shoot apical and oral meristem in Arabidopsis thaliana. Development 125:12531260 Laufs P, Grandjean O, Jonak C, Kieu K, Traas J (1998b) Cellular parameters of the shoot apical meristem in Arabidopsis. Plant Cell 10:13751390 Laux T, Mayer KFX, Berger J, Jurgens G (1996) The WUSCHEL gene is required for shoot and oral meristem integrity in Arabidopsis. Development 122:8796 Lebedeva T (1963) For the distribution of the phenomenon fasciation in cultivated plants. Bot J 48:526535 (in Russian) Lenhard M, Laux T (2003) Stem cell homeostasis in the Arabidopsis shoot meristem is regulated by intercellular movment of CLAVATA3 and its sequestration by CLAVATA1. Development 130:31633173 Leyser H, Furner I (1992) Characterization of three shoot apical meristem mutants of Arabidopsis thaliana. Development 116: 397403 Loennig WE (1980) Fasciation and heterosis in pea: I. Pisum Newslett 12:27 Loennig WE (1981) Fasciation and heterosis in pea: II. Pisum Newslett 13:4446 Long JA, Moan EI, Medford JL, Barton MK (1996) A member of the KNOTTED class of homeodomain proteins enclosed by SHOOTMERISTEMLESS gene of Arabidopsis. Nature 379:6669 Marx GA, Hagedorn DJ (1962) Fasciation in Pisum. J Hered 53: 3143

Plant Growth Regul (2011) 63:115129 McCartan SA, Van Staden J (2003) Micropropagation of the endangered Kniphoa leucocephala Baijnath. In Vitro Cell Dev Biol-Plant 39:496499 Medford JI, Behringer FJ, Callos JD, Feldman KA (1992) Natural and abnormal development in the Arabidopsis vegetative shoot apex. Plant Cell 4:631643 Mendel G (1866) Versuche uber panzenhybriden. Verh Naturf Ver Brunn 4:347 Mertens TR, Burdick AB (1954) The morphology, anatomy and genetics of a stem fasciation in Lycopersicon esmulentum. Am J Bot 41:726732 Meyer V (1966) Flower abnormalities. Bot Rev 32:165195 Meyer KFX, School H, Haecker A, Lenhard M, Jurgens G, Laux T (1998) Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem. Cell 95:805815 Meyerowitz EM, Smyth DR, Bowman JL (1989) Abnormal owers and pattern formation in oral development. Development 106:209217 Mitras D, Kitin P, Iliev I, Dancheva D, Scaltsoyiannes A, Tsaktsira M, Nellas C, Rohr R (2009) In vitro propagation of Fraxinus excelsior L. by epicotyls. J Biol Res 11:3748 Molliard M (1900) Cas de virescence et de fasciation dorigine parasitaire. Rev Gen Bot 12:323328 Mordhorst AP, Voerman KJ, Hartog MV, Mejer EA, Van Went J, Koorneef M, De Vries SC (1998) Somatic embryogenesis in Arabidopsis thaliana is fasciated by mutations in genes repressing meristematic cell divisions. Genetics 149:549563 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15: 473497 Nadjimov UK, Scott IM, Fatkhullaeva GN, Mirakhmedov MS, Nasirullaev BU, Musaev DA (1999) Conditioning of fasciation by gibberellin and genotype in cotton (Gossipium hirsutum L.). J Plant Growth Regul 18:4548 Nestler A (1894) Undersuchungen uber Fasciationen. Osterr Bot Zeitschr 44:343350 Nilsson O, Moritz T, Sundberg B, Sandberg G, Olsson O (1996) Expression of the Agrobacterium rhizogenes rolC gene in a deciduous forest tree alters growth and development and leads to stem fasciation. Plant Physiol 112:493502 Papafotiou M, Balotis G, Louka P, Chronopoulos J (2001) In vitro plant regeneration of Mammillaria elongata normal and cristate forms. Plant Cell Tissue Organ Cult 65:163167 Peyritsch J (1888) Uber kunstliche Erzeugung von gefullten Bluthen und andere Bildungsabweichungen. S. B. kais. Akad. Wiss. Wien Math Nat Kl 97:597605 Poething RS, Sussex IM (1985) The cellular parameters of leaf development in tobacco: a clonal analisys. Planta 165:170184 Poncarova-Vorackova Z, Franova J, Valova P, Mertelik J, Navratil M, Nebesaarova J (1998) Identication of phytoplasma infecting Lilium martagon in the Czech Republic. J Phytopathol 146: 609612 Rajput K (2010) Pattern of secondary growth and formation of asymmetrical successive cambia in Machaerium sp. (Fabaceae). Abstracts, IAWA/IAWS/IUFRO Joint Conference, June 2326, Madison, Wisconsin, USA Rance SJ, Cameron DM, Williams ER (1982) Correction of crown disorders of Pinus caribaea var. hondurensis by application of zinc. Plant Soil 65:293296 Reboredo F (1994) Morphological changes in species (Spartium junceum) collected in areas adjacent to a major motorway. In: Proceedings of 6th international conference on environmental contamination. Delphi, Greece, pp 1012 Reboredo F, Silvares C (2007) Fasciation phenomena and mineral balance in Spartium junceum L. Phyton 47:123132

123

Plant Growth Regul (2011) 63:115129 Reinhardt D, Mandel T, Kuhlemeier C (2000) Auxin regulates the initiation and radial position of plant lateral organs. Plant Cell 12:507518 Reinhardt D, Frenz M, Mandel T, Kuhlemeier C (2005) Microsurgical and laser ablation analysis of leaf positioning and dorsoventral patterning in tomato. Development 132:1526 Riddle LC (1903) Fasciation. Ohio Nat 3(3):346348 Rinne PLH, Van der Shoot C (1998) Symplasmic elds in the tunica of the shoot apical meristem coordinate morphogenetic events. Development 125:14551485 Running MP, Fletcher JC, Meyerowitz EM (1998) The WIGGUM gene is required for proper regulation of oral meristem size in Arabidopsis. Development 125:25452553 Sakharov VV (1986) Polyherbal seeds and fasciation in tetraploid buckwheat. In: Shevchenko VA (ed) Genetic mechanisms of selection and evolution. Moskow, Nauka, pp 1733 (in Russian) Schoof K, Lenhard M, Haecker A, Mayer KF, Jurgens G, Laux T (2000) The stem cell population of Arabidopsis shoot meristems is maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell 100:635644 Sharma VK, Fletcher JC (2002) Maintenance of shoot and oral meristem cell proliferation and fate. Plant Physiol 129:3139 Shavrov L (1961) The fasciations in subtropic. Bul Gl Bot Sada 41:5865 (in Russian) Sinjushin AA, Gostimsky SA (2006) Fasciation in pea: basic principles of morphogenesis. Russ J Dev Biol 37:375381 Sinjushin AA, Gostimsky SA (2008) Genetic control of fasciation in pea (Pisum sativum L.). Russ J Genet 44:807814 Smith RS, Guyomarch S, Mandel T, Reinhardt D, Kuhlemaier C, Prusinkiewicz P (2006) A plausible model of phyllotaxis. Proc Natl Acad Sci USA 103:13011306 Soroka A, Lyakh V (2009) Genetic variability in sunower after mutagen treatment of immature embryos of different ages. Helia 32:3346 Spicer R, Groover A (2010) The evolution of development of vascular cambia and secondary growth. New Phytol 186:577592 Srinivasan S, Gaur PM, Rao BV (2008) Allelic relationship between spontaneous and induced mutant genes for stem fasciation in chickpea. Plant Breed 127:319321 Srivastava PS, Glock H (1987) FPA-induced fasciation of shoots of birch in vitro. Phytomorphology 37:395399 Stange RR, Jeffares D, Young C, Scott DB, Eason JR, Jameson PE (1996) PCR amplication of the fas-1 gene for the detection of viriulent strains Rhodococus fascians. Plant Pathol 45:407417 Steeves TA, Sussex IM (1989) Patterns in plant development, 2nd edn. Cambridge University Press, Cambridge Steiner G (1931) Tylenchus dipsacithe bulb or stem parasitizing strawberry plants in North California. US Dept Agric Bull Plant Rep 15:43 Stimart D, Harbage JF (1989) In vitro proliferation of Pyrus calleryana from vegetative buds. Hortscience 24:2733 Stone JM, Trotochaud AE, Walker JC, Clark SE (1998) Control of meristem development by CLAVATA1 receptor kinase and kinase-associated protein phosphatase interactions. Plant Physiol 117:12171225 Stumm-Tegethoff BFA, Linskens HF (1985) Stem fasciation in Lilium henryi caused by nematodes. Acta Bot Neerl 34:8393 Swiecicki WK (2001) Supplemental data on fasciata genes in Pisum resources. Pisum Genet 33:1920 Swiecicki WK, Gawlowska M (2004) Linkages for a new fasciata gene. Pisum Genet 36:2223

129 Szczesny T, Routier-Kierzkowska A-L, Kwiatkowska D (2009) Inuence of clavata3-2 mutation on early ower development in Arabidopsis thaliana: quantitative analysis of changing geometry. J Exp Bot 60:679695 Taguchi-Shiobara F, Yuan Z, Hake S, Jackson D (2001) The fasciated ear2 gene encodes a leucine-rich repeat receptor-like protein that regulates shoot meristem proliferation in maize. Genes Dev 15:27552766 Tang Y, Knap HT (1998) Fasciation mutation enhances meristematic activity and alters patter formation in soybean. Int J Plant Sci 159:249260 Tang Y, Scorupska H (1997) Expression of fasciation mutation in apical meristems of soybean, Glycine max (Legominosae). Am J Bot 84:328335 Tanskley SD (2004) The genetic, developmental, and molecular bases of fruit size and shape variation in tomato. Plant Cell 16:181189 Thimann KV, Sachs T (1966) The role of cytokinins in the fasciation disease caused by Corynebacterium fascians. Am J Bot 53:731739 Traas J, Vernoux T (2002) The shoot apical meristem: the dynamics of a stable structure. Phylos Trans R Soc Lond B 357:737747 Ueda H, Torikata H (1969) Organogenesis in the meristem culture of cymbidiums III. Histological studies on the shoot formation at thr rhizome-tips of Cymbidium goeringii Reichb. F. cultured in vitro. J Japan Soc Hortic Sci 38:263266 (in Japanese) Vallicelli V (2010) Cactuspedia. http://www.cactuspedia.info/. Assessed 20 May 2010 Van Gelderen DM, Van Hoey Smith JRP (1997) Conifers: the illustrated encyclopedia, vol 1 and 2. Timber Press, Portland Varga A, Thoma H, Bruinsma J (1988) Effects of auxins and cytokinins on epigenetic instability of callus-propagated Kalanchoe blossfeldiana Poelln. Plant Cell Tissue Organ Cult 15:223231 Vernoux T, Kronenberger J, Grandjean O, Laufs P, Traas J (2000) PIN-FORMED1 regulates cell fate at the periphery of the shoot apical meristem. Development 127:51575165 Vitkovskii V (1959) Fasciation of Syringa josikaea shoots. Botanicheskii J 44:505506 (in Russian) Went FW (1944) Plant Growth under controlled conditions. II. Thermoperiodicity in growth and fruiting of the tomato. Am J Bot 31:135150 Werner R (1988) Unusual growth in succulent Euforbias. Fasciation & Dichotomus Branching. Euphorbia J 5:717 White OE (1916) Studies teratological phenomena in their relation to evolution and the problems of heredity. II. The nature, causes, distribution, and inheritance of fasciation with special reference to its occurrence in Nicotiana. Zeits Ind Abst Ver 16:49185 White OE (1917) Studies of inheritance in Pisum: II. The present stage of knowledge of heredity and variation in peas. Proc Am Phil Soc 56:487589 White OE (1948) Fasciation. Bot Rev 14:319358 Williams L, Fletcher JC (2005) Stem regulation in the Arabidopsis shoot apical meristem. Curr Opin Plant Biol 8:582586 Williams RW, Wilson JM, Meyerowitz EM (1997) A possible role for kinase-associated protein phosphatase in Arabidopsis CLAVATA1 signaling pathway. Proc Natl Acad Sci USA 94:1046710472 Yamasaki Y (1940) Studies on the experimental production of fasciation in buckwheat by treatment of seeds with heteroauxin solutions. Jpn J Genet 16:171175 Zielinski O (1945) Fasciation in horticultural plants with special reference to the tomato. Proc Am Soc Hortic Sci 46:263268

123