ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 2: 55 - 60 
Eff ect of sucrose nutrition on the histological structure of carob shoot 
cultures 
Branka VINTERHALTER1, Dragan VINTERHALTER: 
1Institute for Biological Research, 29. novembra 142, Belgrade, Yugoslavia 
2Faculty of Science, Zmaja od Bosne 33-35, Sarajevo, Bosnia and Herzegovina 
Introduction 
Abstract. Analysis of carob (Ceratonia siliqua L.) shoot cultures grown on 2 
and 8 % sucrose in light and darkness (etiolation) showed marked differences in 
histological structure. Etiolated, dark grown shoot cultures were hypolignified on 
both sucrose concentrations. They had highly reduced sclerenchyma and xylem 
production, endodermis with Casparian bands and absence of lenticel formation. In 
light grown cultures shoots in transection showed nearly concentric ring of 
sclerenchyma ( outer) and xylem (inner) ring and the absence of Casparian bands in 
endodermal cells. Increased sucrose nutrition promoted lignification and efficiently 
prevented lenticel hypertrophy characteristic for low sucrose concentration. 
Key words: carob, in vitro, sucrose, light, etiolation, lenticel hypertrophy, 
lignification, hypolignification, Casparian bands 
Effect of sucrose on the growth of plant in vitro cultures bas been subject of numerous studies. 
In most species cultured in vitro optimal concentration of sucrose is 2-3 %. Apart from nutritive 
role sucrose has been recently attributed a regulatory role since changes in sucrose concentration 
can serve in gene activation and signal sensing (KocH & al., 1992, MITRA & al. 1995, SMEEKENS & 
RooK 1997). Increased sucrose nutrition can induce morphogenetic effects as for instance axillary 
bud formation in the rooting stage of Dracaenafragrans (VINTERHALTER & VINTERHALTER 1997). In 
carob sucrose together with light regulates leaf size (VINTERHALTER & VINTERHALTER 2001). It is well 
documented that sucrose stimulates xylogenesis in callus of angiosperms (WETMORE & RIER 1963) 
and in intemodes of Coleus (RIER & BESLOW 1967). WARREN WILSON & al. (1994) confirmed the 
stimulatory effect of exogenous sucrose on xylogenesis. However, the optimal sucrose concentration 
was species dependant. In letuce pith explants it was at 0.2 % and in tobacco pith explants at 3.0 % 
sucrose. 
Carob shoot cultures grown on low sucrose concentrations (2 % sucrose or less) exhibit lenticel 
hypertrophy a rare physiological disorder of in vitro cultures described also in Populus euphratica 
(LLEDO & al. 1995). Histological investigation showed that lenticels appear only on hypolignified 
intemodes (VINTERHALTER & al. 1997). Increased sucrose nutrition promotes the growth of carob 
shoot cultures (VrNTERHALTER 1998) and at the same tirne decreases the frequency of lenticel 
hypertrophy. Another treatment in which lenticel hypertrophy in carob was found to be absen was 
56 Acta Biologica Slovenica, 46 (2), 2003 
cultivation in the darkness which results in fom1ation of etiolated shoot cultures (VINTERHALTER & 
VINTERHALTER 2002). 
In the present study we investigated the histological structure of carob shoot cultures grown in 
conditions of high and low sucrose nutrition both in the light and darkness. We were particulary 
interested in a possible connection between lignification and lenticel hypertrophy. 
Material and methods 
Carob shoot cultures were established and maintained on MuRASH!GE & SKoOG (1962) medium 
with 2.25 µM BA and 0.5 µM IBA as previously described (VINTERHALTER & VINTERHALTER 2001). 
Treatments consisted ofmedia supplemented with 2 % (low) and 8 % (high) sucrose in the light and 
darkness. The light 46.5 µmol m·2s·1 was provided by cool white fluorescent lamps. All treatrnents 
lasted for 35 days. The material for histological study was prepared and stained according to JoHANSEN 
(1940). Shoots were fixed in 1 : 1 : 18 formalin-acetic acid- ethanol (FAA) embedded in the paraffine 
and cut on a rotary microtome in the 8-10 µm thick sections. Sections were double stained with 
safranine-light green or safranine-wasser blau. Presence oflignine was demonstrated in fresh material 
using band sections stained either with an acidified phloroglucinol or aniline sulphate. Hand sections 
of etiolated tissues were stained with Lugol-s reagent (JJK) in combination with acidified 
phloroglucinol or safranine. 
Results 
Experiments conducted in the light: At the end of subculture, shoot explants, initially consisting 
of an apical hud and 2-3 intemodes develop 5-8 new intemodes. Intemodes of the initial explant 
become basal intemodes whilst apical and central intemodes develop in the current subculture. 
Basal intemodes submersed into the medium produce a callus which appears in the endodermis and 
replaces ali cortical and surface tissues developing in a form of pseudo-cortex. 
Apical intemodes in transection have characteristic 5 !obed structure and maturation of vascular 
tissues in not yet complete. Central intemodes are circular in transection and have typical mature 
structure with well differentiated vascular tissues and two massive circumferential rings of lignified 
cells, outer sclerenchyma and inner xylem ring. Difference in color (sclerenchyma is more orange) 
indicates possible difference in lignine composition. Epidermal and subepidermal cells are red from 
accumulation of anthocyanin and resins. 
Basal intemodes of cultures grown on 2 % sucrose, typically undergo lenticel hypertrophy with 
massive surface proliferation of phelloderm cells. Inner tissues of such intemodes show severe 
hypolignification, meaning that the two massive rings of lignified cells (sclerenchyma and xylem) 
are reduced or missing. In conditions of low sucrose concentration lenticel hypertrophy is therefore 
always associated with hypolignification. The analyses of hand sectioned fresh material showed 
that in the earliest stages of lenticel hypertrophy sclerenchyma ring is ruptured or missing (Fig. 1). 
Frequency of lenticel hypertrophy significantly decreases if cultures are grown on the media 
with increased sucrose concentration (Fig. 5). Accumulation of anthocyanins and resins in epidermal 
and subepidermal cells is stimulated same as abundant formation of amyloplasts in all parenchymatous 
cells. In central and basal intemodes sclerenchyma ring is well developed and there is a massive 
formation of both phloem and xylem (Figs. 3, 4). 
B. Vinterhalter, D. Vinterhalter: Effect of sucrose nutrition on the histological structure ... 57 
Figure 1: Early stage of lenticel development on 2 % sucrose in light, basal intemodes, bar= 60 µm. 
Hand section of fresh material stained with Lugol and safranine. Sclerenchyma ring absent, xylem 
organised in srna! groups, prominet lenticel (hl). 
Figure 2: Transection of a dark grown etiolated shoot, 2 % sucrose, bar = 22 µm. Hand section of 
fresh material stained with Lugol and safranine. Endodermal cells with Casparian bands (cb), 
sclerenchyma ring absent and replaced with large, thin-walled parenchymatous cel!s. 
Figure 3: Transection of a basal intemode on 8 % sucrose in light, bar = 60 µm. Paraffine section 
stained with safranine- light green. Typical heavy lignified structure, lenticels absen. Epidermis and 
subpeidermis rich with anthocyanins an resins, circumferential sclerenchyma ring (se), phloem, 
cambal layer and a massive layer of lignified xylem (xy). 
Figure 4: Transection of a basal intemode on 8 % sucrose in light, bar = 22 µm. Hand section of 
fresh material stained with Lugol and safranine. Endodermis with amyloplast (starch sheet, ss), well 
deveoped sclerenchyma (se), phloem (fl) and xylem (xy) . 
58 Acta Biologica Slovenica, 46 (2), 2003 
Experiments conducted in the darkness: After a short transition period cultures became 
etiolated. Paraffine sections of etiolated cultures which were soft and spongy were successfully 
replaced with hand sectioned fresh material. 
Etiolation induced drastic changes in the shoot structure. Etiolated cultures are in general highly 
hypolignified. In the darkness no lignine is produced and rings of lignified sclerenchyma and xylem 
elements (characteristic for light conditions) are never formed. Xylem elements are rare and 
sclerenchyma layer is completely absent, replaced by large parenchymatic cells laying inner to the 
endodermis. Characteristic feature of this endodermal parenchyma are cells with Casparian bands 
which form a continuous or nearly continuous ring around the central cylinder (Fig. 2). 
Hypertrophied lenticels were never observed to appear in etiolated shoot cultures irrespectively of the 
concentration of sucrose in the medium. However, etiolated cultures transferred to light quick:ly became de-
etiolated and in favorable conditions (low sucrose in the medium) lenticel hypertrophy re-appeared. 
Fig 
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100 
90 
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Sucrose, X 
Number of hypertrophied 
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Figure 5: Effect of sucrose concentration on the number of hypertrophied lenticels formed on the 
first intemode. 
B. Vinterhalter, D. Vinterhalter: Effect of sucrose nutrition on the histological structure ... 59 
Discussion 
Hand sectioning of fresh material proved to be an exellent method for rapid investigation of 
lignified tissues in carob shoot cultures grown both in light and darkness. In etiolated shoot cultures 
it enabled us to observe Casparian bands. 
ARZEE & al. (1977) found that in carob seedlings phellogen formation starts at the end of the 
first year. In carob shoot cultures tissue differentiation is very fast and certain conditions may induce 
premature formation and hypertrophy of lenticels which we consider as a physiological disorder. 
What are the factors which induce this phenomenon? 
Our first studies showed that lenticel hypertrophy is connected to cytokinin concentration 
(VINTERHALTER & al. 1992) and restricted ventilation (VINTERHALTER & VINTERHALTER 1992). Absence 
of ventilation triggered lenticel formati on but only if cytokinins were present in the medi um, otherwise 
leaves were sheadeed and cultures perished. 
In some species of trees lenticel hypertrophy is a physiological adaptation to anoxia induced by 
flooding and mediated by ethylene (TANG & KozLOWSKI 1984). Process includes synthesis of ethylene 
precursor (ACC) in submersed tissues, its translocation through the vascular tissues and conversion 
into ethylene in aerated tissues above the line of submersion. 
Although lenticel hypertrophy in vitro has been described only in two species, Ceratonia siliqua 
(VINTERHALTER & AL. 1992) and Populus euphratica (LLEDO & al. 1995) there are notions that it is a 
more widespread phenomenon. Is it possible that in vitro conditions mimic flooding in some plants 
species? In carob lenticel hypertrophy always appears on the basal intemode, first intemode above 
the surface of the medium and than it spreads acropetally. 
Our studies showed that lenticel hypertrophy is affected by sucrose nutrition and light. High 
sucrose nutrition decreases frequency of lenticel hypertrophy whilst growth in darkness (etiolated 
cultures) completelly prevents it (VINTERHALTER 1998, VINTERHALTER & VINTERHALTER 2002). In both 
cases there are changes in the histological structure which affects the abundance of cells with lignified 
walls as reported here. In the first case lenticel hypertrophy does not occur whilst sclerenchyma ring 
is present. Sclerenchyma ring is a barrier which efficiently prevents lateral movement of water in 
stems. It seems that undeveloped sclerenchyma ring characteristic for low sucrose concentrations is 
the factor which stimulates lenticel hypertophy and which the high sucrose nutrition prevents. In the 
second case lenticel hypertrophy is absent although etiolated shoots contain no lignified cells. Here 
sclerenchyma ring although absent is functionally replaced with another barrier, ring of cells with 
Casparian bands which are known to restrict lateral translocation of water (KoLDA 1937). We thus 
suppose that lenticel hypertrophy in carob may be prevented by conditions which limit laterni 
translocation of water and solutes in shoots. 
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