Plant Cell Rep (2005) 23:567572
DOI 10.1007/s00299-004-0869-9
PHYSIOLOGY AND BIOCHEMISTRY
V. M. Jim
nez E. Guevara J. Herrera F. Bangerth
Evolution of endogenous hormone concentration
in embryogenic cultures of carrot during early expression
of somatic embryogenesis
Received: 20 April 2004 / Revised: 29 July 2004 / Accepted: 31 July 2004 / Published online: 16 September 2004

Springer-Verlag 2004
Abstract Embryogenic callus and suspension cultures of
carrot (Daucus carota L., cv. Nantaise), growing on/in
medium including 1 mg/l 2,4-dichlorophenoxy acetic acid
(2,4-D), were transferred to medium with or without this
plant growth regulator, to impair or induce, respectively,
further development of somatic embryos. The endogenous
hormone levels of the cultures were determined over 7
days by means of radio-immunoassay, to characterize their
evolution in the initial stages of embryo development. In
general, levels of indoleacetic acid (IAA) and abscisic acid
(ABA) showed only short-lived differences among treat-
ments during this time in both types of tissue analyzed
(i.e., a peak of IAA in callus cultures in the absence of 2,4-
D, 48 h after medium change, and higher ABA contents
144 h after subculture of suspension cultures in the pres-
ence of 2,4-D). Gibberellins (1, 3 and 20) were detected
only in suspension cultures devoid of 2,4-D, starting 24 h
after subculture. Concerning the evaluated cytokinins
zeatin/zeatin riboside and N6(D2-isopentenyl) adenine/
N6(D2-isopentenyl) adenosinethe most remarkable ob-
servation is that high levels of the former generally coin-
cided with low concentrations of the latter, indicating a
shift from precursor to the active form, and vice versa.
Keywords Callus cultures Daucus carota
2,4-Dichlorophenoxy acetic acid Somatic embryos
Suspension cultures
Communicated by D. Dudits
V. M. Jimnez ()) E. Guevara J. Herrera
CIGRAS,
Universidad de Costa Rica,
2060 San Pedro, Costa Rica
e-mail: vjimenez@cariari.ucr.ac.cr
Tel.: +506-2073502
Fax: +506-2074346
F. Bangerth
Institute for Special Crop Cultivation and Crop Physiology (370),
University of Hohenheim,
70593 Stuttgart, Germany
Introduction
Development of somatic embryogenesis has been grossly
divided into two processes: induction and expression. In
the former, differentiated somatic cells acquire embryo-
genic competence and proliferate as embryogenic cells
and cell aggregates (Komamine et al. 1992), i.e., cells
with dense cytoplasm that divide very rapidly (Kiyosue et
al. 1993; Nuti-Ronchi and Giorgetti 1995). During ex-
pression of somatic embryogenesis, embryogenic cells
and aggregates continue their growth by passing through a
series of developmental stages, namely (in dicots) glob-
ular, oblong, heart-shaped and finally torpedo-shaped
(Schiavone and Cooke 1985). Most biochemical and mo-
lecular studies on development of somatic embryos have
been conducted during the induction process (recently
reviewed by Fehr et al. 2003) or during the more ad-
vanced steps of embryo development, usually beginning
at the late globular stage (Fujii 1997; Kitamiya et al.
2000; Stasolla et al. 2003), the initial steps of expression
being the least investigated.
Carrot embryogenic cultures have been used as a
model system to study somatic embryogenesis in plants
(Komamine et al. 1992). Embryogenic callus and sus-
pension cultures in this species can be easily established
when explants (almost any part of the plant) are cultivated
on medium containing 2,4-dichlorophenoxy acetic acid
(2,4-D). Expression of somatic embryogenesis, on the
contrary, is achieved in this system by transferring the
cultures to auxin-free medium (Fujimura and Komamine
1979).
Since plant hormones are highly related to explant
differentiation in vitro (Grieb et al. 1997), their endoge-
nous levels have been employed to characterize several
developmental processes such as somatic embryogenesis.
However, in most of the few studies conducted in carrot
in which changes in hormone concentrations during pro-
gression of somatic (Kamada and Harada 1981; Noma et
al. 1982; Kiyosue et al. 1992; Michalczuk et al. 1992a,b;
Ribnicky et al. 1996) and zygotic (Ribnicky et al. 2002)
embryogenesis have been evaluated, sampling was con-
568
ducted during or after the first morphological changes had
occurred (usually after 7 days), when biochemical and
physiological determination of embryo development has
already started (Dodeman and Ducreux 1996).
Since, to the best of our knowledge, hormonal evolu-
tion during the very early determination of somatic-em-
bryogenesis expression in carrot has not been thoroughly
analyzed, the aim of this work was to quantify the en-
dogenous concentrations of different hormones during the
first days after expression of somatic embryogenesis in
carrot was triggered. Both callus and suspension cultures
were examined because biochemical differences between
them in another model system have already been reported
(Vu et al. 1993; Jimnez et al. 2001).
Material and methods
Callus cultures
Carrot (Daucus carota L., cv. Nantaise; SomElite, Mechelen,
Belgium) callus cultures were established from hypocotyl segments
as described by Jimnez and Bangerth (2001). They were subcul-
tured every 45 weeks on Murashige and Skoog (1962) medium
supplemented with 3% sucrose and 1 mg l1 2,4-D, at a pH of 5.8
and solidified with 0.8% agar (Kiyosue et al. 1992; Sasaki et al.
1994). In every subculture, embryogenic callus segments were
separated from the non-embryogenic segments following the cri-
teria used by Jimnez and Bangerth (2001), and only embryogenic
segments were further cultivated. The cultures were incubated at
252C in the dark. When the embryogenic behavior was fixed in
the cultures (i.e., there was no further production of non-embryo-
genic cell aggregates), suspension cultures were started, and em-
bryogenic expression was induced.
Suspension cultures
Suspension cultures were initiated according to Seitz et al. (1985).
Briefly, 5 g [fresh weight (FW)] embryogenic callus were trans-
ferred into 100 ml of the medium described above, without addition
of agar, in 250-ml Erlenmeyer flasks. The suspensions were kept at
242C in the dark on an orbital shaker at 120 rpm. Every 2 weeks,
each suspension culture was agitated and divided into two Erlen-
meyer flasks and the volume was adjusted to 100 ml with fresh
culture medium.
Induction of embryogenic expression
To trigger embryo development (expression of somatic embryo-
genesis), callus cultures were transferred into 50 ml of the liquid
medium described above, with or without 2,4-D, in 125-ml Er-
lenmeyer flasks. This procedure was conducted to allow the fresh
medium to reach most of the cells at the same time, which does not
occur if solid medium is used. Cultures were kept under the con-
ditions described above for suspension cultures. To induce embryo
development in suspension cultures, the old medium was carefully
decanted, trying to minimize the lose of cell aggregates, and re-
placed with fresh medium, with or without 2,4-D, up to the usual
volume. Sampling and freezing of tissue in liquid nitrogen for
hormone analysis was conducted at the timepoints described in
Figs. 1, and 2.
Sampling, extraction, purification and quantification
of plant hormones
Determination of indole-3-acetic acid (IAA), abscisic acid (ABA),
gibberellins1+3+20 (GAs), zeatin/zeatin riboside (Z/ZR) and N6(D2-
isopentenyl) adenine/N6(D2-isopentenyl) adenosine (iP/iPA), was
performed as described elsewhere (Jimnez and Bangerth 2001;
Jimnez et al. 2001), with the sole exception that the extract was
dried under low pressure and dissolved in 12 ml 0.01 M ammonium
acetate (pH 7.5), instead of 0.1 M ammonium acetate (pH 9.0).
Hormones were quantified by radio-immunoassay with poly-
clonal antibodies. Cross-reactions of the GA3 antibody used were
determined by Bertling and Bangerth (1995), to be about 90% with
GA1 and GA20. Therefore, the GAs determined by means of this
antibody are expressed as GA3 equivalents and are referred to as
GAs in the following text.
Overall recovery during the described purification procedures
was previously determined with radiolabelled internal standards,
and was found to be between 40% and 70% for IAA, 92% or higher
for ABA, higher than 89% for GAs and higher than 80% for both
cytokinins. Therefore, the IAA concentration was the only one that
had to be adjusted with the corresponding recovery value for each
sample, due to the high variations found. The other hormones were
not corrected for losses.
Statistical analysis
Endogenous hormonal levels were analyzed using at least three
biological replications and the STATISTICA for Windows (Stat-
Soft, Tulsa, Okla.) Version 5.1 Studenten-Version; 97 Edition. The
Post hoc Tukeys Honest-Significant-Difference-Test (HSD) for
unequal N (Spjotvoll/Stoline) was used to determine significant
differences in hormone level means (P<0.05).
Results
Tissue culture
Hypocotyl segments cultured for 7 days in the presence of
2,4-D showed initiation of callus proliferation at both cut
ends, under a dissecting microscope. After 4 weeks of
culture, hypocotyl segments formed mainly non-orga-
nized callus, but the presence of some globular structures
was also detected. After an additional 4 weeks of culture,
two different callus types were observed: a non-organized
type of callus, translucent, smooth and wet (called non-
embryogenic); and an opaque embryogenic callus, with a
highly nodular surface. Selection and subculture of only
the embryogenic calli increased the amount of this callus-
type. After several months, no more non-embryogenic
calli developed from embryogenic callus. Transfer of
cultures to medium devoid of 2,4-D did not induce any
noticeable difference in their external appearance in the
short period evaluated before sampling and freezing were
conducted.
Endogenous hormone analysis
A peak in the IAA concentration in the callus cultures,
observed 48 h after 2,4-D was eliminated from the me-
dium (Fig. 1), was the only significant difference found in
this hormone between cultures transferred to medium with

Fig. 1 Endogenous levels of a indole-3-acetic acid (IAA), b ab-
scisic acid (ABA), c zeatin/zeatin riboside (Z/ZR) and d N6(D2-
isopentenyl) adenine/adenosine (iP/iPA) in carrot callus cul-
tures after transfer to liquid medium with (+) and without ()
2,4-dichlorophenoxy acetic acid (2,4-D). Significant differences
(P<0.05) are indicated with different letters
or without 2,4-D during the period evaluated. Levels of
ABA showed no obvious differences between treatments
or during the evaluation time; values were always close to
2327 ng/g dry weight (DW). GAs were undetectable in
any of the callus culture treatments. Initial levels of Z/ZR
increased significantly after 48 h of culture, particularly in
the absence of 2,4-D, and to an intermediate level in its
presence. This behavior was maintained for the next 24 h,
before returning to the original levels during the later
period of evaluation (Fig. 1). The initial content of iP/iPA
(34 ng/g DW) decreased 24 h after subculture to a very
low level (3.15 ng/g DW) in cultures lacking 2,4-D, while
in its presence the reduction was not so extreme (13.6 ng/g
DW). In the absence of 2,4-D, iP/iPA levels increased 24 h
later and then remained unchanged for another 24 h. In the
presence of 2,4-D, iP/iPA reached a considerably higher
peak 72 h after subculture. Both treatments showed a
drastic reduction in iP/iPA content by the last evaluation
(Fig. 1).
The IAA content of suspension cultures transferred to
medium devoid of 2,4-D remained constant during the
whole period evaluated. However, in the presence of 2,4-
D there was a considerable increase in the IAA concen-
trations starting from 24 h after subculture until about
24 h later (Fig. 2). ABA concentrations were almost
identical in both suspension cultures, except for the last
observation period, when an increase was observed in the
presence of 2,4-D. When 2,4-D was present in the culture
medium, GAs were not detected in suspension cultures
over the entire evaluation time. However, in the absence
of 2,4-D, the level of GAs increased from non-detectable
to 69 ng/g DW after 24 h, and were maintained at that
level during the rest of the evaluation period (Fig. 2).
When cultured in the presence of 2,4-D, the Z/ZR con-
centrations in the suspension cultures remained constant
during the entire period, while an initial reduction was
observed in absence of 2,4-D. Later the Z/ZR levels in-
creased again to those found in cultures containing 2,4-D.
569
Fig. 2 Endogenous levels of a IAA, b ABA, c gibberellins (GAs),
d Z/ZR and e iP/iPA in carrot suspension cultures after transfer to
medium with (+) and without () 2,4-D. Significant differences
(P<0.05) are indicated with different letters
No significant differences were observed among treat-
ments concerning iP/iPA during the first 48 h. Later,
however, considerably higher concentrations were ob-
served in the absence of 2,4-D (Fig. 2).
Discussion
Callus induction from hypocotyls cultivated on medium
containing 2,4-D in this experiment followed the same
pattern described elsewhere for this species (Guzzo et al.
1995). Moreover, the morphological characteristics of
these embryogenic and non-embryogenic callus cultures
were similar to frequently described carrot embryogenic
and non-embryogenic callus cultures (Sasaki et al. 1994;
Jimnez and Bangerth 2001 and references therein).
Transfer of the carrot cultures to medium devoid of 2,4-D
did not result in any evident morphological change since,
on purpose, sampling was conducted before these changes
are usually reported to occur (Komamine et al. 1992;
Dodeman and Ducreux 1996).
The peak in IAA concentration observed in callus
cultures after 48 h in medium devoid of 2,4-D (Fig. 1),
indicates that the tissue increased and then arrested syn-
thesis of this hormone in its active form, viz. IAA. A
similar increase in IAA concentration, but observed later
in the progress of carrot zygotic embryo development,
was attributed by Ribnicky et al. (2002) to the biosyn-
thesis of IAA via the tryptophan-dependent pathway.
These authors consider these high levels of IAA to be
required in ovules for cell proliferation in a differentiated
manner and also to be responsible for the occurrence of
570
adventitious embryony, a phenomenon closely related to
somatic embryogenesis. During somatic embryogenesis,
high endogenous IAA levels have also been positively
correlated to a high embryogenic capacity (Sasaki et al.
1994; Jimnez and Bangerth 2001), but once embryo-
genic development is established and has progressed to
some degree, levels of IAA decrease again (Michalczuk et
al. 1992a; Ribnicky et al. 2002).
In contrast to what was observed in callus cultures, this
IAA surge was not evident in suspension cultures (Fig. 2).
It seems possible that in suspension cultures consisting
mainly of individual cells and small cell clusters, the
surge in IAA level is not induced as quickly as in the
callus cultures. If that were the case, this peak, lasting
24 h at most, could possibly have remained unnoticed if it
occurred between the measurements conducted at 72 h
and 144 h, when no evaluation was performed. Differ-
ences in concentration and evolution of the endogenous
hormones among callus and suspension cultures of the
same genotype have already been reported for Citrus
during initiation of somatic-embryo development (Jim-
nez et al. 2001). One factor that can explain such differ-
ences is the higher osmotic potential of solid culture
medium. It has recently been observed that augmented
viscosity of the culture medium increased plantlet re-
generation from carrot suspension cultures (Nagamori et
al. 2001), a phenomenon strongly regulated by hormones.
ABA seems not to be directly involved in the very
initial stages of embryogenic development of the callus
tissues evaluated, since in our experiment endogenous
ABA levels did not change in response to the presence/
absence of 2,4-D, and an increase was observed only after
144 h (6 days) of evaluation when 2,4-D was present in
the suspension cultures (Fig. 2). These results confirm
previous findings by Kamada and Harada (1981), who
observed that the amount of endogenous ABA in cell
clusters and embryos remained low during the first 7 days
of culture regardless of the presence or absence of 2,4-D
in the medium. Our results also coincide with observa-
tions made by Hatzopoulos et al. (1990), who postulated
that the role of ABA in somatic embryogenesis is exerted
through regulation of certain genes (e.g., DC8) that are
thought to be involved in the desiccation and maturation
phases of embryogenesis, which occur very late in em-
bryo development.
The behavior of GAs is noteworthy, since this hor-
mone was below the level of detection in all callus cul-
tures analyzed, while in suspension cultures, it was evi-
dent only in the absence of 2,4-D. Noma et al. (1982)
observed that high levels of a GA1-like substance and a
reduced ability to metabolise GA1 into GA8 and GA-
conjugates are correlated with the absence of embryo
development, while the opposite allowed further devel-
opment of embryos. They postulated that the removal of
2,4-D may allow a rapid metabolism of GA1, reducing it
to a level permitting embryogenic development. One
hypothesis that might explain the high levels of GAs
found in suspension cultures undergoing embryogenic
development (Fig. 2) is that the increase in the GAs
concentration in the absence of 2,4-D might be caused by
the accumulation of GA20, the precursor of GA1, and not
by the accumulation of GA1 itself, the active form that
Noma et al. (1982) related to inhibition of embryo de-
velopment. This accumulation could be a consequence of
an impairment in conversion of GA20 into GA1, which
might occur at low auxin levels, as observed by Ross
(1998) in pea tissues. Since the antibody used to detect
GAs in our work cannot discriminate GA1 from GA20, it
is not possible to confirm this hypothesis and more re-
search has to be conducted in this direction.
The cytokinin (Z/ZR and iP/iPA) contents analyzed
here exhibited reciprocal behavior, i.e., an increase in the
concentration of one usually coincided with a reduction in
the other. Concomitantly, high levels of a particular cy-
tokinin in a specific treatment in callus cultures generally
coincided with low levels of that cytokinin in the corre-
sponding treatment in suspension cultures (Figs. 1, 2).
There is support for the idea that higher cytokinin con-
centrations, in general, are essential during the initial cell
division phase of somatic embryogenesis, but not for later
stages of embryo development and maturation in carrot
(Fujimura and Komamine 1980; Tokuji and Kuriyama
2003). When analyzing the individual role of the iP-type
and Z-type cytokinins, Centeno et al. (1997) postulated
that the task of the first group in the embryogenic re-
sponse might be as biosynthetic precursors to supply the
large amount of Z-type active cytokinins (Einset 1986;
Chen 1997) required for the stimulation of cell division
prior to somatic embryogenesis. It has been previously
observed that incorporation of zeatin into the medium
during days 3 and 4 of culture, promotes the formation of
carrot embryos to a great extent, probably due to en-
hancement of the cell division that occurs during this
period in the cell clusters that is considered to be one of
the most important events during the embryogenetic
process (Fijumura and Komamine 1980).
Carrot embryogenic cultures are not composed of a
homogenous population of cells, but rather of several
types of cells at different developmental stages and with
different morphogenetic potential (Giuliano et al. 1983;
Nomura and Komamine 1985; Guzzo et al. 2002), which
could have influenced the endogenous hormone contents
measured, as possibly reflected in the relatively high
standard deviation values reported in this work. Use of
synchronized and stage-sorted populations has permitted
a more accurate estimation of the hormone status at
each phase of late-embryo development in other studies.
However, this fractionation is usually conducted through
a series of steps that include culture in medium devoid of
2,4-D, and also sieving and centrifugation to separate the
different morphological stages of embryo development
according to size (Osuga et al. 1999; Sharma 1999). In
practical terms, this is not feasible before morphological
changes have occurred, as in the case of our study.
Moreover, in all other known works in which the very
initial steps of embryo development were analyzed, non-
synchronized cultures were employed (Schrader et al.
1997; Sato-Nara and Fukuda 2000).

To the best of our knowledge, this is the first study in
which endogenous contents of four hormone groups are
simultaneously measured during determination/beginning
of expression of somatic embryogenesis. All known pre-
vious experiments (Kamada and Harada 1981; Noma et
al. 1982; Kiyosue et al. 1992; Michalczuk et al. 1992a,b;
Ribnicky et al. 1996) dealt only with one or two hormone
groups, and, with the exception of Kamada and Harada
(1981), sampling was conducted after the first morpho-
logical signs of embryo development were evident (7 days
post-induction at the earliest), when biochemical deter-
mination has already occurred (Dodeman and Ducreux
1996).
Acknowledgements The authors thank the German Academic
Exchange Service (DAAD) for financial support in the form
of short-term scholarships. The skilled work of A. Azofeifa, E.
Tavares and S. Mass, conducting part of the in vitro culture, is
gratefully acknowledged.
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