Annals of Botany 79: 535-545, 1997
Annals of Botany 79: 535-545, 1997
Ultrastructure of Carrot Seeds during Matriconditioning with Micro-Cel E
Ultrastructure of Carrot Seeds during Matriconditioning with Micro-Cel E
ALINA DAWIDOWICZ-GRZEGORZEWSKA
ALINA DAWIDOWICZ-GRZEGORZEWSKA
The University of Warsaw, Institute of Botany, ul. Krakowskie PrzedmieScie 26/28,00-927 Warszawa, Poland
The University of Warsaw, Institute of Botany, ul. Krakowskie PrzedmieScie 26/28,00-927 Warszawa, Poland
Received: 25 June 1996 Accepted: 21 November 1996
Received: 25 June 1996 Accepted: 21 November 1996
Matriconditioning (MC) of carrot seeds, with Micro-Cel E, improved their germinability by shortening the mean
germination
time and
reducing
the spread.
Cytological
studies
indicated that
catabolic phase
of germination
sensu
Matriconditioning
(MC)
of carrot
seeds, with
Micro-Cel
E, improved
theirthe
germinability
by shortening
the mean
advanced
the 8 dtheperiod
ofCytological
MC, but that
the indicated
observed that
changes
differed in
embryo
and endosperm
stricto
germination
time during
and reducing
spread.
studies
the catabolic
phase
of germination
sensu
tissues.
Completeduring
degradation
storageofprotein
andthat
lipidthebodies,
followed
by starch
were
found to
stricto advanced
the 8 dofperiod
MC, but
observed
changes
differedaccumulation
in embryo and
endosperm
carrotprotein
seeds. and
In the
the catabolic
changes
were restricted
to the
occur
the embryonic
radicles
of of
storage
lipidendosperm,
bodies, followed
by starch
accumulation
were found
to
tissues.inComplete
degradation
micropylar
enclosingradicles
the radicle,
where seeds.
extensive
of storage
cell walls,changes
partial degradation
of protein
occur in thearea
embryonic
of carrot
In breakdown
the endosperm,
the catabolic
were restricted
to the
bodies and area
no storage
lipidthe
hydrolysis
were noted.
Thebreakdown
time course
localization
these changes
suggested
that
micropylar
enclosing
radicle, where
extensive
ofand
storage
cell walls,ofpartial
degradation
of protein
weakening
endosperm
represented
a secondary
effect
ofcourse
priming,
which
was probably
controlled
stimulated
bodies and of
nothe
storage
lipid hydrolysis
were
noted. The
time
and
localization
of these
changes and
suggested
that
by the metabolically
advanced
embryonic
axis. The effect
benefits
of seed matriconditioning
have
been explained
on the
of the endosperm
represented
a secondary
of priming,
which was probably
controlled
and stimulated
weakening
the fine structure
observations.
© 1997 Annals
of Botany
Company
basis
have been
explained
on the
by
theofmetabolically
advanced
embryonic axis. The benefits of seed matriconditioning
basis of the fine structure observations.
© 1997 Annals of Botany Company
Key words: Daucus carota L., seed ultrastructure, matriconditioning, embryo/endosperm relation.
Key words: Daucus carota L., seed ultrastructure, matriconditioning, embryo/endosperm relation.
INTRODUCTION
of the age-related cellular and subcellular damage that
INTRODUCTION
of the accumulate
age-related during
cellularseed
and development
subcellular damage
that
Enhanced speed and synchrony of germination and in- could
and storage
accumulate
during1984;
seed Bray,
development
Enhanced
speed to
and
of germination
in- could
(b) an
(Burgass
and Powell,
1995); and storage
creased
tolerance
lowsynchrony
and high temperatures
are and
the well
andofPowell,
1984;
Bray,
1995);
and (b) lag
an
creased
low andofhigh
temperatures
are the seed
well (Burgass
advancement
various
prehydration
metabolic
events
during
the prolonged
known tolerance
beneficialtoeffects
advancement
of
various
prehydration
seed
of
metabolic
events
during
the
prolonged
lag
known
beneficial
effects
water uptake
treatments (Heydecker and Coolbear, 1977; Bradford, 1986; phase II (germination sensu stricto) of
(germination
sensuDell'Aquila
stricto) of and
the Bewley,
water uptake
treatments
(Heydecker
and Coolbear,
1977;carrot
Bradford,
(GrootIIand
Karssen, 1987;
1989;
Khan,
1992a).
Like other
Umbelliferae,
seed1986;
ger- phase
Karssen,
1987; Dell'Aquila
and Bewley, 1989;
Khan, 1992a).
Like time
otheris Umbelliferae,
carrot Osmoconseed ger- (Groot
Karssenand
et al.,
1991; Smith
and Cobb, 1991).
at harvest
slow and irregular.
mination
al., been
1991;observed
Smith and
Cobb,
1991).
at harvest
time seeds
is slow
and irregular.
Osmocon- Karssen
mination (OC)
ditioning
of carrot
improves
their germination
It has etalso
that
small,
endosperm-conditioning
(OC)
carrot seeds
improves
their germination
It has seeds
also been
observed
small,show
endosperm-con(celery,
carrot, that
tomato)
particularly
performance
by of
shortening
the mean
germination
time and taining
taining
seeds
(celery,
carrot,
tomato)
show
performance
by
shortening
the
mean
germination
time
and
lowering the spread (Brocklehurst and Dearman, 1983). beneficial responses to the OC treatments particularly
(Bradford,
lowering
spread (Brocklehurst
Dearman,
1983). beneficial
the Nonogaki,
OC treatments
(Bradford,
Recently, the
conditioning
of seeds withand
solid
matrix carriers
1986; van responses
der Toorn,to1989;
Matsushima
and
Recently, conditioning
of seeds
with solid
carriers
1986; van der
Toorn,
Nonogaki,
1992);
this1989;
suggests
that a Matsushima
decrease in and
the
(matriconditioning,
matric
priming,
MC) matrix
has been
de- Morahashi,
Morahashi, restraint
1992); this
suggests that
decrease in
the
(matriconditioning,
matric
priming,
been
de- mechanical
of thea endosperm
might
(' weakening')
veloped
(Eastin, 1990;
Khan
et al., MC)
1990, has
1992;
Khan,
mechanical
weakening') of the endosperm might
veloped
(Eastin,
1990;
Khan
et al.,Micro-Cel
1990, 1992;
Khan, explain
the restraint
effects of('priming.
1992). Priming
with
the solid
carrier,
E, synchroexplain
the
effects
of
priming.
1992).
Priming
with
the
solid
carrier,
Micro-Cel
E,
synchroThe objective of this study was to relate cytological data,
nizes and accelerates germination of seeds (including carrot),
The on
objective
of seeds (including
carrot),
of this
study waselectron
to relatemicroscope
cytologicalobserdata,
nizes and
accelerates
light and
transmission
more
effectively
thangermination
osmoconditioning.
As with OC,
the based
based ontolight
transmission
obsermore
effectively
than osmoconditioning.
with
OC, and
the vations,
benefits
of matriconditioning
are retained As
after
drying
the and
changes
occurringelectron
during microscope
the prolonged
lag
benefits of matriconditioning
are retained after drying and vations,
to theare
changes
occurring
the prolonged
lag
phase. There
apparently
onlyduring
two reports
describing
re-hydration
(Khan et al., 1990).
There
are which
apparently
two
reports describing
re-hydration
(Khan
et al.,
1990). of priming as a technique phase.
structural
changes
occuronly
during
osmoconditioning,
in
In spite of the
general
acceptance
changes which
occur
osmoconditioning,
in
spite of the seed
general
acceptance
of priming asbasis
a technique
1983) and celery
lettuce (Georghiou,
Psaras
andduring
Mirrakos,
is not structural
forInimproving
quality,
its physiological
andtocelery
(Georghiou,
Psaras1989),
and Mirrakos,
is not lettuce
for improving
seed It
quality,
its physiological
seeds (van
der Toorn,
and they1983)
relate
the
clearly
understood.
is widely
accepted thatbasis
OC allows
seeds (van and
der not
Toorn,
1989), andtissues.
they Moreover,
relate to the
clearly
It is the
widely
accepted
allows
the embryonic
no
seeds tounderstood.
progress through
prolonged
lag that
phaseOC
(phase
II) endosperm
and not
the embryonic
Moreover, no
seeds
to progress
thethe
prolonged
lag phase
(phasebut
II) endosperm
cytological data
are available
to datetissues.
for matriconditioned
of imbibition
thatthrough
prepares
radicle for
protrusion,
data are available to date for matriconditioned
of imbibition
thatofprepares
radicleprotrusion)
for protrusion,
but cytological
seeds.
phase IIIthe(radicle
(Karssen
prevents
the start
seeds.
of
phase
III
(radicle
protrusion)
(Karssen
prevents
the
start
In this study, attention was focused on the fine structural
et al., 1991). There are also reports of seed metabolic
In this study,
attention
was carrot
focused seeds
on theduring
fine structural
et
al., 1991).
are in
alsoosmotic
reportssolutions,
of seed metabolic
occurring
in the
matriactivity
duringThere
priming
including changes
changes occurring
in the carrot
during
matriactivity during
priming
in osmotic
solutions,
with Micro-Cel
E in seeds
order to
answer
the
of repair
and replicative
DNA,
rRNA,including
proteins conditioning
synthesis
with Micro-Cel
in order to answer
the
of repair
and replicative
DNA,
rRNA,
synthesis
(a) doesE degradation
of storage
following questions:
(Dell' Aquila
and Bewley,
1989; Bino
et al.,
1992;proteins
Ashraf conditioning
(a) embryonic
does degradation
of storage
following occur
questions:
(Dell'
Aquila
and
Bewley,
1989;
Bino
et 1995)
al., 1992;
Ashraf materials
in both the
axis and endosperm?;
and Bray,
1993;
Lanteri
et al.,
1993;
Bray,
and activity
in bothweakening
the embryonic
axis andaendosperm?;
andcertain
Bray, 1993;
Lanteri
et al., 1993;
Bray, protein
1995) and
activity
(b) does occur
endosperm
constitute
primary or
of
enzymes
involved
in sugar,
and
lipid materials
(b)
does
endosperm
weakening
constitute
a primary
or
of
certain
enzymes
involved
in
sugar,
protein
and
lipid
main effect of priming? and (c) can the benefits
of seed
catabolism {Smith and Cobb, 1991).
main
effect
of
priming?
and
(c)
can
the
benefits
of
seed
catabolism
{Smith
and
Cobb,
1991).
There are several indications that the following physio- matriconditioning be explained at the cellular level based on
explained at the cellular level based on
Theremechanisms
are several are
indications
thepriming:
following(a)physiochanges?
the fine structural be
logical
involved that
in seed
repair matriconditioning
logical mechanisms are involved in seed priming: (a) repair the fine structural changes?
© 1997 Annals of Botany Company
b0960370
0305-7364/97/050535 + 11 $25.00/0
© 1997 Annals of Botany Company
b0960370
0305-7364/97/050535 + 11 $25.00/0
Dawidowicz-Grzegorzewska~Ultrastructure
536
MATERIAL AND METHODS
of Matriconditioned Carrot Seeds
tetrazolium chloride (TTC) for 2 h at 25°C (Johnston et al.,
1986) and the intensity of staining visually assessed.
Plant material
Carrot (Daucus carota L. cv. Nantes)' seed' is a schizocarpic
fruit composed of an axillary linear embryo, enclosed by a
thick-walled living endosperm tissue which occupies the
bulk of the seed. Both former components are enclosed by
a thin, non-living seed coat. Primary (P) and secondary (S)
umbel 'seeds' were stored separately in paper bags, at
ambient temperature for no longer than 1 year. Seeds from
tertiary (T) umbels were not studied. The material was
collected in 1991 from Washington State University experimental fields near Pullman, W A (USA) and that in 1993
was supplied by Przedsi~biorstwo Nasiennictwa Ogrodniczego i Szkolkarstwa in Ozarow Mazowiecki, near
Warsaw, Poland.
M atriconditioning
Seeds from P and S umbels were matriconditioned
separately with Micro-Cel E (Manville, Filtration and
Minerals, Denver, Colorado). The main components of this
solid carrier are diatomaceous silica, hydrated lime and
water. Each 1g portion of seed was thoroughly mixed in a
100 ml glass jar with water and the solid carrier, and the jar
loosely capped. The ratio (by weight) of seeds : carrier: water
was 1 :0,5: 1'9, and is slightly modified from that proposed
by Khan et al. (1990). Jars were transferred to 15°C in
continuous white light for 2, 4, 6 and 8 d (2 jars per
treatment time). It was shown by Khan et al. (1990) that at
the end ofmatriconditioning the remaining water content of
the Micro-Cel E (287 %) was in equilibrium with the water
content of the seeds (88 %, on a dry weight basis), and
corresponded to a matric potential of -1,5 MPa.
Germination tests
Germinability was tested over a 10 d period for S umbel
seeds matriconditioned for 0 and 4 d only. For each
treatment, two lots of 100 seeds were incubated in Petri
dishes lined with filter paper and moistened with distilled
water, in constant white light or in darkness at 25°C. The
seeds were considered to have germinated when the radicle
protruded through the enclosing structures. Three parameters, TIO and T50 (time to 10 % and 50 % of final
germination) and final germination percentage, were used to
evaluate the matriconditioning effect. The results are
presented as mean (± s.d.) values from four experimental
replicates (total 800 seeds).
Embryo growth measurements and TTC test
Fifteen embryos (three replicates) were isolated very
gently from the enclosing structures after 0, 2, 4, 6 and 8 d
of matriconditioning from P and S umbel seeds. Their
length was measured using a micrometer and a Zeiss optical
microscope and mean values (±s.d.) presented. The same
embryos were then immersed in 0·2 % solution oftriphenyl
Electron and light microscopy
To compare structural changes during early growth of
seeds conditioned for 6 d and non-conditioned seeds, two
samples of 15 seeds each from S umbels were used for
germination in water at 25°C. Only seeds with the radical
just protruding from the endosperm were used. This required
approx. 24 h of imbibition for seeds conditioned for 6 d
and 72 h of imbibition for non-conditioned seeds. Small
blocks of tissues (1-2 mm 3), containing embryo and surrounding endosperm (without seed coat) were fixed in 3 %
glutaraldehyde in 0·05 M piperazine N-N bis ethanol sulphonic acid (PIPES) buffer (pH 7·2) for 20 h at 4°C,
washed in the same buffer and post-fixed in 1 % OS04 in
0·05 M cacodylate buffer (pH 7·2) for 2 h at room temperature. Dehydration was performed in a graded ethanolpropylene oxide series, followed by embedding in Spurr's
resin. Thin and semi-thin sections were obtained using a
Sorval Porter-Blum MT-2 ultramicrotome. Ultrathin sections on uncoated nickel grids were stained with watersaturated (7-7%) uranyl acetate for 2 hand 0-4% lead
citrate (with a few drops of 10 M NaOH) for 3-5 min or with
1 % KMn0 4 for 1-2 min. Finally, they were viewed with an
Hitachi 600 transmission electron microscope at 100 kV.
For light microscopy, sections were stained with 1%
toluidine blue 0 in 1 % borax, with or without 1 % Azur II
in 1 % borax.
RESULTS
Germinability of primed seeds
The germination rate of seeds was improved by 4 d of
matriconditioning (Table 1). The time to obtain 10% and
50 % of the final germination was considerably shortened in
primed seeds, in both light and darkness. As expected, the
final germination percentage was similar in conditioned and
non-conditioned seeds (Table 1).
Morphology of conditioned and non-conditioned embryos
At harvest time, the embryos differed in size depending on
the location of seeds on the primary (P) or secondary (S)
umbels. Embryos from S umbels were smaller than those
TABLE 1. Time to 10% (T10) and 50% (T50) of final
germination and the final germination percentage of seeds
matriconditioned (MC) for 4 d and control S umbel seeds.
Seeds were incubated in the light (L) or darkness (D) at
25°C over 10 d. Data represent the means (±s.d.) of four
replicates
MCD
MCL
Control D
Control L
T 10 (h)
T 50 (h)
Final germination (%)
9·6
15·8
33·6
36·0
96·0
100·8
61·6±0·80
65'6±0'25
69·0±0·50
66·5±0·20
no
no
Dawidowicz-Grzegorzewska-Ultrastructure of Matriconditioned Carrot Seeds
2. Intraseminal changes in embry o lengthfor P and S
umbel seeds during 8 d ofmatriconditioning at 15 °C in light.
Data represent the mean ( ±s.d.) of three replicates of 15
embry os each
TABLE
Days of
matriconditioning
o
4
o
4
6
8
Umbel order
Length (mm)
Primary
Primary
Secondary
Secondary
Secondary
Secondary
2·1 ±O·26
2·5±O·33
1·4±O·04
1·7±O·16
2·3±O·15
2·7±O·98
from P umbels (Table 2); nevertheless, both were morphologically mature, as indicated by the presence of differentiated cotyledons and an embryonic axis. During 8 d of
matriconditioning the embryos increased in size: both the
axis and the cotyledons of P and S seeds grew in length and
width (Fig. 1A-D). Table 2 shows that after 6 d of
conditioning embryos from S umbels achieved dimensions
typical of P umbels before conditioning.
TTC (Triphenyl- Tetrazolium Chloride) test
The histochemical localization of TTC-test stainability of
embryos isolated from control, non-treated and 2, 4 and 6 dmatriconditioned S umbel carrot seeds is shown in Fig.
1A-D. The data presented show that the intensity and
histological pattern of TIC staining developed gradually
throughout the embryo. All embryos isolated from nonconditioned S umbel seeds remained completely unstained
even after prolonged (8 h) incubation in TTC. Staining was
apparent after 2 d of matriconditioning and was limited to
the embryonic axes. After 4 d of mat ric priming the whole
embryonic axis and cotyledons (at least their basal part)
acquired a deep red colour after 1 h in TTC. Uniform and
A
B
537
intense staining of the whole axes and cotyledons was
observed after 6 d of conditioning. In contrast, embryos
isolated from non-conditioned P umbel seeds, acquired only
a pink colour in the axis or the cotyledons (not shown here).
General appearance of seeds before matriconditioning
Embryo. Three primary tissues: protoderm, procambium
and ground parenchyma were present in the quiescent
carrot embryo. Ground parenchyma in the cotyledons was
differentiated into future palisade and spongy tissues. All
embryo cells had thin primary cell walls enclosing the
protoplast which had characteristics typical of mature,
dormant seeds (Dawidowicz-Grzegorzewska, 1989; Dawidowicz-Grzegorzewska and Podstolski, 1992). Storage protein and lipid bodies occupied most of the cell's volume (Fig.
2). Protein bodies varied in diameter from 0·3 to 2'0,um,
depending on the tissue being studied; the smallest were
found in protodermal and procambial cells, whereas larger
ones were observed in the ground parenchyma of the
cotyledons. Protein bodies in both P and S umbel seed
contained none, one or two globoid inclusions with crystals ;
occasionally, small calcium oxalate crystals were also found,
similar to those shown by Spitzer and Lott (1982). Such
morphology of protein bodies suggests that they had
attained their final, mature stage of development. Lipid
bodies were very abundant and arranged in shells around
the protein bodies and along the plasmalemma (Fig. 2). An
irregularly shaped, condensed heterochromatic nucleus with
a small and dense fibrillar nucleolus was observed between
storage organelles (Fig. 2). Starch was negligible and only
occasionally present in a few amyloplasts. The protoplast
was not abundant in other organelles, such as mitochondria,
ER and dictyosomes, which had a reduced structure. Ribosomes were present as mono somes, previously described
for other dormant seeds (Dawidowicz-Grzegorzewska,
1989 ; Dawidowicz-Grzegorzewska and Podstolski, 1992).
Endosperm. The embryo was surrounded by a thick
c
o
FrG. I. Embryos isolated from S umbel seeds during matriconditioning (M C) and stained with TTC: unconditioned (A) , after 2 (8), 4 (C) and
6 d of MC (D). Note that embryos grow during the treatment and their staining develops, beginning on the second day of MC, from the axis (8)
to the cotyledons (C and D). Magnification x 30.
538
Dawidowicz-Grzegorzewska-Ultrastructure of Matriconditioned Carrot Seeds
FIG. 2. Subcellular details of a ground meristem cell in the embryo axis of a non-matriconditioned S umbel seed. Note the presence of several
storage protein (Pb) and lipid (I b) bodies. The nucleus (N) and nucleolus (Nu) are relatively small and compact and the cell walls (cw) are thin.
Magnification x 10400.
FIG.
3. Subcellular details of the endosperm cell from a non-matriconditioned S umbel seed, showing cell walls thickened with storage materials
(cw), prominent protein bodies (pb) with inclusions (probably globoids) and numerous lipid bodies (Ib). Magnificat ion x 10000.
endosperm (up to 15 cell layers), which was considerably
thinner in the micropylar region around the root cap and
the adjacent part of the radicle (Fig. 7). Unlike the embryo,
the endosperm stored carbohydrates, presumably as /1mannans (Halmer, 1985) in the form of thickened primary
cell walls rather than as starch . Similar to the embryo, most
of the endosperm cell's volume was occupied by storage
protein and lipid bodies (Fig. 3). Protein bodies were larger
than those in the embryo (up to 8 JIm diameter) and
contained two types of inclusions; crystal-containing globoids and prominent druse-shaped calcium oxalate crystals.
as indicated by Spitzer and Lott (1982). Two types of
endosperm cells were observed: near the seed coat side the
protein bodies contained were small. with numerous
globoids but devoid of calcium oxalate. whereas in deeper
layers the protein bodies were large. with few globoids and.
Dawidowicz-Grzegorzewska-Ultrastructure of Matriconditioned Carrot Seeds
539
FIG. 4. The ground meristem cell in the embryo axis of an S umbel seed after 4 d of Me. Note the presence of several degraded protein bodies
(dpb) bearing electron-dense (dark) remnants of storage material (double arrows) and less numerous (in comparison with Fig. 2) lipid bodies (Ib).
Considerable cytoplasmic detail is evident: mitochondria (m) and glyoxysomes (single arrows) in contact with lipid bodies, the appearance of
starch (s) in plastids (P), lobed nucleus with decondensed (in comparison with Fig. 2) chromatin and the presence of ER profiles (arrowheads).
Magnification x 10000.
FIG. 5. The details of a ground meristem cell in the embryo axis of an S umbel seed after 6 d of MC. Note the presence of protein body-derived
vacuole (V), with small remnants of storage protein (double arrowed) and the plastid (P), with starch (s) surrounded by a electron-dense material,
presumably containing the complexes of starch-synthesizing enzymes. Magnification x 50000.
FIG. 6. The same cell (shown in Fig. 5) with nucleus (N) containing diffused chromatin, clearly visible envelope with associated heterochromatin
and prominent nucleolus (Nu) with differentiated (at margins) granular component (gc). Note also the presence of rough ER (arrowhead).
Magnification x 18000.
as a rule, contained one prominent calcium oxalate crystal
(Fig.lO). Throughout, endosperm cell mitochondria and
proplastids had a reduced fine structure and the ER and
Golgi structures were not recognizable. In general, the
whole endosperm had the characteristics of a dormant, but
viable tissue.
540
Dawidowicz-Grzegorzewska-Ultrastructure of Matriconditioned Carrot Seeds
FIG. 7. Longitudinal view of the embryo (Eb) enclosed by the endosperm (En) (half of the endosperm is removed to facilitate penetration of
chemicals) in the non-matriconditioned S umbel seed. Both seed parts are darkly stained with toluidine blue owing to the presence of storage
materials. Magnification x 120.
FIG. 8. Longitudinal view of the embryo axis (Eb) and enclosing endosperm (En) of an S umbel seed after 6 d of Me. Note the weakening of
the endosperm (En) which encloses the radicle tip (both parts are devoid of storage materials and unstained). Magnification x 120.
FIG. 9. A detailed view of the endosperm (En) enclosing the radicle part of the embryo (Eb) S umbel seed after 4 d of Me. Note the presence
of three parts of endosperm responding differently to Me, marked with a single arrow, double arrows and an asterix, and further detailed in Figs
10, 11 and 12 respectively. Magnification x 320.
FIG. 10. Magnified' single arrow' marked part of the endosperm from Fig. 9. This appearance is typical for most of the endosperm, e.g.
accompanying the upper part of the axis and the cotyledons. Note the presence of abundant protein bodies (pb) with globoid (arrowhead) or
oxalate crystal (oc) inclusions surrounded by numerous lipid bodies (grey vesicular background) and of thick storage cell walls (cw).
Magnification x 1600.
FIG. II. Magnified' double arrow' marked region of the endosperm in Fig. 9. Note the presence of thin, hydrolyzed cell walls until the point of
cell separation (hydrolyzed middle lamellae, arrowhead). Protein bodies are also hydrolyzed and converted into vacuoles (V) with remnants of
storage proteins around a limiting membrane (double arrowhead), and lipid bodies are still present (grey, vesicular background). Magnification x 1600.
FIG. 12. Magnified' asterix' marked part of the endosperm (En) in Fig. 9. Note the presence of quiescent-type endosperm cells posse sing storage
protein (pb) and lipid bodies. Note especially the presence of cracks in the radial cell walls (arrowhead) probably arising in response to the
expansion force exerted by the growing embryo. Magnification x 1600.
General appearance of seeds during 6 d of
matriconditioning
Embryo. In S umbel seeds, the first fine structural changes
were observed in the radicle and lower hypocotyl cells, after
2 d of conditioning. Degradation of storage protein and
lipid bodies was initiated and the process progressed
gradually from the protoderm to the ground meristem and
basipetally towards the cotyledons. During the observed
6 d period of conditioning, protein bodies were first converted into several small vacuoles containing some flocculent
residual storage proteins (Fig. 4). Gradually, these vacuoles
Dawidowicz-Grzegorzewska-Ultrastructure of Matriconditioned Carrot Seeds
541
A
B
o
FIG. 13. Time course of embryo-stimulated 'weakening' of endosperm in S umbel seeds during 6 d of MC. Cross-sectional view of embryo axis
(Eb) and endosperm (En). A, 0 d ofMC~note the dark staining of both the endosperm and the embryo, due to the presence of storage materials.
B, 2 d of MC~note that 3 to 4 layers of endosperm surrounding the axis are devoid of storage materials and remain unstained. C, 4 d of
MC~note that endosperm weakening has progressed and that degradation of storage materials on the lower side of the endosperm is more
advanced than on the upper side. D, 6 d of MC~the endosperm is weakened on both sides of embryo. Magnification x 180.
fused and/or disintegrated within the protoplast. As a
rule, disintegration of storage lipid bodies followed that of
protein bodies. During conditioning the number of storage
lipid bodies decreased; concomitantly, glyoxysomes proliferated (Fig. 4) and prominent starch grains appeared in
amyloplasts (Figs 4 and 5). Simultaneously, it was observed
that rough ER, cytosolic polysomes, mitochondria and
Golgi structures were well developed (Fig. 6). Important
changes in the fine structure of the nucleus were also
observed; the size of both the nucleus and the nucleolus
increased compared to those in quiescent seeds (compare
Fig. 6 with Fig. 2), chromatin dispersed, the granular
component in the nucleolus differentiated and, finally,
mitotic divisions appeared in the radicle and hypocotyl
tissues (not shown here). As a result of mitoses, groups of 4,
6 or 8-cells were formed; these cells, however, were not able
to elongate, so the radicle remained entirely enclosed by the
endosperm after the whole period of Me (Figs 8 and 9).
Similar changes to those stated for S umbel seed embryos
were seen in matriconditioned P umbel seeds.
Endosperm. The fine structural changes taking place in the
endosperm during the 6 d period of conditioning were
limited to its micropylar end around the radicle and the
root cap. They were initiated after 3 or 4 d of conditioning,
and after the neighbouring embryonic tissues had already
been 'activated'. The first changes appeared in the cells
located close to the embryo and progressed radially towards
the seed coat (Fig. 3 A-D). They involved the solubilization
of the storage protein body matrix - the remnants of storage
proteins can be seen at their margins (Fig. 11). Such partly
degraded protein bodies sometimes fused and formed
vacuoles, which, as a rule, remained enclosed by a limiting
membrane. The next step observed was the hydrolysis of the
thick primary cell walls and of the middle lamellae, until the
point of cell separation (Fig. 11). It only occurred in those
cells which were already advanced in the course of
proteolysis. The number, morphology and structural characteristics of reserve lipid bodies were not changed during the
whole period of conditioning, unlike those in the embryo.
The described changes were much less advanced at the seed
coat side than in the endosperm closest to the embryo root
tip tissues (Fig. 13B and C). Finally, after the whole period
of condition.ing the endosperm tissue was considerably
'weakened' at the micropylar end (Figs 8 and 9) due to
the solubilization of protein bodies and thinning of the cell
walls, whereas the rest of the cytoplasmic organelles
(including the nucleus) retained characteristics typical of
quiescent seeds (Fig. 10). The fine structure of the cells in the
remaining upper part of the endosperm (which constitutes
about 90 % of the tissue) was not changed during the
priming, except for the presence of ruptured radial cell walls
along the embryo (Fig. 12). The content of such cracked
542
Dawidowicz-Grzegorzewska-Ultrastructure of Matriconditioned Carrot Seeds
FIG. 14. Subcellular details of ground meristem cell from a radicle which is just protruding (at 25 DC, 72 h of full hydration) from a nonmatriconditioned S umbel seed. Note the presence of a vacuole (V) derived from degraded protein body, bearing the remnants of storage
protein (double arrow). Lipid bodies (lb) are abundant, as in quiescent seeds, plastids (P) are devoid of starch and glyoxysomes are absent. The
nucleus (N), with compact chromatin and small, dense nucleolus (Nu), is devoid of granular component. Magnification x 12000.
FIG. 15. Subcellular details of ground meristem cell from the radicle of a germinating (at 25 DC , 24 h of full hydration) S umbel seed following
6 d of matriconditioning. Note that most of storage protein and lipid bodies are degraded, starch (s) is present in plastids (P), and new vacuoles
(V) have formed. Profiles of rER (arrowhead) are present, the nucleus (N) and nucleolus (Nu) are large, the latter with differentiated granular
(gc) and fibrillar (fc) components, and the mitochondria (m) have well developed cristae. Magnification x 16000.
cells was sometimes released through the cell wall's fissures
into the space between the embryo and the endosperm.
Most probably the cell walls cracked in response to the
expansion force exerted by the embryo growing during
conditioning. The number of layers with ruptured cell walls
rose during the matric priming.
General appearance of germinating primed and unprimed
seeds
Two lots of non-conditioned and 6 d-conditioned S umbel
seeds were compared during germination at 25°C. Attention
focused on the beginning of radicle protrusion through the
enclosing endosperm which occurred after 24 hand 72-96 h
Dawidowicz-Grzegorzewska-Ultrastructure of Matriconditioned Carrot Seeds
of imbibition for primed and unprimed seeds, respectively.
The most important differences between non-conditioned
and conditioned seeds concerned the fate of storage
materials in the radicle cells (compare Figs 14 and 15).
Rapid protrusion of the radicle from primed seeds was
possible because matriconditioning had facilitated the
degradation of storage protein and lipids in most of the
radicle cells (Fig. 15), and vacuolation progressed very
rapidly in response to an unlimited supply of water.
Simultaneously, a decrease in the number and size of
previously accumulated starch grains was observed (data
not shown). This observation suggests that the growth
potential of the embryo was created by the accumulation of
osmotically active products of starch hydrolysis in newly
formed vacuoles. Unexpectedly, the degradation of storage
materials at the time of radicle protrusion, was shown to be
incomplete and less advanced in unprimed than in primed
seeds (compare Figs 14 and 15). At the beginning of embryo
growth of unprimed seeds (at 72 h of imbibition) vacuoles
were derived directly from the partly degraded protein
bodies (Fig. 14), and the growth potential created by the
osmotically active products of proteolysis taking place
inside these storage organelles. Contrary to primed seeds,
lipid bodies remained present and were equally abundant as
in the quiescent state, starch was absent, and the nucleus
and nucleolus remained condensed and small (Fig. 14).
DISCUSSION
The results presented here confirm earlier data (Khan et at.,
1990) which indicated that matric priming with Micro-Cel E
accelerates germination of carrot seeds, since the T50 value
of primed seeds was effectively reduced compared to the
non-primed seed controls (Table 1). The final germination
percentage was similar in primed and non-primed seeds as
expected, and variable, due to the frequent (up to 30%)
presence of aborted (embryo-less) seeds.
The large spread of germination times in Umbelliferae
seeds results from the umbel order at harvest time. Taking
into account the fact that germination of primed seeds is
more uniform than that of the unprimed ones, it appears
that priming allows the developmental differences between
primary (P), secondary (S) and tertiary (T) umbel seeds to
diminish. This is confirmed by the observation that the
embryos from S umbel seeds after 6 d of matriconditioning
attained the size typical for P umbel seeds at the moment of
harvest (Table 2). Similar data have already been reported
for carrot (Gray, Steckel and Hands, 1990) and celery
seeds (van der Toorn, 1989) conditioned in polyethylene
glycol solutions: in both cases embryos doubled or even
tripled in size during treatment. Moreover, the data for
carrot seeds presented here indicate that mitotic activity
occurs in the embryo axis tissues during the priming treatment and confirms the earlier data of Gray et at. (1990)
which showed that the cell number per embryo doubled
with priming. In this respect, carrot and celery seeds present
a notable exception, because during the priming of seeds
other than Umbelliferae, cell divisions have not been
observed (e.g. Clarke and James, 1991). It has been shown,
however, that S-phase DNA replicative activity occurs upon
543
priming of tomato and pepper seeds, resulting in an increase
in the number of embryo root tip cells having 4C DNA
content, but such cells remained at the G 2 phase of the cell
cycle and were not able to enter mitosis (Bino et at., 1992;
Lanteri et at., 1993).
The cytological data presented here are consistent with
the hypothesis that MC permits the advancement of some
catabolic events specific to lag phase II (germination sensu
stricto) which prepare the radicle for protrusion; however,
those events which lead to the cell elongation component of
radicle protrusion are prevented.
The morphological observations made on whole carrot
embryos isolated from P and S umbel seeds using TTC gave
an estimate of their viability and an indication of dehydrogenase activity (including malate dehydrogenase),
during 8 d of matriconditioning. The intensity of embryo
staining increased and developed gradually throughout the
embryo, starting in the radicle and eventually reaching the
cotyledons (Fig. 1), suggesting a rise in activity of
dehydrogenases and in respiration rate during the matric
priming. The observation that 100 % of unprimed S umbel
seed embryos remained unstained in TTC in spite of the fact
that they were viable (since such seeds do germinate)
questions the conventionally, and widely used interpretation, of the TTC test as an unequivocal measure of
viability (Matthews, 1981). The possibility cannot be excluded, however, that the system of dehydrogenases is underdeveloped in the seeds dispersed from S, lately maturating
umbels. This supposition is confirmed here by the observation that embryos freshly isolated from unconditioned
P umbel carrot seeds did stain in TTC, at least in their axial
part.
The reported cytological study shows that during matriconditioning of carrot seeds important progress in the
mobilization and degradation of storage materials occurred
in the embryo and endosperm tissues. The fine structural
changes started first in the embryo, where they were limited
to the radicle and to the adjacent lower part of the
hypocotyl. Both types of storage organelle present in the
embryo, e.g. bearing proteins and lipids, underwent degradation (Figs 2-6). Storage protein bodies were converted
into electron transparent vacuoles, which fused or disintegrated. The number of storage lipid bodies decreased;
concomitantly, glyoxysomes and rough ER proliferated in
their close vicinity and finally, the starch grains appeared in
the amyloplasts. The degradation of lipids and the accompanying gluconeogenesis requires extensive de novo synthesis
of enzymes and a substantial part of this metabolic sequence
takes place in glyoxysomes which contain enzymes for fJoxidation and the glyoxalate cycle (Trelease, 1984). The
overall fine structural changes (including those in the
nucleus) suggest that transcriptional, translational and
biosynthetic activities related to the catabolism and interconversions of storage materials are restored in such
partially hydrated, matriconditioned seeds. The structural
changes described were similar to those already reported as
being peculiar to the catabolic phase of germination sensu
stricto in fully hydrated seeds (Dawidowicz-Grzegorzewska,
1989). Thus, during matriconditioning many of the normal
catabolic events of germination sensu stricto are going on,
544
Dawidowicz-Grzegorzewska-Ultrastructure of Matriconditioned Carrot Seeds
with the exception of processes associated with the early
elongational growth of the radicle, which are prevented. It
must also be noted that the lack of other reports on the
cytological changes that take place in the embryo during
seed priming means that a broader discussion of the findings
reported here is not possible.
There are biochemical changes which show that some
processes specific to the catabolic phase of germination
sensu stricto progress during priming. It has been found by
Dell'Aquila and Bewley (1989) that in the embryonic axes of
pea (Pisum sativum L.) seeds, proteins normally associated
with germination are synthesized upon priming in PEG.
Also during the osmoconditioning of endosperm-containing
pepper (Capsicum annuum L.) seeds (Smith and Cobb,
1991), increases in the soluble protein content and in the
activity of enzymes such as isocitrate lyase and glucose-6phosphate dehydrogenase were shown to occur in whole
seeds.
When primed and non-primed S umbel seeds were
incubated in water, the consequences of matriconditioning
were apparent at the cellular level at an early stage of radicle
growth (Figs 14 and 15). This growth occurred sooner in
the metabolically advanced, primed seeds than in unprimed
seeds but, unexpectedly, there were important differences in
the fine structure of mitochondria, in the fate of the storage
materials and in the related mode of vacuole formation in
the earliest cells of the radicle/hypocotyl boundary to
elongate. It was deduced from microscopic pictures that in
primed seeds the water potential for the growth of these cells
resulted from the accumulation in newly formed vacuoles of
osmotically active products of first starch and then lipid
hydrolysis. Such a pattern of early elongational growth is
typical for normally developed, mature seeds (DawidowiczGrzegorzewska, 1981). In unprimed S umbel seeds, however,
the vacuoles in such elongating cells were formed from
partially degraded protein bodies, whereas storage lipid
bodies were not mobilized. It should also be added that the
mode of early growth of unprimed, P umbel carrot seeds
resembled that of the primed, S ones (preliminary data, not
shown). So, it is reasonable to assume that seeds from S
(and probably also from T) umbels are physiologically
underdeveloped at the time of harvest, at least regarding
their ability to catabolize storage lipids. Taking into account
that the majority of Umbelliferae seeds are formed on
secondary or tertiary umbels, such delayed and partial
degradation of reserves at the beginning of seedling
establishment could explain why young seedlings cultured
from unprimed seeds are weaker and grow more slowly than
those from primed seeds (Khan et at., 1992).
The fine structural changes occurring in the endosperm
during the matriconditioning of carrot seeds started later
than those in the embryo and were limited to its micropylar,
radicle enclosing part, which was clearly morphologically
distinct from the rest of the endosperm (Figs 7-13). The first
observed changes involved the partial hydrolysis of storage
protein bodies which, in contrast to the embryo, remained
enclosed by a limiting membrane and never disintegrated.
The following degradation of the thick primary storage cell
walls occurred until the point of cell separation, resembling
the pattern of cell wall hydrolysis found in maturation-
associated fruit dehiscence (Meakin and Roberts, 1990).
The structural features and the timing of the observed
storage protein and cell wall hydrolysis suggested their
autolytic character was probably due to the activity of
enzymes already pre-existing inside the protein bodies and
in the cell wall of the quiescent endosperm cells. Similar
pictures were obtained for the resting date palm seeds
(Chandra Sekhar and DeMason, 1990), where agalactosidase, engaged in the storage cell wall breakdown,
was immunocytochemically localized both inside the protein
bodies and in the cell wall fraction. Moreover, the timing
and localization of the changes described here for carrot
seed endosperm suggest very strongly that the stimulation
comes from the embryo: the changes started at, and were
much more advanced in the close vicinity of the embryonal
radicle. In contrast to the embryo, the biosynthetic activities
related to the storage lipid catabolism which require de novo
enzyme synthesis (Trelease, 1984) were not initiated during
the matric priming period. The latter could be judged from
the unchanged number and appearance of reserve lipid
bodies, the unchanged ultrastructure of the cell nucleus,
which retained characteristics typical of' inactive', quiescent
seeds, as well as by the absence of such organelles as
glyoxysomes and rough ER. In conclusion, some catabolic
changes, which advance the seed in germinative metabolism,
were also taking place in the micropylar part of endosperm
tissue but, in the light of the data presented, seem to be a
secondary effect induced, and controlled by the already
advanced embryonic axis. This conclusion is only partly
consistent with that reported for tomato and celery seeds
(Groot and Karssen, 1987; van der Toorn, 1989; Karssen et
at., 1991; Nanogaki et at., 1992), where storage cell wall
degradation in the micropylar end was also reported (as
a result of induced endo p-mannanase activity), but was
interpreted by the above authors as a main or primary effect
of seed osmoconditioning in PEG solutions. Moreover, in
those studies the evolution of storage materials located in
the embryo tissues during the priming period was not
investigated.
The control of reserve degradation in endospermcontaining dicotyledonous seeds is very poorly understood
with respect to fully or partially hydrated seeds submitted to
different priming treatments (Bewley and Black, 1985).
There are several reports indicating that the pattern of
degradation is determined by hormonal signals produced by
the embryo, the endosperm or both (Jacobsen and Pressman,
1979; Bewley and Black, 1985). The data presented here
seem to suggest that during matric priming the stimulus for
the breakdown of carrot endosperm derives from the
embryo, but it remains to be verified on de-embryonated
endosperms whether the close contact between embryo and
endosperm is an obligatory one and to what extent the
endosperm participates in its own mobilization.
CONCLUSIONS
In the light of the data presented the known benefits of seed
matriconditioning, such as enhanced speed and synchrony
of germination can be interpreted as follows: (a) during
matriconditioning with Micro-Cel E some developmental
Dawidowicz-Grzegorzewska-Ultrastructure of Matriconditioned Carrot Seeds
advance occurs in both the embryo axis and endosperm; (b)
matriconditioned S umbel seed embryos attain the dimensions of those from non-conditioned P umbel seed, thus
explaining the observed uniformity of the early postgerminative growth of seedlings; (c) ultrastructural observations performed during the 6 d period of matriconditioning indicated that breakdown and interconversions
of storage materials, specific to the catabolic phase of
germination sensu stricto, were taking place both in the
embryo axis and in the micropylar part of the radicle
enclosing endosperm. The degree of advancement of these
changes was different in both these parts of the seed and
concerned different storage materials: in the embryo axis a
complete degradation of storage protein and lipid bodies
occurred, whereas in the endosperm only the cell wall
polysaccharides were degraded completely, in contrast to
the storage protein bodies (partial hydrolysis) and to the
lipid bodies (no degradation); and (4) weakening of the
endosperm, one result of matric priming, constitutes a
secondary efffect.
ACKNOWLEDGEMENTS
The author thanks Professors Irena Szumiel and Stanislaw
Lewak for critical reading of the manuscript. This work was
supported by a Grant No.6 P 20400405 from the state
Committee for Scientific Research (KBN), Poland.
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