Journal of Experimental Botany, Vol. 51, No. 343, pp. 167–175, February 2000
Maternal genotype influences pea seed size by
controlling both mitotic activity during early
embryogenesis and final endoreduplication
level/cotyledon cell size in mature seed
Claire Lemontey1, Claire Mousset-Déclas2,4, Nathalie Munier-Jolain3 and Jean-Pierre Boutin1
1 INRA, Laboratoire de Recherche sur le Métabolisme et la Nutrition des Plantes, Route de Saint-Cyr,
78026 Versailles cedex, France
2 INRA, Unité de Recherche en Génétique et Amélioration des Plantes, BV 1540, 21034 Dijon cedex, France
3 INRA, Station d’Agronomie, BV 1540, 21034 Dijon Cedex, France
Received 15 September 1999; Accepted 21 September 1999
Abstract
When reciprocal crosses are made between different
pea genotypes, there is a strong maternal influence
on mature seed size of the reciprocal hybrids, i.e. their
dry weights are similar to that of seeds obtained from
their maternal parents. Reciprocal crosses between
pea varieties having very different mature seed sizes
were used to investigate how the maternal genotype
controls seed development and mature seed size. The
differences in dry seed weight between genotypes and
reciprocal hybrids reflected differences in both cotyledon cell number and mean cell volume, and the maternal control on the establishment of these two traits
was investigated. Using flow cytometry, data relative
to endoreduplication kinetics in cotyledons during the
transition between the cell division phase and maturation were obtained. The appearance of nuclei having
an 8C DNA content indicates the initiation of the endoreduplication phenomenon and thus the end of the cell
division phase. It was shown that the duration of the
cell division phase was the same in the reciprocal
hybrids, its value being intermediate between those
recorded for their maternal parents. This result indicates that the timing of development of the embryo is
not under maternal control, but depends on its own
genotype. Consequently, maternal genotype must
influence the mitotic rate during the cell division phase
to achieve differences in cell number found in the
cotyledons of mature F1-reciprocal hybrids. The final
level of endoreduplication in cotyledons of mature
seeds was also investigated. This study showed that
there is a close relationship (r2=0.919) between the
endoreduplication level in mature cotyledons and seed
dry weight or mean volume of cotyledon cells, suggesting that both maternal and non-maternal factors
could control the number of endoreduplicating cycles
in the cotyledons and, hypothetically, the cotyledon
cell size.
Key words: Cell division, cell size, endoreduplication, flow
cytometry, maternal influence, Pisum sativum.
Introduction
Pea seed development has already been described in many
studies (Bain and Mercer, 1966; Smith, 1973; Hedley and
Ambrose, 1980). It can be divided into three distinct
phases. In the first, the cell division phase, the cotyledon
cells actively divide. In the second phase, maturation, the
cotyledon cells expand, and reserve compounds (starch
and proteins) are stored. The third phase concerns seed
desiccation. At the end of the initial phase of development,
the number of cells in the cotyledons is established (Smith,
1973). In legume seeds, cotyledons represent the major
storage organ, as the endosperm is restricted to a nutrientrich apoplastic liquid which is almost totally resorbed at
the beginning of the maturation (Marinos, 1970). Studies
in different legume species such as faba bean (Davies,
4 To whom correspondence should be addressed. Fax: +33 3 8063 3263. E-mail: [email protected]
© Oxford University Press 2000
168 Lemontey et al.
1977), pea (Davies, 1975) and soybean ( Egli et al., 1981)
have established a positive correlation between cotyledon
cell number and mature seed size. The number of cells
formed in the cotyledons determines the capacity of the
storage organ to accumulate dry matter (Munier-Jolain
and Ney, 1998). Some previous studies of parental effects
have shown that the maternal genotype influences mature
seed mass in pea (Davies, 1975). However, the way by
which the maternal genotype affects seed development is
not understood.
The presence of cells of different ploidy levels in somatic
tissues is called ‘endoreduplication’ or ‘polyteny’. In contrast to dividing cells, endoreduplicating cells are not
believed to undergo mitosis, and in such cells, nuclear
DNA content successively doubles from 2C to 4C to 8C
to 16C etc., where C is the haploid DNA content per
nucleus. Endoreduplication was first described during
seed development. In maize developing endosperm, the
average DNA content per nucleus increases sharply, as
the mitotic index decreases. This increase can reach levels
of 384C in some individual nuclei (Schweizer et al., 1995).
During the early period of field pea (Pisum arvense) seed
development, the DNA content of the cells remains at
the diploid level until cell division is complete, after which
it begins to increase (Smith, 1973). Using microdensitometry, a C-value of 64 has been measured in cotyledon
cells in two pea genotypes (Davies and Brewster, 1975).
In Arabidopsis thaliana, endoreduplication occurs in cells
of the hypocotyl during the elongation of this organ, and
reaches a 16C value in dark-grown seedlings (Gendreau
et al., 1997). Although endoreduplication has been
already described in several plant species, its significance
still remains uncertain. It is often related to nuclear
genome size and/or cellular dimension (Galbraith et al.,
1991; Melaragno et al., 1993; Gendreau et al., 1998). This
suggests that the endoreduplication phenomenon may be
related to the mature seed size, and may be more important in large seeds than in small seeds.
In order to investigate how the maternal genotype
controls seed development and mature seed size, reciprocal crosses between four varieties of pea having different
seed sizes were used. The stage of appearance of the
endoreduplication phenomenon in cotyledon cells was
investigated during the transition period between cell
division phase and maturation using flow cytometry.
Next, the endoreduplication level in the mature seed of
nine pea varieties and hybrids was described in order to
study the relationship between this trait and cotyledon
cell number and cotyledon cell volume.
Materials and methods
Plant material and growing conditions
Garden pea (Pisum sativum L.) is a diploid (2n=14) and
autogamous plant, so varieties used are considered as pure
homozygous lines. Five varieties were used, which show large
differences in their mature seed weights: cvs GSP6, Cation and
Frisson have a low seed weight (respectively 58, 180 and 247 mg
in non-limiting conditions) whereas cvs Solara and Imposant
have a high seed weight (413 and 548 mg). Plants were grown
in pots filled with expanded clay in the greenhouse during the
spring of 1996 and 1997. They were supplied daily with a
complete nutrient solution (Lesaint and Coı̈c, 1983). Reciprocal
crosses were made manually between cvs Frisson and Solara,
and cvs Cation and Imposant, on floral buds before the natural
self-pollination occurred. No more than two pods were grown
on each plant, so that the supply of nutrients to the pod was
never limiting for its growth. Temperature variations were
measured from the beginning of flowering until sampling. Time
was expressed in cumulative degree-days after pollination (°C
DAP), using 0 °C as the base temperature ( Etévé and Derieux,
1982). Seeds were sampled first at 150 °C DAP, and then 1, 2,
3, 4, and 5 d after this date. Seed coats and apoplastic liquid
were removed, and embryos were weighed and immediately
frozen in liquid nitrogen, and then conserved at −80 °C. Some
embryos were fixed in acetic alcohol (ethanol5glacial acetic
acid, 351, by vol.) and preserved in a refrigerator (4 °C ). Some
seeds were left to maturity and stored dry. The cultivar GSP6,
which has a very small seed weight, was only used for studies
carried out on mature cotyledons.
Cell volume
Mature seeds were soaked in distilled water for 1 night at 4 °C,
then the seed coat and the embryonic axis were removed.
Cotyledons were cut in small pieces using a razor blade, and
were immersed in an enzymatic solution (sorbitol 0.45 M;
MgCl 10 mM; KH PO 1 mM; MES 20 mM; MacerozymeB
2
2 4
R-10 1%; pH 5.6) under vacuum conditions for 15 min. The
samples were then macerated at 37 °C for 3 or 4 d. Macerated
cells were separated on a 200 mm nylon mesh to obtain a
homogeneous 10 ml suspension. A 500 ml aliquot was analysed
using a CoulterB Multisizer II (Coulter Electronics Limited ),
which measured the exact volume of the cells, classified the cell
population according to this measure, and calculated the mean
cell volume of the sample.
Cell number
After the cell volume analysis, the remaining cell suspension
was centrifuged, and the volume of the pellet was measured
using a water displacement method. The mean cell number per
cotyledon was estimated according to the two equations below,
supposing that the entire volume of the cells is composed of
insoluble products (cell wall, starch granules, protein bodies):
cotyledon volume=pellet volume×
suspension volume
suspension volume-aliquot
cell number per cotyledon=
cotyledon volume
mean cell volume
Flow cytometry analysis
Immature embryos fixed in acetic alcohol were rinsed in distilled
water for 10 min under vacuum conditions. For mature seeds,
the seeds were immersed in distilled water for 1 night at 4 °C,
then the seed coat was removed. When possible (for embryos
sampled after 190 °C DAP), the cotyledons and the embryo
axis were analysed separately. The material (cotyledons or
Maternal genotype influences pea seed size 169
embryonic axis) was chopped using a sharp razor blade in
about 2 ml of nucleus isolation buffer (Gilissen et al., 1993) to
release the nuclei. The suspension was filtered through a 40 mm
nylon mesh, and DAPI (4∞,6-diamidino-2-phenylindole: A-T
binding specific fluorochrome) was added to the filtrate to a
final concentration of 1 mg ml−1. The DNA content of the
isolated nuclei suspensions were analysed using a Partec PAS-II
flow cytometer equipped with a HBO-100 W mercury lamp and
a dichroic mirror (TK420). The data were plotted on a semilogarithmic scale. In this way, the histogram peaks from 2C to
128C are evenly distributed along the abscissa. Integrals of each
peak in the histograms were obtained using the built-in software
Partec DPAC V2.0. Calibration was conducted with nuclei of
expanded leaves of Pisum sativum cv. Frisson. For each
measurement, at least 3000 nuclei were analysed. For each
genotype and sampling date, at least three seeds were analysed.
At least eight mature seeds were analysed for each genotype.
The mean C value of a sample is calculated according to the
equation:
C ×N
i
i
Mean C value=∑n
i=1 N
sample
with n: number of peaks of DNA content of the sample; C : C
i
value in the nuclei of the peak n ; N : number of nuclei in the
i i
peak n ; N
: number of nuclei in all the peaks of the sample.
i sample
The differences in seed dry weight reflected differences
in cotyledon cell number and mean volume, as shown in
Table 1. Seed size appeared positively correlated with
cotyledon cell number and volume. A strong maternal
influence was noticeable on both traits, as differences in
cotyledon cell number and mean volume between reciprocal hybrids were always significant ( Table 1). Cotyledon
cell number was the factor which appeared the more
tightly correlated to mature seed size (Fig. 1). However,
specificities for the pattern of variation of the two traits
occurred within the two couples of reciprocal crosses.
Cotyledon cell number was the major factor contributing
to the differences between the Frisson–Solara hybrids
because Solara×Frisson had 22% more cells than
Frisson×Solara, whereas its cells were, on average, only
11% bigger. Conversely, cotyledon cell volume appeared
to be the major factor contributing to the CationImposant hybrid differences because Imposant×Cation
had 35% more cells than Cation×Imposant, but its cells
were also on average 59% bigger.
Embryo fresh weight evolution during the transition between
the cell division phase and maturation
Results
Maternal genotype influence on seed mass, cotyledon cell
number and volume
The dry weight of the seeds issued from reciprocal crosses
was examined. The mass of the mature F1 seeds was not
significantly different from that of the maternal parent
( Table 1), although Frisson×Solara and Cation×
Imposant seeds were slightly heavier than Frisson
and Cation seeds, and Solara×Frisson and Imposant×
Cation seeds were smaller than Solara and Imposant
seeds. Maternal effects were also evident with regards to
seed shape (data not shown). The dry weight differences
observed between the mature seeds issued from the
various reciprocal crosses were always significant.
The period of seed development studied, 150–300 °C
DAP, was known to cover the transition period between
the cell division phase and reserve accumulation (maturation) (Ney et al., 1993; Ney and Turc, 1993). Embryo
fresh weight is known to be a good mark by which to
distinguish between these two phases (Fig. 2). During the
cell division phase, embryo fresh weight was too small to
be accurately determined. However, when the cotyledons
began to fill, the embryo progressively became the major
part of the seed. Differences in the seed growth rate
clearly appeared between the parental lines and hybrids
especially for the Cation–Imposant crosses. Time of fresh
weight onset of the embryos showed that maturation
Table 1. Dry weight, mean cotyledon cell number, and mean cell volume in mature seeds issued from reciprocal crosses between the
genotypes Frisson and Solara, and Cation and Imposant±standard error (about 50 measurements for the dry weight, and eight
measurements for cell number and volume)
Columns followed by the same letter are not significantly different at P<0.05 (dry weight: Dunn’s multiple comparison test; cell number and
volume: Student t-test). The two reciprocal cross experiments were analysed separately.
Genotypes
Seed dry weight
(mg)
Cotyledon cell number
(×10−3)
Cotyledon cell volume
(mm3×10−3)
Frisson×Frisson
Frisson×Solara
Solara×Frisson
Solara×Solara
247±3 a
265±3 a
348±10 b
413±6 b
964±44 a
955±17 a
1.171±104 ab
1.243±26 b
384±17 a
409±7 a
457±36 ab
460±30 b
Cation×Cation
Cation×Imposant
Imposant×Cation
Imposant×Imposant
180±3
214±3
460±6
548±7
715±30
969±59
1.309±51
1.649±75
393±18
338±24
540±23
531±23
a
a
b
b
a
b
c
d
a
a
b
b
170 Lemontey et al.
Fig. 1. Relationship between the mature seed dry weight and the
cotyledon cell mean volume (A) or the cotyledon cell number (B). (%)
Frisson×Frisson; (#) Frisson×Solara; ($) Solara×Frisson; (&)
Solara×Solara; (1) Cation×Cation; (6) Cation×Imposant; (+)
Imposant×Cation; (2) Imposant×Imposant. The correlation coefficients are considered very significant (A) and extremely significant (B)
according to the Pearson correlation method (P=0.003 and P<0.001,
respectively).
Fig. 2. Embryo fresh weight evolution during the 150–300 °C DAF part
of seed development of two couples of varieties and their reciprocal
hybrids. (A) (( ) Frisson×Frisson; (---#---) Frisson×Solara;
(---$---) Solara×Frisson; ( & ) Solara×Solara. (B) (—1—)
Cation×Cation;
(---6---)
Cation×Imposant;
(---+---)
Imposant×Cation; ( 2 ) Imposant×Imposant. Values represent the
mean of four replicates±standard error (SE). When not visible, SE
bars are smaller than the symbol.
began between 200 and 225 °C DAP for the two couples
of tested genotypes and their reciprocal hybrids.
Kinetics of endoreduplication in cotyledons during early
maturation
Flow-cytometry analyses were made on homozygous and
heterozygous embryos during the period between 150 and
300 °C DAP. In the early stages of development (from
150 to 200 °C DAP), the cotyledons showed a bimodal
nuclei population, with peaks corresponding to 2C and
4C levels of DNA. From 220 °C DAP, histograms showed
the progressive appearance of peaks corresponding to 8C,
then 16C and 32C levels of DNA. Kinetics of the endoreduplication was quite different among the four pea genotypes: at 280 °C DAP, Cation×Cation embryos showed
about 2.5% of nuclei with 32C level of DNA, whereas
Solara×Solara embryos did not present a DNA level
higher than 8C (data not shown).
Figure 3 shows the evolution of the relative part of the
8C peak in developing embryos. This peak is the first real
endoreduplicating peak; indeed at the end of the cell
division phase, the 4C peak is a mixture of G2 and
endoreduplicating nuclei. Endoreduplication appeared
sooner in small-seeded genotypes (Cation and Frisson)
than in large-seeded genotypes (Imposant and Solara).
Conversely, reciprocal hybrids showed a similar pattern
of appearance of the 8C peak, intermediate between those
of their two parents.
Maternal genotype influences pea seed size 171
Fig. 3. Evolution of the relative part of the 8C peak in the cotyledon nuclei during the 150–300 °C DAF part of seed development of two couples
of varieties and their reciprocal hybrids. (A) (( ) Frisson×Frisson; (---#---) Frisson×Solara; (---$---) Solara×Frisson; ( & ) Solara×Solara.
(B) (—1—) Cation×Cation; (---6---) Cation×Imposant; (---+---) Imposant×Cation; ( 2 ) Imposant×Imposant. Values represent the mean of
three replicates±standard error (SE ). When not visible, SE bars are smaller than the symbol.
Endoreduplication pattern in mature embryos
The proportion of endoreduplicating nuclei was investigated in mature cotyledons among the two couples of
pea genotypes and their reciprocal F hybrids, and GSP6,
1
a very small-seeded pea genotype. Frequency histograms
showing the DNA levels of nuclei of the five homozygous
lines and the four reciprocal hybrids are given in Fig. 4.
Very small seeds (GSP6) presented five DNA levels, from
2C to 32C. Small seeds (Cation, Frisson, and hybrids
where they are the maternal parent) presented six DNA
levels, with 1.5% to 4% nuclei with a 64C DNA content.
Large seeds (Solara, Imposant and hybrids where they
are the maternal parent) presented seven DNA levels,
with about 1% nuclei with a 128C DNA level.
Among these five pea genotypes, the percentage of
nuclei showing a 64C or more DNA content lay in a
range from 0 (GSP6) to 14.23% (Imposant) (data not
shown), and a highly significant positive linear correlation
(r2=0.919) could be found between the mean C-value
and mature seed weight ( Fig. 5). For each cross, the
endoreduplication pattern of the F hybrids were very
1
similar to that of their maternal parents. There was also
a relationship between the mean C-value and the mean
cotyledon cell volume in mature seeds ( Fig. 6). Largest
seeds, which are composed from largest cells, show also
a larger mean C-value.
Discussion
Differences in cotyledon cell number and mean volume
between reciprocal hybrids
The strong maternal influence on mature seed size had
already been shown for many species, including pea
(Davies, 1975). The difference in mature seed weight
between reciprocal F hybrids from the two couples of
1
contrasting genotypes used, indicated such a maternal
effect in the experimental system. However, no hybrid
resembled its maternal parent exactly, suggesting that the
172 Lemontey et al.
Fig. 4. Endoreduplication patterns of mature cotyledons in nine pea varieties and reciprocal hybrids. Patterns are sorted according to the dry
weight of the seeds (see below). DNA content is expressed in arbitrary units (semi-logarithmic scale). (A) GSP6 (58 mg); (B) Cation (180 mg); (C )
Cation×Imposant (214 mg); (D) Frisson (247 mg); ( E ) Frisson×Solara (265 mg); (F ) Solara×Frisson (348 mg); (G) Solara (413 mg); (H )
Imposant×Cation (460 mg); (I ) Imposant (548 mg). Arrows indicate 64C and 128C DNA peaks when present.
genetic constitution of the embryo also had a role in
determining the mature seed size of the F hybrid.
1
Furthermore, no clear heterosis phenomenon was
observed for this character. This confirms the results of
Sarawat et al. who showed positive values of heterosis in
pea over the maternal and paternal parents in all the
agronomic features, except harvest index, seeds per pod,
and seed weight (Sarawat et al., 1994). Previous studies
in pea (Davies, 1975) and soybean (Guldan and Brun,
1985) have shown that seed growth rate and mature seed
size were related to number of cells in the cotyledons. In
maize, it was shown that both endosperm cell and starch
granule numbers are highly correlated with kernel mass
at maturity (Reddy and Daynard, 1983). It has been
determined that the seed growth rate was related to
cotyledon cell volume in common bean (Sexton et al.,
1997). These studies showed that two parameters, namely
cell number and/or cell volume, may explain the maternal
effect on the mature seed size. In the reciprocal crosses
used in this study, it has been shown that cotyledon cell
number and volume can explain the differences that were
observed between reciprocal hybrids. Frisson and Solara
hybrids differ mainly in cotyledon cell number, whereas
differences between Cation and Imposant hybrids are
mainly due to differences in cell volume. These differences
observed between the two couples of reciprocal crosses
suggest that non-maternal genetic factors (for example,
the presence of paternally imprinted genes) are involved
in seed size variation, as suggested for Arabidopsis thaliana
(Alonso-Blanco et al., 1999).
Maternal genotype influences pea seed size 173
Fig. 5. Relationship between mean C-value and the mature seed dry
weight for the nine pea varieties and reciprocal hybrids (see legend of
Fig. 4 for the detail of the genotypes). Values represent the mean of
eight replicates±standard error (SE). When not visible, SE bars are
smaller than the symbol. The correlation coefficient is considered
extremely significant according to the Pearson correlation method
at P<0.001.
Relation between the cotyledon cell number and the
duration of the cell division phase and/or mitotic activity
during this phase
The evolution of embryo fresh weight between 150 °C
and 300 °C DAP provided evidence that this period
represented the transition phase between the cell division
phase and maturation. During this period, the embryo
fresh weight of the various lines began to increase at
different times and showed different rates of increase,
suggesting that the number and timing of cell division
are different. The number of cotyledon cells depends both
on the duration of the cell division phase and on the
mitotic activity in the cotyledons during this period.
Studies on the kinetics of the endoreduplicating phenomenon allowed the detection of differences in cell cycle
evolution between the two couples of pea varieties and
their reciprocal hybrids, and to determine the limits of
the cell division phase.
During the cell division phase, i.e. before 225 °C DAP,
embryo fresh weight stayed very low and cotyledon nuclei
showed a DNA content of 2C and 4C, which represents
nuclei in G1 and G2 phases of the cell cycle, respectively.
Nuclei between the two peaks represent nuclei in the S
phase, that are synthesizing DNA. During this first phase,
there was almost the same proportion of 2C and 4C
nuclei, which indicates a very high mitotic activity.
Early maturation was accompanied by an increase of
Fig. 6. Relationship between mean C-value and cotyledon cell mean
volume between two couples of varieties ( Frisson–Solara and Cation–
Imposant) and their reciprocal hybrids. See legend of Fig. 1 for the
meaning of the symbols. Values represent the mean of eight
replicates±standard error (SE ). When not visible, SE bars are smaller
than the symbol.
embryo fresh weight, and a decrease in the population of
nuclei showing a 2C DNA content. This decrease was
followed by the appearance of nuclei showing an 8C
DNA content which provides unequivocal evidence of
the termination of the cell division phase, using the
observation that cells that have entered endoreduplication
cannot subsequently undergo mitosis (Graffi and Larkins,
1995). The initiation of the endoreduplication is a progressive phenomenon in reserve accumulating organs
during the transition between the cell division and maturation phases ( Kowles et al., 1990). Comparisons of the
patterns of appearance of the 8C peak in Imposant,
Solara, Cation, and Frisson, indicate that the cell division
phase is longer in large-seeded genotypes than in smallseeded genotypes. Conversely, reciprocal hybrids showed
the same pattern of appearance of the 8C peak, intermediate between that of the two parents, which indicates
a normal Mendelian inheritance without dominance for
the character ‘duration of cell division phase’. This means
that the duration of the cell division phase in the embryo
is controlled by the embryo’s own genotype, and is not
under maternal influence. The results of this study suggest
that large-seeded F seeds show a higher number of
1
cotyledon cells than their reciprocal small-seeded hybrids,
and that this difference in cotyledon cell number does not
rely on the duration of the cell division phase. This means
that the difference in cell number must rely on the mitotic
activity during the cell division phase. Thus maternal
174 Lemontey et al.
genotype does not influence the duration of the cell
division phase, but it controls mitotic activity during
this period.
Maternal control on the endoreduplication level in mature
seeds
The study of endoreduplication in pea cotyledons using
flow cytometry is reported for the first time in this paper.
A maximum level of 128C was detected, whereas the
microdensitometry method on Pisum sativum L. allowed
the measurement of a maximum 64C value only (Scharpé
and Van Parijs, 1973; Davies and Brewster, 1975). There
is a clear positive correlation between the endoreduplication level in mature cotyledons and the seed size.
Endoreduplication in plants has often been correlated
with cell growth, and it is widely accepted that it sustains
cell elongation in the absence of mitosis, although it is
not known how these processes are related at the functional and regulatory level (Galbraith et al., 1991;
Melaragno et al., 1993). A correlation was shown between
endoreduplication and cell length in Arabidopsis thaliana
hypocotyl (Gendreau et al., 1998), and evidence was
provided that endoreduplication is initiated before any
growth has occurred, suggesting that endoreduplication
levels could partially determine mature cell size. In this
study, it seems that there is a relationship between cotyledon cell volume and the mean C-value in the mature
seed. In the pea embryo, the control of cotyledonary cell
size could lead to the control of the sink capacity. It may
be hypothesized, by analogy with the Arabidopsis model
(Galbraith et al., 1991; Melaragno et al., 1993, Gendreau
et al., 1998), that seeds presenting a higher C level of
DNA could, therefore, develop a greater growth rate.
Whether the maternal plant controls the endoreduplication level is not clear, because of the apparent contradiction between results obtained on immature and mature
cotyledons. It appears from the above study that there is
no maternal influence on the stage of initiation of the
endoreduplication process in the cotyledons. Conversely,
the final endoreduplication pattern seems to be similar
between heterozygous and homozygous maternal lines,
which is consistent with the results obtained on maize,
where it has been shown that the extent of DNA amplification in F hybrid endosperm tissues is under maternal
1
control ( Kowles et al., 1997).
Conclusion
The maternal influence observed on pea mature seed
weight reflects maternal control on the establishment of
cotyledon cell number, mean cell volume and endoreduplication level in mature seeds. The maternal effect on seedsink capacity and, subsequently, on potential for growth
rate and mature seed weight, could take place during the
early stages of seed development by controlling the mitotic
rate during the cell division phase. After this step, maternal and non-maternal factors could control the number
of endoreduplicating cycles in the cotyledons and, consequently, the cotyledon cell size. These results are in
accordance with those obtained with reciprocal crosses
between two Arabidopsis lines, where it has been shown
that the cell number variation was controlled mainly by
maternal factors, whereas the non-maternal allelic variation affected mostly cell size (Alonso-Blanco et al.,
1999).
Acknowledgements
We would like to acknowledge UNIP ( Union Nationale
Interprofessionnelle des plantes riches en Protéines) for supporting C Lemontey’s PhD thesis. Cvs Frisson and Solara were
supplied by the Station de Génétique et d’Amélioration des
Plantes, INRA, Versailles, and cvs Cation, Imposant and GSP6
seeds were supplied by M Duparque, from the Groupement des
Sélectionneurs de Pois Protéagineux, INRA, Estrée-Mons. The
enzymatic solution for mature seed maceration was elaborated
by Sergio Ochatt, from the Unité de Recherche en Génétique
et Amélioration des Plantes, INRA, Dijon. We sincerely thank
all of them. We also thank Yvette Roux for her excellent
technical assistance, and Dr Christine Rochat and Dr Judith
Harrison for correcting this article.
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