Annals of Botany Page 1 of 15

doi:10.1093/aob/mcp040, available online at www.aob.oxfordjournals.org

Using a model-based framework for analysing genetic diversity during

germination and heterotrophic growth of Medicago truncatula
S. Brunel1, B. Teulat-Merah1, M.-H. Wagner2, T. Huguet3, J. M. Prosperi4 and C. Durr1,*
1

INRA et Agrocampus Ouest, UMR 1191 Physiologie Moleculaire des Semences, 16 bd Lavoisier, F-49045 Angers, France,
GEVES Station Nationale dEssais des Semences, rue Georges Morel, F-49071 Beaucouze, France, 3INP-ENSAT Laboratoire
Symbioses et Pathologies des Plantes (SP2), Avenue de lAgrobiopole, F-31326 Castanet, Tolosan cedex, France and 4INRA
UMR 1097 Diversite et Adaptation des Plantes Cultivees, Domaine de Melgueil, F-34130 Mauguio, France

Received: 17 November 2008 Returned for revision: 8 December 2008 Accepted: 13 January 2009

Background and Aims The framework provided by an emergence model was used: (1) for phenotyping germination and heterotrophic growth of Medicago truncatula in relation to two major environmental factors, temperature and water potential; and (2) to evaluate the extent of genetic differences in emergence-model parameters.
Methods Eight cultivars and natural accessions of M. trunculata were studied. Germination was recorded from
5 to 30 8C and from 0 to 20.75 MPa, and seedling growth from 10 to 20 8C.
Key Results Thermal time to reach 50 % germination was very short (15 8Cd) and almost stable between genotypes, while base temperature (2 3 8C) and base water potential for germination (20.7 to 21.3 MPa) varied
between genotypes. Only 35 8Cd after germination were required to reach 30 mm hypocotyl length with significant differences among genotypes. Base temperature for elongation varied from 5.5 to 7.5 8C. Low temperatures
induced a general shortening of the seedling, with some genotypes more responsive than others. No relationship
with initial seed mass or seed reserve distribution was observed, which might have explained differences between
genotypes and the effects of low temperatures.
Conclusions The study provides a set of reference values for M. trunculata users. The use of the ecophysiological model allows comparison of these values between such non-crop species and other crops. It has enabled
phenotypic variability in response to environmental conditions related to the emergence process to be identified.
The model will allow simulation of emergence differences between genotypes in a range of environments using
these parameter values. Genomic tools available for the model species M. trunculata will make it possible to
analyse the genetic and molecular determinants of these differences.
Key words: Core collection, emergence, Medicago truncatula, modelling, seed, temperature, water potential.

IN T RO DU C T IO N
Germination and heterotrophic growth are crucial steps for stand
establishment of crops. Emergence can be highly variable for
most crops and depend considerably on environmental conditions (Awadhwal and Thierstein, 1985; Durrant et al., 1988;
Benjamin, 1990; Klos and Brummer, 2000; Valenciano et al.,
2004). A precise description of plant functioning during the
early stages before emergence, in relation to environmental conditions, is necessary in order to dissect which seed characteristics, stages and growing conditions lead to differences in
emergence. It is also important to assess the possible genetic
component of variation in emergence results (Eagles and
Hardacre, 1979; Bettey et al., 2000; Cui et al., 2002; Rebetzke
et al., 2007). Over the last decade, model plants have been the
subject of rapid advances in genomics. Exploiting genetic diversity requires increasing knowledge of the phenotypic variability
and there is a huge need for phenotyping collections to take
advantage of the genetic and genomic tools that have been developed on model plants (genetic and physical maps, large collections of genetic resources and mutants).
Ecophysiological models that gather knowledge at the plant
level provide a framework for the analysis of plant behaviour.

They are designed to separate stages and to identify the influence of environmental conditions on biological processes
at the plant-part level (Gutschick and Simmoneau, 2002;
Tardieu et al., 2005; Moreau et al., 2006, 2007). Model
equations can be used to predict processes, which is not the
case when measuring single traits on plants. The parameter
values can vary according to species, genotype or seed lot
and they do not depend on environmental factors. Model
equations and parameter values allow genotype and seed lot
behaviours to be simulated under different conditions. Thus,
ecophysiological models can help in the choice of phenotypic
traits to measure on a wide range of lines at the plant scale.
This is a key step in the analysis of inter- and intraspecific
genetic diversity and could help to advance the analysis of
genetic determinism of agronomic traits (Yin et al., 2000,
2003; Reymond et al., 2003; Tardieu, 2003; Quilot et al.,
2005a, b; Tardieu et al., 2005; Hammer et al., 2006;
Laperche et al., 2006, 2007). Such models have begun to be
used to analyse the genetic diversity of several aspects of
plant, leaf or root growth. However, the very early stages,
from germination to the end of heterotrophic growth, have
rarely been examined in this way (Cui et al., 2002; Zhang
et al., 2005). It is necessary to combine knowledge provided
by analysis of the sources of variation in crop stands, gathered

* For correspondence. E-mail durr@angers.inra.fr

# The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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Page 2 of 15

Brunel et al. An emergence model for phenotyping of M. truncatula

TA B L E 1. Description and characteristics of the studied genotypes

Genotype1

Line number2

Jemalong A17
F83005.5
DZA315.16
DZA045.5
DZA012.J
SA28064
Borung
Paraggio

L000738
L000530
L000734
L000735
L000368
L000174
L000527
Population

Genetic type3
Cultivar
Natural accession,
Natural accession,
Natural accession,
Natural accession,
Natural accession,
Cultivar
Cultivar

CC
CC
CC
CC
CC

Place of origin, altitude (m), latitude, longitude

Seed lot4

Spanish-Moroccan area; unknown

France, Salernes; 261 m; 43.58N- 6.238E
Algeria. Ain El Hadjar; 1070 m; 34.78N, 0.168E
Algeria, Annaba; 100 m; 36.98N, 7.78E
Algeria, Larba; 200 m; 36.58N, 3.28E
Cyprus, Ora; 554 m; 34.88N, 33.28E
Tunisia, Le Kef and Le Krib; unknown
Italia, Calabria; 350 m; 398N, 168E

M-05, M-06, A-06

M-05, M-06, A-06
M-05, M-06, A-06
M-05, M-06, A-06
M-05, M-06
M-05
M-05
Aust-04, A-06

1
F83005.5 France, county number 83, collection site 005; 5 is the reference for the single plant taken within a population that was selfed twice to get
an inbred line. DZA Algeria; 315, 045 and 012 are collection sites; 16, 5, J are the references for the plant used to get an inbred line. SA28064, SARDI
(Australian Collection). Borung and Paraggio are registration names in the Australian register of herbage plant cultivars.
2
Line number as referenced at the Centre de Ressources Biologiques (INRA Montpellier, France, http://www.montpellier.inra.fr/BRC-MTR/).
3
Genotypes labelled CC belong to the first subset of the nested core collections developed at the INRA station in Montpellier.
4
M, Montpellier; A, Angers; Aust, Australia; 04, 05 and 06 are years of production, 20042006.

in emergence models in which seed and seedling functioning

in relation to environmental conditions are described with sufficient detail, with information provided by the tools available
for one model species used in genomic studies.
Emergence models developed for crops (Bouaziz and
Bruckler, 1989; Mullins et al., 1996; Finch-Savage et al.,
1998; Durr et al., 2001) separate the two phases leading to
plant emergence: germination, defined as radicle protrusion
(Bewley and Black, 1994), and seedling growth until emergence. These models take into account the influence of the
main environmental factors in a seedbed, including soil temperature and water content. Differences in their input parameter
values concerning seeds and seedlings can account for differences among genotypes.
We have chosen to study Medicago truncatula, one of the
model legumes (Cook, 1999; Tivoli et al., 2006). Initially
used for investigating the regulation of nodule development
(Cook, 1999; Thoquet et al., 2002), recent work has also provided information about its grain-filling and seed characteristics (Gallardo et al., 2003; Djemel et al., 2005). However,
little is known about its early stages, germination and heterotrophic growth, in relation to environmental conditions
(Buitink et al., 2003; Gallardo et al., 2007; Garcia et al.,
2007) and even less about the possible genetic diversity
during these stages. Our objectives were to measure the ecophysiological parameters concerning germination and emergence for this species and to test the existence of a genetic
influence on these parameter values. A first set of genotypes
was chosen according to three main criteria: (1) originating
from different natural biotopes in order to observe potentially
different responses to environmental conditions; (2) belonging
to a nested core-collection in order to maximize genetic diversity; and (3) being used for the creation of segregating populations, and of associated genetic maps in order to further
analyse the genetic determinism once differences between
genotypes had been observed. Nested core collections of
M. trunculata represent the available germplasm in a limited
sub-sample of accessions (Ronfort et al., 2006), and hence
they can provide a first relevant set of genotypes for possible
phenotypic variation. Due to high synteny of M. trunculata
with other major crop legumes such as pea (Pisum sativum)
and soybean (Glycine max; Yan et al., 2003; Choi et al.,

2004), the genomic regions of interest for M. trunculata are

expected to match those of other related species.
Finally, this ecophysiological analysis of the early stages of
Medicago truncatula should help M. trunculata users before
setting up genetic and genomic analyses in identifying parameters and ranges of environmental conditions related to
emergence processes for which genetic diversity exist and
should be further studied.

MAT E RI AL A ND M E T HO DS
Plant material and seed production conditions

A total of eight genotypes of Medicago truncatula were

studied (Table 1). Jemalong A17 is the reference for
genomic studies and the node of several crosses for the production of recombinant inbred line (RIL) populations.
Borung and Paraggio are cultivars mainly grown in South
Australia. The five other genotypes are derived from natural
populations collected around the Mediterranean basin:
F83005.5, DZA315.16, DZA045.5, DZA012.J and
SA28064. They belong to the first set of the nested core collections (eight accessions, INRA Medicago truncatula
Biological Resource Center Montpellier website: http://www.
montpellier.inra.fr/BRC-MTR/; Ronfort et al., 2006). Very
little information was available regarding their sensitivity to
abiotic stresses: DZA045.5 is known to be frost sensitive,
while F83005.5 is frost tolerant and salt-stress sensitive. As
seed production conditions (maternal environment) can
greatly influence germination and early seedling stages, these
genotypes were characterized for three different seed production conditions (termed M-05, M-06 and A-06; see
below), in order to test whether genotype differences were
observed regardless of seed lot. The number of genotypes
studied varied from five to eight according to seed lot.
M-05 and M-06 seeds were produced in INRA greenhouses
during the spring in 2005 and 2006 near Montpellier (43.618N,
3.878E; south of France, Mediterranean climate). A-06 seeds
were produced in a growth chamber at the National Seed
Testing Station (SNES, Angers). A commercial seedlot of
Paraggio, produced in field conditions in Australia in 2004

Brunel et al. An emergence model for phenotyping of Medicago

(Seedco Australia Co-Operative Ltd), was also studied as an
external reference (Aust-04).
Seeds were stored at a low temperature and 50 % relative
humidity for 8 18 months before the experiments in order
to avoid post-harvest dormancy, commonly observed during
a 3 6 month period after harvest in M. trunculata (Garcia
et al., 2007).
Seed physical and biochemical characters

Seed dry mass (SDM) was measured on a sub-sample of 100

seeds for each genotype of each seed lot. The proportion of
teguments ( percentage of mass of teguments relative to
SDM, %T) was measured on 25 seeds after 24 h of imbibition
to facilitate the separation of teguments. Total carbon and
nitrogen contents (%C and %N) were measured on two replicates of five ground seeds, using a mass spectrometer (VG
SIRA 9) linked to an automatic combustion analyser (Carlo
Erba NA 1500). Sucrose and oligosaccharides contents of
the raffinose family (RFO) were measured as they could be
indicators of the seed maturation state at harvest. Indeed, in
legumes the RFO seed contents differ from other species
(Kuo et al., 1988), and during seed maturation and desiccation
stachyose content progressively decreases while verbascose
content increases (Rosnoblet et al., 2007). Soluble sugars
were extracted from pre-weighed powder (three replicates of
15 20 previously dried seeds) with 1 mL methanol/water
(80:20 v/v), with 40 mL melizitose used as the internal sugar
standard. After heating for 15 min at 76 8C, the liquid was
evaporated under vacuum. The pellet was then dissolved in
1 mL distilled water and centrifuged for 3 min at 14 000 g.
Sucrose (sucrose), stachyose (stachyose) and verbascose
(verbascose) contents were quantified using HPLC
(DIONEX ICS-3000).
Input variables of the model and experimental protocols
for their measurement

The SIMPLE model (SIMulation of PLant Emergence; Durr

et al., 2001) predicts germination and emergence time courses
and final rates in relation to environmental conditions during
sowing. This model has previously been parameterized for a
number of crop species such as wheat, sugar beet, flax,
mustard, bean and oilseed rape (Carrera and Durr, 2003;
Dorsainvil et al., 2005; Moreau-Valancogne et al., 2008),
making it possible to compare different species using the
same set of parameters. The functioning of the SIMPLE
model and the list of equations and input variables have
been described in Durr et al. (2001) and Dorsainvil et al.
(2005). We focused here on the input variables involved in
germination and growth, in relation to temperature and water
potential. Only the equations for calculating these input variables are presented. Experiments were carried out to obtain
base temperature (Tb,germ) and base water potential (cb,germ)
values for seed germination, and base temperature values
(Tb,elong) for seedling elongation for all seed lots and genotypes. Base values are the values below which no germination
or elongation can occur (see calculations below). For germination as well as elongation, data collected at different temperatures were then plotted together: germination and elongation

Page 3 of 15

time courses were fitted to thermal time (sum of temperatures

over the base temperature), as in the model, allowing seed lots
and genotypes to be compared (see calculations below).
Experiments on seed germination

Before each experiment, seeds were scarified to avoid physical seed dormancy. Three replicates of 50 seeds were sown for
each genotype from a given seed lot at each of the tested temperatures or water potentials. Seeds were individually weighed.
When testing temperatures, seeds were sown in plastic
boxes (5.5  12  18 cm) in pleated filter paper (ref. 3236
Whatman), moistened with 80 mL deionised water in order
to obtain non-limiting water conditions for M. trunculata germination. Boxes were kept in incubators at 5, 10, 15, 20, 25
and 30 8C. Temperatures were recorded hourly with sensors
(Testo 177-T3). Observations were carried out 2 5 times a
day depending on the temperature being tested. When testing
water potentials, boxes were incubated at 15 8C, in order to
limit differences in germination time between genotypes due
to temperature. The water potentials tested were 20.25,
20.5 and 20.75 MPa. Seeds were laid onto flat filter paper
(ref. 3645 Whatman) in plastic boxes (5.5  12  18 cm),
watered with 14 mL of osmotic solutions of high molecular
weight PEG (Polyethylene glycol 8000, ref. SIGMA
25322-68-3) used at varying concentrations to control water
potential, after Michel (1983). Seeds were considered to
have germinated when the radicle protruded from the seed
coat (1 mm). Seeds that remained hard despite scarification
were not included in the results.
Seedling growth

Experiments were performed in the dark to mimic preemergence growth. Pots (6.5 cm diameter, 10 cm high) were
incubated in growth chambers at 10, 15 and 20 8C. They
were filled with 500 g of sand (SIFRACO quality NE34:
SiO2 .99.7 %, solid density 2.65 g cm23, mean particle
size 200 mm). The gravimetric water content of the sand was
raised to 0.2 kg kg21 before sowing using a nutrient solution
prepared for young seedling growth (Saglio and Pradet,
1980), and it was maintained constant in order to avoid
water stress by application of deionised water during the experiment. Five seeds per pot were individually weighed, scarified,
and sown at 1.5 cm depth in a known position within the pots
so as to be able to relate each seedlings mass to its initial seed
mass. Three pots (15 seedlings) were observed at each of the
six observation times until seedling length reached a plateau
value (maximal elongation under heterotrophic growth conditions), which was reached at 11, 15 and 30 d at 20, 15 and
10 8C, respectively.
At each observation time, seedlings were harvested, and
hypocotyl and radicle lengths were measured for the seed
batches M-05, Paraggio Aust-04 and A-06. For M-06, only
the final lengths were measured. The masses of the seedling
parts were measured at the same time as seedling elongation
in order to analyse biomass distribution. The seedling parts
were separated, dried at 80 8C for 48 h and weighed for the
experiments performed at 20 8C on seed lots M-05 and
Paraggio Aust-04, and at 10 8C on A-06. An additional

Page 4 of 15

Brunel et al. An emergence model for phenotyping of M. truncatula

experiment was carried out to evaluate the effect of initial seed

mass on the maximum lengths of seedling parts. Seeds from
lots M-05 and A-06 belonging to the same mass range (3.5
4.5 mg) were weighed and then grown in the same conditions
as for the experiments at 20 8C. The final lengths of the seedling parts were measured.
Calculations

A Gompertz function was fitted to the germination rates

obtained for each replicate batch of 50 seeds:
Gt a expb=c expct

where G(t) is the cumulative germination rate at time t from

sowing, a is the maximum cumulative germination rate, and
b and c are shape parameters. Adjustments were made for
each temperature or water potential tested and used to calculate
the time to reach y % (20 up to 80 %) cumulative germination.
Values of Tb,germ or cb,germ are defined as the x-intercept of the
linear regression between the studied factor (temperature or
water potential) and germination rate (Gummerson, 1986;
Dahal and Bradford, 1994). We determined the range of temperatures and water potentials for which a strong linear relationship (r 2 . 0.95) existed between germination rates [1/(time to
reach y % germination)] to calculate the x-intercepts. Base
temperature and base water potential are thus defined as the
fitted values at which no germination occurs.
Weibull functions were fitted to the observed radicle and
hypocotyl lengths:
Lt af1  expbtg g

where L(t) is the length at time t, a is the final length, and b

and g are shape parameters. Time was calculated from the
time needed for 50 % germination onwards in order to separate
the two stages (i.e. germination and heterotrophic growth) and
to eliminate germination rate differences between genotypes.
Elongation rates [1/(time in h to reach z mm); z 20 up to
70 mm) were then calculated from these fittings for the three
temperatures tested: 10, 15 and 20 8C. Base temperature for
elongation (Tb,elong) was the x-intercept of the linear regression
between elongation rate and temperature, and thus the fitted
temperature value at which no elongation occurred.
Hence, time was expressed in thermal time tT, using the following equation:
tT;i

d i
X
T d  Tb

d1

where tT,i is cumulative thermal time on day i, T is mean temperature of day d, and Tb is the base temperature calculated for
each genotype and seedlot. This calculation of time was used
in eqns (1) and (2) to compare germination and growth of the
different genotypes and seed lots. In the case of germination,
day 1 is the sowing date; in the case of elongation, day 1 is
the day at which half the seeds have germinated, in order to
consider elongation time separately from germination.

Statistical analyses

Statistical analyses were performed in order to estimate the

contribution of genotype and seed lot effects on the variability
of results. First, a global analysis of variance was performed
with genotype (G), seed lot (S) and temperature (T) or water
potential (c) considered as fixed factors and all interactions
(G  S, G  T, G  c and G  S  T or G  S  c) considered as random factors. We used the PROC MIXED procedure of SAS (SAS 9.1.3, SAS Institute, Inc.), which
calculates the significance test using the restricted maximum
likelihood (REML) method. Second, genotype effects were
compared within each seed lot. All the mean comparisons
were made using a Tukey test or Bonferronis multiple comparison procedure when samples sizes differed (P , 0.05)
using the STATGRAPHICS Plus 3.1 software.
R E S U LT S
Characteristics of the seeds

Several seed characteristics, expected to differ according to

seed production conditions and genotypes that can influence
germination and growth results, were measured. Seed dry
mass (SDM) and proportion of teguments (%T) varied from
3.3 to 5.2 mg and from 8.8 to 12.1 %, respectively; they were
negatively correlated (Table 2). For the SDM, there were significant effects of genotype and seed lot, whereas no effect was
observed for %T. When considering only the four genotypes
common to the three seed lots, the average SDM and %T of
A-06 (3.9 mg, 10.5 %) were slightly lower than those of M-05
and M-06 (4.15 mg, 10.8 %), which could be related to the
different seed production environments. Whatever the seed lot,
F83005.5, DZA315.16 and DZA012.J had the lowest
SDM values (3.9 mg) whereas DZA045.5 had the highest
(4.5 mg). SDM variations were positively correlated with
time between pollination and natural abscission (data not
shown). For %T, when considering the genotypes within a seed
lot, F83005.5 had high %T values while Paraggio had
among the lowest. Even if %T values remained within a small
range, they were positively correlated with the number of hard
seeds remaining after scarification (data not shown).
The seed carbon and nitrogen contents (%C and %N) were
very similar between seed lots and genotypes, and were 49 %
and 6.8% on average, respectively (Table 2). The average
%N calculated using the four common genotypes of each
seed lot showed that A-06 had slightly higher %N than the
other two seed lots. When considering the genotypes within
a seed lot, differences were observed for %N, with
F83005.5 always having the lowest value.
Stachyose and verbascose contents were high, as expected
for legume seeds, but large variations were observed among
genotypes (Table 2). The seed lot effect was only significant
for sucrose, with higher values for A-06, while the genotype
effect was significant for all the sugar contents. Whatever the
seedlot, verbascose of DZA045.5 and DZA012.J were
higher than for those of the other genotypes. Conversely,
stachyose of both genotypes was lower, suggesting more
conversion to verbascose during seed development.
Finally, several seed characteristics differed for A-06, which
could be related to seed production conditions. Genotypic

Brunel et al. An emergence model for phenotyping of Medicago

Page 5 of 15

TA B L E 2. Description of the genotypes and of their seed characteristics

Sugars (mg mg21 SDM)
Seed lot1
M-052

M-062

A-062

Aust-04
F-values3

Genotype
Jemalong A17
F83005.5
DZA315.16
DZA045.5
DZA012-J
SA28064
Borung
Jemalong A17
F83005.5
DZA315.16
DZA045.5
DZA012-J
Jemalong A17
F83005.5
DZA315.16
DZA045.5
Paraggio
Paraggio
G effect
S effect

Seed dry mass SDM

(mg)

Teguments
(%T)

4.1abc
3.9abc
3.8cd
4.5a
3.6d
4.3ab
3.6d
4.1b
3.9b
3.9b
4.7a
3.6b
3.8b
3.7b
3.3b
4.7a
5.2a
4.1

10.5bcd
11.6a
10.2cd
11.0abc
10.8abc
9.6 d
11.5ab
10.5b
12.1a
10.3b
10.5b
11.4a
9.9b
10.4b
11.3a
10.2b
8.8c
9.5

18.96***
12.12**

2.33 ns
0.28 ns

Carbon content
(%SDM)
49.6a
49.3a
49.2a
49.6a
49.0a
49.2a
48.8a
48.3c
48.3c
49.5ab
50.1a
48.8bc
50.3a
49.2a
46.9a
49.6a
49.1a
50.0
0.51 ns
0.36 ns

Nitrogen content
(%SDM)

Sucrose

7.0a
5.8b
6.4ab
6.9a
6.8a
6.7a
6.9a
7.3a
6.3c
6.9abc
7.1ab
6.5bc
7.3ab
6.3b
6.1b
7.8a
6.9ab
7.6

3.2b
6.0ab
4.6ab
6.0ab
4.5ab
6.5a
3.1b
2.9d
5.3b
5.5b
7.1a
4.0c
4.0c
7.5b
6.3b
9.7a
9.6a
6.4

3.70 ns
1.02 ns

12.38**
9.70**

Stachyose

Verbascose

46.3a
43.8a
43.7a
11.3c
22.8bc
40.5ab
49.3a
56.4a
44.5b
49.3ab
13.5d
21.9c
67.3a
55.8a
53.1a
9.6b
48.6a
53.5

1.9b
6.5b
4.1b
31.9a
38.5a
4.9b
6.8b
1.6b
6.4b
5.1b
38.7a
34.9a
3.6d
10.0b
5.9cd
35.2a
7.0bc
1.7

25.96***
2.92 ns

98.99***
1.63 ns

M, Montpellier; A, Angers; Aust, Australia; 04, 05 and 06 are years of production, 2004 2006.
Means followed by different letters indicate significantly different genotypes within a given production site (P , 0.05; Tukey multiple-comparison test).
Seed lot (S) and genotype (G) were treated as fixed factors; the interaction S  G was considered as a random factor. Analyses were performed by pooling
the data of all the genotypes and seed lots. F-values are followed by level of significance: * P , 0.05, ** P , 0.01 and *** P , 0.001; ns, non-significant.
The denominator degrees of freedom for each effect of each characteristic is 7.
2
3

variations were observed across and within seed lots for seed
mass, teguments, nitrogen and sugar contents.
Germination
Effect of temperature. Seeds were individually weighed to test

for any putative effect of initial seed mass on germination.

No relationships between SDM and germination rate and
final percentage were observed regardless of seed lot, genotype
and temperature (data not shown).
Final percentages of germination remained above 80 % for
all the temperatures tested regardless of the seed lots and genotypes, except at 30 8C. Examples of germination time courses
are presented for two contrasting temperatures, 20 8C and 5 8C,
for M-05 and Paraggio Aust-04, and the external reference
(Fig. 1A). At 20 8C germination was very fast, except
for DZA012.J. At 5 8C, F83005.5, Jemalong A17 and
Paraggio germinated more slowly than the others and
hardly reached about 80 % of germinated seeds. Figure 2A
shows the inverse of the time to reach 50 % germination
(1/tG50) for the whole range of temperatures tested, for all genotypes and for the three seed lots. The optimum germination
temperatures differed among genotypes and seed lots and
were rather low, ranging from 15 to 25 8C. The germination
rates of seed lot M-06 were the fastest, while A-06 had the
slowest and showed a sharp decrease in germination rate
above 20 8C (Fig. 2A). When comparing the genotypes for a
given seed lot, two genotypes differentiated above 15 8C:
DZA012.J in both M-05 and M-06 and DZA315.16

in A-06. Whatever the seed lot, Jemalong A17 and

DZA012.J were greatly affected at the highest tested temperature (30 8C), and 1/tG50 values of F83005.5 were always
significantly lower than those of the other genotypes at the
lowest temperature tested (5 8C).
Base temperature for germination (Tb,germ) was calculated
from the linear zone of the relationship between temperature
and germination rate, i.e. 5 15 8C (Fig. 2A). There was a significant effect of seed lot, mainly due to lower Tb,germ values
for A-06 (0.9 1.9 8C; Table 3) whose germination rates
were much lower than for the two other seed lots. In addition,
DZA315.16 stood out from the other genotypes in this seed
lot with a very low value (0.9 8C). For the two fast-germinating
seed lots (M-05 and M-06), Tb,germ values were similar,
varying from 1.9 to 3 8C. F83005.5 had the highest Tb,germ
for both seed lots, and its value was significantly higher
in M-06.
Germination was plotted against calculated thermal time,
using the genotype- and seedlot-specific Tb,germ. Using this
expression of time, the germination time courses at 5, 10
and 15 8C (the range of temperatures used for Tb,germ calculations) overlapped for a given genotype for each seedlot,
except for the final percentages, which were lower at low
temperatures for some genotypes. Two examples for contrasting genotypes from seed lot M-05 are shown in Fig. 3A,
namely Jemalong A17, the reference in genomics studies,
and F83005.5, the genotype with the highest Tb,germ value.
When all the data obtained at the different temperatures were
pooled, the germination time courses also overlapped for the

Page 6 of 15

Brunel et al. An emergence model for phenotyping of M. truncatula

A

Temperature
100

Germination (%)

80

20 C
Jemalong A17
F830055
DZA31516
DZA455
DZA012J
SA28064
Borung
Paraggio

60
40
20

5 C

0
0

25

50

75

100

125

150

175 0

100

Time after sowing (h)

B

200

300

400

Time after sowing (h)

Water potential

100
075 MPa

Germination (%)

80

60
0 MPa

40

20
0
0

25

50

Time after sowing (h)

75

200

400

600

Time after sowing (h)

F I G . 1. Germination for the seed lot M-05, and Paraggio Aust-04: (A) at 20 8C and 5 8C, and (B) at 0 MPa and 20.75 MPa (at 15 8C). Bars denote s.e., and the
lines are fitted Gompertz functions.

different genotypes of a given seed lot, with no differences

remaining between genotypes in M-06, and differences in
final germination rates only for the other seed lots (for
example, M-05, Fig. 3B). Thermal times for 70 % germination
were very short, ranging from 13 to 16 8Cd for M-05 and
M-06, respectively, but were significantly higher for seed lot
A-06 (16 25 8Cd, data not shown).
Effect of water potential. Final percentages of germination (FP)
mostly remained over 80 % at 20.25 and 20.5 MPa, whereas
the lowest water potential tested, 20.75 MPa, strongly
affected the pattern of germination and final percentages (for
example, M-05, Fig. 1B). Large differences in FP were
observed between seed lots at 20.75 MPa, ranging from 14
to 60 % in M-05, 32 to 99 % for M-06 and 64 to 96 % in
A-06. Considering the genotypes within a seed lot,
DZA315.16 and DZA012.J had the lowest FP and
DZA045.5 the highest. Germination rates (1/tG50) decreased
sharply with decreasing water potentials for all the genotypes
in all the seed lots (Fig. 2B). Germination rates of A-06 were
significantly lower than those of M-05 and M-06. With regard

to the germination rates of the genotypes common to the three

seed lots, DZA045.5 always had the highest germination rate
from 20.25 to 20.75 MPa, particularly in M-06 and A-06.
Base water potential values (cb,germ) were calculated for all
seed lots from the germination rates at 0, 20.25 and
20.5 MPa. For this range, the relationship between water
potential and germination rate was linear (except for
DZA315.16 in A-06 and M-06) and final percentages of germination were not altered. The effect of seed lot was not significant, although the average cb value for A-06 was slightly
high (Table 3). There was a significant genotype effect:
DZA045.5 had markedly lower cb,germ values (21.25 MPa
on average) while DZA315.16 and, to a lesser extent,
DZA012.J, had the highest (20.65 MPa and 20.7 MPa on
average, respectively; Table 3). Further analysis of
DZA045.5 and DZA315.16 seed imbibition (data not
shown) did not reveal any differences in imbibition rate or in
seed water content reached before germination, despite their
contrasting cb,germ values.
Finally, based on all germination results, the effect of seed
lot was mainly observed on germination rates, with A-06

Brunel et al. An emergence model for phenotyping of Medicago

Germination rate (1/tG50, h1)

Temperature

006 M-05 001

Page 7 of 15

Water potential
M-05

0075
004
0005
002

Jemalong A17
F830055
DZA31516
DZA455
DZA012J
SA28064
Borung
Paraggio

Germination rate (1/tG50, h1)

0
M-06

006

M-06

001
0075

004
0005

002

Germination rate (1/tG50, h1)

006 A-06

A-06

001

0075

004

0005
002

0
0

10

15
20
25
Temperature (C)

30

35 0

025
05
075
Water potential (MPa)

F I G . 2. Germination rates (1/tG50; see text) for the three seed lots M-05, M-06 and A-06, and Paraggio Aust-04: (A) as a function of temperature, and (B) as a
function of water potential. Bars denote s.e.

having consistantly lower values irrespective of the environmental factor (water potential or temperature). This maternal
effect was also observed on FP, but with a strong interaction
with genotype: DZA012.J always had low FP under
extreme conditions. For calculated base temperature and
water potential values, cb,germ had a high value, with strong
differences between genotypes; DZA045.5 being relatively
less sensitive to water stress. Tb,germ was less variable,
although F83005.5 had a higher value than the other
genotypes.

Seedling growth in the dark after germination

Hypocotyl and radicle elongation of the M-05 seed lot are

shown at 20 8C and 10 8C in Fig. 4. The average final lengths
(FLs) were calculated from the two last measurements when
the plateau was reached (and the measurements did not
differ statistically). Decreasing temperature significantly
affected FL: the lower the temperature, the shorter the final
lengths of both seedling parts (Fig. 4; Table 4). No effect
of seed lot was observed, even though there was a trend for
shorter FL in A-06 regardless of temperature. Hypocotyl

Page 8 of 15

Brunel et al. An emergence model for phenotyping of M. truncatula

TA B L E 3. Base temperatures and water potentials for the different genotypes

Seed lot1
M-052

M-062

A-062

Aust-04
F-values3

Genotype
Jemalong A17
F83005.5
DZA315.16
DZA045.5
DZA012-J
SA28064
Borung
Jemalong A17
F83005.5
DZA315.16
DZA045.5
DZA012-J
Jemalong A17
F83005.5
DZA315.16
DZA045.5
Paraggio
Paraggio
G effect
S effect

Base temperature for germination

(Tb,germ,8C)
2.5a
3.0a
2.4a
2.3a
2.7a
2.4a
2.5a
2.0ab
2.7b
2.2ab
2.2ab
1.9a
1.8b
1.7ab
0.9a
1.9b
1.9b
2.5
1.34 (7) ns
10.08 (7) *

Base water potential for germination

(cb,germ, MPa)

Base temperature for elongation

(Tb,elong,8C)

21.01b
20.69c
20.72c
21.22a
20.73c
20.92b
20.99b
20.82b
20.88b
20.68c
21.32a
20.57d
20.70bc
20.75b
20.55d
21.21a
20.60cd
20.91
14.21 (7) **
3.32 (7) ns

6.0bc
5.7bc
5.9bc
5.4c
7.1ab
6.5abc
7.4a

7.1a
6.6ab
3.1c
7.0ab
5.6b
5.6
0.58 (3) ns
0.01 (3) ns

M, Montpellier; A, Angers; Aust, Australia; 04, 05 and 06 are years of production, 20042006.
Means followed by different letters are for significantly different genotypes within a given production site (P , 0.05; Tukey multiple-comparison test).
3
Seed lot (S) and genotype (G) were treated as fixed factors; the interaction S  G was considered as a random factor. Analyses were performed by pooling
the data of all the genotypes and seed lots. F-values are followed by the denominator degrees of freedom (in brackets) and the level of significance: * P , 0.05,
** P , 0.01 and *** P , 0.001; ns, non-significant.
2

and radicle FL were significantly different between genotypes

(Table 4): DZA315.16 and Paraggio had the longest radicles and hypocotyls, whereas DZA045.5 and DZA012.J
had the shortest, regardless of temperature and seed lot. In
addition, for the three seed lots, F83005.5 seedling parts
were the most reduced in length at 10 8C (Fig. 4; Table 4).
An additional experiment was carried out to test whether
the differences in FL between genotypes were partly related
to variations in initial seed mass. The radicle and hypocotyl
FL were measured for all the genotypes of all seed lots for
a given range of seed mass (3.5 4.5 mg), and this showed
that the genotype rankings remained the same as for when
the whole range of initial seed masses was used (data not
shown).
Figure 5 shows the relationships between hypocotyl
elongation rates (the inverse of the time needed to reach a
hypocotyl length of 30 mm; 1/tL30) and temperature for
M-05 and A-06 (not measured for M-06). For M-05, a
linear relationship was obtained for the range of temperatures
tested (10, 15 and 20 8C), and this range was used to calculate
the base temperature for elongation (Tb,elong). For A-06, the
linear relationship between 1/tL30 and temperature was
broken above 15 8C for all genotypes. We only used 10 and
15 8C in order to estimate Tb,elong for this seed lot, assuming
there would be linearity between these two temperatures;
thus, Tb,elong values for this seed lot were estimated with relatively less precision. For all seed lots and regardless of genotype, calculated Tb,elong values were always higher than
Tb,germ values (Table 3). There were no significant effects
of seed lot or genotype. Values ranged from 5.4 8C
(DZA045.5) to 7.1 8C (Jemalong A17), except for

DZA315.16 in A-06, where the low value of Tb,germ has

already been noted (see above).
The results were also used to calculate elongation in terms
of thermal time with specific genotype values of Tb,elong, and
examples are shown for Jemalong A17 and F83005.5 in
M-05 (Fig. 6A). Expressed in terms of thermal time, the
curves showing the progress of elongation overlapped for
each genotype irrespective of the temperature at which
growth took place. Differences remained in FL of
F83005.5 because they were shorter at low temperatures.
When the data for the different temperatures were pooled for
each genotype, the elongation curves for all the genotypes of
a given seed lot also tended to be superimposed, although
with differences remaining in final length (Fig. 6B).
Differences also remained in elongation rates; for example,
the thermal time to reach 30-mm hypocotyl length after germination for M-05 varied from 22 to 34 8Cd depending on genotype, with F83005.5 and DZA045.5 having the highest
values. Values for A-06 were generally higher (19 to 48 8Cd,
not shown). Finally, regarding elongation results, A-06 was
also distinguished by having lower FL whatever the temperature and altered elongation rates above 15 8C. Low temperatures had a strong effect on final lengths and there was a
genotype effect, with F83005.5 being more sensitive than
the others. Tb,elong values were higher than those of Tb,germ.
Biomass changes during growth in the dark

The redistribution of the mass from the seed reserves was

analysed in relation to temperature and genotype in parallel
with seedling elongation in darkness. Changes in seedling

Brunel et al. An emergence model for phenotyping of Medicago

the beginning of growth up to 10 % of SDM, which was

reached at about 15 8Cd, and then it slowly decreased. Thus,
the reserves stored in cotyledons were mainly transferred to
the hypocotyl, with no differences between temperatures
despite the differences observed in final lengths.

100

A
% Germination

80
60

DISCUSSION

40

A framework for germination and heterotrophic growth analysis

to help functional-genomics approaches

5 C 10 C 15 C
Jemalong A17

20

F830055
0
100

% Germination

80
60
40

Jemalong A17
F830055
DZA31516
DZA455

20
0

Page 9 of 15

DZA012J
SA28064
Borung
Paraggio

10
20
30
Thermal time after sowing (Cd)

40

F I G . 3. Germination time courses expressed in thermal time (8Cd): (A) for

two genotypes Jemalong A17 and F83005.5 in the M-05 seedlot at 5, 10,
15 8C; and (B) all the temperatures pooled for each genotype of the seed lot
M-05, and Paraggio Aust-04. Bars denote s.e.

mass and distribution over time are shown in Fig. 7 for two
genotypes with contrasting hypocotyl elongation at 20 8C
and 10 8C (Jemalong A17 and F83005.5). Masses of seedling parts are expressed as percentages relative to the corresponding initial seed dry mass (SDM), as they could be
influenced by this factor, and are referred to here as Mc for
the cotyledons, Mh for the hypocotyl, Mr for the radicle and
Ms for the whole seedling. In addition, time is expressed as
thermal time after germination with genotype-specific values
of Tb to allow comparison of experiments on seedling
growth performed at different temperatures and on different
genotypes. Using this method of presentation, no significant
differences in changes in biomass of each seedling part were
observed between genotypes and temperatures. This contrasted
with the differences observed for final length.
The same general trends were observed for changes in
biomass regardless of temperature and genotype. Ms decreased
only slightly with time, to reach 85 90 % of initial SDM at
about 150 8Cd at the end of elongation; these net losses
could only be due to respiration or exudation. In contrast, significant changes were observed for the different seedling parts.
After germination, Mc decreased sharply from .80 % to about
30 40 % of initial SDM, reached at 50 8Cd. This decrease corresponded to the use of the cotyledon seed reserves; thereafter,
Mc remained almost constant. During the same period, Mh
increased sharply up to 40 % of initial SDM, reaching a
plateau at about 40 8Cd. Mr remained low, increasing only at

This study used an ecophysiological model in order to examine

germination and heterotrophic growth of Medicago truncatula,
a species frequently studied for genetic and molecular purposes (Gallardo et al., 2003; Young et al., 2003; Buitink
et al., 2006). This model framework separates the two stages
before emergence and describes the influence of the main
environmental factors during emergence under agronomic conditions. This proved essential in the present study, as differences observed between genotypes depended upon both the
phases of growth and the environmental factors considered.
We took advantage of the existence of nested core collections
(Ronfort et al., 2006), which enabled us to observe genetic
diversity despite the relatively small number of genotypes
we studied. The natural diversity that still remains within
M. trunculata is an advantage compared with working on
more highly bred species, for which genetic variability at the
early stages have been reduced by human selection or have
been influenced by selective breeding for other aspects of
plant growth. An example is the decrease in coleoptile
length observed in some cultivars when breeding for limiting
plant height in wheat (Botwright et al., 2001; Ellis et al.,
2004; Rebetzke et al., 2007).
Genetic and molecular determinism of germination have
been studied on model species such as Arabidopsis thaliana
and Medicago truncatula but rarely with the aim of gaining
knowledge for improving variables related to crop establishment. Many studies haved focussed on seed dormancy (e.g.
Chibani et al., 2006; Lefebvre et al., 2006) as it is an important
physiological trait of seeds and these two model species are
prone to this obstacle to germination. The environmental
factors or metabolic pathways studied are often those influencing dormancy (e.g. nitrate or hormone levels); however, dormancy is more characteristic of wild or weed species than of
cultivated varieties. Early growth after germination has received
even less attention (Gendreau et al., 1997; Cui et al., 2002).
Collecting agro-ecophysiological information on the early
stages for a model species should help contribute to a better
link between ecophysiological knowledge of plant emergence
and studies on genetic determinism of underlying mechanisms.
Main characteristics of Medicago truncatula germination
and heterotrophic growth
Strong effect of seed production conditions and dormancy on
early stages. Species of the Medicago genus, like other

legumes, are prone to having hard seeds (Martin and De La

Cuadra, 2004; Taylor, 2005; Zeng et al., 2005) and they
must be scarified to avoid this supplementary source of variation in the results when studying germination responses to

Page 10 of 15

Brunel et al. An emergence model for phenotyping of M. truncatula

Hypocotyl

100

10 C

20 C

Length (mm)

80

60

40

Jemalong A17
F830055
DZA31516
DZA455

20

DZA012J
SA28064
Borung
Paraggio

0
0

50

100

150

200

250

300

100

200

300

400

500

600

700

Radicle
100
20 C

10 C

Length (mm)

80

60

40

20

50

100
150
200
Time from germination (h)

250

300 0

100

200
300
400
500
Time from germination (h)

600

700

F I G . 4. Hypocotyl and radicle elongation at 20 and 10 8C for the M-05 seed lot genotypes and Paraggio Aust-04. Bars denote s.e., and lines are fitted Weibull
functions.

environmental factors. Moreover, post-harvest embryonic dormancy lasts several months for M. trunculata and can affect
germination studies. Physical seed characteristics (smaller
seeds and higher proportion of teguments, higher sucrose
and stachyose contents), consistant lower germination and
elongation rates distinguished the seed lot produced in a
growth chamber (A-06) under a lower light intensity during
plant growth. This seed lots characteristics suggested lessmature seeds with residual dormancy for some genotypes,
even though the pods had been harvested after natural abscission and been left for the recommended time in order to avoid
this problem. More generally, it was important to study
several seed lots for each genotype in order to be able to distinguish genotypic effects from seed-production conditions.
Maternal effects were observed for germination rates and
final percentages, seedling elongation rates and final
lengths, as could be expected from previous studies (e.g.
Squire et al., 1997; Clapham et al., 2000; Luzuriaga et al.,
2006).
Fast germination and elongation rates but over a narrow range of
environmental conditions. Despite the above considerations it

has been possible to draw general conclusions, and this

study shows that agro-ecophysiological models can be used

to analyse the functioning of non-crop species such as
Medicago. The range of temperatures over which
M. trunculata germinates is wide, but the range in which the
relationship between temperature and germination rate is
linear is narrow (5 15 8C), and the optimum temperature
was found to be rather low. One aim of the emergence
model was to provide a mathematical formalism for comparing
genotypes and seed lots, as well as species. In particular, when
using the calculation of thermal time, which is often used for
crop-development modelling, it appeared that the time taken to
achieve the germination process was very short (15 8Cd to
reach 80 % germination) and did not vary between genotypes;
it was the threshold values above and below which germination no longer occurred that differed. Tb,germ varied from 2
to 3 8C, a range observed for several other species
(Gummerson, 1986; Marshall and Squire, 1996; Tamet et al.,
1996; Dorsainvil et al., 2005), and a value also observed for
leaf development in Arabidopsis thaliana (Granier et al.,
2002), consistent with M. trunculata belonging to the
Galegod cool season growing group of legumes as opposed
to Phaseolids, which have tropical origins and higher temperature requirements (Covell et al., 1986; Moreau-Valencogne

Brunel et al. An emergence model for phenotyping of Medicago

Page 11 of 15

TA B L E 4. Hypocotyl and radicle final lengths at 20, 15 and 10 8C

Hypocotyl final length (mm)
Seed lot

20 8C

Genotype

M-052

Jemalong A17
F83005.5
DZA315.16
DZA045.5
DZA012-J
SA28064
Borung
Jemalong A17
F83005.5
DZA315.16
DZA045.5
DZA012-J
Jemalong A17
F83005.5
DZA315.16
DZA045.5
Paraggio
Paraggio

M-062

A-062

Aust-04
F-values3

75.2ab
67.6abc
76.0a
65.0c
66.6bc
71.6abc
70.0abc
70.2b
64.8b
78.0a
63.8b
67.6b
69.2a
50.5b
51.7b
50.7b
70.1a
69.9

15 8C

10 8C

72.0ab
57.7c
76a
67.2bc
68.7abc
76.3a
74.6ab
73.5a
64.9b
70.9a
70.0ab
74.8a
68.8b
68.2b
59.9b
63.7b
80.9a
72.2

63.6abc
46.2d
69.5a
62.9abc
55.1cd
60.8bc
66.8ab
68.6a
51.4c
73.1a
60.7b
60.9b
63.7a
48.1b
60.8a
48.6b
64.4a
73.8

4.02 (7) *
3.13 (6) ns
5.19 (6) *

G effect
S effect
T effect

Radicle final length (mm)

20 8C
44.4cd
51.7bc
57.5ab
45.6cd
39.1d
60.4a
47.8c
46.0c
54.0b
64.7a
39.2c
42.2c
35.0ab
41.4ab
41.8a
27.8b
45.1a
57.4

15 8C

10 8C

39.3cd
45.2ab
53.5a
40.7cd
34.7d
46.4b
35.6cd
43.4b
41.5b
57.1a
36.2b
37.0b
45.9a
48.2a
50.8a
37.3b
47.8a
41.5

38.7abc
34.0bc
46.1a
33.5bc
30.5c
38.1b
31.5bc
35.0b
34.8b
45.5a
30.8b
34.6b
32.7ab
32.2ab
35.7a
27.5b
34.8a
33.9

5.75 (7) *
0.98 (6) ns
8.37 (6) *

M, Montpellier; A, Angers; Aust, Australia; 04, 05 and 06 are years of production, 2004 2006.
Means followed by different letters are for significantly different genotypes within a given production site (P , 0.05; Tukey multiple-comparison test).
Seed lot (S), genotype (G) and temperature (T) were treated as fixed factors; the interactions S  G, S  T, G  T and S  G  T were considered as
random factors. Analyses were performed by pooloing the data of all the genotypes and seed lots. F-values are followed by the denominator degrees of
freedom (in brackets) and the level of significance: * P , 0.05, ** P , 0.01 and *** P , 0.001; ns, non-significant.
2
3

Elongation rate (1/tL30, h1)

003
M-05
Jemalong A17
F830055
DZA31516
DZA455
DZA012J
SA28064
Borung
Paraggio

002

001

0
003
Elongation rate (1/tL30, h1)

A-06

002

001

0
5

10

15
Temperature (C)

20

25

F I G . 5. Hypocotyl elongation rate (1/tL30; see text) in relation to temperature

for the seed lots M-05 and A-06, and Paraggio Aust-04. Bars denote s.e.

et al., 2007). The very fast germination of M. trunculata is close

to that of bean (Moreau-Valancogne et al., 2007), white
mustard (Dorsainvil et al., 2005) and oilseed rape (Marshall
and Squire, 1996). With respect to water potential,
M. trunculata appeared to be very sensitive to decreasing potentials, and few cultivated species have such high cb,germ requirements. The narrow range of environmental conditions in
terms temperature and water potential requirements within
which germination is both possible and rapid, is evocative of
plant behaviour in the case of wild or weed species, waiting
for favourable germination conditions within the soil. With
respect to seedling growth, elongation (as well as germination)
was very fast (about 30 8Cd after germination to reach 30 mm
hypocotyl length) compared with other studied species (Durr
and Boiffin, 1995; Dorsainvil et al., 2005), but similar to
other legumes (French bean: Moreau-Valancogne et al.,
2007; pea: M. P. Raveneau et al., ESA Angers, France,
unpbl. res.).
The differences in environmental requirements between
species should be kept in mind when comparing the effects
of both temperature and water potential on germination and
growth, as the underlying mechanisms may not be the same
(Nishiyama, 1972; Sung et al., 2003). Thus, results from
studies on reactions to contrasting environmental factors may
only be applicable to similar species.
Low temperatures considerably shortened the length of the
seedling parts with no relationship to the allocation of reserves
to the different plant parts, as previously observed for crop
species (Durr and Boiffin, 1995; Tamet et al., 1996).

Page 12 of 15

Brunel et al. An emergence model for phenotyping of M. truncatula

100
A

Hypocotyl length (mm)

M-05
80
60
40
10 C 15 C 20 C

20

Jemalong A17
F830055

0
100

Hypocotyl length (mm)

M-05
80
60
40
Jemalong A17
F830055
DZA31516
DZA455

20
0
0

DZA012J
SA28064
Borung
Paraggio

50
100
Thermal time from germination (Cd)

150

F I G . 6. Elongation time courses expressed in thermal time (8Cd) after germination: (A) at 10, 15, 20 8C for two genotypes Jemalong A17 and F83005.5
in the M-05 seed lot; and (B) all the temperatures pooled for each genotype of
M-05, and Paraggio Aust-04. Bars denote s.e.

Bouaziz and Hicks (1990) observed similar results with water

stress. Some M. trunculata genotypes were more responsive
than others: many aspects of plant metabolism can be involved
in this effect of low temperatures (Sung et al., 2003;
Chinnusamy et al., 2007; Penfield, 2008) and these need to
be investigated in order to analyse differences between the

Dry mass (% initial seed dry mass)

100

Whole seedling

75

Hypocotyl
50

Cotyledon
Radicle

25

50
100
Thermal time from germination (Cd)

150

F I G . 7. Changes in dry mass of seedling parts over time for Jemalong A17
and F83005.5 at 20 8C and 10 8C. Symbols are as follows: radicle squares;
hypocotyl circles; cotyledons triangles; and whole seedling diamonds.
Symbols for Jemalong A17 are white at 10 8C and black at 20 8C; symbols
for F83005.5 are dark grey at 10 8C and light grey at 20 8C. Lines were
fitted by hand, and bars denote s.e.

genotypes. Cell elongation is sensitive to hormonal changes

(Ellis et al., 2004; Schopfer, 2006; Rebetzke et al., 2007),
and several changes in cell wall and membrane properties
can be involved. For example, extensins are proteins involved
in cell elongation changes at low temperatures (Weiser et al.,
1990), and changes in lipid metabolism can also influence
membrane fluidity (Vaultier et al., 2006).
The other measurements made on seeds of the different genotypes did not reveal significant correlations between seed
mass and germination rate, final percentage germination or
final length. Seed sucrose and raffinose family (RFO) contents
were measured as they may be involved in mechanisms related
to dessication (Bailly et al., 2001; Rosnoblet et al., 2007) and
cold tolerance (Gilmour et al., 2000; Wang et al., 2006).
DZA045.5 had verbascose and stachyose contents that contrasted with other genotypes and it germinated the fastest at
low water potentials. A-06 had higher sucrose contents.
These results suggest that sucrose and RFOs, accumulated
during seed maturation, could be important in germination
and heterotrophic growth, but this requires further study. One
difficulty for gaining more in-depth knowledge of potential
mechanisms underlying genotypic differences in germination
and heterotrophic growth is that the reviews available on
potential mechanisms implied in abiotic stress tolerance (e.g.
Sung et al., 2003; Wahid et al., 2007; Penfield, 2008) rarely
focus on germination and early growth processes.
Existence of genetic diversity on model parameters. Our results
should help users of M. trunculata to choose segregating populations with contrasting parental lines for genetic analysis of
the response to environmental factors. They should also help
to focus on the parameters responsible for this diversity. An
important result is that the genotypic differences varied
according to the environmental conditions and stages considered. During germination, genotypes differed at extreme
temperatures and water potentials: F83005.5 was the least
tolerant to low temperatures, DZA012.J and Jemalong
A17 were less tolerant to high temperatures, and
DZA045.5 better tolerated low water potentials. When time
was expressed as thermal time, the remaining germination
differences between genotypes were low. The main differences
were thus in the values of Tb,germ and cb,germ and in the final
germination percentages. During elongation, F83005.5 was
once again the most responsive to low temperatures. When
time was expressed as thermal time, as for germination, differences between genotypes remained for final length at low
temperatures and, to a lesser extent, for elongation rates.
Paraggio, the cultivar cropped in Australia and chosen for
comparison with the genotypes of the core collection, had
no extreme behaviour during the stages studied (which are
not considered in crop selection). A part of the genetic diversity found for the parameters studied was observed for
environmental conditions not taken into account in the
model as they do not influence emergence. For instance, for
germination the largest differences between genotypes were
observed at 20 8C and above. An increase in genetic variation
is not unexpected under extreme environmental conditions,
such as supra-optimal temperatures not generally encountered
by the ecotypes in their natural biotopes (Swindell, 2006).
These temperatures were not taken into account in the

Brunel et al. An emergence model for phenotyping of Medicago

thermal time calculations as they fell outside the linear
relationship with temperature, and thus were outside of the
limits of the models function. In the field, high temperatures
are rare at crop sowing and thus they do not often limit
model predictions.
Other abiotic and biotic conditions that were not studied
here can influence emergence results. The effects of mechanical obstacles are taken into account in the emergence model
we used, as they have a strong impact under many sowing conditions (Awadhwal and Thierstein, 1985). These effects will be
further studied on the genotypes of M. trunculata considered
here. The effects of salinity or pH variations are not included
in the emergence model we used, and neither are pathogen
effects, and this can limit model predictions when considering
specific sowing conditions. In particular, modelling pathogen
infestations at sowing would be an important further improvement, given the significant decrease that has occurred in the
range of pesticides that can be used, and the need for better
represention of the complex effects of cropping systems on
these infestations.
In spite of these limits, the emergence model can be used to
simulate the emergence of the different genotypes in different
sowing conditions. This should make it possible to evaluate
the extent of variations in emergence in different environments
that are due to genetic variation within the models parameters.
The model can also be used to test virtual ideotypes and to
assist breeding programmes.
ACK N OW L E DG E M E N T S
This research was funded by the Region Pays de Loire and
INRA. We thank M. Delalande and D. Tauzin for information
about the genotypes studied; the technical staff at the SNES,
F. Pantin and E. Thierry for their contribution to these
results; J. Buitink from INRA for her help in measurements
with HPLC; and G. Hunault for his help in the statistical
analyses.
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