Germination of sunflower genotypes with modified fatty acid composition
Raúl González Belo.12, Roberto Benech Arnold, R.34, Jorge Tognetti12, Silvina San Martino5, Natalia
Izquierdo14
1
Laboratorio de Fisiología Vegetal. Facultad de Ciencias Agrarias, Universidad Nacional de Mar del Plata,
Ruta 226 km 73.5, (7620) Balcarce, Argentina.
2
Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, Calle 526 entre 19 y 20, (1900) La
Plata, Argentina.
3
Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET, Av. Rivadavia 1917, (A1033AAJ)
Ciudad Autónoma de Buenos Aires, Argentina.
4
Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, (C1417DSE) Ciudad
Autónoma de Buenos Aires, Argentina.
5
Grupo de Estadística. Facultad de Ciencias Agrarias, Universidad Nacional de Mar del Plata, Ruta 226 km
73.5. (7620) Balcarce, Argentina.
e-mail: nizquierdo@balcarce.inta.gov.ar
ABSTRACT
 Soil temperature at planting date determines germination speed and therefore the quality of plant
stand. In sunflower crops in Argentina, soil temperature can vary with regions, planting dates and tillage
systems. For example, soil temperature in no-tillage systems may be 1-2°C lower than in conventional tillage.
Preliminary evidences suggest that the fatty acid composition of sunflower seeds could modify its
germination, in a possible interaction with temperature. These effects may be of a high applied importance
due to increasing cultivation of sunflower genotypes whose fatty acid composition has been modified for
nutritional and health purposes. However, detailed studies are still lacking. The aim of this work was to study
the effect of temperature on the germination of sunflower genotypes with modified fatty acid composition.
 Experiments were performed with traditional, high oleic (HO, oleic acid >80%), high stearic high
linoleic (HSHL, stearic acid >15%) and high stearic high oleic (HSHO, stearic acid >15% and oleic acid
>60%) sunflower genotypes. In Experiment 1, a traditional hybrid, a hybrid and a line HSHO and a hybrid
and two inbred lines HSHL were used. In Experiment 2, a traditional, a HO and a HSHO hybrid were used. In
both experiments, seeds were incubated in 8-10 constant temperatures within a range 5°C - 38ºC, in darkness,
using growth chambers. Three replications of 20 seeds per genotype were placed in 9 cm diameter plastic
Petri dishes. The absorbent paper of the dishes was moistened with distilled water. Germination was recorded
as protrusion of the radicle (3mm) and it was monitored each 12-24h over 20 days. At the end of the trial, a
tetrazolium test was conducted to the non-germinated seeds to assess their viability. The germination rate was
calculated for each temperature and the base temperature (Tb) was estimated for each genotype.
 In both experiments, the germination rate linearly increased with temperature. In Experiment 1, the
maximum germination rate was 27°C for the traditional genotype, whereas for HSHO and HSHL genotypes,
the highest germination rate was observed between 31ºC and 38ºC. In the three genotypes in Experiment 2,
the highest germination rates were observed around 34ºC. In both experiments, genotype had a significant
effect on base temperature for germination (p=1.55-7 and p=4.01-3, for Experiment 1 and 2, respectively). In
Exp 1, in general, HSHL genotypes presented lower T b than HSHO genotypes (1.98ºC vs. 2.36ºC,
respectively). In both experiments, genotypes with modified fatty acid composition presented higher Tb than
the traditional ones (1.75-2.70°C vs. 0.57°C for Exp. 1 and 2.90-3.29ºC vs. 2.33ºC for Exp. 2). For a given
fatty acid composition, the inbred lines presented a higher Tb than hybrids.
 Temperature increased the germination rate in both traditional and modified fatty acid composition
genotypes. The latter presented a higher base temperature than traditional ones. This higher base temperature
would result in a lower accumulation of thermal time required for germination. The reasons of this higher
base temperature of genotypes with modified composition are still unknown.
 These results suggest that soil temperature at planting time can play an important role in obtaining an
adequate stand of plants in sunflower genotypes with modified fatty acid composition. Thus, selecting the
growing area and sowing date may be key factors for cultivating these genotypes.
Keywords:– stearic – oleic – germination rate - base temperature
INTRODUCTION
Rapid and uniform seed germination in field are essential for improving crop establishment and growth, and
ultimately to obtain high yields. In sunflower, seeds may encounter relatively low soil temperatures
depending on cultivation area, planting date and tillage system. For example, average soil temperatures in
direct sowing can be 1-2°C lower than in conventional planting (Aase and Siddoway, 1980; Potter et al.,
1985). In recent years, different genotypes with modified fatty acid composition have been developed to meet
the growing demand for high quality oils. These oils have better nutritional quality, higher stability towards
oxidation, and are more solid at room temperature than traditional ones, which make them best suited to
particular uses such as frying or fabrication of margarine (Velasco and Fernandez-Martinez, 2002; Crupkin
and Zambelli, 2008). Examples of these are the "high oleic" genotypes that produce >80% of this fatty acid
versus <60% in the traditional, or "high stearic" genotypes containing between 25 and 30% stearic acid
(Fernandez-Moya et al., 2000) versus <10% in traditional ones. There are also genotypes containing both
mutations, such as "high stearic-high oleic" (Pleite et al., 2006), which have 15% and 60% of these fatty
acids, respectively (CAA, 2011). In general, genotypes with modified fatty acid composition have less
percentage of linoleic acid than traditional ones. Modifications in fatty acid composition might have an effect
on seed germination. It is well known that fatty acid composition of cell membranes is closely related to seed
germination performance at different temperatures; i.e. increased proportion of unsaturated fatty acids such as
linolenic acid enables cells to maintain membrane fluidity at low temperatures (Nishida and Murata, 1996;
Murata and Los, 1997; Saeidi and Rowland, 1999). The increase in the unsaturation degree of membrane
lipids is also correlated with the sustained activity of membrane-bound enzymes at lower temperature
(Raison, 1973, 1980). Thus, the unsaturation of membrane lipids is considered to be one of the most critical
parameters for the functioning of biological membranes and, therefore, for the survival of organisms at low
temperatures (Cossins, 1994). Much less is known about the possible influence of change in the composition
of reserve lipids on germination at different temperatures. Lower germination temperatures may prevent or
delay the mobilization of seed reserves in germinating seeds, leading to lower germination (Nykiforuk and
Johnson-Flanagan, 1994).
The thermal time model has been used to characterize the effect of temperature on the germination rate
(Garcia-Huidobro et al., 1982; Gummerson, 1986; Bewley and Black, 1994). This model defines three
cardinal temperatures. These are: i) base temperature (Tb), temperature below which the seed cannot
germinate, ii) optimal temperature (T o), at which germination occurs with the highest speed and iii) maximum
temperature (Tm), for above which germination cannot occur. Thus, germination rate versus temperature has a
bilineal relationship with a positive and negative slope between Tb and To and To and Tm, respectively. The
cardinal temperatures vary between species (e.g. Covell et al. 1986). For example, To germination of
traditional sunflower is 25ºC (Gay et al., 1991), of Cuphea is 21ºC (Berti and Johnson, 2008), of the true seed
potato is 20ºC (Alvarado and Bradford, 2002), and of Jatropha curcas is 30ºC (Windauer et al., 2011). Ellis
et al. (1986) suggested that Tb is constant within the same seed lot and even within the same species.
However, there is evidence that the effect of temperature on the germination rate can vary between genotypes
within a species. For example, Hernandez and Paoloni (1998) observed that sunflower genotypes with
different linoleic / oleic acid ratio, showed different germination rate at low temperatures. These differences
may be due to variations in germination rates, which change cardinal temperatures. There is preliminary
evidence suggesting that the fatty acid composition of the stored lipids could modify seed germination,
possibly in interaction with temperature, but there is no accurate information about it. The objective of this
work was to study the effect of temperature on the germination of sunflower genotypes with modified fatty
acid composition.
MATERIALS AND METHODS
Two experiments were performed. In Experiment 1, we used a traditional hybrid, provided by ACA
(Asociación de Cooperativas Argentinas), a hybrid (HSHO_H) and a line (HSHO_L) with high stearic-high
oleic composition (HSHO) and a hybrid (HSHL_H) and two lines (HSHL_L1 and HSHLO_L2) with high
stearic-high linoleic (HSHL) composition provided by Advanta Semillas SAIC. In Experiment 2, three
hybrids were used, one traditional, one high oleic (HO), and one HSHO, all provided by Advanta Semillas
SAIC. In both experiments, seeds were incubated in 8-10 constant temperatures within 5°C - 38ºC, in
darkness, using growth chambers. Three replications of 20 seeds per genotype were placed in 9 cm diameter
plastic Petri dishes. Seeds were placed on absorbent paper saturated with distilled water. During 20 days, the
number of seeds germinated was recorded every 12-24h (Windauer et al., 2011). A seed with a radicle of at
least 3mm long was considered germinated. At the end of the experiment, tetrazolium test was performed to
non-germinated seeds to assess their viability (ISTA, 1999). For each genotype, the germination rates of the
fraction 30% versus temperatures were plotted (Windauer et al., 2011) and the range of sub-optimal
germination was identified (between Tb and To) as the range with positive slope in the relationship. When no
change point was observed, all temperatures were considered as sub-optimal range. The Tb values were
determined by repeated probit regression analysis (Ellis et al., 1986). Tb data were processed by analysis of
variance procedures (R, 2011). Differences in Tb among genotypes were evaluated with the Tukey test (P <
0.05).
RESULTS AND DISCUSSION
In both experiments, germination percentages were above 90%, except for genotype HSHO of the experiment
2 that reached only 78% germination (data not shown). At 5ºC, in both experiments, traditional genotypes
presented a faster germination evolution compared to HSHO genotypes (Figure 1). At 22ºC and 30ºC minor
differences were observed between genotypes; and in some cases, HSHO genotypes showed a faster
germination evolution compared to traditional genotypes.
Figure 1. Evolution of germination (%) for traditional and HSHO hybrids of Experiment 1 and 2 at three
temperatures. Bars indicate standard error.
In both experiments, the germination rate linearly increased with temperature. In Experiment 1, the maximum
germination rate was observed at 27ºC for traditional genotype. This is consistent with the findings of Gay et
al. (1991), where a traditional cultivar presented an optimum temperature of 25ºC. In Experiment 1, HSHO
and HSHL genotypes showed a maximum germination rate between 27ºC and 38ºC. In the three genotypes in
Experiment 2, the highest germination rates were observed at 34ºC.
In both experiments, genotype effect was found on the base temperature of germination (p=1.55-7 and p=4.013
, for Experiment 1 and 2, respectively). Traditional genotypes presented lower T b than others genotypes
(Table 1). In both experiments, no differences in T b among genotypes with modified composition were
observed, except in HSHO_L which presented the highest T b.
Table 1. Base temperature (Tb, ºC) for the six genotypes of Experiment 1 and the three hybrids of Experiment
2.
Tb
Traditional 0.57 a†
Experiment 1
HSHO_H 2.02 b
HSHO_L 2.70 c
HSHL_H
1.75 b
HSHL_L1 2.01 b
HSHL_L2 2.24 bc
Experiment 2
†
Traditional 2.33 a
HO
3.29 b
HSHO
2.90 b
Means with same letters indicate no significant statistical differences (p<0.05)
The Tb recorded for any genotype was lower than that used by the model OilCrop-Sun (Villalobos et al.,
1996). These results differ from those found by Ellis et al. (1986), who reported that different genotypes of
the same species had similar base temperature.
The higher Tb of seeds with modified fatty acid composition compared to traditional ones could be associated
to the lower unsaturation degree of the lipids stored in the seeds as observed in other species. According to
Dogras et al. (1977), chilling resistance of the seed was positively associated with unsaturation of the fatty
acids in the seed of broad beans (Vicia faba L.) and peas (Pisum sativum L.). Unsaturated/saturated fatty acid
ratio of seed lipids in Pima cottonseed (Gossypium barbadense L.) showed a low correlation with laboratory
germination percentage, but had a strong, positive correlation with emergence under field conditions at low
soil temperatures (Bartkowski et al., 1977). Further research is needed to understand the effect of the fatty
acid composition of the stored lipids in the seed on the germination performance.
Our results suggest that soil temperature at planting time can play an important role in obtaining an adequate
stand of plants in sunflower genotypes with modified fatty acid composition. The optimum temperature was
between 27ºC and 38 ºC for most of the studied genotypes. The traditional hybrids had lower Tb than
genotypes with modified fatty acid composition. A higher Tb delays the emergence in early plantings or
increases the exposure of seeds to fungi and insects, reducing plant stand uniformity. The causes of this higher
base temperature of genotypes with modified fatty acid composition are still unknown.
ACKNOWLEDGEMENT
This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica PICT 941 and
Universidad Nacional de Mar del Plata AGR340-11.
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