It is well known that physiological activity in seeds is affected by water
content (Rupley et al., 1983; Vertucci
et al., 1985). In seeds water binds with varying strengths at different
water concentrations and therefore has different thermodynamic properties (Rupley
et al., 1983). The occurrence of 3 different types of water has been
reported in dry seeds by various researchers (Vertucci and
Leopold, 1984; Vertucci et al., 1985). The
physical state of water in seeds determines the physiological manifestations
connected with imbibition and germination. Ishida et
al. (1987) reported that most of the biological activities cease or
at minimum level in dry seeds including food consumption.
Moisture content reduction in seeds is initiated during maturation of seeds
and orthodox seeds can be dried to low moisture content without any damage (Bewley
and Black, 1994; Mayer and Poljakoff-Mayber, 1989;
When the seeds are subjected to higher temperature, progressive removal of water occurs. Bound water, associated with macromolecules is lost first, resulting in structural and functional deterioration of seeds (Bewley and Black, 1994). Higher temperature induces tighter packing of molecules and increases their structural disorder leading to loss of seed vigour and/or viability. The final water content of dry seeds appears to be important in determining their survival over long periods of storage (Robert and Ellis, 1989).
Seed viability denotes the degree to which a seed is alive, metabolically active
and possesses enzymes necessary for catalyzing metabolic reactions needed for
germination and seedling growth (Basara et al., 2002).
According to Gelmond et al. (1978) the seed vigour
means a high rate of the overall biological activities of the seed, resulting
in a high yield performance. They measured and predicted seed vigour according
to the rate of root emergence of germination or field emergence. Vigour represents
the potential ability of the seed to yield the maximum plant product at the
earliest time under variable environmental field conditions.
In the present study, an attempt was made to correlate how temperature treatment and seed water content affected the viability of pea and soybean seeds.
MATERIALS AND METHODS
Seeds of pea (Pisum sativum L.) c.v. boneville and soybean (Glycine max L. Merr.) c.v. SL. 525 were used to study the correlation of germination percentage, moisture content and seed and seedling vigour. From the seed lots, seeds having uniform size, colour and shape with intact seed coat were selected and were subjected to temperature treatments such as 50, 60 and 70°C for 10 days each.
The temperature treated and fresh, healthy, untreated seeds, used as control
were kept in Petri dishes for germination under laboratory conditions and germination
count was taken daily up to 7 days. Parameters like moisture content, imbibition
percentage, percentage of hard seeds, germination percentage and seed/Seedling
Vigour Index (SVI) were calculated (ISTA, 1985; Copeland
and McDonald, 1995).
Germination percentage: Three replicates of 30 seeds each of treated and untreated or control seeds were placed in sterilized Petri dishes and covered with lid plates which also lined with moistened filter paper. Petri dishes were watered as required to replace evaporation losses. Radicle emergence of 1 mm was scored as germinated and germination percentage was calculated by using the formula:
Moisture content: Thirty seeds from each of the treatment and control
were taken and brought to room temperature by keeping in a desiccator and fresh
weight of these seeds was determined by using electronic balance (Shimadzu AX
120). Then the weighed seeds were kept in a hot air oven at 100°C for 1
h and kept at 60°C until constant weight was obtained. Percentage of moisture
content was calculated, as explained by ISTA (1985):
Seed Vigour Index (SVI): Thirty seeds each in triplicate from the control
and temperature treated seed lot were sampled and sown in garden pots filled
with garden soil, sand and dry powdered cow dung mixed in 2:1:1 ratio. Daily
count of germinated seeds was taken and percentage of germination was calculated.
The seed vigour index was calculated according to Copeland
and McDonald (1995) using the formula given below:
Seedling vigour: Vigour of seedlings of control and temperature treated
pea and soybean was calculated using biomass method. Seedlings after 7 days
of germination were uprooted carefully and washed in running tap water to remove
the sand particles. The seedlings with cotyledon were blotted and weighed using
electronic balance. After noting the initial weight, the seedlings were kept
in hot air oven at 100°C for 1 h and transferred to and kept in 60°C
until constant weight was obtained. The seedling vigour was expressed as biomass
Seeds of P. sativum and G. max are orthodox and the seed lots exhibited 100% germination. When the seeds were subjected to temperature treatments at 50, 60 and 70°C for 10 days continuously, the germination percentage was decreased (Table 1). The high temperature up to 70°C given to the seeds reduced the germination percentage to 10% in pea and 17% in soybean.
Viability of the two legume seeds, P. sativum and G. max is inversely proportional to temperature and above 50°C, the germination percentage is below 50%. Soybean seeds exhibited higher resistance to temperature stress with a higher germination percentage and seed vigour index values than the pea seeds.
For the maintenance of 100% germination, 12.8 and 13.9% of moisture content are found to be essential in pea and soybean seeds, respectively. When the moisture content is reduced to 7.3% in pea and 8.7% in soybean at 60°C, the germination is reduced to 33 and 40%, respectively and this reduction in germination is directly proportional to the temperature (Table 1).
Viability of the two legume seeds, P. sativum and G. max is inversely proportional to temperature and above 50°C; the germination percentage is below 50%. The germination percentage declined to 10 from 33% when the temperature was raised from 60 to 70°C.
The germination is found delayed in both the seed samples subjected to temperature treatment and soybean exhibited more seed vigour in samples treated at 70°C than those of pea seeds.
of Temperature on water content and viability of Pisum sativum and
Glycine max seeds
In the present study, when the seeds were dried gradually, more time was taken
for imbibition as well as germination and this was reflected in the reduced
values of seed vigour index. An important observation in pea seeds was that,
the percentage of hard seeds treated at 60°C was 3.3% and at 70°C, the
percentage was increased to 6.6% (Table 1). But soybean seeds
showed no hardness in any of the temperature treatments.
As a consequence of high temperature treatment, reduction in germination has
been reported in Solanum nigrum (Del Monte and Tarquis,
1997) and in Raphanus sativum (Meng et al.,
2003) seeds. Roberts (1973) opined that orthodox
seeds can be dried to low moisture contents without damaging the embryo and
their longevity increases with decrease in moisture content during storage over
a wide range of conditions. Contradictory to this view, drying at high temperature
leads to significant reduction of moisture content and concomitant loss of viability
in pea and soybean seeds, plausibly due to the continuous treatment for 10 days
at 50 to 70°C (Table 1).
It can be presumed that during this temperature treatment, the tissues lose
the water molecules adsorbed to the molecules of cell membranes. This is in
consonance with the views of Bewley and Black (1994),
who suggested that the adsorbed water or bound water is loosely held by bonding
The reduction of moisture content below 6.4 and 6.8%, respectively in pea and
soybean seeds, when treated at 70°C, resulting in the loss of viability
presumably reveals the loss of all the three types of water as suggested by
Bewley and Black (1994). However, temperature treatment
at 50°C showed above 60% germination, which may be due to the loss of free
water alone. The metabolic activities of the seeds at this temperature treatment
could be maintained almost normal even in the absence of free water. Since,
the rate of viability loss in P. sativum and G. max seeds as a
response to higher temperature treatment and to the rate of water loss is gradual
and more or less continuous, the concepts of bound water cannot be directly
correlated to the reduction of moisture content in these seeds at any particular
temperature. It seems that the entire bound water or type III of bound water
is not completely removed at 70°C because viability is still retained at
least 10% in pea and 17% in soybean.
Bewley and Black (1994) suggested that water content
of seeds exists in a glassy (vitrified) state even at physiological temperatures
and it stops or slows down chemical reactions, assures stability and quiescence
and prevents interactions of cell components. In vitro studies have shown
that vitrification of water retards or prevent denaturation of proteins including
enzymes (Bruni and Leopold, 1991). So, in the present
study, it can be presumed that even the vitrified water in P. sativum
and G. max seeds is being slowly removed by temperatures higher than
50°C, resulting in gradual loss of viability.
The reason for the viability of seeds being maintained at high temperatures
like 70°C may be at least partial maintenance of vitrification or glass
formation of water within the cytoplasm, which is the potential mechanism to
avoid crystallization of proteins and solutes present in the cytoplasm as suggested
by Bruni and Leopold (1991). Williams
and Leopold (1989) explained the glassy nature of water in corn embryos
as a liquid with the viscosity of a solid and its formation from a liquid involves
no chemical and physical change in the solution. The major function of the glassy
state in the dry seeds is its contribution to the stability of the seed components
during storage and thus to the survival during desiccation (Leopold
et al., 1994).
According to Nichols and Heydecker (1968), the rate
or speed of germination is considered as the criteria for seed vigour index
determination. The time taken for imbibition/germination is also very important.
The decline of seed vigour index in P. sativum and G. max seeds
is closely correlated with the loss of moisture content which is the determinant
factor of seed quality. This observation is in conformity with the view of Fu
et al. (1994), who suggested that seed vigour index is an accurate
measure for testing quality of seeds.
A close correlation between moisture content, germination percentage and seed
vigour index was observed in both the legume seeds studied. The temperature
treatments made the seeds more desiccated, resulting in reduction of seed vigour
index, germination percentage and moisture content percentage throughout the
period of study. According to Harrington (1972),
Villiers (1973) and Douglas (1975), seed vigour is
affected by various environmental factors, such as temperature, moisture content
and concentrations of oxygen and carbon dioxide.
In the present study, soybean seeds showed higher resistance to temperature
stress with a higher germination percentage and seed vigour than the pea seeds.
The oil-rich nature of soybean seeds can be correlated to these qualities (Table
1). This observation is in agreement with the view of Pixton
(1967) and according to the researcher, oil content influences seed water
relations and is a major determinant of equilibrium and relative humidity, which
in turn are related to water potential. Roberts and Ellis
(1989) and Ellis et al. (1989) reported the
evidence for a common response of seed longevity to moisture content in oily
and non-oily seeds.
of temperature on seedling vigour in Pisum sativum and Glycine
max temperature treatment
The effect of temperature treatment on pea and soybean seeds reflected on the
rate of germination and growth of seedlings up to 7 days showed that the seedling
vigour was gradually decreased proportional to the increase in temperature (Fig.
Even when conditions are apparently favorable for germination, high temperature
treatment induced dormancy. Ellis and Roberts (1982)
suggested that hard seeded condition is an important consequence of desiccation.
Hard seeded nature has been attributed to both genetic factors and environmental
conditions such as soil fertility, photoperiod, relative humidity and temperature
(Bewley and Black, 1983; Mayer and Poljakoff-Mayber,
1989). So the difference in the distribution of hard seeded condition between
pea and soybean seeds treated to similar higher temperature regimes is presumably
due to the genetic factors.
Hard seeds achieve and maintain a very low percentage of moisture, despite
wide fluctuations in the moisture contents of the surrounding air. Mai-Hong
et al. (2003) observed that in seeds of Peltophorum pterocarpum,
a tree legume, the hard-seededness was induced when seeds were dried to about
15% moisture content. Storage of seeds for extended period of time at high temperature
and humidity resulted in the occurrence of increased hardness in most legumes.
In legume seeds like pea, storage brings about hardening of the seed coat by
oxidation of the phenolic compounds present in the testa (Van
Staden et al., 1989). According to those researchers, the oxidation
of phenolic compounds blocks the small pores in the seed coat and thereby makes
the seeds impermeable to water. Therefore, a large percentage of hard seeds
were seen in stored samples for a long period. However, in the present study,
instead of prolonged storage, high temperature treatment results in enhanced
oxidation of phenolics of the testa of pea seeds. In Glycine max seeds,
there is no sign of decolorization or browning of the seed coat and therefore
no hard seeds were observed and all the seeds imbibed readily and uniformly
when soaked in water.
The first author (KBA) is thankful to the Head of the Department of Botany, University of Calicut for providing necessary facilities and the U.G.C. for granting financial assistance to carry out the study.