Received: 14 July 2021 Accepted: 15 March 2022 Published online: 28 May 2022

DOI: 10.1002/csc2.20750

Crop Science
ORIGINAL RESEARCH ARTICLE
S e e d P h y s i o l o g y, P r o d u c t i o n & Te c h n o l o g y

Is seed coat structure at fault for altered permeability and

imbibition injury in artificially aged soybean seeds?

Vijayan Satya Srii Nethra Nagarajappa Saligrama Nabhirajaiah Vasudevan

Seed Technology Research Center, All India

Co-ordinated Research Project, National
Seed Project, Gandhi Krishi Vignana
Kendra, University of Agricultural Sciences,
Bangalore, India
Correspondence
Nethra Nagarajappa, Seed Technology
Research Center, All India Co-ordinated
Research Project, National Seed Project,
Gandhi Krishi Vignana Kendra, University
of Agricultural Sciences, Bangalore, India.
Email: nethraharsha@gmail.com
Assigned to Associate Editor Veronica
Rodriguez.
Abstract
Ageing induces many deteriorative changes to seeds during storage like genetic
damage, protein degradation, enzyme inactivation and loss of membrane integrity.
In this study, we subject to investigate the alterations in seed coat structure and its
permeability properties due to accelerated ageing affecting the subsequent imbibition
of soybean [Glycine max (L.) Merr.] seeds. Two contrasting seed coat colour geno-
types, JS 335 (white) and Bhatt (black) were selected and artificially aged for 48 and
72 h. The results of seed ageing on permeability, imbibition and germination proved
that permeability and imbibition increased but germination decreased in white geno-
type due to imbibitional injury, whereas germination increased in black genotype due
to breaking of hard seed coat imposed dormancy. The hard seeds in the black geno-
type with a different seed coat structure compared to the white genotype prevented
imbibition injury in aged seeds by regulating permeability. Microscopic studies of
seed coat structure in aged seeds revealed a thick cuticle with small hilar fissure and
compact, dense hourglass cells in black genotype compared to a thin cuticle with
large hilar fissure and loosely packed and distorted hourglass cells in white geno-
type. Thus the altered permeability and imbibition injury in artificially aged seeds is
shown to occur mainly as a result of altered and damaged seed coat structure.

1 INTRODUCTION like genetic damage, protein degradation, enzyme inactiva-

Seed ageing is an irreversible and inexorable process of a pro-
gressive decrease in vigour ultimately leading to the loss of
seed viability (Arc et al., 2011; Lehner et al., 2008; Stew-
art & Bewley, 1980). The rate of seed ageing depends upon
the genotype/ species, the conditions prevailing during stor-
age like moisture content, temperature, humidity and seed
composition (Roberts, 1973). It has been reported that high
moisture content and high temperature accelerate seed deteri-
oration (Ellis & Hong, 1991; Goel et al., 2003) based on which
seeds are accelerated to artificially age by exposing them to
high humidity and temperature of about 40–45 ˚C and 100%
RH (Delouche & Baskin, 1973). Accelerated ageing is shown
to induce many deteriorative changes to seeds during storage
tion and loss of membrane integrity (Arc et al., 2011; Bailly,
2004; Bailly et al., 1996, 2002; Merritt et al., 2003; Ratajczak
& Pukacka, 2005; Ratajczak et al., 2015; Wang et al., 2011)
which ultimately lead to reduced germination (Walters, 1998)
and loss of viability. Though there are numerous studies on
physiology, ROS and its mechanisms of membrane degrada-
tion during ageing, there is less knowledge about the changes
in seed coat structure due to altered permeability properties
and imbibition patterns affecting the process of germination
in artificially aged seeds.
Seed germination of desiccated quiescent seed usually
occurs as a three-step process (Bewley, 1997; Weitbrecht
et al., 2011), commencing with imbibition and terminating
with radical protrusion. However, the rate of water absorption

© 2022 The Authors. Crop Science © 2022 Crop Science Society of America.

Crop Science. 2022;62:1573–1583. wileyonlinelibrary.com/journal/csc2 1573


1574 Crop Science
by seeds during this phase of imbibition depends on the per-
meability properties of the seed coat like its hydrophobic-
ity, thickness, porosity and integrity. It is reported that seed
coats with cracks or pores imbibe water rapidly compared to
intact seed coats (Duke & Kakefuda, 1981; Duke et al., 1983;
Duke et al., 1986; Oliveria et al., 1984; Powell & Matthews,
1979; Powell et al., 1986a; Tully et al., 1981). However, in
Fabaceae, hard seeds with impermeable seed coats pose a
problem for phase I of imbibition, a critical period for germi-
nation as 80% of water absorption is reported to occur during
this phase (Souza & Marcos-Filho, 2001). It is known that
the seeds lose membrane integrity upon ageing or by other
mechanical handling procedures during storage altering the
seed coat’s permeability properties. Veselova and Veselovsky
(2003) reported that aged pea seeds showed imbibitional
injury leading to the production of abnormal seedlings, which
they proposed to be due to permeability changes in the seed
coat to water. However, the changes brought about by acceler-
ated ageing on seed coat structure resulting in altered perme-
ability properties which may be one of the causes of imbi-
bitional injury is not studied. In this study, we subject to
investigate the alterations in seed coat structure and perme-
ability properties due to accelerated ageing affecting the sub-
sequent imbibition of soybean [Glycine max (L.) Merr.] seeds.
Seed coats in Fabaceae have four main layers which include
the waxy cuticle, the epidermis, the hypodermis and the inte-
rior parenchyma (orders of the layers from the surface to
endosperm) (Swanson et al., 1985). The detailed structure and
functions of each layer of seed coat have been reviewed ear-
lier by Souza and Marcos-Filho (2001), the gist of these lay-
ers’ function in permeability to water is presented here. The
outermost cuticle regulates imbibition and is the first barrier
to pests and pathogens; while the epidermis composed of pal-
isade cells (known as macrosclereids) decides permeability.
Both, the hypodermis made of hourglass cells (also called
pillar cells, osteosclereids or lagenosclereids) and the inte-
rior parenchyma made of protoplast-free parenchyma cells are
found all over the seed coat except at the hilum. Thus any vari-
ation/ alteration/ disruption to these layers should impact the
permeability properties of the seed coat.
Soybean was opted for the study as it was proven to be a
more sensitive species for imbibitional injury (Veselova et al.,
2003; Woodstock & Taylorson, 1981). The selection of geno-
types was based on the study of Kuchlan et al.,2010 where
they screened soybean genotypes for the seed coat physical
properties and longevity and concluded that coloured geno-
types had a difference in seed coat structure which contributed
to their longevity difference. Also, studies have reported dif-
ferential regulation of water uptake concerning the colour of
testa and its adherence property to cotyledon in Phaseolus
vulgaris which in turn contributed to vigour difference (Pow-
ell et al., 1986a; Powell et al., 1986b). With this background,
two contrasting colour soybean genotypes JS 335 (white) and
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SATYA SRII ET AL.
Core Ideas
∙ Artificial ageing damages seed coat structure espe-
cially in cuticle and hourglass cells.
∙ The degree of damage differs betweeen genotypes.
∙ The seed coat damage and resulting permeability
changes leads to imbibition injury.
∙ This imbibiton injury is the reason for abnormal
seedlings in artificially aged seeds.
∙ Genotype with hard seed coat escape such damage
and germination loss."
Bhatt (black) were selected for the study assuming that dif-
ferent seed coat structural properties of contrasting genotypes
would provide some insight on permeability concerning sub-
sequent imbibition. To create seed lots of different seed qual-
ities, we opted for accelerated ageing of seeds as it is a proven
method to determine seed vigour (ISTA, 2013). For this, age-
ing treatments of 48 and 72 hours was standardized to study
the seed coat structural changes in aged but viable seeds.
It is to be noted that the International Seed Testing Asso-
ciation protocol (ISTA, 2013) for vigour testing in soybean
also states 72 hours of accelerated ageing as a vigour test
for soybean, supporting our results in the selection of ageing
periods.
The difference in patterns of imbibition concerning alter-
ations in seed coat structure and permeability within and
between two genotypes after accelerated ageing in compari-
son with the control (fresh, non-aged seeds) was investigated.
2 MATERIALS AND METHODS
2.1 Seed material
The two soybean genotypes JS 335 (white) and Bhatt (black)
were selected for the study and the fresh seeds of JS 335
and Bhatt were received from All India Coordinated Research
Project on Soybean, Zonal Agricultural Research Station,
University of Agricultural Sciences, Bangalore, India and
Indian Institute of Soybean Research, Indore respectively.
2.2 Accelerated ageing
Artificial ageing was performed according to ISTA guidelines
(ISTA, 2013). The moisture content of the fresh seed samples
was determined by the hot air oven method (ISTA, 2013) and
was seen to be between 10–14% which was ideal to proceed
with accelerated ageing. For artificial ageing (AA), the plastic
boxes were first sterilized with 5% sodium hypochlorite, dried

SATYA SRII ET AL.
and then each was filled with 40±1.0 ml of distilled water.
Forty-two grams of seeds were placed on the screen one layer
deep to ensure an even uptake of moisture from the humid
environment and the lid was placed on each box. These boxes
were placed on the shelves of the ageing chamber (Manufac-
turer: Thermo Scientific, Model: IGS 60/100/180) allowing
air space of 2.5 cm between boxes to assure temperature uni-
formity. The temperature was set to 41± 0.3 ˚C with 100% RH
and was monitored at regular intervals. After 48 and 72 hours
of ageing, the AA boxes were removed from the chamber.
The seed moisture content of seeds after ageing was measured
using a digital moisture meter.
2.3 Germination percent and viability
Seed germination test was performed within 1 h after removal
from the ageing chamber for 100 seeds in 4 replicates by
between paper method at 25 ˚C for each ageing treatment and
control (fresh, non-aged seeds). The first and final count was
taken on the 5th and 8th day and the seedlings were evalu-
ated as per International Seed Testing Association guidelines
(2010) considering the percentage of normal seedlings to be
germinated.
The viability testing of seeds was performed for 50 seeds in
2 replicates for each ageing treatment using Tetrazolium (Tz)
staining. Tz staining was performed by soaking the seeds in
distilled water for 18 h followed by removal of seed coat and
then soaking the seeds without seed coat in 1% Tz solution for
3 h at 30 ˚C in dark and the uptake of stains was evaluated as
per International Seed Testing Association guidelines.
2.4 Electrical conductivity
Membrane deterioration was measured by an electrical
conductivity (EC) test as per the modified ISTA method
(ISTA, 2013). Fifty seeds in two replicates in each ageing
treatment along with control were soaked in 250 ml of dis-
tilled water for 24 h at 20 ˚C and the conductivity of the elute
was measured using a conductivity meter (Manufacturer: Sys-
tronics, Model: 306).
2.5 Seed imbibition studies
Seed imbibition studies were performed using an electronic
balance approach by measuring the weight difference. Three
replicates of 10 seeds in each ageing treatment and control
were taken for the study. The weight of 10 seeds was measured
using an electronic balance (Manufacturer: ESSAE, Model:
FB 200-172-14666). The values of weight were recorded at
0 h of imbibition and then the seeds were allowed to imbibe
water in petri dishes and the weight at a time interval of 1 h
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Crop Science 1575
was recorded till 3 h to capture the imbibition pattern during
phase I of the triphasic germination curve. The significance
of values for different sample groups at a given time and all
the values within the group at different times were analyzed
using ANOVA (with single factor) with post hoc Tukey Kram-
mer test using Statistical Package for Social Sciences (SPSS)
software.
2.6 Seed coat permeability studies
As it is difficult to visualize the permeability of seed coat
through water uptake, tetrazolium uptake by the embryo in
seeds with intact seed coat was performed to assess the perme-
ability of testa as described earlier by Debeaujon et al., 2000.
The embryo and the aleurone layer stain red upon entry of
the tetrazolium solution into the viable seed, but stay whitish
when the dye does not penetrate. As germination and via-
bility tests of seeds confirmed the seed viability in different
seed ageing treatments, this procedure of Tetrazolium uptake
by embryos proved suitable to visualize permeability neglect-
ing the discrepancies of staining due to seed viability differ-
ence. 10 seeds in three replicates in each ageing treatment and
control was soaked in 1% tetrazolium solution for 3 h with
seed coats and then the seeds were cut open to examine the
penetration of Tz stain into the embryo. Also, the amount
of staining was quantified by measuring the total dehydro-
genase activity in Tz imbibed seeds by a modified method
described by Perl et al., 1978. The seeds imbibed in Tz were
then cut open and washed thoroughly with distilled water, the
red-coloured formazan from the stained regions was extracted
by soaking with 5 ml of 2-methoxy ethanol for 8 h in an
airtight container. The extract was decanted and the colour
intensity was measured in Spectrophotometer (Model Mini
Spec 17) at 480 nm with a suitable blank (Methoxy ethanol).
The total dehydrogenase activity (TDH) was expressed as
absorbance. The significance of values for different sample
groups of two genotypes were analyzed using ANOVA (with
single factor) with post hoc Tukey Krammer test using SPSS
software.
2.7 Microscopy
2.7.1 Light microscopy
Seed coat sections of five to ten seeds in each ageing treat-
ment along with control were examined under the light micro-
scope. Also, the seeds were imbibed in water for 1 h and thin
freehand sections were observed under the light microscope
(Manufacturer: CETI, Model: 0807875). Care was taken not
to create damage to seed coats during sectioning by embed-
ding the seed coats in wax for sectioning. The thickness of
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1576 Crop Science SATYA SRII ET AL.

cuticle, epidermis and hilar openings were measured using

21.721 ± 0.013**
54.568 ± 0.02**
Fiji software (version 1.53c).

Ageing (72 h)
2.7.2 Scanning electron microscopy

Surface features of dry seed coats were examined with SEM.

Electrical conductivity (μScm−1 g−1 )

45.269 ± 0.009**
13.036 ± 1.20**
Ageing (48 h)
Samples from the ventral region were larger (approx. 2–3
mm2 ) and contained the hilum with a narrow surrounding
strip. For each genotype, four seeds were included. All sam-
ples were coated with gold and examined with a Zeiss scan-
ning electron microscope at 15 kV as described by Fengshan

15.008 ± 0.002**
et al. (2004).

8.779 ± 0.014**
Control
3 Results

3.1 Seed quality after ageing

Ageing (72 h)
100 ± 0.00
100 ± 0.00
The moisture content of seeds after ageing showed that JS 335
seeds had higher moisture content (13 and 12% after 48 and
72h of ageing) compared to Bhatt, which had a moisture con-
tent of about 11 and 10% after 48 and 72h of ageing. Results
showed that all the seeds were viable before and after age-

Ageing (48 h)
ing while the germination percent varied between genotypes

100 ± 0.00.
100 ± 0.00
and also within the ageing time (table 1). The initial germi-
Seed quality parameters measured at different ageing periods for two soybean genotypes
nation percent was 100% and 90% and after 72 h of ageing
it was 62% and 100% in JS 335 and Bhatt respectively. To Viability (percent)

**indicates significant difference between ageing times and genotypes at P ≤ .01 (ANOVA with two factors).
confirm the differential response of genotypes related to the
deterioration of seed due to ageing, the damage to seed coats 100 ± 0.00
after accelerated ageing between two genotypes was measured 100 ± 0.00
Control

as Electrical conductivity (EC). The EC in both genotypes

increased with ageing time but there was a difference in EC
values between genotypes. JS 335 and Bhatt had EC values of
Ageing (72 h)

54.56 μScm−1 g−1 and 21.72 μScm−1 g−1 after 72 h of age-

100 ± 0.00**
62 ± 1.29**

ing with Bhatt showing comparatively reduced EC levels than

JS 335. ANOVA with two factors was used to test the signifi-
cance of data and there was a significant difference (P < .01)
in seed quality parameters between genotypes and between
Ageing (48 h)

different ageing treatments.

70 ± 1.25**
93 ± 1.41**
Seed germination (percent)

3.2 Seed imbibition

100 ± 0.00**

Values are expressed as mean ( ±SD).

Ten seeds weight (in mg) measured by electronic balance at 0

90 ± 0.81**
Control

and 3 h of imbibition for JS 335 ranged from 1.11 to 1.49, 1.14

to 1.79, 1.16 to 1.93 while in Bhatt it ranged from 0.85 to 1.12,
0.88 to 1.47 and 0.90 to 1.69 in control, 48 and 72 h aged seeds
respectively (Figure 1). There was a significant difference in
imbibition between genotypes and also for the different ageing
TA B L E 1

Genotype

times within genotypes (ANOVA P < .01). This implies that

JS 335
Bhatt

there is a significant effect of genotypes and ageing time on

the imbibition.
 14350653, 2022, 4, Downloaded from https://acsess.onlinelibrary.wiley.com/doi/10.1002/csc2.20750 by Universitat De Barcelona Biblioteca, Wiley Online Library on [07/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SATYA SRII ET AL. Crop Science 1577

2.25 (a) 2.25 (b)

2 2

1.75 1.75

1.5 1.5

Weight (mg)
Weight (mg)

1.25 1.25

1 1

0.75 0.75

0.5 0.5

0.25 0.25

0 0
0 1 2 3 0 1 2 3
Time (hours) Time (hours)

Control T1 T2

F I G U R E 1 Changes in the seed weight at different time intervals (a) Imbibition in JS 335 (b) Imbibition in Bhatt. Control, fresh seeds; T1,
48 h of ageing; T2, 72 h of ageing. Black bars indicate the standard error

3.3 Permeability of the seed coat Total dehydrogenase(OD @A 480nm) 2

e
1.8

The results showed that the penetration of dye increased as the 1.6
c
f
seed aged for a longer duration compared to control within a 1.4
a
genotype. Between genotypes, Bhatt (black genotype) showed 1.2 d
b JS-335
a greater proportion of hard seeds in control that did not 1
Bha
imbibe and the seeds were not able to be cut open due to 0.8
hardness whereas, in the same aged seeds, the hard seed per- 0.6
cent reduced while in JS 335 (white genotype) there were no 0.4
hard seeds. Hard seeds in Bhatt were confirmed by imbibi- 0.2
tion studies which showed that hard seeds had no significant 0
C T1 T2
change in weight, as they didn’t imbibe water. The intensity
of staining in both genotypes measured as total dehydrogenase F I G U R E 2 Changes in Total Dehydrogenase levels (OD @
activity (OD @ A480 nm) showed that the Bhatt genotype had A480nm) in JS-335 and Bhatt. C, fresh seeds; T1, 48 h of ageing; T2,
significantly less (P < .05) TDH values compared to JS 335 72 h of ageing. Red bars indicate the standard error. Letters above the
(Figure 2). The high levels of TDH values in JS 335 aged seeds bar denotes that TDH levels differed significantly between control and
in comparison to Bhatt is due to the deterioration or damage aged seeds in both genotypes (P < .05)
to the seed coats during ageing which increased its permeabil-
ity to water with stain while hard seed coat in Bhatt resisted
the ageing induced damage. The hard seed number reduced
as seeds aged in Bhatt, but even then 48 and 72 h aged Bhatt
seeds showed significantly less TDH values in comparison to
JS 335 as Bhatt incurred comparatively less damage and per-
meability changes during the same period of ageing due to
hard seed coat.

F I G U R E 3 Seed coat section of soybean showing four layers of

3.4 Structure of seed coat after ageing the seed coat in JS 335 fresh seeds. Scale bar: 20 μm

The sections showed four layers of seed coat ie. cuticle, epi- 0.19 ± 0.03 μm and 0.34 ± 0.07 μm respectively with Bhatt
dermis, hypodermis and interior parenchyma (Figure 3: sec- having comparatively thicker cuticle. The thickness of the epi-
tion of JS 335) as described by Souza & Marcos- Filho, 2001. dermis in 48h aged JS 335 and Bhatt was 0.76 ± 0.04 μm and
The thickness of the cuticle in 48 h aged JS 335 and Bhatt was 1.06 ± 0.1 μm respectively. Thus, the seed coat sections of

1578 Crop Science
F I G U R E 4 Structure of seed coat in 48 h aged seeds. (a)
Cross-section of the seed coat of 48 h aged JS 335 observed under the
light microscope. (b) Cross-section of the seed coat of 48 h aged Bhatt
observed under the light microscope. The black, red and yellow
indicate the cuticle layer, epidermis made of palisade cells and
hypodermis made of hourglass cells respectively. Scale bar: 20 μm
F I G U R E 5 Structure of seed coat in aged seeds (a) Cross-section
of 48 h aged JS 335 seed coat (b) Cross-section of 72 h aged JS 335 seed
coat (c) Cross-section of 72 h aged Bhatt seed coat. The red arrow in (a)
and (b) indicates cracks in the epidermal layer of the seed coat leading
to disruption of palisade cells and the black arrow in (c) indicates
disruption of the cuticle layer of the seed coat. Scale bar: 50 μm
JS 335 and Bhatt aged for 48 h (Figure 4) showed remarkable
differences in their structure and compactness, with JS 335
having a thin cuticle (indicated by black arrow) and epidermis
(indicated by a red arrow) compared to Bhatt. Figure 4a and 4b
also show that the hourglass cells in the hypodermis of JS
335 are loosely packed and have started to collapse after age-
ing for 48 h while Bhatt showed intact and compactly packed
hourglass cells. Also, figure 4a shows the disruption of the
cuticle of JS 335 after ageing for 48 h and further ageing to
72 h, the seed coat of JS 335 ruptured while Bhatt didn’t show
such damage. The seeds aged for 48 hours started accumu-
lating mild disruption in the seed coat cuticle and epidermal
layer (figure 5a) which then disintegrates, collapsing the seed
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SATYA SRII ET AL.
coat structure and integrity in seeds aged to 72 h in JS 335
(figure 5b) while such ruptures in the epidermal layer were
not observed in Bhatt aged for the same duration (figure 5c).
In Bhatt seeds aged for 48 h, there was no much variation in
their morphology (figure 4b) while further ageing for 72 hs
showed a disruption of only the cuticular layer of the seed
coat (figure 5c).
Further the SEM imaging of the ventral region of seed coat
consisting of hilum, micropyle and raphe showed that in JS-
335, a hilar fissure was observed even in the control seeds (7.9
± 0.74 μm) and larger openings of 10.75 ± 0.55 μm and 14.62
± 0.50 μm in 48h and 72h aged seeds respectively (figure 6) on
the other hand in Bhatt genotype the hilum was mostly closed
in control seeds (0.4± 0.04 μm wide opening) while there was
a slight hilar opening (3.92± 0.28 μm) in 48h aged seeds and a
larger opening (5.46± 0.29 μm) in 72h aged seeds. When the
structure inside the hilar fissure was observed, 48h aged seeds
had a compact structure while 72h aged seeds had a more
loosened structure inside the fissure (figure 7). When com-
pared between the genotypes, hilar fissures were observed in
both genotypes aged for 72h, but Bhatt showed a smaller hilar
opening with a more compact cell arrangement compared to
JS 335. The impact of altered seed coat structure after age-
ing in subsequent imbibition was studied by looking at the
seed coat structure 1 h after initial imbibition in aged seeds,
as well as control. This revealed that the seed coat stayed more
stable in fresh seeds of JS 335 after the initial phase of imbi-
bition while there was a loss of seed coat structural integrity
with the seed coats being completely collapsed in aged seeds
(figure 8). On the other hand, in Bhatt, there were no changes
in seed coat integrity, 1 h after imbibition in both fresh and
aged seeds.
4 Discussion
4.1 Role of seed coat structure in seed
quality during ageing
Two genotypes differed in their seed quality after ageing
where JS 335 showed increased deterioration (less germi-
nation, higher EC) compared to Bhatt. The possible reason
why JS 335 deteriorated faster during artificial ageing com-
pared to Bhatt is higher hydration which in turn induced faster
deterioration in the embryo axis. The higher hydration in JS
335 compared to Bhatt during artificial ageing is also con-
firmed by the difference in seed moisture content after age-
ing where JS 335 had comparatively higher moisture content
than Bhatt. The reason for different levels of hydration dur-
ing ageing between the genotypes might be due to the thick
and impermeable seed coat in Bhatt compared to JS 335.
It is reported from other studies that impermeable or hard
seed coat protects the embryo against fluctuations in humidity

SATYA SRII ET AL.
F I G U R E 6 SEM of ventral side of seeds (a) Bhatt control; (b) Bhatt aged for 48h (c) Bhatt aged for 72h (d) JS335 control (e) JS-335 aged for
48h (f) JS-335 aged for 72h. The arrow indicates the hilar fissure in seeds while (a) didn’t show any fissures. Scale bar: 200 μm
and temperature (Mayne et al., 1969) which are the main
variations caused by accelerated ageing. The TDH values for
permeability studies also support this point that there was a
difference in permeability properties of seed coat between
genotypes after ageing where JS 335 had increased permeabil-
ity (high TDH values) on ageing compared to Bhatt. Studies
show that the main cause for impermeability of seed coats to
be the oxidation of phenolic compounds by polyphenol oxi-
dase or peroxidase which in turn, provides impermeability
to seed coats (Loic & Isabelle, 2008; Pourcel et al., 2005).
This might be why dark colour seed coats are less permeable
than light coloured seed coats which is also proven from the
results of the present study where Bhatt with black seed coat is
less permeable than JS 335 with white seed coat. This is sup-
ported by evidence from previous studies in other species like
chickpea (Gvozdeva & Zhukova, 1971), snap bean (Prasad &
Weigle, 1976) and french bean (Powell, 1986a). Besides this,
Bhatt seeds (black seed coats) had hard seeds which imposed
dormancy due to impermeable seed coats while it was not
observed in JS 335 (white seed coat) again confirming the
relation between impermeability and colour. Thus the imper-
meability resulting from oxidation of phenolic compounds in
coloured seed coats is one of the reasons for reduced damage
and deterioration in Bhatt (black) seeds compared to JS 335
(white) after ageing.
Electrical conductivity results showed that EC values
increased as seeds aged. Parrish et al. (1982) reported that
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Crop Science 1579
an increase in electrolyte leakage in deteriorated seeds is an
indication of membrane deterioration leading to the ageing
of seeds. However, Bhatt had comparatively less EC than JS
335 though both genotypes showed increased EC with age-
ing time. This shows that the two genotypes had a different
level of membrane damage during ageing, with black geno-
types having less seed coat damage compared to white. The
reason for the higher integrity of seed coats in black genotypes
could be attributed to the presence of proanthocyanidins with
free radical scavenging activity (Takahata et al., 2001) which
would in turn help in cell repair mechanisms preventing mem-
brane damage (Bailly, 2004).
The results from microscopic analysis of seed coat structure
show that there was a difference in seed coat structure at dif-
ferent ageing times and also between genotypes. JS 335 seeds
had a thin cuticle and epidermis compared to Bhatt (figure 4)
after ageing. The relationship of the cuticle to permeability
in soybean seeds is well established where a thick and intact
cuticle is shown to make the seed coat impermeable whereas
a thin and damaged cuticle with cracks makes it permeable
(Fengshan et al., 2004; Meyer et al., 2007; Shao et al., 2007). It
is to be noted that there was disruption of the cuticle (figure 4)
as permeability increased in Bhatt accompanied by a decrease
in hard seeds as seeds aged which further confirms the role
of cuticle in permeability. The hourglass cells in the hypo-
dermal layer show a marked difference between JS 335 and
Bhatt which is shown in figure 4, where the hourglass cells of
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1580 Crop Science SATYA SRII ET AL.

F I G U R E 7 SEM of internal structure of hilar fissure. (a) JS-335 control; (b) JS-335 aged for 48 h; (c) JS-335 aged for 72 h; (d) Bhatt aged for
48 h; (e) Bhatt aged for 72 h. The arrow indicates the gaps in cell arrangement inside the hilar fissure. The coils are arranged more compact in 48h
aged seeds when compared wth 72-h aged seeds. The asterisk shows the hilar fissure with the inside structure. Control seeds of Bhatt are not shown
as the hilum was intact in it as seen in Figure 6a. Scale bar: 10 μm

ing as previous studies reported that hourglass cells provide

F I G U R E 8 Structure of seed coat 1 h after imbibition in 72 hs
aged seeds of (a) JS 335 and (b) Bhatt. Black arrows show the collapse
of the cuticle, epidermis with palisade cells and hypodermis with
hourglass cells after imbibition leading to loss of seed coat integrity.
Scale: 20 μm
a cushioning effect and prevent damage due to hydration and
dehydration (Pereira & Andrew, 1985). The densely packed
hourglass cells in Bhatt may be the most plausible reason for
the hard seeded nature and prolonged longevity as previous
studies have shown that the densely packed, water-repellent
hourglass cells in physically dormant seeds prolong longevity
by regulating the embryo’s interaction with the environment
(Baskin & Baskin, 2006). So, the results of this study show
that a thicker cuticle, epidermis and intact hourglass cells help
seeds resist artificial ageing induced damage to the seed coat
and thus help the seed coat remain intact preventing hydration
during artificial ageing and thus helping maintain the seed
quality.

4.2 Seed coat structure in imbibition injury

JS 335 are loosely packed and distorted upon ageing whereas
in Bhatt they are compactly packed and intact. This might be The results of viability tests showed that fresh seeds of both
the main reason for the disruption of the seed coat after age- JS 335 and Bhatt were 100% viable but JS 335 and Bhatt

SATYA SRII ET AL.
showed a decrease and increase in germination respectively
after ageing for 72 h. These contrasting results of germination
after ageing between genotypes were because of the increased
fraction of abnormal seedlings produced by aged seeds in JS
335 while because of hard seeds fraction in Bhatt, as both
were considered as not germinated in germination tests as
per ISTA test guidelines. Previous studies in peas and soy-
bean have reported that accelerated ageing increased the pro-
portion of abnormal seedlings which reduced the germination
percent of aged seeds (Veselova & Veselovsky, 2003; Zahra
et al., 2011). However. the reason for abnormal seedlings in
JS 335 can be explained by the changes in imbibition patterns
of aged and control seeds as shown in figure 1 which indi-
cates that the aged seeds imbibe more amount of water at the
same point of time when compared to control leading to imbi-
bitional injury. Hobbs and Obendorf (1972) reported that the
soybean seeds are highly sensitive to the initial stages of imbi-
bition and any variations or stress during this stage could lead
to reduced vigour or prevent germination causing “imbibi-
tion injury”. Imbibition injury is a well-studied phenomenon
in which the sudden flux of a high amount of water into the
embryo would damage the embryo and result in abnormal
seedlings or seedlings with reduced vigour or no germination
in extreme cases (Bramlage et al., 1978). The cause for imbibi-
tional injury in aged JS 335 seeds is mainly due to the change
in permeability properties of seed coats after ageing due to
changes in seed coat structure. Figure 4 and 5 shows disrup-
tion of the cuticle, epidermal layer and also distortion of hour-
glass cells of the seed coat in JS 335 after ageing. Studies have
already established the role of cuticle, epidermis and hypoder-
mis in permeability (Fengshan et al., 2004; Meyer et al., 2007;
Shao et al., 2007). This shows that change in seed coat struc-
ture during ageing altered permeability which in turn led to
the imbibitional injury in aged JS 335 seeds.
The results of permeability, imbibition and germination
percent prove that during artificial ageing there are deterio-
rative changes that occur in seed coats which results in seed
coat structural damage and increased permeability of seed
coat leading to increased imbibition. This increased uptake of
water in the first phase of imbibition by aged seeds resulted in
reduced germination percent of viable aged seeds by produc-
ing more abnormal seedlings.
Besides this, there was a significant difference in seed
hilar opening between genotypes where the hilum was mostly
closed in Bhatt fresh seeds while there was an open hilum
in JS-335 fresh seeds (figure 6). The hilar opening increased
in size as the seeds aged for 48 and 72 h. The cell arrange-
ment inside the fissure showed a marked difference as the fis-
sure enlarged with ageing time. The coils were arranged more
compact in 48 h aged seeds when compared to 72 h aged
seeds in both JS 335 and Bhatt, however, in Bhatt, the coils
were comparatively more compact than JS 335 (figure 7). The
development of hilar fissure may be a crucial factor in decid-
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Crop Science 1581
ing the seed permeability to water and imbibition as studies
show water enters the soybean seeds mostly through the hilum
(Arechavaleta- Medina & Synder, 1981; Meyer et al., 2007).
This also shows that intact closed hilum might be one of the
reasons why fresh Bhatt seeds did not imbibe water while in
JS 335 fresh seeds had an open hilum that imbibed water nor-
mally and germinated. Also, the presence of hilar fissure in 48
and 72 h aged Bhatt seeds and the results of increased imbi-
bition and germination percent in aged Bhatt seeds show that
opening in the hilum region to be regulating factor of seed
coat permeability to water during imbibition. While in aged JS
335 seeds, the wider open hilum may be a contributing factor
for imbibition injury in them as the wide-open hilum in aged
seeds led to an uncontrolled flush of water into seeds leading
to imbibitional injury and producing abnormal seedlings.
Besides the contribution of the hilar opening to permeabil-
ity and imbibition, studies also state that differences in the
structural features of the cell walls and surfaces of aleurone
cells in the top area of the seed may also be responsible for
the difference in the extent of imbibition injury (Sato et al.,
2019). Thus, when JS 335 aged seeds were further subjected
to imbibition, there was deformation of the entire seed coat
structure within 1 h of imbibition as observed in Figure 8
which would be because JS 335 seeds already had less intact
and damaged seed coat with a wider hilar fissure as a result
of ageing which in turn completely deformed upon imbibition
leading to increased permeability and rush of water to embryo
resulting in imbibitional injury. Also, studies show that the
first 3 h of imbibition is critical and the rate of imbibition
depends upon the seed coat thickness (Mc Donald et al.,1988)
and adherence of seed coat to cotyledons (Powell et al.,
1986a). It was reported that seed coats in colourless geno-
types adhered loosely to cotyledon in comparison to coloured
genotypes which in turn regulated water uptake by seeds
(Powell et al., 1986a) which might also be one of the reasons
for Bhatt (coloured) to incur less imbibition damage compared
to JS 335 (colourless). Bhatt genotype with thick, imperme-
able seed coats (hard seeds) incurred less damage even upon
ageing and thus had limited permeability regulating imbi-
bition producing normal seedlings upon germination. Thus,
imbibitional injury is seen to occur as a result of damage to
seed coat structure as seeds age. These changes in seed coat
structure during artificial ageing and their role in inducing
imbibitional injury in artificially aged soybean seeds empha-
sizes the significance of the storage environment in maintain-
ing seed quality as fluctuating temperature and moisture in
storage condition can lead to reduced germination percent due
to imbibitional injury. However further research in naturally
aged seeds is needed to clarify the exact role of seed coat
structure in the imbibitional injury of naturally aged seeds.
The results of this study on artificial ageing between two
genotypes show that the seed coat structure with distorted
cuticle, epidermis, hourglass cells after ageing lead to seed

1582 Crop Science
quality deterioration due to solute leakage while wide-open
hilar fissures in aged seeds lead to altered permeability which
in turn causes imbibitional injury in artificially aged soybean
seeds.
AC K N OW L E D G M E N T
The authors would like to acknowledge the Central Instrumen-
tation Facility, University of Agricultural Sciences, Bangalore
for providing the SEM facility and the Department of Science
and Technology, India for providing INSPIRE fellowship to
V. Satya Srii.
AU T H O R C O N T R I B U T I O N S
Vijayan Satya Srii: Conceptualization; Formal analysis;
Investigation; Methodology; Writing – original draft. Nethra
Nagarajappa: Conceptualization; Funding acquisition; Project
administration; Resources; Writing – review & editing. S.N.
Vasudevan: Methodology; Visualization; Writing – review &
editing.
CONFLICT OF INTEREST
Authors declare no conflict of interest.
ORCID
Vijayan Satya Srii https://orcid.org/0000-0001-6955-1044
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How to cite this article: Satya Srii, V., Nagarajappa,
N., & Vasudevan, S. N. (2022). Is seed coat structure
at fault for altered permeability and imbibition injury
in artificially aged soybean seeds? Crop Science, 62,
1573–1583. https://doi.org/10.1002/csc2.20750