Germination and dormancy of Sida hermaphrodita seeds

Abstract

Sida hermaphrodita seeds produced at two locations in multiple years were investigated for germination and dormancy, including the morphological mechanisms responsible for physical dormancy and changes in the seed coat induced by imbibition and chemical scarification. The imbibing
capacity of S. hermaphrodita seeds immediately after harvest and after one year of storage was high (from 0.69 to 1.08 g H2O g-1 seeds), but varied between years and decreased over time. Seed conditioning and the separation of the heavy seed fraction increased
germination to more than 77%. Each lot of heavy seeds (which sank in water) produced germinated seeds, imbibed non-germinated seeds and hard seeds. The proportion of hard seeds in each batch varied widely between years and locations. An analysis of the morphological and anatomical characteristics
of S. hermaphrodita seeds revealed structures responsible for water absorption. Scarification for 30 minutes with 95% sulphuric acid was most effective in breaking the physical dormancy of seeds (compared with longer periods of scarification) resulting in imbibition without impairing
embryo viability.

Full text

1
DORMANCY OF SIDA HERMAPHRODITA SEEDS
* Author for correspondence
Packa, D., Kwiatkowski, J., Graban, Ł. and Lajszner, W. (2014), Seed Sci. & Technol., 42, 1-15,
http://doi.org/10.15258/sst.2014.42.1.01
Germination and dormancy of Sida hermaphrodita seeds
D. PACKA, J. KWIATKOWSKI*, Ł. GRABAN AND W. LAJSZNER
Department of Plant Breeding and Seed Production, University of Warmia and Mazury in Olsztyn, Plac Łódzki
3, 10-724 Olsztyn, Poland (E-mail: jacekkw@uwm.edu.pl)
(Accepted November 2013)
Summary
Sida hermaphrodita seeds produced at two locations in multiple years were investigated for germination
and dormancy, including the morphological mechanisms responsible for physical dormancy and
changes in the seed coat induced by imbibition and chemical scarification. The imbibing capacity of
S. hermaphrodita seeds immediately after harvest and after one year of storage was high (from 0.69 to 1.08 g
H2O g-1 seeds), but varied between years and decreased over time. Seed conditioning and the separation of
the heavy seed fraction increased germination to more than 77%. Each lot of heavy seeds (which sank in
water) produced germinated seeds, imbibed non-germinated seeds and hard seeds. The proportion of hard seeds
in each batch varied widely between years and locations. An analysis of the morphological and anatomical
characteristics of S. hermaphrodita seeds revealed structures responsible for water absorption. Scarification for
30 minutes with 95% sulphuric acid was most effective in breaking the physical dormancy of seeds (compared
with longer periods of scarification) resulting in imbibition without impairing embryo viability.
Introduction
Sida hermaphrodita (L.) Rusby, a perennial forb, is a member of the family Malvaceae
(subfamily Malvoideae, tribe Malveae) that comprises around 70 genera represented by
more than 1000 species of herbaceous plants, trees and shrubs. The species of the tribe
Malveae have an extensive geographic range and colonise diverse habitats in both tropical
and temperate climatic zones (Tate et al., 2005). The family Malvaceae includes species
that belong to major genera used in farming and horticulture, among them Gossypium
L. (cotton), Hibiscus L. (hibiscus, rosemallow), Abelmoschus L. (okra, gumbo, lady’s
fingers), Alcea L. (hollyhock), Althaea L. (marshmallow) and Malva L. (mallow).
Sida hermaphrodita (Virginia mallow, Virginia fanpetals, Virginia sida, River
mallow), an indigenous plant of North America, was introduced to Poland from the
Ukraine in the 1950s (Borkowska and Molas, 2012). This species has been researched
extensively for more than 50 years, assessing the possibility of using the species in the
production of fibre, fodder and as melliferous plants. Due to its high cellulose content
and a favourable composition of resins and waxes, S. hermaphrodita can be used in the
pulp and paper industry; the species’ ability to accumulate toxic substances from the soil

2
D. PACKA, J. KWIATKOWSKI, Ł. GRABAN AND W. LAJSZNER
makes it suitable for use in phytoremediation of degraded areas; and there is a growing
interest in the plant as an energy crop. At present, S. hermaphrodita, willow (Salix spp.)
and Miscanthus spp. are the main crops grown for energy purposes (Borkowska and
Molas, 2012; Kwiatkowski et al., 2012). Selection and breeding efforts led to the creation
of Sida cultivars that were initially available only in the Ukraine. Cultivars Virdzinia
and Fitoenergia were entered in the Ukrainian register of varieties in 2001 and 2009,
respectively. In 2004-2012, the Polish cultivar Petemi was added to the Polish register of
varieties. The German cultivar SIHEWI0111 was entered in the Register of Community
Plant Variety Rights in 2011 (Anonymous, 2011).
S. hermaphrodita can reproduce both vegetatively and sexually. Vegetative repro-
duction is preferred when establishing small-scale seed plantations as seeds can be
produced in the first year. Seeds are recommended for large-scale commercial plantations
and the rapid development of energy crops increases the demand for high quality seeds.
One of the problems encountered during sexual reproduction is seed dormancy that results
in low germination.
Seed dormancy in the family Malvaceae is caused by an impermeable seed coat that
prevents the embryo from imbibing, thus impairing germination (Rolston, 1978; Kelly et
al., 1992; Baskin et al., 2000). This type of seed dormancy is known as physical dormancy
or hardseededness and is observed in six families of the order Malvales (Baskin et al.,
2000; Baskin and Baskin, 2004). Physical dormancy results from the presence of a palisade
cell layer in the seed coat that is impermeable to water and impregnated with hydrophobic
substances such as suberin, cutin and lignin. The seed coat becomes permeable when the
palisade layer is opened or damaged (Rolston, 1978; Argel and Paton, 1999). In nature,
the seed coat may be damaged by microorganisms, changes in temperature, freezing and
thawing, fire, or enzymes in the digestive tract of animals. In commercial seed production,
seeds may be mechanically, chemically or thermally scarified (Rolston, 1978; Argel and
Paton, 1999).
The objective of this study was to investigate the morphological and anatomical
mechanisms that cause dormancy in S. hermaphrodita seeds and to analyse changes in
the seed coat induced by imbibition and chemical scarification.
Materials and methods
Plant material
Sida hermaphrodita seeds were harvested in 2009 and 2010/2011 from 1-, 2-, 3- and 4-year
old plants from Bałdy plantation and from 7- and 8-year-old plants from Tomaszkowo
plantation. The two stations, separated by a distance of approximately 16 km, are near
Olsztyn (53°46'37'' N, 20°28'29'' E), Poland. Climatic conditions were recorded for both
plantations in 2009 and 2010 as seed quality is affected by the agro-ecological conditions
during development and maturation. Additional evaluations of imbibition capacity were
carried out using seeds harvested 2004-2008 from the plantation in Tomaszkowo and
stored for up to five years.

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DORMANCY OF SIDA HERMAPHRODITA SEEDS
Each year, the seeds were harvested over a one-month period starting in December.
Threshed, mechanically-cleaned and naturally dried seeds were stored in paper bags in a
laboratory at approximately 20-22°C and 40 to 70% relative humidity (RH).
Imbibition capacity
Imbibition capacity was analysed immediately after harvest and after 1, 2, 3, 4 and 5 years
of storage (seeds from Tomaszkowo). Undeveloped seeds, seeds with an atypical colour
and damaged and infected seeds were removed by hand from each lot of mechanically-
cleaned seeds (around 10 g). The material was divided into four samples of 100 seeds
each, and each sample was weighed. The seeds were kept on moistened filter paper for
24 hours, after which they were weighed. The amount of water imbibed is expressed as g
H2O g-1 seeds. The experiment was independently repeated three times for seeds harvested
in 2009 and 2010, and two times for seeds stored for 1-5 years.
Germination
Germination capacity was determined immediately after harvest and after storage (up to
one year). Samples of 6 g of mechanically-cleaned seeds from 1-, 2-, 3-, 4-, 7- and 8-year-
old plants were soaked in 150 ml aerated, distilled water for two hours in daylight. Two
fractions were obtained after the 2-hour soak: heavy seeds (H) sank to the bottom of the
vessel and light seeds (L) floated on the surface. The proportion of H and L seeds in each
lot was determined, and the proportion of H seeds was expressed as the percentage of all
examined seeds. The germination capacity of H and L seeds was determined: H and L
seeds were placed in 20 cm-diameter Petri dishes lined with moistened filter paper. The
dishes were placed in an incubator at 22°C in the dark and seed germination capacity
(visible radicle emergence) was evaluated after seven days.
Morphological and anatomical structure
Seed images were captured using a Nikon SMZ-2T stereo microscope with a camera
and a Jeol-JSM 5200 scanning electron microscope. For anatomical analyses, seeds at
various stages of maturity were fixed in FAA (Formalin-Acetic-Alcohol) and embedded
in paraffin. Freshly harvested seeds and seeds scarified under laboratory conditions with
concentrated 95% sulphuric acid for 30, 60 and 110 minutes were fixed after 24 hours on
moistened filter paper. The specimens were stained with 1% safranin solution (Safranin
O) in 50% ethanol and 0.1% Fast Green FCF solution in absolute alcohol (Gerlach,
1972). After dehydration in isopropyl alcohol, the specimens were embedded in Euparal
and observed under a light microscope and a fluorescence microscope (FM – Labophot
Nikon) equipped with a Nikon B-3A filter. Microphotographs were taken using the Nikon
DS Camera Head DS-5M for colour and fluorescence imaging.
Thousand seed weight
The thousand seed weight of scarified and non-scarified seeds was determined by weighing
eight replicates of 100 seeds each. The mean weight of 100 seeds was calculated and
multiplied by 10, provided that the coefficient of variation for the weights of collected
samples was less than 4%.

4
D. PACKA, J. KWIATKOWSKI, Ł. GRABAN AND W. LAJSZNER
Results
Weather conditions were similar in both stations in both years of the study (figure 1). Total
precipitation in the growing season (April – October) at both stations was approximately
25% higher in 2010 than in 2009. The average temperature in the growing season in
Bałdy in 2010 was approximately 0.3°C higher in comparison with the previous year;
similar values were noted in Tomaszkowo in both experimental years. In Bałdy, total
rainfall was 7-12% higher and average temperatures were 1.4-1.7°C higher in comparison
with Tomaszkowo in both years of the study.
0
20
40
60
80
100
120
140
160
January
February
March
April
May
June
July
August
September
October
November
December
Months
Total rainfall (mm)
-15
-10
-5
0
5
10
15
20
25
30
Average temperature (ºC)
Months
Total rainfall (mm)
Average temperature (ºC)
rainfall 2009
rainfall 2010
temperature 2009
temperature 2010
BAŁDY
0
20
40
60
80
100
120
140
160
January
February
March
April
May
June
July
August
September
October
November
December
-15
-10
-5
0
5
10
15
20
25
30
rainfall 2009
rainfall 2010
temperature 2009
temperature 2010
TOMASZKOWO
Figure 1. Weather conditions at Bałdy and Tomaszkowo in 2009 and 2010.

5
DORMANCY OF SIDA HERMAPHRODITA SEEDS
A B
C
En
R
* C D
E
C R
En
F
1 mm
2 mm 2 mm
1 mm
2 mm 2 mm
Figure 2. Structure of Sida hermaphrodita seeds. A) Seeds at different stages of maturity: non-pigmented
seed coat (left), light brown seed coat (middle), dark grey seed coat (typical of freshly harvested seeds; right).
B) Ripe seeds: grey-coloured seeds (most typical), variously coloured seeds, weakly developed seeds, seeds
with damaged coats. C) Cross-section of an imbibed seed, freshly harvested in 2009. D) Cross-section of a non-
imbibed hard seed. E) Dry seeds scarified with sulphuric acid for 30 minutes showing visible damage to the
seed coat. F) Imbibed seeds scarified with sulphuric acid (left), non-scarified imbibed (middle) and hard seeds
(right). Images taken with a stereo microscope. R, radicle; C, cotyledons; En, endosperm; arrow, chalazal cap;
star, free space between the palisade layer and parenchymal tissue.
Sida hermaphrodita produces schizocarps: dry fruits developed from multiple carpels
that split into mericarps containing single seeds. A mature fruit contains 5 to 8 seeds.
During ripening, initially non-pigmented seeds turn light brown and then dark grey (figure
2A). A random sample of ripened, mechanically-threshed and pre-cleaned mallow seeds
contained: grey-coloured filled seeds, filled seeds of different colours, undeveloped seeds
and damaged seeds (figure 2B). The proportion of filled seeds provides an indication of
seed quality.

6
D. PACKA, J. KWIATKOWSKI, Ł. GRABAN AND W. LAJSZNER
Imbibition capacity
Freshly harvested seeds absorbed water resulting in an increase in their volume (figure
2C), while hard seeds did not absorb water or swell (figure 2D). Differences between
seeds harvested from the two plantations and in different years were apparent. The
average imbibition capacity of seeds obtained from Bałdy was 1.08 ± 0.090 g H2O g-1
seeds in 2009 and 0.85 ± 0.171 in 2010. The material harvested from Tomaszkowo was
characterised by lower water-absorbing capacity (0.76 ± 0.011 and 0.69 ± 0.112 for seeds
harvested in 2009 and 2010, respectively). After one year of storage at room temperature,
the imbibition capacity of seeds harvested from 1-, 2- and 3-year-old plants remained high
with a mean of 1.06 ± 0.058.
The imbibition capacity of seeds decreased with storage time. The average water
absorbing capacity of seeds stored for 1, 2, 3, 4 and 5 years reached 0.94 ± 0.041, 0.78 ±
0.045, 0.72 ± 0.032, 0.66 ± 0.41 and 0.55 ± 0.021, respectively.
Germination
The 6 g samples contained a mean of 1519 ± 94 seeds (from 1310 to 1670 seeds). The
average proportion of heavy seeds was 75% of the seed lot harvested at Bałdy and 83%
of the seed lot harvested at from Tomaszkowo (table 1). Light seeds were characterised
by low germination ranging from 3 to 29% (table 2). The germination capacity of heavy
seeds was greater, ranging from 42 to 77%. In 2010, one-year-old plants grown in Bałdy
did not set seeds. The germination capacity of heavy seeds harvested from 2-, 3- and
4-year-old plants in 2010 varied from 16 to 44%. Higher germination percentages (mean
of 52%) were reported for seeds collected from 8-year-old plants at Tomaszkowo (table
3).
Each lot of heavy seeds produced germinated seeds, imbibed non-germinated seeds
and hard seeds. The percentage hard seeds from Bałdy varied by production year (7%
in 2009 and 20% in 2010), but was more consistent in seeds harvested at Tomaszkowo
(23 and 24% in 2009 and 2010, respectively; table 4). The percentage of imbibed non-
germinated seeds from Bałdy varied between years ranging from 30 to 50%, whereas the
values reported for Tomaszkowo were more consistent (22 to 24%).
Morphological and anatomical structure
Sida hermaphrodita seeds have a well-differentiated, curved embryo, and the radicle tip
and cotyledons are on the opposite ends of the seed, which morphologically corresponds
to two protuberances of the micropylar area (radicle tip) and the chalazal area (cotyledons)
(figure 2C). Images from light and scanning electron microscopes revealed the presence
of residual maternal tissue in the chalazal pole in the form of a chalazal cap that blocked
water uptake (figure 3A). When the chalazal cap was removed from dry and hard seeds,
a 120-150 µm closed slit or groove was exposed (figure 3B). In soft, imbibed seeds, an
opened chalazal slit was visible surrounded by folded and dark-coloured tissue with a
radial arrangement of cells (figures 3C-F, 4A-B). The chalazal slit breaks the continuity
of the palisade layer. When water was absorbed by the seed, a water-filled blister was
formed around the chalazal slit in which the palisade layer was separated from underlying
parenchymal cells (figure 4C, D). The palisade layer can be easily removed (figure 4D).

7
DORMANCY OF SIDA HERMAPHRODITA SEEDS
Table 1. Proportion of heavy seed fractions harvested from 1-, 2-, 3-, 4-, 7- and 8-year-old plants in 2009 and
2010 (mean ± SD) at Bałdy (B) and Tomaszkowo (T).
Plants / years 1-year-old
(B)
2-year-old
(B)
3-year-old
(B)
4-year-old
(B)
7-year-old
(T)
8-year-old
(T)
2009 58 ± 9.0 77 ± 2.4 79 ± 4.8 – 89 ± 2.1 –
2010 – 80 ± 5.2 79 ± 7.4 83 ± 6.3 – 80 ± 7.8
Mean 75 ± 10.6 83 ± 7.5
– no seeds or no plants.
Table 2. Germination (%) of light (L) and heavy (H) seeds harvested from 1-, 2- and 3-year-old plants determined
based on an analysis of a 6 g samples of seeds harvested in 2009 at Bałdy (B).
Time of
analysis
1-year-old 2-year-old 3-year-old mean ± SD
LH LH LH L H
Immediately
after harvest 442 362227610 ± 10.3 60 ± 17.1
After 1 month
storage 558 865297614 ± 13.1 66 ± 9.5
After 1.5 months
storage 4431169267714 ± 11.0 63 ± 17.7
After 2 months
storage 456 460 977 6 ± 2.9 64 ± 11.5
After 6 months
storage 15 66 22 63 16 74 18 ± 3.4 68 ± 5.8
After 1 year
storage 557 853 957 7 ± 1.8 56 ± 2.1
Mean ± SD 6 ± 4.3 54 ± 9.2 9 ± 6.6 62 ± 5.3 19 ± 8.5 73 ± 7.9 11 ± 8.2 63 ± 10.9
Table 3. Germination (%) of H and L seeds harvested from 2-, 3-, 4- and 8-year-old plants determined based on
an analysis of a 6 g sample of seeds harvested in 2010 at Bałdy (B) and Tomaszkowo (T).
Time of
analysis
2-year-old (B) 3-year-old (B) 4-year-old (B) 8-year-old (T)
LH LH LH LH
Immediately
after December
harvest
644 6 42 8 36 2237
Immediately
after January
harvest
12 18 9 32 5 27 27 64
After 2 months
storage 516 5 33 3 24 1655
Mean ± SD 8 ± 3.9 26 ± 15.5 7 ± 2.3 36 ± 5.3 5 ± 2.5 29 ± 6.4 22 ± 5.6 52 ± 13.7

8
D. PACKA, J. KWIATKOWSKI, Ł. GRABAN AND W. LAJSZNER
B
Chp
C D
E F
A
M
Chc
R
M
Figure 3. Hard or imbibed seeds of Sida hermaphrodita. A, B) Chalazal cap (Chc) and chalazal slit (arrow) of
a hard seed after chalazal cap removal. C, D) Chalazal slit in a weakly imbibed seed. E, F) Chalazal slit in a
germinating seed with a strand of parenchymal tissue – the chalazal plug. The slit is surrounded by folded tissue with
radial arrangement of cells. Image taken by scanning electron microscope. Chc, chalazal cap; Chp, chalazal plug;
M, micropylar region; R, radicle.
Table 4. Percentage of germinated seeds, imbibed non-germinated seeds and hard seeds harvested from
plantations in Bałdy and Tomaszkowo in 2009 and 2010.
Year of harvest
Germinated
seeds
(%)
Imbibed non-
germinated seeds
(%)
Hard seeds
(%)
Bałdy 2009 (mean for 1-, 2- and 3 year-old plants)
Bałdy 2010 (mean for 2-, 3- and 4 year-old plants)
63
30
30
50
7
20
Tomaszkowo 2009 (7-year-old plants)
Tomaszkowo 2010 (8-year-old plants)
55
52
22
24
23
24

9
DORMANCY OF SIDA HERMAPHRODITA SEEDS
The chalazal plug, a strand of parenchymal tissue situated directly beneath the chalazal
slit (figures 3F, 4D, E), and a morphologically distinct crescent-shaped area covering the
chalazal plug on one side (figure 4F) are visible on the exposed surface of the tissue.
Palisade cells in the chalazal region of imbibed seeds are in a relaxed state.
Outside the chalazal area, the palisade layer in imbibed seeds showed strong
fluorescence, and this fluorescence was greatest in cells oriented towards the interior of
the seed (figure 5A). Palisade tissue surrounds the entire seed, but has two weak points:
the opening in the chalazal region where it loses continuity and the micropylar area
where the radicle tip is located. Observations under a fluorescence microscope revealed
that in young developing seeds, the chalazal slit was filled with thin-walled cells that
A
F
B
D
E F
R
R
C
*
*
M
M
C
E
1 mm
1 mm
1 mm
1 mm
1 mm
0.2 mm
*
*
*
Figure 4. Chalazal slit in imbibed seeds of Sida hermaphrodita. A) Chalazal slit (arrow) in a freshly harvested
seed. Visible funiculus residue in micropylar area (F). B) Chalazal slit (arrow) in a seed after storage for one
year. Folded tissue surrounds the opening. C) Cross-section of an imbibed seed in a region where the palisade
layer is separated from parenchymal tissue (stars). The chalazal slit is marked with an arrow. Cut by the plane
shown in figure 4B. D) Partially removed palisade layer in the chalazal area. E, F) Completely removed
palisade layer. The chalazal plug (arrow) and crescent-shaped area (star) are marked. Images taken under stereo
microscope. C, cotyledon; E, endosperm; M, micropylar region; R, radicle.

10
D. PACKA, J. KWIATKOWSKI, Ł. GRABAN AND W. LAJSZNER
Figure 5. Palisade layer of Sida hermaphrodita seeds. A) Palisade layer of adjacent, elongated cells showing
strong fluorescence. B) Cross-section of the chalazal slit with palisade ends (arrows) on both sides of the cell
structure connecting the chalazal cap and the chalazal plug. C) Cross-section of the chalazal end with a visible
incision in the palisade layer. D) Slight damage to the palisade layer after 30 minutes of scarification with
sulfuric acid. E) Damage to the palisade layer after 60 minutes of scarification. F) Extensive damage to the
palisade layer after 110 minutes of scarification. Images taken by fluorescence microscopy. Scale bar for all
photos = 100 μm. Plc, palisade layer cells; En, endosperm; Chc, chalazal cap; Chp, chalazal plug.
formed a connection between the chalazal cap of maternal tissue and the chalazal plug
of parenchymal tissue (figure 5B, C). The presence of a chalazal cap and a chalazal plug
was noted in swollen mature seeds, both freshly harvested and stored, but there were no
cellular connections between the two regions. In soft seeds, water can enter the seed via
the chalazal slit to initiate the germination process. The second weak point in the palisade
layer is the micropylar area where the radicle tip is located. Palisade tissue is deprived
of its cohesiveness in the micropylar region and parenchymal tissue located under the
palisade layer is built of thin-walled cells. In soft, imbibed seeds, this structure facilitates
the splitting of the seed coat and the emergence of the radicle.
Chc
Chp
Chc C
A B
D
E F
En
En
Plc
Plc
Plc
Plc
En
En

11
DORMANCY OF SIDA HERMAPHRODITA SEEDS
Seed coat damage caused by sulphuric acid scarification was visible with a stereo
microscope. In comparison with filled and non-treated seeds, acid-scarified seeds were
characterised by a darker and uneven seed coat colour (figure 2E). Non-scarified seeds
differed in their ability to imbibe, whereas scarified seeds imbibed water uniformly (figure
2F).
Observations under a fluorescence microscope revealed damage to the palisade layer
(figure 5). Scarification for 30 minutes proved to be the most effective treatment as slight
damage to the seed coat enabled the seeds to imbibe, and increased their germination
capacity (figure 5D, E; table 5). The longest scarification treatment of 110 minutes caused
the most extensive damage to the seed coat, it lowered 1000 seed weight values and the
germination capacity (figure 5F; table 5).
Scarification decreased the percentage of hard seeds, but prolonged scarification
treatment caused severe seed coat damage and increased the seeds’ susceptibility to
microbial infections that was observed after 30 days of germination (table 5).
Discussion
Morphological basis for seed dormancy in Sida hermaphrodita
In S. hermaphrodita, the seed coat is composed of five layers that develop from the outer
and inner integuments (Savchenko and Dmitrashko, 1973; Chudzik et al., 2010). Outer
integuments produce the outer epidermis of thin-layered cells covered with cuticle, the
mesophyll comprising 2-3 layers of thin-walled cells, and the inner epidermis of cells
with thickened walls. Parenchymal and outer epidermial cells contain pigment that gives
mallow seeds a cinnamon-brown colour. Inner integuments produce the palisade layer
of thick-walled cells, 3-4 layers of parenchymal cells containing tannin, and the inner
epidermis with characteristically thickened cell walls (Savchenko and Dmitrashko, 1973).
Table 5. Thousand seed weight, germination and percentage of hard and infected seeds of Sida hermaphrodita
after 30 days of germination.
Type of seeds Thousand seed
weight (g)
Germinated seeds
(%)
Non-germinated seeds (%)
infected hard
Non-conditioned seeds
Mechanically-cleaned seeds
Seeds scarified with
sulphuric acid for:
30 minutes
60 minutes
90 minutes
110 minutes
Mean for scarified seeds
3.8
4.1
3.8
3.8
3.8
3.7
3.8
33
42
76
71
68
59
69
54
25
11
14
25
39
19
13
33
13
15
7
2
12

12
D. PACKA, J. KWIATKOWSKI, Ł. GRABAN AND W. LAJSZNER
The tissue responsible for seed coat impermeability in S. hermaphrodita is the palisade
layer of adjacent, elongated cells with thickened walls and a clearly manifested light
line. The palisade layer is formed during the transformation of outer epidermis cells of
the internal integument (Savchenko and Dmitrashko, 1973). In Abelmoschus esculentus
(L.) Moench, palisade cells are made of three parts with varied shape and chemical
composition: the prismatic part at the top, the transition part in the middle and the twisted
part at the bottom; the upper part is rich in hydrophilic compounds, whereas the bottom
part is strongly lignified (Serrato-Valenti, 1992). A similar structure of palisade cells is
noted in S. spinosa L. seeds (Egley et al., 1986). A study of S. spinosa revealed that seed
coat impermeability is a trait that develops in the maternal tissues during seed development
(Egley, 1976). This results from the conversion of phenols into insoluble lignin polymers
by a peroxidase enzyme that remains highly active in the palisade layer (Egley et al.,
1983). In our study, the palisade layer showed strong fluorescence, in particular in cells
oriented towards the interior of the seed (figure 5). Cell wall lignification could be
responsible for the strong fluorescence of the inner palisade layer.
Our observations revealed that the palisade layer surrounded the entire seed, excluding
the chalazal area where its continuity was broken (figures 3C-F, 4A-B, 5B). The resulting
discontinuity can be slit-shaped, as is the case in the tribe Malveae (Abutilon theophrasti
Medik., Abelmoschus esculentus (L.) Moench, Malva parviflora L., Sida spinosa, S.
rhombifolia L., S. acuta Burm. f. and S. hermaphrodita (L.) Rusby), or pore-shaped,
as noted in the tribe Hibisceae (Gossypium hirsutum L.) (Simpson et al., 1940; Winter,
1960; Serrato-Valenti et al., 1992; Pipa et al., 2006; Akabari and Salehi, 2008). Plants
of the family Malvaceae have an anatomical structure referred to as a “chalazal cap” or
“chalazal plug” in the chalazal region that prevents water from entering the seed (Baskin
et al., 2000). A chalazal cap sealing the chalazal area from the exterior is a maternal
structure formed during ovule development when the funiculus is fused with the outer
integument (Chudzik et al., 2010). A semi-circular flap at the end of the raphe closes the
opening in the chalazal area. The chalazal cap is connected with the chalazal plug that is
formed by proliferating parenchymal tissue in a developing seed, and it closes the opening
in the palisade layer from the inside (figure 5B). In the seeds of Abelmoschus esculentus,
both the chalazal cap of maternal origin and the exotesta of seeds in the chalazal region
are made of cells that easily absorb water (Serrato-Valenti, 1992).
When the chalazal cap was removed from hard seeds of S. hermaphrodita, a 120-
150 µm groove with tightly adjacent edges was exposed (figure 3A-B). Such seeds do
not absorb water, they do not swell or germinate. The proportion of hard seeds in the
examined batches varied from 4 to 40% (Bałdy) and from 14 to 28% (Tomaszkowo) The
plantation at Bałdy is several times larger than the farm in Tomaszkowo, and it is set on
soil underlain by highly varied types of bedrock (boulder clay, muck soil and gyttja) that
could be responsible for much greater variations in plant growth and seed attributes.
Microscopic examinations showed that soft, imbibed seeds had an opening instead of
a slit that water could enter the interior (figures 3C-F, 4A-B). Seeds were characterised by
a high imbibition capacity at harvest as well as after one year of storage, but the ability
to absorb water decreased with further storage. A study on seeds of Sida acuta and S.
rhombifolia revealed that the chalazal area is responsible for water absorption (Seal and

13
DORMANCY OF SIDA HERMAPHRODITA SEEDS
Gupta, 2000). In S. spinosa and Abelmoschus esculentus seeds, water penetration through
the chalazal slit and the separation of the palisade layer from the sub-palisade layer in
the chalazal region are the key stages that initiate the imbibition process. In the initial
stage of seed swelling (1-3 hours), a kidney-shaped blister is formed between the palisade
layer and the underlying single-cell subpalisade layer, and it initiates morphological and
cytological changes that break the continuity of the palisade tissue (Egley and Paul, 1981;
Egley et al., 1986; Serrato-Valenti et al., 1992). A similar process was observed in soft
seeds of S. hermaphrodita in our study. The palisade layer in the chalazal area was easily
removed after 24 hours of imbibition (figure 4C-D). A crescent-shaped area with changed
cell microstructure was visible on the surface of parenchymal tissue (figure 4E-F). The
above area could be responsible for the formation of a blister between palisade and
subpalisade layers. Further work is needed to validate this hypothesis.
Germination capacity and mechanisms of breaking physical dormancy
According to numerous research findings, freshly harvested seeds of S. hermaphrodita
have a relatively long dormancy period and their germination capacity is reduced to
10-15% for the first six months after harvest. Such seeds are not suitable for sowing
without treatment (Dmitrashko, 1972, 1973; Spooner et al., 1985; Dolin
´ ski et al., 2007;
Dolin
´ ski, 2009). S. hermaphrodita seeds harvested in 2009 had moderate germination
capacity (60 to 68%) for the heavy fraction within six months after harvesting. The highest
germination capacity of 73% on average was reported for seeds collected from 3-year-
old plants (table 2). In the second year of the experiment, seed germination capacity
was approximately 50% lower, reaching 26% for seeds from 2-year-old plants, 36% for
3-year-old plants and 29% for 4-year-old plants (table 3). Three fractions were identified
in each of the studied seed batches: imbibed germinated seeds, imbibed non-germinated
seeds and hard seeds (table 4). The proportion of imbibed non-germinated seeds varied
across the analysed batches. Their presence could indicate that in addition to physical
dormancy that is conditioned by an impermeable seed coat, S. hermaphrodita seeds have
an additional dormancy that is determined by the presence of chemical compounds that
delay or inhibit germination. Physical dormancy combined with physiological dormancy
of the embryo has been identified in some other species of Malvaceae (Finch-Savage and
Leubner-Metzger, 2006). Further work is needed to explore this theory further.
The results of our experiment differ from the findings reported by other authors
in recent years. In a Polish study on the germination of S. hermaphrodita seeds under
optimal conditions, germination was only 4% in freshly harvested seeds, 23% of in seeds
stored for six months, 39% for seeds stored for one year, 44% for seeds stored for 1.5
years and 33% for seeds stored for 2.5 years. Non-germinated seeds did not absorb water
or swell (Dolin
´ ski et al., 2007; Dolin
´ ski, 2009). The above discrepancies could result
from genetic variations in the studied populations, environmental conditions during the
growing period, and seed ripening and treatments applied before germination. In our study,
seeds were imbibed in water and aerated for two hours. Our results suggest that genetic
factors, optimal environmental conditions during seed ripening (2009) and the segregation
of full seeds contribute to the achievement of quality seed material without additional
dormancy-breaking treatments (table 2). Nevertheless, optimal environmental conditions

14
D. PACKA, J. KWIATKOWSKI, Ł. GRABAN AND W. LAJSZNER
are rarely encountered, therefore dormancy-breaking treatments are needed to ensure even
germination. Various dormancy-breaking methods may be considered, including chemical
scarification (concentrated sulphuric acid, caustic soda, alcohol, organic solvents such
as acetone or petroleum ether), physical scarification (high pressure, freezing, heating,
irradiation, scratching, husking) and biological scarification (enzymes that decompose cell
walls, such as pectinase and hemicellulase) (Rolston, 1978; Argel and Paton, 1999).
S. hermaphrodita seeds were chemically scarified using concentrated sulphuric acid
and physically with the use of hot water (Dolin
´
ski et al., 2007; Dolin
´
ski, 2009). In our
experiment, the optimal scarification method was scarification with 95% sulphuric acid
for 30 minutes (table 5). It was the most effective technique because the resulting damage
to the seed coat did not impair water absorption or embryo viability, thus increasing the
germination of S. hermaphrodita seeds.
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