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Content uploaded by Erika Kurucz
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1
Improvement of germination capacity of Sida hermaphrodita (L.) Rusby by
seed priming techniques manuskript
Erika Kurucz
1*
and Miklós Gábor Fári
1,2
1
Department of Plant Biotechnology, University of Debrecen AGTC, Debrecen;
2
Ereky Foundation,
Hungary
*Corresponding author e-mail: era.kurucz@gmail.com
Summary
Sida hermephrodita or Virginia mallow is a perspective perennial herb in the Malvaceae
family able to yield a biomass crop through the last two decades. Additionally, the plants have
a lot of uses and benefits for instance it can use as a fodder crop, honey crop, ornamental plant
in public gardens. It has favourable features like fast growing and resistance against the
disease and climatic fluctuations, etc. Sida is in the beginning phase of domestication
therefore it has a serious disadvantage: the low and slow germination as a big part of wild
plants. Due to the expressly low germination percent the need of seed showing of driller is
should tenfold, 200 thousand seeds/acre instead of 10-20 thousand what is not available and
expensive Therefore practical purposes of our research of seed physiology was to increase
the seed germination percent in a available, basically wild Sida population. In the first stage of
our experiments we examined two factors relating to seed germination percent and seed
germination power during our research: the influence of hot water treatment and the effect of
exogenous or endogenous infection of seed. However, in our germination tests, utilizing
scarified seeds with hot water (65, 80 and 95
o
C), from 29,3% to 46% germinated from those
samples which were collected from the population of S. hermaphrodita in Debrecen. The
average germination for all season was 5-10 % without treatment and rinsed using hot water
up to almost 50%. When physically scarified used, the oldest seeds showed the best
germination (46 %) after the hot water operation in spite of the previous studies. We
discovered that there are close relationship between the seeds collecting time and the ration of
seed infections, as well as germination percentage. Thus, 2009 season was the most
favourable in case of contamination (control: 17,3% and 80
o
C treatment: 0%) as well as
germination percent. It could be concluded that, the best season for our findings was 2009 due
to autumn harvest of Sida seeds. In our opinion, the autumn harvesting should be the best time
to overcome the problem of the low germination and high infection percentage. We also
discovered that apparently there are close relationship between the seed fresh weight or water
uptake capability and the percentage of infection. Following these recognition we modified
our technique, in such a way that we fractionated the seeds based on their fresh weight / or
relative density before we carried out the treatment. When we filtered the floating seeds on
the surface of water, the hot water treatment was performed considerably better on the sunk
seeds after separation. Therefore, by this special priming process we were able to reach 80%
germination capacity of Virgina mallow seeds under laboratory conditions (26
o
C without
illumination).
Keywords
Virgina mallow, Sida hermaphrodita, seed priming technique, seed germination
2
Introduction
Sida (Sida hermaphrodita Rusby, Virginia mallow, Virginia fanpetal, Petemi) is a perspective
perennial herbaceous semishrub plant originating from North America. This endangered
species will probably receive more attention as a temperate climatic biomass crop. Based on
European investigation results, the main value of Sida is that it can cultivate on marginal areas
and the plants can develop under our warming climate conditions with biomass output around
of 10-20 DT ha
-1
. Besides this important point the water content of Sida decrease between 35
and 45% until November preceded the perennial rhizomatous grasses (PRG) and short
rotation coppice species.
Second (bio) generation energy plant are considered as excellent candidates for the future of
bioenergy and they are going to able to serve the biomass supply chain programmes. The
breeding of biomass plants can be economical and sustainable, if the cultivated plant is
adaptable, and it possible to grow calculable and (as it is called), carbon dioxide - positive
field cultivation way. Unavoidable the biotechnology and environmental research of Sida,
which is in a base stage of domestication, before it is, took into cultivation for industrial
biomass purposes. The aim of this and the future investigations are to detect and solve all of
the problems linked closely to the propagation and growing.
S. hermaphrodita is known in the literature as a plant with low seed germination potential. In
Europe, many authors have dealt with this problem because of possible industrial uses of this
plant (Chudzik et al., 2010). For this reason, in Europe this plant is reproduced mainly in a
vegetative way from root cuttings. One of the limiting factor of this method is that currently
not be well known the virological, phytophatological and pests background of the Sida.
Because the lack of investigations of its phytopathological background, the farmers who
planting Sida from root cuts take on considerable environmental and economical risks.
Therefore, it has to give preference to propagate it from seeds than from root cuttings for
industrial-scale bioenergy farms (Kurucz et al., 2012).
In general, there are many reasons which can responsible for the low germination of seeds.
For example, in case of Sida it was observed over 30% of the ovules remained in their
juvenile stage at the time when pollen tubes reached the ovary. These ovules were probably
aborted, did not attract the pollen tubes and underwent degeneration before or during fruit
development (Chudzik et al., 2010). Under Polish conditions, other studies on the biology of
flowering of S. hermaphroditain showed that as many as 95% of seeds were set. From the
results of some previous author, it could be concluded that, the main cause of low germination
is the strong seeds dormancy (Baskin, 2003; Barthodeiszky et al., 1980).
Seeds of the majority of plant species in the world except tropical rainforest and tropical semi-
evergreen forest are dormant at maturity (Smith et al., 2004). According to Smith et al. (2004)
dormant seeds can be classified into one of five classes: physiological (low growth potential
of the embryo); morphological (small undifferentiated or small differentiated, but
underdeveloped, embryo); morpho-physiological (underdeveloped, physiologically-dormant
embryo); physical (water-impermeable palisade or palisade-like layers of cells in seeds, or
fruit coat); and combinational (water-impermeable seed or fruit coat + physiologically
dormant embryo). Other authors divide seed dormancy into seven classes: undeveloped
embryo, embryo is mechanically inhibited by the seed-coat, water-impermeable coats (hard
3
seed), gas-impermeable seed coats, endogen metabolism inhibitors, complex dormancy,
secondary dormancy (Barthodeiszky et al., 1980).
Species with water-impermeable seed or fruit coats, physical dormancy occur in some 15
plant families (Baskin et al. 2003) including the Malvaceae family. Barthodeiszky (1980)
called this phenomenon as “hard seed effect” and this kind of seeds was called “hard seed”.
This impermeability of the coat is caused by the presence of one or more palisade layers of
lignified Malphigian cells (macrosclereids) tightly packed together and impregnated with
water impermeable chemicals (Baskin et al., 2000). An anatomical structure in the
impermeable layer(s) functions as the ‘water gap’, seven types of which have been described
(Baskin et al., 2000).
However, Dolinski (2009) have presented evidence that dormancy-break by heating and by
chemical treatment of Sida seeds may occur through disruption of the seed coats. Immediately
after the harvest, only 3% of control seeds germinated whereas after 6 months, their
germination increased to 14.5% - 35.5%, but after 1.5 years it is decreased (Dolinski, 2009).
Fresh seeds had the best germination (73%) after immersing into the boiling water;
germination capacity decreased along with the water temperature decrease. Water at 70 to
80°C temperature had the most positive effects on seeds (Dolinski, 2009). Spooner (1985)
concluded that scarified seeds, 81% to 99% of the seeds collected from 10 populations of S.
hermaphrodita in Maryland and Ohio germinated, respectively. The average germination for
all 10 populations was 92% (Spooner et al., 1985). They collected the seeds in August and
September. Whereas, the other studies did not mentioned the harvesting date, it is possible
that these good results was owing to the autumnal harvest.
It seems that boiling water has not damaged the germs of fresh seeds, but more and more
seeds imbibed without germination in subsequent examination dates (Dolinski, 2009). The
previous studies revealed that seeds of some species of Malvaceae family scarified using hot
water or concentrated sulphuric acid could have positive effect on seed germination percent
(Dolinski, 2009; Baskin et al., 2003). Even more, it has been stored for a long time with no
fear of their fast loss of germination capacity (Dolinski 2009).
The aim of our present investigation were (1) to study the effect of two hot water pre-
treatment priming methods and (2) to analyse the influence of seed harvesting time on S.
hermaphodita seed germination percent and seed contamination ratio involving four growing
years.
Material and Methods
Collection of seed samples
Germination and, viability tests were conducted during February 2013 on seeds of Sida
collected from our Plants for the Future Biomass Experimental Garden in different years, i.e.,
2009, 2011, 2012 and 2013. In 2009, seeds were collected in autumn. In the following
seasons, we gathered the seeds in spring, in order to ensure the low temperature influence
which is considered necessary. Therefore, in the following seasons 2011, 2012 and 2013, the
4
seeds were collected in spring time. The matured dry fruit clusters were manually harvested
and threshed.
Hot water pre-treatment (HWT)
Seeds of each HWT-samples (except the controls) were immersed into heated water regulated
for the following temperatures and time regime: 65 and 80 °C, for 2 minutes; 95°C for 30
seconds, non-scarified seeds constituted the control and then, part of seeds from every
combination (3×50pcs) were sown on wetted filter papers put into Petri dishes for 26
o
C
without lighting, while others stored for further studies. Seedling counts were performed at 3
and 6 days later (Figure 1), and total percent of germination and contamination rate were
calculated for each treatment.
Floated seed priming technique (FSPT)
The HWT method was complemented with a simple seed priming step to fractionate them
based on their specific weight, and or imbibation / permeability characteristic. First, before we
put the samples into heated water, the seeds were immersed into distilled water of 23-25
o
C for
30 minutes. After this, the hot water treatment was performed on the dipped seeds followed
separation. The sunk but not treated seeds were considered as control. These FSTP seeds were
sown on wetted filter papers and put into Petri dishes for 26
o
C without lighting.
Statistical analysis
For the germination assays each treatment consisted 50-50 pcs seeds and they were repeated
at least three-times. The research results were calculated by Windows Office Excel software
and evaluated statistically by the analysis of variance using the SPSS 14.0 programme.
Results
Germination assays
The effects of various temperatures of water and influence of different growing seasons for
overcoming seed dormancy and the seed contamination rate of Sida hermaphrodita Rushby
are shown in Table 1. It could be notice that dipping seeds into boiling water, the HWT
method significantly broke seed dormancy of Sida hermaphrodita. It was also observed that
while the water temperature regime was higher, the contamination ratio was significantly
lower. By the best combination of HWT, the average of germination ratio was elevated up to
80%. We also observed that from the untreated seeds only 4-5% germinated (Figure 1), except
the growing year of 2009, in which the germination percent was 10%. The seeds of season
2009 have the best germination percent (46%) by means of HWT method. Figure 2. also
5
shows that 80°C HWT resulted the highest germination ratio, almost in each season, except
2013. It was noticed that there were no significant differences between HWT treatment made
at 80
o
C and 95
o
C (Table 2).
Visual observation of seed contamination
Table 1. also clearly shows that the rate of contamination of seeds (or seed coat) is
dramatically decreased by elevating the water temperature up to 65
o
C and 95
o
C. It could be
noticed that the most contaminated season was 2012 in which the contamination ratio reach
52 % in the control (Figure 2). On the contrary, the seeds from 2009 were healthier (17.33%
infected seeds in average). The statistical analysis clearly showed the significant difference
between the control and the treated (65
o
C, 80
o
C, 95
o
C) samples, meanwhile the difference
between 80
o
C and 95
o
C was not significant. The infection ratio in the control seeds of all
seasons fluctuated considerably; these discrepancies can be connected with seasonal and
harvesting date differences.
Results of FSPT-method
Table 2 shows the germination and contamination percentage in water fractionated (dipped
and supernatants) and control seeds. In this FSPT treatment we applied 80
o
C, the most
effective priming temperature. On the basis of our data we can say, that we were able to
dramatically increase the germination percent from 11,3% to 80% in average, and we also
were able decrease dramatically or entirely eliminate the seed contaminations from 32,6% to
0%, under laboratory conditions (Figures 1 and 2).
Conclusion
Hard seeds are common in a number of species in the Malvaceae family (Spooner, 1985). In
the case of Sida spinosa L. it have been shown that water impermeability of the seeds is partly
explicable due to a compact layer of integumentary palisade cells. A similar layer of cells
occurs in seeds of Sida hermaphrodita L, which dormancy can be stopped by hot water
treatment (Chudzik et al, 2010). Spooner et al. (1985) founded that the low germination
percentages obtained apparently due to his failure to scarify the seeds. It should be note that
the seeds was collected in autumn of 1985. However, in this work, germination tests utilizing
scarified seeds germinated from 29,3 to 46% in the case of seeds collected from our S.
hermaphrodita populations. The average germination for all season varied between 4,67%
and 10% without hot water priming. When physically scarified, the oldest seeds (2009)
showed the best germination percent (46%) after HWT treatment.
Our study also revealed that Sida hermaphrodita seeds scarified using hot water treatment
(HWT method) has a positive effect on S. hermaphrodita seeds contamination pattern. The
highest percentage of contamination was observed in 2012. The infection ratio was reduced
by 52% to 0%. The same effect was observed in all seasons. The most favourable temperature
was 95
o
C, but if we take account the germination percent, we should conclude from the data
6
that the most effective water temperature is 80
o
C. However, the germination potential of S.
hermaphrodita seeds showed variability, depending on the growing season. In many
experiments the maximum number of germinating seeds was about 30-40%. For this reason,
this plant actually is reproduced mainly by vegetative way in Europe (Chudik et al., 2010).
We discovered that there are close relationship among the collecting time, the contamination
percentage as well as germination ratio. Thus, 2009 season was the best in case of
contamination as well as germination percent. It could be concluded that, the most favourable
period of seed collecting for propagation is autumn, after the seed ripening. During these
experiments it has been found, that the success of the Sida germination shows correlation not
just with the amount of the endogenous and/or exogenous fungus contaminations, but their
special weight and/or water imbibation / permeable characteristic. By the influence of these
recognitions we modified our seed priming technique, in such a way that we fractionated the
seeds based on their relative density (filtering out the floating supernatant seeds from distilled
water) before we executed the treatment. The hot water treatment (HWT) was performed on
the dipped / sunk seeds after separation; therefore we were able to reach 80% germination in
Petri-dishes under laboratory conditions (26
o
C without lighting). This method is named as
FSPT (floated seed priming technique). However, in our germination tests, utilizing 80% of
separated and scarified seeds collected from Debrecen can germinate, against our previous
results in which the germination reached at maximum 46% by HWT method. Therefore, we
can reduce the amount of sowing-seed requirements in case of Sida hermaphrodita from
200.000-300.000 seeds ha
-1
to 20.000-30.000 seeds ha
-1
. Because of deficient knowledge of
Sida pathological background, the farmers who planting Sida from root cuttings are
confronted by considerable risk of human health and the hazard of environment. According to
our opinion it has to give preference to propagation from seeds that from root cuttings.
From the investigations presented in this work, it could not be concluded clearly that the
features observed during the investigated stage of S. hermaphrodita seeds have physical or
physiological dormancy, or both. Nonetheless, it should be cleared that how can we
characterize and prevent the endogenous or exogenous seed infections. It should be underline
that the present study is the first in the series of studies in which we plan to undertake the
problem of the seed embryology of this promising energetic species. The aim of our further
investigations is that we should obtain more information about seed biology of some other
species of Malvaceae family (Sida hermaphrodita, Kitaibela vitifolia, Kitaibela balansae,
Kitaibela x kovatsii, Althea canabina). If we can increase the seed germination percent of
different mallow species, these achievements may be new complementary material for the
second (bio) generation plants within the biomass supply chain.
Acknowledgement
This work is partly supported by the TAMOP-4.2.2.A-11/1/KONV-2012-0041 project and co-
financed by the European Union and the European Social Fund. Additional financial support
is also gratefully acknowledged for the MOP Biotech Co Ltd. (Nyíregyháza, Hungary) and
Ereky Foundation (Debrecen, Hungary).
7
References
Barthodeiszky A., Czimber Gy. (1980): A magbiológia alapjai (in Hungarian) - ISBN 963-
05-1924-0
Baskin C.C. (2003): Breaking physical dormancy in seeds – focussing on the lens. New
Phytologist 158: 229-232
Baskin J. M.
and Baskin C. C and Xiaojie Li (2000): Taxonomy, anatomy and evolution of
physical dormancy in seeds. Plant Species Biology, 15(2): 139–152
Baskin J. M.
and Baskin C. C. (2003): When breaking seed dormancy is a problem: try a
move-along experiment, Native Plant Journal 4(1): 17-21
Chudzik B., Szczuka E., Domaciuk M. and P. Danail (2010): The structure of the ovule of
Sida hermaphrodita (L.) Rusby after pollination. Acta Agrobotanica, 63 (2): 3–11
Dolinski R. (2009): Influence of treatment with hot water, chemical scarification and storage
time on germination of Virginia fanpetals, Sida hermaphrodita (L.) Rusby seeds,
Buletyn Instytutu Hodowli i Aklimatyzacji Roślin, 251: 293-303
Kurucz E., Szarvas P., Fári M. G. (2012): Alternatives of the multiple use of Virginia
mallow in Acta Agraria Debreceniensis, 46: 51-57
Smith R., Dickie J., Linington S., Pritchard H. and Probert R. (2004): Seed conservation:
turning science into practice - ISBN 1842460528
Spooner D. M. (1985): Observations on the distribution and ecology of Sida hermaphrodita
(L.) Rusby (Malvaceae)
8
Legend
Tables and Figures
Table 1: Influence of hot water treatment (HWT) on germination (%) and contamination (%) of Sida
hermaphrodita L. Rusby seeds (50 pcs seeds per each treatment)
Growing Seasons
HWT
treatment
(
o
C)
Germination
(%)
Contamination
(%)
2009
Collection: autumn
untreated control
8,00
17,33
65
42,00
0,07
80
46,00
0,00
95
30,67
0,00
2011
Collection: spring
untreated control
10,00
21,33
65
31,33
10,00
80
38,67
5,33
95
34,00
4,00
2012
Collection: spring
untreated control
4,67
52,00
65
19,33
18,67
80
36,00
0,67
95
26,67
0,00
2013
Collection: spring
untreated control
4,67
36,67
65
4,00
12,67
80
29,33
2,67
95
30,00
0,00
9
Table 2. Germination and contamination pattern of water-fractionated seeds with or without
HWT priming (50 pcs seeds per each treatment)
Treatments
Repeat
Measurement of germination
Measurement of contamination
Number of
germinated
seeds
Germination
(%)
Number of
contaminated
seeds
Contamination
(%)
FSTP method
(water-
fractionated and
HWT-treated
seeds at 80
o
C)
I
36,00
72,00
0,00
0,00
II
43,00
86,00
0,00
0,00
III
40,00
80,00
0,00
0,00
Mean
39,67
79,33
0,00
0,00
Supernatant
seeds without
HWT
I
1,00
2,00
47,00
94,00
II
0,00
0,00
44,00
88,00
III
0,00
0,00
45,00
90,00
Mean
0,33
0,67
45,33
90,67
Control
(untreated
seeds)
I
7,00
14,00
16,00
32,00
II
3,00
6,00
19,00
38,00
III
7,00
14,00
14,00
28,00
Mean
5,67
11,33
16,33
32,67
10
Figure 1: Effect of water-fractionation on the germination pattern of Sida hermaphrodita Rushby.
Floated seeds= supernatant fraction; sunk seeds= imbibed fraction
11
Figure 2: Influence of hot water treatment (HWT) on germination ( %) of Sida hermaphrodita seeds
harvested in four growing seasons
0,00
5,00
10,00
15,00
20,00
25,00
30,00
35,00
40,00
45,00
50,00
Germination %
Control 65 80 95
Treatment (
o
C)
2009 Collection: autumn
2011 Collection:spring
2012 Collection:spring
2013 Collection:spring
12
Figure 3: Influence of hot water treatment (HWT) on contamination (%) of Sida hermaphrodita seeds
harvested in four growing seasons
0,00
10,00
20,00
30,00
40,00
50,00
60,00
Infection %
Control 65 80 95
Treatment (
o
C)
2009 Collection: autumn
2011 Collection:spring
2012 Collection:spring
2013 Collection:spring
