Seed germination and storage of Chamaerops humilis (dwarf fan palm)

Abstract

Chamaerops humilis (dwarf fan palm) is one of the two Palmaceae species native to Europe, with uses for gardening and revegetation. Its seed germination and conservation (short and medium term) are assessed. Seeds showed dormancy that could be broken by the use of sulphuric acid (15 min), increasing germination percentage from 34% to 68%. However, that initial dormancy disappeared after approximately 10 months storage at 15°C (89% germination). No germination was observed at 15°C, while no differences were found between 20°C, 25°C and 15°C (8-h darkness)/25°C (16-h light) regimens. When seeds were set to germinate after desiccation, final germination percentage decreased compared to that of non-desiccated seeds. However, seeds desiccated and stored (for 85 days) at low temperatures (5°C, -18°C, -80°C and -196°C) did not show lower germination percentages than stored non-desiccated seeds. The only storage temperature that decreased germination compared to that of control seeds was -196°C (liquid nitrogen). T50 values (time to reach 50% of final germination percentage) also increased after storage in liquid nitrogen compared to the other temperatures. Medium-term storage (569 days) was studied at 15°C, 5°C and -18°C. Germination was slower (higher T50 values) when storage was carried out at 5°C and -18°C than at 15°C.

Full text

143
CHAMAEROPS HUMILIS SEED GERMINATION AND CONSERVATION
González-Benito, M.E., Huertas-Micó, M. and Pérez-García, F. (2006), Seed Sci. & Technol., 34, 143-150
© International Seed Testing Association 2006
Seed germination and storage of Chamaerops humilis (dwarf fan palm)
M.E. GONZÁLEZ-BENITO, M. HUERTAS-MICÓ AND F. PÉREZ-GARCÍA
Dpto. Biología Vegetal, E.U.I.T.Agrícola, Universidad Politécnica de Madrid, Ciudad Universitaria, 28040
Madrid, Spain (E-mail: me.gonzalezbenito@upm.es)
(Accepted November 2004)
Summary
Chamaerops humilis (dwarf fan palm) is one of the two Palmaceae species native to Europe, with uses for
gardening and revegetation. Its seed germination and conservation (short and medium term) are assessed.
Seeds showed dormancy that could be broken by the use of sulphuric acid (15 min), increasing germination
percentage from 34% to 68%. However, that initial dormancy disappeared after approximately 10 months
storage at 15°C (89% germination). No germination was observed at 15°C, while no differences were found
between 20°C, 25°C and 15°C (8-h darkness)/25°C (16-h light) regimens. When seeds were set to germinate
after desiccation, final germination percentage decreased compared to that of non-desiccated seeds. However,
seeds desiccated and stored (for 85 days) at low temperatures (5°C, -18°C, -80°C and -196°C) did not show
lower germination percentages than stored non-desiccated seeds. The only storage temperature that decreased
germination compared to that of control seeds was -196°C (liquid nitrogen). T
50
values (time to reach 50% of
final germination percentage) also increased after storage in liquid nitrogen compared to the other temperatures.
Medium-term storage (569 days) was studied at 15°C, 5°C and -18°C. Germination was slower (higher T
50
values) when storage was carried out at 5°C and -18°C than at 15°C.
Introduction
Dwarf fan palm (Chamaerops humilis L.) is distributed in the Western Mediterranean
rim, being found in Europe (Portugal, Spain, France and Italy), Northen Africa and in
Mediterranean islands (Balearic, Sicily, Cerdeña) (Cañizo, 2002). To the East, it can be
found in Malta, although there it is almost extinct (Cañizo, 2002). It is one of the two
native palms in Europe, and the only one found in the Iberian Peninsula. It resists low
temperature and drought and can grow on poor and rocky soils. Due to its rusticity and
recovery after fire, it has a high ecological value for preventing erosion and desertization.
It is also used in gardening and landscaping, both as a decorative element (in cultivation
it can grow up to 4 m) or as soil retainer. The young sprout can be eaten in salads and the
leaves can be used to make baskets and brooms (Merlo et al., 1993, Cañizo, 2002).
Chamaerops humilis can be propagated vegetatively and by seeds. Little is known
about the seed germination and conservation behaviour of this species. Sowing in hot
beds (20-25°C) is recommended after removal of the drupe fleshy tissues (Piotto and
Noi, 2003). Long elapses (3 months) in greenhouse sowing before first seeds germinated
have been reported (Ellis et al., 1985). However, the germination rate can be accelerated
by scarification with 4.5 h in sulphuric acid (Merlo et al., 1993). Seed storage behaviour
has been reported as uncertain (Hong et al., 1996) or long-lived (León, 1961). Due to

144
M.E. GONZÁLEZ-BENITO, M. HUERTAS-MICÓ AND F. PÉREZ-GARCÍA
the great diversity of geographical and ecological distribution of the family (Palmaceae),
seed storage behaviour is very diverse among its species (Hong et al., 1996). In general,
species native to Mediterranean regions, as it is the case of Ch. humilis, would produce
orthodox seeds.
To increase the knowledge about Chamaerops humilis germination biology and seed
conservation, the effects of scarification treatments, temperature, and of short- and medium-
term conservation on seed germination under controlled conditions were studied.
Material and methods
Fruits were collected in September 2001 in the province of Murcia (SE Spain). Epicarp
and mesocarp were removed by means of a coffee-grinder. Although removal of epicarp
and mesocarp has been reported previously by soaking (Merlo et al., 1993; Piotto and
Noi, 2003), the use of water was avoided in this study so as not to increase seed moisture
content. The seed surrounded by endocarp was treated as a unit (hereafter termed as seed,
figure 1). The size of the embryo is much smaller than the seed (0.0017 ± 0.0003 g vs
0.64 ± 0.02 g).
Seeds were stored inside plastic boxes (domestic food containers) at 15°C until use. In
all trials, four replicates of 25 seeds each were tested per treatment for germination. Seeds
were sown in trays with vermiculite, previously autoclaved for 1 h (120°C, 1 atm). Distilled
water was added to the vermiculite when seeds were sown and periodically, so that the
vermiculite was always humid. Incubation took place at 15°/25°C under a 8-h dark/16-h
light photoperiod, unless otherwise stated, in a chamber with relative humidity control (85 ±
5%).
In a first assay, scarification treatments were studied with seeds that had been stored
at 15°C for 80 days before the beginning of the assay. Before sowing, seeds were soaked
in water for 3 days, or in water with few drops of commercial bleach for 1 or 3 days, both
at 25°C. In another treatment, seeds were immersed in H
2
SO
4
(96%) for 15 min, and then
washed thoroughly with tap water.
The effect of incubation temperature on germination was studied in a second assay.
Constant (15°C, 20°C and 25°C) and alternating (15°/25°C) temperature regimens were
employed. Before seeds were sown, they had been stored at 15°C for 170 days and were
scarified for 15 min with H
2
SO
4.
The scarification treatment with sulphuric acid was further studied by immersing
seeds for different periods (5, 15, 30 and 60 min) and using seeds without scarification as
control. Seeds had been stored at 15ºC for 300 days before the beginning of the assay.
For conservation purposes, seeds were desiccated with silica gel at 25°C for 1 month
(RH approximately 5%, replacing the silica gel with desiccated one when it lost its deep
blue colour). Desiccated and non-desiccated seeds were then stored at 5°C, -18°C, -80°C
and -196°C for 85 days. For the first three temperatures, seeds were kept in Kilner-type
glass jars (hermetically closed) and in the jars where desiccated seeds were kept, silica
gel was also included. Silica gel was blue at the end of the storage period, indicating
the maintenance of a dry environment. Seeds stored at -196°C were enclosed in metal
meshes and then immersed in liquid nitrogen. A double amount of seeds was kept at 5°C

145
CHAMAEROPS HUMILIS SEED GERMINATION AND CONSERVATION
and -18°C, so a second group of seeds could be stored for further 484 days (569 days in
total). Seeds were removed from storage and allowed to warm at room temperature for
few hours. They were then scarified with H
2
SO
4
for 15 min before sowing. Before the
beginning of this assay, seeds had been stored at 15°C for 200 days. As control, a group of
seeds was sown after 285 days (200 + 85) and 769 days (200 + 569) conservation at 15°C.
Germination of seeds stored at 15°C for 200 days and desiccated was also studied.
Seed moisture content was determined in intact seeds and in excised embryos,
from non-desiccated (control) and desiccated seeds. Moisture content was determined
gavimetrically (loss of seed weight after 2 h in an 130°C oven; ISTA, 2003). For deter-
mining the moisture content of whole seeds (endocarp, endosperm and embryo), they
were broken into pieces with a hammer before the first weighing. To determine the
moisture content of embryos, they were excised with the help of a clamp and a hammer.
As they were being excised, to minimize water loss, they were placed in a small plastic
container (2 ml) with hermetic closure, until 10 embryos were collected for weighing.
Two replicates were employed per seed or embryo moisture content determination.
Seeds were observed every three-four days and scored as germinated when the radicle
protruded ca. 3mm (figure 1), which occurred generally 4-7 days after the operculum was
lifted. Those seeds were marked by placing a small stick beside them, not to disturb the
growth of the seedling in later counts. The final germination percentage was studied after
100 to 130 days from sowing. ANOVA and Duncan’s multiple range test were performed
on the arcsin transformation of the final germination percentage. T
50
values (days to reach
50% of final germination percentage) were calculated by linear interpolation of the two
germination values closest to the median germination.
Figure 1. (A) Seed of Chamaerops humilis cut (with the help of a saw) transversally (a) and longitudinally
(b). (B) Detail showing embryo (c), endosperm (d) and endocarp (f). (C) State at which a seed was considered
germinated: radicle emerging and operculum lifting. (d) Plantlet 30 days after germination.
1 cm
3 mm
c
d
f
B
C
D
a
b
A

146
M.E. GONZÁLEZ-BENITO, M. HUERTAS-MICÓ AND F. PÉREZ-GARCÍA
Results
For seeds stored at 15°C for 80 days before sowing, the best results were obtained by
soaking in sulphuric acid, as both final germination percentage and germination rate
improved when compared to control seeds (table 1). Soaking seeds in water for 1-3 days
with few drops of bleach did not improve germination. With other scarification procedures
studied, but not reported here, germination was lower than in control seeds, i.e. some
damage must have been produced to the embryos. Those procedures included soaking
seeds in boiling water or in water at 80°C, or mechanical scarification of endocarp with
a metal file or a clamp.
Table 1. Final germination percentages and T
50
values of dwarf fan palm seeds after different presowing
treatments. Final germination percentages recorded after 130 days of incubation at 15/25°C under a 8-h dark/16-
h light photoperiod. Seeds had been stored at 15°C for 80 days before the beginning of the assay. Means
followed by the same letter within columns are not significantly different according to Duncan´s multiple range
test (p< 0.05).
Presowing treatment Duration of
treatment
Germination
(%, mean ± SD)
T
50
(days, mean ± SD)
Control 34 ± 8.2 b 39.2 ± 8.6 a
Soaking in water at 25°C 3 days 38 ± 8.2 b 36.9 ± 8.1 a
Soaking in water with few drops of
commercial bleach at 25°C
1 days
3 days
53 ± 12.4 ab
57 ± 13.1 ab
33.2 ± 5.6 ab
24.9 ± 2.5 bc
Soaking in SO
4
H
2
15 min 68 ± 17.2 a 17.7 ± 3.4 c
Control (non-scarified) seeds that had been stored at 15°C for 300 days showed
higher final germination than previously (table 1 cf. table 2, 34% vs 89%). After 300
days conservation, the final germination and the germination rate were not significantly
different in control and scarified seeds, whatever the duration of the treatment with
sulphuric acid (p < 0.05 and p< 0.25, table 2).
Table 2. Final germination percentages and T
50
values of dwarf fan palm seeds after different scarification times
with sulphuric acid. Final germination percentages recorded after 120 days of incubation at 15°/25°C under a
8-h dark/16-h light photoperiod. Before the beginning of the assay, seeds had been stored at 15°C for 300 days.
Within a column means are not significantly different.
Treatment
Germination
(%, mean ± SD)
T
50
(days, mean ± SD)
Control (untreated seeds) 89 ± 3.3 16.1 ± 1.8
SO
4
H
2
5 min 76 ± 4.9 17.6 ± 3.8
15 min 79 ± 5.2 16.8 ± 2.1
30 min 77 ± 8.7 15.3 ± 3.1
60 min 82 ± 3.5 12.8 ± 1.1

147
CHAMAEROPS HUMILIS SEED GERMINATION AND CONSERVATION
The incubation temperature significantly affected the final germination percentage
(table 3). No germination was observed at 15°C. Germination was similar (74-79%) for
the other temperature regimens. Half of the seeds (two trays with 25 seeds each) that had
been incubated at 15°C were transferred to 15°/25°C. After four months only 3 seeds had
germinated. The non germinated seeds were opened with the help of a clamp, and 14% of
the embryos looked dead (dry and/or dark yellow).
Table 3. Final germination percentages and T
50
values of dwarf fan palm seeds incubated at different temperature
regimens. Final germination percentages recorded after 100 days of incubation at 15/25ºC under a 8-h dark/16-h
light photoperiod. Before sowing, all seed were soaked in sulphuric acid for 15 min. Before the beginning of the
assay, seeds had been stored at 15°C for 170 days. Means followed by the same letter within columns are not
significantly different according to Duncan’s multiple range test (p< 0.05).
Incubation temperature Germination
(%, mean ± SD)
T50
(days, mean ± SD)
15°C 0 b --
15°25°C 76 ± 13.0 a 18.4 ± 1.8 a
20°C 74 ± 7.2 a 20.7 ± 0.4 a
25°C 79 ± 12.8 a 24.5 ± 6.6 a
When comparing germination of non-desiccated and desiccated seeds after short-term
storage (85 days) at low temperature with those of control seeds (stored at 15°C during that
time), significant differences were observed for final germination and T
50
values only after
storage at -196°C (table 4). For the other three cold storage temperatures (5°C, -18ºC and
-80°C), differences were not significant.
For each storage temperature, final germination and T
50
values were similar between
desiccated and non-desiccated seeds (table 4). On the contrary, when seeds that had been
conserved at 15°C for 200 days were set to germinate just after the desiccation treatment,
final germination and T
50
were negatively affected (45 ± 13.4% and 39.2 ± 7.9 days,
respectively) After desiccation, embryo and seed moisture contents were almost half the
original value (from 14.6+ 0.4% to 6.7 + 0.7% for whole seeds, and from 10.6 + 0.0% to
5.2 + 0.8% for embryos).
In the medium term storage (569 days, approx. 1.5 years) at the two temperatures
studied (5°C and -18°C), germination and T
50
values were not significantly different
from those reached by control seeds (stored at 15°C during that same period) (table 4).
Germination of seeds conserved at 15°C had not decreased through time after being stored
for further 484 days (table 4), although their moisture content had decreased during that
time to 11.6±0.2%. A group of seeds, that had been stored at 15°C all that time (769
days), were set to germinate without the sulphuric acid treatment. Their germination was
similar to those treated with the acid (final germination 80 ± 2.8%, T
50
20.4 ± 1.0 days).
When final germination was compared after 569 days in cold storage with that
obtained after 85 days storage, a decrease was only found for non-desiccated seeds stored
at 5°C (table 4). However, it could be observed that the prolonged storage had affected
germination rate for the two temperatures studied (5°C and -18°C) and in both desiccated
and non-desiccated seeds.

148
M.E. GONZÁLEZ-BENITO, M. HUERTAS-MICÓ AND F. PÉREZ-GARCÍA
Discussion
As was observed previously by Merlo et al. (1993), Ch. humilis germination percentage
and germination rate were improved by chemical scarification with sulphuric acid. They
obtained 90% germination seven days after sowing when seeds had been chemically
scarified for 4.5 h with sulphuric acid. However, we found that a duration of 15 min
was enough to improve germination from 34% to 68% and reduce T
50
from 39 days to
18 days. This procedure avoids prolonged immersion times in sulphuric acid that could
damage the embryo and affect the appropriate growth of the seedling.
We found that after approximately ten months storage at 15°C, dormancy had
disappeared and final germination percentage increased. It seems that, during the prolonged
conservation time (80 vs 300 days, tables 1 and 2), the possible causes of dormancy
had disappeared, also reducing the germination rate (T
50
39.2 days vs 16.1 days). The
germination improvement by short-term storage at 15°C has also been observed in seeds
of other Palmaceae species (Pritchard et al., 2004).
Ch. humilis seeds have been reported to germinate (25%) at 15°C (Merlo et al.,
1993); however, no seed germinated at that temperature in our study. The diverse origin
of the seeds could account for those differences, although collection sites are close by.
Table 4. Final germination percentages and T
50
values of dwarf fan palm seeds after storage for 85 and 569 days
at 5°C, -18°C, -80°C and -196°C. Before cold storage, seeds had been stored at 15°C for 200 days. For each
cold storage temperature, a group of seeds was previously desiccated with silica gel at 25°C for one month.
Before sowing, all seeds were soaked in sulphuric acid for 15 min. Final germination percentages recorded after
120 days of incubation at 15°/25°C under a 8-h dark/16-h light photoperiod. Means followed by the same letter
within columns are not significantly different according to Duncan´s multiple range test (p< 0.05).
Storage
temperature
Seed
desiccation
Germination
(%, mean ± SD)
T
50
(days, mean ± SD)
Storage period Storage period
85 days 569 days
P
(2)
85 days 569 days
P
(2)
control
(1)
Non-desiccated 79 ± 5.2 ab 79 ± 10.0 a ns 16.9 ± 2.1 b 18.7 ± 1.6 a ns
5°C
Non-desiccated 70 ± 4.5 bc 57 ± 12.5 a ns 15.7 ± 2.0 b 20.1 ± 1.8 a *
Desiccated 77 ± 8.7 ab 68 ± 8.5 a ns 17.2 ± 2.8 b 22.5 ± 2.4 a *
-18°C
Non-desiccated 83 ± 1.7 a 64 ± 10.2 a * 15.3 ± 0.9 b 22.5 ± 2.0 a **
Desiccated 79 ± 5.9 ab 67 ± 7.7 a ns 17.8 ± 2.1 b 22.5 ± 1.6 a *
-80°C
Non-desiccated 67 ± 9.5 bc -- -- 17.3 ± 1.7 b -- --
Desiccated 66 ± 11.5 bc -- -- 8.8 ± 0.8 ab -- --
-196°C
Non-desiccated 49 ± 5.0 d -- -- 22.5 ± 2.9 a -- --
Desiccated 58 ± 4.5 cd -- -- 21.8 ± 3.3 a -- --
--: trial not carried out
(1)
: Control: seeds stored at 15ºC a total of 285 days (200+85) or 769 days (200+569).
(2)
: For each row and parameter (germination and T
50
), significance of differences between means of 85 days vs
569 days ; * p< 0.05; ** p < 0.01; ns, not significant.

149
CHAMAEROPS HUMILIS SEED GERMINATION AND CONSERVATION
Interpopulation differences in germination behaviour have been reported in several species
(Fenner, 1991; Meyer and Allen, 1999). The other temperatures tested in our work did
not result in significant differences in the germination behaviour. However, the alternating
temperature regimen (15°/25°C) slightly improved germination rate. This could reflect
adaptation of germination in early spring, a strategy show by many Mediterranean species
(Thanos et al., 1995; Pérez-García et al., 1995).
Desiccation reduced the final germination percentage when seeds were set to
germinate immediatly after that treatment, but not when they were stored afterwards at
cold temperature. Lacking more direct evidence, these results could be interpreted as a
possible imbibition damage caused by desiccation (Ellis et al., 1990). Desiccated seeds
were sown directly in humid vermiculite while stored seeds (which presumably had
not rehydrated during storage, as silica gel maintained its blue color) were allowed to
equilibrate to ambient temperature and humidity for some hours before sowing. Presumably,
humidification prior to sowing could overcome part of that damage. Therefore, it could
be said that Ch. humilis has desiccation-tolerant seeds. Like other Palmaceae species with
desiccation tolerant seeds (Phoenix sp. and Syagrus sp.), the treatment slightly reduced
germination (Pritchard et al., 2004). Those seeds were also desiccated with silica gel and
their moisture content was 6-7% for Phoenix species and 11% for Syagrus botryophora.
Desiccation lengthened the germination rate in Ch. humilis, similarly to what was observed
in Hyophorbe lagenicaulis (Wood and Pritchard, 2003).
For conservation purposes, very low temperatures should be avoided as storage in
liquid nitrogen (-196°C), at least at the moisture contents studied (ca. 14% and 7%
f.w.basis), was detrimental for the seeds. Date palm seeds could stand liquid nitrogen
temperatures in a moisture content range of 7.8-16.1% and germination did not decrease
after 546 days storage in liquid nitrogen (Al-Madeni and Tisserat, 1986). In ten palm
species, germination of seeds held for 1 h in liquid nitrogen did not decrease respect to
control seeds, and in one species germination was increased (Al-Madeni and Tisserat,
1986). The results observed in our work could support evidence from other studies where
Palmaceae orthodox seeds (Hyphaene thebaica and Medemia argum, both from drylands)
showed some sensitivity to desiccation and/or freezing stress (Davies and Pritchard,
1998). The damage produced by cryopreservation could be related to desiccation below
an optimum equilibrium relative humidity (Pritchard, 1995).
The results of the present study show that Ch. humilis has long-life, orthodox seeds, as
the germination did not decrease in 1.5 years at 15°C storage and after desiccated storage
at sub-zero temperature (-18°C). However, further work is required to clarify best seed
moisture content for storage in liquid nitrogen.
Acknowledgements
Financial support for this research was provided by the Project RF00-019-C6-D5 of
the Ministerio de Ciencia y Tecnología, Spain. Authors thank Anne Pinder for her help
collecting seeds.

150
M.E. GONZÁLEZ-BENITO, M. HUERTAS-MICÓ AND F. PÉREZ-GARCÍA
References
Al-Madeni, M.A. and Tisserat, B. (1996). Survival of palm seeds under cryogenic conditions. Seed Science and
Technology 14, 79–85.
Cañizo, J.A. del (2002). Palmeras. Ed.Mundi-Prensa, Madrid.
Davies, R. and Pritchard, H.W. (1998). Seed storage and germination of the palms Hyphaene thebaica, H.
petersiana and Medemia argun. Seed Science and Technology 26, 823–828.
Ellis, R.H., Hong, T.D. and Roberts, E.H. (1985). Handbook of seed technology for genebanks, Vol. II
Compendium of specific germination information and test recommendations. IBPGR, Rome.
Ellis, R.H., Hong, T.D. and Roberts, E.H. (1990). Effect of moisture content and method of rehydration on the
susceptibility of pea seeds to imbibition damage. Seed Science and Technology 18, 131–137.
Fenner, M. (1991). The effects of the parent environment on seed germinability. Seed Science Research 1,
75–84.
Hong, T.D., Linington, S. and Ellis, R.H. (1996). Compendium of information on seed storage behaviour.
International Plant Genetic Resources Institute, Rome.
ISTA (2003). International rules for seed testing. The International Seed Testing Association, Bassersdorf,
Switzerland.
León, N.J. de. (1961). Viability of palm seeds. The American Horticultural Magazine 40, 131–132.
Merlo, M.E., Alemán, M.M., Cabello, J. and Peñas, J. 1993. On the Mediterrranean fan palm (Chamaerops
humilis). Principes 37, 151–158.
Meyer, S.E. and Allen, P.S. (1999). Ecological genetics of seed germination regulation in Bromus tectorum L.:
I. Phenotypic variance among and within populations. Oecologia 120, 27–34.
Pérez-García, F. Iriondo, J.M., González-Benito, M.E., Carnes, L.F-, Tapia, J., Prieto, C., Plaza, R., Pérez,
C. (1995). Germination studies in endemic plant species of the Iberian Peninsula. Israel Journal of Plant
Sciences 43, 239–247
Piotto, B. and Noi, A. di (eds). (2003). Seed propagation of Mediterranean trees and shrubs. Agency for the
Protection of the Environment and Technical Services, Rome.
Pritchard, H.W. (1995). Cryopreservation of seeds. In: Methods in molecular biology: Cryopreservation and
freeze-drying protocols (eds. J.G. Day and R. McLellan), pp. 133-144, Humana Press Inc., New Jersey.
Pritchard, H.W., Wood, C.B., Hodges, S. and Vautier, H.J. (2004). 100-seed test for desiccation tolerance and
germination: a case study on eight tropical palm species. Seed Science and Technology 32, 393–403.
Thanos, C.A., Kadis, C.C. and Skarou, F. (1995). Ecophysiology of germination in the aromatic plants thyme,
savory and oregeno (Labiatae). Seed Science Research 5, 161–170.
Wood, C.B. and Pritchard, H.W. (2003). Germination characteristics of fresh and dried Hyphorbe lagenicaulis
seeds. Palms 47, 45–50.