B I O D I V E R S I T A S ISSN: 1412-033X
Volume 6, Nomor 1 Januari 2005
Halaman: 1-5
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Storage and the Use of Peroxydase Enzyme to Detect
Germination Capability of Sandoricum koetjape Merr. Seeds-
A Neglected Tropical Fruit Species
USEP SOETISNA
♥
, DODY PRIADI, SRI HARTATI, ENNY SUDARMONOWATI
Research Centre for Biotechnology, The Indonesian Institute of Sciences (LIPI), Cibinong-Bogor 16911.
Received: 17 September 2004. Accepted: 14 October 2004.
ABSTRACT
Sandoricum koetjape which belongs to the group of mahogany, possesses seeds with sticky white aril, is a neglected local fruit species that
might extinct if conservation efforts are not made. Besides preserving the embryos and embryonic axis on different periods of storage (0, 3,
5, 7 days) on a vacuum glass container containing silica gel, the latter organs were also preserved in liquid nitrogen to study the possibility
of long-term storage. The water content of the preserved organs was measured in relation to the length of storage and the germination rate.
To determine the role of peroxidase in the germination rate of preserved zygotic embryos, the level of peroxidase was measured. Seeds of
control and of 3-day storage were mostly germinated at day-6. The average rate of germination was reduced to 23.33% when the seeds
were desiccated with silica gel for 7 days which resulted in 27.69% water content. This germination capability and the length of hypocotyls
seem to correlate with peroxidase activity in the seeds. In general, the higher the percentage of germination, the longer the length of
hypocotyls, the higher the peroxidase activity, except for seeds desiccated for 7 days. The range of peroxidase activity was 6.81-3856.20
ΔA/2min/mg. When the seeds were desiccated for 7 days, they still could germinate at day-18 which indicated by a very high peroxidase
activity. Peroxidase activity assay could detect the viability within 15 seconds while the TTZ requires 15 minutes. Although the highest
percentage of survived embryonic axis after storage in liquid nitrogen was only 23.42%, the results showed that soaking in 10-20% DMSO
for 20 minutes of prerequisite as without DMSO led to no survival. These results offer an alternative procedure to detect the germination
ability of seeds at early stage and longer period of preservation which could contribute to future ex situ conservation.
© 2005 Jurusan Biologi FMIPA UNS Surakarta
Keywords: recalcitrant, seeds, embryos, desiccation, preservation, peroxidase, Sandoricum koetjape.
INTRODUCTION
Sandoricum which belongs to the family of Meliaceae
comprises 5 species, four of which are restricted to western
Malesia. The fifth, S. koetjape, is commonly cultivated
mainly for its fruit and frequently naturalized from India,
Burma (Myanmar) and Indo-China to Thailand, the whole of
Malesian region, and tropical Australia and even in the New
World Tropics. Timber plantations of S. koetjape have been
established in Burma (Myanmar). The wood of this species
is used for house construction, furniture, cabinet work,
joinery, interior construction, shop fitting, paneling, planking
and decking of boats, scantlings, carvings, butcher
chopping blocks, packing cases, household implements,
and sandals. The wood is also used for the production of
veneer and plywood, blackboard, and for pulp and paper. It
yields a good-quality charcoal and is used as firewood in
Indonesia. It yields a lightweight to medium-weight
hardwood with a density of 290-590 kg/m
3
at 15% moisture
content. The texture is moderately fine to slightly coarse
and even. In Europe, the timber has been applied for
furniture and interior finishing. In Malaysia, the timber is
traded in mixed consignments of medium-weight hardwood.
Thailand which partly exports to Great Britain, considers
this timber is potential (Sosef et al., 1998).
This species is a well-known fruit tree, the fruits being
eaten fresh or processed into jam and chutney. The fruits of
the other Sandoricum are also edible but less palatable. S.
koetjape is also an excellent shade tree with ornamental
value, is planted as an avenue tree, and is suitable for use
in shelter-belts. It has medicinal use as its pounded
leaves are sudorific when applied to the skin and are used
to make a decoction against diarrhea and fever. The
powdered bark is an effective treatment for ringworm,
shows anti-cancer activity, and has been used for tanning
fishing nets. The roots are employed as an anti-diarrheic,
anti-spasmodic, carminative, stomachic and are prescribed
as a general tonic after childbirth. Limonoids isolated from
the seeds showed insecticidal activity (Sosef et al., 1998).
It is semi-deciduous, small to large trees, up to 45-50 m
tall and occurs scattered in primary or sometimes
secondary rain forests, up to 1200 m altitude. S. koetjape
has been reported from lowland dipterocarps forest but also
from kerangas on podzolic soils in both perhumid and
seasonal climates. It can be propagated by seed, but also
by vegetative means like budding, grafting, inarching and
marcotting. Seeds however, can not be stored for any
length of time. The seed with or without the adhering pulp
have 90-95% germination in 16-31 days. Various cultivars
of this fruit tree exist including the tetraploid ones. Important
tree collections are held in the Philippines, Malaysia and
Thailand (Sosef et al., 1998).
There are three main categories of seed storage that
have been recognized. Roberts (1973) defined orthodox
and recalcitrant seeds, as those that survive long term dry
storage and those that can not withstand dehydration,
DOI: 10.13057/biodiv/d060101
BIODIVERSITAS Vol. 6, No. 1, Januari 2005, hal. 1-5
2
respectively. More recently, a third category, intermediate,
was identified comprising seeds that can withstand
dehydration to a certain extent but have reduced longevity.
The first one could be further dried to low moisture contents
(< 5%) without losing viability. The second one are
characterized by the absence of maturation drying and are
shed at moisture contents > 50% (on a Fresh Weight basis).
The so-called intermediate seeds survive drying to
moderately low moisture contents (8-10%) but are often
injured by low temperatures (Ellis, 1991).
Zygotic embryos/embryonic axes have been quite
widely used in attempts at conserving germplasm of plants
with recalcitrant seeds or those with seeds in the
intermediate category. Cryopreservation system which is
based on the reduction and subsequent detention of the
metabolic function, including the cellular division of the
explants, is accomplished when materials are brought to the
temperature of liquid nitrogen (-196°C). Successful
cryopreservation of plant material should be achievable by
the appropriate balance between tissue water content and
freezing rate making the use of cryoprotectants a secondary
consideration. Thus, flash-drying and very fast freezing rate
could be a solution to achieving successful cryopreservation
method as suggested by Berjak et al. (1989).
Peroxidase has been implicated in a various
physiological processes in plants. Plant peroxidases have
been related to several processes such as cell growth,
lignin biosynthesis (Gross, 1977), auxin metabolism
(Gaspar, 1986), disease resistance, and wound healing
(Espelie et al., 1986). Research concerning plant
peroxidase activity has been reported in several species
such as in mung bean (Chabanet et al., 1993), peanut
(Zheng and van Huystee, 1992) and in pedicel of grape
(Perez and Gomez, 1998).
The objectives of the study were to investigate possible
storage method for seeds and embryonic axes and to
determine alternative assay employing peroxidase to detect
the viability of seeds of S. koetjapi.
MATERIALS AND METHODS
Source of plant materials
Seeds were collected from two mature trees grown in
the Germplasm Garden of Research Centre for
Biotechnology-Indonesian Institute of Sciences in Cibinong,
Bogor District, West Java, Indonesia. The trees were
originated from seeds which were collected from Serpong,
Banten Province. The size of the fruits from where the
seeds and embryonic axis were excised was ranging from
5.5 cm (small fruits) to 8.0 cm (larger fruits) in diameter in
average. The aril was removed from the seeds by scrapping
with ash washed and then mixed with fungicide Dithane and
followed with natural drying for 1 hour.
Water content determination and germination test
Fifteen seeds were placed in a container in a desiccator
which also contains 200 g silica gel and kept for either 3, 5
or 7 days. The number of replicates was three. Water
content was measured on day 0 and 3-7 days after storage
by weighing before and after drying in the oven at 100
o
C for
5 hours. Germination was conducted by placing the seeds
on a wetted tissue placed in a germinator at 37
o
C.
Maintenance was conducted by spraying the tissues
containing samples with water every day.
Growth of seeds after desiccation in growing media
Having been maintained in a germinator for one month
during the germination test, the desiccated seeds were
sown either in the soil or in the sand placed in a plastic tray.
They were watered every other day and maintained at room
temperature.
Cryopreservation of embryonic axis
Embryonic axes of S. koetjape were soaked for 20
minutes in either 10% or 20% DMSO (dimethyl-sulfoxide) or
6% sucrose. Prior to freezing, they were placed in cryo
tubes and followed with 2 drops of each related
cryoprotectant solution. After thawing, the axes were
germinated in wetted tissue and watered every day.
Viability test using peroxidase
Enzyme extraction. Enzyme extraction was carried out
by grinding approximately 0.02 g to 0.1 g embryonic axis
with 1 ml extraction buffer consisting of 25 mM Tris-HCl pH
7.5 using a plastic grinder. The homogenate was
centrifuged at 10,000 rpm at 4
o
C for 15 minutes. The
supernatant was used as crude peroxidase.
Protein determination. Protein content was determined
with Bradford solution using the method developed by Grey
(1990) using Bovine Serum Albumin as a standard.
Enzyme assay. Total peroxidase activity was estimated
colorimetrically according to Campbell and Ellis (1992). The
activity was determined by adding 720 μl of 50 mM
potassium phosphate pH 7.0 to 80 μl enzyme extract and
2000 μl of 0.5% guaiacol. The reaction was initiated by
adding 2000 μl of 0.075% hydrogen peroxidase followed by
rapid inversion. The absorbance of the mixture was
monitored at 470 nm after 2 minutes. Peroxidase activity
was expressed as ΔA/2min/mg protein.
Qualitative viability test
Tetrazolium test was conducted by applying 23.4 mM
TTC solution in 0.05 M phosphate buffer pH 7.5 to both
half-cut seeds and embryonic axis. Both germinated and
non germinated ones were soaked in the solution for 15 to
60 minutes. Peroxidase solution used was the same as that
for quantitative assay. Both materials (half-cut seeds and
embryonic axis of germinated and non-germinated) were
either soaked in 1 ml solution or given 20 μl drop of
solution.
RESULTS AND DISCUSSION
Critical water content and germination pattern
Water content of seeds before desiccation was 44.63%
which led to 86.67% germination in average. Desiccating
the seeds for 3 days (35.20% moisture content) did not
seem to have an effect on the germination rate as 90% of
seeds still could germinate. Reducing the water content to
31.19% has caused the germinated rate declined 56.67%
which then dropped to 23.33% when the water content was
reduced to 27.69% which was achieved by 7 days
desiccation. The reduction of the water content not only
caused the decline of the germination rate but also the
delay in the germination. Without desiccation, seeds would
germinate within 6 days while those subjected to
desiccation germinated in 10-18 days depending on the
water content (Table 1.). Figure 1. shows the effect of
desiccation period on water content and germination rate of
S. koetjape seeds.
SOETISNA et al. – Germination of Sandoricum koetjape
3
Table 1. Effect of desiccation period and water content on
germination rate of S. koetjape seeds.
Desiccation
period
(days)
Moisture
content
(%)
Average
germination
rate (%)*
Peak of
germination
time (days)
0
3
5
7
44.63a
35.20ab
31.19ab
27.69b
86.67a
90.00a
56.67ab
23.33b
6
10
14
18
Note: * Observation was conducted at day-20. Figures followed
with different letters indicate significant different at 1% based on
Duncan Multiple Range Test.
Statistical analysis on the relation between water
content and the percentage of germinated seeds showed
that 7 days desiccation has reduced the germination rate
significantly as the reduction of water content dropped from
44.6 to 27.7% (Table 1).
0
10
20
30
40
50
60
70
80
90
100
0357
Desiccation Period (days)
Figure 1. Effect of desiccation period on water content and
germination rate of S. koetjape seeds.
The range of water content level which is considered
high in order to have high germination rate suggests that
this species could be grouped either intermediate or
recalcitrant which requires further confirmation through
series of research in the future. Critical water content of
seeds under this category is varied so preliminary
information has to be searched supported with experiments
prior to drying or desiccation. In certain recalcitrant seeds,
the reduction rate of humidity affected further storage.
Azadirachta indica of Thailand provenance could maintain
viability over 60% after 6 weeks storage if the seeds were
subjected to sun drying for 2-3 days (Chaisurisri et al.,
1986). Seeds of one plant species could behave differently
as reported in neem. This plant seed has been designated
as being recalcitrant (Ezumah, 1986) but also as orthodox
(Tompsett, 1994), while according to Sacande et al. (1997)
the seeds had intermediate storage behavior and were cold
sensitive. The viable seed showed red color after treated by
TTC solution (Figure 2.).
Effect of desiccation and water content on germination in
growing media
The desiccated seeds could still grow and produce roots
and shoots after 64-72 days depending on the desiccation
treatments and growing media. Most seeds could germinate
in the soil regardless desiccation treatments given as the
percentage of seeds producing roots was ranging from 89.9
-100%. Unlike in the soil, those planted in the sand was
ranging from 0-100% depending on desiccation periods
(Table 2.). Although the germination percentage in the sand
was lower than in the soil, the number of the roots was
higher and they were longer in size (Figure 3.). Sand also
resulted shoots to emerge and elongate at 35 days after
planting (64-72 days after desiccation and viability test in a
germinator). Figure 4 shows the shoot growth of S. koetjape
seeds which have been desiccated for 5 days at 2 months
after sewing in sand placed in a plastic container. Those
planted in the soil produced shoot slower as the shoots
emerged not earlier than 55 days after planting. This
indicates that seeds of S. koetjape were still viable for more
than 2 months as long as the growing media is appropriate.
The fact that the growth of roots was excessive which
tends to repress the growth of shoots, suggest that during
germination a growth regulator to stimulate the growth of
shoots such as GA
3
is required.
Table 2. Effect of desiccation period and growing media on the
production of roots and shoots of planted seeds of S. koetjape.
Desiccation
Period
(days)
Growing
media
Seeds
producing
roots (%)
Number
of roots
per
seeds*
Seeds
producing
shoots
(%)
Length
of
shoots
(cm)*
0
3
5
7
Soil 100
100
89.9
100
5
16.5
11.5
15
0
16.7
14.6
0
-
3.0**
1.0**
0.5**
0
3
5
7
Sand 100
50
83.3
0
50
14
19
-
100
16.7
33.3
0
3.0
1.0
4.0
-
Note: *Observation at day 61 after sewing. ** All leaves were not
yet opened.
Cryopreservation of embryonic axis
Embryonic axis which was not treated with DMSO or
high sucrose (control) could not survive after storage in
liquid nitrogen for 6 days. It seems that the effect of the
concentration of DMSO was different to different types of S.
koetjape. The survival rates were 19.67% and 23.42%
which seeds were treated with either 10 or 20% DMSO,
respectively. DMSO seems to be more effective than 6%
sucrose as the percentage of survival was slightly higher
i.e. 23.42% and 10%, respectively (Table 3.).
The results which showed very low survival, suggest
that treatments applied were not yet optimum. DMSO alone
did not seem to be sufficient to protect embryonic axis to
withstand ultra low temperature especially when rapid/direct
freezing in liquid nitrogen is applied. Combination of DMSO
with other cryoprotectants as has been applied to other
plant species might solve the problems. Vitrification or
encapsulation dehydration might be one alternative method
for preserving embryonic axis of S. koetjape. Vitrification
solution which employs 30% glycerol, 15% ethylene glycol,
and 15% DMSO in liquid medium with 0.4 M sucrose could
result in up to 80% survival of tropical tree species tried
after immersion in liquid nitrogen (Sudarmonowati, 2000).
Other cryopreservation protocol has been developed mostly
for various plant species but mainly temperate species such
as Allium cepa (Lakhanpaul et al., 1996). The highest
percentage (62.5%) of surviving embryos of citrus “Garut”
after storage in liquid nitrogen was obtained by dehydrating
the naked embryos for 4 hours in laminar air flow and
soaked in vitrification solution (a mixture between 0.8 M
sucrose and 1.0 M glycerol) for 18 hours (Sudarmonowati et
al., 1998). In other plant species such as Ribes nigrum
(Benson et al., 1996), encapsulation-dehydration has given
a better result than vitrification as it gave a higher survival.
germination rate
water content
BIODIVERSITAS Vol. 6, No. 1, Januari 2005, hal. 1-5
4
On the other hand, embryo axes of “Garut” citrus and
longan fruit were more suitable with vitrification technique
for preservation. This might be because the composition of
vitrification solution tried (0.8 M sucrose + 0.1 M glicerol +
200 ppm citric acid) might not be sufficient. Other causes
might be the high content of phenolic compounds in the
zygotic embryos which triggered with the would due to the
formation of ice crystal which causes the phenolic
compounds accumulated. As different species seems to
require different technique, optimum one for each species
needs to be developed.
Water content of seeds which embryonic axis was
excised was still around 44.63% which considered still high.
Reduction of water content to the optimum one is, therefore,
needed which implies that a technique for a more
appropriate dehydration needs to be optimized. Storage of
the axis at -196
o
C has reduced the survival; further study is
required to optimize the desiccation and freezing
procedures. Embryonic axis will still be used as the use of
this intact organ has the advantage that if cryopreservation
procedures are successful, it should merely be a matter of
manipulating post-freezing conditions to produce vigorous
plantlets.
Peroxidase for detecting viability of stored seeds
The level of peroxidase declined in line with the
reduction of germination rate, although in certain cases it
was deviated. The highest level of peroxidase which was
approaching 4000 U was obtained from seeds that have
been desiccated for 7 days (Figure 5.).
0
500
1000
1500
2000
2500
3000
3500
4000
0357
Desiccation Period (days)
1a
1b
1c
2a
2b
2c
3a
3b
3c
Figure 5. Peroxidase activity of S. koetjape seeds after desiccation
up to 7 days.
0
10
20
30
40
50
60
70
80
90
100
Hyocotyl Length (mm) or Percentage of Seeds
Showing Peroxidase over 1000 U
0357
Desiccation Period (days)
1a Peroxidase ov er 1000 U
1b 1c
2a 2b
2c 3a
3b 3c
Figure 6. Correlation between desiccation periods, hypocotyls
length, and the percentage of germinated seeds of S. koetjape
showing over 1000 U peroxidase level.
This treatment also led to the highest percentage of
seeds containing peroxidase at the level of over 1000 U
(Figure 6.). This enzyme also indicates the ability of rooting
as the highest content was obtained from organs
possessing the longest roots. The higher the hypocotyls of
germinated seeds were indicated by higher peroxidase
Figure 2. The assay of viability using TTC (left to right:
germinated, not yet germinated but viable, dead).
Figure 3. The growth of roots and shoots from seeds of S.
koetjape which have been desiccated for 0, 3, 5 and 7 days (left to
right)
Figure 4. The shoot growth of S. koetjape seeds which have been
desiccated for 5 days at 2 months after sewing in sand placed in a
plastic container.
SOETISNA et al. – Germination of Sandoricum koetjape
5
level. Peroxidase is considered to be the main enzyme
responsible for the catabolism of the phytohormone IAA in
higher plants, thereby, the suggestion of its participation in
the regulation of plant growth as quoted by Zheng and van
Huystee (1992). In terms of IAA catabolism, it can proceed
in the absence (IAA oxidase) or presence (IAA peroxidase)
of H
2
O
2
. The IAA oxidase and peroxidase catalyzed IAA
catabolism was considered as part of auxin regulation in
vivo, hence, a part of growth regulation. The correlation
between auxin, in this case, IAA with peroxidase content
has been proven in peanut as peroxidase content was
twofold in hypocotyls cultured on medium containing high
IAA (4 mg/l) as compared to control (Zheng and van
Huystee, 1992). The fact that the growth of roots was
considered excessive in seeds of S. koetjape after
desiccation which resulted in the repression of shoot
emergence indicated that the concentration of IAA is high
which related to a high peroxidase level.
This result suggests that not only the viability, the length
of hypocotyls of germinated seeds seems to correlate with
the level of peroxidase in the seeds. The length of
hypocotyls of germinated seeds desiccated for 3 days
(35.2% of water content) was almost uniform while the
others varied. In general, the activity of peroxidase was
higher in non desiccated seeds and lower when the
exposure was longer up to 5 days. In mung bean, the
decrease of growth rate of hypocotyls was in parallel with
the loss of cell wall extensibility which suggests changes in
cell wall structure. The cessation of growth might result from
cell wall stiffening processes related to the integration of
diphenyl phenolic cross-linked wall polymer subunits into
the polysaccharide network which is thought to be catalyzed
by specific cell wall peroxidases. It was noted that in mung
bean, the development of peroxidase activities in epidermal
cell wall just at the onset of growth decrease (Cabhanet et
al., 1993). This might explain the increase of peroxidase level
when the seeds of S. koetjape were desiccated for 7 days.
Giving the peroxidase solution only 2 drops seems to be
sufficient for detecting the viability of seeds. The time
required using peroxidase for detecting the viability of seeds
was much shorter than using TTZ, i.e. 15 seconds versus
15 minutes. The viable and germinated seeds had result
much darker solution than the non germinated one (Figure
6.). This technique offers an alternative protocol for
detecting the viability of stored tissues as well as other
growth function. The role of peroxidase in plant species has
been reported such as in Glycine max (Gillikin and Graham,
1991) and in peanut (Zheng and van Huystee, 1992).
CONCLUSIONS
Drying seeds up to 35.20% water content could still
maintain the germination rate of S. koetjape to 90%,
although they have been maintained for 2 months. With this
condition, the longest storage period will be investigated in
the future to confirm whether this water content level is the
most optimum one for seed storage. It seems that there
was a correlation between the viability of S. koetjape seeds
and the level of peroxidase in the seeds. In addition,
peroxidase activity might correlate with the growth of this
species seeds. Detection of viability with peroxidase assay
offers an alternative method to others as it could give faster
result. Effort to cryopreserve the seeds or embryonic axis in
the future would provide a long-term storage of this
recalcitrant species. Various factors affecting the success of
this technique, are, therefore, need to be conducted.
ACKNOWLEDGEMENTS
The authors would like to thank the technical assistance
of Ms. Nurchaedar Rahman and Mr. Nanang Taryana. The
assistance of Mr. Jitno Rijadi in the documentation of the
results was greatly acknowledged.
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