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Breaking Seed Dormancy of Water Lily (Nymphaea Alba

L.) Under In Vitro Conditons
a

S. Sumlu , H.H. Atar & K.M. Khawar

a

University of Ankara, Faculty of Agriculture, Department of Fisheries, Ankara, Turkey

University of Ankara, Faculty of Agriculture, Department of Field Crops, Ankara, Turkey

Published online: 15 Apr 2014.

To cite this article: S. Sumlu, H.H. Atar & K.M. Khawar (2010) Breaking Seed Dormancy of Water Lily (Nymphaea Alba L.)
Under In Vitro Conditons, Biotechnology & Biotechnological Equipment, 24:1, 1582-1586, DOI: 10.2478/V10133-010-0009-3
To link to this article: http://dx.doi.org/10.2478/V10133-010-0009-3

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Article

DOI: 10.2478/v10133-010-0009-3

A&EB

BREAKING SEED DORMANCY OF WATER LILY (NYMPHAEA ALBA L.)

UNDER IN VITRO CONDITONS
S. Sumlu1, H.H. Atar1, K.M. Khawar2
1
University of Ankara, Faculty of Agriculture, Department of Fisheries, Ankara, Turkey,
2
University of Ankara, Faculty of Agriculture, Department of Field Crops, Ankara, Turkey
Correspondence to: Khalid Mahmood Khawar
E-mail: kmkhawar@gmail.com

Downloaded by [University Malaysia Sarawak] at 19:20 12 November 2014

ABSTRACT

Water lily (Nymphaea alba L.) is an important and popular aquatic perenniel plant. It has been used for ornamental and
pharmaceutical purposes in Turkey and various countries. The populations of white water lily have seen rapid erosion in the last
few years due to fast urbanization and industrialization that has produced negative effect on the lilys habitats. It is multiplied
vegetatively primarily through rhizomes, which produce uniform populations. Multiplication of plants through seeds helps to
maintain genetic variability that could be easily used to preserve the species in an effective way. Multiplication of plants through
seeds is difficult due to development of dormancy with the passage of time. The results of the study showed that the fresh seeds of
the species gave highest germination on MS medium containing 1 mg/l BAP + 0.1 mg/l IAA. However, the seeds that were stored
for five months at 40C failed to germinate on medium containing 1 mg/l BAP + 0.1 mg/l IAA, either used alone or combined with
sucrose, IBA and GA3 in different concentrations in the germination medium. After five months these seeds could be germinated
only on germination medium that contained 0.05 to 4 mg/l TDZ, with highest germination on 2 mg/l TDZ with germination
frequency of 51.37%. No significant variation on germination was recorded in the light or dark; however, the seed germinated in
the dark produced 2-3 times longer seedlings compared to those germinated under 16 h light photoperiod. This study signifies the
role of TDZ to break the dormancy of N. alba seeds. As such the results indicate that TDZ could be used effectively to propagate
N. alba from seeds which could help to conserve and multiply this plant species at its natural habitat.
Keywords: aquatic plant, white waterlily, Nymphaea alba L.,
seed germination, TDZ, growth regulators, dormancy
Biotechnol. & Biotechnol. Eq. 2010, 24(1), 1582-1586

Introduction

White water lily (Nymphaea alba L.), family Nymphaeaceae,

synonyms Nymphaea occidentalis, Castalia speciosa (Salisb.),
Castalia alba ((L.)Wood.) is an important aquatic perenniel
plant that produces handsome, floating foliage along with
gorgeous, scented, hermaphrodite, self fertile flowers; which
blooms from June to early August in Turkey. It prefers
warm neutral and basic alkaline water marshes, ponds, slow
moving streams, lakes or canals up to 1.2 m deep, under
sunny conditions (5) and cannot grow in the shade (1). It
is an aphrodisiac, anodyne, antiscrophulatic, astringent,
cardiotonic, demulcent, and sedative. It contains the toxic
alkaloids nupharine and nymphaeine, which are substances
with an effect on the nervous system (6).
In the past these plants were found in almost all slow
moving lakes or ponds throughout Turkey, but with the
increased urbanization, its population is rapidly eroding and
is now found only in limited number of lakes in the provinces
of Antalya, Hatay, Izmir, Konya and Bursa (22). This makes
it clear that if proper and timely measures are not taken to
conserve this plant it will become very difficult to save and
conserve it; and it will be completely lost from the Flora of
Turkey primarily through habitat destruction.
1582

Multiplication of plants through seeds is an effective mean

for conservation of species, as it helps to maintain genetic
variability, which is not possible under asexual methods
of propagation through rhizome, a common practice for N.
alba. Production of white water lily through seeds has proved
difficulties because of the development of dormancy in seeds
with the passage of time (19, 20). This has restricted large scale
natural distribution of the plant with rapid germplasm erosion.
The study was designed for development of an efficient
in vitro seed germination procedure for the white water lily.
The study examined the importance of various plant growth
regulators and the effects of dark and light incubation on the
germination of seeds from N. alba.

Materials and Methods

The seed capsules of white water lily were collected from

the Ulubat lake of the Bursa province, where the plant grows
abundantly. The seeds were embedded in white gelatin like
substances in seed capsules; the latter were removed and the
seeds were wiped with a tissue paper and placed in a cool, dry,
shady place for two days to dry. Ten random samples of 20
seeds each making a total of 200 seeds were taken from these
and tested for seed viability using 2, 3, 5 triphenyltetrazolium
chloride (TTC) (11).
Thereafter, the seeds were divided into two lots. One lot
was tested for seed germination immediately and the seeds in
the other lot were packed in porous containers placed in an air
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tight container. It had another small porous container in it that

contained a rechargeable silica gel, which was blue when dry
and turned to pink when moisture saturated. Aging was slowed
down by keeping the air tight container at 40C for a period of
five months (150 days). The aim was to aid seed vigor and
maintain the ability to germinate seeds for a much prolonged
time.
Both scarified (by cutting the seed coat with sharp blades of
surgical scalpel to encourage germination) and unscarified fresh
seeds were surface sterilized using 70% commercial bleach
containing 5.5 to 6.5% NaOCl (Ace, Turkey). Thereafter,
the seeds were germinated by sowing (1) in sterilized sand
contained in magenta boxes that were submerged in sterilized,
deionised water at pH 7.0. Following was an incubation in 16 h
light or dark photoperiod at 2420C (2), on liquid MS medium
(14) or agar solidified MS medium that contained 10-30 g/l
sucrose submerged in sterilized deionised water at pH 7.0 (3)
and on agar solidified MS medium that contained different
concentrations and combinations of BAP and IAA, both
submerged in sterilized, deionised water at pH 7.0 (Table 1).
The seeds from the second lot, kept at 40C were taken out
after 5 months and both scarified and unscarified seeds from
this lot were germinated using aforementioned procedures
and: 0.5 to 16 mg/l GA3, 0.5 to 16 mg/l KNO3, 0.25 to 4 mg/l
IBA, 0.25 to 4 mg/l IAA and 0.05 to 4 mg/l TDZ in sterilized
deionised water (Table 2). All plant growth regulators
were prepared according to the recommendations of the
manufacturer. All seed germination experiments were carried
out in an incubator at 2420C under 16 h light and 8 h dark
photoperiod or complete darkness.
All experiments contained 20 replications with 10 seeds in
each replication (20 x 10=200 seeds). Data were recorded and
subjected to the statistical analysis using SPSS for Windows

version 12.0. Separation of treatments was made by Duncans

Multiple Range test. Data given in percentages were subjected
to arcsine (X) transformation (21) before further statistical
analysis.

Results and Discussion

The zygotic embryos of 123 out of 200 seeds stained red with
TTC, and showed viability of 61.5% of the seeds.
Scarified or unscarified seeds sown in sand, that was
submerged in sterile deionised water, contained in magenta
vessels (pH 7) incubated in 16 h light or dark failed to
germinate even after 5 months.
TABLE 1
Effects of different concentrations of sucrose on seed
germination of N. alba. L.
Concentrations of sugar
(g/l)

Frequency of seed
germination(%)*

30
25
20
15
10

26.65 b
40.00 ab
20.25 b
40.55 ab
33.40 b

Each value is the mean of 20 replications with 10 seeds

Values within a column followed by different letters are significantly different

at 0.05 level of significance using Duncans Multiple Range Test

No seed germination was recorded from unscarified seeds

in liquid MS medium that contained 10-30 g/l sucrose. Only
5% of the scarified seeds showed seed germination when
grown on liquid MS medium that contained 15 g/l sucrose.
However, the obtained results from agar solidified MS medium,

Effects of different concentrations of BAP-IAA on seed germination of N. alba L.

Seed germination on agar solidified MS medium
BAP (mg/l)
IAA (mg/l)
Sucrose (g/l)
1
30
1
0.1
30
1
0.2
30
2
30
2
0.1
30
2
0.2
30
3
30
3
0.1
30
3
0.2
30
4
30
4
0.1
30
4
0.2
30

TABLE 2

Frequency of seed germination(%)*

20.001 ab2
60.20 a
20.45 b
33.35 b
6.70 c
41.10 ab
0.00 d
20.75 b
33.35 b
33.50 b
40.65 ab
26.75 b

Each value is the mean of 20 replications with 10 seeds

Values within a column followed by different letters are significantly different at 0.05 level of significance using Duncans Multiple Range Test

Biotechnol. & Biotechnol. Eq. 24/2010/1

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Fig. 1. Seed germination of water lily under in vitro condations (a, c); seed germination under dark (b, d); and 16 h light photoperiod (e); very reduced (5-6%)
malformed germinated plantlets on MS medium containing TDZ submerged in deionised sterilised water (f); acllimatisation of plantlets from dark (g); and 16 h
light germinated seeds; (h) flowering and seed set of plants transferred to open ponds.
Bar: Fig. 1a,b,e = 1.5 cm; Fig. 1c,d = 1.1 cm; Fig. 1f,g = 2 cm; Fig. 1h = 10 cm

that contained 10-30 g/l sucrose, indicated inconsistent

behavior of seed germination, as a range of 20.25-40.55% was
documented, with maximum seed germination on MS medium
that contained 25 g/l sucrose (Table 1).
Germination increased on MS medium containing different
concentrations of BAP-IAA. The results (Table 2) showed that
the highest seed germination (60.20%) was on agar solidified
MS medium that contained 1 mg/l BAP-0.1 mg/l IAA; closely
followed by 41.1% seed germination on 2 mg/l BAP +0.2 mg/l
IAA; and 40.65% seed germination on 4 mg/l BAP-0.1 mg/l
IAA.
Seed germination after 5 months of culture
Initially, randomly selected 200 seeds were stored at 40C
and were tested for their viability using TTC, which showed
55.03% seed viability.
These seeds failed to germinate on all combinations of
plant growth regulators and all levels of sucrose described in
Table 1 and Table 2. The seeds also failed to germinate on
0.5-16 mg/l GA3 and KNO3 used alone. No germination was
recorded on either 0.25- 4 mg/l IAA or IBA as well. However,
0.05 to 4 mg/l TDZ applied to seeds in sterilized, deionised
water and incubated in light, broke the seed dormancy that
resulted in seed germination of 25.80 to 51.37%. The highest
seed germination of 51.37% was recorded for seeds treated
with 2mg/l TDZ. The highest germination from TDZ treatment
was partially in confirmation to the TTC test results. No visible
difference in the rate of germination from the seeds germinated
in the dark was observed as well. However, the seedlings
1584

obtained in dark were more vigorous and one to two times

longer, (4 to 5 cm long) (Fig. 1a, c) compared to those obtained
from the seeds germinated under 16 h light photoperiod (1 to
2 cm long) (Fig. 1b, d). A significant reduction (5-6%) and
malformed germinated plantlets were recorded when MS
medium that contained equivalent concentrations of TDZ was
used (Fig. 1e).
The plants germinated in 16 h light and dark were divided
into two lots each. Each lot of germinated seeds from dark
(Fig. 1f) and 16 h light photoperiod (Fig. 1g) was transferred
to aquariums (4 000 lux light intensity). All plants established
under aquarium conditions but were put under stress that
resulted in death of plants after 2 months in the aquariums.
This was primarily due to insufficient light to carry out
photosynthesis.
The other lot, of seeds germinated in 16 h light and dark,
was potted and extrapolated to open ponds under ambient
conditions of temperature and growth. It was expected that the
TDZ treatment could induce malformations of the seedlings.
On the contrary, irrespective of the germination conditions,
the ambient conditions of temperature and light helped the
plantlets to easily establish. The leaves and stems began to
show visible signs of growth after one week after the transfer.
The plantlets that were transferred to ponds showed vigorous
growth and reached a height of 30-40 cm in two months time,
with no abnormalities (April). All of the plants that were
transferred to ponds set flowers and seeds starting from June to
the end of August (Fig. 1h).
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TABLE 3
Effects of GA3, TDZ, KNO3, IBA and IAA (filter paper
moistened in petri dishes) on germination of 5 months old
seeds of N.alba L.
Seed germination medium
Frequency of seed
in liquid
germination**
TDZ (mg/l)
0.05
45.83 ab
0.1
25.80 bc
0.5
31.00 bc
1
42.56 bc
2
51.37 a
4
45.83 ab
1
Each value is the mean of 20 replications with 10 explants
2
Values within a column followed by different letters are
significantly different at 0.01 level of significance using
Duncans Multiple Range Test
Seed germination and dormancy is controlled by large
number of genes and environmental factors (4, 13). The highest
seed germination (60.20%) of fresh seeds on MS medium that
contained 1 mg/l BAP+ 0.1 mg/l IAA is in conformity to the
TTC test showing seed viability of 61.5%. Other combinations
of growth regulators were inhibitory or less promotive and
failed to induce such level of germination. The decrease in
germination that was observed when other combinations
of plant growth regulators were used may be attributed to
metabolic alterations brought about by plant growth regulators
at the seeds and making them difficult to germinate.
No germination from unscarified seeds was recorded on
liquid that was supplied with 10-30 g/l sucrose primarily due
to hard seed coat dormancy. Reduced germination of scarified
seeds compared to the cytokinin + auxin induced germination
may be due to the development of an osmotically enforced
dormancy by sucrose in the medium. This is partially in
confirmity to the findings of Else et al. (7), who found that
mechanical puncturing of the seed coat of N. odorata did not
affect germination. On the contrary, these researchers found
that stratification at 4.40C for 5 months resulted in germination
of crowded seeds in excess of 90% producing ethylene gas that
promoted their germination. They also found that germination
under conditions of seed crowding was inhibited by darkness
and promoted by stratification. The results are also not in
agreement with Smits (19), who found that the innate dormancy
of the seeds of N. alba could be overcome by a cold treatment.
They found that light stimulated the germination of the seeds.
Else et al. (7) found that fragrant water-lily Nymphaea odorata
seeds were dormant at the time of release without any afterripening requirements.
The seeds that were stored at 40C for 5 months showed
viability of 55.03%, and germination of 51.03% after treatment
with 2 mg/l TDZ. The difference in percentage of germination
and viability might be due to sampling error. The results
emphasize that the seeds undergo dormancy rather than losing
Biotechnol. & Biotechnol. Eq. 24/2010/1

viability, in contradiction to Hay et al. (9), who emphasize that

the seeds of N. alba have a significant desiccation sensitivity
and if dried under ambient humidity will lose their viability after
one week. The results are also not in agreement with Baskin &
Baskin (2) and Hay et al. (9) who use stress cold stratification
as the germination requirement for diverse species of this
genus. Instead, the seeds had developed some sort of chemical
dormancy (para dormancy) caused by germination inhibitors,
which had accumulated in the seeds during cold storage.
Cold storage could end dormancy due to morphologically
immature embryos but not dormancy due to the development
of an inhibitor (4). Another reason for lack of germination in
chilled seeds could be the thermo-inhibition when incubation
in the dark at 40C is performed, rather than reduction in seed
viability. Thermo-inhibition was overcome after treatment with
TDZ in line with Berrie et al. (3) who indicated that thermoinhibition of celery seeds is associated with the accumulation
of a germination inhibitor which interacts with cytokinins and
could be overcome by treatment with red light or aplication
of cytokinins or giberellins. Sinska (18) also found that BA
and GA3 counteracted the inhibitory effect of Aminooxy acetic
acid (AOA) and aminoethoxyvinylglycine (AVG) on embryo
germination. He further emphasised that the effect of ethylene
on the removal of embryonal dormancy is related to the action
of BA but not GA3.
The results suggest further that the effect of Thidiazuron on
removal of dormancy is greater in the dark compared to light.
Although the exact role of TDZ in seed germination is not
clear, it is speculated that the inhibitors that are related to the
development of embryonic dormancy could only be removed
when Thidiazuron is used, which is in confirmation to Sinska
(18). He found that the removal of embryonal dormancy is
related to the action of cytokinin (BA). It is further speculated
that the embryonic axis may have supplied thidiazuron to the
cotyledon more vigorously under dark, soon after imbibation,
which mobilized the reserve materials that are required for
germination, in line with Gepstein & Ilan (8), Munoz et al.
(14), Martin et al. (12) and Pino et al. (16, 17).
The magnitude of shoot formation is regulated by the
intensity of light photoperiod and light quality (10). The
seedlings that were germinated with TDZ and in dark were
one to two times longer and vigorous (Fig. 1c, 4-5 cm long)
compared to those obtained from the seeds germinated under
light (Fig. 1d, 1-2 cm long).
White water lily is light loving plant and cannot grow in
dark, low or diffused light (1). It is hypothesized that the usual
slow growth rate of plants leading to their ultimate death in
aquariums was primarily due to inadequate light insufficient
for proper photosynthesis of white water lilies.
In another study Smits et al. (20) found that under anaerobic
conditions cold stratified seeds of N. alba germinated readily
and released ethanol. They found that germination of seeds
in an ethanol solution (350 mM) was generally stimulated
compared to germination in water, but no significant effect was
recorded if seeds had not received a cold treatment.
1585

Conclusions

The research meets its objectives by developing an efficient

in vitro seed germination procedure for the white water lily
(N. alba). The seed germination protocol that was described in
this report could be used as an effective mean for conservation
of the species by maintaining genetic variability. The treated
seeds could also be sown directly in shallow pond nurseries
from where they could be transplanted/transferred to garden
ponds once they reach proper height.

Acknowledgements

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The authors are thankful to Prof. Sebahattin Ozcan, Department

of Field Crops, Faculty of Agriculture, Ankara University,
Ankara for his help during the studies.

REFERENCES

1. Muhlberg H. (1982) The Complete Guide to Water Plants.

A Reference Book, E. P. Publishing Limited, Germany p.
391.
2. Baskin C.C. and Baskin J.M. (1998) Seeds: ecology,
biogeography, and evolution of dormancy and germination,
Academic Press, London, U.K., p. 666.
3. Berrie A.M.M., Don R., Buller D., Alam A., Parker W.
(1976) Plant Science Letters, 3, 163-173.
4. Bewley J.D. and Black M. (1978). Physiology and
biochemistry of seeds. Volume 1, Springer-Verlag, Berlin,
Heidelberg, New York, pp. 229-241.
5. Chiej R. (1984) Encyclopaedia of Medicinal Plants,
Macdonald, London, p. 85.
6. Chopra R.N., Nayar S.L., Chopra I.C. (1986) Glossary
of Indian Medicinal Plants (Including the Supplement),
Council of Scientific and Industrial Research, New Delhi,
p. 329.
7. Else M.J. and Riemer D.N. (1984) Journal of Aquatic
Plant Management, 22, 22-25.

1586

8. Gepstein S. and Ilan I. (1980) Plant Cell Physiol., 21, 5763.

9. Hay F., Probert R., Marro J., Dawson M. (2000) In:
Seed Biology and applications (M. Black, K.J. Bradford,
J. Vazquez-Ramos, Eds.), CAB International, Wallingford,
UK, p. 161-177.
10. Kadkde P. and Seibert M. (1977) Science, 270, 49-50.
11. ISTA (International Seed Testing Association) (1966)
International rules for seed testing, Proceedings of the
international seed testing physiology, 3, 192-106.
12. Martin L., Diez A., Nicolas G., Villalobos N. (1987) J.
Plant Physiol., 128, 141-151.
13. Mayer A.M. and Poljakoff-Mayber A. (1982) The
germination of seeds. Third edition, Pergamon Press, New
York, p. 211.
14. Munoz J.L., Martin L., Nicolas G., Villalabos N. (1990)
Plant Physiol., 93, 1011-1016.
15. Murashige T. and Skoog F. (1962) Physiol. Plant., 15,
473-497.
16. Pino E., Martin L., Guerra H., Nicolas G., Villalobos N.
(1990) J. Plant Physiol., 135, 698-702.
17. Pino E., Martin L., Guerra H., Nicolas G., Villalobos N.
(1991) J. Plant Physiol., 137, 425-432.
18. Sinska L. (1989) Plant Science, 64, 39-44.
19. Smits A.J.M., van Avesaath P.H., van der Velde G.
(1990) Freshwater Biology, 24, 315-326.
20. Smits A.J.M., Schmitz G.H.W., van der Velde G.,
Voesenek L.A.C.J. (1995) Freshwater Biology, 34, 39-46.
21. Snedecor G.W. and Cochran W.G. (1967) Statistical
Methods, The Iowa State University Press, Iowa, USA, p
327-330.
22. Tubives (Turkiye Bitkileri Veri Servisi) (2009) http://
www.tubitak.gov.tr/tubives/

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