Biotechnology & Biotechnological Equipment

ISSN: 1310-2818 (Print) 1314-3530 (Online) Journal homepage: https://www.tandfonline.com/loi/tbeq20

Breaking Seed Dormancy of Water Lily (Nymphaea

Alba L.) Under In Vitro Conditons

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

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: https://doi.org/10.2478/V10133-010-0009-3

© 2010 Taylor and Francis Group, LLC

Published online: 15 Apr 2014.

Submit your article to this journal

Article views: 1168

View related articles

Citing articles: 2 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=tbeq20
 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

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 lily’s 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., Multiplication of plants through seeds is an effective mean
seed germination, TDZ, growth regulators, dormancy for conservation of species, as it helps to maintain genetic
variability, which is not possible under asexual methods
Biotechnol. & Biotechnol. Eq. 2010, 24(1), 1582-1586
of propagation through rhizome, a common practice for N.
alba. Production of white water lily through seeds has proved
Introduction difficulties because of the development of dormancy in seeds
White water lily (Nymphaea alba L.), family Nymphaeaceae, with the passage of time (19, 20). This has restricted large scale
synonyms Nymphaea occidentalis, Castalia speciosa (Salisb.), natural distribution of the plant with rapid germplasm erosion.
Castalia alba ((L.)Wood.) is an important aquatic perenniel
The study was designed for development of an efficient
plant that produces handsome, floating foliage along with
in vitro seed germination procedure for the white water lily.
gorgeous, scented, hermaphrodite, self fertile flowers; which
blooms from June to early August in Turkey. It prefers The study examined the importance of various plant growth
warm neutral and basic alkaline water marshes, ponds, slow regulators and the effects of dark and light incubation on the
moving streams, lakes or canals up to 1.2 m deep, under germination of seeds from N. alba.
sunny conditions (5) and cannot grow in the shade (1). It
is an aphrodisiac, anodyne, antiscrophulatic, astringent, Materials and Methods
cardiotonic, demulcent, and sedative. It contains the toxic The seed capsules of white water lily were collected from
alkaloids nupharine and nymphaeine, which are substances the Ulubat lake of the Bursa province, where the plant grows
with an effect on the nervous system (6). abundantly. The seeds were embedded in white gelatin like
In the past these plants were found in almost all slow substances in seed capsules; the latter were removed and the
moving lakes or ponds throughout Turkey, but with the seeds were wiped with a tissue paper and placed in a cool, dry,
increased urbanization, its population is rapidly eroding and shady place for two days to dry. Ten random samples of 20
is now found only in limited number of lakes in the provinces seeds each making a total of 200 seeds were taken from these
of Antalya, Hatay, Izmir, Konya and Bursa (22). This makes and tested for seed viability using 2, 3, 5 triphenyltetrazolium
it clear that if proper and timely measures are not taken to chloride (TTC) (11).
conserve this plant it will become very difficult to save and Thereafter, the seeds were divided into two lots. One lot
conserve it; and it will be completely lost from the Flora of was tested for seed germination immediately and the seeds in
Turkey primarily through habitat destruction. the other lot were packed in porous containers placed in an air
1582 Biotechnol. & Biotechnol. Eq. 24/2010/1
tight container. It had another small porous container in it that version 12.0. Separation of treatments was made by Duncans
contained a rechargeable silica gel, which was blue when dry Multiple Range test. Data given in percentages were subjected
and turned to pink when moisture saturated. Aging was slowed to arcsine (√X) transformation (21) before further statistical
down by keeping the air tight container at 40C for a period of analysis.
five months (150 days). The aim was to aid seed vigor and
maintain the ability to germinate seeds for a much prolonged Results and Discussion
time. The zygotic embryos of 123 out of 200 seeds stained red with
Both scarified (by cutting the seed coat with sharp blades of TTC, and showed viability of 61.5% of the seeds.
surgical scalpel to encourage germination) and unscarified fresh Scarified or unscarified seeds sown in sand, that was
seeds were surface sterilized using 70% commercial bleach submerged in sterile deionised water, contained in magenta
containing 5.5 to 6.5% NaOCl (Ace, Turkey). Thereafter, vessels® (pH 7) incubated in 16 h light or dark failed to
the seeds were germinated by sowing (1) in sterilized sand germinate even after 5 months.
contained in magenta boxes that were submerged in sterilized, TABLE 1
deionised water at pH 7.0. Following was an incubation in 16 h
Effects of different concentrations of sucrose on seed
light or dark photoperiod at 24±20C (2), on liquid MS medium
germination of N. alba. L.
(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 of sugar Frequency of seed
concentrations and combinations of BAP and IAA, both (g/l) germination(%)*
submerged in sterilized, deionised water at pH 7.0 (Table 1). 30 26.65 b
The seeds from the second lot, kept at 40C were taken out 25 40.00 ab
after 5 months and both scarified and unscarified seeds from 20 20.25 b
this lot were germinated using aforementioned procedures 15 40.55 ab
and: 0.5 to 16 mg/l GA3, 0.5 to 16 mg/l KNO3, 0.25 to 4 mg/l 10 33.40 b
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 1
Each value is the mean of 20 replications with 10 seeds
were prepared according to the recommendations of the 2
Values within a column followed by different letters are significantly different
manufacturer. All seed germination experiments were carried at 0.05 level of significance using Duncans Multiple Range Test
out in an incubator at 24±20C under 16 h light and 8 h dark No seed germination was recorded from unscarified seeds
photoperiod or complete darkness. in liquid MS medium that contained 10-30 g/l sucrose. Only
All experiments contained 20 replications with 10 seeds in 5% of the scarified seeds showed seed germination when
each replication (20 x 10=200 seeds). Data were recorded and grown on liquid MS medium that contained 15 g/l sucrose.
subjected to the statistical analysis using SPSS for Windows However, the obtained results from agar solidified MS medium,
TABLE 2
Effects of different concentrations of BAP-IAA on seed germination of N. alba L.

Seed germination on agar solidified MS medium

Frequency of seed germination(%)*
BAP (mg/l) IAA (mg/l) Sucrose (g/l)
1 - 30 20.001 ab2
1 0.1 30 60.20 a
1 0.2 30 20.45 b
2 - 30 33.35 b
2 0.1 30 6.70 c
2 0.2 30 41.10 ab
3 - 30 0.00 d
3 0.1 30 20.75 b
3 0.2 30 33.35 b
4 - 30 33.50 b
4 0.1 30 40.65 ab
4 0.2 30 26.75 b
Each value is the mean of 20 replications with 10 seeds
1

Values within a column followed by different letters are significantly different at 0.05 level of significance using Duncans Multiple Range Test
2

Biotechnol. & Biotechnol. Eq. 24/2010/1 1583

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).
Biotechnol. & Biotechnol. Eq. 24/2010/1
 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 after-
ripening 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 thermo-
inhibition 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
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, 57-
63.
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/
Biotechnol. & Biotechnol. Eq. 24/2010/1