Conservation of seeds of Crimean endemic species Alyssum calycocarpum Rupr. (Brassicaceae) and Anthyllis biebersteiniana Popl. (Fabaceae) in liquid nitrogen (–196° C)
Preservation ex situ of the seeds of Alyssum calycocarpum Rupr. (Brassicaceae) and Anthyllis biebersteiniana Popl. (Fabaceae), species endemic to the Crimean flora, has been studied using modern cryotechnique. The seeds survived after having been exposed to ultra low temperature (–196°C) during a...
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Інститут ботаніки ім. М.Г. Холодного НАН України
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irk-123456789-1747092021-01-28T01:27:10Z Conservation of seeds of Crimean endemic species Alyssum calycocarpum Rupr. (Brassicaceae) and Anthyllis biebersteiniana Popl. (Fabaceae) in liquid nitrogen (–196° C) Arapetyan, E.R. Судинні рослини: систематика, географія, флора Preservation ex situ of the seeds of Alyssum calycocarpum Rupr. (Brassicaceae) and Anthyllis biebersteiniana Popl. (Fabaceae), species endemic to the Crimean flora, has been studied using modern cryotechnique. The seeds survived after having been exposed to ultra low temperature (–196°C) during a month. Зберігання ex situ насіння Alyssum calycocarpum Rupr. (Brassicaceae) і Anthyllis biebersteiniana Popl. (Fabaceae), ендемічних видів флори Криму, досліджували з використанням сучасного методу кріотехнології. Насіння не втратило життєздатності після перебування його протягом місяця у рідкому азоті. Хранение ex situ семян Alyssum calycocarpum Rupr. (Brassicaceae) і Anthyllis biebersteiniana Popl. (Fabaceae), эндемических видов флоры Крыма, исследовали с использованием современной криотехнологии. Семена сохранили жизнеспособность после хранения их в течении месяця в жидком азоте. 2013 Article Conservation of seeds of Crimean endemic species Alyssum calycocarpum Rupr. (Brassicaceae) and Anthyllis biebersteiniana Popl. (Fabaceae) in liquid nitrogen (–196° С) / E.R. Arapetyan // Український ботанічний журнал. — 2013. — Т. 70, № 6. — С. 747-750. — Бібліогр.: 20 назв. — англ. 0372-4123 http://dspace.nbuv.gov.ua/handle/123456789/174709 en Український ботанічний журнал Інститут ботаніки ім. М.Г. Холодного НАН України |
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Судинні рослини: систематика, географія, флора Судинні рослини: систематика, географія, флора |
| spellingShingle |
Судинні рослини: систематика, географія, флора Судинні рослини: систематика, географія, флора Arapetyan, E.R. Conservation of seeds of Crimean endemic species Alyssum calycocarpum Rupr. (Brassicaceae) and Anthyllis biebersteiniana Popl. (Fabaceae) in liquid nitrogen (–196° C) Український ботанічний журнал |
| description |
Preservation ex situ of the seeds of Alyssum calycocarpum Rupr.
(Brassicaceae) and Anthyllis biebersteiniana Popl. (Fabaceae),
species endemic to the Crimean flora, has been studied using
modern cryotechnique. The seeds survived after having been exposed to ultra low temperature (–196°C) during a month. |
| format |
Article |
| author |
Arapetyan, E.R. |
| author_facet |
Arapetyan, E.R. |
| author_sort |
Arapetyan, E.R. |
| title |
Conservation of seeds of Crimean endemic species Alyssum calycocarpum Rupr. (Brassicaceae) and Anthyllis biebersteiniana Popl. (Fabaceae) in liquid nitrogen (–196° C) |
| title_short |
Conservation of seeds of Crimean endemic species Alyssum calycocarpum Rupr. (Brassicaceae) and Anthyllis biebersteiniana Popl. (Fabaceae) in liquid nitrogen (–196° C) |
| title_full |
Conservation of seeds of Crimean endemic species Alyssum calycocarpum Rupr. (Brassicaceae) and Anthyllis biebersteiniana Popl. (Fabaceae) in liquid nitrogen (–196° C) |
| title_fullStr |
Conservation of seeds of Crimean endemic species Alyssum calycocarpum Rupr. (Brassicaceae) and Anthyllis biebersteiniana Popl. (Fabaceae) in liquid nitrogen (–196° C) |
| title_full_unstemmed |
Conservation of seeds of Crimean endemic species Alyssum calycocarpum Rupr. (Brassicaceae) and Anthyllis biebersteiniana Popl. (Fabaceae) in liquid nitrogen (–196° C) |
| title_sort |
conservation of seeds of crimean endemic species alyssum calycocarpum rupr. (brassicaceae) and anthyllis biebersteiniana popl. (fabaceae) in liquid nitrogen (–196° c) |
| publisher |
Інститут ботаніки ім. М.Г. Холодного НАН України |
| publishDate |
2013 |
| topic_facet |
Судинні рослини: систематика, географія, флора |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/174709 |
| citation_txt |
Conservation of seeds of Crimean endemic species Alyssum calycocarpum Rupr. (Brassicaceae) and Anthyllis biebersteiniana Popl. (Fabaceae) in liquid nitrogen (–196° С) / E.R. Arapetyan // Український ботанічний журнал. — 2013. — Т. 70, № 6. — С. 747-750. — Бібліогр.: 20 назв. — англ. |
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Український ботанічний журнал |
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2025-07-15T11:45:41Z |
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| fulltext |
747ISSN 0372-4123. Укр. ботан. журн., 2013, т. 70, № 6
E.R. ARAPETYAN
Ivan Franko National University of Lviv
Universitetska St. 1, Lviv, 79000, Ukraine
emarapetyan@gmail.com
CONSERVATION Of SEEDS OF CRIMEAN ENDEMIC SPECIES ALYSSUM CALYCOCARPUM
RUPR. (BRASSICACEAE) AND ANTHYLLIS BIEBERSTEINIANA POPL. (FABACEAE) IN LIQUID
NITROGEN (–196° С)
K e y w o r d s : A l y s s u m сalycocarpum, Anthyllis biebersteiniana, Crimea, endemic species, seeds, liquid
nitrogen, cryopreservation, germination
© E.R. ARAPETYAN
Abstract
Preservation ex situ of the seeds of Alyssum calycocarpum Rupr.
(Brassicaceae) and Anthyllis biebersteiniana Popl. (Fabaceae),
species endemic to the Crimean flora, has been studied using
modern cryotechnique. The seeds survived after having been ex-
posed to ultra low temperature (–196° C) during a month.
Introduction
The importance of the preservation of seeds as germ-
plasm resources of the world flora has been emphasized
already by N.I. Vavilov. Black in his report [15] later
noted that large amounts of experimental data were
generated in the field of seed biology during the recent
years and, as a result, have changed our understanding
of the role of seeds in plant conservation. According to
current scientific data, seeds (in particular, their DNA)
contain all genetic information of the genotype formed
during the process of species evolution. Because of that
seed (sexual) reproduction provides the inheritance of
the whole spectrum of species’ genetic variability as
compared to vegetative (asexual) reproduction. Ac-
cording to international standards, an integral part of
the general principle of plant conservation is preserva-
tion of seeds in ex situ conditions, i.e. in special seed
depositories [12]. The conservation of plant diversity
primarily implies wild species conservation. The aim of
the Millennium Seed Bank project by 2020 is to save
25 % of species of world’s wild plants with bankable
seeds [18].
In Ukraine, the seeds of wild and introduced plants
are collected and preserved mainly at botanical gardens.
As a rule, seeds preservation and storage occurs under
the room temperature. These conditions causes the
decrease or loss of the germination ability of seeds.
The fundamental investigations, including those by
Navashin [10], emphasized that preservation under
these conditions has a negative impact on quality of
certain seeds and leads to their germination decrease.
Experiments have shown that the best method of long-
term storage of plant material to ensure genetic stability
within a long period of time is cryopreservation, e.g.
conservation in liquid nitrogen under –196° C. This
method is considered to be an important and up-to-
date tool for plant conservation, especially for the
seed material that forms national natural resources.
Cryopreservation has several important advantages as
compared to other ex situ conservation techniques [16,
20]. It provides a possibility to protect plant material
against the influence of main adverse abiotic and biotic
factors. The first studies in this field in Ukraine were
conducted by Grishenkova for six Carpathian species
[2]. During the past few years, plant cryopreservation
protocols were developed at the Botanical Garden
of Ivan Franko National University of Lviv [14]. The
research mostly concerned rare and endemic species of
the Ukrainian flora.
Material and Methods
The reported research covers selected rare and narrow-
range taxa of the Crimean native flora. Although the
Crimean Peninsula occupies only 4.2 % of the territory
of Ukraine, it is one of the European hotspots of biodi-
versity. The Crimea is considered to be the richest flo-
ristic region of the country. It has the largest concentra-
tion of endemic species (from ca. 120 to 300, according
to various assessments) [4, 5, 19]. The taxa investigated
in the present study were Alyssum calycocarpum Rupr.
(Brassicaceae) and Anthyllis biebersteiniana Popl. (Fa-
baceae) that were introduced in the University Botani-
cal Garden. The species are perennial plants, and the
data of their prior introduction were absent [7, 11].
Alyssum calycocarpum is believed to be a species
restricted to the Crimean-Novorossiysk region [5]. This
species has the «R» status for the European part of the
former USSR in the IUCN Red List [17], i.e. it is under
748 ISSN 0372-4123. Ukr. Bot. J., 2013, 70(6)
the threat of extinction. A. calycocarpum predominantly
grows on limestone and dry slopes of southern beaches
[9]. Iljinska [6] refers to Ruprecht who named the
species, found A. calycocarpum in many places of the
Crimea, and described its location in the Mountain
Crimea and the Novorossiysk floristic region of the
Caucasus. A. calycocarpum is an ornamental plant.
Its shoots are simple or branching, up to 35 cm tall,
petals are yellow, and the silicles are oval or globular.
Its flowering usually starts in May—June [9]. Under the
introduction conditions, flowering starts in late May
and full flowering — at the beginning of June. Seeds
ripen in July.
Anthyllis biebersteiniana is a narrowly endemic
species connected with the alpine elements of the flora
of the Crimean mountain yailas (treeless mountain
tops) [13]. According to Cherepanov, the species also
occurs in the Caucasus [cit. by 5]. It grows on rocky
areas of the mountain steppes of the Crimea [13] and
on meadow steppes of yailas [9]. The flowering of
A. biebersteiniana occurs in June—July under botanical
garden conditions, and seeds ripen in August—
September. Under the introduction conditions, the
species produces vital seeds that have no resting period,
as for the previous species. An average indicator of
laboratory germination of the freshly collected seeds of
A. biebersteiniana is 46.7 %, and the germination ability
under the room conditions decreases every month [8],
equaling 12 % after the period of 6 months.
Cryopreservation of plant material was conducted
in the Cryoconservation Scientific and Technical
Centre of Low Temperature Studies at Ivan Franko
National University of Lviv. The collected seeds of
experimental species that had been kept for several
months in laboratory conditions were then transferred
to the plastic Eppendorf tubes (50 seeds in a threefold
repetition for each species) and were dipped directly into
liquid nitrogen for one month of conservation under
the temperature of –196° C. After the experiment, the
seeds were warmed under laboratory conditions. Slow
warming was performed under the room temperature
by leaving seeds in the Eppendorf tubes for 3 days. The
control seeds were kept under laboratory conditions.
The seeds were then sown and grown in soil boxes.
The survival rate was estimated as the percentage of
seeds that germinated after freezing and thawing. The
significance of differences between various treatments
was evaluated by special software [3]. The Wellington
method [1] was used for the detection of seedlings
growth and development.
Results
Seeds of Alyssum calycocarpum under investigation
preserved their viability after their conservation in liq-
uid nitrogen for a month. The control seeds (unfrozen
ones) and experimental variants were germinated for
two weeks (Table 1).
The final percentage of seed viability in the control
variant (21.4±6.2) was higher than that for the frozen
seeds (18.7±7.9). As the number of investigated samples
was great (n=150), the unreliability of differences
confirms that the germinating ability of the frozen seeds
did not change as compared to the unfrozen ones. The
opening of cotyledons was registered on the 14th day
of the germination beginning both in the control and
treatment variants. The first leaves appeared on the 20th
day of germination. Storage of seeds in liquid nitrogen
did not affect the growth and development of the
cryopreserved diaspores. The seeds stored at –196°C
had the same appearance and growth patterns as the
unfrozen (control) ones. Seedlings were growing
normally showing the same morphology as the plants
recovered from the unfrozen control seeds. The increase
in size and color intensification of leaves was registered
for both variants.
For Anthyllis biebersteiniana, the same strategy of
seed germination was applied. Both in control and
experimental variants, the seed germination in the
was registered on the 4th day after sowing. However, the
period of seed germination in the control variant was
more prolonged than that of the frozen variant (Table 2).
Table 1. Soil germination dynamics of Alyssum сalycocarpum seeds
after cryostorage
Day of ger-
mination
Control Frozen for 4 weeks
Quantity of
germinating
seeds
Percentage of
germinating
seeds
Quantity of
germinating
seeds
Percentage of
germinating
seeds
8th 4.3±1.3 15.5±4.8 6.7±3.8 13.3±7.7
14th 6.0±1.7 21.4±6.2 9.0±4.6 18.0±9.2
16th 6.0±1.7 21.4±6.2 9.3±3.9 18.7±7.9
Table 2. Soil germination dynamics of Anthyllis biebersteiniana
seeds after cryostorage
Day of ger-
mination
Control Frozen for 4 weeks
Quantity of
germinating
seeds
Percentage of
germinating
seeds
Quantity of
germinating
seeds
Percentage
of germinat-
ing seeds
4th 1.3±0.9 2.7±1.8 1.7±1.2 3.3±2.4
11th 1.3±0.9 2.7±1.8 2.7±1.5 5.3±2.8
14th 3.3±1.3 9.3±1.3 5.7±2.0 11.3±4.1
18th 4.7±1.8 9.8±3.5 - -
749ISSN 0372-4123. Укр. ботан. журн., 2013, т. 70, № 6
The cotyledons appeared earlier in control seedlings
than in the frozen ones. The first leaves appeared three
weeks after sowing, simultaneously in the unfrozen
and frozen variants. However, the seedlings of the
seeds treated by low temperatures were delayed in their
development as compared to the control ones, and were
1—1.5 cm tall while the control ones were 2—3 cm tall.
The seeds of both investigated species,
A. сalycocarpum and A. biebersteiniana, preserved
their germinating capacity (Table 3). The germination
of freezing/thawing treated seeds started at the same
time as the germination of non-treated ones. The
seedlings were developing normally and had the same
morphology as those from the unfrozen control group.
All the seedlings produced green leaves. There were
seedlings with well-formed leaves.
The difference between the percentage of seeds
germination in the control and experimental groups
is not statistically reliable. Thus, seed germination in
the experimental variant changes insignificantly as
compared with the control one.
Conclusion
We report the successful cryopreservation of Alyssum
сalycocarpum and Anthyllis biebersteiniana seeds. These
results showed that ultra low temperature (–196° C) did
not negatively influence the seed germination and seed-
lings growth of seeds of A. сalycocarpum and A. bieber-
steiniana.
As shown in this study, conservation of plant material
using liquid nitrogen is consistent with the requirements
of durable preservation of plant genetic information
without negative changes of their gene structure and
biological characteristics. The method is environment-
friendly and cost-effective. The main practical problem
in cryopreserving plant material is the selection of
freezing and thawing conditions for each plant species.
Our analysis of the experimental data suggests that
seeds of introduced species A. сalycocarpum and
A. biebersteiniana are suitable for conservation in
liquid nitrogen by means of direct immersion. The
investigated seeds preserve their viability without the
use of cryoprotective substances. The cryopreservation
protocols were developed for the seeds of these two taxa.
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Percentage of germinating seeds
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Alyssum сalycocarpum 21.4±6.2 18.7±7.9
Anthyllis biebersteiniana 9.8±3.5 11.3±4.1
750 ISSN 0372-4123. Ukr. Bot. J., 2013, 70(6)
Botanical Congress / Vienna, Austria 17—23 July 2005. —
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Recommended for publication Sibmitted 01.04.2011
by E.L. Kordyum
Е.Р. Арапетьян
Національний університет імені Івана Франка, м. Львів,
Україна
ЗБЕРІГАННЯ НАСІННЯ ЕНДЕМІЧНИХ ВИДІВ ФЛОРИ
КРИМУ ALYSSUM CALYCOCARPUM RUPR. (BRASSICACEAE)
ТА ANTHYLLIS BIEBERSTEINIANA POPL. (FABACEAE) У
РІДКОМУ АЗОТІ (–196О С)
Зберігання ex situ насіння Alyssum calycocarpum Rupr.
(Brassicaceae) і Anthyllis biebersteiniana Popl. (Fabaceae),
ендемічних видів флори Криму, досліджували з
використанням сучасного методу кріотехнології. Насіння не
втратило життєздатності після перебування його протягом
місяця у рідкому азоті.
К л ю ч о в і с л о в а: Alyssum calycocarpum, Anthyllis
biebersteiniana, Крим, ендемічні види, насіння, рідкий азот,
кріозберігання, схожість.
Э.Р. Арапетьян
Национальный университет имени Ивана Франко, г. Львов,
Украина
СОХРАНЕНИЕ СЕМЯН ЭНДЕМИЧЕСКИХ ВИДОВ
ФЛОРЫ КРЫМА ALYSSUM CALYCOCARPUM RUPR.
(BRASSICACEAE) И ANTHYLLIS BIEBERSTEINIANA POPL.
(FABACEAE) В ЖИДКОМ АЗОТЕ (–196° С)
Хранение ex situ семян Alyssum calycocarpum Rupr.
(Brassicaceae) і Anthyllis biebersteiniana Popl. (Fabaceae),
эндемических видов флоры Крыма, исследовали с
использованием современной криотехнологии. Семена
сохранили жизнеспособность после хранения их в течении
месяця в жидком азоте.
К л ю ч е в ы е с л о в а: Alyssum calycocarpum, Anthyllis
biebersteiniana, Крым, эндемические виды, семена, жидкий
азот, криосохранение, всхожесть.
МІЖНАРОДНА НАУКОВО-ПРАКТИЧНА КОНФЕРЕНЦІЯ МОЛОДИХ НАУКОВЦІВ
«ПРОБЛЕМИ ТА ПЕРСПЕКТИВИ ДОСЛІДЖЕНЬ РОСЛИННОГО СВІТУ»
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Контактна інформація
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Нікітський ботанічний сад — Національний науковий центр, смт. Нікіта, м. Ялта, АР Крим, Україна, 98648
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