Мікроспорогенез у бобів (Vicia faba L.), вирощених у Мерсіні, Туреччина
Legumes are an alternative to animal foods due to their high protein content. Like other legumes, Vicia faba (faba bean) has high protein content. However, faba bean breeding with classical methods is challenging due to inbreeding depression, self-incompatibility, and abortive embryo formation. The...
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Plant Introduction| _version_ | 1860145132746047488 |
|---|---|
| author | Küçükrecep, Aslı Tekdal, Dilek |
| author_facet | Küçükrecep, Aslı Tekdal, Dilek |
| author_sort | Küçükrecep, Aslı |
| baseUrl_str | https://www.plantintroduction.org/index.php/pi/oai |
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| datestamp_date | 2023-08-26T20:38:45Z |
| description | Legumes are an alternative to animal foods due to their high protein content. Like other legumes, Vicia faba (faba bean) has high protein content. However, faba bean breeding with classical methods is challenging due to inbreeding depression, self-incompatibility, and abortive embryo formation. The inadequacy of classical breeding methods due to the problems in the fertilization biology of the plant limits the production of new varieties. Therefore, the importance of using in vitro haploidization technique in legume breeding is increasing. Anther culture is a widely preferred tissue culture technique for obtaining haploid plants. The initial and most crucial stage in anther culture is the identification of anthers containing microspores in the appropriate developmental stage. In the study, anther samples with single and triple-nucleated microspores were cultured in MS nutrient medium containing 2,4-D (0.5 mg L-1) and Kinetin (2.5 mg L-1). In the first month of culture, embryo and embryoid-like structures were obtained at the heart stage from anthers containing microspores in the mononuclear stage, while anther samples containing microspores in the trinucleated stage were observed to darken and did not develop. |
| doi_str_mv | 10.46341/PI2022017 |
| first_indexed | 2025-07-17T12:54:09Z |
| format | Article |
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© The Authors. This content is provided under CC BY 4.0 license.
Plant Introduction, 95/96, 68–74 (2022)
SHORT COMMUNICATION
Microsporogenesis in faba bean (Vicia faba L.) grown in Mersin, Turkey
Aslı Küçükrecep 1, Dilek Tekdal 2, *
1 Department of Biotechnology, Institute of Science, Mersin University, 33110 Mersin, Turkey
2 Department of Biotechnology, Faculty of Science and Letters, Mersin University, 33110 Mersin, Turkey; * dilektekdal@mersin.edu.tr
Received: 29.07.2022 | Accepted: 22.08.2022 | Published online: 07.09.2022
Abstract
Legumes are an alternative to animal foods due to their high protein content. Like other legumes, Vicia faba
(faba bean) has high protein content. However, faba bean breeding with classical methods is challenging
due to inbreeding depression, self-incompatibility, and abortive embryo formation. The inadequacy of
classical breeding methods due to the problems in the fertilization biology of the plant limits the production
of new varieties. Therefore, the importance of using in vitro haploidization technique in legume breeding
is increasing. Anther culture is a widely preferred tissue culture technique for obtaining haploid plants.
The initial and most crucial stage in anther culture is the identification of anthers containing microspores
in the appropriate developmental stage. In the study, anther samples with single and triple-nucleated
microspores were cultured in MS nutrient medium containing 2,4-D (0.5 mg L-1) and Kinetin (2.5 mg L-1).
In the first month of culture, embryo and embryoid-like structures were obtained at the heart stage from
anthers containing microspores in the mononuclear stage, while anther samples containing microspores
in the trinucleated stage were observed to darken and did not develop.
Keywords: Vicia faba, anther culture, haploidization, microspore
https://doi.org/10.46341/PI2022017
UDC 634.722 : 581.3 : 631.528.2
Authors’ contributions: Aslı Küçükrecep and Dilek Tekdal conceived and designed the experiments, performed the experiments, and
wrote the paper. Dilek Tekdal supervised the study.
Funding: No funding was declared.
Competing Interests: The authors declared no conflict of interest.
Introduction
Leguminosae is the third largest family of
dicotyledons. Legumes have an essential
economic place in human and animal nutrition
with their high protein content. At the same
time, they are significant plants because they
are used as raw materials in the industry
(Duke, 1981; Selva et al., 1989).
In addition to their high nutritional value,
legumes, which can bind the free nitrogen of
the air to the soil, provide the enrichment of
the soil they are planted by attaching the free
nitrogen of the air to the soil, thanks to the
symbiotic relationship they establish with the
Rhizobium bacteria. They have the capacity to
fix about 5 to 20 kg da-1 of nitrogen per year.
This amount varies according to the type of
plant and environmental conditions (Şehirali
et al., 2010).
While the consumption of legumes was
28 million tons in 2000, it increased to 52
million tons (82 % increase) in 2017 due to the
increase in the human population. Worldwide,
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https://orcid.org/0000-0002-4545-9005
Plant Introduction • 95/96 69
Microsporogenesis in faba bean (Vicia faba L.) grown in Mersin, Turkey
in 2017, it decreased by 78 % (64 million tons)
compared to 2005, and the usage increased
by 75 % (51 million tons) (TAGEM, 2019). Most
chickpeas, lentils, beans, and faba beans are
produced commercially in Turkey, followed
by cowpea and peas. The share of agricultural
lands belonging to edible legumes increased
to 3 % in 1980 and 11.2 % in 1990 (Dogan et al.,
2020).
Vicia faba L., which has the most robust
growth feature and nitrogen fixation
capacity among the edible legumes, is known
worldwide as faba bean, brad bean, horse
bean, Windsor bean, tick bean, and fava bean.
Faba beans have medicinal properties. It has
long been known to be used as a medicine to
treat kidney, liver, and ocular sensory diseases
(Sathya Prabhu & Devi Rajeswari, 2018). Faba
bean is the most protein-containing legume,
with a protein content of approximately 21 %.
In addition to its high protein content, it is
ahead of other legumes with its capacity to
bind the free nitrogen of the air to the soil.
The chemical content of faba beans is 51–68 %
carbohydrates, 20–41 % protein (79 % globulin,
7 % albumin, and 7 % gluten), 2.3–3.9 % fat, folic
acid, niacin vitamins, and mineral elements
(Sathya Prabhu & Devi Rajeswari, 2018).
Despite the increase in demand for legumes
in developed and developing countries, a
decrease was observed in the cultivation areas
of legumes, as the cultivation of products such
as sugar cane and corn increased to obtain raw
materials in production areas such as biofuels
(Gülümser, 2016).
As a result of the legume breeding studies
that started in Turkey in 1965, three faba bean
varieties registered in the National Cultivars
List were developed and presented to the
producers (TAGEM, 2019). As a result of the
studies, it has been determined that the
production amount of faba beans was 13.8 %
(5.9 thousand tons), 7.5 % (5.5 thousand tons),
and 8.8 % (5 thousand tons) in 2018, 2019, and
2020, respectively (TAGEM, 2019).
With all these features, the faba bean
should be considered an alternative to meet
the current and predicted increase in protein
needs and enrich the soils where more than
one crop is obtained and impoverished in
organic matter. However, the faba bean is
susceptible to environmental conditions
and biotic and abiotic stresses, and progress
is slow in improving crop varieties due to
difficult pollination control (Bond, 1987).
Therefore, developing new varieties tolerant
to environmental conditions and various stress
factors is crucial. It takes 10 to 14 years to
develop new varieties with classical breeding
studies (Singh et al., 2013; Gülümser, 2016). In
tissue culture practices and breeding studies,
homozygous individuals can be obtained in a
short time, mainly thanks to anther culture,
and the period of obtaining new varieties is
shortened (Singh, 1997).
The microspore development stage is one of
the most important stages affecting success in
anther culture (Grewal et al., 2009). This stage
is defined as the ‘mononuclear microspore
stage’ in many species and refers to the time
from the formation of tetrads after meiosis to
mitosis (Smýkal et al., 2015). In the studies, it
has been stated that the androgenic capacity
of plants with fewer flowering days and small
bud structures is more intense; therefore, it
has been reported that collecting and working
the buds in the early stages of development
affects the success (Smýkal, 2000; Smýkal
et al., 2015).
This study aimed to investigate the effect
of appropriate bud selection and mononuclear
microspore stage on callus induction by using
buds at different developmental stages.
Material and methods
Plant material
In this study, faba bean seeds were obtained
from local growers in Adana (Turkey). Seeds
were sown in plastic pots mixed with 1 : 1 : 1
garden soil, decayed sheep manure, and turf in
the field in Mersin, Turkey. The soil was first
cleared of weeds, and a drip irrigation system
was installed. The soil has been dug up, and the
soil has been sprayed against the possibility
of worms and different organisms eating the
plant roots. The soil was well watered. Ten
seeds were planted directly on the land soil.
Since the optimum temperature value for the
germination of the seeds is 18–20 °C, and these
temperature values are felt in March in Mersin,
the seeds were sown in February.
Microscopic observations
Since the cultured microspore cells being
in the mononuclear or early binuclear
70 Plant Introduction • 95/96
A. Küçükrecep, D. Tekdal
stage increases the success of the culture,
the mononuclear stage in anthers was
determined by 4',6-diamidino-2-phenylindole
dihydrochloride (DAPI) staining. Flower buds at
different developmental stages were collected
and grouped according to size. Anthers were
isolated from buds of different sizes, and crush
preparations were prepared. Preparations
were stained with DAPI and examined under
a fluorescence microscope Olympus BX51
(Japan).
Surface sterilization and in vitro culture
conditions
Suitable-sized flower buds determined for
anther culture experiment were kept under
tap water for 30 minutes to remove soil and/
or dust residues. After standing, it was washed
several times with sterile distilled water. After
standing, it was washed with distilled water.
Explants taken into a sterile cabinet were
soaked in 70 % EtOH for 1 min. Flower buds
were placed in 25 % NaOCl for 15 min., shaken
and kept. Surface sterilization of the samples
was completed by washing four-five times
with sterile distilled water. The anthers of
the sterilized flower bud were isolated under
a stereo microscope Olympus SZ61 (Japan)
and used as the primary material in the tissue
culture study. In order to test the effect of
the microspore development stage on the
success of anther culture, anthers containing
microspores in the late developmental stage
(trinucleate stage) were also cultured. MS
medium containing 2,4-D (0.5 mg L-1) and
Kinetin (Kin; 2.5 mg L-1) was used in anther
culture experiments, and MS medium without
plant growth regulator was used as the control
group. The experiment was carried out with
five replications and 20 anthers in each trial.
Results and discussion
Firstly, seeds obtained from local growers
were germinated in the field area in Mersin,
Turkey. After one week, the seeds germinated,
and the plants were kept for a month to reach
an appropriate size and developmental period
for further analysis (Fig. 1).
Anthers and microspores are used to
obtain haploid plants through androgenesis,
and success in both techniques depends on
the effect of many biotic and abiotic factors
(Murovec & Bohanec, 2011). In anther culture
study, the success of haploidization is affected
by the developmental stages of microspore
cells. The most suitable stage for culture is
the mononuclear or early binuclear stage
of microspore cells. It is stated that this
stage can be determined by evaluating the
buds in terms of size and shape (Zamir et al.,
1980). Therefore, buds of different sizes were
collected to determine the stage in which
mononuclear microspore cells were cultured
and the appropriate flower bud developmental
stage to isolate anthers (Fig. 2).
A B
Figure 1. Two-week-old (A) and one-month-old (B) Vicia faba grew in soil.
Plant Introduction • 95/96 71
Microsporogenesis in faba bean (Vicia faba L.) grown in Mersin, Turkey
Bud size and mononuclear stage images
were determined for the studied faba
bean genotype and are given in Figs. 2
& 3. Accordingly, it was found that the
microspore cells of the anthers taken from
the buds with a size of about 2–3 mm were
in the mononuclear stage (Fig. 3). In the
mononuclear stage, nuclei of microspore
cells were observed to shift from the center
of the cell to the poles of the cell (Fig. 3).
As a result of the analysis, the appropriate
flower bud stage for anther culture was
determined.
Anther culture was performed to
determine the high success rate of culture
studies with anther containing microspores
in the mononuclear stage. For this reason,
anthers containing microspores in the
mononuclear stage and triple nucleated stage
were cultured in nutrient media containing
Kin (2.5 mg L-1) and 2,4-D (0.5 mg L-1) growth
regulators and not containing these plant
growth regulators as a control group. Callus
regeneration is the first and most important
step in anther culture. It was observed that
callus formation started from the second
week of culture in anthers cultured in MS
medium containing Kin and 2,4-D (Fig. 4),
while browning began in anthers with triple
nucleated microspores cultured in the same
media containing. No improvement was
observed in all samples cultured in MS media
without Kin and 2,4-D as a control group. In
a haploidization study performed with three
Figure 2. Flower buds of Vicia faba at different developmental stages analyzed for the determination of the
mononuclear microspore stage.
Figure 3. Determination of mononuclear stage in microspores of Vicia faba by DAPI staining. Images
of flower buds with uni- (1 and 2), bi- (3 and 4), and trinucleate (5 and 6) microspores (filter UMVIBA3).
Numbers refer to the numbers of the flower buds indicated in Fig. 2. Arrows indicate the nuclear number.
72 Plant Introduction • 95/96
A. Küçükrecep, D. Tekdal
wild-types, Phaseolus vulgaris from Colomba
and Mexico and one cultivar of P. coccineus
from Rwanda, it was determined that 2,4-
D and Kin growth regulators, as well as
the microspore development stage, were
important in the success of anther culture
(Muñoz et al., 1993).
It was observed that embryoid structures,
embryogenic callus tissue, and embryo in the
heart stage were formed in the calli of the
anther tissue within a month of the culture
studies (Fig. 6).
Various problems have been encountered
in vitro cultures of legumes, including
V. faba (Fakhrai et al., 1989). In vitro culture
has not been particularly successful with
grain legumes since plant regeneration from
callus has been challenging. Previous studies
reported that calli secreted a significant
amount of phenolic substances and showed
poor growth in tissue culture (Fakhrai et al.,
1989). Many researchers working with V. faba
before have reported failure to observe
shoot organogenesis in culture (Röper, 1979;
Jelaska et al., 1981). In this study, heart shape
embryo was obtained successfully from
the callus regenerated from anthers with
mononucleated microspores.
Conclusions
Anther culture is applied for many
purposes, such as propagation and haploid
plant production. The developmental stage
of microspores in anthers is a key point
for the success of the study. This study
determined that the culture of anthers
at the stage where microspore cells were
mononucleated successfully obtained calli.
It is thought that the findings from this
study are important for the anther culture
method to be applied to obtain pure lines
from the faba bean. Future steps for getting
pure lines include callus culture and plant
regeneration.
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Plant Introduction • 95/96 73
Microsporogenesis in faba bean (Vicia faba L.) grown in Mersin, Turkey
Figure 6. Images of the structures developing from the anther at different developmental stages cultured
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74 Plant Introduction • 95/96
A. Küçükrecep, D. Tekdal
Мікроспорогенез у бобів (Vicia faba L.), вирощених у Мерсіні, Туреччина
Аслі Кючюкрецеп 1, Ділек Текдаль 2, *
1 Відділ біотехнології, інститут науки, Мерсинський університет, Мерсін, 33110, Туречина
2 Відділ біотехнології, факультет науки та літератури, Мерсинський університет, Мерсін, 33110,
Туречина; * dilektekdal@mersin.edu.tr
Бобові є альтернативою тваринній їжі завдяки високому вмісту білка. Як і інші бобові, Vicia faba
також відрізняється високим вмістом білка. Однак селекція бобів класичними методами є складною
через пригнічення інбридингу, самонесумісність та невдале утворення ембріонів. Неприйнятність
класичних методів селекції через проблеми в біології запліднення цих рослини обмежує продукування
нових сортів. Тому інтерес до використання техніки гаплоїдизації in vitro в селекції бобових зростає.
Культивування пиляків є широко застосовуваним методом культивування тканин для отримання
гаплоїдних клонів. Початковим і найважливішим етапом культури пиляків є ідентифікація пиляків,
що містять мікроспори, у відповідній стадії розвитку. Саме тому під час дослідження зразки пиляків з
одно- та триядерними мікроспорами культивували в живильному середовищі MS, що містить 2,4-D
(0,5 мг л-1) і кінетин (2,5 мг л-1). У перший місяць культивування ембріон та ембріоїдоподібні структури
на стадії серця були отримані з пиляків, що містять мононуклеарні мікроспори. В той час як пиляки,
що містили мікроспори на триядерній стадії, темніли та не давали розвитку новим тканинам.
Ключові слова: Vicia faba, культура пиляків, гаплоїдизація, мікроспори
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| id | oai:ojs2.plantintroduction.org:article-1617 |
| institution | Plant Introduction |
| keywords_txt_mv | keywords |
| language | English |
| last_indexed | 2025-07-17T12:54:09Z |
| publishDate | 2022 |
| publisher | M.M. Gryshko National Botanical Garden of the NAS of Ukraine |
| record_format | ojs |
| resource_txt_mv | wwwplantintroductionorg/a0/a8ea94695cfc11ac4248c177ac4243a0.pdf |
| spelling | oai:ojs2.plantintroduction.org:article-16172023-08-26T20:38:45Z Microsporogenesis in faba bean (Vicia faba L.) grown in Mersin, Turkey Мікроспорогенез у бобів (Vicia faba L.), вирощених у Мерсіні, Туреччина Küçükrecep, Aslı Tekdal, Dilek Legumes are an alternative to animal foods due to their high protein content. Like other legumes, Vicia faba (faba bean) has high protein content. However, faba bean breeding with classical methods is challenging due to inbreeding depression, self-incompatibility, and abortive embryo formation. The inadequacy of classical breeding methods due to the problems in the fertilization biology of the plant limits the production of new varieties. Therefore, the importance of using in vitro haploidization technique in legume breeding is increasing. Anther culture is a widely preferred tissue culture technique for obtaining haploid plants. The initial and most crucial stage in anther culture is the identification of anthers containing microspores in the appropriate developmental stage. In the study, anther samples with single and triple-nucleated microspores were cultured in MS nutrient medium containing 2,4-D (0.5 mg L-1) and Kinetin (2.5 mg L-1). In the first month of culture, embryo and embryoid-like structures were obtained at the heart stage from anthers containing microspores in the mononuclear stage, while anther samples containing microspores in the trinucleated stage were observed to darken and did not develop. Бобові є альтернативою тваринній їжі завдяки високому вмісту білка. Як і інші бобові, Vicia faba також відрізняється високим вмістом білка. Однак селекція бобів класичними методами є складною через пригнічення інбридингу, самонесумісність та невдале утворення ембріонів. Неприйнятність класичних методів селекції через проблеми в біології запліднення цих рослини обмежує продукування нових сортів. Тому інтерес до використання техніки гаплоїдизації in vitro в селекції бобових зростає. Культивування пиляків є широко застосовуваним методом культивування тканин для отримання гаплоїдних клонів. Початковим і найважливішим етапом культури пиляків є ідентифікація пиляків, що містять мікроспори, у відповідній стадії розвитку. Саме тому під час дослідження зразки пиляків з одно- та триядерними мікроспорами культивували в живильному середовищі MS, що містить 2,4-D (0,5 мг л-1) і кінетин (2,5 мг л-1). У перший місяць культивування ембріон та ембріоїдоподібні структури на стадії серця були отримані з пиляків, що містять мононуклеарні мікроспори. В той час як пиляки, що містять мікроспори на триядерній стадії, темніли та не давали розвитку новим тканинам. M.M. Gryshko National Botanical Garden of the NAS of Ukraine 2022-09-06 Article Article application/pdf https://www.plantintroduction.org/index.php/pi/article/view/1617 10.46341/PI2022017 Plant Introduction; No 95/96 (2022); 68-74 Інтродукція Рослин; № 95/96 (2022); 68-74 2663-290X 1605-6574 10.46341/PI95-96 en https://www.plantintroduction.org/index.php/pi/article/view/1617/1534 Copyright (c) 2022 Aslı Küçükrecep, Dilek Tekdal http://creativecommons.org/licenses/by/4.0 |
| spellingShingle | Küçükrecep, Aslı Tekdal, Dilek Мікроспорогенез у бобів (Vicia faba L.), вирощених у Мерсіні, Туреччина |
| title | Мікроспорогенез у бобів (Vicia faba L.), вирощених у Мерсіні, Туреччина |
| title_alt | Microsporogenesis in faba bean (Vicia faba L.) grown in Mersin, Turkey |
| title_full | Мікроспорогенез у бобів (Vicia faba L.), вирощених у Мерсіні, Туреччина |
| title_fullStr | Мікроспорогенез у бобів (Vicia faba L.), вирощених у Мерсіні, Туреччина |
| title_full_unstemmed | Мікроспорогенез у бобів (Vicia faba L.), вирощених у Мерсіні, Туреччина |
| title_short | Мікроспорогенез у бобів (Vicia faba L.), вирощених у Мерсіні, Туреччина |
| title_sort | мікроспорогенез у бобів (vicia faba l.), вирощених у мерсіні, туреччина |
| url | https://www.plantintroduction.org/index.php/pi/article/view/1617 |
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