Refinement of a clearing protocol to study crassinucellate ovules of the sugar beet (Beta vulgaris L., Amaranthaceae)

Monika Kwiatkowska , Dariusz Kadłuczka , Maria Wędzony , Beata Dedicova , Ewa Grzebelus


Background: Clearing methods allow relatively quick processing of plant material and examination of cellular structures by rendering tissues and organs translucent. They have been adapted for plant embryology, primarily to study ovule development, megasporogenesis, megagametogenesis and embryogenesis. Such clearing methods overcome several disadvantages of the conventional embedding-sectioning techniques that are arduous and time-consuming. Although numerous protocols with different clearing solutions have been described, there have been no reports to date proposing a reliable method to clear the crassinucellate ovules of the sugar beet (Beta vulgaris L.), an economically important crop. Therefore, this study aims to find a suitable approach to improve the tissue transparency of sugar beet ovules at different developmental stages. Results: We established a methyl salicylate-based protocol that significantly improved the transparency of the B. vulgaris ovule structures, which allowed us to observe the megagameto- and embryogenesis of that species. This was achieved by (1) chemical softening of the tissues; (2) vacuum pump-assisted infiltration step; (3) shaking-assisted incubation with clearing mixtures; and (4) manual removal of the chemically softened seed coat. Conclusions: The effectiveness of our method is due to the strategy combining various approaches at different stages of the procedure aiming at increasing the accessibility of the internal ovule structures to the clearing solution. The results of this study may be applied in sugar beet breeding programs, and it will provide a basis for further investigation of numerous aspects of the species’ embryology. Moreover, that unique approach may be easily adapted to other species developing crassinucellate ovules.
Author Monika Kwiatkowska
Monika Kwiatkowska,,
, Dariusz Kadłuczka
Dariusz Kadłuczka,,
, Maria Wędzony
Maria Wędzony,,
, Beata Dedicova
Beata Dedicova,,
, Ewa Grzebelus (FoBaH / IoPBaB)
Ewa Grzebelus,,
- Institute of Plant Biology and Biotechnology
Journal seriesPlant Methods, ISSN 1746-4811, (N/A 140 pkt)
Issue year2019
Publication size in sheets0.55
Keywords in EnglishDifferential interference contrast (DIC), Embryogenesis, Megagametogenesis, Methyl salicylate, Plant embryology
ASJC Classification1110 Plant Science; 1305 Biotechnology; 1311 Genetics
Internal identifierWBIO/2019/35
Languageen angielski
LicenseJournal (articles only); author's original; Uznanie Autorstwa (CC-BY); after publication
Refinement of a clearing protocol to study crassinucellate ovules of the sugar beet (Beta vulgaris L., Amaranthaceae) of 22-10-2019
1,95 MB
Score (nominal)140
Score sourcejournalList
Publication indicators WoS Citations = 0; Scopus SNIP (Source Normalised Impact per Paper): 2017 = 1.543; WoS Impact Factor: 2018 = 3.17 (2) - 2018=4.46 (5)
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FinansowanieThis research was financed by Syngenta Seeds AB (Sweden), Project No. BZ-928/WBiO/IBRiB/17-18, and the statutory funds for science granted by the Polish Ministry of Science and Higher Education to the Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow.
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* presented citation count is obtained through Internet information analysis and it is close to the number calculated by the Publish or Perish system.
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