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Nassar, Nagib M. A.; Vieira, Marco Andre; Vieira, Clibas; Grattapaglia, Dário

doi:10.1023/a:1018303109798

Cited by 20 publications

(1 citation statement)

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“…Clearing crassinucellate ovules has always been problematic, not only because of the thick layers of the nucellus tissue, but also because of its starchy content, thick integuments and early seed coat formation. The study on apomixis in cassava ( Manihot esculenta Cranz) is a good example of the problem [65]. The authors published molecular data (analysis with RAPD markers) along with anatomic studies to prove apomixis in this species, since pictures of the anatomic analyses conducted by clearing were not convincing.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2019

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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. Electronic supplementary material The online version of this article (10.1186/s13007-019-0452-6) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%