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

Abstract

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.

Production of Gynogenic Plants of Red Beet (Beta vulgaris L.) in Unpollinated Ovule Culture In Vitro

“…The first successful cultivation of female gametophytes to produce haploid and doubled haploid sugar beet plants using an in vitro unpollinated ovule culture was reported by Hosemans and Bossoutrot (1983) [30], D'Halluin and Kelmer (1986) [31], and Van Geyt et al (1987) [32]. Since then, and until now, various in vitro tissue culture techniques, genetic transformation, molecular biology techniques, in vitro selective systems (contributed to obtaining sugar beet plants with high tolerance to abiotic stresses) have been used to improve the characteristics of this crop, propagation, and conservation of valuable forms in practical breeding [33][34][35][36].…”

Vibratome Sectioning and Clearing for Easing Studies of Cassava Embryo Formation

“…Since then, clearing protocols have been used to analyze ovule, ES, and embryo development. Various basic studies aimed at understanding the reproductive biology in several species (Herr, 1982;Herr, 1992;Musiał et al, 2015;Wilkinson and Tucker, 2017;Barke et al, 2018;Hoshino et al, 2018;Colono et al, 2019;Tofanelli et al, 2019); as well as for applied studies for plant breeding (Ogburia and Adachi, 1994;Ogburia and Adachi, 1995;Ogburia and Adachi, 1996;Ponitka and S´lusarkiewicz-Jarzina, 2004;Janowicz and Wojciechowski, 2010;Hoshino et al, 2018;Kwiatkowska et al, 2019). Clearing techniques have also been used to study morphology and gene expression for understanding developmental and other biological processes (Kurihara et al, 2015;Musielak et al, 2016).…”

“…The chemicals used for clearing may affect the consistency of the tissues, making their handling difficult for microscopy observations. Hence, the clearing methodology must be modified accordingly to the tissues due to the diversity of cell sizes, tissue chemistry, and density among different species (Kwiatkowska et al, 2019).…”