An optimised clearing protocol for the quantitative assessment of sub-epidermal ovule tissues within whole cereal pistils.

Abstract

Background Seed development in the angiosperms requires the production of a female gametophyte (embryo sac) within the ovule. Many aspects of female reproductive development in cereal crops are yet to be described, largely due to the technical difficulty in obtaining phenotypic information at the cellular or sub-cellular level. Hoyer's solution is currently well established as a solution for clearing thin tissues samples, such as sections or whole tissues of bryophytes, mycorrhizal fungi, and small model organisms (e.g. Arabidopsis thaliana ). Results Here we report a Hoyer's solution-based clearing method to facilitate clearing of the whole barley pistil, with high reproducibility. The clearing process takes 10 days from fixation to visualisation, whereupon tissue is sufficiently clear to obtain multiple phenotypic measurements from sub-epidermal tissues and cells within the ovule. Conclusion Visualisation of cereal ovules that have not been dissected from the pistil allows an unprecedented capability to collect quantitative morphological information from the developing ovule, integument, nucellus and embryo sac. This will enable comparisons with genetic data to reveal the contribution of pre-fertilisation ovule tissues towards downstream seed development.

Plant phenotyping relevance

全体のオオムギ雌ずいを透明化し、胚珠組織の定量的形態情報を取得する手法の開発が中心であり、植物フェノタイピング手法に該当する。

Code and data availability

The paper describes a Hoyer's solution clearing protocol for cereal pistils with quantitative ovule trait measurements in nine barley cultivars. No public repository deposit, image dataset, or author analysis code with a public URL is provided. The only availability statement directs readers to contact the authors, and

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