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SMALL-SCALE ISOLATION OF MEIOCYTES FROM ARABIDOPSIS THALIANA ANTHERS FOR CYTOLOGICAL ANALYSES

JUNHUA LI1,3,*√, JINHONG YUAN1,3√, KANG JI1, YANLING ZHANG2, JUAN WANG1, SHASHA ZHANG1, SHUBO LIU1, PENGFEI QIU1, NING LI1 AND MINGJUN LI1,3,*

1College of Life Sciences, Henan Normal University, Xinxiang 453007, China

2School of Chemistry and Chemical Engineering, Xinxiang University, Xinxiang 453003, China

3Engineering Laboratory of Green Medicinal Material Biotechnology, Henan Province, Xinxiang 453007, China

*Corresponding author e-mail: Mingjun Li, 041013@htu.cn; Junhua Li, lijh0909@gmail.com; Tel.: +86 0373 3328189; Fax: +86 0373 3328189

√Authors contributed equally to this work.

Abstract

In flowering plants, the meiocytes develop within nourishing tissues – the anthers or the ovaries. Although most plants bear one or more anthers in each bisexual or male flower, the size of anthers that undergo meiosis is relatively small, and each anther contains only a small fraction of male meiocytes. As a consequence, the isolation of plant meiocytes is very challenging due to their relative inaccessibility. Efficient manual dissection of meiocytes from young Arabidopsis anthers for cytological and cytogenetic analyses is technically challenging. Indirect detection of cellular or nuclear phenotypes is impaired by poor penetration of the reagents, and cell type-specific imaging is deminished by the lack of clarity in whole-mount tissues. Here, we describe a method that allows the efficient isolation of male meiocytes of Arabidopsis. Anthers undergoing meiosis were dissected and collected into appropriate isolation/labeling buffer, depending on the downstream application. Meiocytes were released by crushing with a cover slip on a glass slide. The isolated meiocytes could be analyzed using a microscope. The technical skills required are simple, and the procedure does not require any special equipment. Isolated meiocytes are suitable for a variety of dowstream applications, such as cell morphological investigation, reporter gene assays, chromosome analyses, and callose staining.

Key words: Arabidopsis thaliana, Callose, Chromosomes, Pollen mother cells, Meiosis, Reporter assays.