Abstract
Main conclusionThe qPCR assay developed to differentiate haploid and diploid maize leaf samples was unsuccessful due to DNA content difference. Haploid cells are packed more closely together with less cellular expansion.Increased ploidy content (> 2 N) directly correlates with increased cell size in plants, but few studies have examined cell morphology in plants with reduced ploidy (i.e., haploids). To pioneer a scalable new ploidy test, we compared DNA content and cellular morphology of haploid and diploid maize leaves. The amount of genomic DNA recovered from standardized leaf-punch samples was equivalent between these two ploidy types, while both epidermal and mesophyll cell types were smaller in haploid plants. Pavement cells had a substantially smaller size than mesophyll cells, and this effect was more pronounced in the abaxial epidermis. Interveinal distance and guard cell size were significantly reduced in haploids, but the cell percentage comprising stomata did not change. These results confirm the direct correlation between ploidy content and cell size in plants, and suggest that reduced cell expansion predominantly explains DNA content equivalence between haploid and diploid samples, confounding efforts to develop a haploid detection method using DNA content.
Highlights
In both animals and plants, the regulation of cell size is one of the most important and well-studied aspects of development
While haploid leaf samples overall had slightly less DNA, the quantity as input for the real-time PCR (qPCR) analysis showed no significant difference between individual haploid and diploid leaf punches. To ask whether this may be due to denser packing of haploid cells, we looked at the relationship between ploidy content and cell size in equivalent haploid and diploid leaf punches, focusing on the epidermis and mesophyll
Given such a sensitive and specific TaqMan assay for ADH1, the DNA quantifications can be replicated
Summary
In both animals and plants, the regulation of cell size is one of the most important and well-studied aspects of development. Endo-reduplication is a developmentally regulated phenomenon, and cell size is roughly proportional to ploidy content (Roeder et al 2012). This has been well-studied in tissues and cell types with increased (> 2 N) ploidy (Sugimoto-Shirasu and Roberts 2003). Prior studies have investigated stomata length with respect to ploidy determination and doubled haploid (DH) production systems. Stomata guard cell length may be used to differentiate true haploid and doubled haploid plants from false positive and diploid plants as early as the Leaf 2 stage in maize (Choe et al 2012).
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