Short Communication. A robust method for chromosome quantification and ploidy determination in woody species
Harshi K. Gamage A and Susanne Schmidt A BA School of Biological Sciences, The University of Queensland, Brisbane, Qld 4072, Australia.
B Corresponding author. Email: Susanne.Schmidt@uq.edu.au
Australian Journal of Botany 57(2) 87-93 https://doi.org/10.1071/BT09006
Submitted: 6 January 2009 Accepted: 3 March 2009 Published: 11 May 2009
Abstract
Accurate determination of ploidy level of putative polyploid plants is essential for tree breeding and other applications. Methods for ploidy determination include quantification of chromosome numbers in root-tip cells via light microscopy and indirect assessment via anatomical and morphological traits. Flow cytometry is potentially a high-throughput method to quantify nuclear DNA content; however, it does not allow determining chromosome numbers and interfering compounds often prevent its use. Microscopy-based quantification of chromosomes in active root-tip cells remains the most unambiguous method for ploidy determination, although root tips are difficult to obtain from field-grown plants, and light microscopy can result in insufficient resolution in species with many and small chromosomes. Here, we present a robust technique that uses 2, 4-diamidino-2-phenylindole (DAPI) dye and 1000-fold magnification fluorescence microscopy for quantification of chromosomes in root and shoot tips of woody angiosperms and gymnosperms, and overcomes the reported difficulties. Rather than the conventional tip squashing, spreading tips on glass slides resulted in very good chromosome separation in diverse species, with up to 56 chromosomes and a chromosome size of 2–20 μm. Chromosome counts were performed in diploid Agathis robusta, Elaeocarpus angustifolius, Eucalyptus robusta, Paulownia tomentosa, Pongamia pinnata and Toona ciliata, and di- and tetraploid Acacia crassicarpa and Citrus species.
Acknowledgements
We thank Natalie Piperidis from the Bureau of Sugar Experimental Stations for help with methods development. Thanks also go to DPI Bundaberg and ‘Fingerlime Australia’ for providing plant material for this study. The study was funded by The University of Queensland (FirstLink).
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