Short Communication. A robust method for chromosome quantification and ploidy determination in woody species

Abstract

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 highthroughput 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 mm. 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.

Progress in Myrtaceae genetics and genomics: Eucalyptus as the pivotal genus

“…This introductory discussion statement of the first report on chromosome number variation in a set of Myrtaceae species Atchison (1947), provided an early indication of the limited variation in chromosome number in the family, later confirmed by several studies. Furthermore, Atchison (1947) also showed that chromosomes in Myrtaceae are generally small, ranging from 1.2 to 2.5 μm, making detailed cytogenetic analysis difficult, though improved methods have recently been successfully applied to Eucalyptus (Gamage and Schmidt 2009). In that original study by Atchison (1947), species of Psidium showed chromosome counts ranging from 2n022 to 88, strongly suggesting the occurrence of polyploidy.…”

Culture of triploid tissue from the endosperm of an endangered Chilean tree speciesGomortega keule

“…All wild trees and in vitro-cultured genotypes that originated from field shoots or zygotic embryos were diploid. Chromosome numbers were difficult to count due to their small size and high number, as discussed by Gamage and Schmidt (2009). However, the numbers recorded ( Figure 5C,D) supported the ploidy level indicated by flow cytometry.…”

“…Chromosomes could not be counted in cells of other tissue types, including regenerated roots. The procedure followed those of Andras et al (1999), and Gamage and Schmidt (2009) with modifications. Tissue was placed in a solution containing 2 mM 8-hydroxyquinoline (Alfa Aesar, Ward Hill, MA, USA) for 24 h at room temperature, followed by a 3:1 (v/v) ethanol:acetic acid fixative for ≥ 16 h. Hydrolysis was performed in 1.0 M hydrochloric acid (J.T.…”

The eucalypt karyogram resolved

“…The chromosomes 4, 8, and 10 varied in their classification, considering the three different ideograms. (Matsumoto et al 2000;Oudjehih and Abdellah 2006;Gamage and Schmidt 2009) and probably resulted in chromosomes with an over-condensed condition. Thus, cytogenetic analysis most of the eucalypt species studied was limited to chromosome counting.…”

“…To date, the chromosome numbers of 135 Eucalyptus species have been described (Oudjehih and Abdellah 2006), in which, only seven have their karyotype assembled (Matsumoto et al 2000). Improvement in the cytogenetic protocols achieved to date for Eucalyptus (Gamage and Schmidt 2009), has not generated any information beyond chromosome counting. This indicates the limitation of a karyomorphological approach in cytogenetic studies for A few cytogenetic studies have been conducted on Eucalyptus citriodora.…”