the root tip is a common approach for identifying ploidy level, which has been applied in numerous species. However, this method is technically demanding, time-consuming, and laborious. Currently, flow cytometry (FCM) analysis has been widely used in
determined for each DNA content measurement, based on FCM assessments of standards and samples. Fig. 1. Representative cytograms of fluorescence intensity by flow cytometry of G0/G1 nuclei from some C. arabica cultivars and standards. Representative
repeats ( Muranty et al., 2002 ), and flow cytometry ( Kiełkowska and Adamus, 2010 ; Lotfi et al., 2003 ). The first use of DNA flow cytometry in plants was reported by Galbraith et al. (1983) . The nuclei are classified according to their relative
morphological characteristics. This method, however, has not proven always accurate or sufficient. Flow cytometry is the newest technique to determine ploidy level, which appears to become a powerful method. This technique combines the advantages of microscopy
impedance flow cytometry. All experiments were conducted at the World Vegetable Center, Shanhua, Tainan, Taiwan (lat. 23.1°N, long. 120.3°E, elevation 12 m). Before sowing, all seed was treated with trisodium phosphate (TSP) and hydrochloric acid (HCl
cytometry, molecular markers, and biochemical and phenotypic analyses ( Bradaï et al., 2016 ; Harding, 2004 ; Sadat-Hosseini et al., 2011 ). Molecular analyses and flow cytometry have been used in many species to evaluate the trueness-to-type of plantlets
determination. Somatic regenerants were screened by flow cytometry to determine their ploidy level while they were still growing in vitro. The relative amount of nuclear DNA content was measured in leaf samples of regenerated plants using a tabletop ploidy
DNA flow cytometry was used to determine nuclear DNA content in diploid blueberry species, and 3x, 4x, 5x, and 6x ploidy levels. Relative fluorescence intensity of stained nuclei measured by flow cytometry was a function of the number of chromosome sets (X): Y = 3.7X – 2.3 (r2 = 95.1%). DNA flow cytometry should be useful for ploidy level determination in the seedling stage. A significant linear relationship was established between nuclear DNA content and number of chromosomes (x); DNA (pg) = 0.52 x1 (r2 = 99.8%). Based on this equation the haploid genome DNA amount (1C) was calculated as 0.62 ± 0.08 pg, with an approximate haploid genome size of 602 Mbp/1C. The results indicate that conventional polyploid evolution occured in the section Cyanococcus, genus Vaccinium: the increase in DNA was concurrent with increase in chromosome number. DNA content differences among 2x species were correlated with Nei's genetic distance estimates based on 20 isozyme markers. Most of the variation was among species (49%), with 26% between populations within species, and 25% within populations.
Ploidy level in grapevines varies, especially since in vitro techniques are employed in the breeding process and after plants are treated with either chemicals or radiation. Detection of ploidy level in grapevines by microscopic chromosome counting is complicated by their high number and the small size of chromosomes. Flow cytometry provides an accurate and rapid method in determining the ploidy level in plant tissue by measuring the nuclear DNA content in living cells and thus is a very useful tool in plant breeding or genetic studies. The objective of this research was to analyze the ploidy level of a selected group of muscadine vines that were different from normal diploid vines in morphology. These grapes were derived from either chemical treatment of known varieties or from controlled/open pollinations. Among the 26 grapevines investigated, 8 were found to be diploids, 11 were tetraploids, and 7 were chimeric aneuploids. Results of this study indicate that flow cytometry is a quick, reliable tool for determining ploidy levels of grapevines.
Nuclear DNA flow cytometry was used to differentiate ploidy level and determine nuclear DNA content in Rubus. Nuclei suspensions were prepared from leaf discs of young leaves following published protocols with modifications. DNA was stained with propidium iodide. Measurement of fluorescence of 40 genotypes, whose published ploidy ranged from diploid to dodecaploid, indicated that fluorescence increased with an increase in chromosome number. Ploidy level accounted for 99% of the variation in fluorescence intensity (r 2 = 0.99) and variation among ploidy levels was much higher than within ploidy levels. This protocol was used successfully for genotypes representing eight different Rubus subgenera. Rubus ursinus Cham. and Schldl., a native blackberry species in the Pacific Northwest, which has been reported to have 6x, 8x, 9x, 10x, 11x, and 12x forms, was extensively tested. Genotypes of R. ursinus were predominantly 12x, but 6x, 7x, 8x, 9x, 11x, and 13x forms were found as well. Attempts to confirm the 13x estimates with manual counts were unsuccessful. Ploidy level of 103 genotypes in the USDA-ARS breeding program was determined by flow cytometry. Flow cytometry confirmed that genotypes from crosses among 7x and 4x parents had chromosome numbers that must be the result of nonreduced gametes. This technique was effective in differentiating chromosome numbers differing by 1x, but was not able to differentiate aneuploids. Nuclear DNA contents of 21 diploid Rubus species from five subgenera were determined by flow cytometry. Idaeobatus, Chamaebatus, and Anaplobatus were significantly lower in DNA content than those of Rubus and Cylactis. In the Rubus subgenus, R. hispidus and R. canadensis had the lowest DNA content and R. sanctus had the highest DNA content, 0.59 and 0.75 pg, respectively. Idaeobatus had greater variation in DNA content among diploid species than the Rubus subgenus, with the highest being from R. ellipticus (0.69 pg) and lowest from R. illecebrosus (0.47 pg).