Rapid determination of nuclear DNA amounts and ploidy levels in germplasms of Asparagus using flow cytometry

Flow cytometry (FCM) analysis of DNA content was performed by using nuclei isolated from tissues of asparagus and stained with a fluorescence dye (DAPI). Fluorescence intensities have been analyzed using a PARTEC flow cytometer. In general, three ploidy levels are common in Asparagus (x=n=10) 2x, 4x and 6x. Using three different cultivars of Asparagus officinalis L. as reference samples ('Gloria': diploid; 'Hiroshima Green': triploid; 'Purple Passion': tetraploid), the ploidy levels of plants of 'Morado de Huetor' (seedlings or in vitro-cloned material) as well as of several commercial hybrids and of Asparagus species (A. densiflorus Sprengeri, A. acutifolius, A. albus) have been analyzed. The FCM analysis of a population of 'Morado de Huetor' resulted in the detection of nine tetraploid, two triploid, and two (probably) pentaploid plants; one individual of 'PP3' (wild isolation) appeared to be hexaploid. The commercial hybrids: 'Gloria' (used as reference), 'Abril', 'J.Giant', 'Ida Lea', 'Brocks', 'Jacma 2014', ' Jacma 2004', 'Pla 2232', 'Tainan 3', 'Huchels A' and 'Greenwich' were diploid. The populations 'Blanco', 'Limbras', 'Inglaterra', 'URSS', 'India', 'Afghanistan', 'Dinamarca', 'España' and 'California' were also characterized as diploid plant. However, we observed a distinct variation in the DNA content (e.g. 'Dinamarca': 'URSS' = 1: 1.2). 'Violetto d'Albenga' and 'Purple Passion' (used as reference) were tetraploid. 'Hiroshima Green' (used as reference) was triploid. In one plant, Nº 5SB (from anther culture), we observed a ploidy level clearly below the diploid status. Crossing of this plant with a 'normal' diploid parent resulted in an intermediate ploidy level. Plants of Asparagus albus could be characterized as diploid; A. acutifolius as tetraploid, and two plants analyzed of A. sprengeri were triploids. Relative nuclear DNA content which revealed the position of peaks of DNA distribution, representing nuclei in the G1 phase of the cell cycle, differed by the ploidy levels of the plants. The relative frequencies of nuclei within different peaks depended on the analyzed material. Seedlings presented a great variation, probably due to polysomaty, a frequent characteristic of asparagus. FCM analyses of the ploidy status in Asparagus are easier and quicker than the chromosome counts. We will continue our studies with more accurate measurements using intercalating fluorescence stains in order to determine the absolute amounts of DNA.