Nuclear DNA content of Solanum species grown in vitro, as determined by flow cytometry

Abstract

The DNA relative content in nuclei from several Solanum species, which were used as partners for somatic hybridization, were determined using a flow cytometry method. The nuclei were isolated mechanically or via protoplasts from leaves of in vitro grown plants. In the case of S. nigrum as well as S. tuberosum cv. Bzura and dihaploid clone H8105, the nuclei were also obtained from suspension cultured cells by lysis of protoplasts. The source and the method of nuclei isolation affected the pattern of nuclear DNA in the genotypes studied. The mesophyll nuclei showed two distinct peaks on the DNA histograms, whereas the DNA peaks produced by cell suspension nuclei were broad and less distinct. The DNA content in the nuclei, calculated from the DNA histograms of the samples and a DNA standard historgam (Trout Red Blood Cells, having DNA content of 5.05 pg per nucleus), were much lower in mesophyll nuclei than in those obtained from the cell suspension for the same genotypes. The results are discussed in respect of the genetic instability of Solanum genotypes. The usefulness of a flow cytometry approach in somatic hybridization research is also discussed.

Electrofusion of protoplasts from Solanum tuberosum, S. nigrum and S. bulbocastanum

“…In our experiments, however, single fusions between the mesophyll and cell suspension derived protoplasts were frequently observed and the heterokaryons were found to be viable and able to divide after three to four days in culture. As shown previously, the ploidy level of H-8105 suspension cells varied much and many of the cells contained two or three nuclei instead of one (Maciejewska et al 1999) indicating the disturbances in cell division of the cultured cells. Thus, the various ploidy of H-8105 cell suspension derived protoplasts was probably responsible for the abnormalities in regeneration of this protoclone and its putative fusants with mesophyll protoplasts of S. nigrum.…”

“…The ploidy level of the recovered regenerants was determined by flow cytometry method as described by Maciejewska et aL (1999).…”

Nuclear DNA content and chromosome number in somatic hybrid allopolyploids of Solanum

“…The absolute DNA content of the parental species was stable during a year of in vitro propagation, when the measurements by FCM were performed three times in several repeats at several months' intervals. The 2C-values for the diploid S. tuberosum clones examined here were close to those presented in earlier publications (1.77, 1.62 and 1.38 pg; Marie and Brown 1993, Valkonen et al 1994, Maciejewska et al 1999, However, the 2C value for S. nigrum reported before (Maciejewska et al 1999), when trout erythrocytes were used as an external standard and DNA was stained with DAPI, was much lower (4.31 pg) than that obtained here (6.79 pg). However, the use of a base (AT) specific dye (DAPI) and an animal standard with different base composition could generate errors in plant genome size determination (Doležel 1991;Marie and Brown 1993).…”

Molecular and morphological characterization of somatic hybrids between Solanum tuberosum L. and S. etuberosum Lindl.

“…In our experiment, somatic hybrids C-13 (?) S. etuberosum were confirmed on the different criteria, as in earlier reports, such as RAPD (Rokka et al 1994;Thieme et al 1997;Naess et al 2001;Barone et al 2002;Szczerbakowa et al 2003;Greplova et al 2008), SSR (Nouri-Ellouza et al 2006;Gillen and Novy 2007; (Thieme et al 1997;Maciejewska et al 1999;Oberwalder et al 2000;Horsman et al 2001;Szczerbakowa et al 2003;Greplova et al 2008), and phenotypic assessment (Novy and Helgeson 1994a;Thieme et al 2008;Szczerbakowa et al 2003;Greplova et al 2008). Confirmation of somatic hybrids was revealed by species-specific diagnostic bands (RAPD and SSR) from both the parental components.…”