Direct and indirect detection of 2n eggs in hybrid diploid families derived from haploid tbr × wild species crosses

Abstract

The analytic-synthetic scheme makes use of haploidization and 2n gametes-mediated sexual polyploidization to incorporate genetic diversity from wild related species in potato breeding. Whereas 2n pollen can be directly screened by microscopy, 2n eggs are routinely screened indirectly by interploid crosses, a time-consuming and inaccurate technique. In five haploid tbr × wild diploid species families with variability for agronomic characters, 2n eggs were screened indirectly to incorporate this germplasm into a useful form and directly by microscopy to establish the feasibility of this approach. For the indirect screening, at least 15 flowers of each of 49 genotypes/family were crossed with a commercial cultivar. Eighty-five fruits were produced; only 15 of them had seeds (1–25). Chromosome numbers were determined in a random sample of five seeds/fruit, resulting diploid in two genotypic combinations and tetraploid in another two. For the direct screening, eight combinations of staining and destaining (times and concentrations) were evaluated in detached ovules to adjust a clarification technique. The best combination was one day of staining and one day of destaining in 0.5 % acetic acid. In an indirectly detected diplogynoid, the average diameters of the central cell nuclei (CCN) and nucleoli (CCNu) were, respectively, 9.3 μ and 3.4 μ. Gametophytes with diametres 1.26 times the average were considered 2n. By crosses, eleven seeds were obtained in one fruit of this clone whereas the 2n egg frequency determined by microscopy was 7%. The direct technique is easy to carry out once the eye is trained and its results better reflect actual 2n gamete frequencies.