DEVELOPMENT OF A METHODOLOGY FOR THE PRODUCTION OF DOUBLED HAPLOID LINES IN SOLANUM LYCOPERSICUM L.

The present work aimed to develop an efficient methodology to generate doubled haploid lines (DHLs), through in vitro anther culture, in commercial types of Solanum lycopersicum L. Trials were designed to determine the effects of the main factors in the meiocyte induction method (Shtereva et al., 1998), such as the genotype, microspore developmental stage, and the composition of the in vitro induction medium. Flow cytometry and microsatellites (SSRs) were used to determine the origin of the regenerated plants. Hormonal combinations of IAA (0, 1, 2 and 4 mg/L) and 2ip (0, 0.5, 1 and 5 mg/L) were tried to optimize the induction medium. Calli production and plant regeneration were maximized with 4 mg/L IAA and 1 mg/L 2ip. Eight commercial hybrids, and five S. lycopersicum var. cerasiforme, S. pimpinellifolium, and S. lycopersicum accessions were screened in order to select those with best androgenic response. Though some hybrids regenerated plants in the meiocyte induction method, a male sterile line showed the highest regeneration response, as previously reported (Shtereva et al., 1998; Segui-Simarro and Nuez, 2005). The main difficulty encountered with this method was the regeneration of 61% of polyploidy plants. None of the regenerated plants were haploid, and most of the polyploid plants were as heterozygous as the genotypes from which they were derived. A 56% of the 2n plants were partially homozygous. This might be caused by the meiocyte nuclei fusion, during the induction process, or regeneration from somatic cells. In any case, this has lead to the loss of homozygousity in all regenerated plants, the main desirable characteristic of DHLs. Since these plants might derive from the fusion of meiocyte nuclei in the stages between prophase 2 and telophase 2, other trials were done with meiocytes at later developmental stages.