Somaclonal variation of haploid in vitro tissue culture obtained from Siberian larch (Larix sibirica Ledeb.) megagametophytes for whole genome de novo sequencing

Haploids in Conifer Species: Characterization and Chromosomal Integrity of a Maritime Pine Cell Line

“…[19] In the last decade, advances in genome sequencing have prompted the development of protocols to generate true haploid cell lines to be used as a DNA source for massive genome analyses of conifer species. As a result of these efforts, haploid cell lines from megagametophytes of Pinus pinaster [19] and Larix sibirica [18] have been obtained and the haploid status studied using DNA markers. The developmental stage of the megagametophyte is an important factor in these studies, although the optimal stage for the induction of haploid cultures may differ among species and genotypes [6,7,18,19].…”

“…As a result of these efforts, haploid cell lines from megagametophytes of Pinus pinaster [19] and Larix sibirica [18] have been obtained and the haploid status studied using DNA markers. The developmental stage of the megagametophyte is an important factor in these studies, although the optimal stage for the induction of haploid cultures may differ among species and genotypes [6,7,18,19]. Modified Murashige and Skoog [20] or Litvay (as described in [21]) media, supplemented with different concentrations of 2,4-D with or without cytokinins, have been widely used to initiate callus formation and for further proliferation in conifers [6,7,18,19].…”

“…The developmental stage of the megagametophyte is an important factor in these studies, although the optimal stage for the induction of haploid cultures may differ among species and genotypes [6,7,18,19]. Modified Murashige and Skoog [20] or Litvay (as described in [21]) media, supplemented with different concentrations of 2,4-D with or without cytokinins, have been widely used to initiate callus formation and for further proliferation in conifers [6,7,18,19]. However, is well documented that long-term cultures may induce instability of the regenerants [22].…”

“…. ), genic (mutation), or epigenetic [16,18,[23][24][25][26][27]. Since in vitro variation in chromosome sets has been recorded [12,13,16,18,19], it is crucial to validate the suitability of the starting material when approaching de novo whole genome sequencing based on a single haploid tissue.…”

“…Since in vitro variation in chromosome sets has been recorded [12,13,16,18,19], it is crucial to validate the suitability of the starting material when approaching de novo whole genome sequencing based on a single haploid tissue. Different strategies can be used to confirm the presence of all chromosomes in a haploid state and the absence of large deletions involving the loss of chromosome arms [18], including chromosome counting, cytometry, and genotyping using molecular markers [26]. Karyological analysis enables unfailing detection of all modifications in chromosome number, but it is sometimes difficult to obtain a high enough number of cells containing metaphases with chromosomes sufficiently contracted and separated to provide a reliable chromosome count [19].…”