Diploidization in megagametophyte-derived cultures of the gymnosperm Larix decidua

Tested haploid embryogenic lines (n=12) of Larix dedicua Mill, initiated from megagametophyte tissue were maintained on half-strength LM medium without growth regulators. The cultures were analyzed for ploidy level after 1-9 years. All lines tested were found to have doubled (2n=24) their chromosome number at the end of the experiment, though there were a few lines that still gave occasional haploid counts. Flow cytometric data of embryogenic tissue confirmed these results. Protoplasts were stained in ethidium bromide, and cultured human leucocytes and chicken erythrocytes were used as internal standards. Haploid megagametophytes from immature seeds of L. decidua and known diploid culture lines of a related hybrid (L. x eurolepis) were also analyzed by flow cytometry. Haploid reference material had 12.3-13.6 pg DNA per cell, whereas formerly haploid callus lines had an average of 25.0 pg DNA per cell. The one exception was a known, genetically unstable line of L. decidua (34.8 pg DNA per cell). The diploid cell line of L. x eurolepis had 27.6 pg DNA per cell. The results show that spontaneous diploidization of megagametophyte lines is relatively rapid and that both haploid and dihaploid lines are embryogenic in larch.