Polyploidization in embryogenic microspore cultures ofBrassica napus L. cv. Topas enables the generation of doubled haploid clones by somatic embryogenesis

Abstract

Embryogenic microspore and pollen culture followed by subculture of microspore-derived plantlets enabled the production of clones ofBrassica napus cv. Topas. Flow-cytometric analysis revealed that most microspore- and pollen-derived embryos (pEMs) were haploid initially. Spontaneous diploidization occurred at the globular stage of the pEMs, and was expressed as the relative increase of the 2C and 4C nuclear DNA content. Diploidization occurred throughout various organs of the pEMs and resulted in the formation of haploid and doubled haploid chimerics. In some embryos, nearly all cells were doubled haploid. From early cotyledon stage onward, pure haploid embryos were not observed anymore. At late cotyledon and germination stages, pure doubled haploid embryos and plantlets increased in number. Tetraploid pEMs were found occasionally. A culture regime was established to induce somatic embryos on the pEM-derived young plantlets. The ploidy of the somatic embryos varied highly and tended to be the same as that of the tissue at the initiation site on the pEM-plant. The results show that during the embryogenic development ofB. napus microspores, spontaneous diploidization occurs at globular stage, and increases progressively, resulting in the formation of chimerical haploid and doubled haploid plants as well as pure doubled haploid plants; ploidy neither affects pEM development at embryo developmental stages nor somatic embryogenesis, that starts on young pEM-derived plantlets; doubled haploid somatic embryos can be cloned from single pEM-derived plantlets; and doubled haploid embryos develop to fertile plants.