Induction of embryogenesis in isolated microspores of tulip

Abstract

Isolated microspores of tulip ( Tulipa gesneriana) cultivars ‘Leen van der Mark’ and ‘Rosario’ could be induced to form embryos. Microspores of a young developmental stage, i.e. early and mid unicellular, were the most responsive. The first sporophytic divisions, resulting in microspores with three or four vegetative-like nuclei were observed after 1.5–2 weeks in liquid culture. Based on the initial number of microspores, about 0.5–1.0% started to develop sporophytically. Of these induced microspores, 5–20% continued sporophytic development into multicellular structures, which could be observed 5–6 weeks after the start of the culture. Most multicellular structures did not show organisation, contained cells of varying size, and stopped growing after initial divisions. However, 1–5% of the multicellular structures developed into compact globular or embryo-like structures. The latter were elongated and had a smooth epidermal layer, resembling a zygotic embryo. These microspore-derived embryos could be matured by successive transfer to solid culture media with 8% and 3% sucrose, respectively, and were capable of (partial) germination after a 2–3 month cold period to break dormancy. The embryos of Leen van der Mark mainly developed the primary root after germination. Rosario showed a better embryo formation and germination, and a few seedlings were obtained. Although abnormal looking, all structural organs such as the cotyledons, primary roots and the stolon-like droppers were present. Formation of such a dropper is important, because at its end the new bulblet should develop. Green shoots were obtained via regeneration of embryo-derived tissue. Most of the Rosario embryos analysed by flow cytometry were diploid, whereas Leen van der Mark embryos were predominantly haploid. This suggests that chromosome doubling might promote growth and development of microspore-derived embryos.