Abstract
The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Aruga K, Yamamoto K, Nakajima T (1982) Nucleic acids synthesis required for embryogenic induction in pollen grains of Nicotiana tabacum L. In: Fuwara A (ed) Plant tissue culture. Maruzen, Tokyo, pp 537–538
De Paepe R, Koulou A, Pham J, Brown S (1990) Nuclear DNA content and separation of Nicotiana sylvestris vegetative and generative nuclei at various stages of male gametogenesis. Plant Sci 70:255–265
Dolezel J (1989) Improvement of accuracy of scanning absorption measurement of nuclear DNA content in plants. Acta Histochem 86:123–127
Fan Z, Armstrong K, Keller WA (1988) Development of microspores in vivo and in vitro in Brassica napus L. Protoplasma 147:191–199
Hause G, Hause B, Van Lammeren AAM (1992) Microtubular and actin filament configurations during microspore and pollen development in Brassica napus cv ‘Topas’. Can J Bot 70:1369–1376
Hause B, Hause G, Pechan P, Van Lammeren AAM (1993) Cytoskeletal changes and induction of embryogenesis in microspore and pollen cultures of Brassica napus L. Cell Biol Int 17:153–168
Heslop-Harrison J, Heslop-Harrison Y (1970) Evaluation of pollen viability by enzymatically induced fluorescence; intracellular hydrolysis of fluorescein diacetate. Stain Technol 45:115–120
Lacy ER, Kuwayama H, Cowart KS, King JS, Deutz AH, Sistrung S (1991) A rapid, accurate, immunohistochemical method to label proliferating cells in the digestive tract: a comparison with tritiated thymidine. Gastroenterology 100:259–262
Levi M, Sparvoli E, Sgorbati S, Chiante D (1987) Rapid immunofluorescent determination of cells in the S phase in pea root meristems: an alternative to autoradiography. Physiol Plant 71:68–72
Lichter R (1982) Induction of haploid plants from isolated pollen of Brassica napus. Z Pflanzenphysiol 105:427–434
Lilie RD (1951) Simplification of manufacture of Schiff reagent for use in histochemical procedures. Stain Technol 26:163–165
Pechan P, Keller WA (1988) Identification of potentially embryogenic microspores in Brassica napus L. Physiol Plant 74:377–384
Pfosser M (1989) Improved method for critical comparison of cell cycle data of asynchronously dividing and synchronized cell cultures of Nicotiana tabacum. J Plant Physiol 13:741–745
Pretova A, De Ruijter NCA, Van Lammeren AAM, Schel JHN (1993) Structural observations during androgenic microspore culture of the 4c1 genotype of Zea mays L. Euphytica 65:61–69
Stroobants C, Sossuntzov L, Migniac E (1990) DNA synthesis in excised tobacco leaves after Bromodeoxyuridine incorporation: immunohistochemical detection in semi-thin Spurr sections. J Histochem Cytochem 38:641–647
Telmer CA, Simmonds DH, Newcomb W (1992) Determination of developmental stage to obtain high frequencies of embryogenic microspores in Brassica napus. Physiol Plant 84:417–424
Van Lammeren AAM, Keijzer CJ, Willemse MTM, Kieft H (1985) Structure and function of the microtubular cytoskeleton during pollen development in Gasteria verrucosa (Mill.) H Duval. Planta 165:1–11
Wang H, Cutler AJ, Saleem M, Fowke LC (1989) Immunocytochemical detection of DNA synthesis in plant cells. J Plant Physiol 135:15–20
Wang H, Cutler AJ, Fowke LC (1991) DNA replication and the development of preprophase bands in soybean protoplast cultures. Physiol Plant 82:150–156
Zaki MAM, Dickinson HG (1990) Structural changes during the first divisions of embryos resulting from anther and free microspore culture in Brassica napus. Protoplasma 156:149–162
Zaki MAM, Dickinson HG (1991) Microspore-derived embryos in Brassica: the significance of division symmetry in pollen mitosis I to embryogenic development. Sex Plant Reprod 4:48–55
Zarski V, Garido D, Rihova L, Tupy J, Vicente O, Heberle-Bors E (1992) Derepression of cell cycle by starvation is involved in the induction of tobacco pollen embryogenesis. Sex Plant Reprod 5:189–194
Author information
Authors and Affiliations
Additional information
Communicated by G. Wenzel
Rights and permissions
About this article
Cite this article
Binarova, P., Straatman, K., Hause, B. et al. Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L.. Theoret. Appl. Genetics 87, 9–16 (1993). https://doi.org/10.1007/BF00223736
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00223736