REQUIREMENTS FOR DIVISION OF THE GENERATIVE NUCLEUS IN CULTURED POLLEN TUBES OF NICOTIANA

Production of sperm cells by division of the generative cell occurs during growth of Nicotiana (tobacco) pollen tubes through the sporophytic tissue of the style, and is associated with transition to the second phase of pollen-tube growth. When Nicotiana pollen tubes are grown in liquid culture, the extent of generative-nucleus division and the timing of this division depend on the chemical composition of the medium. Addition of reduced forms of nitrogen, either as mixed amino-acids (0.03% w/v of an acid hydrolysate of casein) or as 1 mM ammonium chloride, induces division of the generative nucleus in over 90% of the tubes; 3 mM calcium nitrate does not stimulate division. Individual amino-acids differ in their ability to induce this division. Contaminants in some batches of poly(ethylene glycol), which is a major component of pollen-tube growth media, inhibit generative-nucleus division; this inhibition is greater in the absence of nitrogen, which increases the observed nitrogen-dependence of division. Reduced forms of nitrogen are also required for growth of pollen tubes after division, when callose plugs are deposited. In the absence of nitrogen, growth continues until the point where sperm cell production would normally occur, then ceases. Addition of amino-acids or ammonium chloride thus allows cultured pollen tubes of Nicotiana to progress to their second phase of growth. When Nicotiana pollen is germinated in a complete culture medium at 25-26-degrees-C, sperm nuclei are first observed in the growing tubes after about 10h, and by about 16h most of the tubes have undergone division; at lower temperatures, division is delayed. The timing of division also varies between species of Nicotiana, but division occurs similarly in self-compatible and self-incompatible species. Anaphase in an individual pollen tube is calculated to take less than 4 min. The resultant sperm nuclei usually trail behind the vegetative nucleus, but a variety of arrangements of the three nuclei are observed.