POLAR GROWTH OF HORMOSIRA BÁNKSII ZYGOTES IN SHAKE CULTURE

Zygotes of the fucalean alga Hormosira banksii initiate rhizoidal outgrowths in stationary culture 15 br after fertilization and are then recognizably polar. By 24 hr most embryos are two-celled, and a few are four-celled. In a dark-grown population orientation of the developmental axis, as indicated by the direction of the rhizoidal outgrowth, was random around the vertical axis. In a unilaterally illuminated population the rhizoid usually emerged on the shaded side. Zygotes grown in light or darkness in shake culture, where they were continuously reoriented, usually developed as polar embryos, indicating that gradients of environmental factors are not required for initiation of polar growth. Some apolar embryos developed in stationary and shake cultures, but they were most frequent in dark shake cultures. BROWN ALGAE of the order Fucales typically produce spherical eggs in which the cytoplasm appears to be evenly distributed. On germination a rhizoidal outgrowth is produced, and the first cross wall is formed at right angles to this. The rhizoidal outgrowth defines an axis of developmental polarity, and the cross wall separates two cells which are differentiated in structure and function. Numerous external gradients, including light of certain wavelengths, temperature, acidity, and auxin, cause orientation of the rhizoidaloutgrowth (Whitaker, 1940; Nakazawa, 1962), and Jaffe (1956, 1958) has demonstrated that polarity arises epigenetically. These results appear to implicate environmental gradients in the determination of polarity. However, zygotes grown in darkness, where they are claimed to be as free as possible from gradients of environmental factors (Whitaker, 1940), also develop in a polar manner, and dark development is stated to be "without any evident relation to external factors" (Child, 1941). To account for polar development in darkness it has been inferred that unfertilized eggs must either possess a predetermined, labile polarity, or initially lack polarity, which is determined after shedding by environmental differentials of a minor sort (Whitaker, 1940). It might be questioned, however, whether conditions in dark experiments are gradient-free. Zygotes develop on the floor of containers of sea water, attached to a substratum with which they have a constant one-sided contact and with a constant orientation in the gravitational field. Received for publication June 6, 1966. Supported in part by grant GB 2383 from the National Science Foundation. Thanks are due to Professor V. J. Chapman, University of Auiekland, who made laboratory space and facilities available for this study. Although rhizoids in dark-grown populations are sometimes reported to be randomly oriented (Osborn, 1948), the rhizoid has usually been observed to emerge from the lower hemisphere of the cell, and is in contact with the substratum. The substratum has been considered to form a barrier to diffusion of metabolic products from the zygote and may impose a gradient of acidity on it (Whitaker, 1937a). Furthermore, in zygotes developing embedded in agar-solidified sea water and freed from unilateral contact with the substratum, most rhizoids were produced from the lower hemisphere (Whitaker, 1937b) suggesting a gravitational influence on rhizoid emergence. Thus the interpretation that development in darkness occurs without relation to external factors is open to question, and it is still unresolved whether in the absence of all environmental gradients a polar growth pattern would be established. By growing zygotes in shake culture where they are continuously and rapidly reoriented, it should be possible to examine development in a situation where gradients of external stimuli are eliminated. Thus it should be possible to test the necessity for gradients in the initiation of polar development. This paper reports such experiments with the monotypic fucalean alga Hormosira bacnksii, a native of Australia, New Zealand, and adjacent islandsof the Pacific Ocean. The thallus of Hormosira is perennial and dioecious and consists of alternating inflated receptacles, and connectives. All except the basal receptacle are fertile, and gametes are differentiated and liberated throughout the year. MATERIALS AND METHODS-Plants were collected during tidal exposure from the mid-littoral zone at Kohimarama Beach, Auckland,