The Effect of Sucrose on the Differentiation of Excised Fern Leaf Tissue into Either Gametophytes of Sporophytes

Abstract

Excised juvenile leaves of Microgramma vacciniifolia (Polypodiaceae) develop sporophytic regenerants when grown on mineral agar with sucrose. The ratio of sporo-phytes to gametophytes produced from the leaf tissue increases with higher percentages of sucrose such that at 4%V sucrose, the induction of aposporous gametophytes is a rare occurrence. Experiments varying the osmotic potential with sorbitol and those holding the osmotic potential of the culture medium constant while varying the sucrose level indicate that the effect of sucrose on the differentiation of fern leaf tissue into either gametophyte or sporo-phyte is nutritional rather than osmotic. A significant effect of sucrose in altering the differentialion of fern leaf tissue is the increased rate of senescence promoted by high sucrose concentrations. Both apogamy, the formation of a haploid sporophyte directly from a gametophyte without syngamy, and apospory, the production of a diploid gametophyte directly from sporophytic tissue, circumventing meiosis, can be induced in vitro in ferns and have been shown to be mediated by the concentration of sucrose in the culture medium. High levels of sucrose (>1 %) bring about the formation of apogamous sporophytes (16), whereas the lack of sucrose or low levels of sucrose (< 1 %) in the culture medium promote apospory (3, 13). In addition to producing aposporous gametophytes, sporophytic fern tissue can also give rise to regenerated sporophytic plantlets (4, 10). This has also been found to be dependent on the percentage of sucrose in the culture medium. In the epiphytic fern species Microgramma vacciniifolia, either aposporous gametophytes or sporophytic plantlets can be induced to form in vitro on the laminar surface of the juvenile leaves. Significantly, regenerated sporophytes as well as gametophytes are readily induced on leaves growing in culture medium lacking sucrose. Because both generations of the life cycle can be induced on excised M. vacciniifolia leaves without added sucrose, the effects of increasing sucrose concentrations on gametophytic and I. Paul, Minn. 55108. sporophytic differentiation can be examined. Besides serving as a metabolite, sucrose, like any solute, exerts an osmotic effect. It is therefore necessary to determine whether sucrose is acting as a nutrient or an osmoticum when investigating any sucrose-mediated phenomenon. MATERIALS AND METHODS Cultures of juvenile plants were established from callus tissue of rhizomes of Microgramma vacciniifolia (Langsd. and Fisch.) Copeland (Polypodiaceae) grown on modified Prague's (14) medium (NH4N03, 400 mg/l; Machlis and Torrey [9] micro-nutrients; 2% [w/v] sucrose). Under the greenhouse and culture conditions employed, M. vacciniifolia was completely infertile. To test the effects of sucrose on the differentiation of either sporophytic or gametophytic outgrowths, juvenile leaves 2 to 5 mm in length were excised from young plants growing in vitro and transferred to 125-ml Erlenmeyer flasks containing 50 ml of Prague's nutrient medium, pH 5.7, 0.8% agar, supplemented with various carbon sources or osmotic agents, and autoclaved at 15 lb/in2 pressure for 15 min. Three juvenile leaves were transferred to each flask. The leaves were grown with 16-hr light, 8-hr dark, at 23 C. After 6 weeks, the leaves were harvested and scored for production of either aposporous gametophytes, regenerated sporo-phytes, or intermediates. Intermediates were classed as those structures possessing features of both sporophytes and gameto-phytes and in general were found to have stomates and vascular tissue on a crenulated but slightly thickened prothallus. The number of the growths produced on the excised leaves were totaled with regard to each treatment. The number of leaves with sporophytes produced divided by the number of leaves showing gametophytes was used as a convenient method for comparison of the various sucrose culture media. This number is designated as the s/g ratio. In scoring the percentage of growths produced, only viable leaves were counted since an increase in mortality was evident with increased concentrations of sucrose. RESULTS Effects of Sucrose. When leaves of M. vacciniifolia were excised and transferred to culture medium lacking sucrose, both gametophytes and sporophytes developed on the leaf surfaces (Fig. 1). The addition of sucrose to the culture medium was found to have a profound effect on the promotion of sporophytic over gametophytic development on excised Microgramma leaves. Figure 2 illustrates a leaf which had been cultured on medium with 2% sucrose and which after 6 weeks was covered with new sporophytic plantlets. Increasing the sucrose concentration from 0 to 4% (w/v) greatly favored this increase in sporophyte development (Fig. 3). At 0% sucrose, 46.5%-of the viable juvenile 390