The controlled in vitro differentiation of callus derived from a fern, Pteris cretica L., into gametophytic or sporophytic tissues

Abstract

An attempt was made to control the differentiation of callus, derived from isolated leaves of Pteris cretica L., into gametophytic or sporophytic tissues. In order to provide an adequate supply of callus for these experiments, a study was also made of the conditions promoting the initiation of callus, and its growth in subculture. Several clones of Pteris cretica were established in sterile culture. Callus formation was induced in excised leaves from young sporelings of these clones by culturing the leaves on modified White's medium supplemented with 2% sucrose and 1.0 mg 2,4-dichlorophenoxyacetic acid (2,4-D) per liter. The same treatment induced callus formation in other sporophytic tissues (apogamous buds and stem sections), but no response was obtained from gametophytes. Callus derived from excised leaves could be subcultured and maintained as such, provided that the nutrient medium was supplemented with sucrose and high concentrations of auxin [0.1 mg 2,4-D per liter or 5 mg indole-3-acetic acid (IAA) per liter]. The callus grew equally well when the White's medium was replaced by Moore's medium, or the 0.1 mg 2,4-D per liter by 5 mg IAA per liter. The rate of growth was similar in the light and the dark; under both conditions the callus increased in weight three to four times in 3 weeks. Addition of kinetin (0.001, 0.1, and 1.0 mg/l) or yeast extract (100 mg/l) to the medium did not significantly increase the rate of growth or total yield of callus. When the auxin concentration in the medium was decreased, or when auxin was omitted altogether, differentiation occurred. In the presence of 0.1 g/l or higher concentrations of sucrose, glucose, or fructose, the callus usually gave rise to sporophytes, though the formation of gametophytes was occasionally observed. When the callus was deprived of an exogenous source of sugar, no sporophytes were formed, and the callus instead differentiated into gametophytes. Sugars, therefore, exerted a control over the type of differentiation. This control was not affected by the addition of 0.01 mg 2,4-D per liter to the medium. The part played by sugars in the determination of gametophytic and sporophytic development is discussed in the light of these and previous studies.