Physiological and Morphogenetic Studies of Fern Gametophyte and Sporophyte by Aseptic Culture VI. Notes on the Alternation of Generations

Transcription

1 Bot. Map. Tokyo 78: (June 25, 1965) Physiological and Morphogenetic Studies of Fern Gametophyte and Sporophyte by Aseptic Culture VI. Notes on the Alternation of Generations by Yukio KATO * Received January 20, 1965 The alternation of generations as primarily a morphogenetic problem has been discussed by Belly, De Maggio2>, Wetmore et al3~, and Kato4>. In the life cycle of a fern, the spore grows, on one hand, into a small, essentially two-dimensional thallus i.e. a prothallus, without formation of vascular element. The fertilized egg develops, on the other hand, into a large, three-dimensional plant having leaves, stem and roots. The products developed from these two initials are morphologically quite different. The determination of the factors responsible for each element of the morphological cycle or for the alternation of generations, still remains a basic problem, and there is little direct evidence to account for the difference between the gametophytic and the sporophytic generation. The experimental investigation of the life cycle is largely promoted by means of sterile culture. The study described in the present paper was undertaken to determine the extent to which some antimetabolites influence the gametophytes of the fern in aseptic culture, and an experimental approach on the problem will be considered. It would seem to provide a favourable place for investigations on the causal factors underlying the alternation of generations. The experiments described below were carried out using cultured prothalli of Pteris vittata. Induction of Vascular Strands in the Prothallus The normal prothallial colonies, grown aseptically for about 20 days on the Moore's mineral salt-agar medium supplemented with 1 ml/1 Nitsch's minor element solution, were transferred prostrate on the surface of basic medium containing 10 ;ag /ml mitomycin C (Kyowa Hakko Kogyo Co.) and 1 o sucrose. They were cultured for 2 months under the continuous illumination of white light giving about 1,OOOlux at the plant level. When prothalli are grown on the basic medium alone, they remain thin and flat, and become heart-shaped (Figs. la and 2). If the medium contains mitomycin C and sucrose, the prothallus thickens, becomes pad-like, frequently with thin margin of normal prothallial tissue (Figs. lb, 3 and 4). In longer period culture, the thin prothallial margin disappears almost completely. The pad-like prothallus has a thickness of about 1 mm (Fig. 5), and is extremely solid. The thickening tends to occur in prothalli contacted with the medium, and pad-like prothalli at the top of a colony sometimes reform normal thin prothalli from their margins (Fig. 2b). The thickened prothalli usually bear rhizoids and antheridia on the surface (Fig. 5). The sectioned preparations show that such prothalli possess vascular strands as scattered short elements throughout the length (Figs. 6-8). * Biological Institute, Faculty of Science, Nagoya University, Nagoya, Japan.

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3 June, 1965 KATO, Y. 189 Figs Apogamous sporophyte of Pteris vittata developing from elongated prothalli in sterile culture. 10, An outline of apogamous plant; 11, Enlarged view of the plant of Fig 10; r, rhizoid; h, multicellular hair; 12 and 13, Longitudinal sections of an elongated prothallus; 14 and 15, Longitudinal sections of a center of cell multiplication in the apical region. The lower portion of the wedge-like structure consists of small, vacuolated cells while the upper portion small cells with dense protoplasm. Note multicellular hairs (h) on the surface of the apical region; 16-18, Transverse sections of thickened, elongated prothaliial tissue; r, rhizoid. Figs Vascular tissue induced in prothalli of Pteris vittata by supplying 1% sucrose and 10 ig/ml mitomycin. la, Normal, membraneous prothalli on the basic medium (control) ; lb, Thickened prothalli on a medium containing sucrose and mitomycin C; 2, Heart-shaped prothallus grown on the basic medium; 3 and 4, Prothalli having thin, membraneous margins. mp, membraneous margin; r, rhizoid; 5, Cross section of a thickened prothallus having many antheridia. an, antheridium; 6, Section of a prothallus with vascular strands (a, b, c and d in the figure); tr, tracheid; 7, Enlarged view of the tracheid "a" in Fig. 6; 8, Scalariform tracheid; 9a, Transverse section of a gametophytic callus (see Fig. 9b) grown on a complex medium (Kato, 1963), arrow denoting a tracheid; 9b, A gametophytic callus of the solid type.

4 190 Bot. Mag. Tokyo Vol. 78 The Induction of Apogamy Formation of a vascular sporophyte directly from a non-vascular gametophyte without intervention of sex organs is known as apogamy. The occurrence of induced apogamy, therefore, appears independent of the constitution of the nucleus. The apogamous sporelings frequently occurred in cultures of gametophytes of Pteris vittata. In particular, they have been found on prothalli which were grown on the Moore's mineral salt-agar medium supplemented with 10-5 M thiouracil and 1 o sucrose. Prothalli transferred erect on this medium develop into tall and uprightt cultures which reach about 1 cm in length after several months (Figs. 10 and 11). Rhizoids are abundantly produced on the surface. The thickening of the cultures occurs before the initiation of apogamous plants. The prothalli consist of 10 to 15 cell layers, as judged by sectioned material (Figs. 12 and 13). In the next step, a centre of cell multiplication composed of small cells with dense protoplasm is formed on the top or in the interior of a thickened prothallus. The meristematic state extends, around the centre progressively. Thus the meristematic tissue is wedge-like in shape. Multicellular hairs characteristic of sporophytes arise from the surface of the meristematic tissue (Fig. 15). This formation of sporophytic hairs is a characteristic of the occurrence of an apogamous development. At a certain point of the mass off meristematic cells, an apex was formed, and then the first leaf and the stem appear. The first root is formed adventitiously from the stem. These plants have fully expanded leaves and are able to develop into considerably large plants. The development of apogamous plants found in Pteris vittata is similar fundamentally to those described in other plants by many workers. A Sporophyte-like Gametophyte An abnormal gametophyte was obtained from the cultures grown on the agar nutrient medium containing 10-5 M thiouracil and 1% sucrose. As shown in Figs. 19 and 20, the gametophyte is about 3 cm in length and is quite sporophytic in appearance. It consists of the apical region, stalk and thickened prothallus. Thin, membraneous, normal prothalli arise from certain regions of the stalk (Figs. 20b, c). Such marginal prothalli bear many rhizoids on the surface (Fig. 21). Transversal sections show that the stalk consists of small cells (10-20 in number) surrounded by a jacket of larger cells (ca. 20 in number) (Figs. 22 and 23). In the stalk, vascular strand was not found. It is worthy to note that in the apical region structures. have been observed which have been interpreted as archegonia (Figs ). The archegonium-like structures develop exclusively along the margin of the apical region. The starting of their development is generally noted by the conspicuous protrusion of a marginal cell. In the normal gametophyte of Pteris vittata, the archegonium is the first organ to develop. At a glance, the apical portion of the variant closely resembles that of the developing young sporophyte, but the former is lacking the characteristic mat of multicellular hairs. Fig. 28 represents hairs at the apical portion of the normal sporophyte cultured under aseptic condition, and Fig. 29 tracheid showing lignified secondary wall. It is obvious that the variant is still a gametophyte. General Discussion The present results demonstrate that in the sexually reproducing fern, transitions

5 June, 1965 KATO, Y. 191 from gametophytic to sporophytic morphology can be asexually induced by appropriate treatment. In the past, much effort has been paid to rise the growth rate of plant tissue in culture: better media, the use of growth-promoting substances, liquid media and so on. In the present study, thiouracil, a pyrimidine analogue relating to RNA metabolism, and mitomycin C, which is known to inhibit specially the synthesis of DNA in bacteria, were applied for the investigation of induction of transition from gametophytic to sporophytic morphology. Figs A sporophyte-like gametophyte grown on the Moore's mineral salt-thiouracil-agar medium supplemented with sucrose. 19, An outline; 20, Enlarged view of the plant of Fig. 19; a, thickened prothallus with many rhizoids; b and c, thin, membraneous prothalli developed from the stalk; d, stalk; e, apical region; 21, Section of prothallial tissue arisen from the margin of stalk; 22 and 23, Sections of the stalk, consisting of small cells surrounded by a jacket of large cells; 24 and 25, Transverse sections of the apical region. Note archegonia in the periphery; 26 and 27, Enlarged view of archegonia produced in the periphery of the apical region; 28 and 29, Normal sporophyte and its transverse section. Note multicellular sporophytic hairs.

6 192 Bot. Mag. Tokyo Vol. 78 Wetmore et al.3~ reported that prothalli of Onoclea, Osmunda and Todea planted on the usual Knudson's mineral salt-agar medium with sucrose and auxin possessed vascular tissue. In experimentally induced solid callus tissue of Pteris vittata,, tracheids were frequently found in a medium containing sucrose (Figs. 9a and b). In spite of the presence of tracheids, certain types of solid calli showed no differentiation into sporophyte for a long time. Therefore, tracheids can occur in prothallial tissue without the presence of apogamy. The thickness of the gametophyte is necessary for induction of vascular tissue. This is also a requisite condition for induced apogamy, and is controlled by the sugar in the nutrient medium.5~ The experiment conducted by Whittier et al.6-8> shows that apogamy can be induced in various species of ferns by manipulating the concentration of carbohydrate in the medium. It is suggested that the transition from gametophytic to sporophytic morphology may be promoted by an increase in available energy. Whittier's interpretation, that a high level of energy-providing substrate enables the prothallus to shift from the prothallial pattern to sporophytic pattern, can apply to the in vitro differentiation of fern callus tissues. Bristow9~, using a callus derived from the sporophyte of Pteris cretica, found that the callus regenerated sporophytes and gametophytes on media with high and low concentrations of sugars, respectively. Similar results were obtained in Pteris vittata (Table I). Table I. Differentiation of callus tissue culture (Pteris vittata).

7 June, 1965 KATO, Y. 193 ments in which the initial cells of the two generations germinate. When the zygote itself of the fern, Todea barbara, was excised and cultured, it failed to develop into the usual three-dimensional sporophyte. Instead, it produced an irregular, rather two-dimensional, thalloid structure resembling haploid gametophyte12>. However, the experiments which determine the validity of Lang's hypothesis-to germinate a spore in the conditions in which the zygote begins its life-have not given positive results'. The experiments described in the present paper are still preliminary and fragmentary in nature. Therefore, the author believes the following studies may be required for the elucidation of the mechanism controlling the development of form and the alternation of generations: studies on the difference of fine structure between the zygote and the spore, the facts to support Lang's hypothesis relating to the spore, the conditions inducing apogamy and apospory, the relationship between metabolic pattern andd morphogenetic change in the gametophyte and the sporophyte, immunol_ogical studies for the study of the protein at various developmental_ stages of plants of both generations and so on. Conclusion The author utilized sterile culture techniques as an approach to the morphogenetic problem of the fern life cycle. It has been shown in the present paper that mitomycin C and thiouracil in the medium are effective in alternating the development of fern gametophytes. Although it is too early yet to report any experimental result, three cases obtained with Pteris vittata afford an opportunity to study some of the conditions controlling the development of form. Further experimental studies for the elucidation of the mechanism of the alternation of generations are awaited. I am indebted to Professor M. Kumazawa, for his helpful suggestions and encouragement. This work is supported by a Grant in Aid of Scientific Research of the Ministry of Education (No ). References 1) Bell, P. R., Jour. Linn. Soc. (Bot.). 56: 188 (1959). 2) De Maggio, A. E., Jour. Linn. Soc. (Bot.). 58: 361 (1961). 3) Wetmore, R. H., De Maggio, A. F., and Morel, G., Jour. Indian. bot. Soc. (Maheshwari Comm. Vol.) 42A: 306 (1963). 4) Kato, Y., Biol. Sci. 16: 13 (1964). 5) Whittier, D. P., and Steeves, T. A., Canad. Jour. Bot. 38: 925 (1960). 6) - - and --, ibid. 40: 1525 (1962). 7) --, Amer. Jour. Bot. 51: 730 (1964). 8) --, Amer. Fern Jour. 54: 20 (1964). 9) Bristow, J. M., Develop. Biol. 4: 361 (1962). 10) Lang, W. H., New Phytol. 8: 104 (1909). 11) -, Rep. Brit. Ass. Manchester: 701 (1915). 12) De Maggio, A. F. and Wetmore, R. H., Amer. Jour. Bot. 48: 551 (1961).