Physiological and Morphogenetic Studies of Fern Gametophytes and Sporophytes in Aseptic Culture1 IV. Controlled differentiation in leaf callus tissues

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1 Physiological and Morphogenetic Studies of Fern Gametophytes and Sporophytes in Aseptic Culture1 IV. Controlled differentiation in leaf callus tissues Yukio Kato Biological Institute, Faculty of Science, Nagoya University, Nagoya, Japan Received September 15, 1964 Comparatively little is known about growth and morphogenesis of sporo phytic callus in ferns. Morel (1956) cultured the callus tissues derived from isolated leaves of Adiantum pedatum, and those from stem section of Selaginella and Osmunda cinnamomea. Callus tissues of the last two species were cultured by him for about two years on Knop's medium supplemented with glucose. More recently, Bristow (1962) reported in Pteris cretica L. the controlled in vitro differentiation of callus into gametophytic or sporo phytic tissues. In Pteris vittata, the present author (1963) obtained gameto phytic calli by the dark-culture method. Out of various types of calli obtained, the friable one has already been described in detail (Kato 1964). The purpose of the present paper is to elucidate the fact that calli derived from sporophytic tissues are differentiated into gametophytes and sporophytes. Materials and methods The leaves of Pteris vittata L. were detached from young sporophytes aseptically cultured, and they were transferred to flasks containing culture media. All experiments were carried out under the sterile condition. When the excised leaves were horizontally placed on the agar medium, callus forma tion was initiated at the certain regions of the leaf contacting with the agar surface. Among a large number of complex media tested, most effective one in inducing callus formation was a Moore's medium (Moore 1903) supplemented with 1ml/l Nitsch's trace element solution (Nitsch 1951), 2% sucrose and 0.1mg/l 2.4-D, its ph being 5.8 or so. Powdered yeast extract in the presence of 2.4-D was also effective on the callus growth in con centrations of 0.1 to 1gm/l. In all cases the media were solified with 0.8% agar. When initial tissue masses reached approximately 2mm in diameter, subcultures were made and maintained. Thus obtained calli were grown 1 The main title of this series has been altered as above. This is because of necessity of comparative studies with sporophyte. 5*

2 68 Y. kato Cytologia 30 under the continuous illumination of white light of about 1,000 lux at the plant level, and exceptinally in the dark in a growth chamber where the temperature was maintained at 28 Ž. The calli were transferred to new media every 4 weeks. The subcultured calli grew very well, being three to four times in weight for 3 weeks. Figs Pteris vittata. 1 and 2, cell suspension obtained by a liquid medium culture of callus of somewhat solid type. 3-6, suspended cells obtained from cultured callus of the friable type. Note a great variation of cell size and shape. Experiments were set up to determine whether the type of differentiation is affected by nutritional supplements or not. The culture media employed were free from auxin and/or 2.4-D. As culture vessels, 50 and Erlenmeyer flasks containing respectively 20 and 40ml of the nutrient solution were used. Results In order to know the relationship between the callus-inducing ability of

3 1965 Physiological and Morphogenetic Studies of Fern IV 69 leaves and the age of sporophytes, the leaves detached from young sporo phytes still attaching to the gametophytes and those detached from older ones having leaves of the adult form were compared to each other. Generally speaking, the earier the age of sporophytes was, the stronger the callus inducing ability was. Apospory, direct regeneration of gametophytes from detached leaves, was not found on any medium tested, and the cultured leaves which failed to form the callus finally died without exception. The calli on the complex medium (containing yeast extract) were in definitely proliferative without any differentiation. They were represented by two types; the one by the smooth, round, somewhat solid colony of Figs Pteris vittata. 7, callus tissue of the friable type derived from an excised leaf, propagated for about two months on yeast extract-2.4-d-sucrose medium without being subcultured. 8, sporophytic callus tissue of somewhat solid type grown on agar medium for about two months. 9-11, callus grown on Moore's medium with mineral constituents but without sucrose and 2.4-D. ga..gametophyte (prothallium). ic..isolated cell. tissues (Fig. 8) and the other by the flat, loose, extremely friable masses which were readily broken apart at a touch (Fig. 7). As described by Torrey and Shigemura (1957), the difference of types may be ascribed to the relative amount of 2.4-D and yeast extract in the medium. However, experiments were not carried out to determine the nutrient conditions which induce the friablity of callus. Calli of the friable type were pale green under the white light and tended to turn greenish brown after a month. Accompanied with this colour change, the growth was retarded. When the callus is continuously agitated in the test tube, a dense suspension is readily obtained. Such suspension

4 70 Y. Kato Cytologia 30 can be transferred to agar medium for further cultivation. The cells thus Figs Pteris vittata. Suspended cells obtained by a liquid culture of callus of somewhat solid type , phase-contrast microphotograph. cultivated are extremely irregular in shape-spherical to spiral, lanky, or code-like and different in size-from 10 ~30ƒÊ to over 70 ~800ƒÊ (Figs. 3, 4,

5 1965 Physiological and Morphogenetic Studies of Fern IV 71 5, 6 and 25). The degrees of cell variation differ from callus to callus, depending on the age of the culture. It is evident that callus mass increases its size by random cell division and irregular cellular enlargement. On the other hand, the callus of the solid type is characterized by the unorganized, homogeneous nature of cells (Figs. 1 and 2). However, elongated, giant cells, irregularly shaped cells and cells with protuberance (Figs. 12 and 13) were rarely seen. The callus tissue itself becomes a suspension, to some extent, in the liquid medium if severely agitated. A callus could forcibly be separated into viable single cells and cell aggregates but remained still as tissue pieces. The cells composing a callus are considerably similar to each otheir in size, being 20 ~100ƒÊ in the mean. The following experi ment evidently proves that these calli still possess the ability of differentiation; if separated small pieces of the friable callus tissue are transferred from the complex medium to the agar medium containing merely inorganic salts, growth of the callus itself does not occur, while differentiation of callus tissue into a true gametophyte does occur (Figs. 9, 10 and 11). The first indication of the gametophytic outgrowth was the appearance of many chloro plasts in the cells, the remarkable cell elongation and the formation of rhizoids. The gametophyte thus initiated showed the same developmental pattern with the usual gametophyte germinated from a spore, and the archegonia and antheridia were produced on the cordate prothallium. Thus three tetraploid sporophytes were obtained. There was no difference in appearance between the usual haploid prothallium originated from a spore and the diploid pro thallium developed aposporously from a leaf callus tissue, except for the cytological feature. As shown in Table 1, the diploid gametophyte is generally larger than the haploid in some cytological features. Table 1. Differences of cytological features of the usual haploid gametophyte, gametophytic callus, aposporous diploid gametophyte and sporophytic callus The tetraploid sporophytes have thick and dark green leaves and larger hairs. The dimension of their stomata was measured and compared with that of the diploid ones, the mean values being respectively 23 }1.1ƒÊ and 40.5 }0.7ƒÊ in length. Anomalous stomata and malformed leaves, such as reported by Takahashi (1962), were not found in the present material,

6 72 Y. Kato Cytologia 30 Callus was transferred from the complex medium to a medium consisting of the mineral constituents of Moore's solution, supplemented with 1 or 2% Figs Scaly hairs of Pteris vittata. 23 and 24, scaly hairs on an intact plant , cells of the friable callus, exhibiting their various shapes and sizes. Hair initials are originated by a polar separation of cells with dense protoplasm from cells with less protoplasma (i.e. unequal division). h..scaly hair. hi..hair initial. ic..isolated cell. gc..giant cell. 32, callus tissues derived from germinated spores and obtained by the dark culture method (Kato 1963). left. solid callus of smooth round shape. left. friable callus. sucrose in the absence of 2.4-D. A fragment of callus was scraped and examined after two weeks of culture. On this medium, scaly hairs of various

7 1965 Physiological and Morphogenetic Studies of Fern IV 73 developmental stages were observed in the friable tissues (Figs ). Elon gated giant cells divided frequently into unequal two cells, and one of their daughter cells developed into a normal, well-organized scaly hair (Figs. 29 and 30), the differentiation of which having no correlation to the regeneration of leafy shoots or whole plants. Differentiation of the friable tissue into a whole sporophyte was rather infrequent. Discussion and conclusion Callus-like growths from gametophytic tissues of ferns have arisen spon taneously or have been induced experimentally (Steeves et al. 1955, Partanen 1958, Partanen and Nelson 1961, and Kato 1963, 1964). On the other hand, sporophytic callus was induced from excised leaves of young sporelings on a modified White's medium supplemented with 2% sucrose and 1.0mg 2.4-D per litre (Bristow 1962). Adopting the same methods in Pteris vittata, the present author also obtained some sporophytic calli. The morphological features of cells compos ing the sporophytic callus (Figs. 1-6 and 12-22) are considerably different from those of the gameto phytic callus (Figs. 32, 33 and 34). Tissues of the former consist of elon gated cells exhibiting various sizes, while those of the latter consist mostly of small isodiametic ones with the average diameter of 35ƒÊ. Among the data ob tained in the present ex Figs Gametophytic callus in Pteris vittata. 33, cells from callus tissue grown in the light, exhibiting their isodiametic shape. Note the formation of many chloroplasts. 34, cell aggregates separated from a friable callus, 35 and 36, differenitiation of callus into gametophytic outgrowths (filamentous prothallium) on a defined medium containing inorganic salts. c..cell cluster of an original callus tissue. p..prothallium. r..rhizoid. periment, most interesting fact is that the callus tissue is variously differentiated corresponding to different kinds of culture media: the gametophyte is produced on a medium containing inorganic salts only, while scaly hairs and even whole sporophytes on a medium deprived of an exogeneous source of auxin. In intact plant,

8 74 Y. Kato Cytologia 30 hairs are formed throughout the course of leaf development by repeated periclinal division of epidermal cells (Figs. 23 and 24). At the beginning of crozier development, the characteristic heavy mat of hairs is striking, the hair formation continues throughout the developmental stage of crozier (Steeves and Briggs 1958). In the present case the formation of the hairs has no correlation to that of leafy shoots or whole plants. As shown in Figs. 35 and 36, gametophytic outgrowth occurs only when the callus initiated from spore is transferred to a defined medium containing inorganic constituents only from a complex medium containing sucrose or glucose. Bristow (1962) found that the callus usually gave rise to sporophytes on the media containing low concentrations of auxin and higher concentrations of sugars. Whittier and Steeves (1960) reported that sucrose promoted the occurrence of apogamy, and Steeves et al. (1955) also found that the gameto phytic callus which was apogamous on the sucrose-containing media tended to revert to a perfect gametophyte on the media not containing sucrose. The data obtained from the present experiment and other studies suggest that certain carbohydrates play some part in the determination of gametophytic and sporophytic differentiation. The author wishes to express his gratitude to Prof. M. Kumazawa for his advise and helpful criticism in the preparation of the manuscript. Literature cited Bristow, J. M The controlled in vitro differentiation of callus derived from a fern, Pteris cretica L., into gametophytic or sporophytic tissues. Develop. Biol. 4: Kato, Y Physiological and morphogenetic studies of fern gametophytes in aseptic culture 1. Callus tissues from dark-cultured Pteris vittata. Bot. Gaz. 124: Physiological and morphogenetic studies of fern gametophytes in aseptic culture. III. Growth and differentiation of single cells isolated from callus tissues of Pteris vittata. Cytologia 29: Moore, G. T Methods for growing pure cultures in algae. Jour. Aplli. Microscop. and Lab. Methods, 6: 2, 309. Morel, M. G Nouvelles methodes de culture de tissus. Rev. Gen. Bot. 63: Proliferation des feuilles d'adiantum pedatum cultivees in vitro. Rev. Gen. Bot. 63: Nitsch, J. P Growth and development in vitro of excised ovaries. Amer. Jour. Bot. 38: Partanen, C. R Quantitative technique for analysis of radiation-induced tumorization in fern prothalli. Science 128: and Nelson, J Induction of plant tumors by ultraviolet radiation. Proc. Nat. Acad. Sci. U. S. 47: Steeves, T. A. and Briggs, W. R Morphogenetic studies on Osmunda cinnamomea L. -The origin and early development of vegetative fronds. Phytomorpho. 8: , Sussex, I. M., and Partanen, C. R In vitro studies on abnormal growth of prothalli of the bracken fern. Amer. Jour. Bot. 42: Takahashi, C Cytological study on induced apospory in ferns. Cytologia 27: Torrey, J. G. and Shigemura, Y Growth and controlled morphogenesis in pea root callus tissue grown in liquid media. Amer. Jour. Bot. 44: Whittier, D. P. and Steeves, T. A The induction of apogamy in the bracken fern. C anad. Jour. Bot. 38: