CONSEQUENCES OF ULTRAVIOLET RADIATION ON THE DIFFERENTIATION AND GROWTH OF FERN GAMETOPHYTES

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1 CONSEQUENCES OF ULTRAVIOLET RADIATION ON THE DIFFERENTIATION AND GROWTH OF FERN GAMETOPHYTES BY YUKIO KATO Biological Institute., Faculty of Science, Nagoya University., Nagoya, Japan {Received 9 April 1963) SUMMARY The effects of ultraviolet light (u.v.), principally at 2537 A, on the differentiation and growth of gametophytes in Pteris vittata, Osmunda japonica and Dryopteris varia were investigated. It was ascertained that a single cell may be isolated by u.v.-irradiation and that a normal gametophyte can be regenerated from such a cell. When gametophytes grown under low intensity (150 lux) white light before u.v.-irradiation were irradiated with a high dose, an apico-basal gradient of ability to survive was clearly shown. The survival of each of the cells composing a protonema seems to be related to two different factors; a marked ability for cell division or rapid recovery from the damage of u.v. at the apical region and a high resistance to u.v. at the basal region. Under both culture conditions, cells of a definite length alone survive. General effects induced by the u.v.-irradiation include reversal of the polarity in a protonema and modification of the developmental axis. In addition, swelling of the rhizoid and protonema cells, and occurrence of 'rhizoidal protonema' were also described. INTRODUCTION It has been reported that in fern protonema or protonema cells there is an apico-basal polarity connected witb gradients in physiological properties such as osmotic value, permeability, reaction to dyes, resistance to alcohol (Reuter, 1953), and susceptibility to colcbicine (Nakazawa, 1959). In tbis paper tbe results of tbe appbcation to various developmental stages of gametopbytes of ultraviolet (u.v.) irradiation on tbe gradient of susceptibility and reversal of tbe polarity in tbe protonema bave been studied. In addition, it bas been ascertained tbat a single cell isolated from young gametopbytes of Pteris vittata by tbis treatment may grow into a mature gametophyte in some cases. MATERIAL AND METHODS Tbe source of u.v. ligbt was a 15 W germicidal lamp (made by Toshiba Electric Co.), wbicb produces approximately 95 % of its u.v. energy in a 2537 A band. For irradiation, a Petri disb (5 cm in diameter and 3.5 cm in deptb) was set 30 cm below tbe u.v. lamp. Tbe doses of u.v. applied were cbanged by varying tbe duratio^n of irradiation. Tbe intensity of u.v. at tbe bottom of tbe Petri disb was about 30 Mw/cm2. Tbree fern species, Pteris vittata, Osmunda japonica and Dryopteris varia, were used 21

22 YUKIO KATO as experimental material. Pteris vittata produces small brov^^n spores and germinates easily in daylight or white light 3 or 4 days after sowing.

2 22 YUKIO KATO as experimental material. Pteris vittata produces small brov^^n spores and germinates easily in daylight or white light 3 or 4 days after sowing. The spores of Osmunda japonica contain chloroplasts and germinate within 24 hours after sowing. Dryopteris varia has a gland-hke papilla at the end of the protonema. To inoculate the cuhure, spores were dusted in Petri dishes in some cases on the surface of five-fold diluted Knop's solution, but in most cases on a solid Knop's medium containing 0.8% agar. Cultures were maintained at 28' C under continuous illumination by daylight fluorescent lamps. In a preliminary investigation, an influence of white hght before u.v.-irradiation had been noticed. Therefore, spores were grown under two different conditions of light intensity. A high intensity of approximately lux at the plant level was employed in one series, and a low intensity of lux in the other. The culture medium was renewed once every alternate week. At the outset, the normal process of development in Pteris vittata, from germination to the formation of a mature prothallus, must be described. Under our culture conditions the spores germinate 3 or 4 days after inoculation. Upon germination, the primary rhizoid is differentiated first, and then the initial cell of the protonema appears. The initial cell divides successively into several cells arranged one-dimensionally and thus a filamentous protonema is formed. After a definite period of time, the orientation of cell division of the apical cell of the protonema is converted from longitudinal to transverse. At this point, the growth of the protonema changes from one- to two-dimensional form, and gives rise to a young prothallus. The number of cells making up the filamentous part depends on the culture conditions, especially on the hght intensity. A long filamentous protonema composed of a long, slender cell is formed under low intensity of light, a short protonema composed of isodiametric cells under the high intensity. Subsequent division of these cells takes place in all directions in one plane, and finally a cordate prothallus is formed. RESULTS Irradiation of the dormant spores. Dormant spores irradiated for as much as 8 minutes survived in all species examined. Germination of the treated spores was quite normal, forming one rhizoid cell and one initial protonema cell. In Osmunda spores, however, masses of three or four cells without rhizoids were observed with a fairly high frequency, unlike the control. Fifty per cent germination was obtained in spores irradiated for 15 minutes in Osmunda and for 25 minutes in Pteris. Experiments with Pteris vittata General observation. When spores in the germination stage (having one rhizoid and one initial protonema cell) were irradiated for 1.5 minutes with u.v., the rhizoids alone were frequently injured, as deduced from their reaction to dye and deplasmolysis. The primary rhizoid cells became brownish-white in colour soon after irradiation and lost their turgor completely. These are indications of death. Isolated protonema cells began to regenerate. The onset of the regeneration was characterized by the formation of new rhizoids so that the initial cells possessed one dead and one new secondary rhizoid, as illustrated m Plate i, Nos. i and 2. In a protonema composed of three or four cells, only the death of rhizoids could be induced by irradiation with a low dose. Sometimes each isolated cell thus obtained developed into a normal prothallus. When germinated spores were irradiated for i minute, swelhng and branching of the rhizoids, instead of death, occurred. In addition, on rare occasions, a structure inter-

3 u.v. irradiation of fern prothalli 23 mediate between protonema and rhizoid was observed, i.e. rhizoids with abundant chloroplasts were differentiated. These have been termed 'rhizoidal protonema' by Kato (i957«), because normal rhizoid cells, unlike the protonemal cells, have no chloroplasts. It is interesting that, when irradiated for 2 minutes, a rhizoid developed at the top of the apical cell of young gametophytes (Plate 2, Nos. 11 and 12). This does not occur in the normal development. A similar reversal of the axis of polarity can be induced by other means such as darkness, centrifugation or IAA-treatment (Kato, 19576; Nakazawa, i960). General phenomena mentioned above were observed in cultures which have been kept under both low and high intensities of white hght before u.v.-irradiation. When protonemata, cultured under low intensity of white light, were irradiated with u.v. for 2 minutes, a branching of the protonema occasionally took place (Plate 2, Nos. 13, 17 and 18). This indicates the establishment of a new axis of the development. A similar phenomenon has been induced by ultracentrifugation (unpublished data). These treatments probably result in the loss of apical dominance, as found in the apical and lateral bud relationship of higher plants, and thus an outgrowth of new protonema occurs. In protonemata irradiated with u.v. for 3 or 4 minutes, tumorous types of cell pro- Table I. The percentages of protonemata having survivors in a given region, cultured under low (150 /;/A') and high intensities (2000 lux) of white light before u.v.-irradiation for 5 minutes Class of surviving cells B I A + B B + I A + I A + B + I Culture condition 150 lux 2000 lux A: protonemata with survivors ot the apical cell alone in a protonema. B: basal cell alone. I: intervening cells between apical and basal regions. A + B: both apical and basal cells. B + I: both basal and intervening cells. A + I: both apical and intervening cells. A + B + I: survivors present in all the regions of a protonema. liferation were frequently found. Although most of the cells composing a protonema are not killed with relatively low doses, they lose their polarity. The orientation of cell division then becomes almost random, and the cell arrangement becomes extremely irregular. Generally speaking, all the abnormal types observed tend to revert to normal prothallial growth. Ability to survive. For this investigation the protonemata were grown under two different light conditions. The results of the experiments with filamentous protonema stages, irradiated for 5 minutes, are shown in Table i. According to the position of surviving cells, affected protonemata could be classified into the following types: A (survival of the apical cell alone), B (basal cell alone), and I (intervening cells between apical and basal cells), and their combinations, A + B (both apical and basal cells), B + I (both basal and intervening cells), A + I (both apical and intervening cells) and A + B + I. The last includes the survival of some of intervening cells in addition to the apical and basal cells. The values in Table i represent the frequencies of occurrence of protonemata classified as described. In protonemata cultured under the low intensity of white light before u.v.-irradiation, the apical cells tend to survive (Plate i, Nos. 3, 4 and 8), and, by contrast, in those under the high intensity the apical or basal cells or both tend to survive (Plate i, Nos. 5-7). Under the former condition, the basal or intervening cells, as shown in Plate i, Nos. 9 and 10, survive in comparatively low frequency. Thus a gradient of ability to survive in the cells forming a

4 24 YuKio KATO protonema was clearly demonstrated, the apical cells having the highest ability and the basal cells the lowest. Variable results were obtained under the high intensity light conditions and thus the gradient was not clear cut. However, it seems that the abihty to survive may be connected with two opposing factors. This point will be discussed later. Single cells, isolated by this treatment, may regenerate to produce normal mature gametophytes in some cases. But the beginning of regeneration is considerably delayed, and days is necessary for it. Isolated single cells frequently cease to grow for a long time before their chloroplasts and nuclei degenerate and they finally die. Sometimes, 10 (a) (b) i (c) > 0-1 Length of surviving cells (/A) Fig. I Variation m length (^) of surviving cells from irradiated protonemata oipteris vittata (a), (c) and (e) Data rrom gannetophytes grown under the low intensity of white light before uj.-irradiation. (b) (d) and (f) Data from gametophytes under the high intensity, (a) and however, they divide several times and then disintegrate without further development or segmentation. Under the low intensity of white light, one of the cells at the basal region, the basal cell itself in most cases, was induced to cut off a new daughter cell laterally which developed into a new protonema and finally into a prothallus (Plate i No 7) General processes of development of isolated cells resemble those from spores. It is worthy of note, however, that, even in the culture under weak white light, the length of the filamentous part (termed 'stalk' by the author in a separate paper) of regenerating prothalli was usually shorter than that obtained by germination from spores. A similar phenomenon has been seen in single cells isolated by pricking their neighbouring cells with a fine glass needle (Ito, 1962). => & 6

5 U. V. irradiation of fern prothalli 25 Fig. I shows the variations in length of surviving cells taken from irradiated protonemata. It is very evident from this figure that the type of surviving cells is similar in protonemata grown both under low and high intensities of white light. In general, the shorter cells, 300 [x or less in length, tend to survive. Table 2 shows that the more apically cells are situated, the shorter becomes their length. These data indicate why the apical cell is apt to survive since cells of 300 M or less in length tend to survive under both cultural conditions. However, almost all the cells of protonemata grown under a high intensity of light are less than 300 \x in length and should be qualified to survive. Therefore, other factors responsible for survival must be considered. When the u.v.-irradiation was given to prothalli at 'spoon-like' or nearly mature stages, the localization of the survivors was very intricate. For instance, one marginal cell of the prothallial wing (Plate 2, No. 20), a basal cell, or some cells in a meristematic region, etc., were capable of surviving. It was impossible to find any general principle concerning the ability to survive in different regions of the prothallus. Table 2. Lengths (li) of cells composing a protonema grown under two different light intensities Cell number from base to apex I ' s * Means Culture D lux ± 62* ± 33 ± 49 ± 24 ± 30 ± 19 ± 23 ± 34 ± 12 ± S.E. condition 2000 lux 240 ± ± ± ± ± 6 Experiments with Osmunda japonica Dormant spores of Osmunda seem to have a high resistance to u.v.-irradiation (although spores of Equisetum arvense which are similar in containing abundant chloroplasts were killed by short u.v.-irradiation for 3-5 minutes). Two or three days after germination, the gametophytes are composed of four to five cells. All the experiments were made with the material at these stages. The duration of u.v.-irradiation was 2.5 or 4.5 minutes. It is interesting that rhizoid formation was much promoted by these treatments. Irradiated protonemata bore, precociously, three to six secondary rhizoids, with four as the mean (Plate 2, No. 16), while untreated ones usually bore one or two. Of these rhizoids induced by u.v., the primary rhizoid, was dead. Increase in number of rhizoids per protonema may be a result of compensation for injury to the primary rhizoid. Rhizoids were formed on the lateral, basal or even apical parts of a protonema, often abnormally at the tip of an apical cell as observed in Pteris vittata (Plate 2, No. 15). Experiments with Dryopteris varia The experiments with this species were concerned mainly with the formation of the terminal papilla of the protonema. Only one papilla usually appears at the tip of the apical cell at the five- or six-cell stage. The sporelings of the germination stage were irradiated with u.v. for 2 minutes. The resulting terminal papilla was differentiated

6 26 YuKio KATO preociously e\en at the one- or two-cell stages. In addition, even at the one-cell stage the terminal rhizoid was formed in a few cases on the apical side of an initial cell (Plate 2, No. 14). DISCUSSION Regeneration from a single cell of the protonema or prothallus. I can find only two reports on the regeneration of a fern gametophyte from isolated, single cells. Meyer (1953) reported regeneration from isolated single cells in gametophytes of Aspkmum adiantum-nigrum. He obtained his isolated cell from the gametophyte because most cells were damaged by accidental fungus contamination. Ito (1962) also described how a single cell can be isolated by pricking, freehand, its neighbouring cells with a fine needle. In this investigation it was found that a single cell may be isolated by u.v.-irradiation and that, in some cases, a mature gametophyte may be regenerated from it. Ability to survive. There are remarkable differences in the abilities to survive among protonemal cells associated either with the regions from which they are isolated, or with the cultural conditions before u.v.-irradiation. In gametoph3^tes grown under a low intensity of white light, the existence of an apico-basal gradient of survival was clearly shown, i.e. basal or intervening cells of a protonema tend to be injured, while the apical cells survive. This result, however, is not parallel with that of the resistance to alcohol (Reuter, 19^3) or with that of the polar susceptibility to colchicine (Nakazawa, 1959). Susceptibility to colchicine along the morphological axis is highest at the apical region of a protonema and lowest at the basal region. It has been reported that in fern gametophytes there are apico-basal gradients in permeability, isoelectric point, plasmolytic properties, stainability to dyes and DNA content. The relation between these gradients and the abilities revealed in the present case is still unknown. However, it seems that these gradients could play an important role for survival of each cell in a protonema. From the present observations, it is suggested that apical cells have a marked ability to divide and elongate, and might readily recover from partial injury, soon after isolation, and finally regenerate. The length of each cell composing a protonema is an essential factor for survival. On this point, injury of cells caused by u.v.-irradiation is considerably different from that caused by colchicine or alcohol. In the latter hypertrophied apical cells can neither develop nor segment further. In protonemata grown under a high intensity of white light before u.v.-irradiation, both apical and basal regions may survive while the intervening cells are more hkely to be injured. It is supposed that survival is connected with at least two different gradients found in a protonema. On the one hand, the ability to regenerate is higher in the apical region than in the more basal region, on the other hand, the 'resistance' to u.v., by which IS meant non-sensitivity, is higher in the more basal cells of a protonema. Thus, the ability to survive will be understood as a result of the interrelation of these factors. It would not be unreasonable to suppose that the intervening cells might have the lowest activities. ACKNOWLEDGMENTS It is great pleasure to acknowledge my gratitude to Professor M. Kumazawa and I. Harada for helpful advice and encouragement during the course of this work.

7 THE NEW PHYTOLOGIST, 63, PLATE I dr K > >'o 1 '"^^^V"!?' EXPLANATION OF PLATE i Isolation of a single cell by u.v.-irradiation {Pteris vittata). Times in parentheses indicate the duration of u.v.-treatment, s, spore membrane. Nos. I and 2. Dead primary rhizoids (dr) and newly formed rhizoids (nr). p, protonema. (3 minutes.) Nos. 3, 4 and 8. Isolated apical cells (a) and their subsequent division. (8, 5 and 5 minutes, respectively.) The cell below the arrow in No. 4 is dead. Some single cells grew up to mature gametophytes. Nos. 5 and 6. Isolated basal cells (b). (8 minutes.) Nos. 7. Outgrowth of a new prothallus from a surviving basal cell, rp, regenerating prothallus. op, dead original protonema. (6 minutes.) Nos. 9 and 10. Isolated intervening cells (i). The cells above and below the arrows are dead. (5 minutes.) YUKIO KATO--C7.F. IRRADIATION OF FERN PROTHALLI (facing p. 26)

8 THE NEW PHYTOLOGIST, 63, i PLATE 2 16 YUKIO KATO U.V. IRRADIATION OF FERN PROTHALLI (facing p. 27)

9 u.v. irradiation of fern prothaui 27 REFERENCES ITO, M. (1962). Studies on the differentiation of fern gametophytes. i. ReKeneriilion (JI'single cells isolated from cordate gametophytes oi Pteris vittata. Bot. Mag. (Tokyo), 75, 19. KATO, Y. (19570). Experimental studies on rhizoid differentiation of certain ferns. Plivlon (Ari^eiilina}, 9, 25. KATO, Y. (19576). Some experiments on the polarity of spores in Diyo/iteiis eiylhiosoia and Eijuisetiim arvense. Cytologia, 22, 328. MEYER, D. E. (1953). Ijber das verhalten einzelner isolierter Prothalliumzellen und dessen Bcduutunt; fur Korrelation und Regeneration. Plaiita, 41, 642. NAKAZ.^WA, S. (1959). Morphogenesis of the fern protonema. i. Polar susceptibility to colchicine in Dryopteris varia. Phyton (Argentina), 12, 59. NAKAZ.'^WA, S. (i960). CytodifTerentiation pattern of Drvopteris protonema modified by some chemical agents. Cytologia, 25, 352. REUTER, L. (1953). A contribution to the cell physiologic analysis of gr(jvvth and morphogenesis in fern prothallia. Protoplasma, B, 42, i. EXPLANATION OF PLATE 2 Effects of u.v.-irradiation on the growth and differentiation of gametophytes in Pteris vittata, Osmunda japonica and Dryopteris varia. Times in parentheses indicate the duration of u.v.-treatment, s, spore membrane; pr, primary' rhizoid; tr, terminal rhizoid. Nos. II and 12. Formation of rhizoids at the end of young protonemata and outgrowth adjacent to them (Pteris vittata). (2 minutes.) Nos. 13, 17 and 18. Extreme branching or outgrowths resulting from the loss of apical dominance (P. vittata). (2 minutes.) No. 14. Formation of rhizoid at the top of an initial protonema cell (Dryopteris varia). (2 minutes.) No. 15. Formation of rhizoid at the top of an initial protonema cell (Osmunda japonica) (4.5 minutes.) No. 16. Extra-formation of secondary rhizoids. Primary rhizoids (pr) dyiing (Osmunda japonica). (2.5 minutes.) No. 19. A dead marginal cell (dc) in a young prothallus (Pteris vittata). (8 minutes.) No. 20. Isolated cells (ic) in the young prothallial stage (P. vittata). Most of these single cells grew up into 'teaspoon-like' gametophytes. (5 minutes.)

APICAL DOMINANCE IN FUCUS VESICULOSUS

APICAL DOMINANCE IN FUCUS VESICULOSUS BY BETTY MOSS Department of Botany, University of Newcastle upon Tyne (Received 2 December 1964) SUMMARY Apical tips of Fucus vesiculosus L. were grown in sterile