CESSATION OF VASCULAR ACTIVITY CORRELATED WITH APOSPOROUS DEVELOPMENT IN PTERIDIUM AQUILINUM (L.) KUHN

Transcription

1 New Phytol. (1981) 88, CESSATION OF VASCULAR ACTIVITY CORRELATED WITH APOSPOROUS DEVELOPMENT IN PTERIDIUM AQUILINUM (L.) KUHN, BY E. SHEFFIELD* AND P. R. BELL Department of Botany and Microbiology, University College London, Gower Street, London WC1E6BT, U.K. {Accepted 30 October 1980) SUMMARY Apospory has been found to occur on leaves oi Pteridium aquilinum still attached to parent plarits, although previous work had indicated that detachment was a prerequisite for regeneration. It is clear, however, that such recrudescence occurs only in leaves where active vascular flow has ceased, and which are also in contact with a moist surface. It is proposed that the cessation of an active vascular supply has an inductive effect. The inability of mature fronds to behave aposporously is attributed to the presence of a thick cuticle external to the superficial cells, which is thought to prevent the exchange of metabolites between frond and medium. INTRODUCTION The experimental induction of apospory has usually involved the detachment of organs or parts of organs from the remainder of the plant. From this general experience, and from the results of specific experiments it has been inferred that in the case of leaves the interruption of vascular continuity between parent plant and frond is a prerequisite for aposporous behaviour. Reports have appeared, however, which describe the development of aposporous outgrowths on leaves which were still attached (see Steil, 1939 for review; Hurel-Py, 1950; Sheffield and Bell, 1981). It has been shown by Whittier (1966) that the natural apospory observed in Pteridium by earlier workers was merely the production of abnormal outgrowths, lacking gametophytic characters, from aborted sporangia resulting from infestation by mites. Takahashi (1962) reported that no apospory was observed in Pteridium unless the sporophytic organ was detached from the plant. However, we have reported the development of unambiguously gametophytic outgrowths from juvenile fronds of Pteridium still attached to the plant (Sheffield and Bell, 1981) under conditions similar to those in which Hurel-Py (1950) observed outgrowths from the leaves of Gymnogramme. In both these instances the leaves in question had grown down into the agar medium, and in the Pteridium example the petiole was found to be partly necrotic and it seemed likely that vascular continuity had ceased. The present investigation concerns more recent observations in Pteridium which have shown that outgrowths occasionally arise on laminae which appear to possess full vascular connection with the rest of the plant, as in the example reported by Hurel-Py. * Present address: Department of Cryptogamic Botany, University of Manchester, Oxford Road, Manchester, Ml3 9PL, U.K. ()02S-646X/81/ $02.00/0 (C) 1981 The New 18 2

2 534 E. SHEFFIELD AND P. R. BELL MATERIALS AND METHODS Spontaneous apospory Sporophytes of Pteridium aquilinum were raised in aseptic conditions as described by Sheffield and Bell (1981). After several weeks of culture in tubes, the plants were transferred to medium in 250 ml wide-necked flasks stoppered with foam bungs and covered with foil. When at least 7 weeks old the plants wer^ inspected for fronds bearing aposporous outgrowths. Twenty plants bearing such fronds were placed in a 1 % solution of eosin (Gurr) so that only the roots and parent gametophyte were submerged. After 1 to 2 h fronds were detached, mounted on slides and examined with a Zeiss photomikroskop II using incident blue illumination with filters BP , FT KIO, LP520 and KP560. Aposporous fronds were detached from 10 similar plants and incubated in a 0*01 "o solution of fluorescein diacetate (Widholm, 1972) in Moore's medium for 5 min at room temperature. These fronds were examined in the same microscopic conditions. (The native fluorescence of unstained fronds, and also that of heat-killed fronds was found to be negligible). Ten 4-week-old plants growing on slants of medium were submerged in liquid Moore's medium. The juvenile fronds from 10 similar plants were detached and placed in liquid Moore's medium at the same time. After 5 days all juvenile fronds were examined microscopically. Experiments with detached fronds Fifteen fronds were detached from plants less than 7 weeks old and the petioles pushed into medium so that the laminae were raised above the surface. Control fronds were detached and placed flat upon the medium. The laminae were examined microscopically after 4 days of culture and stained with fluorescein diacetate after 14 days. Electron microscopy Juvenile fronds and portions of fronds taken from plants growing in the field were fixed in 3 % glutaraldehyde in 0*05 M phosphate buffer ph 6 0 for 3 h at room temperature. The material was washed twice and left overnight in ice-cold buffer. Post-fixation was in 2 % osmium tetroxide for 2 h at 0 C and dehydration in acetone. The material was embedded in Epon 812, sectioned at about 30 nm and the sections mounted on grids before staining with uranyl acetate and lead citrate. The sections were examined in a Hitachi HS9 electron microscope. ^^^ RESULTS Examination of at least 400 plants revealed that approximately 15% of those cultured for 7 weeks or more bore outgrowths with all the features characteristic Fig. 1. Frond attached to a 3-month-old plant lying flat upon the surface of the medium. The margins of the pinnules are bordered by gametophytic outgrowths, x 3. (b) Frond attached to a 3-month-old plant in which only the pinnae on one side were in contact with the medium. Outgrowths are present oiily on those pinnules lying on the medium, x 3. (c) Frond of an 8-week-old plant supplied through the roots with eosin for 1 h, viewed in transmitted light. Only one lobe (A) was in contact with the medium and producing outgrowths. The vascular tissue is clearly evident. A fork of vascular tissue has differentiated in the median part of the leaf (arrow) unconnected with the main vascular supply, x (d) The same frond as in (c) viewed with epi-fluorescent illumination. Eosin has not entered the aposporous lobe, nor the fork unconnected with the main supply, x 112,

3 Vascular Inactivity and Pteridium apospory

4 536 E. S H E F F I E L D A N D P. R. BELL of gametophytic tissue (see Sheffield and Bell, 1981). Such outgrowths appeared most frequently on juvenile laminae, but also occurred on more mature foliage. The tissue giving rise to outgrowths was invariably found to be in close contact with a surface; normally that of the culture medium, but occasionally the moisture-laden sides of the culture tubes or flasks. Both juvenile and mature fronds lying flat on the surface of the medium produced large numbers of cordate gametophytes all around the periphery and often from the surface of the leaves [Fig. 1 (a)]. Fronds which had grown so that only one half of the leaf touched the surface gave rise to outgrowths only from those pinnules in contact with the medium [Fig. l(b)]. ji; Juvenile fronds detached and cultured so that the laminae were not in contad with the medium were never found to bear aposporous outgrowths. After 14 days of culture, sych laminae were found to be strongly fluorescent when stained with fluorescein diacetate and examined in the fluorescence microscope. Controls cultured in contact with the medium gave rise to outgrowths with frequencies similar to those previously observed (see Sheffield and Bell, 1981). Fragments of laminae from mature fronds in the field, surface-sterilized and placed on medium, remained alive for at least 8 weeks, but produced no aposporous outgrowths. Juvenile laminae of plants cultured for 5 days submerged in liquid medium showed no signs of aposporous outgrowths. Control laminae, detached and cultured in liquid medium, regenerated freely. The thinness of the fronds and the dark colour of the solution made it easy to follow the passage of eosin. The tracer was transported in the vascular system, and quickly rose to the extremities of all free-standing fronds. In wholly aposporous fronds, although in every instance dye was clearly visible in the midrib, it did not enter the branches running to the pinnules, even when the outgrowths were at the earliest possible stage of recognition. Figure l(c) shows a frond which had produced outgrowths in only one region; Figure l(d) illustrates the extent to which the dye penetrated this frond. It can be seen that the dye had not entered the vascular supply to the aposporous lobe. It was also found that in some fronds wholly in contact with or immersed in the medium, some regions had lost active vascular connection with the midrib, although outgrowths were not visible. An example is shown in Figure 2(a). Eosin failed to penetrate to the extremities of the veins in one part of the lamina. Aposporous fronds from plants ranging in age from 7 to 12 weeks were found to be strongly fluorescent when stained with fluorescein diacetate and then viewed with the fluorescence microscope. The fluorescence of the sporophytic tissue was as intense as the new gametophytic tissue produced from both the marginal and superficial cells [Fig. 2(b)]. Electron microscopy revealed that the outer walls of the epidermis of juvenile frond cells were not more than 01 fim thick, and that no significant depth of cuticle could be distinguished [Fig. 2(c)]. The corresponding walls of plants growing in thefield[fig. 2(d)] were at least 27 fim thick, and covered by a cuticle approximately 60 nm thick. DISCUSSION It is clear the aposporous outgrowths may be produced on fronds of Pteridium attached to plants, contrary to the contention of Takahashi (1962). This phenomenon, however, occurs only when the tissue is in contact with a moist surface and active tracheary connection with the parent plant has ceased. The question

5 Vascular Inactivity and Pteridium apospory i^ig. 2. (a) Frond of a 9-week-oId plant in contact with the medium, but not showing apospory, viewed with epi-fluorescent illumination 2 h after supplying the plant with eosin. Eosin has not reached the tips of the veins supplying the lobes in the lower part of the photograph, x 12. (b) Aposporous frond attached to an 8-week-old plant, stained with fluorescein diacetate and viewed with epi-fiuorescence. x (c) Electron micrograph ofthe peripheral region of a juvenile frond. X (d) Electron micrograph of the cell wall of a mature frond. A significant depth of cuticle can be clearly seen, covering the wall, x

6 538 E. SHEFFIELD AND P. R. BELL naturally arises of whether the cessation of xylem flow is a consequence of the water potential in the area of leaf concerned being satisfied by direct absorption through the epidermis, or whether there is actual blockage of the tracheids. If not the primary cause of this stoppage, blockage seems soon to develop since the eosin experiments were done with leaves rising into unsaturated air. This should have re-established the water potential in the mesophyll cells and restored transpirational flow. The absence of vascular flow into regions of fronds showing apospory, observed in every instance, strongly suggests that failure of xylem function has causal significance. Certainly a few instances were observed where flow had ceased into regions of laminae in contact with medium without regeneration being evident. Nevertheless these do not contradict the hypothesis, since if the cessation of vascular influx is inductive it must occur before visible regeneration. The extensive observations'made in this investigation are thus all in line with the view expressed earlier on other grounds that deprivation of some influence from the vascular system pre-disposes the cells of the leaf to a switch in gene activation (Sheffield and Bell, 1981). The experiments with detached leaves inserted into the medium so that the laminae remained elevated show that contact with medium is essential for this switch in gene activation to be expressed. It seems likely that the medium acts as a sink for substances leaching out of the cells, but which are replaced by the parent plant if active xylem flow is maintained, as evidenced by the regular absence of regeneration of attached laminae when these were submerged in liquid medium. It is these substances which are envisaged as being responsible for maintaining activation of the sporophytic genes and the correlative inhibition of further cell divisions in the sporophytic mode. To quote Takahashi (1968) they 'maintain and fortify the sporophytic nature' of the frond. F'irm evidence for the existence of these putative substances, and their nature, is now being sought. The reluctance of older leaves to regenerate can be reasonably attributed to the thick cuticle preventing exchange of metabolites between the leaf and the medium. Fluorescein diacetate staining demonstrated that no cell death occurred, confirming the earlier view (Shefifield and Bell, 1981) that cell death is not a prerequisite for apospory, although in some instances it may accompany it. ACKNOWLEDGEMENTS We are most grateful to Ms S. M. Laird for excellent technical assistance, and to Mr R. Packer, Birkbeck College, for the loan of a low power objective. REFERENCES HrREi.-Fv, C;. {1950). Recherches preiiminaires sur la culture asceptique des prothalles de P^ilicinees. Rerue (ienerale de Botanique, 57, Sfu-FFiKi.D, E. & BEM., P. R. (1981). Experimental studies of apospory in ferns. Annals of Botany, 47, STI:II., W. N. (1939). Apogamy, apospory and parthenoj^enesis in the Pteridophytes. Botanical Review ' ' TAKAMASMI, C. (1962). Cytolo^ical study on induced apospory in ferns, ('vtologta, 27, TAKAHASHI, C. (1968). Correlative effect amon«organs in a fern sporophyte on tht' induction of aposporv Botanical.XJafiazine (Tokyo), 81, (){}() ()()2. ' UiDHOL.M, J..\I. (1972). The use of Huorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technoloftx, 47, WHITTIER, D. P. (1966). Natural apospory in Pteridmm} American Fern Journal, 56,

7