J . Lima. SOC. (Bot.), 58, 373, p . 377 With 1 text-figure Printed in Qrecct Britain

377

Developmental patterns of adventitious sporophytes in Phlebodium aureum J. Sm.

BY MAX WARD

Glenwille State College, GlenwiUe, Weat Virginia, U.S.A.

I”IB0DUCTION

Following the hypothesis of Lang (1909, 1915), few experiments were made to test his contention that the gametophytic and sporophytic forms of typical pteridophytes might be determined by the environment peculiar to their normal development.

!Crials by Ward (Ward & Wetmore, 1954) gave tentative support to Lang’s hypothesis when formative effects on the young sporophyte from encapsulating and restricting tissues were thought to be indicated following experimental manipulation. Work by Jayasekera & Bell (1959) and more recently by DeMaggio & Wetmore (1961) in certainother ferns has illuminated developmental aspects of the young sporophyte under diverse con- ditions-in the latter case, free and unattached to parental tissues from the time shortly after fertilization.

The work of Steward (1958) has shown that small groups of cells far removed in time and space from an initial fertilized egg, still capable of unrestricted meristematic activity, may elaborate tissues which culminate in maturity in a normal plant of the species in question. Support has thus been lent to contention that any 2 n cell of appropriate vital capacity might, a t any stage remote from a fertilized egg, or zygote, be totipotent in reconstituting a mature individual plant. It could be assumed that an isolated callus cell (or any meristematic and undifferentiated cell) of the pteridophyte sporophyte might thus be a sound starting-point for experimental production of a second sporophyte, of the form and pattern ordinarily presumed from a fertilized germ cell. Such sporo- phyte could thus develop in artificial culture completely uninhibited by tissues other than products of its own proliferations.

DEVELOPMENTAL PATTERNS IN ADVENTITIOUS SPOEOPHYTES

In the present work attempts have been made to induce formation of callus tissues in Phlebodiurn aureurn J. Sm. from leaf and stem segments through culture procedures widely employed in recent years in other phyla of plants. From these trials have come distinctive patterns of reconstituting both the gametophytic and the sporophytic form in this species.

Petiole segments maintained in culture under a variety of media compositions have regularly given rise to gametophytic forms, exemplised in Fig. 1 A. The adventitious tissues involved have arisen from the inner parenchyma cells, probably from near the vascular bundles. Surface protrusions are always filamentous at first, and the two-dimen- sional form, characterized by apical notch and appended rhizoids, follows regularly. These gametophytic plants have followed a normal growth pattern, culminating in the production of sex structures in later subculture. The chromosome count is being sought. No exact knowledge about fertility is yet available.

Some filamentous regenerants were observed to proceed directly to an orderly aggregate of three dimensions without an obvious intermediate thalloid form (Fig. 1B). This latter entity did not arise from a structure with characteristics of the gametophyte, nor from any previous regular accumulation of cells. The growth was aerial, completely unrestricted by parental or elaborated extraneous tissues. The sporophytic plant arose, therefore,

378 MAX WARD

amid an unrestricted environment, decidedly different from the encapsulated, sexually- derived spomphytic form.

Adjacent regenerants, h t of a filamentous nature, elaborated the gametophytic form. Thus, on the aame segment, similarly looking regenerant plants with obviously the same quality of origin followed without delay diverse growth patterns to produce on one hand the gametophytic form, and on the other the more complex sporophytic form.

From stem (rhizome) segments in similar culture media, regular production of both the gametophytic and the sporophytic plants have been obtained (Fig. 1C). Regenerant growths occur regularly on either the sides or the cut ends of the segments, and the occur- rence of the elaborated forms is about equal in number. The forms occur immediately adjacent to each other, but the pattern of origin is Merent. Gametophytic forms are preceded by filamentous uprisings ; the sporophytic is regularly preceded by accumulation of a cellular mwa and followed by the origin of the spomphytic form in three-dimensions as though from a previously existing 'bud'.

Fig. 1. Diagrammcltic repmaentation of adventitious gametophytm and aporophytea of Phlihdkm w- pmduced in culture. (A) Adventitious (approus) gemetophyte on petiole segment; (B) sporophyte developing from &mentous outgrowth on similar petiole segment ; (C) gametophytic and sporophytio p h t a arising in proximity on section of rhizome ; (D) p b t a of sporophytic form on intaut leaf of apogamous sporophyte; (E) aerially-formed sporophyte on v & i d y extended three-dimeneiond outgrowth of apogamous sporophyte in subculture.

Yet, it is distinctly clear that these new plantlets are not mere buds or their consequent shoots on a parent stem. They form their own mots as well and readily become complete planta, quite -able of separate existence. An identity of form of the plant newly derived from regenerant tissue and that of the plant derived from a fertilized egg in distinctly different surroundings is thus apparent.

Regenerant sporophytea obtained from rhizome segments and placed in subculture (in tubes maintaining high humidity) have shown a marked propensity for initiating secondary sporophytea on their intact and functioning leaves (Fig. 1 D). These plants are of regular, three-dimensional form, and seem to be products of previously normal plants and are not produced as a result of disturbance, such as that which induced their pro- genitors from excised rhizomes. Their nuclear constitution is not yet known. Similar fonns have not been found arising from either normal, sexually produced sporophytes or from apogamous spomphytes of gametophytic origin.

Leaves of the subcultured sporophytm which bear new, apogamous sporophytes gradually show signs of debility and loss of vitality. Such decadence does not appear to be the causative factor in the induction of the new aporophytic forms, for the initiation of the secondary sporophyte may be observed on apparently normal and fully intact leaves. These apogamously derived sporophytes from spomphytes (themselves of apogamous origin) present developmental ";lpecta for further study.

Developmental p23attems of adventitious sp0rophyte.s 379

Elongated, cylindrical outgrowths from apogamously-produced sporophytes in culture are common, particularly under crowded growing conditions. Organized sporophytic forms, with leaf and stem regions, frequently appear aerially on the surface of these three-dimensional vertical structures (Fig. 1 E). Here, again, is a case wherein the sporo- phytic type of plant forms aerially, unencumbered and unrestricted by extraneous cellular accumulations. No gametophytic entities have been observed resulting from such formative patterns.

In Phkbodium aureum, under conditions of cultivation regularly employed for growth of experimental plants, the apogamous production of sporophytes from normal gameto- phytes is also commonly found, perhaps a result of ageing and of characteristics of the nutritional medium not yet fully determined.

Fern sporophytes, long thought of as peculiarly the product of two haploid germ cells, may therefore be had under experimental conditions from both haploid and diploid tissues and under formative conditions far removed from those incident to products of a fertilized egg cell. The community of origin of such typically diploid forms and the obviously kindred and typically haploid forms dims the classical distinction about the essential differences in origin of the two life cycle forms and casts doubt on the genetic constitution classically ascribed to each.

The completely uninhibited origin of the sporophytic form from the filamentous derivatives of parent tissue seems to represent a step toward an understanding of problems raised by earlier contentions that the three-dimensional form is a product of its restricted environment. Development of such form from individual cells of induced fern callus is presently a desirable and enticing objective.

Comparable testing of the original hypothesis may also be done when successful techniques are arrived at for allowing the pteridophyte spore to develop within an artificial plrtstic, but yielding, encasement. Possible development of such a spore, under restriction, into the two dimensional or three-dimensional form will be of much interest in relation to the classical expectations regarding the haploid and diploid tissues of the pteridophyte life cycle.

CONCLUSIONS

These developments, although far from complete, may lead one to wonder whether the sporophyte ww indeed significantly modified in the earlier experimentation (Ward t Wetmore, 1954), and whether or not the manipulation had indeed altered the morphology.

It is clear in the present experiments that the formative controls are neither under- stood nor successfully manipulatable. Production of the sporophytic form or the gametophytic comm with ba&g randomness under the regenerant behaviour so far observed. Occurrence of the two forms side by side, on the same sporophytic segment and under the same apparent conditions, suggests identity of origin of the two.

In the origin of the sporophytic form in three dimensions, aerially and unencumbered, on Glamentous outgrowths, lies the suggestion that the forces of restraining tissues are not essential to the elaboration of the three-dimensional form. Further, the ready formation of the gametophytic plant from filamentous outgrowths, even adjacent to the former, and in a manner quite similar to the elaboration from a spore, makes certain that the developmental patterns and forces are not yet understood.

The grand hypothesis of Lang is thus explored stiU further, but there is left the conclusion at present that much more than physical factors of the environment peculiar to either plant in the classical alternation of generations scheme are a t work in a complex developmental pattern.

380 MAX WARD

BEFEEENCES

DEMACWIO, A. E. & WETMOEE, R. H., 1961. Morphogenetic studies on the fern Todeo b a r a .

JAYASEXEXLA, R. D. E. & BEIJ,, P. R., 1969. The effect of veriow experimental treatments on the

L ~ G , W. H., 1909. A theory of alternation of generation in archegonhte plants based upon the

LUG, W. H., 1916. Preeidential Address: Phyletic and c a d morphology. Rep. Brit. Am., Nan-

STEWARD, F. C., 1958. Growth end organized development of cultured cells. III. Interpretations of

WARD, M. & WETMORE, R. H., 1954. Experimental control of development in the embryo of P a -

III. Experimental embryology. A m . J . Bot., 48: 651-65.

development of the embryo of the fern The&piwi8pbtrie. Pluto, 54: 1-14.

ontogeny. New Phytd., 8: 1-12.

Chsstg*: 707-18.

the growth from free cell to carrot plant. A m . J . Bot., 45: 709-13.

bodiwn a u r m J.Sm. A m . J . Bd., 41: 420-34.