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The experimental investigation of the Pteridophyte life cycle. By P. R. BELL.
Department of Botany, University College, London
(With Plate 7 and 3 Text-figures)
INTRODUCTION
The identification of the factors responsible for the morphological cycle in Pteridophyta remains one of the most perplexing problems in causal morphology. It is some
50 years since Lang first suggested, arguing from the existence of isomorphic life cycles in
the algae, that the reasons for the profounddifferencebetween the simpleparenchymatous
gametophyte and the complex differentiated aporophytemust be sought in the differences
in the environments in which the initial cells of the two generations germinate (Lang,
1909). I n those algae, such m Dictyota, where the initial cells of both the sexual and
asexual generations germinate freely, unenclosed in any of the parent tissue, the two
generations are identical in form and can be recognized only by their reproductive
behaviour. As Lang realized, his hypothesis is open to experimental veritlcation, but
surprisingly little has hitherto been done in this field, largely because of the technical
diBculties involved. An alternative hypothesis, advanced more or less simultaneously
with Lang’s, ascribed the difference in form between the two generations of the Pteridophyta to different determinants in the nuclei of the two generations (Blackman, 1909).
Such a general hypothesis is not so open to verification as Lang’s. A discussion of the
relative importance of the nucleus and cytoplasm in the control of form follows after a
review of the evidence a t present available bearing upon Lang’s hypothesis.
EXPERIMENTS
UPON QERMINATINQ SPORES AND ZYQOTES
By the use of pure culture techniques, facilitating the experimental treatment of gametophytes and embryos, it has been possible to go some way towards testing Lang’s hypothesis, but much of the evidence for and against it is still indirect. The two crucial experiments which would determine the validity of the hypothesis once and for all are, f h t , to
germinate a spore in the conditions in which the zygote begins its life, and, secondly, to
isolate and allow a zygote to germinate in a free condition.
The first experiment would ideally involve injecting a spore into an archegonium, but
it is difficult to see how this can ever be done for very simple reasons of size. Most fern
spores are 30-4011 in diameter or least dimension, and the canal in the neck of an archegonium only about 15p across. Rupture or removal of the neck and implanting the spore
directly into the egg cavity, although not impossible for reasons of size, would hardly
afford a legitimate test of Lang’s hypothesis, since, as will be mentioned later, experiments have recently shown that removal of or damage to the neck above an ordinary
zygote modifiesthe embryogeny in a remarkable manner. It is possible to inject a spore
into the archegonial cushion of a gametophyte, but this is very far from providing it with
the environment normally enjoyed by the zygote. The experiment has been tried many
times with Pteridium aquilinum, (L.) Kuhn, but so far, and not unexpectedly, with no
results whichwould support Lang’shypothesis. Outgrowths of cylindricalform, eventually
broadening to a cordate lamina, have often been obtained from injected gametophytes
at or near the site of injection, but it has never been possible to show that these have
originated from the injected spore, or even to identify the spore with certainty in the
necrotic tissue bordering the site of injection. Moreover, outgrowths of this form frequently occur spontaneously from normal gametophytes or from gametophytes injected
with water alone.
Albaum (1938) has provided good evidence for the existence in normal cordate gameto-
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phytes of gradients of growth-regulatingsubstances. A strong gradient runs posteriorly
from the base of the apical notch, and most of the archegonia arise directly in the
path of this gradient. It has been argued that zygotes germinating in concentration
gradients of this kind, and possibly also of a nutritive nature, are polarized by them and
in consequence their development from the very beginning is switched into the path of
sporophytic complexity. Not sufficient is yet known of the nature of the metabolites
diffusing within the gametophyte to reproduce the natural gradients exactly in vitro.
Nevertheless, it is known that a concentration gradient of indole-3-aceticacid reproduces
some of the effects of those occurring naturally within the gametophyte (Albaum, 1938;
Jayasekera & Bell, in preparation). Experiments were therefore set up in which spores
of Pteridium were' sown in a gradient of indole-3-acetic acid diffusing from a central
source on an agar plate (Text-fig. I). The results gave little of interest in relation to the
problem discussed here. The concentration of indole-3-acetic acid adjacent to the ring
wholly inhibited germination; midway to the periphery germination was poor and the
Text-fig. 1. Pteridium aquilinum. An experiment to determine the effect of germinating spores in a
gradient of indole-3-aceticacid. (A) Petri dish containing 20 ml. Moore's medium solidified with
1.5% agar. (B) Glacis ring plaoed on top of the original medium after solidification and containing
1.0 ml. of the same medium supplemented with 40 mg./l. indole-3-aceticacid. Further explanation in text.
differentiationdisturbed, so that spores gave rise to nodules of cells instead of filaments
(a well-known effect of indole-3-acetic acid in sublethal concentrations on germinating
spores (Mohr, 1956 ; Soussontzov, 1957)). At the periphery germination and development were normal. Despite the fact that the concentration of indole-3-aceticacid must
have fallen progressively along the radii in which the spores were sown, no constant
relation between the direction of the gradient and the pattern of the development of the
spores lying in it could be observed.
Comparative morphology also yields an argument against the morphologicalcomplexity
of the sporophyte being dependent ab initio upon concentration gradients within the
gametophyte. In some species of the Schizaeaceae and Hymenophyllaceae the gametophytes are filamentous and clusters of archegonia are borne laterally quite removed
from the direct path of any gradients within the body of the gametophyte. Nevertheless,
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far as is known, the embryogeny of these ferns is similar to that of ferns with the
familiar cordate gametophyte (Stone, 1958).
Lang’s hypothesis, so far as it relates to the spore, remains unproven. The second crucial
experiment the hypothesis demands, the isolation of a zygote, is certainly technically
possible, given patience and skill. Although it has not yet been completely achieved, it
has been possible to go a long way towards it. The experiments in question have been
carried out on Thelypteris palustris Schott , Individual specimens of gametophytes were
raised in pure culture and taken for experimentation when about 0.75 cm. or more long
and fully archegoniate. An area of the archegoniate cushion, bearing 3-5 mature turgid
archegonia and 0.5-1.0 mm. behind the apical notch, was selected under the microscope
and dissected out from each gametophyte with the aid of a micromanipulator. The
archegoniate surface of the portion of gametophyte so removed waa approximately
square and of the order of 0 . 2 4 - 5 mm. a t the edge. These minute blocks were immediately transferred to suspensions of antherozoids obtained from thickly sown cultures of
the same species. After 24 hr. each block was transplanted on to an agar slope and the
behaviour followed. From fourteen blocks, about a third of those treated in this way,
embryos developed. Even though the zygotes were removed from the influence of all but
a minute portion of the parent gametophyte, the embryos were immediately recognizable
as sporophytes, and no growth from these blocks not recognizable as sporophytes could
be traced to a zygote. The development of the embryos arising in these isolated blocks
differed from the normal only in minor features. For example, the rate of development
(as measured by the emergence of the embryo from the calyptra) was reduced by about
half as a consequence of the treatment, and the appearance of the first root was delayed
even more in relation t o that ofthe first leaf and stem. Undifferentiated basal tissue (the
‘foot ’) also occupieda significantlylarger portion ofthe volume of the young experimental
embryos than normally (Jayasekera & Bell, in preparation).
I n another series of experiments with Thelypteris palustris, the neck was removed from
above a zygote before its first cleavage, but the gametophyte was not in any other way
damaged. It is possible to recognize an archegonium in which fertilization has occurred
by the cells a t the base of the neck which, following fertilization, enlarge and possibly
undergo some division, so that a collar is formed above the zygote, on top of which stands
the unchanged upper part of the archegonial neck. The earliest at which it was possible
to identify this collar with certainty in T. palzlstris was the third day after fertilization,
2 days before the average time of cleavage of the zygote. I n three gametophytes the
neck was successfully removed by a clean transverse cut at the level of the top of the
collar, without any damage to the zygote beneath. I n each of the gametophytes so
treated the zygote developed into a parenchymatous mass, on the upper part of which
leaves and then stem apices appeared. These structures are not dissimilar to those regenerated from the parenchyma of the cut rhizome of Onoclea by Wardlaw (1946) and
by the author from portions of the rhizome of sporelings of T. palustris placed on agar.
This remarkable modification of the embryology, also obtained in similar circumstances
in Phlebodium aureum (L.)J.Sm. by Ward & Wetmore (1954),was attributed t o the partial
release, by the removal of the neck, of the constraint placed upon the expanding zygote by
the tissues of the gametophyte. The results of these experiments are discussed in detail
elsewhere (Jayasekera & Bell, in preparation) ; the point to be observed here is that although differentiation of the tissue deriving from zygote was delayed by the removal of
the archegonial neck, it was not prevented, nor were the tissues ultimately differentiating
anything other than clearly sporophytic in morphology.
At present, therefore, it can be said that although it is possible to modify in form and
rate the development of a zygote by various treatments, normal sporophytic morphology
is fairly rapidy obtained by the products of its division. Although, when the neck of the
archegonium is removed, there is a conspicuous intermediate, more or less amorphous,
parenchymatous stage before differentiation sets in, this in no way resembles the characSO
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teristic morphology of the gametophyte, as might be expected from Lang’s hypothesis,
nor has any tendency been observed for gametophytic tissue to arise from it. The conclusion seems inescapable that, contrary to Lang’s hypothesis, the development of the
zygote is in some way determined so that given adequate nutrition, it yields tissues which
rapidly acquire the morphological complexity of the sporophyte. I n those ferns with cordate gametophytes, the orientation of the dividing wall of the zygote, and consequently
the spatial arangement of the first organs, almost certainly depends upon gradients within
the gametophyte (cf. Wardlaw, 1955), but there is no evidence that these gradients are
responsible for investing the zygote with the capacity for producing tissues able t o acquire
a higher grade of morphology. The manner in which complexity of development may be
regulated is considered later.
THE STABILITY
OF THE UAMETOPHYTIC AND SPOROPHYTIU MORPHOLOGY
Although in the sexually reproducing ferns transitions from the morphological level of
the gametophyte to that of the sporophyte and vice versa probably rarely occur in natural
conditions, they can be experimentally induced.
A generally successful method of inducing apogamy is t o allow the gametophytes to
age without fertilization. The occurrence of induced apogamy (but possibly not the
ability of the outgrowths to grow to maturity) appears independent of the constitution
of the nucleus; for example, apogamous sporelings arise not infrequently in cultures of
ferns, such as Thelypteris 5alustris and Pteridium aquilinum, which being simple diploids in the sporophytic phase cannot be suspected of hybrid origin. So far it has not been
possible t o cultivate these sporelings beyond a few leaves, but in Phyllitis scolopendrium
(L.) Newm. a haploid apogamous sporeling has been successfully reared until it produced
sporangia, when, as expected, meiosis failed and no spores were produced (Manton,
1950). Some species of Lycopodium, capable of normal sexual reproduction, give rise to
sporelings apogamously very readily in agar cultures, but it has not yet been possible to
raise these to the spore-bearing condition (Freeberg, 1957). There is no reason t o believe
that their behaviour a t sporogenesis would differ from that of the ferns arising similarly
without fertilization.
The reverse process, the aposporous production of gametophytes from sporophytes
has also been frequently reported, but with the possible exception of Osmunda regalis L.
(Lang, 1924), these observations appear to have been made principally on plants taken
from collections, rather than from the wild (see, for example, Beyerle, 1932). Plants in
collections may have genetic or physiological unbalance, resulting either from the selection of some decorative feature, such as cresting of the leaves, or from their being subjected to environmental stresses not normally encountered, and in consequence their
behaviour may be abnormal. Accordingly, observations have been made on Thelypteris
palustris and Pteridium aquilinum raised from spores collected from wild populations.
Fully expanded leaves from young sporophytes were detached and placed in sterile
conditions on mineral agar slopes (Bell &, Richards, 1958). Almost without exception
these leaves have eventually given rise to outgrowths of stable gametophytic form. It
seems likely from these results that material tested previously was not abnormal and that
the capacity to produce gametophytes aposporously is general in the ferns.
I n certain conditions, a detached leaf will give rise to an outgrowth with sporophytic
morphology (Text-fig. 2) (see also, Beyerle, 1932). The exact conditions in which this
occurs, rather than the production of gametophytic tissue, cannot yet be specified, but
it can be said that in the numerous experiments which have beencarriedout,notlessthan
fifty in number, outgrowths from those parts of leaves in intimate contact with a n agar
surface have never been other than gametophytic in form. The one sporophytic outgrowth
was obtained from the petiole rising from an inverted lamina, itself producing numerous
gametophytic outgrowths. Duncan (1941), working with Doodia caudata also observed
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that gametophytes of normal form were obtained only from those parts of leaves in
actual contact with damp Sphagnum; elsewhere the outgrowths were cylindrical and
atypical. The significance of these results is discussed later.
It isof interest to note that the young sporelingof Dryopteris borreriNewm., anobligately
apogamous fern, appears to be less stable morphologically than the young embryo of a
sexually reproducing fern. The spores of D. borreri develop quite normally in sterile culture and the young sporophyte appears when the gametophyte is about 0.5 cm. long.
Text-fig. 2. Thelypterie pdwtria, Regeneration of both gametophytic and sporophytic tissue from
the fifth leaf of a sporeling. (A) Position of the sporophytic outgrowth ( 8 )in relation to the lamine
which is lying on the agar surfme and giving rise freely to gametophytesaposporously. (B) Detail
of the sporophytic outgrowth: T , root arising endogenously.
Six young sporophytes of this fern, each bearing at least one fully expanded leaf, were dissected from the parent gametophytes and each placedupon 20 ml. of mineral-agarmedium.
Sexually produced embryos, if cut out at this stage, or even earlier, continue to grow
quite normally on an agar surface, and any gametophytic tissue adhering to them undergoes no or very little proliferation. The behaviour of the D. borreri sporelings proved to
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be conspicuously different. Growth of the sporophytic tissue was very slow and eventually
ceased, the successive leaves remaining juvenile in form and eventually dying. At the
same time sheets of gametophytic tissue grew out from the base of the sporeling (Textfig. 3). This gametophytic tissue produced no antheridia nor did it show any tendency to
revert to the sporophytic form until it had been in culture for almost a year. A possible
explanation of these results is that the morphology of the young apogamously produced
sporophyte is not, like that of the sexually produced, firmly established. Both the upgrading and the down-grading of the morphology occur more smoothly in D. borreri,
and possibly in other obligately apogamous ferns, than in the sexually reproducing.
Text-fig. 3. Drgopter& borreri. Behaviour of young sporeling placed on Moore’s medium
solidified with 1.5% agar. Explanation in text.
The facts of apogamy and apospory in the sexually reproducing ferns demonstrate not
only that the transition from gametophytic to sporophytic morphology can be induced
by appropriate treatment, but also that the transition can be reversed. Since these
transitions can be brought about quite independently of sexual fertilization or sporogenesis, it follows that these morphological ohanges are independent of chromosome
number. Moreover, measurements have shown that there is the same amount of deoxyribonucleic acid in the nuclei of aposporously produced gametophytes as in those of
the parent sporophyte (Bell & Richards, 1958); consequently any change in the number
of strands in the chromosomes, such as is known to occur in some regions of higher plants
(Duncan & Ross, 1950; Hasitschka, 1956),accompanying the degrading of the morphology can be ruled out.
It is noteworthy that the gametophytes produced aposporously from sporophytes by
experimental treatments show no tendency to revert spontaneously to the sporophytic
form. Neither Lang (1924)nor Manton (1950)report any tendency for the diploid or triploid
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gametophytes of Osmunda to produce sporophytes apogamously. One example of an
apogamous sporophytic outgrowth has occurred in a diploid gametophyte of Pteridium
after 6 months’ culture. This outgrowth, a simple leaf of limited growth, is quite similar
t o those produced occasionally by normal haploid gametophytes. We can conclude that
the new level of morphology imposed by experimental means is no less stable than that of
normal gametophytes.
TEERELATIONSHIP
BETWEEN METABOLISM AND MORPHOLOQICAL
CHANQE
It might be expected that one of the most effective ways of modifying morphology would
be to interfere drastically with metabolism, either by stimulating or by retarding it.
It is certainly very easy to disrupt the normal sequence of development in gametophytes
by including various substances, such as sugars, indole-3-acetic acid and adenine, in various concentrations in the media on which they are grown, or conversely b y omitting from
the media any source of nitrogen. The gametophytes instead of developing in normal
sequence from a filament t o a cordate plate, either remain filamentous or develop into
irregular nodules of cells. There is no firm evidence, however, that such disruption permanently impairs the morphological potentialities of the gametophytic tissue ; similar
aberrations occasionally appear in ordinary mineral-agar cultures and outgrowths of
normal form can often be obtained from them on subculturing on the same standard
medium (the ‘type I’proliferations of Steeves, Sussex & Partanen (1955)). Occasionally
irregular growth is continued on subculturing and the ability to produce outgrowths of
normal form lost (the ‘type I1 ’ proliferations of Steeves et al. 1955). This more permanent
impairment of growth is accompanied by aberrations in mitosis, so that the nuclei in
different cells have different chromosome numbers (Partanen, Sussex & Steeves, 1955).
These variations in development in culture, although of considerable interest, are clearly
not directly related to the regular alternation of form in the life cycle.
Although both Lang (1898) and Duncan (1941) believed that good illumination, and
consequently enhanced photosynthesis was a contributory factor, it has not yet been
possible to discover with certainty any nutritional treatments which will promote the
apogamous development of sporelings in sexually reproducing forms. The coralloid
proliferations described by Steeves and his co-workers (1955) of the gametophytes of
Pteridium apuilinum, the main source of apogamous sporelings in this fern, appeared
sporadically in cultures and the factors inducing them could not be identified, except
that they became more frequent with the age of the culture. Numerous cultures of this
fern have been raised by the author on media containing sugars, the precursors of nucleic
acid, and indole-3-acetic acid, but with no evidence that any of these substances, singly or
in various combinations, promoted apogamy. There is, however, a suggestion from Thelypteris palustris that apogamous outgrowths are stimulated not only as the culture ages,
but also as the medium dries. A gametophyte of T . palustris, about a year old, was
accidentally left on an agar slope open to the air of the laboratory. Some 4 weeks later,
when the slope had contracted t o about half its original volume, the gametophyte was
found to be covered with sporophytic outgrowths apogamous in origin and more numerous than these had ever been observed before on a gametophyte of this fern (Pl.7). It has
not yet been possible to repeat this under controlled conditions, but the observations are
of particular interest in view of Wettstein’s (1942)investigations of analogous behaviour
in the diploid gametophyte of the moss Phascum cuspidatum Hedw. The leaves formed
when the culture was allowed to dry came to resemble sporogonia, and rudimentary
sporangia were produced a t their tips. On remoistening, subsequent leaves were of the
normal gametophytic form. The experiment was repeated on the same plant so that the
relation between the complexity of the morphology and the hydration of the culture
was clearly established. Bauer (1956)has more recently shown that dryness of the medium
promotes the &pogamousdevelopment of sporogonia in a diploid race of another moss,
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INVESTIGATION O F THE PTERIDOPHYTE LIFE CYCLE
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Georgia pellucidu Rabenh. It is known that substances with growth-regulating properties
accumulate in the media upon which the gametophytes of both ferns and mosses are
grown (Bell, 1958; Gorton & Eakin, 1957), and some sort of equilibrium must exist
between the concentrations of the diffusing substances in the medium and in the tissues
of the gametophytes. Partial drying of the medium may cause a shift in this equilibrium
SO that substances with morphogenetic properties re-enter the gametophytes and affect
their development. It seems unlikely that the drying effect is a simple osmotic one, at
least, in the ferns, since were it so it would be much more readily reproducible.
Substances with direct morphogenetic effect are known in higher plants (see, for
example, Skoog & Miller, 1957). One of these substances, kinetin, is also known t o
promote nuclear division in the primordia which arise on the protonema and give rise to
the leafy shoots in the gametophyte of the moss Tortella caespitosa (Schwaegr.) Limpr.
(Gorton & Eakin, 1957). The existence of substances able to enter into the metabolism of
plants and influencetheir morphology, together with the preceding circumstantial evidence,
makes it not improbable that a substance or substances exist, naturally occurring in the
metabolism of the fern, which will promote the apogamous development of sporophytic
outgrowths from gametophytes. Such substances may be discovered when those which
accumulate in the media upon which the gametophytes are grown have been isolated and
identified.
As with the sexually reproducing ferns, no substances have yet been discovered which
will promote morphological transition t o the sporophyte in the gametophytes of the
obligately apogamous ferns. With Pteris cretica L., the addition of a sugar, such as glucose
or sucrose, t o the medium, causes the sporophytes to appear earlier than in controls,
but no evidence has been found of a diminution in the actual quantity of gametophytic
tissue produced before the development of the protuberance leading t o the sporophyte.
Although attempts to stimulate the production of the sporophyte directly from the
gametophyte by nutritional means have'met with little success so far, a sharp diminution
in the supply of nutrients to the sporophyte certainly promotes the degrading of the
morphology. The experiments already described in which the aposporous development of
the gametophytes has been regularly induced, involve a treatment of this kind. The young
leaves, detached and placed on agar, are not only dependent entirely upon absorption
from the medium for their supply of minerals, but, since they are closely appressed t o
the medium, possibly subject to outward diffusion of metabolites and enzymes. It is
very striking that in these cultures the outgrowths of gametophytic tissue have not
appeared until the leaf or fragment of leaf was near death. By this time not only must
correlation of the cells of the leaf have broken down, but also considerable autolysis of
protein and modification of the cytoplasm must have occurred. It will be recalled, too,
that Lang (1924) obtained gametophytes aposporously from Osmunda by drastically
reducing the nutrition of the sporelings.
The fact that regeneration of sporophytic form has never been obtained from sporophytic tissue in intimate contact with an agar surface, but only from that some distance
from it, becomes significant in view of the nutritive conditions which can be envisaged.
The situation is perhaps comparable with that obtaining in orchid protocorms growing
on an agar surface. The protocorm itself consists only of undifferentiated tissue and
Went (1954) observed that the bud from which the fully differentiated plant arose always
appeared in the protocorm a t the point furthest from the surface of the medium. This,
the point which would be least deprived of essential metabolites by diffusion into the
medium, appears to be the only region capable of growth at a higher level of morphology.
Also relevant is the recent observation by Bauer (1957) that the morphology of regeneration from the seta of the moss Physeomitrium pyriforme placed upon an agar medium
depends upon the hydration of the medium, a factor which would influence the speed of
outward diffusion from tissue placed upon it. With high concentrations of agar (4 yo),
the tissue regnerated assumed the form of a young sporogonium or remained as an
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LVI
undifferentiated mass of cells ; only with low concentrations of agar (1.5yo)did the regeneration assume directly the protonemal form of the gametophyte.
An explanation in terms of nutrition probably also holds for the results obtained with
the young sporelings Dryopteris borreri. Removed from the gametophyte and placed on a
relatively large volume of a simple mineral agar medium, the curtailment of their nutritive supply and the loss of metabolites by diffusion may be such that the level of metabolism associated with an advanced morphology can no longer be supported and the
morphology is consequently degraded. That the young sporelings of D. borreri should
react to metabolic stress in a manner different from that of the young embryos of a
sexually reproducing fern must reflect a difference in physiology of great morphological
importance.
Recently Hotta & Osawa (1958)have provided direct evidence of a relationship between
themetabolismofthegametophytesofD. erythrosora(Eat.)0.Ktze., asexuallyreproducing
species, and the complexity of their morphology. From the analysis of samples of gametophytes at successive stages of development, they have been able to demonstrate &
sharp rise in the amount of protein per unit dry weight of the gametophytes at the point
a t which the cordate plate begins to form a t the tip of the simple filament issuing from
the spore. Moreover, if the gametophytes are grown in the presence of certain amino
acid analogues, such as ethionine and 5-methyl-tryptophane, which probably interfere
with normal protein synthesis, the transition from the filament to the cordate lamina is
completely prevented. This indicates that the protein synthesis is intimately involved
in the change of morphology and that the increase in protein content does not merely
reflect an increase in the number of small non-vacuolated cells at this stage. Other
experiments show that the addition of 8-azaguanineto the culture medium also causes
the gametophytes to remain in the filamentous form. This result is ascribed to the
interference of the 8-azaguaninewith the synthesis of nucleic acid, probably of ribonucleic acid. There is, therefore, evidence that the change from the filament to the cordate
lamina in the gametophyte of this species involves rapid synthesis of protein, and possibly also of ribonucleic acid. The increase of protein is of such magnitude that it must
accumulate predominantly in the cytoplasm and not in the nuclei.
An analysis of the treatments which bring about morphological change in the ferns
(and probably in other Pteridophyta), together with the direct evidence provided by
Hotta & Osawa of metabolic change accompanying morphological change, provides a
background upon which a new hypothesis can be formulated to account for the Pteridophyte life cycle.
AN
ASSESSMENT OF THE FACTOR8 CONTROLLING MORPHOLOQICAL UOMPLEXITY I N
THE PTERIDOPHYTA
A review of the experimental evidence available has led to the view that the development
of the spore and the zygote must be in some way determined, even though the normal
course of development can be disrupted by external treatments. It is now necessary to
consider whether the innate control of development derives from the nucleus or the
cytoplasm.
Although the ability of the nucleus to control growth and development is well established, there is evidence that the cytoplasm may also influence the form assumed by
developing tissues, independently of the nucleus. For example, in both archegoniate and
higher plants the products of reciprocal crosses sometimes differ in appearance (Wettstein,
1937) and even in the pairing behaviour of the chromosomes at subsequent meioses
(Wangenheim, 1957).These differences can be attributed to the different maternal cytoplasms going to form the initial zygotes. There is clearly every reason to expect that the
chemical and physical properties of the cytoplasm will have direct morphological consequences, for the cytoplasm contributes to the formation of the spindle, the apparatus
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INVESTIQATION OF THE PTERIDOPHYTE LIFE CYCLE
197
which determines the plane of cell division (for a review of the origin of the spindle
in plant cells, see Tischler, 1951). The directions in which cells divide and build up a
tissue are of profound anatomical and ,morphological importance, because distinctive
shapes, degrees of ramification, dimensions, and symmetry, all depend as much upon the
direction of cell division as its extent. Size, in turn, may, by influencing%
the distribution
of metabolites, determine the pattern of differentiation within a tissue (Bower, 1930;
Wardlaw, 1952). Consequently, the accumulating evidence (see, for example, Mather
& Jinks, 1958) that some variation in the properties of the cytoplasm independently of
the nucleus is permitted in somatic tissue is of great importance for the student of
growth and form.
There are grounds, therefore, for inquiring whether features of the cytoplasm may not
control the complexity of morphology which a plant is able t o achieve. This is not t o
deny the nucleus ultimate control, but t o suppose that in somatic development the immediate control is exercised by the cytoplasm. There is some evidence from higher plants
that this may be the situation. I n a species of Gardenia described by Stewart (1924),for
example, the lateral branches have a morphology markedly different from that of the
main shoot. If the laterals are struck as cuttings, they form squat, irregularly branched,
bushes, quite unlike the normal plant. The morphology imposed upon the lateral shoots
must, therefore, follow from a permanent change in the functioning of their meristematic
cells. This does not, however, appear t o result from any modification of the nucleus,
since the flowers are normally borne on the lateral shoots, and the seeds so far as is
known yield plants of the normal composite morphology. The inference is that in normal
development the cytoplasm of the cells of lateral meristems becomes in some way
altered and that this alteration is reflected in the different symmetry of the lateral
members.
The recent demonstration by Steeves & Sussex (1957) and Sussex (1958) that the primordia of the leaves of ferns continue t o develop into leaves if they are detached and
placed on a nutrient medium indicates that the characteristic form of these members is
not due to continuous influences from the main axis. A change must have occurred in the
cells of the primordium in its initiation so that their progeny build up an organ of dorsiventral, instead of radial, symmetry. Since the spores, which are produced on the leaves,
ultimately reproduce normal plants, this change can not be in the nuclei. The most
satisfactory hypothesis is that the change in the cells of the primordium leading t o the
dorsiventral symmetry is confined t o the cytoplasm, and that the properties of the
cytoplasm may be vaned by internal factors.
There are reasons for believing that the alteration of morphological levels occurring
naturally, or induced, in the ferns also has its immediate origin in cytoplasmic, rather
than nuclear changes. The arguments can be grouped as follows. First, the estimates
of deoxyribonucleic acid in the nuclei of gametophytes arising aposporously and in
those of the parent sporophyte give no evidence of gross nuclear change accompanying
the morphological degradation. Secondly, the evidence points to experimentally induced
apogamy and apospory resulting from metabolic situations not normally encountered in
the life cycle. There is no firm evidence that metabolic stress in any plant, applied either
directly or by grafting, can induce nuclear change. Moreover, the experiment with
Thelypteris palustris in which different parts of the one leaf regenerated sporophytic and
gametophgrtic tissue would suggest a mutability of the nucleus quite contrary t o the
generally accepted view of its stability in the face of environmental fluctuation. Thirdly,
the existence of the obligately apogamous ferns, in which the transition from gametophytic to sporophytic morphology is a normal feature in development, is a strong
argument against the morphological changes being directly initiated by the nucleus.
The up-grading of the morphology of these ferns after the gametophyte has reached a
certain size is a smooth and continuous developmental process, not suggesting any abrupt
nuclear intervention. That the morphological transition can be readily reversed in
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P. R. BELL:
[J.L.s.B.LVI
experimental conditions in D yopteris borreri and other species (Steil, 1939 ; 1944) with
subsequent regeneration of the sporophyte is further evidence against the nucleus
being immediately involved.
On the other hand, it is very probable that by its general control of the cytoplasm
the nucleus allows the morphological transitions of apospory and apogamy to take place
with greater or less facility. It is well known that horticultural varieties of ferns often
show these phenomena very readily, probably a result of genetic unbalance following
artificial selection. The curious form of Phyllitis swlopendrium described by AnderssonKotto & Gairdner (1936) is an excellent example of this effect. Also,the fact that hybrids
between sexually reproducing and obligately apogamous ferns are themselves obligately
apogamous shows the profound influence of the nucleus of the obligately apogamous species
upon the cytoplasm of the sexual with which it becomes associated a t fertilization.
Nevertheless, it seems more in accord with experimental evidence to regard the function
of the nucleus in somatic development as permissive, rather than determinative. It
is envisaged that limits are set to the functioning of the cytoplasm, but within these
limits variation, spontaneous or induced, may occur with quite striking morphological
consequences.
The foregoing considerations lead to the conclusion that the cytoplasm determines the
gross nature of the morphology acquired by developing fern tissue. It is not denied that,
as hybridization experiments show, the nucleus is responsible for the fine features of
each morphological state which distinguish individual species, but it is proposed that the
alternation of morphological levels in the life cycle of the Pteridophyta is a reflexion of
the different states and properties of the cytoplasm in each generation.
A RE-EXAMINATION OE THE EVENTS IN THE PTERIDOPHYTE
LIFE UYCLE
If, as concluded in the last section, the cytoplasm is of importance in determining the
level of morphological complexity, then, in the sexually reproducing ferns, there must be
some mechanism by which the state of the cytoplasm is changed in the process of reproduction. The two stages of sexual reproduction, gametogenesis and fertilization, are
examined separately, and it will be seen that the former has several features of significance to this inquiry.
So far as spermatogenesis is concerned, there is little that appears relevant. The antheridia are produced very early upon normal gametophytes, a t a time when there is no tendency for sporelings to appear apogamously. Antheridia may even be present upon filamentous gametophytes, where, if the results of Hotta & Osawa (1958), already referred
to, are of general validity, the protein metabolism is of a very low order. The spermatozoids themselves consist of little more than nuclear material. Oogenesis, on the other
hand, is accompanied by several features of interest. First, archegonia do not usually
appear until after the production of antheridia, when the gametophyte is more mature,
and its protein metabolism probably more complex (Hotta & Osawa, 1958). Moreover,
in cordate gametophytes, the archegonia appear just behind the apical notch, and it is
significant that this is the region from which develop, in Pteridium aquilinum, the coralloid proliferations from which arise most frequently the apogamous outgrowths (Steeves
et al. 1955). I n the obligately apogamous ferns, the process which develops into the
sporophyte also arises from the gametophyte in the region corresponding to the archegoniate cushion in the sexually reproducing ferns. Secondly, in oogenesis itself there is
evidence of cytological phenomena which occur nowhere else in the life cycle. In
P. aquilinum and Thelypteris palustris the nucleus of the egg in the very young archegonium is clearly visible and stains with Feulgen’s reagent with a n intensity only a
little less than that of the somatic nucleus. As the archegonium matures, the nucleus
of the egg expands to an irregular oblate spheroid with a major diameter of the order
of 18p and a minor (in the axis of the archegonium) of lop. As this expansion occurs
J.L.S.B. LVI]
INVESTIGATION O F THE PTERIDOPHYTE LIFE CYCLE
199
the nucleus stains progressively less with Feulgen’s reagent and at maturity the
staining cannot be identified with certainty (Pl. 7). The volume of the nucleus of
the mature egg cell is some twenty-five times that of the somatic nucleus, so that if
the amount of deoxyribonucleic acid in it, and its capacity for taking the stain,
were unchanged the staining would be considerably diluted. However, experiments
with basic fuchsin in aqueous solution of an intensity adjudged by eye equivalent
to that of the stain in the somatic nuclei show that it is still clearly visible when
diluted twenty-five times. A subjective element inevitably enters into the matching
of colours in a minute opaque object under the microscope and in aqueous solution,
but it does look as if either the amount of deoxyribonucleic acid in the egg nucleus
is less than that in the somatic nucleus, or that it has undergone some change so
that its capacity to take Feulgen’s stain is lessened. There is evidence of a similar situation in the egg nucleus of certain Angiosperms (Rowlands, 1954; Krupko & Denley,
1956). Whether deoxyribonucleic acid is in fact absent from these egg nuclei has yet t o
be demonstrated incontrovertibly, but there is clearly evidence of nuclear activity
peculiar to the egg. It may be that the expansion of the egg nucleus (presumably due t o
the incorporation of cytoplasmic material), and the dilution and possible modification
of its deoxyribonucleic acid are an indication of an intense interaction at this point in the
life cycle of the sexually reproducing fern of the nucleus and cytoplasm. It is not unlikely
that it is here that the cytoplasm begins to be modified and its capacity for supporting
an up-graded morphology established. No cell, however, can multiply and give rise to a
differentiated tissue unless it has an organized nucleus but the fact that the nucleus of
the egg is strikingly ill-defined in comparison with the,somatic nucleus suggests that the
modifying of the cytoplasm of the egg is achieved at the expense of nuclear organization.
The entrance of the compact nucleus of antherozoid, and its intimate association and
fusion with the nucleus of the egg, should perhaps be looked upon as the event which is
essential for the completion of the modification of the cytoplasm and the reorganization
of the nuclear component. This accomplished, continued growth is possible (cf. Rowlands,
1954), and the zygote gives rise to tissues of sporophytic complexity.
On this hypothesis development of a sporophyte from an unfertilized egg (parthenogenesis) would be a very unlikely event, since the cytoplasm in the modified condition
and an organized nucleus would not occur together. This would account for the striking
fact that, although known in the Angiosperms, parthenogenesis is almost unknown in
the homosporous ferns. It has been recorded only in certain horticultural varieties
(Farmer & Digby, 1907), the life cycle of which is altogether abnormal. If material
similar to that examined by Farmer & Digby can be traced, it will be of interest t o see
whether the staining of the egg nucleus with Feulgen’s reagent is in fact similar t o that
of the sbmatic nucleus. No recent studies have been made of the parthenogenesis reported
in Marsilea drummndii A.Br. (Strasburger, 1907) and Selaginella (Bruchmann, 1919;
Goebel, 1915).
Relevant t o the discussion of the events in the egg cell is a recent report by Mather &
Jinks (1958) of experiments on Aspergillus glaucus Link, in which they have compared
the products of sexual and asexual reproduction. Sincethey find considerably less variation
amongst the products of sexual reproduction than amongst those of asexual, they conclude
that in sexual reproduction the nucleus has a profound effect upon the cytoplasm, so that
in each reproduction cycle the cytoplasm is restandardized, and many or all of the alterations that have occurred in it in somatic development eliminated. Although these
authors are not concerned with the determination of morphological complexity, it is
noteworthy that an entirely different line of inquiry should have led t o the view that it is
predominantly in sexual reproduction that the nucleus imposes organization upon the
cytoplasm, whereas in somatic tissue the cytoplasm is permitted some variation in its
properties and functions.
If gametogenesis and fertilization are the occasions when, in the sexually-reproducing
200
P. R. BELL:
[J.L.s.B. LVI
ferns, an up-grading of the morphology is made possible by changes in the cytoplasm,
then the converse process must occur at sporogenesis. The physiology of sporogenesis is
as yet hardly investigated, but there is some evidence that it is encouraged by increasing
the amount of carbohydrate avdable to the tissue. It is often observed,for example, that
ferns in situations where insolation is considerablemay be depauperate, but abundantly
fertile. Also Sussex & Steeves (1958), working with a number of ferns, have shown that
whether or not sporangia appear on leaves developingfrom isolated primordia in culture is
markedly influenced by the sucrose content of the medium. Although the evidence is still
conflicting, there are grounds for believing that the onset of the reproductive phase may
depend upon the balance of the carbohydrate and nitrogen metabolisms, promotion of
the former rather than the latter favouring reproduction. If the nitrogen metabolism is
relatively depressed in the fertile fronds of ferns, then meiosis may well lead to spores
containing a cytoplasm of which the proteins and ribonucleicacid are reduced both quantitatively and qualitatively to such an extent that recovery in the tissues developing from
the spores is not immediately possible. This reduction would, on the hypothesis advanced
earlier, be reflected in the simple morphology of these tissues.
If the foregoing be the situation in the sexually reproducing ferns, then in the obligately apogamous, since there is no indication of an interaction between nucleus and
cytoplasm similar to that in the egg cell of the sexually reproducing, the cytoplasm
deriving from the spore must be capable of progressive elaboration, facilitating a progressively more complex morphology of the tissues containing it. It is possible that the
eEciency with which the cytoplasm is able to evolve in this fashion varies with the specks,
but comparative studies of this nature have yet to be made.
The number of sets of chromosomes in the nucleus of the spore appears to influence
the ability of apogamous outgrowths to reach maturity. Gametophytes whose nuclei
contain only one set of chromosomes rarely produce viable sporophytes apogamously,
whereas in at least two species, Dryopteris a u s t r i m (Jacq.) Woynar and D.Jilix-mas(L.)
Schott, the nuclei of the gametophytesofwhich containtwo dissimilarsets of chromosomes,
comparatively vigorous plants have been produced in this way (Manton & Walker, 1954).
Since these two species are allotetraploids, the comparative vigour of the apogamously
produced sporophytesmay be facilitated by the interaction of similar,but not homologous,
chromosomes in the gametophytic nucleus. That obligate apogamy in D. borreri and some
other ferns is associated with general genetic unbalance and is not determined by a
simple genetic factor was shown by Manton’s (1950) investigations. Although hybridizing experiments show that the apogamous condition is dominant, there is no form of
Mendelian inheritance.
No explanation can be offered at this stage for the cytological irregularity, the formation of the restitution nucleus, which accompanies obligate apogamy and ensures that
viable spores are produced having the same chromosome number as the parent. There is,
however, some evidence that ribonucleic acid has an effect upon dividing somatic nuclei
similar to that of colchicine (Allen, Wilson & Powell, 1950). If the metabolism of the
obligately apogamous ferns is such that ribonucleic acid is progressively accumulated by
the cytoplasm, it is possible that a continuation of this process might lead to effects
upon meiosis. It is noteworthy that the behaviour of the sporogenous cells is very variable in the obligately apogamous ferns; in only a proportion of the sporangia are eight
spore-mother cells formed, in the remainder various other irregularities occur generally
leading to a breakdown of meiosis and abortive spores. Such a range of behaviour would
be expected were the factor controlling it a component of the cytoplasm variable in
quantity.
J.L.S.B. LVI]
20 1
INVESTIGATION OB THE PTERIDOPHYTE LIFE CYCLE
CONCLUSIONS
The purpose of this paper has been to indicate a new approach to the morphological
problems of the Pteridophyte life cycle. The gametophyte and the sporophyte may be
looked upon as two levels of morphological complexity. I n proposing that they reflect
different states of the cytoplasm, which can be accounted for in terms of cell chemistry,
a hypothesis is advanced which, although speculative, accommodates the experimental
evidence at present available, and may serve as a stimulus to future research.
Two Lines of investigation immediately suggest themselves and both are a t present
receiving attention, although it is too early yet to report results. First, in the obligately
apogamous ferns, since the transition from spore to sporophyte is not interrupted by
sexual fertilization, a study of the protein and nucleic acids of the cytoplasm a t various
stages of development becomes a matter of great interest and importance. Those concerned with the problems of embryogenesis in the higher plants have also reached the
conclusion that apogamous forms may yield information of great value (Steward &
Pollard, 1958). Secondly, an elucidation of the events in the egg cell is imperative for it
involves not only problems of development, but also those of the transmission of the
genetic information by the female nucleus.
Causal morphology is entering an exciting phase, for the problems of growth and form
are beginning to be resolved in terms of proteins and nucleic acids, the complex molecules
of the living cell of especial biological significance. Because of the isolation of the gametophyte from the influences of the parent sporophyte in the Pteridophyta, members of
this group may provide rather simpler developmental situations than the higher plants,
and prove particularly suitable for investigation by the cell physiologist. Almost a
century ago Hofmeister’s elucidation of the morphology of the life cycle of the Pteridophyta proved the key which unlocked the mysteries of the life cycle of the higher plants.
There are indications that the Pteridophyta may play a similar role today, and the discovery of the causal factors governing the morphological developments in the familiar
Pteridophyte cycle facilitate the investigation of the rather more intricate cycle of the
Angiosperms.
ACKNOWLEDGEMENT
for much stimulating criticism and
The author is indebted to Prof. D. Lewis, F.R.S.,
discussion in the preparation of this paper.
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EXPLANATION OF PLATE 7
(a) Thdypte7ia pduatris. Portion of old prothallue aovered with aporophytio outgrowths arS1~
apogmoualy. (b) and (c) Pteridkm qw’Zinm. Stages in the maturation of the arohegonium
stained in Feulgen’s reagent. Each section, cut at 12p, contains h o s t the whole of the egg
nucleus (en.). The intensity of the staining of the egg nuoleua diminishes as the mhegonium
appromhes maturity.
9-2
P. R. BELL
Journ. Linn. SOC. Bot. Vol. LVI, PI. 7
(Facing p . 203)