J . Linn. Soc. (Bot.),58, 373, p . 361
With 1 plate and 3 text-jigurm
361
Ptinted in &eat Britain
Morphogenetic factors influencing the development
of fern embryos
BY A. E. DEMAGGIO
Rutgers, The State University-CoUege of Phamnacy,
Newark, New Jersey, U.S.A.
INTRODUCTION
The orderliness with which a fertilized egg develops through a series of stages, characteristic for a given species, to the adult form is unquestionably one of the most remarkable
of all natural phenomena. It is obvious that this one cell, initial of the embryo, seemingly
so simple in organization, must be endowed with all the prerequisites necessary for performing the numerous and often diverse functions associated with growth and increased
structural complexity : e.g. cell division, cell enlargement, and cell differentiation. However, it is equally obvious that the problems involved in determining the precise manner
by which the prerequisites are transmitted to the egg and there organized,distributed, and
utilized during embryogenesis are so complex that up to the present time relatively little
information concerning these processes has been obtained. Nevertheless, by virtue
of its natural or inherited totipotency, the fertilized egg should be recognized as the
logical starting point for developmental and morphogenetic studies, despite the many
problems often posed in obtainjng, storing, and utilizing it for experimentation.
Progress in plant embryogenesis, especially the experimental phase, has moved into
prominence in the past decade, largely because of the development of manipulative as
well as culture techniques on the one hand, and the recent introduction of analytical
methods and equipment which permit approachesto biochemical changes in unbelievably
small amounts of material on the other hand. The electron microscope has opened the
cell to examination both for its cytology and its organization. Moreover, autoradiography and tracer studies have given a o w s to movements and accumulations of
materials, thus yielding information on nutrition and metabolic change which had hitherto not been open to the study of embryology ( W d a w , 1955; Steward & Mohan Ram,
1961). Although in none of these investigations has it been possible to isolate the unicellular, fertilized egg and nurture it to maturity in Witro, recent progress in this direction
clearly demonstrates that this ultimate goal is within reach.
Much of our knowledge concerning the relationship of the developing embryo to its
external and internal environment has been derived from studiea in culturing embryos
and other reproductive structures of angiosperms and gymnosperms (Steward & Mohan
Ram, 1961). From the information presently available it is evident that a variety of
morphogenetic factors, genetic and environmental, are operating to control various
stages in the growth and development of any particular plant embryo. The accumulated
data not only indicate that these fadom are responsible for normal orderly development,
but also evidence has become available to show that modification of these factors, even
temporarily, often results in drastic alterations in morphological and physiological
characteristics. The manner by which these factors influence the sequence of events in
einbryogenesis is not known despite considerable work on this problem (Sinnott, 1960).
Results from certain of the studies support the contention that the normal sequence of
events occurring during embryogenesis is not predetermined simply by the genetic
constitution of the plant ; rather that developmental steps may be dependent as well on
the regulated influences of environmental factors, physical and/or chemical. Therefore,
a primary aim of embryological studies must be to determine the relative importance
362
A. E.DEMAWIO
and the interrelations of genetic, physical, and chemical factors in regulating the growth
of a simple homogeneous*cell, whether it be embryo, spore, or h e vegetative cell;
and in providing the specific subatanma necemiwy for that cell to attain ita morphological and physiologicalheterogeneity.
APPROACH TO THE PROBLEM
In the lower archegoniate p h t a , bryophyta and vascular cryptogams, a double
opportunity exislts for assessing the degree to which various morphogenetic factors
affectsingle-celledinitids (fertilizedegg or spore)and contribute to the orderly formation
of the two characteristicp
h in the life cycle. It seems reasonable to assume that the
spore is as much an embryonic cell as the fertilized egg since under the proper conditions
both germs, to use Profegsor w
s inclusive term,give rise to characteristicplants and
both initiate a separate phase in the life hietory. A study in embryonic development
conceivably wuld involve the utilization of either one of them two germs.
While it is obvious that th0 products obtained on the germination of the spore and the
fertilized egg we quite distinct morphologically,there is little direct evidence to account
for the Werence between gametophytio and spomphytic generations. During the years,
fundamental quegtionshave been raised concerningthe potentialitiea of the two initi&ing
cells in the life W r y (La.ng, 1909,1915;Blackman, 1909);nevertheless, the determination of the factors reeponeible for alternation of generations still remains a perplexing
and baaic problem in morphogeneais.
The work described in the present paper ww undertaken to determine the extent to
which selected environmental factom d u e n c e the development of fern embryos in
their natural habitat, wntained within the amhegonial venter, or isolated and grown
free in sterile culture. Recent studies on fern sporeg (Partanen, 1957; Partanen &
Nelson, 1961) indicate that environmental changes often produce deviations in the usual
pattern of development. Similar observations have been made in studies reported here
utilizing fertilized eggs and older fern embryos. Some of the experimental data bearing
on the development of fern spores and embryos have been reviewed by Wetmore (1959)
and Bell (1969).The present report will attempt to include more recent observations on
this topic in the hope that they may further stimulate studies in the area,of alternation
of generations.
ANALYSIS OF ALTERNATING GENERATIONS
Since the elucidation by Hofineister in 1851 that the life cycle in the higher plants
(Tracheophyta)consists of a regular alternation of an asexual with a sexual generation,
this alternation of generations has become widely recognized as a basic pattern of reproduction. Strasburger (1894)pointed out that the alternating of a sporophyticphase with
a gametophytic phase was associated with a reduction in chromosome number from the
diploid (2n) to the haploid ( I n ) level. Experimental evidence from a variety of sources
(seeWetmore, 1959) indimtea that the differencein chromosomenumber between a spore
(112) and a fertilized egg (2n)is not adequate in itself to account fully for the morphological
differences between plants produced from these initial cells. How then can we account
for and interpret the origin of these dissimilar generations?
Prom a phylogenetic point of view, two theoriea attempting to explain the origin of
the sporophyte have received considerable attention in the past. The ‘antithetic’
theory, suggested by clilakovw (1868, 1874) and developed by Bower (1890; see
Origin of a Land F‘lora, 1908, for summary of arguments) was based on the concept
that the sporophyte was essentially a new and progressively vegetative structure elaborated and interpolated into the life history in cormexion with the migration of plants from
water to land. Opposed to this theory, the proponents of the ‘homologous’ theory of
Pringsheim (1878;staked anew by Scott, 1896) maintain that the sporophyte is not a new
ikforphogenetic factcn-8in$uewi?q the devebpnzent of fern embryos
363
etructure, but is to be regarded merely as a mod%ed expression of the gametophyte
when developed under different environmental conditions. It is of interest to recall that
these two points of view on the alternation of generations were the basis of a Discussion
before the h e a n Society in 1909 in which such eminent htanista aa b n g , Bower,
Scott, Farmer, Oliver and Tansley participated. While a detailed consideration of the
palaeobotanical and historical evidence which supports or refutea these theories is
beyond the scope of this paper, it should be noted h t the experimental evidence,
primarily from studiea on apogamy and apospory, tends to favour the latter hypothesis
(Whittier, 1961 ; Lal, 1961).
It is noteworthy that Lang (1909, 1915) postulated that environmental conditions
(physical and nutritional) must greatly influence the course of development in the fertilized egg and spore. He reasoned that reatrainta or pressures, exerted on the fertilized
egg by adjacent prothalkd cells,were in some way influencing the characteristic growth
pattern of this germ as contrasted with the spore germinating freely on some substrate
external to the plant.
Experimental investigations, made possible by advances in surgical techniques and
by the introduction of methods for plant tissue culture, have yielded indirect evidence
which seems to support this hypothesis.
The surgical experiments of Ward & Wetmore (1954) demonstrated that the young
fertilized egg of the fern Phlebodium (Polypodium)
aureurn,growing in its natural environment within the archegonium,was apparently subjected to considerablepreaeure from the
surrounding prothallial cells, 88 Lang had previously theorized. When the young,
undivided egg waa relectsed partially from these pressures by cutting away portions of the
surrounding tissue, the usual sequence of embryological stages did not occur and various
abnormalitieswere noted. Such surgically treated embryos usually developed cylindrical,
or somewhat irregular, aggregations of cells, all of which produced leaves and axes and
some which developed ultimately to normal sporophytes.
Jayasekera & Bell (1959) also have found that the developing fern embryo is under
the influence of mechanical pressure supplied by surrounding cells. When the neck of the
archegonium was removed from the unicellular embryos of Thelypterk palustris, the
pressure appeared to be released and profound changes were observed in the usual
development of the zygote. For the most part, these changes in embryo development
were quite similar to those noted by Ward & Wetmore (1954).
Some of the surgical experiments of Ward & Wetmore and Jayasekera & Bell have
been repeated using Todea barbara (DeMaggio,unpublished)to determine if abnormalities
similar to those produced in the polypodiaceous ferns could be induced to form in the
geologically older Osmundaceae. Results from this study similarly have shown that
removal of the neck of the archegonium and/or portions of surrounding prothallial tissue
caused the embryo to develop into a cellular tissue mass and delayed the eventual formation of organs. However, if the incisions were made after the embryo had attained
considerable size (15O-250,4, development proceeded normally, though in several
instances more slowly, until the usual sporophyte was produced.
From these limited studiea it is evident that physical pressures or restraints are
active as a factor which must be reckoned with in any investigation of early development
of fern embryos. Release of this restraint, even partially as in the experiments cited,
results in a temporary deviation from the usual developmental pattern, afbr which normal
plants ultimately develop. Older embryos do not appear to be influenced to any appreciable extent by the restraining environment. Removal of portions of surrounding
tissue did not seriously limit the potentiality of already partially developed embryos
in the completion of their development in a normal period of time. Nevertheless, it
seems reasonable to conclude that restraint or pressure may be one important morphogenetic factor, at least temporarily, in the development of fern embryos contributing to
the regular alternation of generations. It is to be noted, however, that Blackman (1909)
364
A. E. D E ~ ~ W I O
early suggested that the origin of the two dissimilsr generations WM probably the result
differencesinherent in the two initial germs and not due to the action of
of c-riatic
environmental influences. Them chacteristic differenceshe proposedwere not differences
in chromome number,but rather diffsrenoes in some other nuclear component.
A modification of Blackman’s hypothesis has been advanoed recently by Whittier
(1961). On the baais of studies on induced apogamy in €’tedium aqudinum (1960),
Whittier & Steeves proposed that in the Merenoes in growth and morphological expression betwean gametophyte and sporophyte, energy supply normally available to the two
generations must be recognized.When the level of respiratory substrate (sugar)supplied
to the pmthdua ia increased, morphogenetic changes are observed which often lead to
the establishment of a thr?ee-dimemiondgrowth pttern. Further, it has been shown that
the conthe apogamous production of prophytes can be controlled by &pulating
centration of ‘energy yielding substrate’.!Chew obeervations, and the results of current
studies on induced apogamy in haploid end diploid gametophytee, have led Whittier
(1961) to postulate that although the number of genomes may definitely influence
the morphological expression of,the two generations, the ultimate response is not a
direct effect; rather it may be due to the interaction of nuclear and nutritional factors.
These studies have introduced two modifications in Blackman’shypothesis:
(1) The characteristic Werenoes between the two generations may be found in the
lessened availability of some cell substrate in the gametophyte at its haploid level which
doee not permit the development of the more complex growth pattern of the sporophyte.
(2) The expression of genetic potentialities may be influenced by environmental factors, and that of them nutrition, for example, carbohydrate nutrition, may govern
partially the behaviour of developing hiti&.
An undoubted more sharply defined thesis hsd already been suggested by Bell (1959),
when he proposed that the development of a fern spore or zygote must be determined
by the Merent etatea and potentialities of the cytoplasm in each generation.
More recently Bell & Zafar (1961), employing an apogamously reproducing fern,
Dryopi%& bowek, attempted to determine whether the attainment of morphological
levels of development (gaznetophyteand sporophyte) could be correlated with changes in
the proportion of protein to non-nitrogenousmaterids in their growing systems. n e i r
investigations discloaedthat an exponentid relationship actually exists between amounts
of growth of the gametophyte, from spore to the emergence of the sporophyte, and the
amounts of nitrogen present. Reports of Hotta, Osawa & Sakaki (1959) had
suggested that the transition from one-dimenaionalto two-dimensional growth of the
fern profhallus wm related to rapid incresse in protein synthesb in the presence of a
special type of RNA. In fact, a re-examination of the data of Hotta et d.(1958, 1959)
uncovered a similar relation for protein and RNA during the transition from the onedimensional to two-dimensionalgrowth of the protha,llw. Bell & Zafar (1961) concluded
that there is little evidence to substantiate a correlation of morphological change with
gross incremes in rates of protein or R S A synthesis. Instead, these authors point out
that transitions in morphological complexity, such M those occurring during alternation
of generations, are probably the result of a qualitative change in the nature of protein
and RNA formed during the transitional periods of development.
These studies suggest that an ultimate. answer to the question of why the morphology of the gametophyte should be so merent &om that of the sporophyte resides in
a thorough evalurttion and analysis of the biochemical ‘building blocks ’ of living matter,
proteins and nucleic acids; moreover, that the role of vmious morphogenetic factors is
incidental to and will modify only the pre-determined p t t e r n of development.
While the precedhg mcounts have been limited intentionally to a consideration of
selected studies utilizing ferns &B experimental material, the interesting work of Bauer
(1956, 1957) and others who are approaching the problem of alternating generations
by utilizing other archegoniate plants should not be overlooked. However, even from
Norphoqenetic factors ~nJluencingthe development of fern embryos
365
the brief analysis of alternation of generations presented here, certain generalizations
regarding the underlying causes can be made.
(1) No single factor, or combination of factors, is known whioh will allow for the artificial control of alternating generations.
(2) Much experimental evidence indicates that genetic factors do not exert complete
and unyielding control over the development of fertilized egg and spore.
(3) Environmental factors have been shown to exert considerable control on development of fern initials and influence the ultimate form attained.
(4) The environmental factors which so far are known to play a part in the development of those vascular plants experimented on are physical restraint and nutrition.
It was in an attempt to determinethe extent to which physical restraint and nutrition
influence the development of fern embryos and thereby contribute to the regular alternation of generations, that the experimental work now to be described was undertaken.
We shall consider frrst those morphogenetic effects of restraint, and, second, those
morphogenetic effects produced aa a result of nutritional influences.
MORPHOGENETIC FACTOR I-RESTRAINT
Admittedly much of the available evidence indicating restraints or pressures as a
significant factor in the early organization of embryos is only indirect. Although changes
in pattern of development have been noted and have been duplicated at will in experimentally treated embryos, generally normal plants ultimately resulted. It should be
noted, however, that in these reports the total influence of restraint or pressure was not
removed completely from the developing embryos. Only a portion of the surrounding
tissue was removed. In no instance reported was the early, unicellular embryo ever
freed completely from physical contact with surrounding cells.
Bell (1959))in commenting on the diversity of form occurring in the fern life cycle and
presenting experimental methods for determining the causal factors involved, indicates
that a crucial experiment would be to isolate m p l e t e l y an undivided fertilized egg and
allow it to grow in a free condition. I n this manner restrictive influences could be better
evaluated. This approach has been used in the work now to be reported.
A recenk study (DeMaggio & Wetmore, 1961a) disclosed that the osmundaceous fern
Todea burbura is admirably suited for investigationsinvolving completely freed embryos.
Employing this species we found it possible, by the use of suitable manipulative techniques, to remove the embryo from its position within the venter at all stages in development from the 4th to the 20th day following fertilization. It was readily apparent that
here was an ideal plant for comparing the development of ‘contained’ and ‘freed’
fern embryos and for attempts at assessing the degree to which physical restrictions
contribute to what we ordinarily consider the orderly development of the sporophyte.
Develqment of the ‘contained’ ernbryo
Prothalli of Todea barbarawere grown in culture from sporeson a simple nutrient medium
and the initiation and development of sex organs was followed closely (DeMaggio, 1961a).
Considerable variation was found to exist in the timing and sequence of reproductive
events when various stages were compared with those of polypodiaceous ferns. However,
the overall course of development was representative of the leptosporangiate type. At
maturity, the archegonium is a relatively large structure composed of 6-8 tiers of neck
cells, each tier made up of four cells, a long densely protoplasmic neck canal cell with two
nuclei, the ventral canal cell, and the flattened egg cell containing a large centrally
located nucleus embedded in the protoplasmic matrix (Pl. 1, fig. 1). From its inception
the egg cell appears to be surrounded by a thin membrane which adheres closely to the
surrounding cells of the venter. Repeated attempts to remove the egg cell a t this stage
JOURN. LINN. S0C.-BOTANY,
VOL. L M I
24
A. E.DEMAQQIO
366
in developmenthave been unsuco888fu1as the delicate membrane a p p r s unquestionably
to be coherent to cells of the adjacent jacket and usually is damagedduring manipulation.
It has been suggested (Bell, 1961) that changes take place in the bounding layer of the
cytoplasm of the fern egg on fertilization. In Toderc these chmgea, although not visible
mimoscopidy, may well occur aleb for it has been possible to ex& fertilized eggs
undamaged as early as 3-4 days after fertilization (Pl. 1, @. 2). Furthermore, it is quite
possible that even younger fertilized eggs also can be excised once the available techniques and operative skills have been refined. Apparently the act of fertilization changes
the character of the outside of the egg for it seems to be much le&s adherent to surrounding cells,as evidenced by the eaae with which embryos can be excised. While the acquisition of a ‘fertilization membrane’ is known for the eggs of many invertebrate animals
(Rothechild, 1956), the occurrenoe of a aimilar phenomenon in embedded plant zygotes
ha3 yet to be demonstrated conclusively.
950
-
Emerge from
calyptra
900850
-
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Enlarging
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650-
-
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400
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Procambiu m
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200 150 100 -
Stem and root
Leaf
250
Foot
32-celled
50-
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Unicellular
I
5
1
10
I
15
I
20
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25
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30
Days following fertilization
Text-@. I. Growth of ‘contained’ embryos of T&
barbara grown on a solidSed,
nutrient. medium with 1 yo sucrose.
Once fertilization has been succeasfully inithted, the embryo develops through a
series of charaoteristic stages until it bursts the d y p t r a of surrounding cells and soon
becomes established as a self-sufticient sporophyte. The major changes in embryo size
and morphological complexity following fertilization are diagrammatically illustrated
in Text-fig. 1. A detailed description of the growth and development of Todea embryos
will be found in a previous publication (DeMaggio, 1961b).
During the first few days following fertilbmtion the embryo increwes only slightly in
dlorphogenetic factors in$wncing the development offern embryos
367
size (25-45~)and with the exception of rearrangement of chloroplasts no changes are
visible under a light microscope. The &st division occurs about 6 days after fertilization and succeeding divisions follow closely so that by the 13th day most embryos
examined consist of approximately 32 cells. Differentiation in the contained embryos
is not evident until the 15th day following fertilization when anticlinal divisions are
noticeable in the foot region. The foot is the first organ found in T& and its large
cells are eaaily discernible in embryos sectioned when 17 days of age. Prior to the initiation of leaf, stem, and root, internal differentiation of the embryo characterized by the
formation of a central core of provascular tissue, is noted. Eighteen days after fertilizetion, leaf initials can be recognized, followed in 1-2 days by the formation of stem and
root initials. The remaining period of contained development is characterized by a considerable increme in size of the embryo, the formation of procambial tissue and its extension inta the enlarging organs, and f h d y the emergence from the calyptra of the embryo
now bearing recognizable organs.
One recognizes that although the duration of contained development for Todea
embryos lasts approximately 30 days, rapid increase in size is associated mainly with
those activities occurring during the latter stages of development, e.g. cell division, cell
enlargement, and cell differentiation. Duringthe h t half of its contained development,
the embryo exhibits only a threefold increase in size; the formative activity during this
time being largely cell division. It is during the last half of its development, when differentiation and organ formation take place, that a tenfold increase in size is noted.
For the purposes of comparing growth of normal, ‘contained’ embryos with completely
‘freed’ embryos, stages in contained development have been designated arbitrarily as
‘pre-division’, ‘division’, and ‘differentiation’ phasea. The ‘pre-division’ phase of
growth is considered as that period of embryo development from fertilization up to the
first division of the embryo aa indicated, about 6 days in Todm. The ‘division’ phase of
growth is characterized by embryos 7-15 days old. The ‘differentiation’ phase begins
with internal differentiation, h t noted, in 17-day-old embryos and continues through
the formation of organs, culminating for our purposes in the breaking of the calyptra.
Deve,!qment of ‘freed’ embryos
Considerable variation was seen to exist in the development of embryos which were
completely isolated from the venter of the archegonium and grown free in sterile culture.
In some instances these variations could be accounted for in terms of the nutritional
requirements for embryos isolated a t the different stages of growth. This is particularly
true for embryos isolated during the ‘differentiation’ phase of their growth. However,
variations in the development of excised ‘pre-division’- and ‘division’-phase embryos
were found to result from factors other than nutritional ones. Throughout this study,
we observed that the older the embryo on excision, the less its growth pattern deviated
from that of ‘contained’ embryos; and the younger the excised embryo, the grmter the
degree of variation in its further development (DeMaggio & Wetmore, 1961b).
Experiments were initiated by excising older ‘differentiation’-phaseembryos, 20 days
aftm fertilization, and allowing them to develop on an agar-solidSed medium consisting of
a modified Knudson’s solution (Steeves, Sussex & Partanen, 1955) supplemented with
trace elements (Nitsch, 1951) and 1% sucrose. Although in this medium development
progressed very slowly, most embryos in about 10 months’ time did attain a size and
complexity comparable to 30-day-old ‘contained’ embryos. When 20-day-old embryos
were excised and placed in the =me mineral medium, but lacking a solidified substrate,
growth proceeded a t a much faster rate and advanced sporelings were recognizable after
a 1 month culture period. While this rate of growth still is not optimal, when compared
to ‘contained ’ development, the results clearly indicate that isolated embryos can develop
to normal sporophytesprovided the proper nutritional supplementa are provided.
24-2
368
A. E. DEMAWIO
These prehinary results were codrmed when younger ‘dif€erentiation’-phase
embryos were excised, 17 days after fertilization,and planted in the aame liquid nutrient
which fostered adequately the growth of older isolated embryos. On this simple medium
these embryos, showing an immature foot region and bearing no obvious signs of internal
differention, could not be induced to develop beyond the stage of organ formation.
One or 2 % sucrose concentrations were found to be necessary for optimal growth, but
in no instancedid development continue unless coconut watefl waa added to the medium.
With the single exception of root formstion, the addition of 10% autoclaved coconut
water allowed development to proceed at a normal rate. This rate of growth could not
be duplicated by the addition of varying concentrations of amino acids in placeof
coconut water, but could be duplicated, and in fact exceeded, by supplying equal
concentrations of sorbitol and inositol (5Omg./l.), both known components of the
‘neutral’ fraction of coconut water (Shantz, Pollard & Steward, 1958;Steward & Mohan
Ram, 1961).The stimulatory effect of these and other cyclitols on plant tissue cultures
has been demonstrated more recently by Steinhart, Anderson & Skoog (1961).
The s u d attempts to grow essentially undifferentiated embrvos of Todea to
maturity led us to conclude: (1) that the nutritional requirements of younger embryos
are more complex than those of older embryos; and (2) that embryos entering the
‘differentiation’phase of growth, or elready in advancedktagesof growth, are not subsequently influencedin their development by the earlier restrictionsof surroundingprothallial thue.
The first conclusion is supported by data obtained from a number of sources (Rijven,
1952 ; Ziebur & Brink, 1951 ; %ppport, 1954) which indicate early dependence of the
embryo on nutrient-supplying maternal tissue, and in later stages of development the
evolution of a conaiderabledegree of metabolic autonomy. The second conclusion implies
that if restraint is a -1 factor affecting growth and development of fern embryos, and
moreover, if restraint actually doee supply some directive effect in the production of a
sporophyte, this stimulus must be active during the very early stages in development
prior to the attainment of multicellular complexity and apparent metabolic selfsufficiency.
To test the hypothesis tht reetraint is a morphogeneticfactor, active in early embryo
development, young, unicellular, embryos (35-55~)were excised 4 4 days after fertilization, prior to the hitid segmentation and allowed to develop in a liquid nutrient
medium. Of the many Merent nutrient formulae employed for culturing unicellular
embryos of To&u none has proved to be as effective aa a Bimple mineral aalt (Knudson’s)
trace element (Nitsch) solution modified by the addition of 3% sucrose and supplied
with sorbitol and inositol at a concentration of 50 mg./l. each.
In this medium, unicellular embryos were noted to undergo several divisions during
the Grst week in culture. These divisions occurred in the usual sequence characteristic
for ‘contained’ embryos with the basal wall forming h t , followed in regular succession
by the median and transverse walls. During the second week in culture most embryos
continue active divisions; however, these divisions, unlike those occurring in the normal,
‘contained’embryo, are without any regularor charaderistic order. Asgrowth continues,
the free-growing embryo loses its globular, three-dimensionalappearance and begins to
develop into an unorganized, two-dimensionalcellular maas, in many respects resembling
proliferations or oalli art%cially induced in a variety of cultured tissue. The small
embryonic mass did not continue proliferating in thismanner indefinitely. After 1 month
in culture no further increase in size of the embryo waa noted and no other visible signs of
growth and cellular activity could be detected. Examination of embryos at this stage
Tulecke, Weinstein, Rutner & Laurencot (1961) have urged thst a clear distinction be made
between the tern coconut milk and coconut water. Since the materiel used in this study consisted
of the liquid endosperm of the coconut, the term coconut water ie appropriate and is used throughout
this papea,
Morphogenetic factors influencing the develqnnent of fern embryos
369
shows them to be composed of many, thin-walled, parenchyma cells loosely cemented
together to form a structure still recognizable as the embryo. When these embryos are
grown in liquid culture on a rotating shaker one finds in the medium single cells and
clumps of two, three, or more cells, which seemingly have been loosened from the original
embryonic mass. Although attempts to nurture these individual embryonic cells and
cell aggregates to duplicate the stages in normal embryology so far have been without
success, work in this direction is continuing.
If these embryos grown for 1 month in a liquid medium are not rotated but left
stationary and examined after an additional culture period of 3 4 months, evidence of
continued activity in the embryo is detected. The embryos do not develop in the usual
manner characteristic of those ‘contained’ within an archegonium but begin to produce
outgrowtbs which give irregular, asymmetrical, two-dimensional, thalloid plants (Pl. 1,
fig. 3). These thalloid plants are mentially gametophytic-like in nature. Not only do
they resemble prothalli produced routinely from germinating spores but after long
periods in culture some have produced structures remarkably similar to antheridia
(Pl. 1, fig. 4). These antheridia-like structures, which develop on the thalloid body in a
position where one ordinarily expects antheridia to form, have remained immature and
have not produced sperm cells or progenitors of sperm cells. For reasons unknown to us
a t this time, these thalloid bodies emerging from fertilized eggs do not continue to develop
on the simple nutrient media which have proved adequate for other fern tissue of both
gametophytic and sporophytic origin. Nor has it been possible to culture them beyond
the stage where antheridia-like outgrowths are produced. Conclusive proof that these
twoxhensional structures arising from cultured embryos are capable of giving rise to
functional antheridia and archegonia remains to be obtained.
The demonstration that completely excised, undivided, embryos of Todea produce in
free culture growth forms morphologically similar to the prothalli might suggest that
these thalloid structures arise as a result of the complete absence of restraining surroundings. The characteristic proliferating cells of the free-growing embryo further serve to
indicate a lack of limiting and restraining boundaries. In the normal course of ‘contained ’
development, unorganized cellular proliferation of the embryo is never observed. Certainly the embryo must be capable of this type of abnormal and unlimited growth, as is
evidenced by the response in free culture, yet the tissue surrounding ‘contained’
embryos evidently presents a restrictive environment which limits the capacity for
undirected or unoriented growth. This may occur through orienting long-chain molecules
of the material comprising spindle fibres during mitosis and so have a relation to planes
of cell diviaion, whereas this necessary restriction is missing when the fertilized egg is free
and growth is observed largely to be marginal and/or terminal. If this assumption is
correct, then the observed dependence of very young plant embryos on parent tissue,
prothallus or endosperm, is necessary not only for supplying specific nutrient and growth
substances but also for providing a period of contained development during which the
embryo acquires three-dimensional multi-cellular, complexity and some degree of biochemical stability.
Evidence for this train of thought has been obtained by growing older excised embryos
of l’hto maturity in culture. These embryos, identified as being in the ‘differentiation’
phase of growth, had already acquired multicellular complexity before they were
removed and placed in free culture, By supplying simple nutrients, they grew normally,
being able to adjust to the changing environment and also apparently being able to
utilize the nutrients and energy-supplying materials in the medium. Similar results also
have been reported recently with embryos of Pteris longifolia (Rivihres, 1959).
The marked difference in growth habit between isolated ‘differenthti0n’-phase
embryos and ‘pre-division’-phase embryos could be explained by visualizing early
development as a time during which the embryo acquires the necewry enzymes and
enzyme systems for utilizing the photosynthetic materials which will sustain its later
370
A. E.DEMAWIO
independent growth. During this period of adjustment, the young embryo of necessity
is being nutritionally supported by the parent prothallus. Possibly the necessary substrata which would support the induction of these enzymes is missing h m the medium.
Nevertheless, our mulk indicate that in Todea embryos these enzyme systems are not
fully developed until growth has progressed to a point where differentiation is about to
begin for it has not been possible to nurture earlier excised embryos in vitro to produce
the charaderistic sporophyte. Analogousresultshave been reported h m studiesongrowth
of excised proembryos of Cdrua m k r m r p in culture by Ranga Swamy (1961). More
recently, Nomtog (1961) has succeeded in culturing young proembryos of barley
which first p w as simple spherical forms before becoming differentiated. Very young
proembryos (Sop in size) grow very slowly and without normal differentiation. It
thus would seem that the acquisition of the neceasmy prerequisite materials which allow
the embryoto attainnormdsporophyticmorphology-whether these materials be proteins,
nucleic acids, or hormones-is in some manner related to the development of a threedimensional, multioellular embryo. It is quite possible that this type of development
is necessary for the initiation of ‘gradients’ such as those suggested by Wardlaw (1955)
and more recently by Wetmore (1959) and by Steward (1958). The ‘contained’ embryo,
therefore, appears to be conditioned by the restraining and restricting environment to
develop into a three-dimensional tissue mass whereupon differentiation and organ formation ensue with the subsequent formation of the sporophyte. This would explain why
older embryos, partially or completely excised, develop directly to normal sporophyles
while younger embryos when released from containment fail to attain the normal course
of development.
Although in this interpretation, emphasis is placed on the morphogenetic effects of
restraint in guiding early embryo development, one should bear in mind that theseeffects
cannot be separated completely from the inherent metabolic capabilities of the embryo.
Together, i n t e d and external fadom contribute to the orderly development of the
embryo and the production of the sporophyte. Only when the internal or external
environment is d r a s t i d y altered is the pluripotentidity of this growing germ
expressed.
MOEPEOGENETIC FACTOFt II-NUTBITION
In the preceding discussion evidence has been presented to show that restraint may be
a very important morphogenetic factor in determining the regular alternation of gametophytic and sporophytic stages in the reproductive cycle. An alternative explanation to
account for the mults obtained might appropriately indicate that removal of the embryos from the normal position on the prothallus, a t any stage during their development,
deprives them of certain nutritional substances, the lack of which seriously impairs their
subsequent development. It would be an overeimplification of the problem to suggest
that other factors, internal aa well &s external, were not involved in the morphological
transition from the simple, two-dimensionalprothallus to the complex, three-dimensional
sporophyta Although a t present little information is available concerning the exact
nature of these nutritional factors and their function in alternating generations, many
recent studies have demonstrated that nutritional influences exert considerable control
on the morphological form attained by the growing plant or plant part and could be
implicated as a major morphogenetic factor in development.
Miller & Miller (196l), invwtigating the effect of different light conditions and sucrose
on the growth end development of onoclea sensibilia gametophytes, find that a specific
light requirement must be satisfied for growth to take place. They state, however, that
morphogenesie depends on the carbohydrate supply. Other studies by Wetmore (19Sa),
Sussex & Clutter (1960), and Allsopp (1953), have shown that varying the sugar concentration available to isolated fern apices, excised leaf primordia, or sporeling plants,
musea profound changes in the morphology of the developing leaves. Whether we agree
Morphogenetic factors inJluencing the devebpnent of fern embryos
371
that these changes result primarily from the action of sugars on the mitotic activity in
cells of the leaf, as Wetmore has suggested may be the case, or are produced because of
nutritional changes taking place directly in the shoot apex as proposed by Allsopp, the
interesting fact remains that these alterations have resulted as a consequence of changes
in available carbohydrate nutrition.
Not only are vegetative phases of development affected by carbohydrate concentration but also the induction of reproductive phases of development may be dependent
on an adequate supply of sucrose or other carbohydrate materials, as indicated by recent
investigators. The production of sporangia on excised fern leaves growing on a medium
supplied with high concentrations of sucrose has been reported by Sussex & Steeves
(1958). Although the production of sporangia on leaves of Todea barbara was found to
be enhanced by an increase in organic nitrogen, in addition to an increase in carbohydrate supply alone, on the basis of their total experiments these workers conclude
that a high concentration of sucrose was an essential requirement for fertility. The
interesting series of experiments conducted by Whittier & Steeves (1960) show that
apogamy can be induced in various species of ferns by manipulating the concentration
of carbohydrate in the nutrient medium. To explain how an increase in sugar concentration causes a thalloid gametophyte to give rise directly to a vascular sporophyte these
authors postulate that the two-dimensionalgrowth pattern of the prothallus may result
from an insuscient energy supply. Increasing the energy supply, by addug higher concentrations of carbohydrate, allows the gametophyte to attain a more complexpattern of
development, resulting in the production of an apogamous sporophyte. It is suggested
that the transition from gametophytic to sporophytic morphology may be fostered by
an increase in available energy. How this relates to kinin activity and cell division is
not as yet clear.
The results of these studies clearly indicate that many phases in the life history of
the fern plant are dependent for their normal development and functioning on the presence of an adequate supply of carbohydratea. To determine whether nutritional changes
in carbohydrate concentration also influence the usual developmental sequence in fern
embryology, thereby affecting the production of a normal sporophyte and the regular
alternation of generations, the following experiment was performed.
Prothalli of T. barbara bearing fertilized eggs were placed on the simple nutrient
medium previously described supplemented with varying concentrations of sucrose
ranging from 0 to 10%. Cultures were maintained in a regulated growth room a t a
temperature of 25”f2 “C. and kept under 12 hr. of light supplied by a combination of
0uorescent and tungsten lamps. At frequent intervals, several prothalli were removed
from each of the experimentalmedia ;Gxed, sectioned, and stained according to the usual
procedure for examination ofthe developing embryos. All results reported were obtained
utilizing normal, ‘contained’ embryos.
On examination, size determinations of the embryos were made by averaging length
times width measurements for no less than ten embryos of the same treatment. Stages
in embryo development also were recorded a t each interval as was the age of the embryo,
determined as days following fertilization. The most significant variations in embryo
growth and development were noted on prothalli grown without sucrose, or those supplied with 1,2,4 and 10 % sucrose (Text-fig. 2). The mults presented here &refrom these
samples only; a more detailed report will a p p r at a later date (DeMaggio, in
preparation).
An examination of these data reveals that 1% or 2 yo sucrose supplied to cultured
prothalli appears to produce conditions for optimal growth of the ‘contained’ embryo.
Those embryos on prothalli furnished with 1yoor 2 yosucrose grew quite rapidly and a t
the end of 30 days in culture had reached considerable size. Embryos on prothalli
grown without a source of externally supplied carbohydrate grew much more slowly,
and a t the end of a 45-day growing period were only half as large as embryos on prothalli
A. E. DEMAWIO
372
950
-
900-
800750 700 a50
650-
g 600E
-c m-:: 500450-
n
2
E
400350300-
250
-
200-
-
150
100-
M5
10
15
20
25
30
35
Days following fertilization
40
45
I
50
Text-@. 2. Growth of ‘contained’embryos of Todea barbara supplied with varying concantretions of sucmae. Sucroee concentretion: O,OyO;0, 1 yo and 2 yo; 0 , 4 yo; 0 , 10%.
Enlarging organs
Procambial tissue
Stem and root initia
Leaf initialsinternal differentiati
Initiation of foot
Fifth division 32-cell
Fourth division
16-celled
Third division 8-celled
Second division
4-celled
First division 2-celled
Days following fertilization
Text-%. 3. Stagea in development of ‘contained’embryos of Todsa bwbara supplied
with varying concentrationsof SUCMBB.
Morphoqe&ic factors infEuencing the devehpnent of fern embryos
373
supplied with optimal concentrations of sucrose. A similar growth response wm noted
for embryos on prothalli supplied with 4 yosucrose. When an examhation a t the various
time intervals was made of prothalli grown on a medium with 10 % sucrose it was found
that embryos 4ad increased only shghtly in size. After 45 days’ growth these embryos
barely had doubled in their initial sue and even after a prolonged culture period had
displayed little evidence of achieving an increased rate of growth. The inhibitory action
of high concentratioas of sucrose on ‘contained’ embryos parallels the results obtained
when ‘freed’ embryos of Todea.were grown on media with elevated sucrose concentrations
(DeMaggio t Wetmore, 1961b) and supports previous work which demonstrated inhibitory action of high sugar concentrations for growth of angiosperm embryos (Rappaport, 1954). Results wing various concentrations of mannit01 suggest that this is not
an osmotic effect.
The observed difference in the growth rate of ‘contained’ embryos grown while subjected to varying states of carbohydrate nutrition is a strong indication that the normal
growth of Todea embryos, as reflected by increases in size, can be influenced by nutritional
factors. However, an answer to the question of whether these nutritional influences are
able to alter significantly sporophytic development, and in this manner impede alternation of generations, cannot be ascertained until the relationship between the stages in
embryogenesis and the nutritional status is better understood. Additional data were
acquired and the stages of embryo development were correlated with the concentration
of carbohydrate supplied to the respective prothalli as illwtrated in Text-fig. 3.
During the ‘division’ phase of embryo growth, 7-15 days following fertilization,
‘contained’ embryos grown without the addition of sucrose to the medium were noted
to be almost as advanced in their development as embryos nourished with optimal
concentrations of 1 yoor 2 yosucrose. The same situation was not observed for embryos
from prothalli grown on higher sucrose concentrations. In this same time period embryos
on prothalli supplied with 4 % or 10 % sucrose were found to be delayed considerably in
their morphological development.
The developmental pattern observed in these ‘contained’ embryos during the ‘differentiation’ phase of their growth, 15-30 days following fertilization, differed somewhat
from that observed during the earlier growth period. The most noticeable difference
wm seen in embryos on plants supplied with a 4 % concentration of sucrose, These
embryos, once they entered the ‘differentiation’ phase of growth, rapidly acquired the
characteristics associated with this level of development and after 30 days in culture had
developed to a greater degree than embryos not provided with external sucrose. At
the end of a 30-day culture period, prothalli supplied with 1% or 2 % sucrose bore welldeveloped embryos, many beginning to emergefrom the calyptra and surrounding gametophytic tissue. Embryos on prothalli grown on the other media appeared normal in
almost all respects, even though they had not achieved the same level of morphological
complexity as embsyos on plants grown with 1 % or 2 % sucrose. When prothalli bearing
embryos in various stages of development were allowed to remain on their respective
nutrient media for extended periods of time, most embryos were observed to give rise to
the regular sporophyte characteristic for this species. The only exceptions were a few
embryos on plants nourished with 10 yosucrose which aborted early in their development
and did not mature.
During this study it was apparent that tt direct relationship exists between embryo
size in T&a and the complexity of morphological development. The inhibitory influence
on growth of embryos (increme in sue) noted both in the absence of a carbohydrate
supply or with increased concentrations of cmbohydrate was reflected in a similar
inhibition on various embryonic stages of development. However, in most instances this
inhibition did not succeed in totally prohibiting the embryo from further size increase
and associated morphological development. Rather, the nutritional inhibition only
served to delay the eventual successionin the usud matmation pattern. It would appear
374
A. E.DEMAGGIO
then, that the effeot of additional carbohydrate nutrition on the developing ‘contained’
embryo of Todea ia one of interferiq with the proceseee regulating size increase, organ
initiation, and the attainment of maturity. Carbohydrate concentrations, within the
range employed in this investigation, caused no extreme variation in the expected
sequenceof embryologicaletages ;only the timing was aEected. “herefore, it seems rewonable to postulate that in Todea the morphogenetic effects of carbohydrate nutition on
embryo development do not appear adequate to cause a drmtict change in form; and
that carbohydrate nutrition, in itself, could not be responsible for altermation of generations. In this role of wbohydrate nutition in determining morphological form, one is
led to agree with Sussex & Clutter (1960) who conclude from their studies on leaf morphology that unidentified interactions in the plant which regulate the rate of develop
ment may also affect the subsequent shape of the growing plant or organ.
Very recent studies, still in the experimental phase, indicate that when prothalli
bearing fertilized eggs me nourished on media supplemented with sucrose (1%) and
napblacetic acid ( N U )at 1 0 d ~
concentration, the embryos remain in the ‘division’
phase of their growth indehitely. These embryos (Pl. 1,fig. 6)are characterized by rapid,
unorganized cell divisions and an enlarged calypha. Our studies thus far indicate that
the addition of this hormonal substance interfern not only with the timing of embryological events but also with the morphological form, by affecting certain essential stages
in development. While premature, the suggestion is advanced that hormonal influences
play a substantial role in orderly embryo development. A detailed study of their
interaction with environmental influences may lead to a new working hypothesis with
which to expand investigations of alternating generations.
CONC!LVSIONS
The present analysis has been restricted to a consideration of only two of the many
morphogenetic factors effective in influencingthe development of fern embryos, restraint
and nutrition. The experiments described in this paper, still essentially exploratory in
nature, have demonstrated that restrictive and nutritional influences play active p a h
in regulating functional proceases leading to the establishment of recognizable and
characteristicphases in development. Early, complete removal of the natural, restrictive
limitations imposed on the growing embryo has been shown to limit seriously its potentiality for three-dimemiom1 growth and normal spmphyte production. Whether this
single modification, leading to what looks like a r e v e d of regular phases in alternation
of generations, results in a loss of control over the planes of cell division, as previously
theorized (DeMaggio & Wetmore, 1961a),or succeeds in depriving the embryo of essential
growth substancea necessary for orderly sporophyte production, remains to be seen.
While it has been shown that in this species changes in carbohydrate nutrition do not
account for permanent alterations in morphological form, the possibility cannot be
overlooked that nutritional and hormonal interadions can give rise to profound changes
in embryo development of a magnitude to suggest that these substances exert considerable control in determining morphological levels of development.
The fnrther elucidation of the mechanisms, externalor internal, controllingthe development of form and altermation of generations is eagerly awaited; for the Signiscance and
validity of the views expressed here necessarily are dependent on future experimental
studies.
ACKNOWLEDOEME~S
The author is gratefully indebted to Professor Rdph H. Wetmore, Harvard University, Cambridge, Massachusetts, for much stirnulatin@;
discussion of this problem during
the course of several years and for his critical reading of the manuscript ;to Dr Dean P.
m t t i e r , Virginia Polytechnic Institute, Blacksburg, Vbghia, for the use of his unpub-
Morphogenetic jmtops influencing the development of fern embryos
375
lished data; and to Mr M. Loss and Mr J. Petrolino, Rutgers, College of Pharmacy,
Newark, New Jersey, for their invaluable technical assistance.
A portion of the work described was supported by Grant no. 549 from the Rutgers
Research Council.
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EXPIANATION OF PLATE
1
Toderr barbam
Fig. 1. Section of mature archegonia. x 600.
Fig. 2. Undivided, fertilized egg isolated early in development. x 720.
Fig. 3. F'mtWoid-like body produced from cultured embryo. x 100.
Fig. 4. Antheridial-like outgrowtba developing on W o i d structure produced from cultured embryo.
x loo.
Fig. 6. Section of prothallua and embryo grown for 46 b y 8 on 8 medium supplemented with
NAA. ~400.
Journ. Linn. SOC.Bot. Vol. 58, S o . 373
A. E. DEMAGGIO
Plate 1
(Pacing p . 376)