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Methods in plant morphogenesis. By C. W. WARDLAW,
Department of Botany,
University of Manchester
1. INTRODUCTION
The term morphogenesis connotes the genesis or origin of form-or, in the words of the
Oxjord English Dictionary, ‘the origination of morphological characters ‘-and, by extension, the related structural details. Both parts of the word are therefore of high significance : together they convey, to the biologist, the idea of the coming into being of visible
and tangible shapes or patterns, as in the growth of an organism from a fertilized egg to
the full manifestation of its distinctive characters in the adult state. Morphogenesis is
a comprehensive subject, admitting of many approaches and therefore of many investigational techniques. Indeed, if we hold that the eventual aim in morphogenesis is t o
give as full an account as possible of the progressive manifestation, or expression, of the
distinctive characters of organisms during their development from the zygotb onwards,
it is well nigh impossible to say where the study of morphogenesis begins and where it
ends. I n practice, however, and with some necessary reservations, science tends to
advance more rapidly when individual projects are clearly defined and separated off for
special study. My task this morning is to indicate some of the methods which have
have enlarged our understanding of morphogenesis.
I n general terms, one might say of the several disciplines that contribute to morphogenesis that :
(i) morphology is concerned with the visible evidence of formal and structural developments in embryonic, growing and maturing regions;
(ii) physiology is concerned with the phenomena of metabolism and growth: in
particular with the identification of the specificsubstancesinvolved, and with the patterns
of distribution of those metabolites which precede and determine the earliest visible
developments;
(iii) the specific nature and characteristic sequence of metabolic patterns during ontogenesis are, directly or indirectly, determined by elements in the genetical constitution;
(iv) all morphogenetio processes have physical and mathematical aspects of the kind
expounded by D’Arcy Thompson.
Some progress has been made along each of the lines indicated above. Nevertheless, it
is important to recognize that the results of individual analytical or experimental investigations do not, in themselves, enable us to explain the inception of form, i.e. how the
many processes involved are so timed and integrated that a living entity, exemplifying
developmental harmony, comes into being ; how specific and distinctive organization,
and seemingly appropriate functional activities are evident at every stage ; in short, how
anindividual of 8 particular species,recognizable ‘by head’, results. Thus although we are
specially concerned with experimental investigations in this symposium, it Beems to me
that approaches other than experimental ones are both valid and desirable in this
introductory paper.
2. MORPHOLOGICAL
OBSERVATIONS
Although we have a very considerable fund of information on the form, structure and
histology of plants, including their embryology, there is still need for further work. This
point deserves some emphasis; for there can be no true understanding of morphogenesis
unless there is precise observation of the size, shape and relationships in time and space,
of nascent organs in embryonic regions. This work is often considerably more difficult
to carry out than is sometimes thought; and, accordingly, it is sometimes neglected or
insufficiently well done. But without this exact preliminary information, there can be
no proper formulation of the relevant physiological problems nor can an adequate experimental or analytical programme be designed. If, for example, the relationship of a very
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young leaf primordium t o the apical meristem is inaccurately observed, the interpretation of certain experimental observations is likely t o be less sound, or less accurate, than
it might be. There are also experiments in which it is important t o know whether a
young leaf primordium is in early or in late plastochrone. The basic method, indeed the
sine qua mn, in morphogenesis, therefore, is the precise observation of embryonic regions.
These are usually small, delicate, and sometimes rather inaccessible; but they must be
brought under direct observation and kept alive if an experimental programme is t o be
carried out.
3. SURQIUAL
TREATMENTS AND THE RELATIONSHIPS OF ORGANS AND TISSUES
Among the most distinctive features of plants are their size and shape, and the relationships in space of the component organs: thus shoots may be long or short, simple or
much branched; leaves may be small or large, simple or compound, and variously disposed on the stem ; and comparable remarks cpuld be made of flowers and inflorescences.
There may also be more or less conspicuous morphological changes during ontogenesis,
e.g. heteroblastic development, or the transition from the vegetative t o the reproductive
phase. I n each instance, the primary pattern of development, i.e. the primary shape and
relationships of organs, and the pattern of their tissue systems, is determined a t an
apical meristem. As these morphogenetic activities usually take place with considerable
regularity, the growth of the plant from the zygote to the adult state exemplifies what
Thoday (1939)has aptly described as developmental harmony. These simple observations
invoke a host of fundamental questions. They have often been stated and need not be
repeated here (see Wardlaw, 1952). But if, for example, our aim is to investigate factors
which may determine the inception, growth, and form of particular organs, their mutual
relationships, regulated development and the differentiation of their tissues, what experimental procedures are available ? I n general, the experimental method requires the
separation of organs one from another, e.g. of a leaf primordium from the apical meristem
or from adjacent primordia, followed by observation of the nature and course of development in the new situation thus created. I n this connexion the use of surgical techniques
has proved t o be of considerable value. Although this approach to the investigation of
embryonic tissues has its disadvantages and limitations, and is, indeed, open to various
objections, nevertheless, it is a simple fact that surgical procedures have yielded information which, t o date, has not been forthcoming from any other experimental procedure.
Here reference may be made to the experimental investigations of phyllotaxis by M. & R.
Snow (1947), and of the various organogenic and histogenic activities of the shoot apex
by Ball, Wetmore, Sussex, Cusick, Cutter, Steeves, Wardlaw and others. I n parenthesis,
I have long been aware of the limitations of this approach: there must, it would seem,
be a limit t o the number of ways in which the shoot apex, leaf primordia, etc., can be
incised with useful results. It is surprising, however, how many instructive experiments
can be based on surgical techniques, the more so when one bears in mind the possibility
of combining these with localized chemical or more general nutritional treatments. Here
I would call your attention to the very interesting experiments of Camus (1943-5) and
of Wetmore and Sorokin (1955) on the induction of vascular tissue in undifferentiated
tissue-culture callus by grafting in young buds, with and without applications of auxin,
and indeed, by the application of auxin alone to the graft incision. Gregory & Veale
(1957) have shown that the classical experiment of removing the shoot apex to induce or
promote the development of lateral buds, can be made to yield yet further information
by simultaneous auxin or nutrient treatments. There are also the recent studies of
regeneration of vascular tissues after incision by Jacobs (1952) and Jacobs & Morrow
(1957)-work pioneered by Simon in 1908-in which he has demonstrated a close quantitative relationship between the downward movement of auxin and xylem differentiation.
Surgical techniques have also been used, with good results, in the investigation of roots
and of the gametophytes and young embryos in hepatics and ferns.
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4. DEVELOPMENTAL
PHYSIOLOGY
The physiology of development covers a vast field of which only the most general indications can be given here.
Biochemical investigatiolwr. Since all morphogenesis involves metabolism, there is
virtually no limit to the relevant biochemical problems ; and similarly, other branches of
plant physiology are more or less directly involved. This is necessarily so, since, a t base,
all morphological development results from the functioning of genetically determined
reaction systems in particular environments. What is commonly described as Developmental Physiology is the subject of a considerable book by Biinning (1948) and has
many contemporary adherents.
From the time of Sachs, who laid the foundations of biochemical morphogenesis in
plants, attempts have been made to discover the nature and functional significance of the
substances, both general and specific, that are present in embryos and in the apical
growing points of shoots, leaves, roots and flowers. Considerations of time and space
do not admit of reference being made to the many valuable chemical analyses of embryos
and apices in which the presence of carbohydrates, amino acids, auxins, enzymes, etc.,
have been reported. Reference might here be made to the work of Steward and of R.
Brown and their collaborators. It is, of course, evident that this kind of information is
essential to a more adequate understanding of apical growth and morphogenetic activity,
though, in the present stage of knowledge, only some of it has an immediate application.
One direct use of the new information in morphogenetic investigations, namely the
improvement of tissue and organ culture media, may be mentioned (Wetmore, 1953,
1954,1957).
Tissue culture. Although this is now the subject of a vast literature, only a relatively
small part of it has so far shed any direct light on morphogenetic processes. The most
interesting and illuminating discoveries include those in which the formation of either
roots or buds has been induced by the addition to the standard medium of certain substances, e.g. adenine and indole-3-aceticacid (IAA),in particular concentrations (Skoog,
1954; Skoog & Miller, 1957; Skoog & Tsui, 1948).The aseptic culture of single cells, or of
small groups of cells, in media of known composition,and their further growth into organized structures, which has now been achieved, is not only a realization of Haberlandt’s
classical concept of tissue culture, but opens up fascinating and important fields for
further exploration (Steward, Mapes & Smith, 1958). It has also been shown by several
investigators, Rijven (1952,1956),Haccius (1955),Rietsema, Satina & Blakeslee (19534),
Rietsema, Blondel, Satina & Blakeslee (1955),“ukey (1938),and others, that the normal
course of embryonic development can be modified by excising young embryos and growing them under controlled conditions, or by treating them with physiologically active
solutions while they are still in the seed. The growth and further development in pure
culture of young fern sporophytes, excised fern and other apices, young leaf primordia,
roots, immature fruits, and ovules, as reported by various investigators, open up virtually inexhaustible fields for new work on morphogenesis (refs. in Steward et aE. 1958).
Before leaving this topic I ought to mention the very interesting investigations by
Wetmore & Morel (1951)and Freeburg & Wetmore (1957)on the pure culture of gametophytes of Lgco@ium spp., and by Allsopp & Mitra (1958) on the pure culture of mosses.
Direct and indirect chemical treatments. Because of the minute size and inaccessibility
of the embryo or the shoot apex, many experiments on chemical morphogenesis consist
in applying growth-regulatingsubstances to the soil or to leaves of different ages, in the
hope, or expectation, that some of the substance applied will be translocated to the shoot
apex and there induce morphogenetic effects of a novel kind. Such experiments are now
the subject of a very considerable literature in which many anomalous developments,
usually in the adult stems, leaves, flowers, etc., have been illustrated. This experimental
approach has shown that many specific substances, both natural and synthetic, disturb
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the normal metabolism and morphogenetic activity of embryonic regions; but the work
would undoubtedly gain in value if the earliest changes effected in the apex were more
closely observed. Many physiologically active substances have also been incorporated in
tissue-culture media, but to date, this procedure has added rather less to our knowledge
of morphogenetic processes than had perhaps been expected.
It may be that the specific substance, though present in the medium, does not reach
those regions of the tissue culture, e.g. its upper surface, which are capable of morphogenetic activity. Wetmore (1953,1954),for example,foundthat excised apicesof Adiantuum
pedatum L. could be induced to grow more slowly or more rapidly by changing the composition of the culture medium ; but none of the specific substances tested altered the
fundamental morphogenetic pattern of the apical meristem. Contemplation of this fact
led me to observe the action of various physiologically active substances when they were
applied directly to apices of ' Dryopterie aristata' (D.dilatata (Hoffm.)A. Gray) which had
been laid bare by the removal of all older primordia and scales. I n fact when substances
such asIAA, IAN, vitaminB,etc., were appliedinthismanner,the normalorganization and
morphogenetic activity of the apical meristem were more or less considerably modified.
In different instances, the more conspicuous results included the inception of double leaf
primordia,the formation of buds in leaf sites,the development of a sunken meristem by the
upgrowth of the subapical region, the cessation of primordium formation, and the parenchymatization of the meristem, culminating in the outgrowth of scales from the former
site of the apical cell (Wardlaw, 1957a). In short, several of the characteristic modifications obtained by surgical techniques resulted from these direct chemical treatments.
Along the same general lines, interesting observations have been recorded by M. & R.
Snow (1937) ; Ball (1948); Gorter (1951) and Wardlaw & Mitra (1958); and by Rietsema
et at., Haccius, and others, on young embryos. Of course, it is one thing to induce these
interesting morphogenetic developments : it is a task of considerably greater magnitude
and difficulty to explain them. These observations all relate to the vegetative apex, but
chemical treatments applied to plants a t the onset of flowering, as demonstrated by
J. & Y. Heslop-Harrison (1957a, b) and others, have also yielded results of very considerable interest. It need hardly be said that there is virtually unlimited scopefor further work
on experimental morphogenesis in the rich assemblage of materials afforded by the
flowering plants.
THEGENETICAL APPROACH
A n extensive literature testifies to the fact that the distinctive form and structure of the
individual organism are fundamentally determined by its genetical constitution, though
factors in the environment are always a t work and may also have more or less conspicuous effects. This general statement introduces a vast and complex subject; for although
all the elements of the genetical constitution are present in the zygote, different genes,
or group0 of genes, may differ greatly in their time of action and in the magnitudes of their
effects. Contemporary investigators of physiological-genetics and of morphogenesis
are concerned with such searching questions as : in embryonic regions, how and when do
particular genes and their products, reacting in the cytoplasm, determine, or contribute
to, the primary morphogenetic pattern and its subsequent amplifications?This kind of
inquiry is obviously complex and difficult, but at least a beginning can be made by close
comparative observations on the growth, morphology, anatomy and histology of primary
embryonic regions, e.g. shoot apices, in related materials which have been studied genetically ; and, better still, where characteristic differences in metabolism and/or growth
can be referred to particular genes. In shoot apices of diploid and tetraploid Vinca rosea,
the distal embryonic cells of the tetraploid have the same anticlinal dimensions as the
diploid, but 1.5 times the periclinal dimension. Differences in the size and cellular constitution of the shoot apex in diploid and tetraploid maize, and some consequential
growth and morphogenetic differences, have also been reported. The periclinal chimeras
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in Datura, described by Blakeslee and his colleagues, are too well known to need special
mention here (see Wardlaw, 1957 b, for refs.). Morphogenetic studies of the shoot apex
and its primordia in Nywqhaea and Nuphar by Cutter (1957) have shown that certain
relationships, important in taxonomy, are evident during early organogenesis but would
almost certainly escape observation during the later development. Different genetical
effects in early organogenesis are well illustrated by studies of leaf formation; for whereas
many leaf primordia are very much alike at their inception on the apical meristem,
specific and distinctive differences can already be observed in the subapical region. I n
some species, the base of the primordium remains small and discrete, whereas in others
it extends round the axis to form a sheathing organ as in the Umbelliferaa and many
monocotyledons. The evidence to date is that differencesin the genetical constitutions of
related species and varieties may be reflected in the size of the embryonic cells and in the
differential or allometric growth pattern. The diversity in floral structure, indicated in
taxonomy by contrasted terms such as polypetaly and gamopetaly, hypogyny and
epigyny, apocarpy and syncarpy, eta., can all be referred to differential growth during
floral organogenesis. How genes determine the pattern of growth, and how mutant genes
may modify that pattern, are complex problems, but we can, I think, see how a beginning
can be made.
The effects of ionizing radiations and of mutagenic substances would be included under
the general heading of this section (Gunckel, 1957; Gunckel & Sparrow, 1954; Sparrow,
Gunckel, Schairer & Hagen, 1966).
MNTHEMATICAL
ANALYSES
I n aJ1 but the simplest organisms, the ontogenetic development, from the zygote to the
adult state, is characterized by changes in size, form and structure. Roots apart, all the
primary developments in vascular plants can be referred to the activity of the shoot
apical meristem. These developments usuaUy take place with very considerable regularity and fidelity. In some species the successive and characteristic changes in form
during ontogenesis have been related to the nutritional status of the apex and analysed
in terms of the distribution of growth. According to thinkers such as DArcy Thompson,
the whole of this regular morphogenetic development lends itself to mathematical treatment; and more recently, investigators such as Schuepp (1952), Abbe & Stein (1954)
and Richards (1948,1961) have shown how much new light may be shed on the growth,
morphogenetic activities and regulated development of the apex by a percipient mathematical approach.
APPLIED MORPHOOENESIS
It is probable that morphogenesisis generally regarded as a somewhat academic study.
Yet a little reflexion will show that it has, or could have, an important applied side.
Many horticultural practices, such as pruning, fertilizing a t particular times, vernalization, photoperiodic treatments, the production of seedless fruits, the prevention of fruit
and leaf fall, etc., are fundamentally concerned with morphogenetic processes. Time does
not admit of more than this passing mention of the possibilities of a still wider application of a knowledge of morphogenesis to agriculture and horticulture.
ORGANIZATION
Whatever the species, the orderly ontogenetic process culminates in an individual of
characteristic size, shape and distinctive appearance. In its organization, which we may
define as the summation of its genetical constitution and its specific,orderly development,
one species usually differs in certain relatively minor characters from a closely related
species, and more or less considerably from a distantly related species. There is, however,
the familiar but remarkable fact that closely comparable, major organizational featurea
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are present in organisms which, on taxonomic grounds, are quite unrelated. All vascular
plants, for example, are characterized by an axis surmounted by an apical meristem
which gives rise to the lateral members, leaves, buds, etc., in a regular manner. The same
pattern of phyllotaxis may be observed in ferns and higher plants. Solenostelicvascular
systems are known in Selaginella, ferns and flowering plants ; heterospory has evolved
independently in unrelated groups ; and ao on. These close similarities of development,
whether in related or unrelated species, have been described as homologies of organization, and it has long been recognized that they are widespread in the Plant Kingdom.
So we have to account for the fact that organisms in different lines of descent, and presumably with quite different genetical constitutions, may have major organizational
features in common. This is not only a phenomenon of great interest in evolutionary
biology : it is clearly of great importance in causal investigations of form and structure.
Some botanists may perhaps regard notions such as those of organization and of holismthe concept that organisms develop as integrated wholes-as being self-evident truths
and beyond the reach of practical investigation. Nevertheless, they are fundamental
phenomona and some attempt to understand how the specific organization of individual
species, and how homologies of organization are determined, is of the very essence of
biological inquiry. Since homologies of organization have a real existence, it seems
probable that common factors determining systems of a general kind may be involved.
In this connexion the diffusion reaction theory of morphogenesis advanced by Turing
(1952) may be mentioned as being of the kind that is of special interest. Among other
points, he indicated that comparable patterns may be produced by reaction systems of
quite different constitution. The validation of this, or indeed of any other similar theory
which rests on a physico-chemicalbasis, will depend on evidence of its generality and on
experiments in which the reaction system can be disturbed in characteristic ways with
predictable results.
The results of the normal morphogenetic activities in any species, beginning with the
zygote, are seen in the appearance of its taxonomic characters, and in the progressive
manifestation of its specific organization and holistic nature during ontogenesis. To this
fascinating and endlessly diversified field of inquiry there are clearly many approaches.
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