MERISTEMS, GROWTH, AND DEVELOPMENT IN WOODY PLANTS An Analytical Review of Anatomical, Physiological, and Morphogenic Aspects By J. A. Romberger U.S. Department of Agriculture, Forest Service (The author is Plant Physiologist at the Forest Physiology Laboratory, Plant Industry Station, Beltsville, Md.) Technical Bulletin No. 1293 October 1963 12 88725 PREFACE Apical meristems are small. Lateral meristems are thin. Together they constitute a physically insignificant fraction of the total mass of a tree or shrub. Yet the whole future of the plant depends upon the activity of its meristems. Growth and morphogenesis, and the control of these processes, are largely localized in the meristems proper and in their ancillary regions of occasional cell division, con- tinuing cell enlargement, and cell differentiation. The subject area encompassing meristems, growth, and development is basic to a wide range of research problems in forestry and horticulture. This bulletin is intended for students and research workers, in plant physiology, horticulture, and the forest sciences, who are inter- ested m the control of growth and development in woody plants. It is not a textbook. nTustrations duplicating those readily avail- able in texts have not been provided. Headers are assumed to have knowledge of the basic principles of the anatomy, physiology, and biochemistry of plants, and to have access to textbooks on these sub- jects. I have attempted to go beyond the textbook level in analyzing complex problems, in searching for interrelations between the various islands of research information, and in providing a guide to the early as well as the more contemporary literatuijB. The approach is nonauthoritarian. Many questions are asked. Few are answered. Eeaders are encouraged to speculate and to doubt and question my interpretations as they see nt. I wish to be regarded not as an expert, or a teacher, but as a fellow student. Although growth control in woody plants has many special aspects, it cannot be considered as a subject completely separate from that of growth control in herbaceous species. Much, or most, of the ex- perimental work on growth regulators, photoperiodism, and photo- morphogenesis was done with herbaceous species. Some of the evi- dence discussed and literature cited in this review is not directlj concerned with trees or shrubs, but such citation and discussion is nonetheless prerequisite to intelligent consideration of the specific problems of growth control in woody plants. Throughout the review, emphasis is put upon lines of work spe- cifically aimed at increasing our basic knowledge of meristems and the control of their activities. The voluminous literature concerning purely empirical experimentation aimed at early application in the field IS not stressed. As a knowledge of political and social history is indispensable to social scientists, a knowledge of the history of biology is likewise indispensable to the biological theoretician and experimenter. With- out the past, without an appreciation of past successes and failures, and their significance to us, our future advance would be wavering in direction and lacking in momentum. Such considerations, and the belief that discussions of sincere attempts to arrive at truth are IV PREFACE never obsolete, prompted use of the historical method of exposition in most sections of this review. Plant names are generally the Latin names given in the works cited. Many original sources give no authorities for the names em- ployed. None are given here. Some of the names used herein are not current or are in dispute. Readers who need current names and authorities must seek information in the papers cited, and elsewhere. No review of this type can cover all related areas in addition to the central subject. The very important and closely related subjects of the control of flowering in woody plants, and the physiology of seed dormancy and the germination process, are treated only mci- dentally. Also outside the area of immediate concern are breaking of dormancy by deliberate wounding of plants or by applications of any of a great variety of chemicals having no known relation to any endogenous regulators. This review is not exhaustive even within the subjects covered. The goal was to provide access to important lines of work rather than to cite all significant papers. Some references were intention- ally omitted because they are included in bibliographies of other works cited. Some important papers were undoubtedly overlooked, and numerous recent ones came to my attention too late to be included. Covera^ of some subject areas was modified because of the existence of relatively recent and readily available reviews by other authors. With these limitations imderstood, I hope that these discussions will encourage and facilitate further work on the fascinating subject of meristems and their activity or dormancy in woody plants. A written discussion is Imear. Only one aspect of a subject can be presented at a time. Words, sentences, and paragraphs follow one another. Each separate fact or idea in turn briefly commands the reader's attention. But the realm of ideas is not one dimen- sional. The numerous facts and ideas embodied in this review are related to each other more like various points within the volimie of a sphere than like points on a straight line through space. To pro- mote escape from linearity, numerous cross references have been provided in the text. These are indicated in italics within parenthe- ses, either alone or separated from citations to other works by a semicolon. I sincerely appreciate the assistance and advice received from many people during the preparation of this bulletin. Particularly helpful were Edward R. Moser, Librarian, Division of Biology, California Institute of Technology, and the staff members of the National Agricultural Library in Washington, D.C., and Beltsville, Md. Drs. Bruce M. Pollock, Harry A. Borthwick, Thomas O. Perry, and Robert M. Allen made many constructive suggestions after reading all or parts of the manuscript. CONTENTS Pace Part I. Anatomy and physiological morphology 1 Organization of meristems 1 Development of the meristem concept 1 The origin of cells 1 The apical cell theory _ 3 The histogen theory of apical organization 4 Transition to modern concepts 5 Organization of gymnosperm shoot apices 6 Cytohistological zonation 6 Zone apicale, anneau initial, and méristèm médullaire 12 Organization of angiosperm shoot apices 13 Tunica-corpus theory 13 Cytohistological zonation 16 Méristèm d'attente, anneau initial, and méristèm médullaire 18 Metrameristem 20 Synopsis on shoot apices 20 Organization of root apices 21 Root apex versus shoot apex 21 Ajpical cells and histogens 22 Körper-Kappe theory 23 Many-celled promeristems versus central cells 24 The quiescent center 27 Physiological morphology of shoot meristems and buds 30 Reactivity of shoot meristems 30 Metabolic differentiation within the meristem 30 Culture of isolated apices 31 Morphogenic regions of the apex 32 Special significance of the subapical region 34 Origin of leaves, cataphylls, and vascular tissue 35 Initiation of primordia 35 Procambium ^ 38 Development of leaves 41 Development of cataphylls 44 Vegetative buds and the morphogenic cycle 46 The bud concept 46 A peculiar anatomical feature—the crown 47 Terminal buds 49 Axillary buds 54 Adventitious buds 58 Physiological processes in buds 59 Shoot tip abortion 62 Inability to form terminal buds 62 Physiology of apical abortion 63 Physiological anatomy and development in the cambium 65 Developmental anatomy 65 Morphogenic cycles in the vascular cambium 68 Part II. Episodic growth and dormancy of shoots 71 Concepts, nomenclature, and definitions 71 The dormancy concept and its development 71 Kinds of dormancy—definitions 73 . Alternate growth and dormancy 76 Implications of episodic growth 76 Associated anatomical and cytological changes 78 Analysis of the control problem 80 Internal physiological factors 80 Why summer growth inhibition? 80 Possible root influences 81 Correlated inhibition and apical. dominance 81 v VI CONTENTS Part II. Episodic growth and dormancy of shoots—Continued Analysis of the control problem—Continued Page Experimental control of growth and dormancy in various species 84 An introduction to photoperiodism in woody plants g4 Pinits sylvestris 86 Faaus aylvatica 89 Robinia paeudoacacia. _ 92 Catalpa bignonioides 93 Weigela florida .- — 94 Comtí8 florida ._ 95 ÄÄtis typhina 96 Acer p8ev4oplatantL8 96 The significance of photoperiodism 97 Are photoperiodic receptor and response mechanisms general? 97 Mechanistic implications of photoperiodic responses 99 Possible mechanisms of growth and dormancy control 100 Photomorphogenesis 100 Early work on light intensity and spectral quality 100 Duration of light — _-.- 102 Etiolation - 104 Phytochrome—a photomorphogenic receptor 106 Responses to light of limited spectral regions 111 A second photomorphogenic receptor? 115 Some kinetic aspects of photomorphogenesis and photoperiodism 117 Circadian rhythms in relation to photo- and thermoperiodism 119 Endogenous circadian rhythms 119 Circadian rhythms and photoperiodism 121 Circadian rhythms and thermoperiodism. 124 Endogenous growth regulators 125 Introduction __> 125 The auxin concept 126 Auxins in buds and shoots.. 128 Auxins in developing long shoots versus short shoots 130 Auxins and cambial activity 133 The significance of auxins in dormancy control 138 Gibberellins 140 Kinins 146 Other possible regulators 148 Endogenous growth inhibitors 150 Interactions 154 Nonperiodic temperature effects . 157 Chilling requirements . 157 Unsatisfied chilling requirements and dwarfing 161 High temperature and rest induction 163 Warm baths as rest breaking agents 164 Part III. Episodic growth and dormancy of roots 166 Growth and dormancy in roots 166 Definition of the problem 166 Seasonal and episodic root growth 166 Anatomical and physiological aspects. 171 Control of root growth 175 In retrospect 177 literature cited 180 PART I. ANATOMY AND PHYSIOLOGICAL MORPHOLOGY ORGANIZATION OF MERISTEMS Development of the Meristem 0)acept The Origin of Cells The concept of meristems is a relatively recent one. Its formula- tion depended upon prior evolution of ideas concerning the cellular structure of organisms and the origin of cells. The evolution of those ideas was slow. Truths which seem obvious to us now were arrived at by the efforts of generations of dedicated men. There were undoubtedly many brilliant minds among the bota- nists and microscopists of the I7th and 18th centuries. They did all they could do with the instruments available to them. But the results of their efforts advanced knowledge of cells and tissues only a little beyond the levels attained by Grew, Malpighi, and Leeuwen- hoek. It was known that cork and wood are cellular in structure, but the cell was not recognized as the basic structural unit of all plant parts. Nothing was known about the origin of either cellular structure or of cells. The great barrier to progress was chromatic aberration in lenses. Objects under the microscope shimmered with all colors of the rain- bow. Details were blurred out and misinterpretation was easy. The development of achromatic lens systems was a breakthrough of great significance to biology. Achromatic microscopes became gen- erally available to biological research institutions in about 1830. A resurgence of interest in plant anatomy and development began im- mediately and a great wave of progress followed shortly thereafter. In 1830 the fact that wood is composed of mostly empty cells was generally accepted, although some question remained about the cel- lular origin of vessels. That other plant parts also consist of cells was, however, still not widely recognized. Modification of the cell concept to include not only the empty, thick-walled chambers of wood and cork, but also thin-walled structural units filled with liq- uids and gels came quickly after achromatic microscopes were in use. On the basis of detailed studies of the structure of mosses and other plants Mirbel (1837)^ maintained that the cell is the funda- mental unit of structure in the plant kingdom. Treviranus (1835), Mirbel (1837), and Mohl (1845a, b) removed objections to the cel- lular structure of wood vessels by observing that vessels arise from files of cells which lose their end walls. ^ Names and dates in parentheses refer to Literature Cited, p. 180. 2 U.S. DEPT. OF AGRICULTURE, TECHNICAL BULL. NO. 1293 A great series of further contributions was made by Mohl. Bast, bark, and other plant parts were all found to be cellular. Mohl was a very conscientious and careful observer who accurately described what he saw but did not engage in philosophical speculations. His papers, characteristically short and to the point, are still interesting and significant (see early volumes of Botanische Zeitung), Mohl's work overcame all objections to the cellular theory of plant struc- ture and led to its acceptance as undisputed fact. Solution of the problem of cell origin was also made possible by the achromatic microscope. The new knowledge that cells are the structural units of organisms did not answer the question of cellular origin. It was not at first obvious that cells are produced only by division of preexisting cells. Progress, however, was rapid during the two decades after 1830. Brown (1831) published evidence that every living plant cell con- tains a nucleus, but did not realize its significance. Schleiden in- volved the nucleus in his explanation of cell origin, but only as a vesicle which somehow arises in a generative center and then pro- duces the remainder of the cell. Schleiden (1842) summarized his work in a textbook which shows philosophical romanticism remi- nescent of Goethe's botanical writings. Nevertheless, the book had a profound effect upon botanical research because it convinced young botanists of the need for developmental studies and insisted that they use inductive methods. Schleiden's theory of cell origin was further developed by Schwann (1839). He believed the cell to be coagulated or precipitated from sap, first the nucleolus, then the nucleus, and finally the remainder of the cell. The Schleiden-Schwann theory assigned no role to the nucleus after cell formation, and certainly did not anticipate the possibility of nuclear division. The theory enjoyed a short ascend- ency, but then went into decline because it could not accommodate the further findings of observers. Leadership in the field soon passed to Mohl and Nägeli. Both Mohl and Nägeli were influenced by the Schleiden-Schwann theory, but they did not accept it as doctrine. Mohl continued his painstaking observational and descriptive work. In numerous short papers he described vacuoles, chloroplasts, and starch granules. He also described and named the protoplasm and recognized it as the essence of living matter, not merely unorganized slime. Mohl con- sidered nuclei in the embryo sac to be derived from vesicles in the protoplasm, perhaps as envisioned by Schleiden and Schwann, but he also mentioned cell division as the normal method of cell repro- duction in the vegetative parts of plants. Other botanists became convinced that cells in growing plant parts are formed by cell division. Meanwhile Nägeli (1842, 1844) made very careful observations of cell division during pollen formation and elsewhere and described the process, including nuclear division, with great accuracy. Yet even Nägeli continued to believe in the possibility of the spontaneous generation of life and of cells through- out his entire lifetime. Even before the concept of apical meristems was well established it was obvious that lateral zones of cell formation must be respon- sible for stem thickness growth. Mirbel (1837), writing at a time MERISTEMS, GROWTH, AND DEVELOPMENT IN WOODY PLANTS 3 when cells were thought to be coagulated from sap, used the term "cambium" in the sense of a sap or juice saturating the growing parts of plants. The term remained in use, but with new meaning, after the origin of cells by division was established. Anatomy and development in the cambium was a relatively noncontroversial sub- ject. The outlines of our present knowledge of the cambial meristem were already evident in Nägeli's (1864) "Dickenwachsthum des Stengels." Valuable contributions were also made by Sanio (1872) and Mischke (1890), but the mechanism of girth increase in the cambial meristem itself was not well understood until later (see Bailey 1923). The Apical Cell Theory The large, single, apical cells of various mosses and algae were discovered and described by Nägeli (1845a, b). In apices of these plants it was obvious that all new cells were derived from preexist- ing cells by division. The concept of a single apical cell, dividing in a regular and predictable manner, and giving rise to all other cells of these plants, was enthusiastically accepted by the majority of botanists. The idea seemed inherently logical and at the time a working assumption that higher plant apices also possess single apical cells was a reasonable one. Understanding of cell origin and ñirther improvements in micro- scopes and in sectioning techniques had by 1850 made it possible to undertake meaningful studies of the organization of apical meri- stems of higher plants. The term "meristem" (from the Greek meristos^ meaning divided) seems to have been introduced by Nägeli (1858). Hofmeister (1852) published the first description of the organiza- tion of an apical meristem of an angiosperm. He reported a unique initial cell in Zoster a marina (eel-grass), this cell being visible in early stages of development and dividing like the single apical cell of Equiseturrh. Later he reported Acer and Fraxinus to have cune- iform terminal cells and some other tree species to have tetrahe- dronal apical cells (Hofmeister 1857). Hofmeister's apical cell theory received strong support from Pringsheim (1869), Nägeli (1878),Korschelt (1884), Dmgler (1886), and Douliot (1890). The theory held that there were no fundamen- tal differences in mode of origin of apical tissue between vascular cryptogams and phanerogams because it was supposed that, in both groups, all cells could be traced to divisions of a single apical cell. The applicability of the theory to any but embryonic apices of higher plants was soon questioned by some workers and a long con- troversy arose, the details of which are given by Koch (1891) and also by Schüepp (1926). Gymnosperms received considerable attention because of their phylogenetic position between vascular cryptogams and angiosperms. Various workers reported single tetrahedral or prismatic apical cells in gymnosperm apices. A few careful observers, such as Strasburger (1872) and Groom (1885), could see no evidence for single apical cells. These dissenters were vindicated in later decades. The fact that others continued to report and describe single apical cells illus- 4 U.S. DEPT. OF AGRICULTURE, TECHNICAL BULL. NO. 1293 trates the powerful effects which preconceived ideas can have upon observers. Reviewing the situation Douliot (1890) concluded gymnosperms, like vascular cryptogams, to have apical cells, sometimes pyramidal, sometimes prismatic, but always unique. Angiosperms he believed to have usually three, but sometimes only two, apical initial cells. AVliile formalistically neat and satisfying, Douliot's position was not favored by time. Most of the early work was strongly charac- terized by formalism with little regard for the dynamic aspects of tissue development and cell function. The Histogen Theory of Apical Organization Meanwhile Hanstein (1868), working mostly with angiosperms, had evolved and published his histogen theory of apical organiza- tion. His ideas were based upon studies of 46 genera, including AlrmSj Populus^ Platanus^ Aesculus, Sambucus^ and Robinia. In contrast to the apical cell theory, Hanstein's histogen theory main- tains that the shoot apex in angiosperms consists of a central core of irregularly arranged cells covered by a variable number of man- tlelike layers. It proposes that each layer, and the core, is derived from a distinct initial cell or small group of cells (the histogens or tissue formers). Thus the origin of different parts of the apex can- not be traced to a single cell, but each part can be traced to one of a series of vertically superimposed initials or groups of initials. Hanstein attached less importance to the behavior of individual cells than to the general distribution of growth in the apex as a whole. He did, however, attempt to assign specific destinies to vari- ous regions of the meristem, regions which in turn were derived from the series of superimposed initials. The surface layer, or "dermatogen," Hanstein believed, produced only the epidermal sys- tem ; the underlying layer or layers, which he called the "periblem," produced the cortex; and the central core, or "plerome," produced the procambial and pith tissue of the axis. Hanstein originally ap- plied his terms to zones of meristematic tissue in the early stages of development from initials, but in later literature the same terms were sometimes applied to the initials themselves. The predestination aspect of Hanstein's theory drew a great amount of criticism which was reviewed and discussed hj Schmidt (1924). A further difficulty was that in many apices periblem and plerome were not distinguishable, and in others where they were distinguishable their respective roles did not conform to Hanstein's ideas. These weaknesses were noted and discussed repeatedly (Koch 1891; Schmidt 1924; Korody 1937). The histogen theory was applied to root as well as shoot apices. The availability of precision microtomes made it possible by 1870 to prepare good median sections of apical meristems. This led to many studies of root meristems and reports concerning their histo- gens. Janczewski. (1874a, b) introduced a fourth histogen, the "calyptrogen," in his descriptions of roots of grasses and other plants which have a rootcap of independent origin. With regard to root apices the histogen theory attained general acceptance. In fact, Hanstein's ideas and terminology are not yet totally obsolete