STUDIES IN THE MORPHOGENESIS OF LEAVES III. PRELIMINARY OBSERVATIONS ON VEGETATIVE GROWTH IN LEMNA MINOR AND E. J. WINTER*

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1 [74] STUDIES IN THE MORPHOGENESIS OF LEAVES III. PRELIMINARY OBSERVATIONS ON VEGETATIVE GROWTH IN LEMNA MINOR BY ERIC ASHBY, ELISABETH WANGERMANN Department of Botany, The University, Manchester AND E. J. WINTER* (With 4 figures in the text) INTRODUCTION Farlier papers in this series (Ashby, 1948 a, b) refer to the fact that successive leaves on the stems of fiowering plants are not all alike, but show regular changes, from node to node, in cell size and number and in leaf shape. This fact raises the problem as to whether these regular changes merely refiect changes in the external environment during development, or are due to internal changes which occur even in a constant external environment. This problem is being studied in this laboratory using Ipomoea. For a critical analysis, however, it is necessary to use a plant which can be grown under closely controlled environmental conditions, and in which the points of origin of successive leaves are as close together as possible. These conditions are satisfied by Lemna minor. Some unpublished observations on Lemna, made by two of us (F. A. and F. J. W.) eleven years ago, form the basis of our present investigations, and our combined data are used in this paper. It is known from studies of populations of Lemna minor (Ashby, Bolas & Henderson, 1928; Ashby & Oxley, 195) that when growth is unrestricted, the number of fronds increases exponentially with time. But these studies throw no light on the contribution made by each frond-meristem to the growth of the whole population. What is the expectation of life of a Lemna frond? Do fronds continue indefinitely to produce daughter fronds? Are the successive daughter fronds produced from one mother frond all alike in size and morphology? These questions have a direct bearing on the problems of heteroblastic development and physiological age which are the subject of the present series of papers; and they can be studied in Lemna with the assurance that environmental conditions are under control and morphological complexities are at a minimum. Morphologists do not agree about the way to interpret the frond of Lemna, but for the purpose of this study it is unnecessary to hold an opinion as to what a Lemna frond is. Whether fronds are regarded as leaves (Goebel, 1891-), or phyllodes (Arber, 1919), or cladodes (Hegelmaier, 1868), or shoots (Caldwell, 1899), they are morphological units of the same organism separated by intervals of time, and therefore subject to any process of senescence the organism may undergo. * Some of the observations recorded in this paper were made by two of us (E. A. and E. J. W.) in the University of Bristol in 197. Our thanks are due to Prof. M. Skene for the facilities afforded to us at that time.

2 Studies in the morphogenesis of leaves 75 The adult frond of Lemna is approximately pear-shaped, with its acute end proximal to the mother frond. On either side of the proximal end, in the body of the frond, there is a 'pocket' from which daughter fronds are produced (Fig. i a, b). Daughter fronds do not appear simultaneously on both sides of the mother frond: they are produced alternately from one side and from the other, and clones of Lemna seem to be consistent in respect of the side of the mother frond from which the first daughter frond is produced. In Lemna minor (unlike L. trisulca) each mother frond produces more than two daughter fronds; successive fronds from the same pocket arise from almost contiguous meristematic zones (Fig. i c, d), so that daughter fronds, although considerably separated in time, are very little separated in space. Under the conditions of the experiments, a daughter frond became detached from the mother frond before the next daughter from the same pocket began to enlarge. Fig. I. Diagram of a frond of Lemna minor, from above, with flaps of pockets cut away. In the left pocket the first daughter frond has separated from the parent frond; only its stalk remains (a). Above a is the third daughter frond (c) just beginning to enlarge, and the fifth (e), still a primordium. In the right pocket is the half-grown second daughter frond (b), with its own first daughter frond (b'), and above the second daughter is the fourth daughter (d), still a primordium. EXPERIMENTAL PROCEDURE Experiments were carried out to obtain answers to the following questions: (i) How long does any one frond live? (ii) How many daughter fronds does each mother frond produce? (iii) What differences are there between successive daughter fronds from one mother frond? (iv) What is the relation between cell size and frond size during growth? The experimental procedure was simple. Small colonies of Lemna were grown in constant conditions of light, temperature, and nutrient solution by a technique already fully described (Ashby & Oxley, 195*). Fronds selected for observation were marked as soon as they appeared with specks of indian ink (which had been shown in preliminary trials to have no effect on growth rate). In earlier experiments one marked frond was distinguished from another by the position of the indian ink speck; in later experiments each marked frond occupied a numbered hole (1-5 cm. in diameter) in a perforated sheet of perspex, floated on the surface of the culture solution. The date of appearance and position of the daughter fronds from each marked mother frond were then recorded. Observations were made every 24 hr. Each daughter frond was recorded when it first appeared beyond the rim of the mother frond. Since the mother frond does not grow in * Details of the technique are as follows. In all experiments Clark's (1926) culture solution was used. Its composition is 0-4 m.mol. Ca as CaH2(PO4)a, 8-o m.mol. K as KNO, 10 m.mol. Mg as MgSOi; O-OI m.mol. Fe as FeC^; to i 1. of water. The ph was adjusted to 4-8. The light intensity in all experiments was 400 i.e., and the temperature was 26-5 C. (in 197 experiments), and 25-5 C. (in 1948 experiments). In 197 colonies were grown in continuous light, and in 1948 under a 14 hr. day.

3 76 ERIC ASHBY, ELISABETH WANGERMANN AND E. J. WINTER width after its first daughter frond appears, these records are vahd measures of the intervals between successive fronds. The side of origin of daughter fronds is described as follows: if a frond fioats with its point of attachment to the mother frond toward the observer, then daughters which appear to the right are described as 'right fronds' (R.), and those to the left as 'left fronds' (L.). VEGETATIVE LIFE HISTORY The data from three experiments are summarized in Tables i and 2. Table i. length of life of fronds, with standard errors (), and mean intervals between successive daughter fronds Clone I (197), mean II (1948) Ill (1948) length of life of mother fronds (days) Position of first daughter frond interval (days) between daughter fronds: O-I IO-II L '5 5-O 41 L. 5-O S R O-I Table 2. Distribution of number of daughters from mother fronds Percentage of fronds producing: Only I daughter Only 2 daughters Only daughters Only 4 daughters Only 5 daughters Clone II 0 a II-I Clone III Although these data are not extensive they lead to three simple conclusions, which are supported by numerous other experiments: (a) Lemna fronds do not remain alive for long periods. Under the conditions of the experiments each frond lived for about 5-6 weeks. Natural death of a frond follows a gradual yellowing, which is readily distinguished from death due to other causes. (6) During its life each frond gives rise to a limited number of daughter fronds, the number depending upon the clone of Lemna used. Each mother frond, together with its daughters, may therefore be regarded as a shoot of determinate growth, in which a limited number of nodes are separated in time, though not in space; and, broadly speaking, each daughter frond repeats its mother's history. The pattern of development shown in Fig. I may be interpreted, therefore, as a sympodial system of shoots, each shoot bearing a number of nodes corresponding to the mean number of daughters characteristic of the clone (Fig. 2).

4 Studies in the morphogenesis of leaves 77 (c) Lemna minor, already known to exist in physiologically different clones (White, 196), exists also in morphologically different clones. There is some indication that the pattern of development may be changed by changing the environmental conditions, but confirmation of this must await further experiment. Fig. 2. Diagram of morphological interpretation of clone II of Lemna minor. The base of the model represents the mother frond, which bears five daughter fronds in succession at a meristematic zone here shown elongated into a shoot. Each daughter frond repeats the history of the mother frond. Growth is therefore sympodial. Lettering as in Fig. i, except that a represents a frond, not the stalk of a frond which has become detached. MORPHOLOGICAL DIFFERENCES BETWEEN SUCCESSIVE DAUGHTER FRONDS In the course of observations on the vegetative life history of Lemna it was noticed that the areas of successive daughter fronds produced from one mother frond progressively diminished. It is this circumstance which makes the morphogenesis of Lemna relevant to work already published (Ashby, 19486) on Ipomoea: for in Lemna such a trend as this in frond area is not likely to be due to changes in the environment, or to increasing distance from the source of nutrient supply, or to increasing differentiation of the plant body. Accordingly, it is reasonable to suppose that the trend of diminishing area is related either to some nutrient deficiency or to some process of ageing at the meristems of the mother frond; it is unlikely to be due to nutrient deficiency, because the average area of fronds in a colony does not diminish with time, but remains approximately constant. This indicates that late daughter fronds with sub-average area must in their turn give rise to early daughter fronds of more than average area. This behaviour has indeed been noticed and is now being thoroughly studied. ' Data on the areas of successive daughters were collected as follows: while experiments on the life histories of clones II and III were in progress, daughters of the fronds under observation were segregated in separate holes in the floating perspex sheets. These daughter fronds were kept in culture for about 11 days, until they in turn had produced at least two daughter fronds: this was to ensure that they were fully grown. They were then fixed in formalin-acetic alcohol. For observation under the microscope the preserved fronds were prepared as follows: they were cleared in Eau-de-Javelle for about hr., washed and dehydrated to 50% alcohol, left for min. in a mixture of equal parts of 50% alcohol and glycerine jelly, and mounted in glycerine jelly. This treatment provides transparent mounts, in which every cell layer can be seen by focusing at different levels. Areas of the fronds were then determined by tracing micro-projections of them

5 78 ERIC ASHBY, ELISABETH WANGERMANN AND E. J. WINTER and measuring the areas of the tracings with a planimeter. The data are given in Fig- - It is evident from Fig. that in both clones there is a highly significant trend of diminishing area among successive daughter fronds from the same mother frond. It remained to determine whether the smaller areas are due to smaller cells in the fronds, or to fewer cell divisions; this was done by comparing the sizes of epidermal cells. It is impossible to estimate from random samples a reliable average cell size for the epidermis E Daughter frond Fig.. areas of fully grown daughter fronds in clones II and III. The vertical bars represent twice the standard errors of the values entered in the figure. There is no standard error for the fifth daughter frond of clone II. Table. Numbers of cells per mm."^ from three regions of the lower epidermis of Lemna minor, in successive daughters ( represents the standard errors of the means of the samples of fronds.) Clone II Clone III Daughter... I 2 I Region A B C III of a Lemna frond, because there are steep gradients of cell size from tip to base of the frond (see p. 79). Therefore three regions of the lower epidermis were chosen, which could be located regardless of its size and shape: one near the tip {A), one near the point of emergence of the root {B), and one near the base of the frond (C); all three just to one side of the midrib. Comparisons were made of the cell size from corresponding regions of successive fully grown fronds. The results are entered in Table. Clearly there are no significant differences in cell size between corresponding regions of first to third daughter

6 Studies in the morphogenesis of leaves 79 fronds. Therefore the highly significant diminution in area of successive fronds (Fig. ) must be attributed to a decrease in the number of cells per frond: this means either that there is a progressive diminution in the size of successive meristems, or successive meristems undergo progressively fewer cell divisions. There is no evident external cause for this trend, neither is it due to any evident 'position effect' of successive fronds. The third daughter frond develops in the same position as the first; but the first daughter breaks away from the parent frond while the third daughter is still a primordium, so that by the time the third daughter frond begins to enlarge there are no other developing fronds in the same pocket. The same applies to all subsequent daughters. The only evident factor which alters as successive daughter fronds appear is the age of the mother frond which produces them. For these reasons morphogenesis of Lemna is now being studied in relation to the ageing of the mother frond. A NOTE ON CELL SIZE AND MORPHOGENESIS IN LEMNA A preliminary study has been made of the changes in the size of epidermal cells which accompany the enlargement of a Lemna frond. The data for this study were obtained as follows. Ninety fronds of different ages were fixed and cleared as described above (p. 77). The youngest fronds were little more than primordia and had to be squeezed out of the pockets of the parent fronds. Microprojections of the outlines of the cleared fronds were traced on paper. The length of each tracing from base to tip was measured. The fronds were then divided into nine approximately equal classes according to frond length, ranging from i-oo to -0 mm. The purpose of this classification was to discover the mean length of epidermal cells corresponding to frond length at different stages of enlargement. It was found that, although epidermal cell size was approximately constant in transects at right angles to the long axis of the frond, it was by no means constant in transects parallel to the long axis; it is for this reason that reliable estimates of mean cell size cannot be made. The relation between frond length and cell length was accordingly worked out by taking samples of cell size from base to tip of every frond at intervals of 0-22 mm., along one side of the midrib. Samples were taken from the lower epidermis because it is free from stomata. Cell counts were made, using a square field mm.^ in area and a \ in. objective, by traversing cleared fronds with the aid of a mechanical stage. The number of whole cells in each sample field was counted, and a correction was added for the number of cells partly included in the field (see Ashby, 1948 b). Since the cells are, apart from convolutions on their radial walls, approximately isodiametric in surface section, the square roots of the reciprocals of these counts were used as estimates (for comparative purposes only) of cell length. The results are summarized in Table 4 and Fig. 4.* The interrelationships between frond length, cell length, and position on the frond, are illustrated by a three-dimensional model constructed from smoothed data (Fig. 4). The following points are noteworthy: (i) Even in the youngest fronds which were examined (fronds about one-third of their adult length) there is a steep gradient in epidermal cell size from base to tip (a): cells at * Clone III was used for these determinations. It is evident from Tables i and 2 (p. 76) that in a population first and second daughters greatly outnumber third and fourth daughters. The differences in cell number hetween successive daughter fronds therefore do not obscure the trend of the data.

7 80 ERIC ASHBY, ELISABETH WANGERMANN AND E. J. WINTER the base of these fronds are small and actively dividing, whereas cells at the tip have reached almost their full size, their walls are convoluted, and fully developed stomata are present in the upper epidermis. (ii) In older fronds {b) the gradient in cell size is reversed. It appears that during enlargement of the frond a wave of cell expansion moves from the tip toward the base, and the region of maximum cell size in a mature frond is at c, about a quarter of the distance from base to tip. Table 4. Square roots of areas of cells in lower epidermis {mm.) (Estimate of average standard deviation of each entry, assuming variances are normally distributed = 12 % of each mean. There are apparent coincidences among the entries because they are derived from the reciprocals of wbole numbers.) Distance along frond (mm.) I I-S7 Class modes of lengtb of frond (mm.) i-io 1-2 i's o-oio ? Distance along frond (mm.) Fig. 4. Relationships between cell lengtb and frond length in clone III. Ordinates: cell length; left-toright abscissae: distance along ironds; front-to-back abscissae: stage of enlargement of fronds (in classes of frond length). L = class mode of frond length. An approximate estimate of the coefficient of variation of ordinates is c.v. = 12%; therefore differences exceeding about 24% of the ordinates are significant, a = smallest fronds, witb small meristematic cells at tbe base and almost fully grown cells at the tip. 6 = large fronds, witb largest cells near the base and smallest cells at the tip. c = region of maximum cell size in mature frond.

8 Studies in the morphogenesis of leaves 81 SUMMARY Papers in this series are contributions to an analysis of the changes in leaf morphology from node to node on herbaceous plants. The present paper offers evidence that similar changes in morphology occur between successive fronds from the same meristem in Lemna minor. Preliminary experiments establish four facts, namely, that in adequate conditions of light, temperature, and mineral nutrition: (i) Lemna fronds do not remain alive for more than 5-6 weeks; (ii) during its life each Lemna frond gives rise to a limited number of daughter fronds, the number being characteristic of the clone used; (iii) the areas of successive daughter fronds produced from one mother frond progressively diminish; (iv) this diminution in area is not associated with any reduction in epidermal cell size, but is due to the fact that there are fewer cells in late-formed than in early-formed daughters from the same mother frond. The authors have pleasure in thanking Mr George Breckon for technical assistance and Mr F. H. Roberts for drawing Figs, i and 2. REFERENCES ARBER, A. (1919). Vegetative morphology of Pistia and the Lemnaceae. Proc. Roy. Soc. B, 91, 96. ASHBY, E. (1948a). Studies in the morphogenesis of leaves. I. New Phytol. 47, 15. ASHBY, E. (19486). Studies in the morphogenesis of leaves. II. New Phytol. 47, 177. ASHBY, E., BOLAS, B. D. & HENDERSON, F. Y. (1928). The interaction of factors in the growth of Lemna. I. Ann. Bot., Lond., 42, 771. ASHBY, E. & OXLEY, T. A. (195). The interaction of factors in the growth of Lemna. VI. Ann. Bot., Lond., 49, 09. CALDWELL, O. W. (1899). On the life history oi Lemna minor. Bot. Gaz. 27, 7. CLARK, N. A (1926). Plant growth promoting substances, hydrogen ion concentration, and the reproduction oi Lemna. Plant Physiol. i, 27. GOEBEL, K. ( ). Pflanzenbiologische Schilderungen. Teil II. Marburg. HEOELMAIER, F. (1868). Die Lemnaceen. Leipzig. WHITE, H. L. (196). The interaction of factors in the growth oi Lemna. IX. Ann. Bot., Lond., 50, 827. WINTER, E. J. (197). Growth of Lemna minor. Nature, Lond., 19, {Received 8 December 1948) New Phytol. 48,

OF THE LEMNA FROND MORPHOLOGY

OF THE LEMNA FROND MORPHOLOGY MORPHOLOGY OF THE LEMNA FROND FREDERICK H. BLODGETT (WITH PLATE XIV AND ONE FIGURE) In the case of structure simplified by reduction, it is sometimes necessary to trace the development of the parts through