STUDIES IN BRITISH PRIMULAS

Transcription

1 STUDIES IN BRITISH PRIMULAS VII. DEVELOPMENT OF NORMAL SEED AND OF HYBRID SEED FROM RECIPROCAL CROSSES BETWEEN P. VULGARIS HUDS. AND P. VERIS L. BY S. R. J. Botany School, University of Oxford* {Received 3 Jime 1959) (With Plate 8 and 4 figures in the text) INTRODUCTION In previous papers in this series, Valentine has described the nature of the seeds produced in reciprocal crosses between Primula vulgaris, P. elatior and P. veris. The degree of success of seed development (seed compatibility) was analysed and a genetical hypothesis put forward to explain the results. In its latest version (Valentine, 1956) this suggests that success or failure of the developing seed is determined by the genetical constitution of the tissues concerned, and that the interaction between maternal tissues and endosperm is of major importance. Valentine's conclusions were based mainly on measurements of seed size and weight, on seed dissection and on germination data. It was felt that a more detailed study of the early development of the seeds and of the interactions of the tissues was needed, and accordingly the present investigation was undertaken. In this paper, development of the seeds of the species themselves and of one of the interspecific crosses is described; in a further paper, the other two crosses will be dealt with, and the results as a whole discussed. MATERIALS AND METHODS The plants used were of British origin, from the collection maintained by Professor D. H. Valentine at Durham. They were kept out of doors during the winter, and in early spring brought into an insect-proof cool greenhouse. They were treated with a fungicidal dust to prevent attacks by Botrytis. Only legitimate crosses were made. Thrum flowers were emasculated before pollination by removal of the corolla tube with the attached anthers before they were mature. Pin flowers were not emasculated, as this was found not to be necessary in the absence of insects. Pollinations were heavy. For each cross, the female parent was selected carefully, in order that all the pollinations for the one cross could be done at once; this ensured that all the capsules from a single cross were developing at as near as possible to the same time and under the same conditions. This excluded possible effects of environmental variation. In each case an unpollinated ovary was fixed at the time of pollination of the rest. Thereafter the developing capsules were fixed at 10, 20, 24 days, and each fourth day after that. In the first season the capsules were fixed with Karpechenko's Fixative under reduced pressure, and infiltrated by means of a chloroform-ethyl alcohol series. Later a * This work was carried out while the author was in the Botany Department. Durham Colleges in the University of Durham. 302

2 Studies in British Primulas VII 303 more satisfactory method was adopted. The capsules were split at the apex to allow more rapid penetration of the fixative, which was formalin-acetic-alcohol. Infiltration M'as effected by means of a normal butyl alcohol series. Sections were cut at 15 M, and mounted serially. They were stained in either Heidenhain's haematoxylin, or safranin and fast green (Maheshwari, 1939), the latter stain being especially useful for integument structure. DEVELOPMENT OF SEEDS (a) The ovules before fertilization The ovules are anatropous, borne on a free central placenta. They have two integuments. Fig. I is a diagrammatic representation of the unpouinated ovule, and it shows that each integument has two distinct layers. Outer integument i and inner integument 2 are very similar in appearance, being composed of small regular cells, with brown Fig. I. Diagrammatic representation of unpouinated ovule (X approx. 375): (a) Outer integument i. (b) Outer integument 2. (c) Inner integument i. (d) Inner integument 2. (e) Chalazal region, (f) Chalazal pocket, (g) Micropyle, contents. This brown coloration is said to be due to the presence of tannins in the cells (Decrock, 1901). Inner integument 2 has been the subject of some discussion in the past. In Primula the nucellus is ephemeral, so the embryo sac is in direct content with this inner layer of the inner integument from a very early stage. This has led to the idea that it serves some kind of nutritional function, and to its being termed a 'tapetum' and a 'nutritive layer'. Brink and Cooper (1939) called it the endothelium, and this term has been used by others. In the present account it will be referred to as inner integument 2. This does not suppose any special function for the layer. The inner layer of the outer integument (outer integument 2), is also one cell thick. The cells are very regular, cubical, and filled with dense contents. The outer layer of the inner integument (inner integument i) on the other hand, is made up of several layers of cells, rather irregularly shaped, with rather less dense contents. This layer is in contact with the embryo sac in two places, where the inner integument 2 is interrupted: C N.P.

3 304 S. R. J. WOODELL at the micropyle, in the region of which the embryo is located in its early stages of growth, and at the so-called 'Chalazal Pocket', at the opposite end of the chalazal region. Between these two gaps, the inner integument two cells are large and rather irregular, as compared with those of the remainder of this layer. The embryo sac is rather large and highly vacuolated. At maturity, there are three antipodals, a single primary endosperm nucleus, and an egg and two synergids. (b) Post-fertilization development in the species The development of the three species was studied, and there is a remarkable similarity between them. The description of any one would serve for all, and even after some experience it is difficult to distinguish a particular ovule section as belonging to a particular species. The main and most obvious differences are in timing of the various stages, and in the final size of the seeds. The variation in timing may be purely due to environmental differences, as not all the crosses were started at the same time. The graphs of seed and embryo length in Fig. 2 give some indication of just how slight this variation is. The description below refers mainly to the cowslip (P. veris). Dahlgren (1916) studied early development in several members of the family Primulaceae, including Primula. He made some useful observations on the very early postfertilization stages, and some of his remarks have been used to supplement this account, since I did not attempt to make any detailed investigation of the early post-fertilization stages. After fertilization the zygotic division is delayed, and after it has occurred growth of the embryo is slow (Fig. 2b). Dahlgren has counted over 1000 endosperm nuclei in an embryo sac in which the zygote had not yet divided. He states that the primary endosperm nucleus remains adjacent to the egg apparatus at the first division; after this division one daughter nucleus migrates in the thin cytoplasmic layer towards the chalazal end of the embryo sac, the other remaining in its original position. A large number of free endosperm nuclei arise, the first few divisions being simultaneous throughout the endosperm. Outer integument. During the early stages of development of the seed the outer integument goes through a period of very rapid growth (Fig. 3, a-e). By the 20th day after fertilization the cells of outer integument i have elongated outwards, their walls tapering and the outer end being rounded. These cells will later form the papillae with which the mature seed is covered. The inner walls of these cells become heavily thickened, and the contents shrink and eventually disappear. By the 24th day the cells of outer integument 2 show a very thin layer of cellulose thickening on their inner walls, and this extends slightly up the lateral walls (Fig. 3d). This thickening increases steadily and rapidly, and by the 36th day it already half fills the cells. It continues to increase (Fig. 3, e and f) until in the mature seed the whole of the cell except for a very small area on the outer side is filled. The ultimate result of this course of development of the outer integument is that the mature seed has an outer wall which is heavily thickened and very hard. Inner integument. The course of development of the inner integument is markedly different from that of the outer. The picture is one of progressive degeneration, first of inner integument i, and then of the innermost cell layer. At 10 days, signs of degeneration are already apparent in inner integument i (Fig. 3b). The inner cells are losing their shape and becoming compressed; their contents are

4 Studies in British Prirnttlas VII Time in days after pollination Time in days afler pollinacion (a) 10 M SO 60 (c) Fig. 2. (a) and (b) the three species, graphs of (a) seed length and (b) embryo length with time, (c) seed length and (d) embryo length with time. P. veris; P. vulgaris; P. veris 9, P. vulgaris,j and P. vulgaris $ :< P. veris rj. Scales for seed and embryo lengths in arbitrary units.

5 3o6 S. R. J. WOODELL shrunken. By 20 days this breakdown has spread to all but the innermost layer (Fig. 3c). The process is less rapid in the chalazal region (Plate 8, a). By the 28th day all that remains of the inner integument i is a thin layer of empty cells; the contents have degenerated and the cells are collapsed. Meanwhile the inner integument 2 has been undergoing a similar degeneration. The cells become less regular, and are progressively compressed (Fig. 3, b, c and d). By the 44th day the inner integument i has completely broken down, even in the chalazal region, and the inner integument 2 has been reduced to a thin layer of compressed cells. Fig 3. Sections of integuments of P. t'em. X 320. Outer integument uppermost in each case, (a) unpollinated ovule, (b) 10 days, inner integument already showing signs of degeneration, (c) 20 days, further degeneration apparent, (d) 24 days, thickening of outer integument two beginning, (e) 32 days, thickening increasing, inner integument nearly fully broken down, (f) 44 days, thickening nearly complete, inner integument a thin layer of cell debris. The result of this breakdown of both layers of the inner integument is that the mature seed is hned with a thin dark layer of cell debris, compressed between the outer integument and the endosperm (Plate 8, c). The endosperm. From an early stage the number of endosperm nuclei increases rapidly, and they are soon fairly evenly distributed throughout the thin layer of cytoplasm that lines the seed cavity. There is a greater concentration in the chalazal region, and here the cytoplasm layer is thicker and rather more granular. Cell formation commences between 20 and 24 days, in the chalazal region; this is not unexpected since

6 Studies in British Primulas VII 307 the inflow of nutrients reaches this region first. Often by the 24th day there is a complete single layer of cells, with the beginnings of a second in the chalazaj region (Plate 8, a). Successive layers of cells are built up on the first, in parallel rows. By about 36 days the Fig. 4. Embryos oi P. veris in longitudinal section, (a-e, h / 415, f-g, i-j ). (a) 10 days, (b) 20 days, (c) 24 days, (d) 28 days, (e) 32 days, (f) 36 days, (g) 40 days, (h) 36 days, (i) 44 days, (j) 60 days. seed is loosely filled with endosperm (Plate 8, b) and it becomes more closely packed after this time. Food reserves, mainly in the form of oil droplets, appear in the cells at about 40 days, and rapidly increase from then on, until the cells are packed w ith reserve nutrients (Plate 8, c). The embryo. It has already been pointed out that the embryo gets off to a very

7 3O8 S. R. J. WOODELL slow start and very little growth takes place for a prolonged period (see graph. Fig. 2b). There then ensues a period of rapid enlargement. From about eight cells at 24 days (Fig. 4c), it increases to about sixty cells at 32 days (Fig. 4e). Henceforth grovi'th is even more rapid (Fig. 2b). Between 36 and 40 days it loses its spherical shape and starts to elongate, and in another 4 days the young cotyledons are apparent, the embryo being heart-shaped. The embryo elongates until it is nearly as long as the seed cavity. The cotyledons are well developed, and the embryo becomes packed with food reserves (Plate 8, c). The overall picture that is given by this process of seed development is one of a series of phases following closely upon each other. There is a primary phase of rapidly growing integument, with its corollary of rapid increase in the overall size of the seed; the second phase is one of very intense endosperm development, with an accompanying breakdown of the inner integument; this is followed by the sudden access of activity in the embryo which undergoes a period of rapid growth and differentiation, at least partly at the expense of the endosperm. A disruption of one of these phases, would, presumably, disrupt those which succeed it. It is with this pattern of normal development in mind that we should view the development of hybrid seed. Finally, it should be pointed out that in any of the species there is a good deal of variation between seeds in the same capsule, and between different capsules on the same or different plants. These differences are minor, and are not likely to affect the final seed production very much. They are probably caused partly by slight differences in the time of fertilization; one would not expect simultaneous fertilization of all the ovules in a capsule. A small time lag will be enhanced in its effect by the fact that those seeds which are fertilized first will, by virtue of their slightly greater maturity, be able to compete more successfully for the available nutrients. This would not matter where supplies of food are adequate, but might be vital in a case where they are not, or where their intake is, for some reason or another, impeded. Between different plants, more factors come into play. Minor genetical and environmental variations can all be expected to play their part in varying the rate of seed development. (c) Seed development in an interspecific cross There are three possible pairs of interspecific crosses involving the three species with which we are concerned. They are: P. vulgaris x P. veris and its reciprocal P. vulgaris x P. elatior and its reciprocal P. veris x P. elatior and its reciprocal One of these pairs will be described in this paper, and the other two will be discussed in a later account. As Valentine has shown, all set seed and produce a good yield, though not all the seed is viable. The pair of crosses that will be described in this paper is that involving the cowslip, P. veris, and the primrose, P. vulgaris. The most unusual and interesting point concerning these crosses in Primula is that the type of seed produced is dependent on the direction on which the cross is made. The phenomenon is not uncommon in cases where the parents have different chromosome numbers, but it is rarely recorded when they are of the same number. The two types of seed produced have been described by Valentine (1955); suffice it to say that when one

8 Studies in British Primulas VII 309 species (that with the higher 'genetic value') is used as the female parent, so-called 'type A' seed is produced, and in the reciprocal cross the seed is of 'type B'. Type A seed is smaller than normal, but generally well filled with endosperm. The embryos are much smaller than those in normal seed. Germination varies from cross to cross; in some it is very good, in others poor. Type B seed is large, but in most cases there is little or no endosperm, the embryos fail to reach maturity, and germination is poor or nil. (i) P. veris? x P. vulgaris S {cowslip xprimrose) {type A) Integuments. Outer integument i develops in an apparently normal way until about the 28th day, after which it practically ceases to grow or to lay down thickening on its inner side. The resuh is that the cells are not so robust at maturity as those of the species' seeds. The cells of outer integument 2 are rather late in commencing thickening, the thin layer on the inner walls does not become apparent until about 28 days from pollination. Thenceforward thickening is rapid, and is completed by about 50 days. Inner integument i breaks down as rapidly as in normal seeds, and by 24 days only the outermost layer remains. The cell contents of this layer are shrunken; the cells are breaking down in a few more days, and breakdown is complete by the 40th day. This is a little slower than normal. However, the delay in breakdown has caused a gap to remain between the inner integument 2 and outer integument 2, and in this gap are seen the remains of the cells of inner integument i (Plate 8, e). Inner integument 2 shows signs of becoming thicker than normal by 20 days, and as the seed develops it becomes an increasingly prominent layer of tissue (Plate 8, d, e). Its cells do eventually become somewhat compressed, but, unlike the same layer in the normal seed, it does not degenerate into a layer of cell debris. Endosperm. The early stages of endosperm development are similar to those in non-hybrid seed, but it soon becomes evident that there are less nuclei at a given stage in the hybrid. Cell formation, however, often commences by 20 days, and at this time there is sometimes even a second layer in the chalazal region. Some seeds show no signs of having cellular endosperm until much later. By the 24th day it may be up to three or four cells thick, and after another 8 days or so it half fills the seed. There are, scattered irregularly throughout the tissue, occasional large nuclei. Food material starts to accumulate after about 36 days, and after this time the quantity of endosperm increases little. The amount in the mature seed is variable. Even at best, although it may fill the seed, it is loosely packed. Most seeds are not quite full of endosperm. Embryo. Division of the zygote generally does not take place before the 20th day after pollination. Thereafter the embryo is very slow growing. After 32 days it is still a very small sphere, and only after about 40 days does it begin to elongate (Plate 8, e). Elongation is less rapid than in normal seeds. Throughout development the size of the embryo appears to be directly dependent on the amount of endosperm present in the seed, but even in well-developed seeds it is less than half the length of that in a nonhybrid seed. There are found in this cross occasional seeds that never proceed beyond the early stages of development. After about 24 days they are smaller than the rest, their inner integument 2 is very thick, and their embryo-sac contents are degenerate. It is not certain that these have been pollinated; it is possible that they are unfertilized seeds that have been stimulated to some development by diffusion of growth substances from adjacent fertilized seeds.

9 310 S. R. J. WOODELL The seed produced from this cross between P. veris and P. vulgaris with the former as female parent, is smaller than that of the three species. It differs from normal seed in several other characteristics. The outer integument is normal in appearance, but the inner integument is markedly overdeveloped. The later degeneration of inner integument I has left a gap between the outer and inner integuments, and inner integument 2 is very thick, forming an even, solid, slightly compressed layer. The micropyle and chalazal pockets are not in any way occluded, however. The endosperm is less in quantity than that of non-hybrid seeds, and it contains occasional aberrant nuclei. The embryo is rather small. Not all seeds reach maturity. In Valentine's experiments, the germination of seed from this cross varied from o to 37.5% and averaged about 14%. (ii) P. vulgaris $ X P. veris (^ {primrose x cowslip) (type B) Integttments. Outer integument i cells grow rapidly in size from the start, but their walls are very thin, and only after about 32 days does any sign of thickening appear on their inner walls. The amount of thickening is slight, and at maturity the layer consists of large thin-walled cells, irregular and flimsy. Outer integument 2 is equally slow in development. The cells become irregular in shape and arrangement, and the characteristic cup-shaped thickening that appears early in the cells of this layer in normal seeds does not commence until about 36 days after pollination. Here too, it does not amount to much, and never more than about half fills the cells. The result is that the outer integument is a very flimsy layer in the mature seed. The two layers of the inner integument degenerate rapidly soon after pollination. By the 28th day degeneration of these layers is nearly complete, except in the chalazal region, where there is a persistent group of inner integument 2 cells. In a welldeveloped seed (Plate 8, g) this is still apparent, but the inner integument in the rest of the seed has practically disappeared. Endosperm. Endosperm development is variable, but even in the most advanced seeds it is small in quantity and abnormal in appearance (Plate 8, f, g). In some cases cell formation begins at about the 28th day, but frequently it is delayed beyond this time. The endosperm grows very slowly after this, and has not been observed to fill more than about one-third of the seed (Plate 8, g). It is abnormal in appearance; the cells are irregular in size, shape and arrangement; tbe nuclei are abnormal in appearance and variable in size; vacuoles are sometimes seen in the cytoplasm before cell formation commences. In many seeds the endosperm starts at some stage to degenerate, often leaving a formless mass of unidentifiable seed contents. In no case has a well-developed, normal-looking endosperm been seen in this cross. Embryo. The embryo appears to have its fortunes linked very closely to those of the endosperm. In the most favourable circumstances it remains small for a long time and never passes beyond the spherical stage. In one case it was observed at 32 days to consist of about sixty small, dense cells. As the seeds develop further the embryo remains as a dark spherical object, with no apparent differentiation (Plate 8, f). The seed from this cross is thus highly abnormal. The outer integument remains virtually unthickened, even in the mature seed. Degeneration of the inner integument is even more rapid and complete than in non-hybrid seed. The endosperm is small in quantity, often non-cellular, and abnormal in appearance, and it often degenerates fairly early in the course of seed growth. The embryo remains small and undifl^erentiated, never exceeding about one-flfth of the normal embryo in length (see graph of embryo

10 Studies in British Primttlas VII 311 length. Fig. 2d). In Valentine's experiments, no germination was obtained of seeds from this cross. DISCUSSION The development of the two types of seed, type A and type B from this pair of crosses, shows remarkable differences, reflecting those differences that Valentine pointed out in gross morphology, seed set and germination. There are still two reciprocal pairs of crosses to be described, those between P. vulgaris and P. elatior, and between P. veris and P. elatior. It would be premature to attempt to put forward an hypothesis explaining the seed failure in Primula before discussing the developmental course in these pairs of crosses; detailed discussion will therefore be left to the next paper. There are some points that are worth eonsidering here in the light of what has already been described. The development of the three species follows a pattern that can be considered as typical for the Primulaceae (Dahlgren, 1916). There has been, in the past, some controversy over the possible function of the innermost layer of the inner integument (inner integument 2). This layer of cells, with its deposits of tannin, is in intimate contact with the embryo sac from an early stage, since the nucellus is ephemeral. The majority of workers have ascribed a nutritive function to the tissue. Dahlgren (1916) has pointed out that the position of the tissue, regularity of the cells, richness of cytoplasm and large nuclei suggest that this is its role. However, it is dangerous to infer physiological function of cells from their morphological appearance. Hegelmaier (1889) argued that the tissue acted as a protection against 'toxic chemical influences' from the surrounding degenerating tissues. Schmid (1906) supported this view, but Magnus (1913), discovering that in some families the cells had a cuticle, suggested that the layer hindered loss of water and nutrients to the surrounding tissues. The early degeneration of this layer would seem to suggest that it does not serve a very important function in seed development. I think it unlikely that the cells of this layer serve a direct nutrient function. It is possible that in some way it acts as the region through which nutrients are passed from the placenta to the embryo sac. However, the vascular supply to the seed appears to end behind the chalazal pocket, where this layer is absent, and during normal development suflicient food supplies may reach the embryo sac from this area alone. If this is so then this layer of cells will not act as a transfer region. It seems likely that when these cells degenerate, as they do fairly early in development, their contents are absorbed and serve as a source of nutrient for the young endosperm but this can hardly be said to be their function. The possible role of this layer is of interest, since it is the layer of the integument that appears to be most affected in the hybrid seeds. Its hypertrophy in the type A seed, and its almost complete disappearance in the type B seed are remarkable, and would seem to indicate that it plays more than a passive role in seed growth. Just what this role is may become clearer in the light of seed development in the remaining crosses. It may be noted here that hypertrophy of this layer has been noticed by several workers in other genera. Brink and Cooper (1945) found gross hypertrophy in crosses between tetraploid and diploid races of Lycopersicon pimpinelufolium. Fagerlind (1948) noted a similar phenomenon in Rosa. Sansome, Satina and Blakeslee (1942) and Sachet (1948) have described endothelial hypertrophy in Datura. In every case, however, it is accompanied by rapid breakdown of the endosperm. In Priniida, at least in the case described here, hypertrophy of the inner integument 2 is not accompanied by such breakdown;

11 312 S. R. J. WOODELL indeed the opposite is the case; when tbe inner integument 2 degenerates even more rapidly than in normal seeds, the endosperm is also degenerate. This point will be discussed further after the other crosses bave been described. SUMMARY 1. The development of seed of the three species P. vulgaris, P. veris and P. elatior is essentially similar, though minor differences in timing and seed size occur. The cells of the outer integument, two cells thick, are progressively thickened and form the hard coat of the mature seed. The inner integument, several cells thick in the unfertilized ovule, steadily degenerates as the seed develops and in the mature seed remains only as a layer of compressed cell debris. The persistent endosperm develops slowly at first, but as the growth of the integument slows down it enters a period of rapid growth, filling the entire seed. This is followed by very rapid development of the embryo, which has increased in size very little for several weeks. At maturity the endosperm and embryo are packed with food materials. 2. In the reciprocal crosses P. veris '4 / P. vulgaris S and P. vulgaris '4 A P. veris S the course of development is very different. In the former the outer integument is normal, but the inner integuments' innermost layer becomes thickened and remains as a prominent layer in the mature seed. The endosperm is reduced in quantity, but is fairly normal in appearance. The embryo is small and later in development. The seeds are small. In the reciprocal, the seeds are large, but the outer integument is highly abnormal, very little thickening taking place. The inner integument degenerates rapidly and almost completely disappears. The endosperm rarely develops very far, and at best is only small in quantity. It is irregular in appearance; the cells being variable in shape and size, and the nuclei often being abnormally large. The embryo is very small and never shows any sign of differentiation. Degeneration of the embryo sac contents is often apparent. 3. The function of the inner layer of the inner integument is discussed. It is possible that it serves some kind of passive role in the nutrition of the embryo-sac contents. Its abnormal development in hybrid seeds is possibly of some significance in the problem of seed failure. ACKNOWLEDGMENTS My thanks are due to Professor D. H. Valentine, under whose supervision this work was carried out, and who has offered valuable criticism of this manuscript. The Department of Scientific and Industrial Research financed this investigation and their support is duly acknowledged. REFERENCES COOPER, D. C. & BRINK, R. A. (ii;)45). Seed collapse following matings between diploid and tetraploid races of Lycopersicon pimpinellifolium. Genetics, 30, 376. DAHLGREN, K. V. OSSIAN (1916). Zytologische und Embryologische Studien iiber die reihen Primulales und Plumbaginales, Kungl. Svensk Vetenskap. Handlingar, 56, i. DECROCK, E. (1901). Anatomie des Primulacees. Ann. Sci. Nat. Bot., 3, 2. FAGERLIND, F. (1948). Compatibility, Eu- and Pseudo-incompatibility in the genus Rosa. Acta. Hort. Berg., IS, I. HECELMAIER, F. (1889). Uber den Keimsack einiger Compositen und dessen LTmhullung. Bot. Zeit., 47, 8a5. MAGNtjs, W. (1913). Die atypiscbe Embryonalentwicklung der Podostemaceen. Flora, 105, 275. MAHESHWARt, P. (1939). Recent advances in microtechtiique IL Cytologia, io, 270. SACHET M, (11)48). Fertilization in six incompatible species crosses of Datura. Avi. y. Bot., 35, 302.

12 THE NE;W PHYTOLOGIST, 59, 3 PLATE 8 (Facing p. 313)

13 Studies in British Primulas VII 313 SOME, E. R., SATINA, S. & BLAKESLEE, A. F. (1942).^ Disintegration of ovults in tetraploid-diploid and in incompatible species crosses in Datura. Bull. Torrey Bot. Club, 69, 405. ScHMiD, E. (1906). Beitrage die Entwicklungsgeschichte der Scrophulariaceae. Beih. z. Bot. Centralblatt, 20, 175. VALENTINE, D. H. (1953). Evolutionary aspects of species differences in Primula. Svmp. Soc. Exp. Biol., VALENTINE, D. H. (1954). Seed incompatibility. Proc. Sth Internat. Bot. Congr., Section 9, 170. V^ALENTINE, D. H. (1955). Studies in British Primula.': tv. Hybridization between Primula vulgaris Huds. and P. I'eris L. NeiL- PhytoL, 54, 70. EXPLANATION OF PLATE 8 Plate S, a-g. Longitudinal sections of developing seeds of P. veris (a-c); P. veris.? - P. vulgaris cj (d, e) and P. vulgaris $ X P. veris S (f, g). a. 24 days: Cell walls forming in endosperm, thickening of cells of outer integument 2 commencing. b. 36 days: Inner integument compressed between endosperm and outer integument. Note that at this late stage the embryo is still small. c. 60 days: Seed nearly mature. Endosperm cells packed with food reserves. This section passes mainly through one of the cotyledons, but the other is visible. d. 36 days: Inner integument i persists as a distinct layer (compare Fig. i). Inner integument 2.IS a thick, well-defined layer. Thickening is commencing in cells of outer integument i. Note small size of seed, shrunken endosperm. e. 48 days: Note the even thickness of inner integument 2. Note also that the persistence of inner integument i until the outer integument was thickened has left a gap between outer integument and thick inner integument 2. Endosperm does not fill the seed completely, but food material is contained in the cells. Embryo healthy in appearance. f. 40 days: One of the best developed seeds from this cross. Endosperm small in quantity, irregular and abnormal in appearance. Embryo minute. Note the lack of thickening of the integuments. g. 48 days: F,ndosperm semi-degenerate. Seed collapse and almost total breakdown of the seed contents follow. Figs, a, c 43; b, g 27: d, e, f 47.

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