A PRELIMINARY NOTE ON THE POLLEN
DEVELOPMENT OF LATHYRUS ODORATUS*
BY JOAN LATTER, B.Sc,
CONTENTS.
1. I n t r o d u c t o r y . . . . 199 2. M a t e r i a l a n d M e t h o d s . . . 199 3. D e s c r i p t i o n 2 0 0
P A O l
5. Tapetal Development . . . 207 6. Sterility 207 7. Summary 209 4. Meiosis 202
1. Introductory.
A CYTOLOGICAL study of the pollen development of Lathyrus
odoratus was primarily undertaken to determine whether this
form afforded any evidence which might serve as a physical basis of crossing over. From the work of Bateson and Punnett, the phenomenon is known to occur genetically in
L. odoratus, and may be explained by the assumption
that at some period in the reduction divisions interchange of chromatin occurs between the homologous paternal and maternal chromosomes.
The general fixation of the material proved to be exceptionally good. Hence after particular attention had been given to points bearing on the basis of crossing over, the work was extended to a fuller study of all stages in the development of the pollen.
It is proposed in this paper to state briefly the results which have been obtained since the study was started in October 1923, and to point out the chief lines of interest along which this work is being carried further.
2. Material and Methods.
The material was of Cretin stock originally obtained from Prof. R. C. Punnett. Seeds from both light- and dark-axilled plants with normal flowers were sown in Regent's Park Botanic
Gardens in June 1923 and buds collected during September and October.
At the first collection of material two fixing fluids were used : 1 per cent, chrom-acetic solution, and Allen's modification of Bouin's fluid (75 c.c. picric acid-saturated aqueous solution, 25 c.c. commercial formalin, 5 c.c. glacial acetic acid, 2 gms. urea crystals, 1.5 gms. chromic acid). The latter was found to give very good results for Lathyrus, and was subsequently adopted to the exclusion of other fixatives. The material was embedded in paraffin, cut at 12 M and stained with iron-alum-haematoxylin.
3. Description.
The resting archesporial nuclei present a faintly staining very delicate reticulum, which appears rather as a mass of fine granules than as a continuous network of threads. In the centre of this is a single large spherical deeply-staining nucleolus.
The first indication of approaching prophase is the contraction of the granular mass from all parts of the nuclear membrane, leaving a perfectly clear area surrounding the mass and the embedded nucleolus. The formation of this clear zone was found by measurements to be due to con-traction of the granular material and not to expansion of the nuclear membrane.
Pollen Development of Lathyrus Odoratus
this stage affords ample opportunity for new arrangements of chromatin particles to occur. In fact it seems remarkable that there should be any constant reappearance of linked characters, unless some influence other than chance is controlling this linear arrangement. It is possible that in a gel the spacial relationships of particles might be maintained. In order, therefore, to bring the facts into line with genetical observations, it must be assumed either that some mechanism is at work which controls the linear arrangement of the granules during thread formation, or more probably, that the entirely granular appearance is due to differential staining, the granules in reality being distributed on a linin reticulum.
Meanwhile changes take place both in position and appearance of the nucleolus. This large central spherical body gradually passes to the periphery of the reticulum, becoming more oval in form as it does so. The whole reticulate mass approaches the nuclear membrane on the side towards which the nucleolus is travelling. When in contact with the nuclear membrane, the nucleolus becomes flattened along it on one side and assumes an elliptical appearance.
Certain nucleoli exhibit points of special interest about this time. Amoeboid forms were observed, but seem to be only of rare occurrence. From these, dark staining processes are put out, usually tapering towards the end where they penetrate the reticulum, while the central part of the nucleolus appears vacuolate. In no case was a definite continuity of nucleolar process and reticulum thread observed at this stage; but it seems possible that actual transference of chromatin material is taking place from the nucleolus, particularly as in these stages the thread-like character is becoming qu'te distinct. A few instances of nucleolar "budding" were also observed, but the ultimate fate of the material thus budded off has not yet been ascertained. It is hoped that further observations will throw light on the functions of the nucleolus at this stage and on its subsequent history.
It may be suggested here that if the nucleolus is a reservoir of chromatin material during the resting stages, then in order that the individuality of the chromosomes may
be preserved from generation to generation it seems that the important or "factor bearing" part of the chromosomes is the linin rather than the chromatin. The material passing from the nucleolus to the thread might be considered to alter the acidity of the thread substance, and so cause it to stain as "chromatin." On this conception the core of linin would contain the genes, while the material derived from the nucleolus would surround and overlay this and so thicken the thread. Otherwise it must be assumed that there is some orderly arrangement of chromatin in the nucleolus: that it is absorbed from the chromosomes in regular succession at the reconstitution of daughter nuclei, and extruded again in a similar way. It is difficult to see how such a fluid substance in the nucleolus could serve as the physical basis of inherit-ance of Mendelian differences, with crossing over of groups of genes, as is now known to occur in various plants and animals.
4. Meiosis.
The now deeply staining chromatin thread becomes tightly coiled in the synizetic knot at one side of the nuclear cavity. In every case one or two clear connecting strands remain in contact with the nucleolus, and stretch from it to the knot. The thread at this stage is a. very fine structure, later becoming conspicuously thickened. Possibly therefore this constant connection with the nucleolus is of importance in the transference of chromatin material to the spireme. The nucleolus is very deeply staining at this time, thus making it impossible to determine whether the connecting threads actually penetrate it, or whether they are merely closely applied to the surface. It is usually an elliptical body in contact with the nuclear membrane. The thread appears to be a perfectly continuous structure at this stage. In the synizetic knot the continuity is hard to trace, but no free ends are distinguishable.
Pollen Development of Lathyrus Odoratus
established. Possibly they are parallel stages in two independent series. Usually the knot leaves its peripheral position and becomes central in the nuclear cavity. The thread then forms other definite loops which radiate out from the central mass of material. The centre is often occupied by a second nucleolus, a spherical body, while the one against the nuclear membrane has assumed the typical thin crescent shape. This central nucleolus probably originates from the elliptical one, as spherical "buds" were seen separating off from the large nucleolus during early synizetic stages. It appears that the looped thread is con-tinuous at this stage, but a definite statement cannot be made on this point, owing to the presence of a deeply staining mass of material at the centre through which the thread cannot be traced.
Another appearance which occasional nuclei assume after synizesis is as follows. The knot retains its peripheral position, and the thread is loosened into definite loops which reach across the nuclear cavity and are polarised with respect to the nucleolus. The apices of the loops lie free from the latter structure, which seems to be definitely penetrated by the proximal ends. The number of ends entering the nucleolus was in one instance found to be thirteen or fourteen. This indicates that the number of independent loops is probably seven. The thread is so delicate at this stage, and the loops so twisted amongst one another that an exact count is rendered difficult. In this arrangement it is uncertain whether the loops are entirely independent of one another, the arms of each being joined at the distal ends while the proximal ends enter the nucleolus, or whether the thread is still a continuous structure. Probably the loops are free from one another, as their independence has been observed at this stage in other plants. This point cannot be determined here, however, on account of the great stain-ing capacity of the nucleolus, which obscures the course of the thread in this region.
It seems that this stage is followed by a rearrangement of the loops causing them to radiate out from the .centre of the nucleus. No early diakinetic stages were seen to follow
directly on the polarised condition. It is hoped that further observations will show the relation between this and the more typical radiating arrangement of loops. So far, transition stages from one to the other have not been found.
When the thread has loosened out from synizesis, it soon becomes evident that seven loops are persisting and becoming thickened, probably at the expense of the others, which appear to be drawn into the centre. This number is of importance as it corresponds with that of the haploid chromosomes in Lathyrus and is obvious while the spireme is still a delicate structure. The split character of the thread at this stage has been observed in a few cases, the split corresponding to that seen in the V-shaped chromosomes at the heterotypic anaphase, and which is finally consummated on the homotypic spindle.
In the early stages of this looping, an indefinite tangle of spireme remains at the centre, A true nucleolus may be present also. In many cases it was impossible to determine whether the central mass was composed of spireme and nucleolus, or of spireme only. An extremely dense aggregation of threads would when stained appear as a solid structure. As the seven persisting loops become more definite this tangle in the majority of nuclei gradually disappears, probably being used up in thickening the surrounding threads. It has been suggested by Professor Gates that the term " broxonema" be applied to this stage of radiating loops (ftpoxo? = a noose or slip knot). It corresponds to the stage usually known as the second contraction.
In later stages of this looped condition, it can be seen that in a certain number of loops the arms or two sides become intimately twisted round one another for a considerable length toward the proximal end. The general appearance of nuclei in this condition is that of a seven-spoked wheel. The stages intervening here before diakinesis are definite and have been clearly followed. From them it is obvious that each loop represents two homologous chromosomes, one maternal and one paternal in origin, united end to end at the apex of the loop. This arrangement combined with the twisting of the two arms about each other, undoubtedly affords opportunity for interchange of segments between the two chromosomes
Pollen Development of La thy r us Odoratus
of each pair at points where the threads cross, and gives a possible physical basis for crossing over in conjunction with telosynapsis. Though a certain amount of thickening and contraction has taken place in the thread up to this stage, it is still a delicate and plastic structure.
In the earlier stages of the broxonema the twisting of the two arms of each loop about each other appears to be the usual condition. Condensation and contraction of the chromatin then takes place, the loops become further thickened and the twisting now disappears. This presumably happens usually by untwisting, but if the thread breaks and rejoins at any cross this would lead to crossing over. Further condensation of the loops leads gradually to diakinesis. The homologous chromosomes still remain united at the distal end. The thickened chromosomes at first appear in a more or less continuous chain, rough and irregular in outline, indicating that condensation of chromatin is not complete. Thin connecting strands may be seen joining the ends of adjacent pairs of chromosomes. Transition stages between this and typical diakinesis are observed, in which the chromosome pairs become entirely detached and separated, the bivalents assuming different forms, and lying free periph-erally in the nuclear cavity. The intimate contact of the two arms of each bivalent is brought about in different ways, but no indication of a split was seen which could cause crossing over at this time. The chromatin is very condensed at this stage, thus rendering exchange of material far less probable than in the thin plastic thread.
The nucleolus has meanwhile become vacuolate or fragmented, and in late diakinesis frequently no definite trace of it is found. At this time also the nuclear membrane disappears. Sometimes fragments of the nucleolus remain after the membrane has vanished. Occasionally, however, nuclei were seen with definite nuclear membranes, but no trace of any nucleolar material.
Extremely few cases of multipolar spindles have been found. This may be due to the fact that the multipolar stage is rapidly passed over and the normal bipolar spindle quickly formed. It may in part be due to the treatment, as the
definite multipolar spindles were found only in material fixed in chrom-acetic fluid. On the cytoplasm around the spindle numerous spherical lightly staining bodies are present. These would appear to be fragments of nucleolus, but it is not clear what forces could be at work to bring about such a scattered distribution. Also the total volume of these bodies seems greatly to exceed that of the original nucleolus.
Observations on the actual reduction divisions have yielded no results worthy of special comment, and will be briefly outlined.
At metaphase the chromosomes are very compact con-densed bodies. Bivalency is usually apparent, but there is no indication of the homotypic split. This becomes evident in anaphase, when seven V-shaped chromosomes travel to each pole. There is no definite organisation of resting daughter nuclei after the heterotypic division, though occasion-ally connecting strands of deeply staining material were observed between adjacent chromosomes.
Typical interkinesis stages are found in which the univalent chromosomes have become more elongated. The halves remain in contact with one another only at the centre—the arms swinging out at right angles causing the chromosomes to assume the form of a + . They take up the usual peripheral position in the nuclear cavity.
The homotypic spindles are usually at right angles or parallel to one another. The anaphase is apparently passed through rapidly as very few instances were observed.
The homotypic telophase shows the characteristic densely grouped deeply staining chromosomes, usually three groups only seen in one plane, due to tetrahedral arrangement. At this stage occasional signs of an evanescent cell plate formation were observed. No spindle fibres remain between the nuclei, though the cytoplasm is arranged in regular striations, similar in arrangement to spindle fibres. Across these striated areas are deeply staining zones, corresponding in position to the equatorial plate of the original spindle. The nature of these zones has not yet been determined. Apparently they are temporary structures as at later stages no traces of them are visible.
Pollen Development of Lathyrus Odoratus
The daughter nuclei enlarge, the chromosomes becoming much elongated and apparently anastomosing one with another. The staining power becomes less, and three to four nucleoli arise in each nucleus, presumably by absorption of chromatin from the chromosomes.
The only feature in the further development of the pollen worthy of special note is that of cell wall formation. The study of this phenomenon has not yet yielded any very satisfactory results. The temporary appearance of cell-plates has been mentioned above. Later, at the time when the chromosomes lose their definite outline and anastomose with one another, there are no indications of constrictions in the cytoplasm, or of plates. A few cases of furrowing were observed, but in these the daughter nuclei were of abnormal appearance, small and shrunken, with the chromatin present merely as an indefinite mass. There appeared to be a space between the mother cell wall and the contracted cytoplasm. Further study will be made on this point if similar stages are found in normal material, and staining tests applied to determine whether this area is in reality a space, or whether it is occupied by cell wall material, which pushes its way into the cytoplasm, causing the appearance of a narrow furrow.
5. Tapetal Development.
The study of the tapetum has at present revealed no multinuclear stage in the individual cells. They remain uninucleate throughout the whole process of pollen develop-ment, with the exception of the very occasional occurrence of binucleate cells during the synaptic stages' of the pollen mother cells.
Other curious features of the tapetum are the great elongation of its cells toward the pollen mother cells, and occasional outgrowths of tissue into the pollen sac. These latter were in one case seen to grow right across the loculus, dividing it into two parts.
6. Sterility.
At various stages in the development of the pollen certain individual cells may be seen displaying abnormalities or
complete disintegration. The chromosomes may become very pale-staining and vacuolate or densely clumped, these appear-ances being assumed at different stages of the homotypic and heterotypic divisions. Another abnormality is the distribution throughout the loculus of dark staining droplets. These spherical bodies seem to be derived from the wall substance of the pollen mother cells, as in most cases a dense aggre-gation of droplets replaces the walls. In such loculi the tapetal cells also are replaced by dense masses of these dark staining bodies.
In later stages of development complete sterility has been noted. All four cells of a tetrad may become shrunken and have no distinct nuclei, and remain enclosed in a thick mother-cell wall. Again, one or two members only of a tetrad may disintegrate, the others retaining completely normal appearance. Sometimes no sign of sterility is apparent till the pollen grains are fully formed: they then become irregular in shape and much shrunken.
In comparing the sterility of genetically "fertile" plants such as those described above with that of plants genetically "sterile" for pollen, three conditions have been noticed in the latter which are not observed in the former type. These are, firstly, bipartition of the pollen mother cells after heterotypic division ; secondly, fragmentation of the chromosomes; and thirdly, encroachment of the tapetum to the centre of the loculus.
In conclusion it may be stated that Lathyrus odoratus conforms essentially to the telosynaptic method of chromo-some pairing, and that nevertheless a possible basis for crossing over has been observed in the post-synaptic thread. Crossing over was at one time thought to be associated with parasynapsis only, but here we have evidence of its occurring in a typical telosynaptic form.
It is hoped that further study will reveal the function and ultimate fate of the nucleolus, the nature of the temporary cell plates and details of cell wall formation. A knowledge of these facts, together with a fuller understanding of tapetal development and the causes of sterility, is necessary before an account of the pollen development in Lathyrus odoratus
Pollen Development of Lathyrus Odoratus
can be considered complete. A fuller account of this work will be published later.
This work is being carried out at King's College, London, while in receipt of a grant from the Department of Scientific and Industrial Research.
The problem was suggested to me by Professor Gates, and I wish to take this opportunity of thanking him for his interest and helpful criticism during the investigation.
7. Summary.
1. In this paper the main points in the pollen development of Lathyrus odoratus are briefly described.
At the beginning of the meiotic phase the reticulum contracts from the nuclear membrane and exhibits an entirely granular appearance.
2. During thread formation, occasional amoeboid nucleoli are observed. Nucleolar "budding" is also seen.
3. Connecting strands are constantly found between the synizetic knot and the nucleolus.
4. The thread at all stages appears usually to be a continuous structure.
5. The synizetic knot is followed by a stage in which the thread is thrown into seven definite loops which radiate out from the centre of the nucleus. For this stage, which has frequently been called the second contraction stage in cyto-logical literature, the name broxonema is proposed.
6. The haploid number of chromosomes in Lathyrus
odoratus is seven. Each loop represents one pair of
homo-logous chromosomes joined distally end to end.
7. The arms of each loop are twisted round one another at one period. This affords opportunity for exchange of segments of chromosomes, and gives a possible physical basis for crossing over in a telosynaptic form.
8. These stages are followed by typical diakinesis and heterotypic divisions.
9. During homotypic telophase evanescent cell plates occur between the daughter nuclei. Later, the cell walls are formed by furrowing.
10. The tapetum remains uninucleate throughout.
