706 [Vol. 34, 162. Somatic Syn.desis in Daphne odora.11*' The Chromosome Behavior in Meiosis

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1 706 [Vol. 34, 162. Somatic Syn.desis in Daphne odora.11*' The Chromosome Behavior in Meiosis By Tosisuke HIRAOKA Botanical Institute, College of Science, Kyoto University (Comm. by Y. KUWADA, M.J.A., Dec. 12, 1958) In the first paper, the behavior in mitosis of the chromosomes in somatic syndesis in Daphne odora was reported (Hiraoka, 1958). In the present paper, the behavior in meiosis will be reported. Material and methods. The observations were made in sporogenous cells, pollen mother cells, and pollen grains by the methods of acetocarmine smear and paraffin section preparation (Hiraoka, 1958). In some cases, fresh pollen mother cells were observed in situ in the anther with liquid paraffin as mounting medium. Observation. 1) Premeiotic mitoses and the last interphase just preceding meiosis. In the premeiotic mitoses including the last one, the chromosome behavior is similar to that observed in the ordinary somatic mitosis. The somatic syndesis takes place in anaphase and the complete or incomplete liberation from the syndesis is observed in prophase (cf. Hiraoka, 1958). When the last interphase approaches, the mitotic process proceeds synchronously in all cells in the loculus. In interphasic nuclei, the chromosomal bodies are found distributed along the nuclear periphery. The chromosomal bodies are somewhat irregular in outline and show a vacuolated structure. The number of these bodies observed in each nucleus varies to some extent, but the number of highest frequency is 14, a number which is the haploid (Table I). One or two nucleoli of different sizes are found in the nucleus. The nucleolus is spherical in shape and frequently shows a vacuolated structure (Fig. 1). Table I. Last premeiotic interphase 2) Meiosis. When the meiosis commences, the chromosomal bodies become more irregular in outline than in the interphase. In some cases they show several fine threads projecting out of their surface. The vacuolated structure of the bodies which is observed in the in- *) Studies on the mechanism of nuclear division, IV. This investigation is supported by a Grant from the " Jimbun Kagaku Kenkyuhi " of the Ministry of Education.

2 No. 10] Somatic Syndesis in Daphne odora. II 707 terphase is now transformed into a thread structure irregularly entangled. As the thread structure is more distinctly observed in the bodies, the bodies become more indistinct in their outline (chromonema stage, Fig. 2). At this stage, some of the chromosomal bodies may join together with their projecting threads, but in the majority of cases they maintain their individuality. In the next stage the irregular outline of the bodies is rendered smooth again, and the chromonema threads which appeared to form the reticular structure are transformed into a form of more or less regular spiral or zig-zag. In some favourable cases, two spirals which lie side by side are discriminable in the body (Fig. 3). As the spirality becomes distinct in the bodies, the bodies are stained more intensely with the nuclear stains. These bodies are gradually condensed, finally to form the densely stainable massive bodies or prochromosomes (prochromosome stage, Hiraoka, 1941, Fig. 4). In the course of the prochromosome formation, a tendency of these bodies of gathering on one side of the nucleus is recognizable. These prochromosomes are in size nearly equal to or somewhat smaller than the chromosomal bodies in the interphase. Their number is estimated at 14 on the fact that this number occurs most frequently (Table II). The prochromosomes are, therefore, in reality, double chromosomes in this case of somatic syndesis taking place in the premeiotic mitosis. In this stage the part of the nuclear membrane on the side opposite to the localized chromosome group is often found separated from the surrounding cytoplasm, indicating that the pollen mother cells are extremely sensitive to the fixative. In the following stage, the prochromosomes become loosened, and very frequently, two loosely coiled spirals, sometimes twisting around and sometimes parallel with each other, are observable coming out of the prochromosome body (Fig. 5). The formation of the prochromosomes and this unravelling process do not always take place synchronously in all the prochromosomes in the nucleus, so that some of the prochromosomes remain massive, while others have been unravelled into two spirals (unravelling stage, Hiraoka, 1941). The two spirals are then drawn out from a loosely coiled state into two uncoiled threads, and the threads of a pair become dificultly distinguishable from the threads of other pairs. To speak in other words, it becomes hardly recognizable which two are the original two in pair. The leptotene nucleus is thus formed (Fig 6). Table II. Prochromosome stage

3 708 T. EIRAOKA [Vol. 34, In this plant, in the stages from late leptotene to early pachytene, it is very difficult to obtain well fixed figures. In the majority of cases, the so-called extrusion of nuclear substances occurs. It takes place in regular order from the top to the base of the anther, while in the region near the basal part of the anther the pollen mother cells usually show no extrusion at all. When living pollen mother Fig. 1. Interphase just preceding meiosis. Fig. 2. Chromonema stage. Fig 3. Early prochromosome stage. Fig. 4. Prochromosome stage. Fig. 5. Unravelling stage. Fig. 6. Leptotene. Fig. 7. Synaptic stage. Fig. 8. Pachytene. Fig. 9. Strepsitene. Fig. 10. Diakinesis. Fig metaphase. Fig. 12. Interkinesis. All from paraffin section preparations x

4 No. 10] Somatic Syndesis in Daphne odora. II 709 cells in these stages are observed in situ in the anther, no such extrusion is recognizable to occur. This result of observation points to the conclusion that the extrusion phenomenon is an artifact caused by fixation. Therefore, the following description is based on the result of observation of the pollen mother cells in the basal part of the anther where no extrusion takes place. In the leptotene the chromosome threads come to gather in some degree to form a mass (the so-called synizesis, Fig. 6). The chromosome threads are distributed evenly in the mass, but later they show some tendency of parallel arrangement (Fig. 7). Some of them are found looped out of the mass, and occasionally some indications of side by side pairing of two threads are recognizable. The threads then become distinctly thicker, and as the contraction en masse is rendered looser, the thick spiremes appearing to be of the structure of a series of disks are found distributed evenly in the nucleus, the pachytene stage (Fig. 8). In the diplotene, the duality of the spiremes is not clearly discernible along the whole length, but recognizable in places. In the strepsitene, the spiremes are much thicker and come together, to some extent, to locate in the central region of the nucleus (Fig. 9). Then the stage diakinesis follows, at which 14 bivalents are found distributed in the peripheral region of the nucleus. One or two of the bivalents are of ring shape and the rests are of dumbbel shape (Fig. 10). After the nuclear membrane and the nucleolus have disappeared, these bivalent chromosomes form a nuclear plate (Fig. 11). In acetocarmine preparations, there are some indications of the spiral structure of the chromosomes. The disjunction behavior of chromosomes at anaphase is not always normal, in some cases there occurring non-disjunction or chromosome lagging. The anaphasic chromosome pairing which is observed in somatic mitosis is not recognizable to occur in this case of meiosis. In the telophase, the polarized orientation of the chromosomes disappears rapidly, and in the early telophase the chromosomes are evenly distributed along the periphery of the nucleus, no trace of the polarization being recognizable at all. One or two nucleoli are formed in symmetrical positions in the sister nuclei. When the telophasic internal change takes place in the chromosomes, the chromosomes become irregular in outline, and the internal structure is rendered more or less looser, but each chromosome remains being individually distinguishable from others even in interkinetic nuclei (Fig. 12). The chromosome counting in the daughter nuclei in this stage shows that the number varies to some extent in accordance with the observed fact that here in the anaphase the non-disjunction and chromosome lagging occasionally occur (Table III). The high sterility of this plant may originate from these abnormalities in

5 710 T. HIRAOKA [Vol. 34, chromosome behavior (Osawa, 1913). A comparison of Table III with Table IV (II-metaphase) shows that the chromosomal elements appear in haploid number more frequently in interkinesis (61.10/c) than in second metaphase (49.0 o ). This fact may be taken as due to the fact that some non-disjunction chromosomes remain still in paired state during interkinesis. Table III. Interkinesis In the second metaphase the chromosomes are spherical or more or less ellipsoidal, and show no marked difference in shape from the chromosomes in the first anaphase. The numbers of chromosomes obtained in the second metaphase are given in Table IV, which also vary to some extent. In the anaphase, the chromosomes, logitudinally divided, are separated to poles. No chromosome pairing was observed in the anaphase. However, in view of the facts that the chromosomes of second metaphase are free from pairing, and that the haploid number is found more frequently in pollen nuclei (61.4%) than in second metaphase plates (49.0%), it seems probable that pairing might occur at the second anaphase between certain chromosomes, as a result of introduction of some extra chromosomes by non-disjunction. When the chromosomes reach the pole, their polarized orientation disappears rapidly. In the tetrad nuclei thus formed, the chromosomes are evenly distributed in peripheral regions. Table IV. Second metaphase 3) Pollen grains. The mature pollen grains vary in size. A small grain may appear to have only 10 chromosomal bodies contained in its nucleus, but in the majority of cases where pollen grains are of large sizes the number of these bodies amounts to 14, the haploid number, a result showing the free state of chromosomes from pairing. The statistical data are given in Table V. Table V. Pollen grains

6 No. 10) Somatic Syndesis in Daphne odora. II. 711 Conclusion.. While in some Diptera (Stevens, 1910; Metz and Nonidez, 1921; Metz, 1927; and others) it has been reported that the somatic syndesis in the last premeiotic ana- and telophase is maintained throughout the growth period, the synaptic stage being in these cases considered ineffective (Culex) or observably completely omitted (Asilus and Drosophila), in Daphne the result of observation appears to show that the pairing at the last premeiotic anaphase is cancelled at leptotene stage, and that in the following stage, the meiotic syndesis takes place afresh between homologous chromosomes. In view of the fact, however, that in leptotene and zygotene stages the nucleus is in a highly hydrated state, in different degrees in different species (Hiraoka,it rather seems more likely that the cancellation of pairing at leptotene stage in Daphne is a mere result from nuclear hydration in this stage or a temporary phenomenon of seeming dispairing due to swelling of chromosome matrix (old matrix) by hydration, not involving in it any fundamental change in synaptic activity. While in mitosis chromosome pairing takes place in anaphase, in meiosis it does not, if we disregard a possibility of occurrence of non-disjunction. However, we observed some cases of non-disjunction, and the results of chromosome counting suggest that while in the first anaphase no active pairing occurs there is a certain possibility of pairing in the second anaphase taking place between the chromosomes originating from non-disjunction. The non-disjunction is an abnormal behavior, however, and we should like to conclude that in Daphne, the behavior in meiosis of chromosomes in somatic syndesis is conforming as in mitosis with Darlington's view on chromosome association (Darlington, 1937). References Darlington, C. D. (1937): Recent Advances in Cytology, London. Hiraoka, T. (1941): Cytologia, 11. (1952) : Cytologia, 17. (1958): Proc. Japan Acad., 34, 700. Metz, C. W. (1927): Ztschr. Zellf. mikr. Anat., 4. Metz, C. W., and Nonidez, J. F. (1921): Jour. Exp. Zool., 32, Osawa, I. (1913): Jour. Agr. Imp. Univ. Tokyo, 4. Stevens, N. M. (1910): Jour. Exp. Zool., 8.

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