Cytologia 51: 731-736, 1986

Cytoplasmic Channels and Chromatin Migration in Mulberry

R. C. Verma, A. Sarkar and B. C. Dos1

Central Sericultural Research and Training Institute , Berhampore (W. Bengal), India

Accepted October 9, 1985

Since the discovery of cytomixis in Oenothera biennis and O. gigas by Gates (1911), it has been observed in some other plants belonging to families

Alismaceae, Poaceae, Fabaceae and others (Bell 1964). Though, the origin, development and function of cytoplasmic channels and chromatin migration has been discussed by many workers (Sarvella 1958, Takats 1959, Kamara 1960, Heslop-Harrison 1966, others), the phenomenon is still not very clear. The occurrence of cytomixis was reported for the first time in mulberry by Verma et al. (1984).

Mulberry (Morus) belongs to the family Moraceae. The leaves of this plant are fed to silkworm which produces silk. Triploid mulberry was evolved at Central Sericultural Research and Training Institute, Berhampore, by crossing artificailly induced tetaploids with diploids (Das et al. 1970). Triploids are reported to be superior to other varieties in leaf yield and nutritiveness (Mustafaev 1970) but they are highly sterile. One male line of evolved triploid was studied meiotically, which showed cytomixis reported in this paper.

Material and methods

Young flower buds were fixed in 1:3 (acetic-alcohol) mixture for 24 hours. Anthers were stained and squashed in 1% iron acetocarmine. Photographs were taken from temporary preparations.

Observations

In all 286 cases were recorded where cytomixis was observed at different stages

Table 1. No. of cases and cells involved in cytomixis at different stages of meiosis

1 Present address: Division of Sericulture, Sher-e-Kashimir University of Agricultural

Sciences and Technology, Mirgund, Kashmir, India.

Onagraceae, Asteraceae,

732 R. C. Verma, A. Sarkar and B. C. Das Cytologia 51

Figs. 1-12. 1-3, prophase I showing cytomixis involving 2, 3 and 4 cells respectively. 4, metaphase

I showing cytomixis, involving 3 cells. 5, anaphase I showing cytomixis. 6, metaphase II showing

cytomixis involving 3 cells. 7, telophase II showing cytomixis through double bridge. 8, cy

tomixis between anaphase I and anaphase II cells. 9, pentad and tetrad together. 10, diakinesis

showing only one bivalent attached to nucleolus. 11, metaphase I showing 26 bivalents. 12,

cytomixis involving 7 cells, also showing multinucleolate cells. •~1700.

1986 Cytoplasmic Channels and Chromatin Migration in Mulberry 733

of meiosis.Prophase I: In all 121 cases were recorded at prophase I, of which 2 cells

were involved in 75 cases (Fig. 1), 3 cells were involved in 25 cases (Fig. 2), 4 cells were involved in 15 cases (Fig. 3) and 5 or more cells were involved in 6 cells in cytomixis (Table 1).

Metaphase I: At metaphase I, 33 cases were recorded of which 2 cells were involved in 30 cases while 3 cells were involved in 3 cases (Table 1, Fig. 4). The migration of chromosomes could clearly be seen in some cells (Fig. 4).

Anaphase I: Fifteen cases were recorded at anaphase I and in all only two cells were involved (Fig. 5).

Metaphase II: At metaphase II, 6 cases were recorded showing cytoplasmic connections, in one of them 3 cells were involved (Fig. 6).

Anaphase II: As many as 99 cases were recorded at anaphase/telophase II. In all of them 2 cells were involved. Sometimes instead of one cytoplasmic bridge, two bridges were found connecting the cells (Fig. 7).

Besides the above cases where cytomixis involved same stages of meiosis, some cases were also observed where two different stages of meiosis were involved (Table 1). Anaphase I and metaphase II cells were found connected in 4 cases, and anaphase I and anaphase II cells were found connected in 8 cases (Fig. 8).

Table 2. No. of bivalents at diakinesis/metaphase I

Other abnormalities like multinucleolate cells (Fig. 12), anucleate cells, unusually large cells, hexapolar anaphase II, triads, pentads (Fig. 9) and hexads were also observed. Diakinesis/metaphase I cells not exhibiting cytomixis were also studied for chromosome number in 20 cells. The number of bivalents ranged from one to 26 (Table 2, Figs, 10, 11), while the diploid chromosome number in mulberry is 2n=28. Pollen sterility was recorded using aceto-carmine staining method and 47.91 pollen grains were found to be sterile.

Discussion

Though cytomixis has been observed in many plants, the causes leading to it

are not very clear. However, two commonly accepted explanations about cytomixis

734 R. C. Verma, A. Sarkar and B. C. Das Cytologia 51

are, i) it is generally observed in genetically unbalanced types such as hybrids, haploids, triploids and otherwise disturbed plants and ii) earliest stages of meiosis are particularly favourable for giving rise to cytomixis (Levan 1941).

In the present case, cytomixis was observed in a triploid which was raised by crossing artificially induced tetraploid and diploid. The tetraploid used in the above cross was itself a hybrid between M. indica 'X' and M. alba var. Mandalaya which created an 'innate unbalance' in heterozygous cytoplasm favourable for cytomixis as postulated by Woodworth (1931). The resultant triploid might be genetically unbalanced and making it more favourable for cytomixis. Therefore, the cytomixis observed in the present case is because of the initial hybrid nature of the material coupled with triploidy both creating a favourable condition for the above

phenomenon.It can be seen from the Fig. 3 that in some cases formation of cytoplasmic con

nections started very early in meiosis i.e. at leptotene/zygotene. Since, the cytoplasmic bridges seem to be very well established by these stages, they might have been formed before the onset of meiosis by nonsynchronous premeiotic mitotic division where cytokinesis and karyokinesis were out of phase resulting in incomplete cell wall formation and subsequent production of chromatin bridges between PMCs

(de Nattancourt and Grant 1964). However, not all the cells entered into cytomixis early in meiotic prophase I, in some of them (Fig. 1) migration took place at a later stage. In Fig. 1 almost all the chromosomes have migrated from one cell to another leaving only nucleolus behind, while in Fig. 2 all the chromosomes have completely migrated to another cell. In such cases the recipient cells would be hexaploid while the donor cells would be anucleate. Such anucleate cells would usually degenerate. The recipient hexaploid cells after completing meiosis would form triploid gametes which after fertilization with a female gamete of a triploid

plant would give rise to different combination of chromosome complements. In Fig. 2 where three cells are involved, all the chromosomes have migrated from one cell to the middle one which would be a hexaploid cell now, but this cell is again connected with another cell. Though the direction of chromatin migration is not very clear between these two cells, there could be two possibilities, either the content of third cell would migrate to the middle cell producing a 9-ploid cell or the content of middle cell would migrate to third cell producing a normal triploid cell and a hexaploid cell. In cases where more than 3 cells were involved, the chromatin migration would give rise to many more complicated situations.

At the advanced stages of meiosis too i.e. at diakinesis/metaphase I, cytoplasmic

connections and chromosome migration were observed (Fig. 4). This again would result in deficient and excess chromosome numbers. In Fig. 10 there is only one bivalent left while in Fig. 11 as many as 26 bivalents could be seen. The cells with deficient chromosome number, if complete the meiosis would produce deficient

gametes and some of them are expected to be sterile or nonfuctional. It can be seen from the Table 2 that the bivalents ranging from one to 26 were observed, implying

thereby that the chromosome migration was broken at different intervals right from the very initiation of the process upto the complete migration of chromatin material. Similarly cytoplasmic connections in other stages of meiosis such as

1986 Cytoplasmic Channels and Chromatin Migration in Mulberry 735

anaphase I (Fig. 5), metaphase II (Fig. 6), and anaphase/telophase II (Fig. 7) were also observed. The frequency of cytomixis at anaphase II was very high (Table 1).

The fact which emerges from the above study is this that all the stages of meiosis are equally susceptible to cytomixis, contrary to the general belief that early stages are more favourable (Maheshwari 1950, Levan 1941).

Another very peculiar situation encountered in the present study was the cytoplasmic connections between the two different stages of meiosis such as between anaphase I and metaphase II, and anaphase I and anaphase II (Fig. 8, Table 1). In a condition where anaphase I chromosomes which are still undivided move to another cell of meiosis II division such as anaphase II where the chromosomes have already divided, what would be the behaviour of anaphase I chromosomes in anaphase II cell? The anaphase I chromosomes might be left as laggards or might behave in some other way. It is difficult to ascertain the fate of those chromosomes till their behaviour is traced in subsequent stages.

The cytomixis gave rise to other abnormalities. Some cells were found to have many supernumerary nucleoli (Fig. 12). As many as 30 small and big nucleoli were present in a cell (Fig. 12). It is well known that some latent nucleoli appear when the cell environment is changed due to excessive hybridity (Verma and Raina 1981). In the Fig. 12, 7 cells are connected with each other through cytoplasmic connections and the content of 3 cells have migrated more or less into two cells. This made the cytoplasm of these cells more heterozygous, favourable for the appearance of latent nucleoli because of the accummulation of nucleolar organizing DNA fragments which were earlier present in various chromosomes of other cell. In comparison to normal cells, some unusually large cells, 4-6 times larger than normal cells with or without nucleus were observed. This could be because of excess chromatin material resulting from the migration and accumulation follwed by their remigration to other cells. That the migration took place at anaphase II and later stages was also evident from the presence of hexapolar anaphase II, triads, pentads (Fig. 9) and hexads. Pollen stainability was found to be only 52.08%. Most of the pollen were shrunken and sterile.

Though triploids as such are supposed to be sterile owing to their abnormal chromosomal behaviour, in the present case the extent of sterility was such that it was not attributed to the above irregularities alone. The cytomixis observed was also responsible for the sterility because 1) the number of pollens would be considerably reduced because of degeneration of cells with no or very little genetic material and 2) among the cells completing all the meiotic stages, most of them would be genetically imbalanced beacuse of less or more number of chromosomes than the normal ones. The sterility in a way is useful in mulberry since it leads to high yield (Abdullaev et al. 1964) and nutritiveness.

Summary

Cytomixis was observed in triploid mulberry. The cause of this has been attributed to initial hybrid nature of tetraploid and triploid. The cytomixis involved many cells at a time and was observed in all the stages of meiosis, contrary to the

736 R. C. Verma, A. Sarkar and B. C. Das Cytologia 51

common belief that only earlier stages are favourble. In some cases two different

stages of meiosis were also involved in cytomixis. Cytomixis resulted in other

abnormalities like anucleate cells, cells with more or less chromosome number, mul

tinucleolate cells, unusually large cells, triads, pentads and hexads. Cytomixis

leads to sterility in mulberry resulting in higher leaf yield and nutritiveness, useful

for silkworm feeding.

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