Genetic control of meiosis in rice, Oryza sativa L. I. Classification of meiotic mutants induced by MNU and their cytogenetical characteristics

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1 Jpn. J. Genet. (1983) 58, pp Genetic control of meiosis in rice, Oryza sativa L. I. Classification of meiotic mutants induced by MNU and their cytogenetical characteristics BY Kunio KITADA, Noni KURATA, Hikaru SATOH and Takeshi OMURA Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University, Fukuoka 812 (Received December 9, 1982) ABSTRACT Twenty-five meiotic mutants were selected from 281 sterile mutants induced by the treatment of the fertilized egg cells of rice with N-methy-N-nitrosourea. Of these, 19 were desynaptic and asynaptic mutants. In addition other meiotic mutants showing vacuolation of cytoplasm, incomplete cytokinesis, arrest of meiosis and degeneration and fused meiocytes were also obtained. Each of these mutants was governed by a single recessive gene. 1. INTRODUCTION Meiosis is a complex process, which ensures the reduction of chromosome number and genetic recombination through cytological phenomena, such as, homologous chromosome pairing, formation and maintenance of chiasmata, disjunction of bivalents, etc. Therefore, meiotic events may be controlled by several genes. Many genes causing various meiotic irregularities have been reported in various animals and plants (Baker et al. 1976; Golubovskaya 1979). These meiotic mutants are useful materials for understanding how meiosis is controlled by specific genes. Irregular meiosis results in reduced pollen and seed f ertilities in higher plants. Therefore, meiotic mutants are selected from the sterile mutants. The systematic analyses of meiotic mutants were made by the use of mutants induced by X-rays or chemical mutagens in Pisum sativum (Gottschalk and Jahn 1964; Gottschalk and Klein 1976), Vicia faba (Sjodin 1970) and Zea mays (Golubovskaya and Mashnenkov 1975,1976,1977; Golubovskaya and Sitnikova 1980). To elucidate the genetic control of meiosis in rice, meiotic mutants have been selected from the sterile mutants induced by the treatment of the fertilized egg cells with N-methyl-N-nitrosourea (MNU). Although most of the meiotic mutants obtained were asyndetic, some mutants exhibited cytoplasmic abnormalities. This paper reports the cytogenetical analysis of the mutants exhibiting cytoplasmic or cytokinetic abnormalities in the pollen mother cells (PMCs).

2 232 K. KITADA, N. KURATA, H. SATOH and T. OMURA 2. MATERIALS AND METHODS Selection of meiotic mutants: The fertilized egg cells of rice variety "Kinmaze" were treated with MNU according to the method of Satoh and Omura (1979). Mutants with sterile character were selected in the M2 generation. The plants with poor growth habit and morphological abnormality were excluded from the materials. The mutants, in which the sterile characters were fixed in the M3 generation, were classified as sterile mutants. Each sterile mutant was examined for the presence or absence of meiotic abnormality in the PMCs. The sterile mutants resulting from irregular meiosis were classified as meiotic mutants (MM). Cytological observation: PMCs were fixed in 1: 3 aceto-alcohol solution for 24 hours and stored in 70% ethyl alcohol in a refrigerator. Observation of chromosomes was made in aceto-carmine stained squash preparations and that of the cells was made in aceto-carmine stained no squash preparations. To determine the extent of sterility, the percents seed set and the stainable pollen grains were recorded in the self ed plants of the mutants. Inheritance: Each mutant plant was crossed with the original variety "Kinmaze". F2 segregation for meiotic mutants was investigated according to seed fertility. 3. RESULTS AND DISCUSSION Of the 281 sterile mutants, 28 exhibited meiotic abnormalities in the PMCs (Table 1). Among them, two were due to translocations and one due to trisomic. Therefore, 25 mutants examined were meiotic. Meiotic mutants were contained in higher frequency in completely sterile mutants than in partially sterile ones. Table 2 indicates the classification of 25 meiotic mutants. Nineteen meiotic Table 1. Selection of meiotic mutants from sterile mutants Two translocations, one trisomic.

3 Genetic control of meiosis in rice (j) 233 Table 2. Classification of meiotic mutants obtained containing abnormal behavior of chromosomes. mutants exhibited abnormalities in chromosomal behavior. The various number of univalents occurred at metaphase I in these mutants. Of these, 16 mutants were desynaptic and other three were asynaptic. This suggests that there are relatively many genes, which control chromosome pairing, formation and maintenance of chiasmata of meiosis in rice. Cytogenetical characteristics of these desynaptic and asynaptic mutants will be reported in another paper. Six different meiotic mutants exhibited cytoplasmic or cytokinetic abnormalities: vacuolation of cytoplasm (MM-22), incomplete cytokinesis (MM-23), arrest of meiosis and degeneration (MM-25, 28), fused meiocytes (MM-26) and other plural abnormalities (MM-24). Cytological characteristics and inheritances of these mutants were as follows. Cytological characteristics MM-22 The behavior of chromosomes and cytoplasm were normal until diakinesis. Apparent aberration was observed at diakinesis. Bivalents were irregularly distributed to one side of nucleus in larger half cells of the mutant compared to the uniform distribution in a normal (Fig. la). Vacuole was observed in part of the cytoplasm in almost all cells at metaphase I (Fig. 1b). The vacuole persisted at anaphase I, telophase I and even during the second division (Figs. lc, d, e). The vacuole of cytoplasm caused division apparatus to get off from the center of the cell, however, the formation of spindle and the behavior of chromosomes were normal. Cytokinesis was incomplete in the portion of vacuole at telophase I and II. Many daughter cells were observed as connected cells at dyad and tetrad stage (Figs. if, g). Fertile pollen grains were all individual (Fig. 1h). Microspores containing

4 234 K. KITADA, N. KURATA, H. SATOH and T. OMURA Fig. 1. Meiotic behavior in PMCs of MM-22 showing vacuole in cytoplasm. (a) Diakinesis: un-uniform distribution of bivalents in the nucleus. (b) Metaphase I: vacuole beside the spindle. (c) Anaphase I: spindle is got off from the center of the cell by vacuole. (d) Prophase II: incomplete formation of cell-plate at the portion of vacuole. (e) Metaphase II: vacuole causes non-parallel direction of spindles in daughter cells. (f) Tetrad: two tetrad cells are connected in the portion of vacuole. (g) Tetrad acuole in a tetrad cell. (h) Pollens: note sterile and fertile grains.

5 Genetic control of meiosis in rice (I) 235 vacuole in cytoplasm degenerated during microsporogenesis. Pollen fertility ranged from 51.0% to 60.5%, and seed fertility from 55.5% to 68.4%. MM-23 The behaviors of chromosomes and cytoplasm were normal until anaphase I, however, aberration was observed at telophase I. Cytokinesis was incomplete in almost all cells (Figs. 2a, b, c). Cells with incomplete cytokinesis and no cytokinesis were 90.2% and 6.1%, respectively, judging from the formation of cell wall at dyad stage. Almost all dyads were partially connected through cytoplasm. The behavior of chromosomes was normal at the second division, but cytokinesis at telophase II was incomplete similar to telophase I (Fig. 2d). Almost all tetrads were fused (Fig. 2e). Frequently, the cells containing four nuclei were observed, but fused nuclei in these cells were not observed. Fig. 2. Meiotic behavior in PMCs of MM-23 showing incomplete cytokinesis. (a) Telophase I: incomplete cytokinesis showing only slight cell-plate formation. (b) Prophase II: incomplete cell-plate between daughter cells due to incomplete cytokinesis at T-I. (c) Metaphase II: cell plate between the daughter cells is incomplete. (d) Telophase II-Tetrated: incomplete cytokinesis in the second division. V-shaped tetrated due to the connection in the part of cytoplasm. (e) Tetrad: ring-like tetrad due to incomplete cytokinesis in both divisions. (f) Pollens: note connected pollen grains.

6 236 K. KITADA, N. KURATA, H. SATOH and T. OMURA Besides individual pollen grains, connected grains were also observed (Fig. 2f). Individual pollen grains, two, three and four connected grains were 34.3, 59.2, 4.8 and 1.7%, respectively. Fifth percent of individual grains were sterile. Of two connected grains, 37.8, 44.6 and 17.6% were both fertile, only one fertile and both sterile, respectively. Almost all three and four connected grains were sterile. Seed fertility ranged from 4.4% to 6.2%. The above results suggest that cytokinesis and karyokinesis in meiosis are under different genetic control. Similar results have been reported in potato (Mok and Peloguin 1975), barley (Sharma and Reinbergs 1972) and Datura (Satina and Blakslee 1935). MM-25, 28 Aberration observed was similar in MM-25 and MM-28. Chromosomes as thin threads were pulled together into the synizetic knot beside the nucleolus at synizetic stage (Figs. 3a, b). But homologous chromosome pairing did not Fig. 3. Meiotic behavior in PMCs of MM-25 showing the arrest of meiotic progress and degeneration at prophase I. (a-b) Synizetic stage: chromosome threads congregate into the synizetic knot but no homologous chromosome pairing occurs. (d, e) Meiocytes at pre-zygotene: degeneration of cytoplasm and chromosomes occurs.

7 Genetic control of meiosis in rice (I) 237 occur (Fig. 3c). Meiosis did not progress at synizetic stage. Cytoplasm enlarged and degenerated and the degeneration of chromosomes occurred (Figs. 3d, e). No pollen grains were formed. The seeds were obtained by cross with normal pollens, therefore, female gamete would be fertile. In the case of male sterile mutants, starting stage and situation of breakdown were different. The initial stage of breakdown in MM-25 and 28 was pre-zygotene. The mutants of prophase I breakdown similar to MM-25 and 28 have been reported in ms-1 and 10 of tomato (Rick i945, i948), ms-8, 9 and 17 of maize (Beadle 1932) and Mutant 69 and 38B of pea (Gottschalk and Kau11974). MM-26 Pollen mother cells were not separated by cell-plate at early prophase I. Cytoplasm of several cells was connected (Fig. 4a). During the progress of meiosis, the degree of cell connection decreased. Separate cells were 15.8% at diakinesis. Two, three and more connected cells were 30.5, 23.6 and 30.0%, respectively (Fig. 4b). The spindles in connected cells were normally formed (Fig. 4c). Connected cells in part of cytoplasm were observed at dyad and tetrad stage (Figs. 4d, e). Cytokinesis in meiotic division was normal. In addition to individual pollen grains, two to six connected grains were also observed (Fig. 4f). The individual grains were 19.1% and about two third of them were sterile. The percentage of two connected grains was In most cases two connected grains were all sterile. All three and more connected grains were sterile. Seed fertility ranged from 10.7% to 18.7%. Aberrant chromosomes were observed in 15.5% of cells at metaphase I and anaphase I. One third of them was tetraploid (Fig. 4g) and the other one third was aberration of chromosome configuration, that is, the chromosomes were not condensed and shortened as in normal plants (Fig. 4h). In the remaining proportion, both aberrations occurred in the same cells (Fig, 4i). No aberration was observed at any other stage except these stages. The partial fusion of pollen mother cells in MM-26 may be caused by the abnormal or incomplete cytokinesis in premeiotic mitosis. This suggests that the premeiotic mitosis is different in some respect from other mitosis. The mutants of incomplete or absent cytokinesis, specifically in premeiotic mitosis, similar to MM-26 have been found in maize (Debdeff 1940). barley (Smith 1942) and Helianthemum (Snoad 1954). Smith (1942) observed the polyploidy and the chromosome elongated configuration at metaphase Tin the mutant similar to MM-26. MM-24 exhibiting plural abnormalities in meiosis was different from the above-mentioned meiotic mutants. Detailed cytogenetical analysis of MM-24 is in progress.

8 238 K. KITADA, N. KURATA, H. SATOH and T. OMURA Fig. 4. Meiotic behavior in PMCs of MM-26 showing fused meiocytes. (a) Early prophase I : several connected meiocytes by incomplete cell-plate formation at premeiotic mitosis. (b) Diakinesis: two connected meiocytes. Distribution of bivalents in the nucleus is normal. (c) Telophase I: two connected meiocytes. Division apparatus is normally formed. (d) Second division: several daughter cells are connected. (e) Tetrad: connected cells between two tetrads. (f) Pollens: note two, three and more connected grains besides individual grains. (g-i) Aberrant chromosomes: tetraploid at anaphase I, long and tenuous chromosomes at metaphase I and tetraploid and tenuous chromosomes at metaphase I.

9 Genetic control o f meiosis in rice (j) 239 Table 3. Fertility in Fl and its segregation in F2 of crosses between meiotic mutants and a cultivar "Kinmaze" Inheritance The fertility of Fl and its segregation in F2 of the crosses between mutants and their original cultivar, Kinmaze, are shown in Table 3. All the Fl hybrids were normal in seed fertility and the segregation in F2 showed a good fit to the expected 3: 1 ratio. These results indicate that the meiotic irregularity in each meiotic mutant is controlled by a single recessive gene. We wish to thank Dr. S. Shanmugasundaram, The Asian Vegetable Research and Development Center, TAIWAN, for his kind reading of the manuscript. REFERENCES BAKER, B. S., CARPENTER, A. T. C., ESPOSITO, M. S., ESPOSITO, R. E. and SANDLER, L. (1976) The genetic control of meiosis. Ann. Rev. Genet. 10, BEADLE, G. W. (1932) Genes in maize for pollen sterility. Genetics 17, GOLUBOVSKAYA, I. N. (1979) Genetic control of meiosis. Int. Rev. Cytol. 58, GOLUBOVSKAYA, I. N. and MASHNENKOV, A. S. (1975) Genetic control of meiosis. I. Meiotic mutation a f d in corn (Zea mays L.) causing absence of the first division of meiosis. Genetika 11, GOLUBOVSKAYA, I. N. and MASHNENKOV, A. S. (1976) Genetic control of meiosis. II. A desynaptic mutant of corn, induced by N-nitroso-N-methylurea. Genetika 12, GOLUBOVSKAYA, I. N. and MASHNENKOV, A. S. (1977) Multiple disturbance of meiosis in corn, due to a single recessive mutation pam A-A 344. Genetika 13, GOLUBOVSKAYA, I. N. and SITNIKOvA, D. V. (1980) Three meiotic matations disturbing chromosome segregation at the first meiotic division in corn. Genetika 16, GOTTSCHALK, W. and JAHN, A. (1964) Cytogenetische Untersuchungen an desynaptischen and mannlich-sterilen Mutanten von Pisum. Z. Vererbungslehre 95, GOTTSCHALK, W. and KAUL, M. L. H. (1974) The genetic control of microsporogenesis in higher plants. The Nucleus 17, GOTTSCHALK, W. and KLEIN, H. D. (1976) The influence of mutated genes on sporogenesis. A survey on the genetic control of meiosis in Pisum sativum. Theor. Appl. Genet. 48, LEBDEFF, G. A. (1940) Failure of cytokinesis during microsporogenesis in Zea mat's following heat treatment. Cytologia 10, MOK, D. W. S. and PELOGUIN, S. J. (1975) Three mechanisms of 2n pollen formation in diploid potatoes. Can. J. Genet. Cytol. 17, RICK, C. M. (1945) A survey of cytogenetic causes of unfruitfulness in the tomato. Genetics 30, RICK, C. M. (1948) Genetics and development of nine male-sterile tomato mutants. Hilgardia 18,

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