, .- m 7 L3"??- éag’é} fiflfcmg am 3mm University OVERDUE FINES: 25¢ per day per item RETURNIM LIBRARY MATERIALS: \ Place in book return to remove charge from circulation records A CYTOGENETIC INVESTIGATION OF MULTIPOLAR CELL DIVISION AND CYTOMIXIS IN DIPLOID CRESTED WHEATGRASS, AGROPYRON CRISTATUM By Li-Ching Wang Linkous A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1982 ABSTRACT A CYTOGENETIC INVESTIGATION or MULTIPOLAR CELL DIVISION AND CYTOMIXIS IN DIPLOID caasrao WHEATGRASS, AGROPYRON CRISTATUM By Li-Ching Wang Linkous Multipolar cell division and cytomixis are abnormal cytological phenomena occurring during meiotic or mitotic process. They can occur spontaneously, or can be induced by chemicals and/or physical means. They play very important roles in chromosome number evolution. Agropyron cristatum, #CC-37-119, was obtained from a seed treated with 0.1% colchi- cine solution for 12 hours. Different types of cytological irregularities were observed in the clones of CC-37-119, i.e., multipolar cell division, cytomixis, chromatin bridges, lagging chromosomes, unequal segregation, precocious division, early segregation and chromosomal fragments. Col- chicine—induced irregularities were found to be inheritable. Multipolar cell division appears to be the main cause of gametes with reduced chromo- some numbers. Cytomixis seems to give rise to microspores with an in- crease or decrease in chromosome number. All the observed irregularities appear to reduce the pollen viability. They appear to provide possible mechanisms for the production of aneuploid gametes which in turn may lead to aneuploid evolution. DEDICATION I want to dedicate this thesis to my beloved parents, Mr. and Mrs. Wang Sen-Dean. Without their love. care, encouragement and support, this work would not have been possible. I also wish to express my deep appre- ciation to my advisor, Dr. William Tai, for his assistance and direction in the preparation of this thesis. Last, but not least, I wish to thank my dear husband, Clovis. ii TABLE OF CONTENTS Page Ii‘trOductionOOOOOOOOOOOOOOOOOOOOOOOOOOIOOOOOOOOOOOOOOOOOOOOO 1 Literature Review on MUItiPOIar cell DiViSionOOOOOOCOOOOOOOOOOOOOOOIOOOOOOOOOO cytomiXiSOOOOI00.000.000.00...00.0.0000...OOOOOOOOOOOOOOO 6 Materials and Methods origi‘l Of MaterialSOIOOOOOOOOO0.0...OOIOOOOOOOOOOOOOOOOOO 9 CYtOlogical MethOdSOOOC.0...0.0....COOOOOIOOOOOOOOOCOOOOO 9 Results MiCOSiSOOOOOOOOOOCOO....0...OOOOOOOOOOOOOOOOOOOO000...... 11 Premeiotic MitOSi-SOOOOOOOOOOOIO...OOOOOOOOOOOOOOOOOOOOOOO 11 MeiOSiSOOOO00.0.0000...0.00.00.00.000000000000IIOOOOOOOOO 11 Normal Meiotic BehaVicr. O O I O O O O I O O O O O O O I O O I O O I O O O O O I I O O O O 14 Abnormal Mei°tic BehaVior. O O O O I O O O O O O O O O I O O I I I I O O O O O O O O O 0 20 P0118“ MiCOSi-SOOOOOO0......O..0...OOOOOOOOOOOOOOOOOOOOOOO 35 P0118“ ViabilitYOOO....00...00......OOOOOOOOOOOOOOOOOOOOO 40 Discussion Inheritance of Colchicine-Induced Irregularities......... 46 MUltiPOIar C811 DiViSionOOOOOOOOOOOOOOOOIOOOCCOOOOOOOOOOO Q7 cytomiXiSOOO...OOOOOCOOOOOOOOOOOOOOOOCOOOOOOOOIOOOIOOOOOO 52 summary.OOOOOOOOOOOOOOOOO0.0...0.0...OOOOOOOOOOOOOOOOOOOOOOO 55 List Of References.‘00....OO...000......OOOOOOOOOOOOOOOOOOOO 56 iii Table Table Table Table Table Table Table Table Table Table Table Table 11. 12. LIST OF TABLES Chromosome Behavior at Diakinesis in Clones Of CC-37—11900000000.0.0....0....00...... Chromosome Behavior at Metaphase I in Clones Of CC-37-119.00000000000......O00...... Chromosome Behavior at Anaphase I in Clones Of CC-37-119000OOOOOOOOOOOOOOO..00...O... The Number of Nuclei at Telophase I in Clones Of CC-37-11900000000000OOOOOOOOOOOOOOOO Chromosome Behavior at Metaphase II in Clones Of CC’37-119000000000eoeeeeoeeoeeoeeeo Chromosome Behavior at Anaphase II in Clones Of CC-37-119000000000OOCOOOOOOOOOOOOOO Multipolar Cell Division (MPD) at Different Meictic Stages in Clones Of CC-37-119.IO0..OOOOOOOOCCOOOOOOOOOOOOOOOOOOOOOOO The Number of Nuclei at Quartet Stage in Clones Of cc-37-119eoeoeoeooee000000000000 The Number of Nuclei at Microspore Stage in Clones Of 00-37-119.0000000000000000...... Chromosome Behavior at Metaphase of Pollen Mitosis in Clones of CC-37-119.............. Chromosome Behavior at Anaphase of Pollen Mitosis in Clones of CC-37-119.............. Viability of Pollen Grains in Clones Of CC-37-11900IOOOOOOOOOOOIOOOOOOOO0.000......0.... iv 23 27 28 32 33 34 48 38 39 Al 42 45 LIST OF FIGURES Figure 1. Metaphase of mitosis with a normal chromsome number 2n=4 in Clones Of CC-37-119 (2415X)00000000000 Normal Meiotic Behavior Figure 2. Pachytene (785x)..................................... Figure 3. Diakinesis (661x).................................... Figure 4. Metaphase I (661x)................................... Figure 5. Anaphase I (537x).................................... Figure 6. Telophase I (827x)................................... Figure 7. Prophase II (1042x).................................. Figure 8. Metaphase II (1419x) ................................ Figure 9. Anaphase II (1273x) ................................. Figure 10. Quartet (66IX) I.000......OOOOOOOOOOOOOOCOOOOO0...... Abnormal Meiotic Behavior: Multipolar Cell Division (MPD) Figure 11. Chromosomes congregated into 2 groups; one group with 4 bi-valents, the other with 3 bivalents (4-3) at diakinesis. (751x) .............................................. Figure 12. Chromosomes congregated into 2 groups: one group with 6 bivalents, the other with 1 bivalent (6-1) at diakinesis. (821x) ................ Figure 13. Chromosomes congregated into 4 groups with 4-1—1-1 at diakinesis. (765x) ....................... Figure 14. Cell with 7 bivalents separated into 2 groups (4-3) to form two "micrometaphase" plates at metaphase I. (748x) ................................. 13 16 16 16 16 16 16 16 16 16 22 22 22 22 Figure Figure Figure Figure Figure 15. 16. 17. 18. 19. vi Cell with 3 groups (4-2-1) of bivalents at metaphase I. (680x) 00000000000000...ooe......... Cell with 4 groups (3-2-1-1) at metaphase I. (680x)eoeeeeco00000000.000000000000000eoeoeeeeoee Abnormal groupings of dyad chromosomes (4-3) at anaphase I. (595x)ee00000000000000.0000...eoeooeo Abnormal groupings of dyad chromosomes (3-2-2) at anaphase I. (776x)eeeeoeeeeoeeeeeeoeeceeeooeooooe Chromosomes separated into 2 groups (6-1) at prophase II. (850x)...oeeee00.000000000000000.ooooee Abnormal Meiotic Behavior: Multipolar Cell Division (MPD) Figure Figure Figure Figure Figure Figure Figure Figure Figure 20. 21. 22. 23. 24. 25. 26. 27. 28. Cell had two metaphase plates (5-2) at metaphase II. (IOIZX)OOOOOOOOIOOO00.0.0000...OOOOOOOOOOOOOOOOO Two microcells at metaphase II, one with 4 dyads, the other with 3 dyads. Arrows showed cytokinesis. (867x)---------------------------------------------- One cell had normal metaphase II. The other cell had 6-1 grouping and began supernumerary cytokinesis (arrow) at metaphase II. (881x)------'°- One microcell with only one X-shaped chromosomes at metaphase II. (1098x) ooeeoeeeooeooeeeeeoeeeoeeoo Chromosomes separated into 2 groups (4-3) and supernumerary cytokinesis occurred (arrows) at anaphase II. (867x) eeeeeeeeeeeeeoeeeeeeeeeooeeeeeee Multipolar Cell Division (MPD) at anaphase 11 (5-2) and supernumerary cytokinesis occurred (arrOWS). (867X) 0.0...OOOOCOOCOOOCOOOOCCIOOOOO...O. Chromosomes separated into 3 groups (3-3-1) at anaphase II. (728x) eeeeeoeeeeeeeeeeeeoeooeoeoeoe Cell with 6 daughter cells at quartet stage. (867x) oeeeoeoeeeeooeoeeeeeeeoeeoeooeeeoeone. Cell with 5 daughter cells and some micronuclei at the quartet stage. (535x) eeeeooeeoeoeeeeeoeeoeee 22 22 22 22 22 31 31 31 31 31 31 31 31 31 vii Abnormal Meiotic Behavior: Cytomixis Figure Figure Figure Figure Figure Figure Figure Figure Figure 29. 30. 31. 32. 33. 34. 35. 36. 37. Part of the nucleus (arrow) of donor cell migrated into the adjacent recipient cell at early prophase I. (1195x) ................... Cells were connected by several spiralized chromosomal threads (arrows) at early prophase I. (1017X) OCOOOUOOOOOOOOOOCOOOOOI0.0.0.0... Additional chromosomal material (arrow) in the cell at early prophase I. (759x).............. Additional chromosomal materials (arrow) in the cell at early prophase I. (759x) ............. Cell had 7 bivalents with one extra coil of prophase chromosome (arrow) at diakineSi-s. (825x) 0.......CCCCOOOOOOOOOOOOCOOCOOO Cell had 7 bivalents with one extra coil of prophase chromosome (arrow) at diakineSis. (458x) .CCOCCCOOOCOO'COOOCOOOOOOOCOOOC Cell had 7 bivalents with one extra coil of prophase chromosome (arrow) at diakineSiSe (1017X) 0............00.000.000.000... Cell had 7 bivalents with a big mass of double-stranded thick chromosomal threads (arrow) at diakinesis. (903x) ............... Cell had 7 bivalents plus 2 univalents at diakineSiSe (788X) COCOCOOCOOOOOOOOOOO00.0.0000... Abnormal Meiotic Behavior: Figure Figure Figure 38. 39. 40. Cell had 6 bivalents at diakinesis. (729x) .......... Cell had 5 bivalents with one prophase chromosomal threads (arrow) at metaphase I. (46BX)OOOO00.000000000000000000000000.000000000000 Cell with 7 bivalents to form a single metaphase plate had extra chromosomal materials (arrow) at metaphase I. (579x)............. 19 19 19 19 19 19 19 19 19 26 26 26 Figure Figure Figure Figure Figure Figure 41. 42. 43. 44. 45. 46. viii Multipolar cell division and cytomixis occurred in the same cell. Cell with 7 bivalents to form multiple microplates (2-2-2-1) with extra chromosomal materials (arrOW) at metaphase I (695X)oeeeooooeoeoeooeeeooooo. Cell had 8 bivalents and separated into 2 groups (6-2) at metaphase I. (1028x)ooeoeooooooooo Cell had 6 bivalents and separated into 2 groups (5-1) at metaphase I. (897x) .............. Cell had extra chromosomal material (arrow) at anaphase 1' (440x)........0.00.0...0000.00.0.0... Multipolar cell division and cytomisix occurred in the same cell. Cell separated into 3 groups (3-2-2) had extra chromosomal material (arrow) at anaphase I. (637x).............. Cell had a lot of extra Chromosomal materials at anaphase 1‘ (672x)I.00....OOOOOOOOOOOOOOO00.0.0.0... Abnormal Meiotic Behavior Figure Figure Figure 47. 48. 49. Four daughter cells at quartet stage. One daughter cell had extra chromosomal materials (661x) OOOOOOOOOOOOOOOOOOOOOO0.0.0.000...O Chromatin bridge at anaphase I. (758x).............. One microspore had a normal sized nucleus. The other had three small nuclei (799x)............. Normal Pollen Mitosis Figure Figure 50. 51. ‘One Inicrosporocyte had normal 7 X-shaped chromosomes at metaphase (1447x)........... One ndcrosporocyte had normal 14 single-stranded chromosomes at anaphase (951x) ............................................. 26 26 26 26 26 26 37 37 37 37 37 ix Abnormal Pollen Mitosis Figure 52. Figure 53. Figure 54. Figure 550 One ndcrosporocyte had one X-shaped Chromosome (951x).CCOCCCOCCCOCCOCCOOOCOCCC...0.000.... One ndcrosporocyte had 11 single-stranded chromosomes (882x) C...OCCOOOCCCOCOCOOOCOOCOOCCOOOOOOO One ndcrosporocyte had 3 single-stranded chromosomes, two with 2 single-stranded chromosomes. One had 5 X-stranded chromosomes and 5 single-stranded chromosomes (882x) ,,,,,,,,,,,,, One ndcrosporocyte had 8 X-shaped Chromosomes (882x) O...0.0.0.0...OOOOOOOOOOOOOOOOOOOOO Abnormal Pollen Mitosis Figure 56. Figure 57. Figure 58. Viability of Figure 59. Figure 60. Figure 61. One udcrosporocyte had 16 single- stranded chromosomes and one chromosome fragment (IIZZX) 0.00.0000...0.0.0.0...OOOOOOOIOOOOOOO Miltipolar mitosis in microspore- cyte (67SX) 0.0000IOOOIOOOOOOOOOOOOO0.0.0.....0.00.... Multipolar mitosis in microsporo- cyte (900X) .0.0.0..0.0.0.0....OOOOOOOOOOOOOOOOOOOOOO. pollen grains Normal sized, viable pollen grain.was darkly stained by I z-KI SOIUtion (591x) OOCOOOOOOOOOOQ Small sized, viable pollen grains were darkly stained by I -KI solution (591x) 2 Non-viable pollen grains were not stained by I 2 -K1 SOIUtion (591x) 0.000000000000000000...0.0... 37 37 37 37 44 44 44 44 44 44 INTRODUCTION Multipolar cell division (MPD) has occurred spontaneously and can be induced artifically in many plant and animal species. Tai (1970) observed that the chromosome complement became subdivided into several groups in the meiotic division of colchicine-treated diploid Agropyron cristatum. The grouping seemed to be based on the origin of the genomes in hybrids and polyploids. He proposed that multipolar cell division provided a pos- sible mechanism for changes in chromosome numbers, which evolutionarily led to aneuploidy or polyhaploidy. Tai (1970, 1971) also suggested that in plant breeding, induction of haploidy by MPD and doubling of the chro- mosome complement in the haploids could produce an individual homozygous for every gene locus. Cytomixis appears to be another mechanism for change in chromosome number, which in turn results in aneuploid evolution (Romanov and Orlova 1971; Tai and Vickery 1972; Cheng 1974). The migration of chromosomal ma- terials from the donor cell to the recipient one may have the consequence of an increase or decrease of chromosome numbers. It has been observed in many plants including diploid (Kosova 1973), hybrid and polyploid (Kihara and Lilienfeld 1934; Katterman 1933; Tai and Vickery 1972) and mutants (Kamra 1960) during mitotic or meiotic processes. The present investigation was designed to make further and detailed observations of colchicine-treated Agropyron cristatum. The objectives of this investigation are: To observe the chromosome behavior of a colchicine-treated diploid Agropyron cristatum plant which has shown MPD and cyto- mixis. To examine the inheritance of these induced irregularities. To analyze the significance of multipolar cell division in chro- mosome number evolution. To evaluate the significance of cytomixis in chromosome number evolution. LITERATURE REVIEW Multipolar Cell Division Multipolar cell division is a phenomenon in which the chromosome com- plement is separated into two or more groups in either meiosis or mitosis. Each group functions more or less independently and has its own chromosome movement. Multipolar cell division results in variable chromo- some numbers in the daughter cells (Tai 1970). The same phenomenon has been described in various materials by different terms, such as "incom- pact spindle" (Darlington and Thomas 1937), "double plate metaphase" (Upcott 1939; Vaarama 1949), somatic meiosis (Huskins 1948), reductional grouping (Wilson 1950), multipolar Spindle (Therman and Timonen 1950; Walters 1958), split spindle (Neilsen and Nath 1961) and complement frac- tionation (Thompson 1962). Multipolar cell division has been found to occur spontaneously in animal species and tissue culture. After analyzing multipolar mitosis in tumors, Boveri (1888) concluded that the phenomenon was pathological. Baltzer (1909) observed that tri-polar mitosis in the sea-urchin eggs which may produce daughter cells with a reduced number of chrcmoscmes. In the analysis of rat liver cells, Glass (1956, 1957) found that multipolar mitosis occurred in all haploid, diploid, triploid and tetraploid clones of rat liver cells. The haploid and triploid chromosome groups existed simultaneously within the same tetraploid cells. Haploids observed to have 10+11 chromosome grouping and triploids had 2n-1n and 1n-1n-1n distinct genome groups. Therefore, he suggested that multipolar cell division is very important to genome separation and to lower the ploidy level. Knudson (1958) discovered that the formation of multipolar spindles during the first meiotic division caused the majority of sterile bulls. Rizzoni _£ 21° (1974) pointed out that in mammalian cell culture, euploid segregation occurred through multipolar cell division, and that the frequency of multipolarity increased linearly with the age of cul- ture. Also the human cancer cells are often characterized by the occur- rence of multipolar cell divisions and multinucleated cells (Therman and Timonen 1950, 1954; Timonen and Therman 1950; Ofterbo and Wolf 1967). More reports about spontaneous multipolar cell division come from plants including haploid, diploid, polyploid, hybrids and tissue culture. In haploid Ulva mutabilis, the chromosomes segregated randomly following multipolar meiosis forming viable zoospores (Hoxmark and Nordby 1974). Vasek (1962) found that multipolar cell division occurred in the micro- sporogenesis of diploid Clarkia exilis. He suggested a recessive gene for the occurrence and persistence of multipolar cell division. The presence of multipolar cell division in polyploid plants is very common and frequent, and has been described by many authors (Upcott 1939; Vaarama 1949; Weimarck 1973). In the study of Rubgs hybrids, Thompson (1962) and Bammi (1965) proposed that complement fraction resulted in chromosome grouping in meiotic as well as mitotic cells. Dewey (1974) ob- served chromosome grouping and multiple metaphase plates in hybrids and induced amphiplcids of Elymus conadensis x Agropyron libanoticgm. Based on the study of Daucus carata culture Bayliss (1973) suggested that multi- polar cell division might result from lack of physical organization of the tissues or the chemical constituents of the medium. Multipolar cell division can also be induced artificially by differ- ent means. Induced multipolar cell division is always associated with lagging chromosomes, chromatin bridge, chromosomal fragments, unequal segregation, micronueclei, supernumerary cells, etc. Many inorganic and organic chemicals which are dangerous to human health have the ability to induce multipolar cell division. Colchicine is one of the many excellent inducing agents. From the observations on onion cells, Ostergreen (1950) suggested that different concentrations of col- chicine would give different degrees of mitotic Spindle abnormalities, and that low concentration of colchicine will induce multipolar cell divi- sion. Radiation, e.g., X-rays and gamma-rays, has also been reported to induce multipolar cell division (Rustard 1959; Levis and Martin 1963; Amer and Farah 1974). The percentage of multipolar cell division and associated abnormalities appears to increase with the dosage and duration of radiation. Both extremely high and low temperatures will suppress the start of cell division and increase the frequency of multipolar cell divi- sion (Huskins and Cheng 1950; Mazia 1961). Cytological irregularities can also be induced by microorganisms. Nimnoi (1974) reported that potato spindle tube virus induced multipolar cell division in potato pollen mother cells. Abraham (1974) found that spindle abnormalities and lagging chromosomes in the stamen hairs of Tradescantia clone 02 growing in cowdung sand. The significance of multipolar cell division in chromosome evolution has been discussed by many authors. It appears to be a source of aneu- ploidy. Thompson (1962) and Tai (1970, 1971, 1972) suggested multipolar cell division will produce gametes with extremely variable chromosome num- bers. If too many chromosomes are gained or lost, the gametes may not live and function. However, if only a small number of chromosomes are involved, the gametes may survive and be functional as well. They also pointed out that multipolar cell division seems to provide a mechanism to reduce the ploidy level from polyploids to polyhaploids. Multipolar cell division is also responsible for genome separation (Tai 1970; Cheng 1976), and thus can be used as a tool for plant breeding (Thompson 1962; Tai 1970, 1971). Cytomixis At the beginning of the 19th century, the phenomenon of the migra- tion of nuclei or chromatin from one cell to the neighboring one was dis- covered in plant somatic and reproductive cells (Miehe 1901; in Cheng Kuo-chang 1974; Kornike 1901). Later it was called chromatin extrusion or "cytomixis" (Gates 1911). Currently the term "cytomixis" is used in a much broader rather than the narrower sense. In the present study the term "cytomixis" is used to describe the migration of chromatin or parts of a nucleus from one cell to another, and the presence of cells with additional chromosomal materials. Cytomixis is fairly widespread. It has been described in many species including diploids (Kosova 1973; Syemyarykhina abd Kuptsow 1974; Cheng 1974), polyploid hybrids (Katterman 1933; Kihara and Lilienfeld 1934; Romanov and Orlova 1971; Tai and Vickary 1972; Shkutina and Kozlov- skaya 1974; Syemyarykhina and Kuptsow 1974), and mutants (Sarvella 1958; Kamra 1960). It was observed in somatic tissues (Tarkcwska 1960) but more frequently in pollen mother cells (Kihara and Lilienfeld 1934; Katterman 1933; Romanov and Orlova 1971; Tai and Vickery 1972; Shkutina and Kozlov- skaya 1974). The cause of cytomixis still remains unsolved. Some believed it is an abnormal phenomenon. Tarkowska (1960, 1965, 1966) suggested that some mechanical stimuli creates sufficiently large pressure differences bet- ween cells and induces cytomixis from cells with higher internal pressure to those with lower. Katterman (1933) and Takats (1959) believed that it was caused by methods of fixation, and that different types of fixative would have different frequencies of cytomixis. Some thought that it is the results of abnormal environmental conditions, i.e., a change of temperature, rainfall, drought and radiation (Katterman 1933). Cytomixis also had been discovered in diseased plants, therefore it was proposed that extrusion of chromatin was caused by pathogens (Tischler 1934) or by the weakening of plants (Fraser 1914). Woodworth (1929) and Kamra (1960) suggested that it is the result of hybridization. Based on the experiments and observations of lily and Lysium chinense, Cheng (1974) strongly proposed that cytomixis is a normal physiological condition and may be enhanced by external environmental factors, e.g., temperature fixation, mechanical changes, etc. (Gates 1911; Kihara and Lilienfeld 1934). Migration of chromosomal materials from the donor cell into the ad- jacent recipient cell has been observed most frequently in the early meiotic prophase I (Katterman 1933; Kihara and Lilienfeld 1934; Bopp- Hassenkamp 1959; Romanov and Orlova 1971; Tai and Vickery 1972; Shkutina and Kozlovskaya 1974). It also occurred rarely at metaphase I and ana- phase I (Romanov and Orlova 1971). In somatic cells, it usually occurs during interphase but not other mitotic stages (Cheng 1974). Romanov and Orlova (1971) suggested that chromatin migrates from the donor cell through the cytomictic channels into the cytoplasm of the recipient cell. Usually it migrates in multiple directions, but sometimes only in a single direction (Cheng 1974; Heslop-Harrison 1966; Weiling 1965). The significance of cytomixis has been analyzed by a number of authors. Sinotc (1922) suggested that cytomixis is an artifact, there is no need to consider its significance in genetics. Some authors proposed that cytomixis may result in an increase or decrease of the chromosome numbers in pollen mother cells, and provides a mechanism for aneuploidy evolution (Katterman 1933; Kihara and Lilienfeld 1934; Kamra 1960; Sadasivaiah and Magoon 1965; Romanov and Orlova 1971; Tai 1967; Tai and Vickery 1972; Cheng 1974; Kosova 1974). Cheng (1974) discovered double and multinucleated protOplast in culture, and suggested that cytomixis could cause the formation of multinucleated cells, which in turn would produce pclyplcids (Romanov and Orlova 1971). MATERIALS AND METHODS Origin of Materials During a study of colchicine-treated diploid (2n=14) Fairway crested wheatgrass (ggropyron cristatum), Tai (1964) discovered a plant (CC-37-119) to have different types of cytological irregularities. Plant CC-37-119 was obtained from a seed treated with 0.1% aqueous solution of colchicine for 12 hours. The plant has been maintained at Evans Farm, Utah State University by Dr. Douglas R. Dewey. Clones were sent to Dr. Tai at Michigan State University. These materials were used in this cur- rent research. Cytolggical Methods For mitotic studies, root tips from newly transplanted CC-37-119 plants were excised and pretreated in 0.01% colchicine solution for four hours in the spring of 1978, followed by fixation in Farmer's solution (3 parts absolute ethanol: 1 part glacial acetic acid) overnight. Root tips were stored in the same solution in the refrigerator (at 3°C). The aceto-carmine smear technique was used to prepare slides for observation. If the Feulgen stain technique was used, the root tips were hydrolyzed in 1 N H01 at 60°C for 10-11 minutes. In the spring of 1978, young spikes were collected for meiotic anal— ysis from plants maintained in the greenhouse and the open field. When 10 spikes were collected from 4—8 a.m., the pollen mother cells showed the best separation of chromosomes and therefore best for cytological obser— vations. The spikes were fixed immediately in Farmer's fixative and stored in the same solution under refrigeration (3°C). The aceto- carmine smear technique was used again to prepare slides. Premeiotic Mi- tosis, all stages of the meiotic division and pollen mitosis were exam- ined for the study of chromosome behavior. All cytological observations were made with a Zeiss photomicroscope II under phase contrast illumination. Photomicrographs were taken with 35mm Kodak Panatomic-X film or with 4x5" Kodak Contrast Process Ortho film. Pollen viability was determined on the basis of the staining reac- tion with the IZ-KI solution. Pollen grains were considered viable if they were round and stained darkly under bright field observation. Shrunken and lightly stained pollen grains were recorded as non-viable. RESULTS Mitosis All cells examined for mitosis appear to be fully normal with no abnormalities observed at any mitotic stage. The chromosomes behave as normally as any typical diploid species during the entire mitotic pro- cess. Plant CC-37-119 exhibits a normal diploid chromosome number of 2n=14 in its root tip cells (Fig. 1). Its karyotype shows a pair of acrocentric ’chromosomes with satellites. Schulz—Schaeffer and Jurasits (1962) ob- served no satellite chromosome in diploid Fairway crested wheatgrass. Perhaps, the staining and squash technique that they used resulted in chromosomes too condensed to determine fine details. The rest of the com- plement have similar morphology as was described by them with two pairs ofnmtacentric chromosomes and four pairs of acrocentric chromosomes. Premeiotic Mitosis Chromosome behavior of premeiotic mitosis appeared to be normal. There are 14 chromosomes at metaphase and a 14-14 equal disjunction at anaphase. No chromosome pairing was observed at premeiotic mitosis. Meiosis With a total of 5800 pollen mother cells and another 1587 observed at the quartet stage, 62.48% and 60.24% appeared to be cytologically 11 12 Figure 1. Metaphase of mitosis with a normal chromsome number 2n=4 in clones of CC-37—119 (2415x) 13 0‘ 14 normal, respectively. The rest of the cells showed different types of cytological irregularities e.g., congregation of chromosomes into groups (multipolar cell division), lagging chromosomes, chromatin bridges, un- equal segregation, early segregation, chromosome fragments, cytomixis, micronuclei, etc. Normal_ygiptjg_3ehavior Prophase I Leptotene: The chromosomes are not tightly packed together. They are visible as very thin, long and uncoiled threads, single stranded and unpaired. Zygotene: The chromosomes start to shorten and thicken. Homologous chromosomes come slowly together, align side by side and begin to pair. The sister chromatids of each homologue are no longer visible Pachytene: The paired homologous chromosomes continue to shorten, condense and remain tightly twisted around each other (Fig. 2). Diplctene: Terminalization of chiasmata begins at this stage. There- fore, homologous chromosomes start to pull apart from each other and holes appear in bivalent configurations. The bivalents are usually randomly distributed. In some cells, the nucleolus remains visible and the nuclear envelope remains intact. In other cells, they both may disintegrate and disappear. Diakinesis: The chiasmata continue to terminalize and still hold the chromosomes together at the end. The chromosomes reach maximum shortening and condensation. The nucleolus disappears and the nuclar envelope is 15 Normal Meiotic Behavior Figure 2. Pachytene (785x) Figure 3. Diakinesis (661x) Figure 4. Metaphase I (661x) Figure 5. Anaphase I (537x) Figure 6. Telophase I (827x) Figure 7. Prophase II (1042x) Figure 8. Metaphase II (1419x) Figure 9. Anaphase II (1273x) Figure 10. Quartet (661x) 16 17 disintegrated completely. Seven bivalents are randomly distributed all over the cytoplasm. The chromosomes show typical morphology with no over- lapping. Usually, this is the best stage at which one can analyze the pairing behavior of chromosomes (Fig. 3). Metaphase I Chromosomes reach the maximum degree of condensation. Seven bivalents move towards the equatorial position and align themselves to form a single metaphase plate (Fig. 4). Anaphase I and Telophase I At anaphase I, the homologous chromosomes start to separate from each other with seven dyad chromosomes moving towards each pole (Fig. 5). After the chromosomes reach the poles, a new nuclear envelope is formed at each pole to form a new nucleus. Cleavage of cytoplasm at the equator separates the primary microsporocyte into two daughter cells. Each has a haplOid chromosome number (Fig. 6). Meiosis II Second meiosis takes place independently in two secondary microsporocytes. Once again the chromosomes start to condense at prophase II and each dyad shows a typical X shape (Fig. 7). The seven X-shaped dyads align themselves at the equator during metaphase II (Fig. 8). At anaphase II, the chromatids separate from each other (Fig. 9). After separation, each chromatid is termed as a single-stranded chromosome and seven of them migrate towards the opposite poles. Eventually, cytokinesis takes place and a pollen mother cell gives rise to four daughter cells, a quartet. Each daughter cell has seven chromosomes (Fig. 10). 18 Abnormal Meiotic Behavior: Cytomixis Figure Figure Figure Figure Figure Figure Figure Figure Figure 29. 30. 31. 32. 33. 34. 35. 36. 37. Part of the nucleus (arrow) of donor cell migrated into the adjacent recipient cell at early prophase I. (1195x) Cells were connected by several spiralized chromosomal threads (arrows) at early prophase I. (1017x) Additional chromosomal material (arrow) in the cell at early prophase I. (759x) Additional chromosomal materials (arrow) in the cell at early prophase I. (759x) Cell had 7 bivalents with one extra coil of prophase chromosome (arrow) at diakinesis. (825x) Cell had 7 bivalents with one extra coil of prophase chromosome (arrow) at diakinesis. (458x) Cell had 7 bivalents with one extra coil of prophase chromosome (arrow) at diakinesis. (1017x) Cell had 7 bivalents with a big mass of double-stranded thick chromosomal threads (arrow) at diakinesis. (903x) Cell had 7 bivalents plus 2 univalents at diakinesis. (788x) l9 20 Abnormal Meiotic Behavior Early Prophase I The occurence of cytomixis was observed at early prophase I, in which parts of the nucleus of some donor cells migrated into the adjacent recipient cells (Fig. 29). Some cells were seen to be connected by several spiralized chromosomal threads (Fig. 30). Donor chro- mosomes were present in the plasma of the adjacent cell. About % out of a total of 800 early prophase I cells observed were found to have additional chromosomal materials in the cells (Fig. 31 and Fig. 32). They appear to be the consequence of cytomixis. Diakinesis Out of a total of 1114 cells observed, 66.97% (746 cells) showed no unusual chromosome behavior at diakinesis. The rest of the cells exhibited various types of cytological abnormalities (Table 1). 28% of the total 1114 cells showed a tendency for the chromosomes to congre- gate into two or more groups (Fig. 11, Fig. 12 and Fig.13). 1.08% of the cells had 8 bivalents or 7 bivalents plus 2 univalents (Fig. 37), 3.5% of the cells were observed to have 6 or 5 bivalents (Fig. 38). Also the results of cytomixis were observed in which the recipient cells had seven bivalents with one extra coil of prophase chromosome or with a big mass of double stranded thick chromosomal threads (Fig. 33, Fig. 34, Fig. 35 and Fig. 36). It appeared that a certain amount of chromosomal material was lost by some cells and gained by others. Metaphaseml’ At metaphase I, multipolar divisions first became con- clusively evident. Instead of aligning themselves to form a single 21 Abnormal Meiotic Behavior: Multipolar Cell Division (MPD) Figure 11. Figure Figure Figure Figure Figure Figure Figure Figure 12. 13. 14. 15. 16. 17. 18. 19. Chromosomes congregated into 2 groups; one group with 4 bi-valents, the other with 3 bivalents (4-3) at diakinesis. (751x) Chromosomes congregated into 2 groups: one group with 6 bivalents, the other with 1 bivalent (6—1) at diakinesis. (821x) Chromosomes congregated into 4 groups with 4-1-1-1 at diakinesis. (765x) Cell with 7 bivalents separated into 2 groups (4-3) to form two "micrometaphase" plates at metaphase I. (748x) Cell with 3 groups (4-2-1) of bivalents at metaphase I. (680x) Cell with 4 groups (3-2-1-1) at metaphase I. (680x) Abnormal groupings of dyad chromosomes (4-3) at anaphase I. (595x) Abnormal groupings of dyad chromosomes (3-2—2) at anaphase I. (776x) Chromosomes separated into 2 groups (6-1) at prophase II. (850x) ‘ j”. I 43$ 3. fa } " '«l a a. “ J mgr)? i O Q b.‘ . a ‘9 s. r‘ffl/’.I -.."t.". ‘. ' "s _ .‘ ‘ " I. V (s. *-, f8" 4 a; -‘ a 51* rrfil‘ . . -' i ' m ' O .. .. ‘ Q. J. 23 Table 1 Chromosome Behavior at Diakinesis in Clones of CC-37-119 Number of Bivalents Number of Cells Percentage 7 746 66.97% 6-1 101 9.07% 2 groups 5-2 71 6.37% 21.36%fi 4-»3 66 5.92% 28.10% ~—5-1—1 17 1.53Z—W (313 4—2-1 27 2.42% cells) 3 groups 3—3-1 15 1.35% 5.93%- L—3—2-2 7 0.63%-J 3-2-1-1 6 0.54% 4 groups[ 3 0.81%4 4-1—1-1 3 0.27% 8 12 1.08% 6 32 2.87% 5 7 0.63% Cytomixis 4 _--- 0.36%_‘ Total Number of Cells 1114 24 equatorial plate, the seven bivalents were separated into two or more groups and formed more than one metaphase plate (Table 2). Each group con- tained one to six bivalents. Grouping of chromosomes at metaphase I and later stages can be divided into four categories: (1.) normal cells with seven bivalents forming a single metaphase plate (Fig. 4); (2.) cells with seven bivalents separated into two groups (6-1, 5-2 or 4-3) to form two "micro- metaphase" plates, (Tai, 1970) (Fig. 14); (3.) cells with three groups (5—1-1, 4-2-1, 3-3-1 or 3-2—2) of bivalents (Fig. 15); (4.) cells with four groups (4-1-1—1, 3—2-1-1 or 2-2-2-1) (Fig. 16). Two-group separa- tions were more frequent than three-group or four-group separations. The overall frequency of multipolar cell division observed at metaphase I is 29.99%, or 413 of 1377 cells observed. Irregular chromosome numbers were observed in 2.48% of the cells which had 8, 6 or 5 bivalents line up on the equator (Table 2) (Fig. 42 and Fig. 43). They oriented on one or more metaphase plates. Occasionally cells with abnormal chromosome numbers exhibit multipolar cell division. Out of 1377 cells, 0.44% in this stage exhibited cytomixis. In addi— tion to seven bivalents, cells with extra chromosome materials were ob- served. The seven bivalents may form a single metaphase plate or multiple microplates. (Fig. 39, Fig. 40 and Fig. 41). Early segregation of bivalents into dyads were observed at this stage. Irregular chromosome behavior which occurred at earlier stages con— tinued at anaphase I (Table 3). Multipolar cell division and the 25 Abnormal Meiotic Behavior: Figure Figure Figure Figure Figure Figure Figure Figure Figure 38. 39. 40. 41. 42. 43. 44. 45. 46. Cell had 6 bivalents at diakinesis. (729x) Cell had 5 bivalents with one prophase chromosomal threads (arrow) at metaphase I. (463x) Cell with 7 bivalents to form a single metaphase plate had extra chromosomal materials (arrow) at metaphase I. (579x) Multipolar cell division and cytomixis occurred in the same cell. Cell with 7 bivalents to form multiple microplates (2-2-2-1) with extra chromosomal materials (arrow) at metaphase I (695x) Cell had 8 bivalents and separated into 2 groups (6-2) at metaphase I. (1028x) Cell had 6 bivalents and separated into 2 groups (5-1) at metaphase I. (897x) Cell had extra chromosomal material (arrow) at anaphase I. (440x) Multipolar cell division and cytomisix occurred in the same cell. Cell separated into 3 groups (3-2-2) had extra chromosomal material (arrow) at anaphase I. (637x) Cell had a lot of extra chromosomal materials at anaphase I. (672x) a v, --‘@ “i 8"!” . o .o Ino'W so 26 o i}. ' ' for... \ 4' via- . I ‘ . . C - h h. ' ‘2‘ ’r Q '~ \ O ‘5'. ', . - @ © 27 Table 2 Chromosome Behavior at Metaphase I in Clones of CC-37-119 Number of 3.121299 “399233.925..- Percentage. 7 867 62.96% 6-1 160 11.62% 2 groups 5-2 89 6.46% 24.47%-[ 4-3 88 6.39% 29.99% r—B-l—l 15 1.09%“ (413 4-2-1 27 1.96% cells) 3 groups 3—3—1 7 0.51% 4.65%'~ -—3-2—2 15 1.o9°/.-J 4-1-1-1 2 0.15% 4 groups 3-2-1—1 5 0.36% 0.87%-J 2—2-2-1 5 0.36% 8 6 0.44% 6 25 1.82% 5 3 0.22% Cytomixis 6 0.44% Early Separation 57 4.14% Total Number of Cells 1377 28 Table 3 Chromosome Behavior at Anaphase I in Clones of CC-37-119 Numberrpf_pivalents Number of Cells Percentage 7 678 60.37% 6-1 151 13.45% 2 groups 5-2 77 6.86% 25.30%‘ 4-3 56 4.99% 28.05% ,_5_1-1 5 0.45%‘T (315 4—2—1 10 0.89% cells) 3 groups 3-3—1 9 0.80% 2.59%‘ L-3-2-2 5 0.451-9 4 groups C3—2—1—1 2 0.18%] 0.18%" 8 10 0.89% 6 22 1.96% Cytomixis 4 0.36% Unequal Segregation 26 2.32% Precocious Division 12 1.07% Chromatin Bridge 41 3.65% Lagging Chromosome 15 1.34% Total Number of Cells 1123 29 cytomixis were both observed at this stage. Approximately 28.05% of the total 1123 cells examined at anaphase I had abnormal groupings of dyad chromosomes (Fig. 17 and Fig. 18). The grouping patterns seem to follow those observed at metaphase I. Five cells or 0.36% of the total contained extra chromosomal materials which may have resulted from cytomixis at earlier stages (Fig. 44, Fig. 45 and Fig. 46). Other types of cytological abnormalities at anaphase I include: chromatin bridge (3.65% out of the total 1123 cells observed) (Fig. 48), lagging chromosomes (1.34%) (Fig. 46), unequal segregation (2.32%) and precocious division (1.07%) (Table 3). Extra daughter nuclei are the main taype of irregularities observed at telophase I (Table 4). The presence of extra daughter nuclei may be related to earlier irregularities, such as multipolar cell division and cytomixis. Chromatin bridges sometimes remain intact through telophase I. A prophase II, chromosomes showed a tendency to separate into several groups in some cells (Fig. 19). Chromosomal fragments were also observed. A total of 957 cells were analyzed at metaphase II (Table 5). Two hundred eighty eight cells or 30.09% exhibited multipolar cell division with supernumerary metaphase plates in pretty much the same patterns as metaphase I (Fig. 20, Fig. 21, Fig. 22 and Fig. 23). Some microcells had one to six chromosomes, but showed typical X-shaped metaphase II chromo- some morphology and formed the typical metaphase plates (Fig. 21, Fig. 22 and Fig. 23). The loss of chromosomes may be the results of multipolar 3O Abnormal Meiotic Behavior: Multipolar Cell Division (MPD) Figure Figure Figure Figure Figure Figure Figure Figure Figure 20. 21. 22. 23. 24. 25. 26. 27: 28. Cell had two metaphase plates (5-2) at metaphase II. (1012x) Two microcells at metaphase II, one with 4 dyads, the other with 3 dyads. Arrows showed cytokinesis. (867x) One cell had normal metaphase II. The other cell had 6-1 grouping and began supernumerary cytokinesis (arrow) at metaphase II. (881x) One microcell with only one X-shaped chromosomes at metaphase II. (1098x) Chromosomes separated into 2 groups (4-3) and supernumerary cytokinesis occurred (arrows) at anaphase II. (867x) Multipolar Cell Division (MPD) at anaphase II (5-2) and supernumerary cytokinesis occurred (arrows). (867x) Chromosomes separated into 3 groups (3-3-1) at anaphase II. (728x) Cell with 6 daughter cells at quartet stage. (867x) Cell with 5 daughter cells and some micronuclei at the quartet stage. (535x) .m- #x ‘0 0 A" J‘\" o '5 o ‘ d .‘1. ~" 0 3‘ I '. '\ . r‘ . 3 1 ) u.- ’1 IN (1‘ 32 Table 4 The Number of Nuclei at Telophase I in Clones of CC-37-119 Number of Nuclei Number of Cells Percentage 2 (Normal) 211 65.73% 3 48 14.95% 4 35 10.90% 5 17 5.30% Chromatin Bridge 10 _. _._§;AJZL__ Total Number of Cells 321 33 Table 5 Chromosome Behavior at Metaphase II in Clones of CC-37-119 Numbergof Dyads __._"Ngmbgr_2fugg}]§______-_.-_Pgrcentage 7 588 61.44% 6—1 119 12.43% 2 groups 5-2 71 7.42% 25.70%1 4-3 56 5.85% 30.09% 7‘5-1-1 7 0.73%‘7 (288 4-2-1 11 1.15% cells) 3 groups 3-3-1 11 1.15% 4.18%~ L—3-2—2 11 1.15%-J 4-1—1-1 1 0.10% 4 groups I: :1 O.20%-J 3-2-1—1 1 0.10% 8 11 1.15% 6 16 1.67% Chromosomal Fragment 9 0.94% Early Sggregation 40 4.17% Total Number of Cells 957 34 Table 6 Chromosome Behavior at Anaphase II in Clones of CC-37-119 Number of Dyads Number of Cells Percentage 7 745 60.62% 6-1 180 14.65% 2 groups 5-2 78 6.35% 25.96%1 4-3 61 4.96% 27.91% r-5-1-1 9 0.73%“ (343 4-2-1 6 0.49% cells) 3 groups 3-3-1 6 0.49% 1.87%“ L--3—2--2 2 0.16%" 1. groups [4-1—1-1 1 0.087.] 0.08%-J 8 11 0.90% 6 23 1.87% 5 1 0.08% 4 1 0.08% Unequal Segregation 10 0.81% Chromatin Bridge 53 4.31% Lagging Chromosome 34 2.77% Chromosomal Fragment 8____ 0.65% ........... Total Number of Cells 1229 35 cell division and supernumerary cytokinesis which occurred at metaphase I and/or metaphase II and grouping of chromosomes may proceed into anaphase 11. Early segregation of chromatids and chromosomes fragments were also observed at metaphase II. Multipolar cell division continued at anaphase II (Fig. 24, Fig. 25 and Fig. 26). Chromosomes separated into several groups and migrated toward opposite poles. Out of a total of 1229 cells analyzed at anaphase II, different types of irregularities were recorded as follows: multi- polar division 27.91%, chromatin bridge 4.31%, lagging chromosomes 2.77%, fragmentation 0.65% and unequal segregation 0.81% (Table 6). Sometimes more than one type of irregularity may occur in the same cell. Most telophase II cells had two micronuclei. The remains of chroma- tin bridges were observed in some cells. A total of 1587 quartets were analyzed. Approximately 39.76% contained either micronuclei or more than four daughter cells (Table 8) (Fig. 27, 28 and Fig. 27). Out of a total of 4802 microspores examined after the quartet stage, 59.18% of them had a normal sized nucleus, 36.26% contained 2 to 10 nuclei of different sizes and another 3.94% carried very small nuclei (Table 9) (Fig. 49). Pollen Mitosis With a total of 266 metaphase cells and 88 anaphase cells analyzed, 50.00% and 38.64%, respectively, were observed to have normal pollen mito- sis. Cells had normal seven X-shaped chromsomes at metaphase (Fig. 50), 36 Abnormal Meiotic Behavior Figure 47. Figure 48. Figure 49. Four daughter cells at quartet stage. One daughter cell had extra chromosomal materials (661x) Chromatin bridge at anaphase I. (758x) One microspore had a normal sized nucleus. The other had three small nuclei (799x) Normal Pollen Mitosis Figure 50. Figure 51. One Inicrosporocyte had normal 7 X-shaped chromosomes at metaphase (1447x) One ndcrosporocyte had normal 14 single-stranded chromosomes at anaphase (951x) Abnormal Pollen Mitosis Figure 52. Figure 53. Figure 54. Figure 55. One nficrosporocyte had one X-shaped chromosome (951x) One ndcrosporocyte had 11 single-stranded chromosomes (882x) One uncrosporocyte had 3 single-stranded chromosomes, two with 2 single-stranded chromosomes. One had 5 X-stranded chromosomes and 5 single-stranded chromosomes (882x) One nficrosporocyte had 8 X—shaped chromosomes (882x) 38 Table 8 The Number of Nuclei at Quartet Stage in Clones of CC-37-119 Number of Nuclei Number of Cells Percentage (%) 4 956 60.24 5 122 7.69 6 245 15.44 7 52 3.28 8 163 10.27 9 15 0.95 10 12 0.76 11 7 0.1.4 12 8 0.50 13 1 0.06 14 4 0.25 15 l 1 0.06 16 0 0.00 17 1 0.06 Total Number of Cells 1587 39 Table 9 The Number of Nuclei at Microspore Stage in Clones of CC-37-119 Number of Nyelei __ Number of Cells Percentage (%) 1 (Normal) 2842 59.18 2 1082 22.53 3 344 7.16 4 160 3.33 5 92 1.92 6 37 0.77 7 9 0.19 8 12 0.25 9 2 0.04 10 2 0.04 11 1 0.02 One Small Nuclei 189 3.94 Chromatin Bridge 3Quu....--.____...-_--- 0.62 Total Number of Cells 4802 40 and normal 14 single-stranded chromosomes at anaphase (Table 10 and Table 11) (Fig. 51). The other cells might have different numbers of chromo- somes or chromosome fragments (Table 10 and Table 11) Fig. 52, Fig. 53, Fig. 54, Fig. 55 and Fig. 56). Multipolar mitosis was also observed in pollen grains. (Fig. 54, Fig. 57 and Fig. 58). Pollen Viability Pollen viability was examined by the staing reaction widi Iz-KI solu— tion. Out of a total of 2783 pollen grains, 58.07% of them were stained darkly and believed to be viable (Table 12) (Fig. 59 and Fig. 60). They were arbitrarily classified into 2 sized groups: the large sized group presumably are those with a normal or more than normal number of chromo- somes (Fig. 59); and the small sized group are presumably those to have fewer than the normal number of chromosomes (Fig. 60). 41 Table 10 Chromosome Behavior at Metaphase of Pollen Mitosis in Clones of CC-37-119 Number of X—Shaped Chromosomes Number of Ce}j§’_fer§egtage (fig 1 15 5.64 2 5 1.88 3 6 2.26 4 6 2.26 5 7 2.63 6 , 25 9.40 7 (Normal) 133 50.00 6 X-Shaped + 2 Single-Stranded Chromosomes 24 9.02 5 X-Shaped + 4 Single-Stranded Chromosomes 17 6.39 3 X-Shaped + 8 Single-Stranded Chromosomes 1 0.38 8 12 4.51 6 X-Shaped + 1 Single-Stranded Chromosomes 3 1.13 2 X-Shaped + 1 Single-Stranded Chromosomes 1 0.38 7 X-Shaped + 1 Single-Stranded Chromosomes 1 0.38 7 X-Shaped + 2 Single-Stranded Chromosomes 1 0.38 6 X-Shaped + 3 Single-Stranded Chromosomes 1 0.38 8 X-Shaped + 1 Single-Stranded Chromosomes 2 7 X—Sheped + Fregmept‘-_____’---..-_-_~_ 6 2.26 Total Number of Cells 226 42 Table 11 Chromosome Behavior at Anaphase of Pollen Mitosis in Clones of CC-37-119 Number of Single-Stranded Chromosomes __Number of Cells__Pereeg£ege_(%Q 1 5 5.68 2 4 4.54 3 4 4.54 4 a 4.54 5 3 3.41 6 2 2.27 7 2 2.27 10 1 1.14 11 2 2.27 12 5 5.68 13 9 10.23 14 (Normal) 34 38.64 15 3 3.41 16 6 6.82 16 + Fragment 2 2.27 14 + Fragment 2 2.27 Total Number of Cells 88 43 Abnormal Pollen Mitosis Figure 56. Figure 57. Figure 58. Viability of Figure 59. Figure 60. Figure 61. One Inicrosporocyte had 16 single- stranded chromosomes and one chromosome fragment (1122x) Miltipolar mitosis in microsporo- cyte (675x) Multipolar mitosis in microsporo- cyte (900x) pollen grains Normal sized, viable pollen grain was darkly stained by Iz-KI solution (591x) Small sized, viable pollen grains were darkly stained by Iz-KI solution (591x) Non-viable pollen grains were not stained by Iz-KI solution (591x) 44 45 Table 12 Viability of Pollen Grains in Clones of CC-37-119 Number of Cells Percentage (%) Fertile Pollen Grains Normal Size . 1314 47.22%'————1 58.07% Small Size 302 10.82% Non-Fertile Pollen Grains Normal Size 683 24.54%‘————j 41.93% Small Size 484 11:231-4- ’---»-----_--w----‘- - Total Number of Cells 2783 DISCUSSION Inheritance of Colchicine-Induced Irregularities During his study of colchicine-treated diploid Fairway crested wheat- grass, Tai (1964, 1970) discovered two plants CC—37-119 and CB-9-85, to have exceptional irregular cytological behavior, e.g. multipolar cell division, supernumerary cytoplasmic cleavage, unequal segregation, chroma- tin bridges, lagging chromosomes, and micronuclei at different stages of meiosis. Both plants had been treated with 0.1% colchicine solution for 12 hours. Perhaps due to low colchicine concentration and short duration of exposure, polyploidy did not occur; instead both plants exhibited a high degree of spindle abnormalities (Sakharov, e£_al, 1969). In his study of progeny of CB-9-85, Chen (1976) observed the same types of ir- regularities. Plant CC-37-119 has been propagated asexually since 1963. Through the years, some types of cytological irregularities persisted in its clones. Effects of colchicine treatment seem to be permanently main- tained in the plants without restitution. They are inheritable, transmit- ted from generation to generation. Tai (1970) proposed that to guide chromosome migration in angiosperm plants, an organelle which he termed "spindle organizer" would move toward opposite sides of the cell and establish themselves as poles. If "spindle organizer" is broken, multi- polar cell division (MPD) results. The current study seems to further 46 47 strengthen this model. Since "spindle organizer" is a physical entity, it -can be broken by physical or chemical treatments. Attraction between broken pieces of "spindle organizer" and the chromosomes would result in multipolar cell division. Multipolar Cell Division Multipolar cell divisions have been reported to occur spontaneously in both animals or plants or can be induced artificially. In the present study of clones of CC—37-199, multipolar cell division occurred at every meiotic stage from late diakinesis through the quartet stage, and during pollen mitosis, too. Thompson (1962) suggested that after "complement fractionation", each group of chromosomes is an independent unit. Tai (1970) also empha- sized that the behavior of each micrometaphase plate in a cell with multi- polar meiosis is an independent event. Their points are further mani— fested in the present study. All possible numerial combinations of chromo- some groupings have been observed in this study. Multipolar cell division (MPD) has been observed at every meiosis stage. From the number of cells exhibited, MPD seems to be dropped from MI and MII to AI and A11. How- ever, a X2 test showed this difference is not statistically significant (Table 7). This indicates that there has been no restitution of "spindle organizer". Even the chromosomes migrate toward the poles together and become situated close to one another. They remain as a separate group and do not regroup. The chromosome movement of different groups showed cer- tain degrees of asynchronization. One group may remain in metaphase II, 48 Table 7 Multipolar Cell Division (MPD) at Different Meiotic Stages in Clones of CC-37-119 5.5183“ _. _- _._.C.eJ.1.s- raise- 24394331} .8. xi 521293.822. Fssausmsrgf ., 99.1.1.5. Diakinesis 313 801 1114 Metaphase I 413 964 1377 Anaphase I 315 808 1123 Metaphase II 288 669 957 Anaphase II ‘_ 343 886 1129 Total 1672 4128 5800 _ 1672 X2 = 1.99‘< 9.448 (Non-significant) 49 while another group proceeds to anaphase II (Fig. 24). One cell had an intact chromosome complement and 2 microcells as results of MPD (Fig. 22). They all came from the same primary microsporocyte, however, multi- polar cell division occurred in one secondary microsporocyte without influencing the other (Fig. 22 and Fig. 27). All the above phenomena pro- vide further evidence to substantiate that multipolar cell division in each group is an independent event, and that each micrometaphase plate be- haves as an independent unit with its own movement. As the number of groupings increases, the grequency of the occurrence decreases. Two-group separation occurred more frequently than three-group or four-group sep- aration. The independent behavior of multipolar cell division also suggests the same conclusion as Tai's (1907), namely, an organelle exists which functions analogously to the centriole of animal cells. Darlington and Thomas (1937) proposed that "pole determinants" exist as diffuse parti- cles. They congregate when spindle poles are normally formed, but remain separate at other times. They have the function of centrosomes and pos— sess their continuity as single and coherent bodies. The spindles are developed through the cooperation between centromere and pole determi- nants. Swanson and Nelson (1942) considered the extra-pole determinants to be a prerequisite for multipolar meiosis. Walters (1958) described the "spindle organizer" that may be a compound structure, usually single and following the regular division cycle. However, it may undergo super- numerary division under extraordinary conditions into substructures. Based on the observations of "complement fractionization", Thompson 50 (1962) proposed two possible origins of multipolar cell divisions. One is that the chromosomes are guided by a split spindle; the other is that the chromosomes are first grouped and then form their own split spin- dles. The microtubule-organizer-center described by Pickett-Heaps (1969) is a diffuse, amorphous, osmiophilic and differentiated cytoplasmic re- gion active in microtubule formation. It has the ability to initiate polymerization and depolymerization of microtubules and to determine their orientation. All above models seem to be in agreement with and can be used to describe our own hypothetical organelle, the spindle organizer (Tai 1970). Tai describes the "spindle organizer" as a single organelle which controls the formation of spindle apparatus and the migration of chromosomes. There is only one spindle organizer in a male or female gamete. The spindle organizer is genome specific. After fertilization. spindle organizers from male and female gametes may either fuse, differentially disintegrate, or remain disjoined (Orton and Tai 1977). If they do not fuse or disinte- grate, multipolar cell division arises. Also, the spindle organizer can be broken spontaneously or artificially. When it is broken, multipolar cell division also occurs. It is also a possible mechanism for the occurrence of lagging chromosomes, unequal segregation and early separation which were observed in this investigation and occur frequently in hybrids. Lack of attraction between chromosomes of one species and spindle organizer of a different origin will result in large numbers of laggards. The significance of multipolar cell division in the evolution of chromosome number has been discussed by several authors. "Split spindle" 51 are known to be a mechanism responsible for variation in chromosome num- bers in somatic tissue of many species. (Li and Tu 1947; Huskins 1948; and Vaarama 1949). Thompson (1962) pointed out that "complement fractionation" produces gametes with extremely variable chromosome numbers. If gametes have lost or gained too many chromosomes, they appeared to be aborted. However, some of these gametes will be functional and kept in the popula- tion due to a tendency for the formation of balanced or less unbalanced genome. Thompson also proposed that multipolar cell division provides an evolutionary mechanism for decreasing the ploidy level. After careful observation of microsporogenesis of colchicine-treated diploid.Ag£gEy£gn cristatum and inter- and intra-specific hybrids of Mimulus glabratus, Tai (1970), Tai and Vickery (1970, 1972) recognized the same evolutionary significance of multipolar cell division. Tai (1970, 1971) further pointed out the possibilities of using multipolar cell division in the studies of plant genetics and plant breeding. Induction of haploidy by multipolar cell division followed by doubling of the chromosome complement may possibly produce individuals which are homozygous for every gene locus. The present observations of CC-37-119 clones show that multipolar cell division is the major irregularity which occurred in 1672 of a total of 2176 abnormal cells (76.84%). This indicates that multipolar cell divi- sion is the main contribution of variable chromosome numbers during pollen mitosis. Figure 60 shows darkly stained, but smaller sized pollen grains. They represent pollens having fewer chromosomes but which may be viable. All these explain that multipolar cell division appears to provide a possible mechanism for the production of aneupolid microspores. 52 Cytomixis Cytomixis is a relatively widespread phenomenon. It has been re- ported in various plant species by many authors (Katterman 1933; Kihara and Lilienfeld 1934; Sarvella 1958; Kamra 1960; Takowska 1960, 1965, 1966; Romanov and Orlova 1971; Tai 1972; Kosova 1973; Syemyarykhina and Kuptsow 1974; Shkutina and Kozlovskaya 1974; Cheng 1974). In pollen mother cells, cytomixis occurs most frequently in the early prophase I, and rarely in metaphase I and anaphase I (Katterman 1933; Romanov and Orlova 1971; Tai and Vickery 1972; Shkutina and Kozlovskaya 1974). Romanov and Orlova (1971) suggested that chromosomes or parts of nuclei of donor cells migrate through the "cytomictic channel" (Weiling 1965; Heslop-Harrison 1966) into the adjacent recipient cell, and remain in the form of that stage. The migration could be in multiple directions or some- times in a single direction (Cheng 1974). In the present study, the actual processes of cytomixis -— the migra- tion of chromosomal materials from the donor cell into the adjacent cell - was also observed at early prophase I, usually before pachytene (Fig. 29, Fig. 30, Fig. 31 and Fig. 32). The consequences of cytomixis, i.e., the addition or reduction of chromosome materials were observed at vari— ous meiotic stages (Figures 33-46). The extra threads have been proved to be chromosomal materials by their positive reaction toward the Feulgen stain. They usually appear as long threads with different degrees of con- densation at early meiosis. As meiosis proceeds in the host cells, these extra materials also assume regular division progress, e.g. further con- densation, migration and lining up at the equator. Usually the regular 53 and extra chromosomes move asynchronously within the same protoplast (Fig. 45 and Fig. 46). While with the extra materials move at a slower pace, sometimes, they can catch up and show typical chromosome morphology at metaphase I and/or anaphase I. Subsequently, cells with various chromo- some number at metaphase II, anaphase II and pollen mitosis were found. The presence of micronuclei at the quartet stage (Fig. 47) and in micro- spores was also observed. Darkly stained, different sized pollen grains, representing more or fewer than normal chromosome numbers, may be viable. However, aneuploid gametic chromosome number can be caused by other pro- cesses in addition to cytomixis. The importance of cytomixis in the evolu- tion of chromosome number in plants has been recognized by many authors. (Katterman 1933; Kihara and Lilienfeld 1934; Persival 1930; Sarvella 1958; Kamra 1960; Romanov and Orlova 1971; Tai 1967; Tai and Vickery 1972; Cheng 1974). Generally, the chromosomes, transferred through cytomitic channels, revealed severe structural damage, with subsequent disintegration and de- generation (Katterman 1933, Weiling 1965, Tarkowska 1966; Romanov and Orl- ova 1971; Shkutina and Kozlovskaya 1974). Similar phenomenon was observed in this study. However, it is possible that the chromosomes pass through usually the regularthe cytomitic channels undamaged and intact and are in- corporated into the nucleus of the recipient cell (Romanov and Orlova 1971) (Fig. 36, Fig. 37 and Fig. 42). Thus, cytomixis is a possible mech- anism that increases and decreases the number of chromosomes of future ga- metes. Presumably a large number of chromosomes were involved in the do- nor and recipient cells. They appear to degenerate or form aborted 54 gametes. However, if just a small number of chromosomes are lost, the hypocells still may survive and form unbalanced but functional gametes. Therefore, cytomixis is a possible mechanism for the cause of aneuploidy evolution; i.e. hyperploid and hypoploid (Romanov and Orlova 1971; Tai 1967; Tai and Vickery 1972). Romanov and Orlova (1971) and Cheng (1974) also suggested that cytomixis could be the cause of multinuclear cells which may in turn give rise to polyploids. The cause of cytomixis remains controversial and unsolved. Some sug— gested the phenomenon of cytomixis is an abnormal condition which could be caused by mechanical damage or environmental pressure (Takats 1959; Tarkowska 1960, 1965, 1966), by fixing solution (Takats 1959), by abnor— mal environment conditions (temperature, rainfall, drought, radiation) (Tung, e£_al 1973 in Cheng 1974), by disease and weakening (Fraser 1914), or by plant hybridization (Woodworth 1929). However, Cheng (1974) strong- ly suggested that cytomixis is a normal physiological phenomenon; other external factors, e.g., temperature, fixation -- etc., may enhance its oc- currence. The results of this study cannot unequivocally explain the cause of cytomixis. Further investigations have to be done. SUMMARY The cytology of colchicine-treated, diploid Agropyron cristatum, CC-37-119, was studied. The effects of colchicine treatment were found to be inheritable. Different types of cytological irregularities were ob- served, such as multipolar cell division, cytomixis, chromatin bridges, lagging chromosomes, unequal segregation, precocious division and others. These abnormalities appear to result in the reduction of pollen via- bility. They may provide mechanisms for the production of aneuploid ga- metes which could in turn lead to aneuploid plants. 55 LIST OF REFERENCES LIST OF REFERENCES Abraham, 8., Cytologia, 32, 537 (1974). Amer, S.A., and O.R. Farah, Cytologia, 32, 507 (1974). Bammi, R.R., Nature, 293, 608 (1965). Baltzer, F., Aich Zellforsch, 2, 549 (1909). Bayliss, M.W., Nature, 222, 529 (1973). Bopp—Hassenkamp, G., Exptl. Cell Res., 33, 182 (1959). 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