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EX KLOTZSCH presented bg meu L A. BEMPOMC— has been accepted towards fulfillment of the requirements for Mdeqree in II DET- 4 2g. KENM’W C SINK Major professor nateJuw (214 1% 0469. ABSTRACT KARYOMORPHOLOGICAL AND CYTOLOGICAL INVESTIGATIONS OF POINSETTIA, EUPHORBIA PULCHERRIMA WILLD. EX KLOTZSCH by Maxwell A. Bempong The primary objectives of the investigation reported in this thesis were (1) to evaluate genetically the results of reciprocal crosses between 28 and 42 chromosome cultivars of poinsettia, (2) to determine the karyomorphology of 28 chromosome types, and (3) to study the meiotic chromosome associations in 42 chromosome plants. The reciprocal crosses yielded three 35 chromosome progeny, char- acterized by reduced rate of vegetative growth and floral development. Pollen from 28 chromosome cultivars (EcEe White) failed to effect seed formation when 42 chromosome plants were employed as the pistillate parents. No seed was realized from selfing 42 chromosome cultivars. To determine the basic chromosome number of E, pulcherrima, metaphase plates were analyzed in terms of length of chromosomes, position of kinetochore, ratio of long—arm to short—arm, percent total comple— ment length, percentage of long—arm, mean of absolute length of each set of chromosomes in a cell and presence or absence of secondary constric— tion and trabant. On the basis of cytological analyses, it may be inferred that the basic chromosome number is seven. However, additional genetic evidence is needed to support the cytological findings. For the explanation of the segregation of parental genetic traits, such as bract color, in characteristic Mendelian ratios, the following Page 2. M. A. Bempong reasons were advanced: (l) the progenitors of our current commercial cultivars might have arisen as allotetraploids, (2) as a result of autosyndesis the original species parents behaved as amphidiploids, and (3) the progenitors had many loci which were alike or similar. Subsequently, interactions of these similar genes may produce diploid segregation of certain parental traits, such as bract color, while at the same time, segregation for other traits may suggest complex gene— tical behaviour. The meiotic chromosome pairing of the 42 chromosome plants showed a higher frequency of multivalents than bivalents. Meiotic irregu— larities such as irregular distribution of chromosomes, resulting from abnormal disjunction of multivalents, and lagging of chromosomes at both anaphase I and II were observed. These abnormalities in meiosis are indicated as one of the factors contributing to low seed set, probably through production of nonviable, unbalanced gametes. Major Professor: Dr. Kenneth C. Sink KARYOMORPHOLOGICAL AND CYTOLOGICAL INVESTIGATIONS OF POINSETTIA, EUPHORBIA PULCHERRIMA WILLD. EX KLOTZSCH By \>~ gt Maxwell AI Bempong A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1967 ACKNOWLEDGEMENT The author extends his appreciation to Dr. Kenneth C. Sink, advisor, for his helpful suggestions and counsel in the planning and writing of this work. Special thanks are also extended to Dr. G. B. Wilson of the Department of Botany and Mr. David A. Gilbart, a colleague in the Department of Horticulture, for their suggestions and advice. For her assistance in processing research data and prepara- tion of manuscript, the author expresses his gratitude to Marie Antoinette Madjinou. Finally, the author expresses his indebted— ness to the Government of Ghana for financing his graduate studies. ii TABLE OF CONTENTS PAGE ACKNOWLEDGEMENT.............. ..................... . ......... ii TABLE OF CONTENTS ..................................... . ..... iii LIST OF TABLES.......... ............ ..... ........... .. ...... v LIST OF FIGURES ...... . .................. ....... ............ . vi INTRODUCTION ............ . ......... ... ....................... 1 LITERATURE REVIEW ....... ..... ..... ...... ........... . ........ 5 Chromosome counts.. ...................................... 5 Evolutionary changes in the poinsettia ................... 6 Meiotic chromosome pairing ............................... 8 Hybridization ............. . ...... . ............... . ....... 9 Bract color inheritance ................... . .............. ll EXPERIMENT I. 28 x 42 chromosome cultivar and reciprocal cross.. ....................................... . .......... 13 Materials and methods ......... . ........... . .............. 13 Results ......................... . ........................ l6 Gross morphology ................ . ...................... 20 Cytological analysis... ................................ 20 EXPERIMENT II. Karyotype studies on 2n=28 cultivars........ 25 Materials and methods .................................... 25 Results ......... . ........... .. ............. . ........ ..... 25 EXPERIMENT III. Meiotic chromosome associations in 2n=42 cultivars.. .................................... . ......... 36 Materials and methods .................................. .. 36 Results ..... . ............. . ....................... . ...... 36 iii Table of Contents (Cont.) PAGE Nucleolar contents.. ....... ..... ....... ..... ........... 36 Chromosome associations. ............ ... ............. ... 44 Meiotic irregularities ................ . ......... ....... 46 DISCUSSION Reciprocal crosses between 2n=28 and 2n=42 cultivars and selfing ........ ... ..... .... ...... . ....... .... ............ 49 Karyomorphological analysis in 2n=28 cultivars ........... 54 Meiotic chromosome associations in 2n=42 cultivars....... 57 SUMMARY AND CONCLUSIONS........ ....... . ..... .. .............. 62 LITERATURE CITED ...... . ....... . ........... .. ........... ..... 66 iv TABLE LIST OF TABLES PAGE Results from reciprocal crosses between 28 and 42 chromosome cultivars and self-pollination........... 16 Mean of lengths in mm. of each set of 'Ruth Ecke' chromosomes and percentage of long arm length in 20 cells .................. . ............... . ......... 26 Mean of total length, percent of long arm, long arm- short arm ratio, percent of total complement length and absolute length of chromosomes in 20 metaphase plates in'Ruth Eckel..... ............. ....... ....... 28 Duncan's multiple range test for total mean length of chromosomes in 20‘Ruth Ecke'metaphase plates........ 29 Duncan's multiple range test for percent long arm ratio of chromosomes in 20'Ruth Ecke'metaphase plates..... 29 Distribution of chromosome associations in 2n=42 culti— vars ......................... ... .................... 44 LIST OF FIGURES FIGURE PAGE 1. Comparative heights of two parent plants with different levels of ploidy and their progeny ........ . ............. 15 2. Comparative heights of two parent.plants with different levels of ploidy and their progeny ............... . ...... l9 3. Comparison of floral development of 6—month-old plants with different levels of ploidy ...... . ........... .. ..... 22 4. Somatic chromosome complements of cultivars 66-376, 66-377, and 66—378 ....................... . ..... . ........ 24 5. Metaphase plates, photokaryotype and idiogram for 'Ecke WhiteC X6,000. ..... . ..... ....... ..... ..... ............. . 33 6. Metaphase plates, photokaryotypes and idiogram for 'Ruth Ecke' and 'White Eckefl X8,000... ..... ............ ....... 35 7. Number of nucleoli in six nuclei from cultivars 64—8 (2n=42). X8,000... ..... . .......... . .................... 39 8. Pollen mother cells from 64—5, with varying number of chromosome associations. X5,000... ..................... 41 9. Pollen mother cells from 64—5 with varying number of chromosome associations. X3,000 ...... .. ........... ..... 43 10. Observed number of types of chromosome associations in 60 pollen mother cells of 64—5..... ............ .. ....... 45 ll. Cytological aberrations, lagging and irregular chromo- some separations from pollen mother cells of 64-5. X6,000........... ............. .... .......... . ........ ... 48 vi INTRODUCTION Many genetic processes are reported (Darlington 1937; Stebbins 1950; Camp and Gilley 1943; Swanson 1964) to be operative in the evolution of plant species. Among these processes are: (a) gene mutations, (b) structural changes in chromosomes which may result from reciprocal translocations, centric fusions, pericentric inver— sions, and other forms of chromosome aberrations (White 1954), (c) inter-specific hybridization, polyploidy, and (d) apomixis. Morphological changes in chromosomes are considered to be one of the primary processes (Riley 1958) which have contributed greatly to speciation. While these changes may not be the direct causes of speciation, they are believed (Babcock 1942; Babcock gt. a1. 1942; Babcock and Camaron 1934) to create an interspecific sterility, in some species, that acts as a reproductive isolating mechanism. Morphologically, they affect the shape, number, and size of chromo— somes, either through gain or loss of chromocenters (Darlington 1937). In addition to morphological changes in chromosomes in relation to evolution of species, cytological investigations have revealed that many of our agronomic and horticultural crops evolved as hybrids many years ago. Assuming that amphidiploidy or allopolyploidy is followed by structural changes in the chromosomes as reported in the case of Crepis, particularly 9, neglecta and C, fuliginosa (Tobgy 1943; Camp 1945), creation of interspecific sterility will be the result culminating in a reproductively isolated species. The evolved species, either from changes in chromosome morphology or from interspecific hybridization followed by such chromosomal changes, or vice versa, may undergo direct increase of the genomic number. This may involve either somatic or reproductive tissues. Doubling of the chromosomes in the somatic tissues will result in a polyploid branch, which, when asexually propagated will yield a polyploid shoot. Euph: orbia pulcherrima, varieties Paul Ecke, Indianapolis Red, and Improved Albert Ecke are reported (Ecke 1963) to have arisen in such a manner. If the polyploid branch gives rise to germinal tissue, the gametes formed on the branch will have more than the n chromomsome number. A similar phenomenon has been reported by Stewart (1961) to occur in many 2n=28 chromosome poinsettia cultivars. He obtained nine "tetraploids" from root cuttings taken from "diploid" cultivars. If these two conditions occur in the original parent or parents to produce polyploids, more polyploids of higher magnitude can be obtained through the same fundamental processes, or by self—pollination of the polyploids or other crosses in which the polyploids are one or both of the parents. From cytological analyses in small grain and some horticultural crops it seems reasonable to infer that diploid condition is an indica- tion of primitivity and that specialization is enhanced by polyploidy derived from the original diploid parents through somatic or germinal, or both, increase in chromosome number. Haber (1925) emphasized that poinsettia, with its reduction in the number of glands, a well defined stelar, and the complexity of the vascular supply, make it one of the highly developed plants. Stebbins (1950) reported that about 50 percent of the known species of the Euphorbiaceae are polyploids. The question then is, is the species, Euphorbia pulcherrima, composed of only polyploids? The answer will call for cytogenetic evidence to support all possible theories and assertions. Currently all we know is that a large number of the cultivars of E. pulcherrima, in addition to other genetic variations such as bract—color, leaf shape inheritance, differential susceptibility to photoperiodism and tempera— ture for flowering, vary in terms of the degree of ploidy. Whether 2n=28 cultivars are diploids or tetraploids is not known. The lack of this information resides in the fact that there is no cytolo— gical or cytogenetic evidence to substantiate any claim that may be made in terms of the genome of the species. The present work on cultivars Ruth Ecke and White Ecke, with 2n=28 and eight other cultivars possessing a somatic chromosome complement of 42, was undertaken to determine the karyotype of the species and the mode of chromosome pairing in meiosis. To achieve these ends, three principal objectives were advanced. 1. To study the karyomorphology of the cultivars which are currently designated as diploids. 2. To make directional crosses between the 28— and 42-chromosome types and to cytologically analyze the resultant progeny, emphasizing the somatic chromosome complement they possess. 3. To determine the meiotic chromosome associa— tions in a 42-chromosome cultivar. LITERATURE REVIEW The term "so called" is often used to prefix diploid and other polyploid types in E, pulcherrima. The uncertainty as to the correct ploidy of the various cultivars stems from the fact that most of the genetic and cytogenetic investigations involving poinsettia have been directed toward the determination of chromosome number (Moyer 1934), inheritance of bract color (Stewart 1960, 1967) and embryogenesis (Sink 1963 and Milbocker 1966). There is also the possibility that small size of the chromosomes has given vent to lack of interest shown in karyomorphological studies of this species (Perry 1943). Chromosome Counts The cultivated poinsettias are composed of cultivars whose chromosome numbers are 28, 42, or 56. Cytological studies of the various cultivars since the early 1900's have clearly elucidated chromosome numbers in multiples of seven. Carano (1925) reported that E. pulcherrima had 10 chromosomes in the haploid cells. Moyer (1934) repudiated Carano's work by reporting that chromosome counts from the root tips of four species and three varieties of the genus Euphorbia, section poinsettia, were characterized by a diploid number of 28. In his studies on chromosome number and phylogenetic relationships in the Euphorbiaceae, Perry (1934) not only confirmed Moyer's (1934) report but emphasized that the morphology of the chromosomes in terms of number and size of the many sub-genic divisions of Euphorbiaceae revealed close relationship within and equally between the different sections and others showed complex and unrelated forms. 0n the basis of his findings, Perry (1943) classified the Euphorbiaceae into two groups. The first group he called the primary system, which consisted of n=8 and the second group or secondary system consisting of n=6, 7, 9, and 10. Sections such as Anisophyllum, Adenopelatum, and Poinsettia were grouped under the secondary system (Perry 1943). Improvement of smear technique poineered by Ewart (1957) heralded cytological studies of poinsettia on a large scale. Ewart and Walker (1960) and Pai (1960) reported 56 chromosome numbers in 'Mrs. Paul Ecke,‘ 'Indianapolis Red,‘ 'Improved Albert Ecke,‘ and 'Barbara Ecke Supreme.‘ For some of the 28-chromosome types, Ewart and Walker (1960) and Pai (1960) reported 'White Ecke,‘ '0ak1eaf,‘ 'Saint Louis,' 'Ruth Ecke,l and 'Henrietta Ecke.l Milbocker (1966) made crosses between 'Barbara Ecke Supreme' and 'White Ecke.‘ The resultant progeny from the cross possessed a somatic chromosome com- plement of 42. Evolutionary Changes in Poinsettia Haber (1925) points out that E, pulcherrima embodies both primitiveness and specialization. She goes on to emphasize that the reduction in number of glands from four to one and a well defined stelar nature, complexity of the vascular supply, and the disappearance of the vestigial traces are suggestive of the species' highly evolved nature. It is not known if polyploidy augmented this specialization as reported by Haber. However, Stebbins (1950) recognized as a mutational change which enhances formation through "concatenation of gene and chromosomal changes" with a concomitant reconstruction of the genotype of the ancestral species to yield derived species. Phylogenetic development of E, pulcherrima, compiled by Stewart (1957) showed that as a result of sport mutation, 'Mrs. Paul Ecke' and 'Ruth Ecke' were obtained from 'Oakleaf' in 1929 and 1932 respectively. 'Indianapolis Red,l 'Improved Indianapolis Red,‘ and 'Barbara Ecke Sup— reme,‘ each possessing a somatic complement of 56 chromosomes, are somatic mutations of 'Oakleaf,' a 28-chromosome type and the main trunk of the phylogenetic tree of E, pulcherrima. It must be mentioned that 'Oakleaf' arose as a chance seedling. Whether or not the progenitor of 'Oakleaf' was a 14— or 28—chromosome cultivar, we have no way of knowing. Stebbins (1947, 1950), Darl— ington, and Janaki Ammal (1945) point out that polyploids constitute 30 to 35 percent of the angiosperms found in the temperate zone. As high as 75 percent of the Gramineae (Stebbins 1940, 1949) are reported to be largely polyploids and a higher frequency is reported to exist among the Rosaceae, Polygonaceae, Malveraceae, and a host of families. Perry (1943) reported that the family Euphorbiaceae, contains about fifty percent polyploidy types. While polyploidy may predominate in one family, a diploid condition may have a high frequency in another. In Fagaceae (Swanson 1957), Moraceae, Cucubitaceae, and Polemoniaceae, polyploidy had not been reported to occur naturally. Such distribution of polyploidy among angiosperms, according to Stebbins (1938), correlates with the growth habit. Data on chromosome counts (Stebbins 1938) point to the direction that polyploidy is most prevalent in herbaceous perennial and equally infrequent among woody forms, whose base numbers range from 11 to 16. The chromosomes of species of Euphorbiaceae (Darlington and Wylie 1955) are reported to exist in multiples of 6, 7, 8, 9, and 10. There is therefore, the possibility that the poinsettia, a herbaceous perennial species, mostly propagated asexually, and with its somatic chromosome complement in multiples of 7 was highly susceptible to chromosome mutation changes. This could have been a direct genomic increase of one type or a derived polyploid initiated through the hybridization of some of the original species, probably a hybrid of 2n=12 and 2n=16, followed by a direct genomic increase of the resul— tant hybrid. If the latter course was the case, an allotetraploid would ensue. Meiotic Chromosome Pairing Chromosome associations as reported by Ewart and Walker (1960) and by Pai (1960) revealed the occurrence of multivalent formation. From the observed multivalent formation in pollen mother cells in both 56- and 28-chromosome types, Ewart and Walker (1960) suggested 7 and not 14, as was previously conceived, as the basic chromosome number of poinsettia. The basis of their suggestion was that the observed multivalents in diakinesis and subsequent movement of bivalents toward the poles in anaphase I in the 28—chromosome type could only indicate that more than two homologous chromosomes were present. Two explana— tions were offered by these two workers for the behaviour of the chromosomes. The first explanation was that the bivalent could have resulted from earlier quadrivalent association. The second explana— tion advanced to account for the behavior of the chromosomes supposed that a precocious separation during the first division could have accounted for the movement of bivalents toward the poles. Ewart and Walker (1960) favored the latter as the possible explanation. A confirmation of Ewart and Walker's finding, in terms of chromo— some association came from Pai (1960). She reported the presence of multivalents in the 28—chromosome types. Among the 56—chromosome cultivars, bivalent, quadrivalent, and hexavalent associations were found with the latter association being at a lower frequency. Hybridization Moyer (1934) crossed Oak to white and the reciprocal, and obtained a hybrid with a somatic complement of 28 chromosomes. From the result of these crosses, Moyer (1934) reported that E, pulcherrima possessed 28 chromosomes. Perry (1943) confirmed Moyer's previous counts. Ewart and Walker (1960) attempted directional crosses and selfing on a large scale. According to their data selfing produced the expected results, whereas directional crosses yielded mixed results. When the cultivar with the higher number of chromosomes was used as the pistil— late parent, i.e. 56 x 28, no progeny were obtained. Progeny with either 10 28 or 56 chromosomes, and not the expected 42 were obtained when the order was reversed. However, Milbocker (1966) reported 42 chromosome cultivars from 28 x 56 crosses using‘Ecke White'and'Barbara Ecke Sup- -reme'respectively. He observed that the 42 chromosome cultivars were vigorous and segregated for both parental characteristics in terms of bract color and leaf shape. One of the questions raised by Ewart and Walker (1960) was that seed, formed as a result of apomixis, show evidence of leaf shape seg— ragation? Stebbins (1950) expressed the view that a large number of apomicts are pseudogamous and require pollination for effective and successful production of seed just as much as sexual species. He also added that facultative apomicts may produce occasional hybrids when pollinated by different species. As an added explanation to the production of seed when 28 x 56 crosses were made, Ewart and Walker (1960) suggested that a nonreduc- tion or normal reduction could have occurred followed by doubling coupled with fertilization by a diploid pollen grain to yield 56 chromo— somes. Bremer (1962) attempted to explain Ewart and Walker‘s (1960) results in terms of endo—duplication mechanism, which he had found to be effectively operative in Saccharum species. Implicit in Bremer's explanation was that in hybrid species of Saccharum the increase in chromosome number could arise from doubling of chromosomes at the time of female sex cell formation within the female parents. Subsequently, 11 following meiosis I in the embryo sac mother cell, increased chromosome number could be expected in the embryo sacs and egg cells, arising as a result of endo—duplication. Sink (1963) reported that embryo sac formation occurred regardless of the direction in which crosses involving 28— and 56-chromosome cul— tivars was made. He attributed failure of seed formation when a 28— chromosome cultivar was used as the male parent to degeneration of the maternal ovule tissue. Histological observations revealed (Sink 1963) differentiation of normal embryo up to the plumular and the cotyledon- ary stage. At this time the ovule tissue appeared degenerated and shrunken. Antithesis of the above phenomenon is reported by Philippi (1960) in pelargonium. Here, notwithstanding the direction of crossing, no viable progeny were obtained. The explanation was that the embryo died while the endosperm remained normal. Since a normal embryo was found to be present up to a certain developmental stage, the possible cause of the degeneration was attributed to endosperm - maternal tissue incompatability (Sink 1963). Bract Color Inheritance Bract color inheritance is one plant characteristic of poinsettia which has been studied extensively. Contrary to the confusing state— ments on the phenomenon, as reported by Robinson and Darrow (1929) and Perry—Lancaster (1935), Stewart (1960) and Stewart and Arisumi (1967) presented genetic and histological evidence for bract color inheritance. 12 Stewart (1960) reported that results from testcrosses between red and white bracted poinsettias indicated a single recessive factor as the difference between white and red bracts, which he designated as wh and Wh respectively. In their genetic and histogenic determination of pink bract color in poinsettia, Stewart and Arisumi (1967) reported that pink bract color was due to a single, pk, recessive to the factor (Wh) for normal red pigmentation. They further indicated that the pk locus assorted independently of the wh locus, and that the double recessive, whwh/pkpk produced a white bracted plant. EXPERIMENT I. 28 x 42 CHROMOSOME RECIPROCAL CROSSES MATERIALS AND METHODS Rooted cuttings of 'Ecke White,' a 28 chromosome cultivar and of six 42 chromosome cultivars, 64—4; 64—5; 64—7; 64—8; 64—13; and 65—2, were grown in the greenhouse. Since pollination was made between November and January, no precautionary measures were taken against possible insect contamination. Patterned after Stewart's method (1960), terminal pistillate flowers in cyathia located in clusters which lacked fully developed staminate flowers were selected for crossing without emasculation. Where both pistillate and staminate flowers appeared simultaneously, emasculation was performed before crossing. Removal of the staminate flower parts was continued until the stigma was no longer receptive. For selfing, every flower part was left intact and the selected pis- tillate, as in the case of crossing, was hand pollinated several times within a period of three days as suggested by Pai (1960). One hundred and five days after pollination, when seeds began to ripen, the seed pods were covered with Kraft paper bags to prevent the loss of seeds when the pod expelled them. Greenhouse temperatures were kept at 70 F day and night during the experiment. Seeds obtained from these crosses were germinated in petri dishes after the removal of the lower tips of the hard shell or seed coat adjacent to the radicle. Seeds germinated within five days and were subsequently transplanted to three-inch clay pots, containing a 1:2:1 mixture of sand, soil, and German peat, respectively. 13 Figure 1. 14 Comparative heights of six—month old parent plants with different levels of ploidy and their progeny. A. 'Ecke White,' a 28 chromosome cul— tivar used as the pistillate parent in a directional cross with a 42 chromo- some cultivar. B. 64-8 cultivar with a somatic chromo— some complement of 42. C. 66—376 is the progeny of a cross between 'Ecke White' and 64—8. The somatic chromosome complement is 35. 15 16 Meristematic shoot apices were taken from the progeny of the above crosses. For temporary squash preparations, a mixture of 0.002 M solution of 8-hydroxyquinoline and a saturated solution of p-dichlo— robenzene mixed in a 1:1 proportion was used. In some cases materials were not prefixed in this solution. aThe treatment was carried out for approximately twenty—four hours at room temperature. Following treat- ment, the shoot apices were dehydrolized and stained in a mixture of two percent aceto—orcein and normal hydrochloric acid (9:1) as presc— ribed by Sharma (1963) and heated over a flame for about three seconds. The slides were properly sealed with vaseline and stored in a refrig— erator at2 C for approximately 24 hours for observation. RESULTS Table I. Results from reciprocal crosses between 28 and 42 chromo— some cultivars and self—pollination. Treatment Seed produced Number of Number of partially aborted developed ovaries seed 28 x 42 16 86 383 42 x 28 ~ 29 319 42 self - - 710 Sixteen seeds were harvested from 1, 428 crosses of 28 x 42 and reciprocal crosses, made between November and January, 1966. l7 Twelve out of sixteen seeds germinated but only three survived. Two of the three plants obtained have rose bracts, and the other is white. Two of these plants are shown in Figures 1, 2, and 3. 18 Figure 2. Comparative heights of two parent plants different levels of ploidy and their progeny. A. 'Ecke White,' the pistillate parent (2n=28). B. 64—13, the staminate parent (2n=42). C. 66—377, the progeny (2n=35). 19 20 The two rose—colored plants exhibited a slow rate of growth when compared with either of the parents. The white plant had a mode- rately slow rate of growth but not as pronounced as the rose plants. When a limited number of cuttings were taken from the three plants without pretreatment with a rooting hormone, no roots were produced by the rose colored plants. They produced only callus tissues. Cut- tings from the white plant produced roots after remaining in the sand medium and under mist system for approximately ten weeks. When root— ing hormones were applied to the cut ends, cuttings of the rose plants produced roots earlier than the white plants. Cytological analysis of the meristematic shoots of the three plants indicated that they all had a constant somatic complement of 35 chromosomes. Figure 4 shows metaphase chromosomes with 2n=35. In the red plants there was at least one dividing nucleus per over 300 cells. In the white plant, on the other hand, the ratio was less than 1:300. In addition to this observation there was a large number of binucleate and multinucleate cells in the three plants, particularly, the red ones. These two factors could account for the reduced amount of growth because the rate of cytokinesis was reduced. Globular materials were found scattered within the cytoplasm. More of these bodies were found in the red plants than in the white plant. They were aceto—orcein positive but feulgen negative. On the basis of their differential reaction to the two stains, they were con— sidered to be plastids. The number of the globular bodies ranged from as low as 16 to as high as 35. Figure 3. 21 Comparison of floral development of six—month—old plants with different levels of ploidy. A. 'Ecke White' and 64—8 showing well developed pistillate and stam— inate parts. B. 'Ecke White' and 64-13 in an advanced stage of floral development. Partially enlarged cyathia may be seen on both plants. C. 66—376 and 66—377 showing no perceptible floral development. Figure 4. 23 Somatic chromosome complements of cultivars 66—376, 66—377, 66-378. A. and B. Original and retouched metaphase plates of 66-376 ('Ecke White' x 64—8) 2n=35. C. and D. Original and retouched metaphase plates of 66—377 ('Ecke White' x 64—13) 2n=35. E. and F. Original and retouched metaphase plates of 66—378 ('Ecke White' x 64-8) 2n=35. EXPERIMENT II. KARYOTYPE STUDIES IN 2n=28 CULTIVARS MATERIALS AND METHODS Shoot tips were taken from cultivars whose somatic complement is 28 chromosomes. Ruth Ecke'and'White Ecke'were used for this study but the former was studied more extensively. Stock plants selected for the experiment were pruned to ensure active growth of the lateral buds. Newly formed and actively growing buds were removed and fixed in a mixture of 0.002 M solution of hydroxy- quinoline and a saturated solution of para—dichlorobenzene mixed in the same proportion as in experiment 1. After 24 hours of treatment, the procedure outlined in the previous experiment was followed. Per- manent slides were made by allowing the slides to remain in tertiary- butyl alcohol for about six hours followed by application of balsam mixed in xylene. Photo—micrographs were taken with Zeiss photomicro— scope at X1,000. For analysis of percentage of total complement length (TCL), relative and absolute lengths, long arm—short arm ratio, and percent of long arm, chromosome or set of chromosomes were made from an exploded picture, magnified thirteen times, in accordance with the method des— cribed by Rothfels and Siminovitch (1958). RESULTS The somatic chromosomes of poinsettia stained very well with aceto—orcein, except for the constrictions at the position of the cent— romere. This differential staining was clearly seen in early metaphase, 25 26 when the chromosomes were large and had not attained their maximum contraction. In late metaphase the entire lengths of the chromosomes are uniformly stained. The mean lengths, absolute lengths, percent of total complement lengths, percent of long arm, and long arm—short arm ratios measured during the metaphase stage for 'Ruth Ecke' are given in Tables 2 and 3. Table 2. Mean of lengths in mm of each set of 'Ruth Ecke' chromo- somes and the percentage of long arm length in 20 cells. Chromosome Number Cell 1 2 3 4 5 6 7 Total 1 19.8 13.4 11.6 12.2 9.5 8.2 5.1 79.8 58.2 54.4 54.6 62.3 57.6 53.1 57.1 397.3 2 20.5 16.3 12.5 13.0 9.3 7.8 6.3 85.7 57.1 55.3 54.5 78.3 69.8 52.8 59 3 427.1 3 20.3 18.4 14.3 13.1 9.0 8.4 6.6 90.1 59.2 52.5 57.1 69.2 62.5 55.4 59 3 415.2 4 22.6 16.5 14.2 12.4 11.1 9.8 7.9 94.5 56.7 55.3 57.8 59.2 58.6 51.8 56 5 395.9 5 15.7 13.2 12.1 11.8 10.6 9.4 6.2 79.0 58.3 54.4 59.2 62.5 59.8 50.9 59.4 404.5 6 16.1 13.4 12.0 11.5 9.5 7.9 7.0 77.4 60.0 57.5 59.1 60.0 60.0 56.0 57 1 409.7 7 17.2 14.7 12.3 10.1 8.9 7.4 6.1 76.7 61.5 55.2 58.3 60.0 59 4 54.5 62 3 411.2 8 25.4 21.2 18.9 16.8 14.5 12.3 8.2 117.3 60.0 50.8 56.7 59.2 66.6 53.1 59.3 405.7 9 26.4 23.0 19.5 18.7 15.1 12.5 10.2 125.4 60.5 58.2 56.7 65.2 73.3 58.3 59 2 431.4 27 Table 2. (continued) Chromosome Number Cell 4 5 Total 10 21.2 18.1 17.7 14.8 12.6 11.5 8.4 104.3 61.1 54.5 56.3 63.8 66.6 54.5 62.5 4.9.3 11 21.2 18.6 17.3 15.4 13.2 10.6 8.8 105.1 56.2 54.5 58.1 65.0 61.8 51.8 55.5 402.9 12 22.3 20.1 19.6 15.4 13.3 11.4 8.7 110.8 57.5 53.2 55.0 66.6 61.5 54.5 53.0 401.3 13 24.6 17.4 14.6 13.1 12.7 10.2 7.3 99.9 58.2 54.5 57.1 69.2 61.5 52.3 57.1 409.9 14 15.4 13.2 10.5 10.1 8.7 7.9 7.2 73.0 63.2 54.5 60.0 70.0 62.3 52.2 58.2 420.4 15 14.4 13.4 9.0 11.3 9.5 8.4 6.2 72.1 58.8 53.2 55.2 53.6 62.5 51.8 58.3 403.4 16 13.8 11.4 10.2 9.8 9.3 8.5 6.7 69.7 57.3 52.3 53.6 68.5 55.0 51.3 56.5 394.5 17 16.3 14.8 12.6 12.8 10.4 8.6 6.2 81.7 54.5 53.8 54.4 59.1 70.0 51.6 62.4 405.8 18 16.2 14.7 12.5 11.9 11.1 9.3 7.4 83.1 57.5 55.5 57.2 63.4 61.4 53.6 58.1 406.7 19 17.0 14.5 12.3 10.8 9.5 8.7 6.4 79.2 58.3 53.8 55.6 61.2 60.1 52.4 57.3 398.7 20 16.7 14.2 13.8 11.9 9.6 8.7 6.4 81.3 58.2 51.6 53.4 67.3 66.6 51.6 59.2 407.9 28 Table 3. Mean of total length, percent of total complement length, absolute length, percent of long arm and long arm to short arm ratio in mm from 20 metaphase plates of 'Ruth Ecke.‘ Chromosome 1 2 3 4 5 6 7 Mean of total 19.15 16.02 13.85 12.84 10.87 9.37 7.16 length Percent of TCL* 21.44 17.94 15.53 14.37 12.17 10.49 8.02 Absolute length** 2.67 2.23 1.93 1.79 1.51 1.30 1.00 Percent of long 58.61 54.25 56.54 64.68 62.68 53.17 58.38 arm Long arm to short 1.35:1 1.07:1 1.22:1 1.64:1 1.61:1 1.06:1 1.24:1 arm ratio * TCL — Total complement length ** Absolute length was obtained by dividing the mean of the lengths of chromosomes 1, 2, 3, 4, 5, and 6 by the mean length of chromo— some 7. Table 4. Table 5. 29 Duncan's Multiple range test for total mean length of chromosomes in 20 metaphase plates of 'Ruth Ecke.‘ (2n=28) One percent significance of chromosome length in mm. Chromosomes l 2 3 4 5 6 7 19.15 16.02 13.87 12.84 10.87 9.37 7.16 Duncan's Multiple range test for percent long—arm ratio of chromosomes in 20 metaphase plates in 'Ruth Ecke.’ (2n=28) One percent significance of long-arm ratio Chromosomes 1 2 3 4 5 6 7 58.61 54.25 56.54 64.68 62.84 53.17 58.38 I? 30 The 28 chromosomes are arranged and numbered according to Hu's (1958) method, i.e. in the order of their lengths. The differences found in the chromosome length might possibly have arisen from their degree of contraction in the mitotic cycle. The 28 chromosomes are graded in size and vary in length from 0.012 mm to 0.032 mm. A gen— eral description of the chromosome set of seven based on the length, location of the kinetochore and the presence or absence of trabants or secondary constrictions is outlined below. Chromosome 1. The longest chromosome. Submedian with a sec— ondary constriction. The middle segment of the chromosome is thicker than the other adjacent arms. Chromosome 2. Long chromosome. Median with a secondary cons- triction. Chromosome 3. Medium chromosome. Submedian. Chromosome 4. Medium, slightly shorter than chromosome 3. Subterminal. The longer arm is oval shaped at the base. Chromosome 5. Small to nearly medium chromosome. It is subterminal. Chromosome 6. Small chromosome with median primary constric— tion. Chromosome 7. Smallest chromosome with submedian primary constriction. Photomicrographs of cells at metaphase are shown in Figures 5 and 6. The morphology of the chromosomes as outlined above may be 31 fully recognized in some of the figures and in some others may be partly recognized. Metaphase and photokaryotype from both'Ruth Eckd and White Ecke can be seen in Figures 5 and 6. Figure 6C shows the idogram of Ruth Ecké. 32 Figure 5. Metaphase plate, photokaryotype and idiogram for 'Ecke White.‘ X6,000 A. Photodrawing of metaphase plate B. Photodrawing of metaphase plate C. Photokaryotype D. Idiogram 33 :o‘.«, " § .r :«33'3 . " ...... . time- ’c ’ 3‘ . .4 . s 9‘" I!" in! uuuu uni-u C D Figure 6. 34 Metaphase plates, photokaryotypes and idiogram from shoot apices of 'Ruth Ecke and White Ecke'(2n=28). X8,000. A. Metaphase plate of Ruth Ecke. B. Metaphase plate of'White Ecké, C. Idiogram of'Ruth Ecke somatic chromosomes. D. Photokaryotype from'Ruth Ecke shoot apex. E. Photokaryotype from White Ecke shoot apex. 35 3,: ...: ...: 8.3 on”: :5- 3: .1. ...: ...: 2.3.»..- 3: use. EXPERIMENT III. MEIOTIC CHROMOSOME ASSOCIATIONS IN 2n=42 CULTIVARS MATERIALS AND METHODS Plants with a somatic chromosome complement of 42 were grown in clay pots in the greenhouse and allowed to flower. Partially developed cyathia were selected. White anthers located between the immatured and light yellow pollens were killed and fixed in modified Carnoy's solution from four to twenty four hours, after evacuation. Following fixation, the anthers were kept in 70 percent alcohol. Smearing technique involved placing anthers on a slide and cutting them in half. A drop of aceto—carmine was added and the con— tents of the anthers were squeezed out by pressing with the flattened end of a glass rod. With the material under a dissecting microscope, the debris was removed, a cover slip was placed on it and a drop of stain was added from the side. The material was slightly heated and flattened gently and finally observed microscopcally. RESULTS Nucleolar Contents A comparison of nuclear contents in terms of number of nucleoli can be seen in Figure 7. The number of nucleoli ranged from a mimi— mum of 4 to a maximum of 12. The size of the nucleus, from many observations did not seem to influence the number of nucleoli rather the number of nucleoli in a nucleus seemed to affect the size of the nucleoli. When 12 nucleoli were found in a nucleus (Figure 7A & B) 36 37 at least half the number were large and the remaining half extremely small. On the other hand, if 6 or 9 nucleoli were observed they seemed to be fairly large. In some of the nuclei containing less than 12 nucleoli there appeared to be juxtaposition or overlapping or fusion of two or possibly more nucleoli. Figure 7. 38 Number of nucleoli in six nuclei from cultivar 64-8 (2n=42). X8,000. A. 12 nucleoli. B. A nucleus containing 12 nucleoli of which 5 are comparatively large and the remaining 7 very small. C. A nucleus containing 4 large and 4 small nucleoli. D. Fusions of nucleoli are clearly seen in two places. E. The nucleus seems to contain only two nucleoli, however, small ones may be seen. F. Four nucleoli are located in the nucleus. There are four large ones and four small nucleoli, which are barely conspicuous. 39 Figure 8. 4O Pollen mother cells from cultivar 64—5 (2n=42) with varying number of chromosome associations. X5,000. A. and B. show 31V 1 4 III 1 6 II 1 6I = 42. C. and D. show ZIV l 7 III 1 4 II 1 51 = 42 E. and F. show 1V 1 41V 1 4 III 1 311 1 3I = 42. 41 Figure 9. 42 Pollen mother cells from 64—5 with varying number of chromosome assoc- iations. X3,000. A. and B. show 21V 1 8111 1 511 = 42 C. and D. Show 21V 1 7III 1 411 1 51 = 42. E. and F. show 1V 1 31V 1 SIII 1 511 = 42. 44 Chromosome Associations Meiotic chromosome pairing as shown in Figures 8 and 9 revealed the occurence of pentavalents, quadrivalents, trivalents, bivalents, and univalents. The maximum number of pentavalents, quadrivalents, trivalents, bivalents, and univalents were 1, 4, 6, 8, and 4 respec— tively. The frequencies of the various valencies per cell based on sixty cells were 0.44 for the pentavalents, 2.14 quadrivalents, 5.05 for the trivalents, bivalents, 6.15, and 1.02 univalents. Table 7 shows the range and mean chromosome associations based on twenty cells. Figure 10 shows a histogram of the distribution of associations of chromosomes per cell. Table 6. Distribution of Chromosome Associations in 2n=42 Cultivars No. of Hexa— Penta— Quadri— Tri— Bi- Uni- cells valents valents valents valents valents valents R M R M R M R M R M R M 60 0—0 0 0—7 0.23 1—6 2.63 3-8 5.01 3—8 6.0 0—8 2.93 No hexavalent association was found in the cells observed. Pollen mother cells with varying numbers of chromosome associations are pre— sented in Figures 8 and 9. Ring, chain, and cross—type quadrivalents, chain and Y-shaped trivalents were noticed. The pentavalents were either associated in mZQESDQWWV MEQmQEOQID .10 WMQxc Q2? mmmmssz QmSQmme MFZMJ<>SOZBO N mHZmJ<>_mQ_m._. mpzwsdzm mhzw44>0202 vmm_ wwwOVm wwwva mnomvmm O _ N _ Q _ w _ 5773.9 :10 HE’S/AWN 46 the form of Y or in a chain. Movements toward the poles at anaphase I were not strictly in pairs as reported by Ewart and Walker (1960). Multiple associations involving more than two chromosomes were observed. Meiotic Irregularities Certain meiotic irregularities in terms of number of chromo— somes in a cell were observed. In some instances, 14 chromosomes were counted in anaphase II and in others, 42 chromosomes were counted in telophase I. Figure 11 shows some of the lagging and irregular chromosome separations which were observed in PMC's from the cultivar 64—5, 2n=42: Figure 11. 47 Cytological aberrations, '1agging and irregular separations in pollen mother cells of 64—5, 2n=42. X6,000. A., B., C., and D. show early telophase I with 42 or more chromosomes. The presence of more than 42 chromosomes in C. and D. could have resulted from fragmentation. E. and F. Late telophase II with 17 chromosomes lying in the centre of the tetrads. The size and position of the chromosomes suggest metaphase I with different co—orientation. G. and H. Metaphase II plate containing 30 chromosomes. The chromosome associa- tions range from univalent to quadrivalent. 48 DISCUSSION Reciprocal Crosses Between 2n=28 and 2n=42 Cultivars and Self—pollination. Crosses involving the 28 chromosome plants as the female parents were the most successful, whereas the reverse crosses or selfing prod—' uced shrivelled or germless seeds. This behavior, however, does not agree with the general experience as reported by Thompson (1930) that inter- or intraspecific crosses are usually successful when the higher polyploid is the female parent. Two questions arise from the results of this study. The first has to do with the unsuccessful seed production when the higher poly— ploids were the female parents and the second pertains to the low quantity of seeds produced. It is realized that no specific and clear-cut answers can be offered at this juncture; however, it may be stated that any one of the factors listed below could have influenced the two phenomena. These factors include female sterility, pollen sterility, cross- incompatibility, self-incompatibility, meiotic mechanisms such as pairing and chromosome or chromatid separations, sensitivity to cer— tain environmental conditions and physiological or biochemical defic- iencies. The poinsettia is generally propagated vegetatively and for that reason little or no attempt has been made to establish lines, differing in degree of fertility, through a selection program. And as a poly— ploid, coupled with its vegetative propagation, it stands a very good 49 50 chance of accumlating a tremendous array of meiotic irregularities, and possibly certain changes on the somatic level. Subsequently, in the absence of mutation the cultivars will continue to perpetuate their inherent genetic systems. In maize, Fischer (1941) reported that some tetraploids were found to be self—sterile and that cross-incompatibility also existed among some stocks. In the original diploid lines from which these polyploids were derived, however, self—sterility and cross-incompati— bility were exhibited. The difference in the behavior of the polyploid and the diploid maize may suggest that incompatibility can possibly arise as a result of changes in the genetic balance of the species. Through selection, the breeder can eliminate plants which possess the undesired genetic combinations. Since the genetic constitution of poinsettia has been left virtually unchanged, except artificial or natural changes in the chromosome numbers, the incompatibility, if the situation exists in poinsettia, either self— or cross—incompatibility, will continue to appear in all breeding programs. Fischer's work on maize may further infer that as the ploidy of a plant changes from one level to another, particularly from diploid to polyploid, a certain percentage of pollen abortion is encountered, which will subsequently reduce the plants' previous percentage of fert- ility. This seems to be the situation existing in poinsettia. In the absence of the original diploid one has no way of testing this hypo— thesis and obtaining cytogenetic evidence to substantiate it. However, 51 the literature abounds of reports on behavior of diploids and poly— ploids in terms of fertility (Howard 1942, Giles 1942 a and b, Swaminathan and Sulba 1959, Rosa and Boyes 1946). The results of the karyological studies reported elsewhere in this study, seemed to indicate that all the known cultivars of poin— settia are polyploids. On the strength of this, the 2n=28 and 42 chromosome cultivars, which were used in the breeding experiment, may be designated as tetraploid and hexaploid respectively. Hence it may be plausible to suggest that the behavior of maize (Fischer 1941, Buraham 1962) obtains in poinsettia culminating in reduced percentage of seed set. The experiment on meiotic chromosome pairing in 2n=42 chromo— some cultivars showed a higher frequency of multivalents, than bivalents. The sum of quinquivalents, quadrivalents, and trivalents in every cell exceeds the sum of bivalents. Implicit in such multivalent association is that at anaphase I or II unequal separation and distribution of chromo— somes and chromatids respectively to either of the poles is likely to occur. In fact, such was the case in some of the cells observed. Three of five chromosomes were found in some instances to remain in the equa— torial plane in anaphase I. Gilles and Randolph (1951) showed that frequency of quadrivalents and bivalents are capable of influencing self-and cross—incompatibility. Their data reveal that a shift from higher to lower number of quadri— valents and a corresponding shift from lower to higher number of bivalents 52 increased the percentage of seed set. A similar relationship between quadrivalent and bivalent associations and fertility is reported by Swaminathan and Sulba (1959). Their data on Brassica campestris var. toria showed higher frequency of quadrivalent and lower frequency of bivalent associations associated with reduced fertility and the reverse associations for increased fertility. Rosa and Boyes (1946) found that the same phenomenon obtains in flax, where diploids out—yield the te— traploids. An antithesis of this phenomenon is found in Dactylis glomerata, a polyploid species, which exhibits a higher frequency of multivalents coupled with high seed set or production (Brix and Quadt 1953). While the effect of multivalent associations may be expressed quite differently in different species in terms of fertility or percentage of seed set, there is a possibility that the low seed set in poinsettia, a polyploid species with a high frequency of multivalent associations, may be a concomitant of the multivalency coupled with certain detrimental envir— onmental conditions and physiological or biochemical deficiencies. Physiological deficiency observed in any organism may be genic or environmentally controlled. As it occurs in some plants, physiolog— ical incompatibility can play a role in the precocious abscission of cyathia in crosses between the 2n=28 and 2n=42 chromosome cultivars. In the event of this the two parents may have similar functions but a particular biological synthesis which attends seed formation and mat- urity will have to be carried out by different pathways in the two 53 parent plants. Such physiological activities are capable of augmenting a higher frequency of cyathia abscission or shrivelled and non—viable seeds. Harland (1933) observed a similar phenomenon in crosses invol— ving cotton. The literature abounds of evidence to the effect that environ— mental changes are capable of modifying or suppressing genic effects even though the genes for that character may be present. On the basis of this premise there is the possibility that precocious abscission of cyathia encountered in the greenhouse experiments may be the result of biochemical changes taking place in the plants and influenced by the environment. Auxin has been reported to prevent leaf and fruit abscission (Sacher 1957) by maintaining the integrity of cellular membranes. These biological changes which attend abscission and senescence of leaves and fruits have been prevented to a certain degree, and at least in some fruit trees, by the use of auxin. The mechanism involved here is the maintenance of the level of RNA, DNA, and protein (Sacher 1965). Ewart and Walker (1960), and Pai (1960) reported that an exogenous supply of auxin to enhance seed set in poinsettia yielded favorable results in terms of quantity, but the number of viable seeds was not very encouraging. From Pai's work and that of Ewart and Walker (1960), one fact has, at least, been established and that is during the course of development, the level of endogenous auxin or a growth factor in poinsettia dwindles u 54 and that exogenous auxin can hold the cyathia on the plant to mat— urity. How much of this is genic controlled and how much is environ— mentally controlled is not known yet. There is an indication, however, that in a situation where fertilization fails due to differential grow— th of the pollen tube, cross—incompatibility, self—sterility, and abor- ted pollen, stimulation of auxin synthesis (Nitsch 1962) is reduced, with concomitant abscission of the flower. Hence the cause, event and result of ovary abortion can be both, or either genic or biochemical phenomenon and both are influenced, to a certain degree, by environ— mental factors. Assuming that one or all of these factors numerated above were operative in the genetic and physiological systems of the plant, the production of twelve viable seeds from which three plants were obtained with somatic complement of 35 chromosomes, there occurred, to a lesser degree, normal meiosis. Karyomorphological Analysis in 2n=28 Cultivars The description of the karyomorphology of the 28 chromosomes of Ruth Ecké and'White Ecke'was determined on the basis of length, posi— tion of kinetochores, presence or absence of satellites or trabants, percentage of total complement length and arm ratios. Regarding the lengths of the 28 Chromosomes of the cultivars studied, eight were large and long, four medium to nearly long, eight medium, and the remaining eight small. The long chromosomes were about 55 1.5 to 2.2 times longer than the medium and about three times as long as the small ones. The lengths of the metaphasic chromosomes of RutH Ecke'were longer than those of'White Ecke} However, with the exception of the shape of chromosome 7 in Ruth Ecke, the two cultivars have com- parable karyotype. On the basis of position of kinetochore and constrictions, the fourteen pairs of chromosomes were classified into four groups, which included (1) metacentric, (2) submetacentric to nearly metacentric, (3) submetacentric, and (4) subterminal. Chromosomes possessing med— ian kinetochore were considered metacentric, where one or two chromo- somes possessed median kinetochore, and the others submedian, the whole set was considered submetacentric to nearly metacentric. Eight chromosomes were included in the metacentric category, 4 submetacen- tric to nearly metacentric, 8 were consistently submetacentric and the remaining eight subterminal. Four of the large and long chromosomes were metacentric and sat— ellited and the other four submetacentric and also satellited. Among the eight medium chromosomes, four were submetacentric and the remain— ing four subterminal. The eight chromosomes described as small, four were consistently metacentric and the other four submetacentric. The medium to nearly long chromosomes had nearly netacentric constrictions. Analysis of variance of the data for the lengths, percentage of total complement length, arm ratio of individual chromosomes, coupled with the presence or absence of satellites revealed that the fourteen 56 pairs of chromosomes could be divided into four classes of different lengths represented by chromosomes 1, 2 —— 3 -— 4, 5 —— 6, 7, and four classes of arm—ratios by chromosomes 2, 6 —— 3 —— 1, 5, 7 —— 4 and two classes on the basis of presence of satellites —— 1 —- 2, 3, 4, 5, 6, 7, the last five chromosomes being without secondary con- strictions or satellites. Considered collectively, the three different classes as outlined above could be employed to group the fourteen pairs of chromosomes into seven different classes as described below. Chromosome Size des— Arm ratio Presence or cription absence of satellite 1 Long Submedian Satellited 2 Long Median Satellited 3 Long Nearly median None 4 Medium Subterminal None 5 Medium Submedian to nearly terminal None 6 Short Median None 7 Short Submedian None These cytological findings suggest that the basic chromosome number is seven and not fourteen as previously suggested; however, genetic evidence would be required to substaniate it. 57 It may be mentioned, however, that even though the basic number of n=7 has been shown by idiogram, the 28 chromosome cultivars segre— gate some of their parental genetic traits (bract color, Stewart 1961) or differences in characteristic diploid mendelian ratios instead of tetraploid ratios. On the basis of this segregation pattern it may be postulated that the causes underlying the behavior could be (a) that the 28 chromosome cultivars could have arisen as allopolyploids, (b) that as a result of autosyndesis the original species parent be~ haved as amphidiploids, (c) the progenitors has many loci which were alike or similar. Subsequently, interactions of these similar genes may produce diploid segregation of certain parental traits, such as bract color, while at the same time, segregation for other traits may suggest complex genetical behaviour (Elliot 1958). Meiotic Chromosome Associations in 2n=42 Cultivars The idiogram of the somatic metaphase chromosomes showed the presence of eight satellited chromosomes in the 2n=28 cultivars. Thus, in the 42 chromosome cultivars used in the study of meiotic chromosome associations, the presence of twelve satellited chromo— somes are to be expected. After the first meiotic division and in the subsequent telophasic stage, and assuming that these satellited chromo— somes were equally distributed, six of these chromosomes would constit— ute part of the haploid complement. From leptotene to at least the beginning of the first division, twelve of the above mentioned chromo— xomes would make up part of the total chromosomes in each nucleus. If 58 If the presence of a satellited chromosome accounts for the presence of nucleolus or vice versa, then twelve and six nucleoli may be counted before the end of the meiotic prophase and at the end of the second division. McClintock (1931) reported that secondary constrictions observed in somatic metaphase chromosomes generally come about as a result of nucleolar formation. The nucleolar organizing region is reported by Steward and Bamford (1942) to associate with heteropycnotic regions which, in somatic metaphase chromosomes, will reveal only constrictions. From these two reports it may be deduced that if twelve nucleoli were counted in the prophase stage and six in the telophasic stage then there were twelve and six secondary constricted chromosomes res— pectively in these stages. Figure 7 shows distribution of nucleoli in the pollen mother cells of a 42 chromosome cultivar. The number of the nucleoli ranged from five to twelve. The difference in size may be due to the action of individual organizers. A cytological evidence regarding the differential action of individual organizers was offered by Navashin (1934) when he reported that a differential nucleolar size was a measure of nucleolar — organizer's competition for available material. Thus, the size of the nucleolus is dependent upon the rate of action of the chromosome bearing the satellite. The karyomorphological analysis of the chromosomes as shown in the idiogram points out that there existed among the satellited chromosomes differences in arm ratio, length and width of the arms 59 bearing the trabants. There is therefore, the possibility that the nucleolar differences as depicted in Figure 7 may be a reflection of the gross morphological differences among the SAT chromosomes culmin— ating in their differential competitive ability for nucleolar organiz— ing material. By cytological definition, homologous chromosomes will synapse at zygotene. However, in polyploids, more often than not, there is a deviation from 100 percent valency. Factors such as kinetochore dam— age, desynapsis, interlocked chromatids, environmental shock and lack of homology will influence the degree of meiotic association. Chromo- somes may be reasonably homologous, but due to lack of homologous re— gions pairing may be inhibited and subsequently segregation may lead to deficiency resulting in partial sterility. Furthermore, the size and number of chromosomes influence to a large degree the number of associations expected. Small size and large numbers of chromosomes have limited pairing blocks; subsequently there is a limitation to maximal valency. In poinsettia, and particularly the 42 chromosome type, the probability of getting the maximum association of six chromo— somes is remote since the largest chromosome is about 0.03 mm. long. According to Swanson (1964) a study of triploid organisms possessing three sets of chromosomes showed that although all three homologous chromosomes could synapse with one another, at no place along the chromosomes did an association of more than two—by-two occur. The three—by—three arrangement, if it did occur, should do so in zygotene 60 where synapsis occurred, however, contraction and repulsion of chromo— somes and terminalization of chiasmata would not maintain the associa— tion of the initial three-by—three (Serra 1965). Pai (1960) and Ewart and Walker (1960) did report the occurrence of bivalents, quadrivalents, and hexavalents in a 56 chromosome culti— var. In the 28 chromosome types, univalents, bivalents, and a very small frequency of quadrivalents were reported by Pai (1960). While a reduced frequency of maximal association was obtained in the 28 chromosome type, a maximal association of eight or even seven was not reported in the 56 chromosome type. The multivalent associations observed in the pollen mother cells of the 42 chromosome type of poinsettia agree with the findings of Pai (1960) and Ewart and Walker (1960) except that more trivalents were counted in this work than have been reported previously. This work also recognizes the presence of chromosome fragmentations in telo— phase I and other meiotic irregularities such as lagging of chromosomes, unequal distribution of chromosomes, resulting from abnormal or impro— per disjunction of multivalents in anaphase I and II. The author assumes the position that the lack of seed set which was pronounced in the 42 chromosome cultivars could have been due to (1) production of nonviable unbalanced gametes resulting from either (a) irregular distribution of chromosomes caused by abnormal disjunc- tion of multivalents (Darlington 1937), or (b) other meiotic irregular- ities such as false univalents and lagging of chromosomes (Dawson 1962); 61 (II) genetic-physiological sterility, which has not been elucidated, and which may or may not be associated with meiotic irregularities, but may be possibly involved in this phenomenon (Randolph 1941). Dawson (1962) explains that the behavior of trivalents, quad- rivalents, and quinquivalents will depend upon the positions of the chromosome centromeres, relative to one another and to the orientation of the spindle. In trivalent formation three possible types of co— orientation, namely linear, convergent, and indifferent will result in unequal distributions of chromosomes. Thus the Y—shaped configur— ation, an example of indifferent co—orientation, will result in either one chromosome moving to each of the poles leaving a false univalent at the equatorial plate or two chromosomes moving to one pole and one to the other. In either of the two cases, a lagging chromosome or unequal distribution of chromosomes will ensue respectively. Chromo— somes of quadrivalents with indifferent co—orientation may show many different patterns of segregation. Dawson (1962) suggests that (1) two chromosomes may move to one pole — one to the other and a false univalent left lagging on the equatorial plate; (2) one chromosome may move to each pole leaving two false univalents. The lagging of chromosomes arising from improper disjunction of multivalents will greatly influence the fertility of the cultivars in which the phenomenon obtains (Sparrow, gp.lgl. 1942). SUMMARY AND CONCLUSIONS 1. Plants possessing a somatic complement of 35 chromosomes were realized from the reciprocal crosses. These are the first reported cultivars with 35 chromosomes. Pollen from lower poly- ploid plants failed to effect seed formation when the pistillate parent plants were of a higher level of ploidy. The realization of 35 chromosome plants was indicative of occurrence of normal meiosis but at an extremely reduced frequency. 2. All the 42 chromosome cultivars selfed during the investigation did not produce seed. This behavior may be attributed to: a) irregular distribution of chromosomes resulting from abnormal disjunction of multivalents. b) various meiotic abnormalities such as lagging of chromosomes at both anaphase I and II. c) meiotic irregularity of physiological nature presumably gene controlled and possibly augmenting self—incompatibility. One or all or any combinations of the above may be responsible for the production of nonviable unbalanced gametes. The production of such gametes may inevitably culminate in precocious abscission of cyathia. 3. Reduced growth in length coupled with slow rate of floral develop- ment, as demonstrated by the 35 chromosome plants may have been due to slow or reduced rate of cytokinesis. Cytological observa— tions revealed that there was about one dividing cell per 500 non-dividing cells. In the parent plants the ratio of dividing 62 63 cells to non—dividing cells was not as wide as reported above. 4. Cytological analysis of the 28 chromosome types indicates a basic number of seven. Based upon length of chromosomes, posi- tions of kinetochore, presence or absence of secondary constric- tions and trabants, the chromosomes may be classified as follows: Chromosome 1 — the longest of the chromosomes. Submedian with a secondary constriction. The middle segment is thicker and longer than the adjacent arms. Chromosome 2 — a long median chromosome possessing a second- ary constriction Chromosome 3 - Medium in length with a submedian to nearly median primary constriction. Chromosome 4 — It is medium in length, slightly shorter than chromosome 3. It has a subterminal primary constriction, the longer and larger arm being oval in shape at the base. Chromosome 5 — Small to nearly medium chromosome possessing subterminal primary constriction. Chromosome 6 - Small and short chromosome with a median primary constriction. Chromosome 7 — The smallest and shortest chromosome, with a submedian primary constriction. 5. On the basis of the karyomorphological studies the current termino— logy in terms of the ploidy of poinsettia. The following termino- logy is proposed. However, this is a tentative proposal until a 64 2n=l4 cultivar is reported and establishment of tetraploid inheri— tance in 2n=28 cultivars. a) tetraploid — to refer to all 28 chromosome cultivars. These, among others, may include'White Ecke; Ruth Ecké and bakleaff b) pentaploid — to designate all cultivars possessing somatic complement of 35 chromosomes. e.g. M.S.U. (produced by the author) 66—376, 66—377, 66—978. c) hexaploid — to be considered as all poinsettia cultivars possessing somatic chromosome complement of 42. e.g. M.S.U. (produced by Milbocker) 64—13, 64—8, 64—7, 64—5, 65—2, etc. d) octaploid - this term may be used to designate such culti- vars as'Barbara Ecke Supreme, which possesses a somatic complement of 56 chromosomes. The nucleoli observed in different cells were not uniform in terms of number and size. The variation may be due to fusion of two or more nucleoli or suppression of others as a result of differential activity of the nucleolar organizers and/or differential competition for nucleolar organizing materials. Multivalents were observed to be more frequent than univalent formations in 2n=42 cultivars. The occurrence of trivalents and quadrivalents exceeded the combined frequencies of univalents and bivalents. In the presence of these multivalents, the co-orienta— tion of chromosomes may possibly enhance the frequency of unequal 65 distribution or disjunction of chromosomes in anaphase I and II coupled with lagging of others in the equatorial planes. The resultant unbalanced and nonviable gametes may concomitantly give birth to the incessant precocious abscission of cyathia, which confronts many poinsettia breeders and geneticists. 10. ll. 12. 13. 14. 15. LITERATURE CITED Babcock, E. B. 1942. 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