Chromosome Botany (2013) 8: 69-74 © Copyright 2013 by the International Society of Chromosome Botany

Cytomorphological Studies in some species of L. ()

Raghbir C. Gupta, Santosh Bala1, Shilpa Sharma and Manish Kapoor

Department of Botany, Punjabi University, Patiala-147002, Punjab, India

1Author for correspondence: [email protected] Received May 8, 2013; accepted August 6, 2013

ABSTRACT. Meiotic studies, chromosome pairing and chiasma frequency were performed on 12 species of Chrysanthemum (Asteraceae). All the species studied are based on x=9, the commonest base number of the genus. The present record of 2n=36 for C. maximum is a new tetraploid cytotype for the species. C. koreanum (2n=54) and C. paludosum (2n=18) are the two species studied cytologically for the first time. Chromosomal counts on Indian accessions of species viz. C. coccineum, C. indicum, C. multicaule, C. pyrethrum and C. segetum are made for the first time. Chiasma frequency ranges from 1.80-1.99 per bivalent in diploids, 2.47-3.01 per bivalent in tetraploids and 1.37 in hexaploid. Multivalents are noticed in both diploids and tetraploids.

KEYWORDS: Chiasma frequency, Chrysanthemum, Meiosis, Multivalents

Chrysanthemum (Compositae or Asteraceae) is cosmo- fixed in Carnoy’s fixative. Meiotic studies were made by politan in distribution and comprises of approximately using standard acetocarmine technique. Pollen fertility 200 species with four in India, majority of which are was estimated by their stainability in glycerol-acetocarmine cultivated. Chrysanthemum is believed to be native of (1:1). Photomicrographs were taken from freshly prepared China where it was being cultivated more than 2,000 slides using Nikon 80i Digital Imaging system. years ago. It is characterized by the annual or perennial herb, alternate, dissected, long peduncled, RESULTS arranged in corymbose clusters, ray floret pistillate, disc Observations on chromosome number, meiotic behaviour florets fertile and perfect, involucre scales are imbricated and chiasma frequency are given under each species. and appressed. Amongst the ornamental of Asteraceae, the Chrysanthemum is one of the widely Chrysanthemum carinatum All the plants with different cultivated and popular composit and ranks third among colour of capitula revealed to be diploid with 2n=18 (Fig. commercial flowers in the world (Prasad and Kumar 1A). Meiosis is characterized by 0-2 quadrivalents besides

2000). Chrysanthemum morifolium is the imperial normal bivalents in most of the PMCs with 1IV+7II as the of Japan and considered as one of the most important most common configuration (Fig. 1B). The quadrivalents herbs used in traditional Japanese medicine. are either ring shaped with four chiasmata or chain shaped Cytologically, the genus is based on x=9 and has many with three chiasmata and show successive type of diploid and high polyploid (2x-25x) species. The genus is orientation at Metaphase-I. The bivalents are mostly ring quite interesting from cytogenetic point of view due to shaped with two chiasmata with terminal localization or a large sized chromosomes, occurrence of reciprocal trans- few may be rod shaped with one chiasma. The chiasma locations, polyploidy and localization of chiasmata. frequency varies from 14-18 per PMC with an average of Further, lots of hybridization and mutation work on orna- 16.2 per PMC which works out to be 1.8 per bivalent. mental species has resulted in large numbers of . Further meiotic course is normal results into high pollen Keeping in view the occurrence of diversity and many fertility (82%). interesting cytogenetic phenomena in Chrysanthemum, the present cytological studies were carried out in 12 C. coccineum kakko (Pyrethrum coccineum) The species of Chrysanthemum (C. coccineum, C. carinatum, species is found to be hexaploid with 2n=54. Most of the C. coronarium, C. indicum, C. koreanum, C. leucanthemum, bivalents (19-22) per PMC are with two chiasmata besides C. maximum, C. morifolium, C. multicaule, C. paludosum, some having single chiasma (Fig. 1C). Mostly there is C. pyrethreum and C. segetum). terminal localization of chiasmata. Meiotic abnormalities such as bridges and laggards are noticed in 5-7 PMCs out MATERIALS AND METHODS of 32 cells observed. Microsporogenesis is normal leading Materials for present study were collected from cultivated to 70% pollen fertility. individuals growing in Botanical Gardens of Punjabi University, Patiala; Punjab Agriculture University, Ludhiana C. coronarium ( coronarium) Cytological and G. B. Pant University of Agriculture and Technology, screening in many cultivated plants with various colour Pant Nagar. The chromosome numbers were determined capitula revealed all these as diploids with 2n=18 (Fig. through meiotic studies for which young capitula were 1D). Most of the plants are structural hybrids as 4-12 70 GUPTA ET AL. chromosomes involved in reciprocal translocations. The is normal with high pollen fertility (79%). number of quadrivalents varies from 0-3 per PMC with an average of 0.85. The number of bivalents varies from 3-9 C. morifolium ( ‘Garden Chrysanthemum’) The per PMC with an average of 6.90. The most common present study deals with 29 varieties of the species configuration is ofIV 1 +7II found in 6 PMCs of total 21 collected from PAU, Ludhiana. All these varieties are observed. Most of the quadrivalents show successive type perennial with great diversity in flowers. All the varieties of orientation with equal distribution at Anaphase-I. The are hexaploid with 27 bivalents (Fig. 1M) and subsequent quadrivalents are mostly ring shaped with four chiasmata balanced course of meiosis. But a few cells in some while most of the bivalents with two chiasmata have distal varieties are found to have laggards and micronuclei localization. Chiasma frequency per PMC varies from 16- (Figs. 1N-Q). Pollen fertility varies from 80-90%. 18 with an average of 17.33 per PMC. Thus there is a tendency for formation of two chiasmata in each C. multicaule ( myconis or Coleostephus chromosome at terminal ends. Microsporogenesis is multicaulis) Cytologically, the species is diploid (2n= normal with considerable good number of fertile pollen 18) with 0-2 quadrivalents. The most common association grains. is of 1IV+7II noticed in 60% PMCs (Fig. 1R). Quadrivalents are either ring shaped with four chiasmata or chain shaped C. indicum The cultivated plants of the species are with three chiasmata. The number of bivalents with two found to be hexaploid with 27 bivalents at diakinesis/ chiasmata varies from 4-6 whereas with single chiasma Metaphase-I in most of the PMCs (Fig. 1E). However, are 0-4 per PMC. The chiasmata frequency per cell varies some are observed to have 1-2 quadrivalents besides the from 14-18 with an average of 14 per PMC and 2.33 per bivalents. The average association comes out to be bivalent. PMCs with laggards, bridges, dyads, triads with

0.35IV+26.3II. The most common association is of 27II or without micronuclei are also observed (Fig. 1S). Low (75%). The number of bivalents with two chiasmata varies pollen fertility (65%) is noticed. from 1-10 per PMC whereas those with single chiasma per cell varies from 29-39 per PMC with an average of C. paludosum The species is diploid with 2n=18. 34.30 per PMC which works out to be 1.37 per bivalent. Besides the formation of 0-7 bivalents, 0-1 hexavalents Most of the PMCs have some meiotic abnormalities in the and 0-2 quadrivalents are also reported with an average form of laggards and bridges (Fig. 1F). Microsporogenesis frequency of 77.77, 6.66 and 16.22, respectively (Figs. is also mostly normal but few cells are observed with 1T, U). Among these the most common association is triads leading to low reproductive potential (52%) (Fig. found to be of 9II (40%) followed by 1IV+7II (33.33%). The 1G). multivalents account for about 23% of chromosomes. Hexavalents are ring shaped whereas ring shaped C. koreanum The species depicts hexaploid chromosome quadrivalents are more common than chain type. Among count of 2n=54 (Fig. 1H). Chromosomal segregation is the bivalents there is relatively high frequency of those normal in majority of PMCs but a few show laggard with single chiasma than those with two chiasmata. The formations (Fig. 1I). Further during microsporogenesis, chiasma frequency ranges from 11-18 with an average of dyads, triads, tetrads with or without micronuclei are 13.93 per PMC and 1.99 per bivalent. In spite of noticed. Pollen fertility gets reduced to 70%. occurrence of multivalent at Anaphase-I, distribution is normal with high pollen fertility (83%). C. leucanthemum (Leucanthemum vulgare) The species bear tetraploid chromosome number of 2n=36 (Fig. 1J). C. pyrethrum Cytologically the species is diploid with Meiosis is characterised by the occurrence of 0-3 nine bivalents at diakinesis/Metaphase-I (Fig. 1V). The quadrivalents per PMC besides bivalents. Ring or chain bivalents are mostly ring shaped with two chiasmata each. shaped quadrivalents are observed in almost equal The chromosomal distribution during anaphases is normal frequency. Relatively the bivalents with two chiasmata with perfect microsporogenesis which resulting into 75% are more common than ones with single chiasma. The pollen fertility. chiasma frequency varies from 24-32 per PMC with an average of 29.64 which comes out to be 2.47 per bivalent. C. segetum (Glebionis segetum) The meiotic studies During anaphases and telophases, a few PMCs are found the species to be tetraploid (2n=36). Meiotic course reported with chromatin bridges and laggards, and triads was quite abnormal with the formation of 4-7 quadrivalents during microsporogenesis (Fig. 1K). Pollen fertility is and 1-10 bivalents (Figs. 1W, X). The average association substantially reduced (50%). come out to be 4.94IV+7.66II. More than 50% chromosomes were involved in multivalent formation. Quadrivalents C. maximum (cultivar ‘Shasta Daisy’) Meiotic study were either ring shaped with four chiasmata or chain reveals the to be tetraploid (2n=36) with 4IV+10II as shaped with three chiasmata with 2-5 and 1-3 per PMC, the most common configuration (Fig. 1L). Further meiosis respectively. Bivalents are mostly with two chiasmata and CYTOMORPHOLOGICAL STUDIES IN CHRYSANTHEMUM 71

Fig. 1. Meiotic chromosome numbers in 12 species of Chrysanthemum. C. carinatum. A. PMC at diakinesis showing 9II (8 ring + 1 rod shaped). B. PMC at diakinesis showing 1IV+7II (ring shaped). C. coccineum. C. PMC at diakinesis showing 27II. C. coronarium. D. PMC at diakinesis with 9II. C. indicum. E. PMC at diakinesis showing 27II. F. PMC at Anaphase-I showing laggards. G. Triad. C. koreanum. H. PMC at Metaphase-I showing 27II. I. PMC at Anaphase-I showing laggards. C. leucanthemum. J. PMC at Metaphase-I showing 18II. K. PMC at Anaphase-I showing bridge. C. maximum. L. PMC at Metaphase-I showing 4IV+10II. C. morifolium. M. PMC at metaphase-I showing 27II. N-P. PMCs at anaphase-I, telophase-I and anaphase-II showing laggards, respectively. Q. Tetrad with micronucleus. C. multicaule. R. PMC at diakinesis showing 1IV+7II. S. PMC at anaphase-I showing laggard. C. paludosum. T. PMC at metaphase-I showing 1VI+2IV+2II. U. PMC at metaphase-I showing 1VI+1IV+4II (ring shaped). C. pyrethreum. V. PMC at diakinesis showing 9II (ring shaped). C. segetum. W. PMC at diakinesis showing 2IV+14II. X. PMC at metaphase-I showing 6IV+6II (all ring shaped with 1 chain shaped quadrivalent). Bar=10µm. 72 GUPTA ET AL.

3-10 were with single chiasma. The number of chiasmata Verma and Chandel (1994), C. coronarium by Vogt and per PMC varied from 30.16 per PMC with an average of Oberprieler (1993), Kaul and Kaur (1995), C. leucanthemum three per bivalent. In spite of high frequency of multivalent, by Dowrick (1952), Bala and Gupta (2011), C. morifolium the chromosome distribution is almost normal in most of by Nazeer and Khoshoo (1985), Gupta and Gill (1983) are the PMCs. Further course of meiosis was also normal with confirmed both from India and outside. 80% pollen fertility. No chromosomal record is available for these species from India however known from outside India viz. C. DISCUSSION coccineum (Dowrick 1952), C. indicum (Kamaiaka and Morphology In the present study 12 species of Yonezawa 1989), C. multicaule (Harling 1951), C. Chrysanthemum were investigated for their morphological pyrethrum (vide Fedorov 1969) and C. segetum (Murín and cytological parameters. For C. morifolium most 1993). previous data on morphological characteristics seem to The present record of 2n=36 for C. maximum is a new support the view of evolution from simple to complex tetraploid cytotype for the species. Chrysanthemum form. The various cultivars of this species are differentiated koreanum (2n=54) and C. paludosum (2n=18) are the two on the basis of flower size. The flowers are differentiated species studied cytologically for the first time. on the basis of shape of ray florets. For colour, yellow is primitive, while purple and red are thought to evolve from Multivalent formation and heterozygosity Newly pro- yellow sequentially through orange and pink (Wang et al. duced polyploids are less productive than their diploid 2010). C. carinatum was typical species with white colour parents. Autopolyploids are characterised by meiotic ray florets with yellow tinge at the base and the disc florets chromosomal irregularities in the form of high frequency have purple color forming three distinct rings of the colour of multivalent formation leading to unequal segregation thus, giving the name of tricolor to the species. The plants of multivalent, random chiasmata and cytologically of the species were found to have lot of variation not only imbalanced gametes are produced. The produced gametes in colour pattern of capitula but also with respect to either non-functional or produce unbalanced offspring. and achene characters. Some of the plants are typical of C. All the investigated species showed maximum number carinatum type whereas others were not having the typical of bivalents followed by quadrivalents and hexavalents. colour of capitula and other features of keeled involucral Quadrivalents and hexavalents are present in tetraploids bracts and flat winged achene. Therefore, making these and hexaploid species. Overall 36.7% plants were plants more near to C. coronarium. It showed that the homozygous with nine bivalent formation, 27.9% have plant might be intraspecific hybrid. Similarly, C. one hexavalent with five bivalent, 11.76% have two coronarium showed radiate and ligulate type of capitula. quadrivalents with five bivalents and the remaining 16.1% However, in some plants there is tendency of change of have one hexavalent with one quadrivalent with four two to three whorls of disc florets into ligulate whorls. bivalents. Rana (1965) was able to synthesize an Some of the plants with radiate capitula have two to three interchange stock of the species in which 12 of 18 whorls of ray florets. In open pollinated populations the chromosomes were involved in rearrangement and form a frequency of plants with radiate capitula is much more single multiple association during meiosis. It has been than those with ligulate heads. The rest of the species of suggested by many investigations that the frequency of Chrysanthemum worked out in present investigation multiple formation and disjunction are under gene control showed similar morphological characteristics with little (Lawrence 1958; Rees 1961). Rana and Jain (1965) variation. reported involvement of same two pairs of chromosomes in the different populations of C. carinatum, whereas Chromosome numbers Chromosome size and form Paria and Pardhan (1971) reported the presence of vary little between or within species. The genus shows a different pairs of chromosomes in reciprocal translocations. polyploid series between 2x-25x types. In diploid species, Structural heterozygosity or chromosomal rearrange- the chromosome size ranges from 6-8 µm. There is ments involved in translocation and inversions. The gradual size diminution through the polyploid series to presence of multivalent in diploids indicated the possibility Chrysanthemum lacustre (2n=98) whose average chro- of heterozygote translocations between two pairs of mosome size is under 3 µm. Chrysanthemum sonare chromosomes. Newly formed structural changes bring out (2n=80) has its largest chromosome size 4 µm long with genetic variability in the gametes by forming new genetic majority being about 3.5 µm. Increase in chromosome linkage groups. Presently, multivalents have been noticed number (ploidy) accompanied by decrease in chromosome in all the diploids except C. pyrethreum. The presence of size. The basic chromosome number in all the species was multivalent in tetraploids; C. morifolium and C. segetum found to be 9 (Dowrick 1952; Watanabe 1977). may be due to partial homology of the two genomes The chromosome numbers in nine species of present present might be due to structural hybridity. study are confirmed by previous reports. Chromosome Studies reveals that all species of present study have counts in Chrysanthemum carinatum by Murín (1993), maintained a significant amount of interchange heterozy- CYTOMORPHOLOGICAL STUDIES IN CHRYSANTHEMUM 73

Table 1. Chiasma frequency in the species of Chrysanthemum studied Species name Range of chiasma frequency Chiasma frequency per per PMC bivalent C. carinatum (2n=18) 14-18 1.80 C. coronarium (2n=18) 16-18 1.92 C. indicum (2n=54) 29-39 1.37 C. leucanthemum (2n=36) 24-32 2.47 C. multicaule (2n=18) 14-18 2.33 C. paludosum (2n=18) 11-18 1.99 C. segetum (2n=36) 30-45 3.01 gosity in natural populations with clarity establish the CONCLUSION adaptive role of chromosomes rearrangement. The adaptive The present study on 12 species of Chrysanthemum value of structural heterozygosity has been reported by mostly show genome homologies reflected in the form of Burnham (1956) and Rees (1961). multivalent. Besides, there is reciprocal translocation in Meiotic analysis of all cultivars of C. morifolium several varieties/ species. Therefore, the study may act as studied in present investigation revealed only bivalents. a platform for future breeding programmes and applied Similar observations were made by earlier workers research in this ornamentally important genus. (Dowrick 1953; Nazeer and Khoshoo 1985). Dowrick (1953) reported multivalent in only two cells of the ACKNOWLEDGEMENTS. The study was supported by financial grant under DRS SAP-III Programme and Rajiv Gandhi National cultivars examined and noted that cultivars with 54 Fellowship Scheme under UGC, New Delhi. chromosomes invariably formed 27 bivalents. Nazeer and Khoshoo (1985) found higher frequencies of multivalent LITERATURE CITED but concluded that hexaploids and higher aneuploids Baker, B. S., Carpenter, A. T. C., Esposito, M. S., Esposito, R. showed diploidized meiotic behaviour. Parallel studies E. and Sandler, L. 1976. The genetic control of meiosis. with polyploid native Japanese species of Chrysanthemum Annual Rev. Genet. 10: 53-134. also showed very high frequencies of bivalents formation Bala, S. and Gupta, R. C. 2011. In Marhold, K. (ed.). IAPT/ IOPB Chromosome data 12. Taxon 60: 1784-1786. (95.5-99.8%) of those possible, which were maintained in Burnham, C. R. 1956. Chromosomal interchanges in Plants. tetraploids and hexaploids hybrids but not in triploids, Bot. Rev. 22: 419-552. pentaploids and heptaploids hybrids (Watanabe 1977). Dowrick, G. J. 1952. The chromosomes of Chrysanthemum, I: The species. Heredity 6: 365-375. Fedorov, A. A. (ed.). 1969. Chromosome numbers of flowering Chiasma frequency A chiasma is a point where two plants. Izdatelstvo “Nauk,” Leningrad. homologous non-sister chromatids exchange genetic Gupta, R. C. and Gill, B. S. 1983. Cytology of family Compositae materials during chromosomal crossover at some point in of the Punjab plains India. Proceedings of the Indian National Science Academy, Part B Biological Sciences meiosis. The chiasmata become visible during the 49: 356-366. diplotene stage of prophase I of meiosis but actual crossing Harling, G. 1951. Embryological studies in the Compositae. over thought to occur at pachytene stage. When each 1-3. Acta Hort. Berg. 16: 1-120. Kamaiaka, H. and Yonezawa, Y. 1989. Hexaploid Chrysanthemum bivalent begins to split, the only points of contact are at indicum L. (Compositae) newly found in the Shikoku the chiasmata. district, western Japan. Chrom. Inf. Serv. 46: 14-17. Meiotic recombination has long history investigation Kaul, M. L. and Kaur, J. 1995. Checkered prophase I in crown by cytological and genetical methods and more recently daisy. Cytologia 60: 195-203. Latos-Bielenska, A. and Vogel, W. 1990. Frequency and by molecular approaches. In cytological method, distribution of chiasmata in Syrian hamster spermatocytes recording of numbers and locations of chiasmata at late studied by the BrdU antibody technique. Chromosoma prophase I and metaphase I of meiosis demonstrate the 99: 267-272. Lawrence, M. J. 1958. Genotypic control of crossing-over on physical exchange between homologous non-sister the first chromosome ofDrosophila melanogaster. Nature chromatids (Tease and Jones 1978; Latos-Bielenska and 182: 889-890. Vogel 1990). This represents an efficient approach, by Murín, A. 1993. Karyologické atúdium okrasn@3ych rastlín rapidly scoring large number of meiocytes, to genome flóry Slovenska. Biologia (Bratislava) 48: 441-445. Nazeer, M. A. and Khoshoo, T. N. 1985. Meiotic variations in wide levels of recombination as well as the distribution of Chrysanthemum morifolium complex. Nucleus 28: 35-41. recombination events within chromosomes. The frequency Paria, P. and Pradhan, K. 1970. Maintenance of interchange and distribution of chiasmata is under genetic control heterozyotes in annual Chrysanthemum. Cytologia 36: 627-632. (Rees 1961; Baker et al. 1976; Ouick 1993). Prasad, S. and Kumar, U. 2000. Commercial floriculture. Agro. Bios. Publ. Jodhpur. Quick, D. L. J. 1993. Principles and techniques of contemporary 74 GUPTA ET AL.

. Blackie Academic & Professional, Glasgao. p. a new and novel material for the study of radiation 298. induced chromosomal aberrations at mitosis. Cytologia Rana, R. S. and Jain, H. K. 1965. Adaptive role of interchange 59(3): 339-343. heterozygosity in the annual Chrysanthemum. Heredity Vogt, R. and Oberprieler, C. 1993. Mediterranean chromosome 20: 21-29. number reports 3 (171-176). Fl. Medit. 3: 338-340. Rees, H. 1961. Genotypic control of chromosome form and Wang, H. B., Chen, F. D. Chen, S. M., Fang, W. M., Zhu, X. R. behaviour. Bot. Rev. 27: 288-318. and Li, F. T. 2010. Investigation of standard Chrysanthemum Tease, C. and Jones, G. H. 1978. Analysis of exchanges in cultivars in six cities of China. Journ. Plant. Genet. differentially stained meiotic chromosomes of Locusta Resour. 12: 570-574. migratoria after BrdU-substitution and FPG staining I. Watanabe, K. 1977. Successful ovary culture and production Crossover exchanges in mono-chiasmata bivalents. of F, P-hybrids and androgenic haploids in Japanese Chromosoma (Berl.) 69: 163-178. Chrysanthemum species. Heredity 68: 317-320. Verma, R. C. and Chandel, S. 1994. Annual :