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

Cytomorphological Studies in some species of Chrysanthemum L. (Asteraceae)

Raghbir C. Gupta, Santosh Bala1, Shilpa Sharma and Manish Kapoor
Department of Botany, Punjabi University, Patiala-147002, Punjab, India
1

Author for correspondence: santosh_gamo@rediffmail.com 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 cosmopolitan in distribution and comprises of approximately 200 species with four in India, majority of which are cultivated. Chrysanthemum is believed to be native of China where it was being cultivated more than 2,000 years ago. It is characterized by the annual or perennial herb, leaves alternate, dissected, long peduncled, flowers arranged in corymbose clusters, ray floret pistillate, disc florets fertile and perfect, involucre scales are imbricated and appressed. Amongst the ornamental plants of Asteraceae, the Chrysanthemum is one of the widely cultivated and popular composit and ranks third among commercial flowers in the world (Prasad and Kumar 2000). Chrysanthemum morifolium is the imperial flower of Japan and considered as one of the most important herbs used in traditional Japanese medicine. Cytologically, the genus is based on x=9 and has many diploid and high polyploid (2x-25x) species. The genus is quite interesting from cytogenetic point of view due to large sized chromosomes, occurrence of reciprocal translocations, polyploidy and localization of chiasmata. Further, lots of hybridization and mutation work on ornamental species has resulted in large numbers of cultivars. Keeping in view the occurrence of diversity and many interesting cytogenetic phenomena in Chrysanthemum, the present cytological studies were carried out in 12 species of Chrysanthemum (C. coccineum, C. carinatum, C. coronarium, C. indicum, C. koreanum, C. leucanthemum, C. maximum, C. morifolium, C. multicaule, C. paludosum, C. pyrethreum and C. segetum). fixed in Carnoys fixative. Meiotic studies were made by using standard acetocarmine technique. Pollen fertility was estimated by their stainability in glycerol-acetocarmine (1:1). Photomicrographs were taken from freshly prepared slides using Nikon 80i Digital Imaging system.

Observations on chromosome number, meiotic behaviour and chiasma frequency are given under each species. Chrysanthemum carinatum All the plants with different colour of capitula revealed to be diploid with 2n=18 (Fig. 1A). Meiosis is characterized by 0-2 quadrivalents besides normal bivalents in most of the PMCs with 1IV+7II as the most common configuration (Fig. 1B). The quadrivalents are either ring shaped with four chiasmata or chain shaped with three chiasmata and show successive type of orientation at Metaphase-I. The bivalents are mostly ring shaped with two chiasmata with terminal localization or a few may be rod shaped with one chiasma. The chiasma frequency varies from 14-18 per PMC with an average of 16.2 per PMC which works out to be 1.8 per bivalent. Further meiotic course is normal results into high pollen fertility (82%). C. coccineum kakko (Pyrethrum coccineum)The species is found to be hexaploid with 2n=54. Most of the bivalents (19-22) per PMC are with two chiasmata besides some having single chiasma (Fig. 1C). Mostly there is terminal localization of chiasmata. Meiotic abnormalities such as bridges and laggards are noticed in 5-7 PMCs out of 32 cells observed. Microsporogenesis is normal leading to 70% pollen fertility. C. coronarium (Glebionis coronarium)Cytological screening in many cultivated plants with various colour capitula revealed all these as diploids with 2n=18 (Fig. 1D). Most of the plants are structural hybrids as 4-12

RESULTS

Materials for present study were collected from cultivated individuals growing in Botanical Gardens of Punjabi University, Patiala; Punjab Agriculture University, Ludhiana and G. B. Pant University of Agriculture and Technology, Pant Nagar. The chromosome numbers were determined through meiotic studies for which young capitula were

MATERIALS AND METHODS

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chromosomes involved in reciprocal translocations. The number of quadrivalents varies from 0-3 per PMC with an average of 0.85. The number of bivalents varies from 3-9 per PMC with an average of 6.90. The most common configuration is of 1IV+7II found in 6 PMCs of total 21 observed. Most of the quadrivalents show successive type of orientation with equal distribution at Anaphase-I. The quadrivalents are mostly ring shaped with four chiasmata while most of the bivalents with two chiasmata have distal localization. Chiasma frequency per PMC varies from 1618 with an average of 17.33 per PMC. Thus there is a tendency for formation of two chiasmata in each chromosome at terminal ends. Microsporogenesis is normal with considerable good number of fertile pollen grains. C.indicum The cultivated plants of the species are found to be hexaploid with 27 bivalents at diakinesis/ Metaphase-I in most of the PMCs (Fig. 1E). However, some are observed to have 1-2 quadrivalents besides the bivalents. The average association comes out to be 0.35IV+26.3II. The most common association is of 27II (75%). The number of bivalents with two chiasmata varies from 1-10 per PMC whereas those with single chiasma per cell varies from 29-39 per PMC with an average of 34.30 per PMC which works out to be 1.37 per bivalent. Most of the PMCs have some meiotic abnormalities in the form of laggards and bridges (Fig. 1F). Microsporogenesis is also mostly normal but few cells are observed with triads leading to low reproductive potential (52%) (Fig. 1G). C. koreanum The species depicts hexaploid chromosome count of 2n=54 (Fig. 1H). Chromosomal segregation is normal in majority of PMCs but a few show laggard formations (Fig. 1I). Further during microsporogenesis, dyads, triads, tetrads with or without micronuclei are noticed. Pollen fertility gets reduced to 70%. C. leucanthemum (Leucanthemum vulgare) The species bear tetraploid chromosome number of 2n=36 (Fig. 1J). Meiosis is characterised by the occurrence of 0-3 quadrivalents per PMC besides bivalents. Ring or chain shaped quadrivalents are observed in almost equal frequency. Relatively the bivalents with two chiasmata are more common than ones with single chiasma. The chiasma frequency varies from 24-32 per PMC with an average of 29.64 which comes out to be 2.47 per bivalent. During anaphases and telophases, a few PMCs are reported with chromatin bridges and laggards, and triads during microsporogenesis (Fig. 1K). Pollen fertility is substantially reduced (50%). C. maximum (cultivar Shasta Daisy) Meiotic study reveals the plant to be tetraploid (2n=36) with 4IV+10II as the most common configuration (Fig. 1L). Further meiosis

is normal with high pollen fertility (79%). C. morifolium (cultivar Garden Chrysanthemum)The present study deals with 29 varieties of the species collected from PAU, Ludhiana. All these varieties are perennial with great diversity in flowers. All the varieties are hexaploid with 27 bivalents (Fig. 1M) and subsequent balanced course of meiosis. But a few cells in some varieties are found to have laggards and micronuclei (Figs. 1N-Q). Pollen fertility varies from 80-90%. C. multicaule (Coleostephus myconis or Coleostephus multicaulis) Cytologically, the species is diploid (2n= 18) with 0-2 quadrivalents. The most common association is of 1IV+7II noticed in 60% PMCs (Fig. 1R). Quadrivalents are either ring shaped with four chiasmata or chain shaped with three chiasmata. The number of bivalents with two chiasmata varies from 4-6 whereas with single chiasma are 0-4 per PMC. The chiasmata frequency per cell varies from 14-18 with an average of 14 per PMC and 2.33 per bivalent. PMCs with laggards, bridges, dyads, triads with or without micronuclei are also observed (Fig. 1S). Low pollen fertility (65%) is noticed. C.paludosum The species is diploid with 2n=18. Besides the formation of 0-7 bivalents, 0-1 hexavalents and 0-2 quadrivalents are also reported with an average frequency of 77.77, 6.66 and 16.22, respectively (Figs. 1T, U). Among these the most common association is found to be of 9II (40%) followed by 1IV+7II (33.33%). The multivalents account for about 23% of chromosomes. Hexavalents are ring shaped whereas ring shaped quadrivalents are more common than chain type. Among the bivalents there is relatively high frequency of those with single chiasma than those with two chiasmata. The chiasma frequency ranges from 11-18 with an average of 13.93 per PMC and 1.99 per bivalent. In spite of occurrence of multivalent at Anaphase-I, distribution is normal with high pollen fertility (83%). C. pyrethrum Cytologically the species is diploid with nine bivalents at diakinesis/Metaphase-I (Fig. 1V). The bivalents are mostly ring shaped with two chiasmata each. The chromosomal distribution during anaphases is normal with perfect microsporogenesis which resulting into 75% pollen fertility. C. segetum (Glebionis segetum) The meiotic studies found the species to be tetraploid (2n=36). Meiotic course was quite abnormal with the formation of 4-7 quadrivalents and 1-10 bivalents (Figs. 1W, X). The average association come out to be 4.94IV+7.66II. More than 50% chromosomes were involved in multivalent formation. Quadrivalents were either ring shaped with four chiasmata or chain shaped with three chiasmata with 2-5 and 1-3 per PMC, respectively. Bivalents are mostly with two chiasmata and

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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=10m.

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3-10 were with single chiasma. The number of chiasmata per PMC varied from 30.16 per PMC with an average of three per bivalent. In spite of high frequency of multivalent, the chromosome distribution is almost normal in most of the PMCs. Further course of meiosis was also normal with 80% pollen fertility.

Morphology In the present study 12 species of Chrysanthemum were investigated for their morphological and cytological parameters. For C. morifolium most previous data on morphological characteristics seem to support the view of evolution from simple to complex form. The various cultivars of this species are differentiated on the basis of flower size. The flowers are differentiated on the basis of shape of ray florets. For colour, yellow is primitive, while purple and red are thought to evolve from yellow sequentially through orange and pink (Wang et al. 2010). C. carinatum was typical species with white colour ray florets with yellow tinge at the base and the disc florets have purple color forming three distinct rings of the colour thus, giving the name of tricolor to the species. The plants of the species were found to have lot of variation not only in colour pattern of capitula but also with respect to leaf and achene characters. Some of the plants are typical of C. carinatum type whereas others were not having the typical colour of capitula and other features of keeled involucral bracts and flat winged achene. Therefore, making these plants more near to C. coronarium. It showed that the plant might be intraspecific hybrid. Similarly, C. coronarium showed radiate and ligulate type of capitula. However, in some plants there is tendency of change of two to three whorls of disc florets into ligulate whorls. Some of the plants with radiate capitula have two to three whorls of ray florets. In open pollinated populations the frequency of plants with radiate capitula is much more than those with ligulate heads. The rest of the species of Chrysanthemum worked out in present investigation showed similar morphological characteristics with little variation. Chromosome numbers Chromosome size and form vary little between or within species. The genus shows a polyploid series between 2x-25x types. In diploid species, the chromosome size ranges from 6-8 m. There is gradual size diminution through the polyploid series to Chrysanthemum lacustre (2n=98) whose average chromosome size is under 3 m. Chrysanthemum sonare (2n=80) has its largest chromosome size 4 m long with majority being about 3.5 m. Increase in chromosome number (ploidy) accompanied by decrease in chromosome size. The basic chromosome number in all the species was found to be 9 (Dowrick 1952; Watanabe 1977). The chromosome numbers in nine species of present study are confirmed by previous reports. Chromosome counts in Chrysanthemum carinatum by Murn (1993),

DISCUSSION

Verma and Chandel (1994), C. coronarium by Vogt and Oberprieler (1993), Kaul and Kaur (1995), C. leucanthemum by Dowrick (1952), Bala and Gupta (2011), C. morifolium by Nazeer and Khoshoo (1985), Gupta and Gill (1983) are confirmed both from India and outside. No chromosomal record is available for these species from India however known from outside India viz. C. coccineum (Dowrick 1952), C. indicum (Kamaiaka and Yonezawa 1989), C. multicaule (Harling 1951), C. pyrethrum (vide Fedorov 1969) and C. segetum (Murn 1993). The present record of 2n=36 for C. maximum is a new tetraploid cytotype for the species. Chrysanthemum koreanum (2n=54) and C. paludosum (2n=18) are the two species studied cytologically for the first time. Multivalent formation and heterozygosity Newly produced polyploids are less productive than their diploid parents. Autopolyploids are characterised by meiotic chromosomal irregularities in the form of high frequency of multivalent formation leading to unequal segregation of multivalent, random chiasmata and cytologically imbalanced gametes are produced. The produced gametes either non-functional or produce unbalanced offspring. All the investigated species showed maximum number of bivalents followed by quadrivalents and hexavalents. Quadrivalents and hexavalents are present in tetraploids and hexaploid species. Overall 36.7% plants were homozygous with nine bivalent formation, 27.9% have one hexavalent with five bivalent, 11.76% have two quadrivalents with five bivalents and the remaining 16.1% have one hexavalent with one quadrivalent with four bivalents. Rana (1965) was able to synthesize an interchange stock of the species in which 12 of 18 chromosomes were involved in rearrangement and form a single multiple association during meiosis. It has been suggested by many investigations that the frequency of multiple formation and disjunction are under gene control (Lawrence 1958; Rees 1961). Rana and Jain (1965) reported involvement of same two pairs of chromosomes in the different populations of C. carinatum, whereas Paria and Pardhan (1971) reported the presence of different pairs of chromosomes in reciprocal translocations. Structural heterozygosity or chromosomal rearrangements involved in translocation and inversions. The presence of multivalent in diploids indicated the possibility of heterozygote translocations between two pairs of chromosomes. Newly formed structural changes bring out genetic variability in the gametes by forming new genetic linkage groups. Presently, multivalents have been noticed in all the diploids except C. pyrethreum. The presence of multivalent in tetraploids; C. morifolium and C. segetum may be due to partial homology of the two genomes present might be due to structural hybridity. Studies reveals that all species of present study have maintained a significant amount of interchange heterozy-

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Table 1. Chiasma frequency in the species of Chrysanthemum studied

Species name C. carinatum (2n=18) C. coronarium (2n=18) C. indicum (2n=54) C. leucanthemum (2n=36) C. multicaule (2n=18) C. paludosum (2n=18) C. segetum (2n=36) Range of chiasma frequency per PMC 14-18 16-18 29-39 24-32 14-18 11-18 30-45 Chiasma frequency per bivalent 1.80 1.92 1.37 2.47 2.33 1.99 3.01

gosity in natural populations with clarity establish the adaptive role of chromosomes rearrangement. The adaptive value of structural heterozygosity has been reported by Burnham (1956) and Rees (1961). Meiotic analysis of all cultivars of C. morifolium studied in present investigation revealed only bivalents. Similar observations were made by earlier workers (Dowrick 1953; Nazeer and Khoshoo 1985). Dowrick (1953) reported multivalent in only two cells of the cultivars examined and noted that cultivars with 54 chromosomes invariably formed 27 bivalents. Nazeer and Khoshoo (1985) found higher frequencies of multivalent but concluded that hexaploids and higher aneuploids showed diploidized meiotic behaviour. Parallel studies with polyploid native Japanese species of Chrysanthemum also showed very high frequencies of bivalents formation (95.5-99.8%) of those possible, which were maintained in tetraploids and hexaploids hybrids but not in triploids, pentaploids and heptaploids hybrids (Watanabe 1977). Chiasma frequency A chiasma is a point where two homologous non-sister chromatids exchange genetic materials during chromosomal crossover at some point in meiosis. The chiasmata become visible during the diplotene stage of prophase I of meiosis but actual crossing over thought to occur at pachytene stage. When each bivalent begins to split, the only points of contact are at the chiasmata. Meiotic recombination has long history investigation by cytological and genetical methods and more recently by molecular approaches. In cytological method, recording of numbers and locations of chiasmata at late prophase I and metaphase I of meiosis demonstrate the physical exchange between homologous non-sister chromatids (Tease and Jones 1978; Latos-Bielenska and Vogel 1990). This represents an efficient approach, by rapidly scoring large number of meiocytes, to genome wide levels of recombination as well as the distribution of recombination events within chromosomes. The frequency and distribution of chiasmata is under genetic control (Rees 1961; Baker et al. 1976; Ouick 1993).

The present study on 12 species of Chrysanthemum mostly show genome homologies reflected in the form of multivalent. Besides, there is reciprocal translocation in several varieties/ species. Therefore, the study may act as a platform for future breeding programmes and applied research in this ornamentally important genus. ACKNOWLEDGEMENTS.
The study was supported by financial grant under DRS SAP-III Programme and Rajiv Gandhi National Fellowship Scheme under UGC, New Delhi.

CONCLUSION

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