Pawan Kumar Rana, Himshikha, Puneet Kumar*, Vijay Kumar Singhal and Raghbir Chand Gupta

Transcription

1 2012 The Japan Mendel Society Cytologia 77(2): 1 7 Impact of Reciprocal Translocations and Non-Synchronous Disjunction of Chromosomes on Pollen Fertility in Astragalus Chlorostachys from the Northwest Himalayas (India) Pawan Kumar Rana, Himshikha, Puneet Kumar*, Vijay Kumar Singhal and Raghbir Chand Gupta Department of Botany, Punjabi University, Patiala , Punjab, India Received September 28, 2011; accepted January 26, 2012 Summary Cytological investigations carried out on a population basis in Astragalus chlorostachys Lindl. which exists at diploid level (n=8) revealed the presence of quadrivalents, the late disjunction of 1 4 bivalents and some pollen sterility (34%) in 1 accession collected from Koksar, at 3,140 m in the Lahaul Valley (Lahaul-Spiti, Himachal Pradesh). The presence of quadrivalents in this diploid species seems to be a consequence of reciprocal translocations. The occurrence of structural heterozygosity in the species has been reported here for the first time. All of the other populations showed normal bivalent formation and regular sporad generation and 100% pollen fertility. The paper here discusses in detail the course of meiosis and the effect of multivalent formation and non-synchronous disjunction of bivalents on meiotic course and pollen fertility. Key words Lahaul-Spiti, Pangi Valley, Chamba, Parvati Valley, Meiosis, Structural heterozygosity, Chromosome number, Cold region. Astragalus chlorostachys Lindl., known variously as Astragalus altissimus Rech. f., Astragalus nuristanicus Kitam. and Hedysarum strobiliferum Baker, belongs to the tribe Galegeae of the family Fabaceae. It is a tall woody herb with sparsely pubescent stems. Leaves are imparipinnately compound and with leaflets oblong, obtuse and glabrescent. Yellowish-white flowers are produced in axillary, peduncled racemes. The species is native to Afghanistan, Bhutan, India, Nepal and Pakistan. In India it grows in moist shady places between altitudes of 2,000 3,600 m in the states of Assam, Himachal Pradesh, Jammu-Kashmir, Sikkim and Uttarakhand (Chowdhery and Wadhwa 1984, Naithani 1984, Sharma and Jamwal 1988, Wenninger 1991, Sanjappa 1992). Local people in the Kumaon Himalayas use a whole plant infusion as a tonic and febrifuge; decoction of the root is given in tuberculosis (Trivedi 2006). During cytomorphological surveys of the species in various cold regions of the Northwest Himalayas, the authors have detected 1 population which displayed the presence of multivalents, late disjunction of some bivalents and some pollen sterility. All of the other populations showed normal bivalent formation and regular sporad generation and 100% pollen fertility. In this paper, the aim was to study in detail the male meiotic course and to investigate the effect of multivalent formation and non-synchronous disjunction of bivalents on meiotic course and pollen fertility. Materials and methods Materials for male meiotic studies were collected from the different localities falling in the * Corresponding author, puneetbotcyto@gmail.com DOI: /cytologia.77.xxx

2 2 P. K. Rana et al. Cytologia 77(2) Fig. 1. a) A section of a world map showing places (marked with a star) where chromosome counts were previously made in A. chlorostachys. b) Map of Jammu and Kashmir and Himachal Pradesh states showing previously (marked with star) and presently (in square) studied localities for chromosome numbers in A. chlorostachys. c) Collection sites (marked with asterisks) in the Chamba, Lahaul- Spiti and Kullu districts of Himachal Pradesh for the present study. districts of Lahaul-Spiti (Lahaul Valley), Chamba (Manimahesh hills, Pangi Valley) and Kullu (Parvati and Malana Valley) of Himachal Pradesh, India (Fig. 1a c, Table 1) in July, The young developing floral buds from healthy plants were fixed in freshly prepared Carnoy s fixative (1 glacial acetic acid : 3 chloroform : 6 ethanol, v/v/v) for 24 h and subsequently stored in 70% ethanol in a refrigerator. Developing anthers from floral buds were squashed in 1% acetocarmine and preparations were studied for chromosome counts, and detailed meiotic behavior in pollen mother cells (PMCs) at the prophase-i, metaphase-i (M-I), anaphases-i/ii (A-I/II), telophases-i/ii (T-I/II) and sporad stages. A total of PMCs were examined to determine chromosome counts while slides were prepared from different anthers/flowers for the analysis of chromosomal associations. Pollen fertility was estimated through stainability tests for which anthers from mature flowers were squashed in glycerol acetocarmine mixture (1 : 1) and 1% aniline blue dye. Well-filled pollen grains with uniformly stained cytoplasm were scored as fertile/viable while shrivelled pollen with unstained or poorly stained cytoplasm were counted as sterile/unviable. Photomicrographs of chromosomes, sporads and pollen grains from temporary mounts were taken using Nikon Eclipse 80i microscope. Results and discussion Meiotic chromosome number Male meiotic studies were carried out on the basis of plants collected from different localities of the Lahaul Valley (Lahaul-Spiti district), the Manimahesh hills and the Pangi Valley (Chamba district) and Parvati Valley and Malana Valley (Kullu district). All these accessions shared the same diploid meiotic chromosome count of n=8 as confirmed from the presence of 8 bivalents at M-I (Fig. 2a) and 8 : 8 chromosomes distribution at A-I (Fig. 2b). The present chromosome count of n=8 in the species is in agreement with the earlier records of 2n=16 from Ferozpur Nullah

3 2012 Reciprocal Translocations in Astragalus chlorostachys 3 Table 1. Locality, habitat, latitude and longitude, altitude, accession number, meiotic chromosome number, ploidy level, meiotic behaviour, pollen fertility of different accessions and previous chromosome reports in A. chlorostachys S. No. Population Locality, habitat, latitude and longitude and altitude Accession number (PUN) Meiotic chromosome number n Ploidy level Meiotic behaviour Pollen fertility Previous counts 1 Lahaul Valley Near Koksar, Lahaul Valley in Lahaul-Spiti, Himachal Pradesh, growing on moist slopes, N; E, Alt.: 3150 m Koksar, Lahaul Valley in Lahaul-Spiti, Himachal Pradesh, growing among small stones slopes, N; E, Alt.: 3140 m Sissu, Lahaul Valley in Lahaul-Spiti, Himachal Pradesh, growing on moist slopes, N; E, Alt.: 3170 m Keylong, Lahaul Valley in Lahaul-Spiti, Himachal Pradesh, growing around agricultural fields, N; E, Alt.: 3340 m x Normal 100 2n=16 Ledingham, x Multivalents & ; Gohil et late disjunction al., 1981; of 1 4 bivalents Ledingham & x Normal x Normal 100 Rever, 1963; Ashraf & Gohil, 1989; Kumari et al., Manimahesh hills Dunali, Manimahesh hills, Chamba, Himachal Pradesh, around forest area, N; E, Alt.: 2780 m x Normal Pangi Valley Muhani, Pangi Valley, Chamba, Himachal Pradesh, growing in moist places around Corylus colurna and Juglans regia, N; E, Alt.:2500 m x Normal 98 4 Malana Valley Malana, Parvati Valley, Kullu, Himachal Pradesh, along water streams, N; E, Alt.: 2640 m 5. Parvati Valley Kasol, Parvati Valley, Kullu, Himachal Pradesh, on moist slopes, N; E, Alt.: 1610 m x Normal x Normal 99

4 4 P. K. Rana et al. Cytologia 77(2) Fig. 2. a) PMCs showing n=7 at metaphase-i. b) A PMCs showing 8 : 8 chromosomes distributions at anaphase-i. c) PMCs in a group showing the late disjunction of chromosomes at anaphase-i (arrowed). d) PMCs showing configuration of 1 IV (ring, arrowed) +6 II at metaphase-i. e) A PMCs showing configuration of 1 IV (chain, arrowed) +6 II at metaphase-i. f) Apparently fertile stained and transparent sterile (arrowed) pollen grains. Scale bar=10 μm (Figs. a e); 20 μm (Fig. f). (1,950 m) locality in the Kashmir Himalayas (Gohil et al. 1981, Ashraf and Gohil 1989) and other parts of the Himalayas from India (Kumari et al. 1989) and some parts of southwest Asia (Ledingham 1960, Ledingham and Rever 1963, for Iraq and Iran) (Figs 1a c). Ashraf and Gohil (1989) have also studied the chiasma frequency in the species, and found a positive correlation between recombination indices and the total chromatin length.

5 2012 Reciprocal Translocations in Astragalus chlorostachys 5 Meiotic behaviour The course of meiosis was almost normal in the accessions worked out for plants from Dunali (Manimahesh hills), Near Koksar, Sissu, Keylong (Lahaul Valley), Muhani (Pangi Valley), Malana and Kasol (Parvati Valley). In a few PMCs, 1-4 bivalents were noticed as showing non-synchronous disjunction during A-I (Fig. 2c). However, these bivalents were ultimately included at the poles and the subsequent results showed normal sporad formation and 100% pollen fertility (Table 1). One of the accessions, scored from Koksar at 3140 m in the Lahaul Valley, showed an abnormal meiotic course in 43.14% of the observed PMCs due to the presence of multivalents (ring and chain type), as well as the late disjunction of 1 5 bivalents and pollen sterility (34%). Data on the number of PMCs with ring or chain type quadrivalents and the percentage of chromosomes involved in the formation of multivalents are given in Table 2. An analysis of 204 PMCs in the accession revealed that 43.14% of the observed PMCs showed normal 8 bivalent formation and that the rest of the PMCs showed 1 quadrivalent (ring or chain type, Figs. 2d, e, Table 2). The frequency of PMCs having a ring type quadrivalent was noticed to be much higher (44.61%) in comparison to those having a chain type quadrivalent (12.25%). The percentage of chromosomes involved in multivalent formation due to reciprocal translocation works out to be of which 11.15% are involved in the ring type quadrivalents while 3.06% formed chain type quadrivalents. Besides, the late disjunction of 1 3 bivalents during A-I had also been noticed in 28.90% PMCs. Consequent to the presence of quadrivalents and non-synchronous behaviour of some bivalents in disjunction, high Table 2. Chromosomal associations in the Koksar accession in A. chlorostachys Configurations PMCs observed No. of PMCs with one IV ring type No. of PMCs with one IV chain type No. of bivalents not involved in multivalent formation No. of PMC with 8 bivalents Total PMC % age PMC Total bivalents Total no. of chromosomes % age of chromosomes involved in reciprocal translocations

6 6 P. K. Rana et al. Cytologia 77(2) pollen sterility (34%) resulted in this accession (Fig. 2f). The presence of quadrivalents in this diploid species seems to be a consequence of reciprocal translocations. Reciprocal translocations are a type of chromosomal aberration which involves the exchange of segments from 2 nonhomologous chromosomes (Mahama and Palmer 2003). The orientation of multivalents at M-I decide whether a translocation heterozygote causes sterility of gametes or not (Gohil and Koul 1978, Sharma and Gohil 2003). The sterility of pollen grains in a translocation heterozygote is due to the presence of duplications and deficiencies in gametes because of an adjacent disjunction. On the other hand, alternate orientations of multivalents result in viable gametes. In many plant species, individuals with reciprocal translocations are usually semi-sterile and 50% of the meiocytes display the alternate configuration of the interchange complex at M-I (Burnham 1956, Ghaffari et al. 2009). Plants completely sterile as a result of reciprocal translocations have also been reported in Allium consanguineum (Gohil and Koul 1978) and Allium roylei (Sharma and Gohil 2003). In the present study, the djacent orientation of multivalents at M-I seems to be the probable cause of some pollen sterility in this species. The occurrence of structural heterozygosity in the species has been reported here for the first time. The non-synchronous disjunction of chromosomes, which sometimes result in a bridge-like appearances at anaphases, is quite distinct in its origin from actual bridge formation, which is the result of inversion heterozygosity. Nonsynchronous behaviour of chromosomes during their segregation is generally attributed to the interlocking of chiasmata. The non-synchronous behaviour of chromosomes may result in laggards during anaphase/telophase and consequent pollen sterility (dela Viña and Ramirez 1995). The nonsynchronous disjunction of chromosomes in this accession also seems to be responsible for causing pollen sterility as has been reported earlier in Solanum melongena (Roxas et al. 1995), Caltha palustris (Kumar and Singhal 2008), Meconopsis aculeata (Singhal and Kumar 2008), Artemisia parviflora (Gupta et al. 2010), Clematis orientalis (Kumar et al. 2010), Plantago lanceolata (Bala and Gupta 2011) and Ranunculus laetus (Kumar et al. 2011). Acknowledgements The authors are grateful to the University Grants Commission, New Delhi for providing financial assistance under the DRS SAP I, II and III and ASIST programmes and CSIR for providing a Senior Research Fellowship to Dr. Puneet Kumar. Further support was provided by the UGC under the Dr. D.S. Kothari Post-Doctoral Fellowship Scheme (Award Letter No. F.4-2/2006 (BSR)/13-427/2011(BSR)) to Dr. Puneet Kumar. Thanks are also due to the Head, Department of Botany for the necessary laboratory and library facilities. References Ashraf, M. and Gohil, R. N Studies on the cytology of legumes in Kashmir Himalaya. IV. Meiotic behavior in 21 species of Astragalus L. Cytologia 54: Bala, S. and Gupta, R. C Chromosomal diversity in some species of Plantago (Plantaginaceae) from North India. Int. J. Botany 7: Burnham, C. R Chromosomal interchanges in plants. Bot. Rev. 22: Chowdhery, H. J. and Wadhwa, B. M Flora of Himachal Pradesh: An Analysis. Vol. 1. Botanical Survey of India, Howrah. Ghaffari, S. M., Karimzadeh, G. and Najafi, A. A Occurrence of reciprocal translocations in Lathyrus boissieri Sirj (Fabaceae) from Iran. Cytologia 74: Gohil, R. N. and Koul, A. K Structural hybridity in Allium consanguineum. Cytologia 43: Gohil, R. N., Ashraf, M. and Raina, R Cytotaxonomical conspectus of the flora of Kashmir II. Chromosome numbers of 51 dicotyledonous species. Herba Hungarica 20: Gupta, R. C., Himshikha, Rana, P. K., Kumar, P., Singhal, V. K First report of structural heterozygosity in Artemisia parviflora (Asteraceae) from Parvati Valley in Kullu District (Himachal Pradesh, India). Bot Serb 34:

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