173-179
Cytologia 77(2): 1–7
© 2012 The Japan Mendel Society
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–147002, 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, e-mail: [email protected]
DOI: 10.1508/cytologia.77.xxx
GALLEY
PROOFS
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, LahaulSpiti 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, 2007–2011.
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 20–50 PMCs were examined to determine chromosome counts
while 30–60 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
Lahaul Valley
Manimahesh hills
Pangi Valley
Malana Valley
Parvati Valley
2
3
4
5.
Population
56149
Malana, Parvati Valley, Kullu, Himachal Pradesh, along water
streams, 32 3′51′′N; 77 15′38′′E, Alt.: 2640 m
Kasol, Parvati Valley, Kullu, Himachal Pradesh, on moist slopes,
32 0′46′′N; 77 19′22′′E, Alt.: 1610 m
56049
56147
Muhani, Pangi Valley, Chamba, Himachal Pradesh, growing in
moist places around Corylus colurna and Juglans regia,
32 59′48′′N; 77 35′30′′E, Alt.:2500 m
8
8
8
8
8
51256
51259
8
8
51226
51127
8
Meiotic
chromosome
number n
51263
Accession
number
(PUN)
Dunali, Manimahesh hills, Chamba, Himachal Pradesh, around forest area, 32 25′ 38′′N; 76 36′14′′E, Alt.: 2780 m
Sissu, Lahaul Valley in Lahaul-Spiti, Himachal Pradesh, growing on
moist slopes, 32 28′46′′N; 77 7′37′′E, Alt.: 3170 m
Keylong, Lahaul Valley in Lahaul-Spiti, Himachal Pradesh, growing around agricultural fields, 32 34′13′′N; 77 49′02′′E , Alt.:
3340 m
Near Koksar, Lahaul Valley in Lahaul-Spiti, Himachal Pradesh,
growing on moist slopes, 32 24′26′′N; 77 14′50′′E, Alt.: 3150 m
Koksar, Lahaul Valley in Lahaul-Spiti, Himachal Pradesh, growing
among small stones slopes, 32 24′46′′N; 77 14′11′′E, Alt.: 3140 m
Locality, habitat, latitude and longitude and altitude
2x
2x
2x
2x
2x
2x
2x
2x
Ploidy
level
Normal
Normal
Normal
Normal
Normal
Multivalents &
late disjunction
of 1–4 bivalents
Normal
Normal
Meiotic
behaviour
99
100
98
100
100
100
66
100
Pollen
fertility
2n=16
Ledingham,
1960; Gohil et
al., 1981;
Ledingham &
Rever, 1963;
Ashraf &
Gohil, 1989;
Kumari et al.,
1989.
Previous
counts
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
1
S. No.
Table 1.
2012
Reciprocal Translocations in Astragalus chlorostachys
3
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 1IV (ring, arrowed) +6II at metaphase-I. e) A PMCs showing
configuration of 1IV (chain, arrowed) +6II 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
Reciprocal Translocations in Astragalus chlorostachys
2012
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 14.21. 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
Total PMC
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
42
24
18
18
42
36
30
36
60
78
48
24
36
54
18
36
42
18
36
7
8
6
7
4
4
5
9
5
7
6
–
2
9
3
3
–
3
–
14
12
9
10
11
10
10
15
15
20
14
4
8
18
6
9
7
6
6
5
1
3
1
7
4
4
5
9
12
8
3
4
9
1
4
4
2
5
2
3
–
2
–
2
1
1
1
1
–
1
2
–
2
2
3
1
1
204
91
25
88
44.61
12.25
43.14
% age PMC
Total bivalents
1632
182
50
696
704
Total no. of chromosomes
3264
364
100
1392
1408
% age of chromosomes
involved in reciprocal
translocations
11.15
3.06
42.65
43.14
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)/13427/2011(BSR)) to Dr. Puneet Kumar. Thanks are also due to the Head, Department of Botany for
the necessary laboratory and library facilities.
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