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Meiotic Configurations and Secondary Chromosome Associations in

Indigofera tinctoria L. (Family: Fabaceae)

Article  in  Cytologia · September 2016

DOI: 10.1508/cytologia.81.291

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© 2016 The Japan Mendel Society Cytologia 81(3): 291–294

Meiotic Configurations and Secondary Chromosome

Associations in Indigofera tinctoria L. (Family: Fabaceae)
Bapi Ghosh1, Animesh Kumar Datta1*, Aninda Mandal2,
Debadrito Das1 and Divya Vishambhar Kumbhakar1
1
Department of Botany, Cytogenetics, Genetics and Plant Breeding Section, University of Kalyani,
Kalyani 741235, West Bengal, India
2
Department of Botany, A.B.N. Seal College, Cooch Behar 736101, West Bengal, India

Received September 16, 2015; accepted June 4, 2016

Summary Meiotic analysis performed in Indigofera tinctoria L. (Family: Fabaceae) reveals n=8 chromosomes
with an average chromosomal association of 7.79II+0.44I at metaphase I (MI). Predominance of 8II formation
(87.86%) has been noted. The bivalents form rods (mean per cell – rod: 5.88 0.21; ring: 1.87 0.19; associa-
tion: 7.75II+0.50I) mostly at diplotene with a mean chiasma of 9.62 0.21 per cell. Anaphase I cells (94.12%)
are mostly cytologically balanced (8/8) with an average pollen fertility of 93.60%. A persistent feature (89.39%)
in MI cells has been the presence of secondary association of chromosomes and the chromosomes assort them-
selves into variable groups of two (6.04%), three (7.04%), four (25.50%), five (56.71%), six (4.02%) and seven
(0.67%). Out of 25 different associations studied among the group classes, 3II(1)+2II(1)+1II(3) under the five
group class (25.83%) was the most frequent. Statistical analysis of cytological data reveals that the probable basic
chromosome number in the species is x=5, suggesting polyploid lineage.

Key words Indigofera tinctoria, Meiosis, Secondary grouping of chromosomes, Basic chromosome number,
Polyploid lineage.

Secondary pairings of chromosomes or secondary chromosome association in Indigofera tinctoria L. (Fam-

associations refers to pairs of bivalents and univalents
lying in close proximity without possessing any distinct
material connections (Darlington 1965). Such associa-
tions were attributed to modifications in chromosomal
rearrangements such as duplications, interchanges, etc.
(Stebbins 1950), heterochromatin distribution in the
genome facilitating alignment and synapsis of non-ho-
mologous regions (Jelenkovic et al. 1980), and outcome
of residual pairing of those chromosomal segments that
failed normal synapsis at zygotene but were sufficiently
identical to attract each other without the formation of
chiasmata (Malgwi et al. 1997).
The phenomenon of secondary association of chromo-
somes was reported first in Oryza sativa (Kuwada 1910)
and then in Dahlia variabilis (Ishikawa 1911). Since
then, it is often encountered in meiotic processes of dif-
ferent plant species in tracing basic chromosome num-
bers and the polyploid nature of the species (Kempanna
and Riley 1964, Gupta and Roy 1973, Agarwal 1983,
Sengupta and Datta 2003, Mukherjee and Datta 2005,
Iqbal and Datta 2007, Das et al. 2009, Bhattacharya and
Datta 2010, Mandal and Datta 2011, Halder et al. 2012,
Kumar and Singhal 2013). The present investigation de-
scribes meiotic configurations documenting secondary
* Corresponding author, e-mail: dattaanimesh@gmail.com
DOI: 10.1508/cytologia.81.291
ily: Fabaceae), an annual, biennial, or perennial shrub,
based on the climate under which it is grown (Gabori-
aud-Kolar et al. 2014). The species possesses immense
therapeutic significance (root/leaves/stem) in traditional
systems of medicine (Saraswathi et al. 2012), apart from
its commercial importance (leaves) as a dye-yielding
plant (Siva 2007).
Materials and methods
Plant material
Seed samples of Indigofera tinctoria L. were obtained
from Medicinal Plant Garden Narendrapur Ramkrishna
Mission, Govt. of West Bengal, India (germplasm
source for medicinal plant species). Seeds were sown
and the plants were raised in the Experimental Garden
of Department of Botany, University of Kalyani during
the months of April to December (West Bengal plains,
22.989100, 88.447844, elevation 48 feet above sea level).
A voucher specimen has been deposited in the Herbari-
um, Botany Department, University of Kalyani.
Meiotic analysis
Young inflorescences of 2–3 cm (floral buds are nearly
sessile in racemes) were collected from five randomly
selected plants (flowering period ranged from July to
October) and fixed (9 a.m. to 10 a.m.) in Carnoy s solu-
292 B. Ghosh et al. Cytologia 81(3)

Figs. 1–12. Meiotic configurations (n=8) in Indigofera tinctoria. 1) Diplotene with rod and ring bivalents. 2) Metaphase I
with eight bivalents. 3–12) MI cells with secondary groups. 3) Two groups. 4) Three groups. 5) 3II(2)+1II(2)–four
groups. 6) 3II(1)+2II(2)+1II(1)–four groups 7–9) 2II(3)+1II(2)–five groups. 10) 3II(1)+2II(1)+1II(3)–five groups.
11) 3II(1)+1II(5)–six groups. 12) 2II(1)+1II(6)–seven groups. (ring = right handed arrow; nucleolus= dotted arrow)
Scale bar =10 µm.

tion for 48 h (one change was given in the fixative after
24 h) and preserved in 70% alcohol under refrigeration.
Pollen mother cells (PMCs) and pollen grains obtained
from anther squash preparations were stained in 2% pro-
pionocarmine solution. Fully stained pollen grains were
considered fertile (Marks 1954). Meiotic data recorded
at diplotene–diakinesis, metaphase I (MI), and anaphase
I (AI) were pooled over the plants and represented in the
text.
Photomicrographs of meiotic plates were taken from
temporary squash preparations and suitably magnified.
Results and discussion
Meiocytes of I. tinctoria show 2n=16 chromosomes
always (Figs. 1–12) as reported earlier (Bairiganjan and
Patnaik 1989). The bivalent configuration studied per
cell at diplotene (Fig. 1) has been predominantly rod
(rod: 2–7, 5.88 0.21; ring: 0–4, 1.87 0.19; chaisma per
cell 9.62 0.21) with mean association of 7.75II+0.50I
per cell. The univalents (2–8) are marked in close prox-
imity to each other at diplotene, and such juxtaposed
arrangements represent the residual attraction between
homologues and their recent separation.
Mean chromosome association per cell at MI was
7.79II+0.44I (420 cells scored) with predominance of 8II
(87.86%) formation (Fig. 2). Rest of the chromosomal
associations are 7II+2I (6.90%), 6II+4I (1.90%), 5II+6I
(1.90%), and 4II+8I (1.43%). AI cells mostly (94.12%;
170 PMCs observed) show equal (8/8) segregation of
chromosomes (rest cells: 9/7-2.94% and 8/1/7-2.94%).
Pollen fertility was 93.60% (1511 pollen grains scored).
Well-scattered meiocytes (341 cells assessed) at MI
(Figs. 3–12) are used to analyze secondary grouping of
2016 Meiotic Configurations and Secondary Chromosome Associations in Indigofera tinctoria L. (Family: Fabaceae)
chromosomes. About 298 PMCs (87.39%) show distinct
secondary groupings of chromosomes. Bivalents and
univalents tend to form variable groups of two (6.04%),
three (7.04%), four (25.50%), five (56.71%), six (4.02%)
and seven (0.67%). A total of 25 different types (two
groups class–four types; three groups–four types; four
groups–five types; five groups–eight types; six groups–
two types; and seven groups–two types) of associations
are observed among the group classes. The predomi-
nant associations found are 3II(1)+2II(2)+1II(1) under
the four group class/Fig. 6 (15.43%) and 2II(3)+1II(2)/
Fig. 7–9, (22.81%) and 3II(1)+2II(1)+1II(3) under the
five group class/Fig. 10 (25.83%). Group class of six–
2II(2)+1II(4)–3.02% and 3II(1)+1II(5)–1.00%/Fig. 11
and that of seven–2II(1)+1II(6)–0.33%/Fig. 12, are
rarely noted.
Computation of χ2 test analysis among the group
classes reveals heterogeneity (χ2 = 412.03, DF 5,
p<0.001), thereby indicating that the bivalents and uni-
valents tend to assort themselves more preferentially
into groups of certain numbers (more predominantly
into the five group class) than others much against ran-
dom distribution. Further, the total frequency of the five
group class is found to be significantly higher than the
expected pooled frequency of the rest of the group class-
es (observed: five group class–169, other classes–129;
expected: five group class–149, other classes–149,
total–298, χ2 =5.36, DF 1, p<0.05).
Persistent occurrences of secondary association of
chromosomes suggest a secondary polyploid nature of
the species. A statistical analysis of the cytological data
reveals that the basic chromosome number (x) is prob-
ably five (selective doubling of chromosomes possibly
as x=5, n=8). Gupta and Agarwal (1982) evaluated the
genus Indigofera on karyomorphology and suggested
the base number was four. In the present investigation,
the meiocytes show a total lack of multivalent formation
and variable number of associations indicating possible
allopolyploid lineage of the species. Chenuil et al. (1999)
reported that a cytological approach involving the ob-
servation of meiotic cells is conventional to ascertain
whether the species is auto- or allopolyploid in origin.
Polyploid origin with diploid like meiotic behavior
is a significant aspect of the studied species. Jiao et al.
(2011) opined that most flowering plant lineages have
undergone at least one polyploidization event during
the evolutionary history. Datta et al. (2015) reviewed
polyploidy in angiosperms and raised a few questions,
of which the pairing behavior of homologues as the
consequence of genome duplication was one of them.
Cytological diploidization in polyploid lineages has been
explained differently in different plant species (Riley
and Kempanna 1963, Corredor et al. 2007, Cifuentes et
al. 2010, Greer et al. 2012, Yant et al. 2013) but still the
phenomenon lacks uniformity and universality.
293
Acknowledgements
The authors are thankful to DST-PURSE Pro-
gramme, University of Kalyani for financial assistance.
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