Mutagenic Studies in male Meiosis in Chromium

treated accessions of Vigna Angularis.

A. KUMAR

Botany Department, Chaubattakhal, Distt.Pauri Uttarakhand

SUMMARY
The mutagenic effect of heavy doses of Cr on male gametophyte development
were investigated in two accessions of genus Vigna angularis (V1 and V2) by
using parameters like chromosome configuration at metaphase I/diakinesis,
chiasmata/chromosome frequency, number of spores/tetrad, pollen size and
pollen sterility. Both the accessions had 2n=22. The frequency of
chiasmata/chromosome was found to be >0.6 and <0.8 in the treated plants. The
meiotic course was found to be abnormal in all the treatments and their
frequency of anomalies increased with the increase in Cr concentration.
Abnormalities noticed were restitution nucleus, clumping, chromatin bridges at
telophase I/II, micronuclei at telophase I and II, etc. The spore arrangement was
found to be tetrahedral. Triads and polyads were also found along with tetrads.
The pollen sterility in V1 treatment were found to be 14 to 34 % and in V2 from
20 to 31%. The pollen/anther in V1 different treatments varied from 145 to 390
and in V2 from 80 to 313. The pollen diameter in V1 was found to be 54 to
115μm and in V2 from 54 to 109μm.
Keywords: Chromium effects, Male meiosis, Vigna angularis.

INTRODUCTION
The legumes have been under cultivation throughout the world since time
immemorial. They occupy a significant position among food crops as source of
pulses, vegetables, oils, etc., (Khanna and Gupta, 1988). The legumes have a
unique property of maintaining and restoring soil fertility through bacterial
nodules, which are formed on their roots. India has the distinction of being
world’s largest producer of legumes (pulses and oil yielding) occupying about
13 % of area under cultivation and producing 22-23 million tons of grains
annually (Tiyagi and Alam, 1992). The Vigna angularis is known as soonthiya or
rayans in Garhwal. It belongs to the family Fabaceae. Its English name is Adzuki
bean. Vigna flowers and fruits from August to November. The genus Vigna has
about 150 species in tropics and 25 species in India (Gaur, 1999). In V. angularis
the chromosome number (2n=22), chromosome size (1.6 µ m and 3.7 µm; Sen and
Bhowal, 1960) and karyotype (Galasso et al., 1992; 1993; Zheng et al., 1991) has
been worked out earlier. Chromium is important for metallurgical industry.
Chromium is the second most common heavy-metal contaminant in
groundwater, soil and sediments. Chromium phytotoxicity has been considered
to be inhibitory for plant growth, its presence in excess amount within the plant
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can cause stunted shoot growth and poor root development. Much
research has been conducted to determine the toxic effects of chromium on
different plant species. The plants growing near industries emitting liquid
pollutants or air pollutants or solid waste do not survive. This is because the
heavy metals cause changes in their genetic contents thus leading to abnormal
phenotype and irregular meiosis. These plants do not produce normal seeds. In
the present work two accessions of Vigna were subjected to heavy Cr doses and
the mutagenic changes induced in their male gametophyte development were
studied.
MATERIALS AND METHODS
For the present cytogenetic analyses two accessions of V. angularis were
procured from local sources Chaubattakhal (1600m asl) and Uttarkashi (1200m
asl). To see the effect of Cr on the development of male gametophyte various
concentrations of Cr were prepared (10-1 M to 10-10M from stock solution of
potassium dichromate). The seeds of the accessions were kept in all the
prepared concentrations for 24 and 48 hours. The seeds after treatment were
sown in polybags having vermiculite. The plants obtained were then transferred
to agriculture field. For the male meiosis floral buds of desirable age were
collected from the mature plants and fixed in Carnoy’s fluid II (6:3:1:: absolute
ethanol: chloroform: glacial acetic acid) for 24 hours and then transferred in
70% ethanol and stored in refrigerator. Anthers were smeared and squashed in
1.5% aceto-carmine for studying meiosis. All the observations and
photomicrographs were taken from unsquashed and squashed temporary
preparations.
Various cytological parameters that were analyzed are:
(1) Chromosome configuration at metaphase I/diakinesis, including the
chiasmata frequency,
(2) Types and frequency of various meiotic anomalies,
(3) Number of spores per tetrad,
(4) Pollen size,
(5) Pollen sterility, and
(6) Number of pollen per anther.
Pollen sterility was measured by staining the pollen grains with 1% aceto-
carmine for overnight. The pollen grains were considered viable only if they
were uniformly stained.

RESULTS AND DISCUSSION

Out of 24 and 48 hours treatment only 24 hours treated seeds grew and no
germination was found in 48 hours treatment. Details of male meiosis were
worked out in all control and treated sets of both the accessions of Vigna
angularis. The Vigna angularis had 2n = 22 established at metaphase I and
anaphase I (fig. 1 a). Data related to frequency distribution of chromosome
configuration at metaphase I, chiasmata per PMC and chiasmata per
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chromosome are listed in table 1. Mostly bivalents were observed. The

chiasmata per chromosome in V1 varied from 0.65 to 0.83 and in V2 from 0.63
to 0.81 in treated sets.
In treatments a number of abnormalities were observed and the percent
distribution of various anomalies is given in table 2. The anomalies observed
were as follows:
(1) Restitution nucleus formation at first meiotic division,
(2) Retarded movements of bivalents for metaphase I alignments and
chromosomes for metaphase II,
(3) Clumping of chromosomes at metaphase I (fig.1 b) and metaphase II,
(4) Random grouping of bivalents at metaphase I (fig. 1 c),
(5) Late disjunction of bivalents at metaphase I and chromatids at metaphase
II,
(6) Laggards at anaphase I and anaphase II (fig. 1 d, e),
(7) Chromatin bridges at telophase I and telophase II (fig. 1 d) and
(8) Micronuclei at telophase I and telophase II.
Microspores were arranged in tetrahedral tetrads. The polyads were also
observed. The number of pollens per anther in control of V1 accession was
found to vary from 330 to 400. W ith the increase in Cr concentration, number of
pollens per anther decreased up to 145. Similarly in V2, the number of pollens
per anther in control varied from 285 to 350. The number of pollens found to
-2
decrease with increase in Cr concentration. In 10 M, the number of pollens per
anther was found to be 80.
In V1 control, the pollen size varied from 90 to 123 µm. The pollen size
also decreased with the increase in Cr concentrations. In 10-2 M, pollen size was
lowest (55 µm). In V2 control, the pollen size varied from 95 to 121 µm and on
increasing Cr concentration level the pollen size was reduced to 54 µ m. The
maximum pollen sterility in both V1 and V2 was found in higher concentrations
of Cr (10-2 M). It varied from 14 to 32% in V1 and 23 to 28% in V2 with
increasing level of Cr.
During the present study the effect of high concentration of Cr was
observed in both the accessions of Vigna angularis. The chiasmata frequencies
per chromosome in control varied from 0.77 to 0.91 while at 10-2M it varied
from 0.55 to 0.68 in accessions V1 and V2. In higher doses of Cr various
meiotic anomalies like: restitution nucleus formation, retarded movement of
bivalents/chromosomes for metaphase I and II alignment, clumping of
chromosomes at metaphase I and II, late disjunction of chromosomes/
chromatids at metaphase I and II, laggards at anaphase I and II, chromatin
bridges at telophase I and II and micronuclei at telophase I and II were
observed.
The restitution nucleus was formed due to chromatin agglutination. The
chromatin agglutination also resulted in clumping of chromosomes at
metaphase, anaphase and chromatin bridges. Late resolution of
chromosomes/chromatids resulted in late disjunction. Random grouping of

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chromosomes/chromatids resulted in late disjunction. Random grouping

of chromosomes in more than one group at metaphase I may be due to
‘longitudinal splitting’ of spindle. The retarded movement of bivalents and
chromosomes for metaphase I and II alignments respectively may be due to
malfunctioning of microtubules responsible for the displacement of
chromosomes. Lagging of chromosomes during anaphase I and II may be due
to:
(1) Delayed resolution of chromosomes/ chromatids of the bivalents/
chromosomes , or
(2) Malfunctioning of the spindle apparatus.
The bridges were chromatin bridges produced because of the stretching of
chromosome segments between poles. The stretching of the chromosomes was
due to chromatin agglutination in the chromosome segments. Lagging of the
chromosomes resulted into micronuclei formation.
Triads and polyads were also observed. Triads were formed due to arrest
of meiosis in one of the two sister dyad cells after first meiotic division. The
polyads were formed due to cytoplasmic partitioning of additional nuclei at the
end of meiosis. The reason for these additional nuclei is the formation of
micronuclei from laggards.
The pollen size varied to a large extent and the percent sterility also
increased with the increase in Cr concentrations.

Table 1. Mean data related to rod and ring bivalents, chiasmata per cell and chiasmata
per chromosome in two accessions of V. angularis.
V1 V2
Rod Ring Rod Ring
Conc. Xma/cell Xma/cell
II II Xma/chro II II Xma/chro
Co 3.60 7.40 18.40 0.84 3.80 7.20 18.20 0.83
-10
10 M 3.80 7.20 18.20 0.83 4.20 6.80 17.80 0.81
10-9M 4.60 6.40 17.40 0.79 4.40 6.60 17.60 0.80
10-8M 5.00 6.00 17.00 0.77 5.20 5.80 16.80 0.76
-7
10 M 4.40 6.60 17.60 0.80 4.80 6.20 17.20 0.78
-6
10 M 5.60 5.40 16.40 0.75 5.60 5.40 16.40 0.75
10-5 M 6.40 5.20 16.80 0.76 6.80 4.20 15.20 0.71
-4
10 M 6.60 5.00 16.60 0.75 6.80 4.40 15.60 0.71
10-3M 7.20 3.80 14.80 0.67 7.60 3.40 14.40 0.65
10-2 M 7.80 3.20 14.20 0.65 8.20 2.80 13.80 0.63

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Table 2. Data showing percent distribution of various anomalies in different treatments

of two accessions of V. angularis.
10 - 10 -
Co 10 -10 M 10-9M 8 10-7M 10 -6 M 10 -5 M 10 -4M 3 10 -2 M
M M
Restitution nucleus - - - - - - - - - 20.00
Retarded movements - 5.17 8.69 8.22 11.11 12.02 8.57 8.00 12.50 17.69
Late disjunction - 4.50 - - - - - - - -
Laggar ds 4.54 - - - 2.56 6.34 5.35 21.73 23.00
Chromatin bridges - - - - 2.50 - - - - -
Micr onuclei 2.5 - - - 2.50 4.25 4.54 5.26 14.00 15.01
Retarded movements - - - - - - 6.45 6.12 20.00 3.85
Late disjunction - - - - - - 3.55 2.25 12.42 7.69
Laggar ds 3.63 - - - - - - - - -
Chromatin bridges 2.22 2.22 - - - 2.77 7.50 4.68 15.00 17.00
Micr onuclei 2.00 2.70 - - - 5.12 5.18 3.65 14.63 20.50
Polyspor y 3.00 4.00 - - 2.00 5.00 3.01 4.89 8.01 15.10
Restitution nucleus - - - - - - - - - 18.30
Retarded movements - 8.19 9.09 15.38 5.67 2.54 9.00 4.84 6.89 4.55
Laggar ds 7.98 - - 11.12 - 5.45 3.33 2.13 3.28 16.67
Micr onuclei - - - - 11.65 2.88 5.26 1.95 2.50 12.15
Retarded movements - 4.14 2.10 - 1.90 - 2.10 - 2.28 9.11
Late disjunction - 2.50 2.07 - 2.10 - 1.07 - 2.10 4.10
Laggar ds 5.20 4.25 - 2.25 9.65 3.45 - 3.45 14.56
Micr onuclei 1.05 - - 4.08 2.30 5.10 1.00 4.34 6.40 10.20
Chromatin bridges 0.95 - - 3.10 2.68 4.55 0.55 2.20 4.10 5.10
Polyspor y 2.00 - - 4.00 9.00 8.00 20.00 17.00 18.00 24.00

Table 3. Data showing mean values of Psize: pollen size (µm), P/A: pollen per anther
and PSt: pollen sterility in different treatments of two accessions of V. angularis.
V1 V2
Psize P/A PSt Psize P/A PSt
Co 110.82 349.30 11.88 318.80 110.06 23.08
10-10M 101.68 326.20 14.37 297.90 96.81 20.75
10-9M 97.27 294.00 17.78 289.30 93.92 26.48
-8
10 M 94.38 296.10 19.09 274.80 90.88 25.53
-7
10 M 91.33 288.50 24.64 261.10 87.83 28.16
-6
10 M 88.29 270.60 29.62 246.00 84.33 27.24
-5
10 M 84.64 267.90 30.02 202.50 79.16 27.73
-4
10 M 81.13 258.20 32.77 148.80 68.35 29.57
-3
10 M 67.28 193.70 32.17 107.70 65.46 28.52
-2
10 M 60.43 181.30 34.2 101.10 60.28 31.65

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(A)

(B)

a. b.

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(C)

(D)

c. [[52]]
d.
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(E)

a. normal metaphase I showing bivalents; b. Stickiness at metaphase I; c.

Random grouping at metaphase I, d., e. Laggards at anaphase I d. Chromatin
bridge at telophase I.

REFERENCES
1- Galasso, I., D. Pignone and P. Perrino (1992). Cytotaxonomic studies in
Vigna, I. General
technique and Vigna unguiculata C-banding. Caryologia 45: 155-161.
2- Galasso, I., D. Pignone and P. Perrino (1993). Cytotaxonomic studies in
Vigna .2.
HeterochromatinCharacterization in Vigna unguiculata and three Related
Wild Species.
Caryologia 46: 275-282.
3- Gaur, R.D. (1999). Flora of the district GarhwalNorthwest Himalaya (with
ethno botanical
notes). Pp. 302.and 662.
4- Khanna, S.S. and M.P. Gupta, (1988). Raising production of
pulses.Yojana, 32:4-8.
5- Sen, N.K. and J .G.Bhowal (1960). Cyto taxonomy studieson Vigna.
Cytologia. 25: 195-
207.
6- Tiyagi, S.A. and M.M. Alam. (1992). Pulse cultivation in India.
Everyman’s Science, 27: 82-
84.

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7- Zheng, J., M. Nakata, H. Uchiyama, H. Morikawa and R. Tanaka

(1991). Giemsa C-banding patterns in several species in Phaseolus L., and Vigna
Savi, Fabaceae. Cytologia.
56: 459-466.

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