CARYOLOGIA Vol. 63, no.

1: 62-72, 2010

Cytogenetic characterization of induced autotetraploids in grass

pea (Lathyrus sativus L.)
Talukdar* Dibyendu
Department of Botany, University of Kalyani, Kalyani, Nadia-741 235, West Bengal, India

Abstract — Induction of polyploidy was successfully achieved in grass pea (Lathyrus sativus L.) variety BioR-
231 using 0.10% (eight hours three days), 0.20% (six hours two days and eight hours two days), 0.30% (eight
hours one day) and 0.40% (six hours one day) colchicine solution by apical shoot tip treatment of young seed-
lings. Compared to diploid variety the tetraploid plants were morphologically distinguished by luxuriant vegeta-
tive growth showing broader and thicker leaflets and stipules, flattened stems, increased number of branches,
enhanced length of tendril as well as peduncle, delayed flowering and reduced pollen fertility. Number of sto-
mata decreased but its size increased significantly in tetraploids. Presence of 7II and 7-7 separation at meiosis-I
were the usual feature in diploids (2n=2x=14) while occurrences of 28 chromosomes in different combinations
of multivalents, bivalents and univalents at metaphase-I and unequal separation of chromosomes toward the
spindle poles, bridge formation, laggard at anaphase-I distinguished tetraploid (2n=4x=28) from diploid plants,
cytogenetically. All these meiotic irregularities through the formation of unbalanced gametes have been ascribed
for pollen sterility and reduced seed yield in tetraploid plants. Nature of origin, morphology and formation of
trivalent as well as quadrivalent indicated autopolyploid nature of present tetraploids in grass pea. Occurrence
of majority pairing as bivalents has also been discussed.

Key words: cytogenetics; induced autotetraploid; Lathyrus sativus.

INTRODUCTION ing to its improvement as an ideal and alterna-

Grass pea (Lathyrus sativus L.) belonging to
the family Leguminosae is an annual, well adapt-
ed and protein rich winter pulse crop and is
strictly diploid with chromosome number being
2n=2x=14 (VERMA 1979; BATTISTIN and FERN-
ANDEZ 1994; BISWAS 1998; TALUKDAR 2008a).
Isolation of high yielding lines with low seed
neurotoxin, ODAP (β-N-oxalyl-L-α, β-diamino
propionic acid) is one of the prime objectives of
this crop for which a wider base of germplasm is
essential (TADSEE and BEKELE 2003; GRANATI et al.
2003). Induction of polyploidy may create wider
amplitude of genetic variation which is necessary
to study various cytogenetic backgrounds relat-
*Corresponding author: e-mail: dib_tal@rediffmail.
com or dibyendutalukdar9@gmail.com
tive pulse crop in different geographical regions.
In grass pea, cytogenetic consequences of differ-
ent types of trisomics and tetrasomics have been
reported (TALUKDAR and BISWAS 2007; TALUK-
DAR 2008b). Using these aneuploid stocks link-
age mapping of different morphological traits
has been initiated (TALUKDAR 2009). Polyploids
have immense importance in maintaining these
aneuploids in population. Successful crossing
between tetraploids and diploids often results
in triploid plant which can produce different
trisomics, 3n+1 or 3n–1 individuals in progeny
(BELLING and BLAKESLEE 1924; NAGESH and SUB-
RAHMANYAM 1995). In addition to increase in the
size of different vegetative and reproductive or-
gans in tetraploid plants there are evidences that
doubling the chromosome number may change
magnitude of sterility, pattern of sexuality, plant
habit and increase winter hardiness (BLAKESLEE
and AVERY 1937). Distribution of polyploid spe-
cies of Lathyrus and other tetraploid genera in
CYTOGENETICS OF INDUCED AUTOTETRAPLOIDS IN LATHYRUS SATIVUS L .
cold and humid regions of northern hemisphere
and diploid species in xeric regions agrees with
the hypothesis that polyploids are more tolerable
and adaptable to a wider range of environmen-
tal conditions (STEBBINS 1980; SHAO et al. 2003).
The ability to induce chromosome doubling,
therefore, is of importance to practical as well as
to theoretical genetics and in evolution. Analysis
of meiotic chromosome association in autopoly-
ploid plants can give precise information on or-
ganization of chromosome pairing and synapsis
during meiosis (SYBENGA 1975) and has practi-
cal value in understanding of undesirable effects
of polyploidy on fertility and stability (GILLIES
1989). Grass pea is a suitable plant for cytoge-
netic research but very few attempts have been
made to characterize induced autopolyploids in
this crop (BISWAS 1998). Meiotic consequences of
artificial polyploidization and its effect on differ-
ent phenotypic traits can be studied to enhance
the knowledge base of chromosome doubling
in grasspea. In this background, endeavor was
made in the present investigation to induce de-
sirable variations in Lathyrus sativus L. through
artificial polyploidization to explore cytogenetic
consequences of these polyploids in this crop.
MATERIALS AND METHODS
Induction of polyploidy - Fresh seeds of a local
cultivar ‘BioR-231’ were sown in earthen pots
with 25.46cm diameter during winter season of
2005-06 to raise the seedlings. Emerging shoot
apices of each of the 20 young seedlings were
treated with 0.10%, 0.20%, 0.30% and 0.40%
aqueous solution of colchicine by cotton plug
method for one day and two and three con-
secutive days with a variation of six hours and
eight hours in each case. Small cotton wads were
soaked in different concentrations of colchicine
solution by adding the chemical drop wise with
a dropper and were placed on the shoot apex.
The wads were kept moist throughout the treat-
ment period by adding respective colchicine so-
lution at regular intervals. The treated seedlings
were maintained under the same conditions of
growth. After specific duration of the treatments
the cotton wads were removed and shoot api-
ces were washed well with distilled water. A set
of 20 plants was treated with water as controls.
Side branches were removed to facilitate growth
of the treated buds only. Preliminary morpho-
logical screening for putative tetraploids was
conducted on the basis of stomatal size and its
63
feequency at lower surface of leaflets in treated
seedlings. The epidermal layer of the lower sur-
face of the 30 leaflets of the first formed primary
branch in each plant was peeled off, stained with
a 1% w/v I2-KI aqueous solution and observed
under a light microscope. Similarly, leaf sam-
ples taken from five control seedlings were also
studied. Fifteen slides were prepared for each
seedling and the length and breadth of 30 sto-
mata were measured on each slide with an ocu-
lar scale. Stomatal frequency was determined by
counting number of stomata in 20 microscopic
fields for each seedling and compared with that
of control plants. Significant differences between
control and treated populations for stomatal size
and frequency was ascertained by simple t-test
at p<0.05 level. Mean values of different mor-
phological and yield related characters were
estimated in tetraploid plants at C1 and C2 gen-
erations and compared with control by simple
t-test. Meiotic analysis confirmed the tetraploid
number of chromosomes in each generation.
Meiotic studies - Meiotic analysis were carried
out on suitable sized flower buds, collected from
diploid control plants and colchicine treated
plants, fixed between 9.00-10.00A.M in propio-
no-alcohol (1:2) for 6 hours and then preserved
in 70% alcohol for future studies. After wash-
ing the fixed buds in distilled water anthers were
smeared in 1% propiono-carmine solution to
analyze meiosis in the microsporocytes. Phot-
omicrographs were taken from well scattered
plates. Size and sterility of pollen grains from
randomly selected anthers were studied follow-
ing staining with 1% acetocarmine solution. At
C0 generation preliminary meiotic analysis was
performed to identify and confirm suspected
tetraploid plants. Detail cytological analysis was
however carried out at C1 and C2 tetraploid gen-
erations along with diploid control plants. Aver-
age frequency of different chromosome configu-
rations per PMC was estimated from different
associations.
RESULTS
Among the 480 colchicine treated seedlings of
Lathyrus sativus L. cultivar ‘BioR-231’ polyploi-
dy was detected in samples treated with 0.10%
(eight hours three days), 0.20% (six hours two
days and eight hours two days), 0.30% (eight
hours one day) and 0.40% (six hours one day)
colchicine solution (Table 1). Two to three days
old seedlings attaining heights of 2.00-3.00cm
64 TALUKDAR

were found to be more responsive to colchicine
treatment giving rise to mature tetraploid plants.
In the treated and survived population, ten vari-
ant plants (C0) showing different plant morphol-
ogy and significant departure from control values
in stomatal nature were primarily selected. Out
of these ten putative tetraploids, seven plants
were confirmed as tetraploids by preliminary
cytological tests. These seven plants producing
a total of 18 seeds were harvested separately
and sown to raise C1 generation. Eleven plants
were survived at C1 generation and grown well
to produce 36 seeds. Out of 29 seedlings grown
in the following C2 generation, 6 plants died
prematurely. Mixoploid plants with tetraploid
shoot and diploid roots were encountered only
in one-day treatment with 0.10% colchicine for
8 hours. Rest of the treatment exhibited either
high degree of mortality or found to be ineffec-
tive to induce variation in diploid chromosome
number (Table 1).
Morphology of tetraploid plants - Initially, sto-
matal number and size were used to distinguish
tetraploid plants from diploids and eventually
confirmed by chromosome association at meio-
sis at C0 generation. In seven C0 tetraploid plants,
mean value of number of stomata from epider-
mal layer at the lower surface of leaflets varied
between 47.63±1.81 and 48.03±2.48 while it
was estimated as 69.40± 4.50 in diploids. Length
and width (mean) of guard cell (µm) was be-
tween 40.54±0.09 and 41.22±0.03 and between
11.60±0.10 and 12.92±0.07, respectively in
seven tetraploids. Mean of length and width of
stomatal aperture (µm) varied from 24.77±0.08
to 25.17±0.06 and from 6.05±0.01 to 7.99±0.05
in C0 plants. In diploid plants, length and width
(mean value) of guard cell has been calculated
as 31.30± 0.19µm and 9.86±0.14 µm, and those
values of stomatal aperture were 17.39±0.23 µm
and 3.09±0.17 µm, respectively. However, differ-
ent morphological traits were studied in detail on

TABLE 1 — Effect of colchicine treatments to apical meristems on seedlings of Lathyrus sativus L.

Colchicine Duration Number of seedlings Number of

concentrations (%) Hours Days Treated Survived tetraploids*
0.10 6 1 20 18 0
0.10 6 2 20 16 0
0.10 6 3 20 13 0
0.10 8 1 20 17 0
0.10 8 2 20 15 0
0.10 8 3 20 12 1

0.20 6 1 20 17 0
0.20 6 2 20 14 1
0.20 6 3 20 14 0
0.20 8 1 20 15 0
0.20 8 2 20 12 2
0.20 8 3 20 10 0

0.30 6 1 20 12 0
0.30 6 2 20 12 0
0.30 6 3 20 11 0
0.30 8 1 20 11 2
0.30 8 2 20 10 0
0.30 8 3 20 09 0

0.40 6 1 20 11 1
0.40 6 2 20 11 0
0.40 6 3 20 09 0
0.40 8 1 20 09 0
0.40 8 2 20 07 0
0.40 8 3 20 05 0
* confirmed by cytological analysis.
CYTOGENETICS OF INDUCED AUTOTETRAPLOIDS IN LATHYRUS SATIVUS L . 65

cal bud, swelling of stem and thickening of first

Fig. 1 — A flowering twig of diploid variety ‘BioR-231’
of grass pea (Lathyrus sativus L.).
Fig. 2 — A tetraploid plant at C2 generation.
Fig. 3 — Seeds of diploid (D) and tetraploid plants.
diploid control plants, tetraploids at C1 and at C2
generation (Table 2). In general, an increase in
the size of stomatal aperture as well as guard cells
and reduction in number of stomata observed in
tetraploid as compared to diploid plants (Table
2). The tetraploids were distinguished from dip-
loid plants by stunted or arrested growth of api-
formed leaves at early vegetative stage but in the
later stages of development rate of growth in-
creased vigorously. The leaves and stipules were
dark green and thicker in tetraploids. Both line-
ar-lanceolate shape of leaflets and semi-sagittate
stipules in diploids were modified to ovate-lan-
ceolate in tetraploids giving a lower leaflet index
(Figs. 1, 2). In contrast to diploid plants stems
were somewhat fasciated bearing increased
number of branches, longer tendrils and com-
paratively large sized flowers but the length of
anther filament and style remained comparable
with those of diploids. Other basic features like
prostrate habit, alternate arrangement of leaves,
opposite orientation of leaflets, occurrence of
terminal tendril, solitary axillary flower and
number of floral parts also remained unmodi-
fied in tetraploid although peduncle tended to
be rigid with increase in length. Flowering was
delayed as compared to diploid plants. Pollen
sterility significantly increased over diploids in
different magnitude and seed setting reduced
drastically in tetraploids but seeds were slightly
heavier and larger than diploids (Fig. 3). Pol-
len fertility and number of seed yield per plant

TABLE 2 — Major phenotypic characteristics of induced autotetraploids at C1 and C2 generations and their diploid
control variety in Lathyrus sativus L.

Characters Tetraploid Diploid

C1 generation C2 generation
mean ± SE mean ± SE mean ± SE
Plant height (cm) 82.71±0.79* 69.87±0.92* 63.55±0.44
Internode length (cm) 4.31±0.19 4.22±0.10 3.22±0.06
Leaflet length (cm) 7.22±0.05 8.60±0.11 6.10±0.02
Leaflet width (cm) 0.62±0.07 0.59±0.05 0.54±0.01
Number of branches 20.30±0.04* 18.09±0.16* 12.40±0.06
Stipule length (cm) 2.82±0.29* 2.80±0.18* 1.64±0.07
Stipule width (cm) 1.10±0.31* 1.20±0.35* 0.54±0.13
Tendril length (cm) 5.92±0.44 5.56±0.37 3.39±0.52
Number of stomata (lower surface) 48.12±4.89* 48.70±1.31* 69.40±2.81
Length of guard cell (µm) 40.67±0.57* 39.89±0.25* 31.29±1.29
Width of guard cell (µm) 11.95±0.43 10.86±0.23 9.86±0.69
Length of stomatal aperture(µm) 24.54±0.56* 24.88±0.67* 17.42±1.07
Width of stomatal aperture(µm) 6.11±0.39* 5.89±0.23* 3.11±0.43
Peduncle length (cm) 6.11±0.61* 6.20±0.39* 3.26±0.16
Days to flowering 80.0±0.59* 79.27±0.49* 50.0±0.61
Days to maturity 149.70±0.22* 142.09±0.27* 129.00±0.95
Pollen diameter(µm) 62.15±5.70* 69.90±2.20* 40.19±3.25
Pollen sterility (%) 40.46±0.09* 23.39±0.05* 1.57±0.07
Seed yield/plant (gm) 0.24±0.69* 5.88±0.77* 16.60±0.89
Biological yield/plant (gm) 49.65±1.26* 42.11±1.75* 26.65±0.86
* mean value significant at 5% level at C1 and C2 generations as compared to diploid control variety.
66 TALUKDAR

Figs. 4-15 — Meiotic-I chromosome association of diploid and tetraploid plants. (4-5) 7II at diakinesis showing one
bivalent attached with one nucleolus and at metaphase-I, respectively in diploid plants. (6) 14II in C2 tetraploid plants.
(7-8) 11II+1IV+2I showing one ring quadrivalent and univalents (→) at diakinesis and metaphase-I. (9-10) 5IV+4II
at diakinesis and metaphase I. (11) 12II+1IV indicating (→) one quadrivalent at diakinesis. (12-13) 12II+1III+1I
association showing one trivalent at diakinesis and metaphase–I (→). (14) Clumped bivalents at metaphase-I. (15)
Binucleolate (→) condition.

however found increased in C2 progeny. Pollen
diameter increased over diploids but not varied
considerably between generations (Table 2).
Meiotic analysis of tetraploid plants - Lathyrus
sativus var. ‘BioR-231’, used in the present study,
is strictly diploid with 2n=2x=14 chromosomes
as confirmed in root tip karyotype as well as in
meiotic studies (TALUKDAR 2008a). At diakinesis
and metaphase-I presence of seven bivalents (n =
7) was the regular feature in the present diploid
material (Figs. 4, 5). In tetraploids (2n=4x=28),
homologous chromosomes have equal oppor-
tunities to pair at meiosis. At metaphase-I, 28
chromosomes have been observed in different
associations in the present tetraploid plants
showing maximum pairing as bivalents and were
CYTOGENETICS OF INDUCED AUTOTETRAPLOIDS IN LATHYRUS SATIVUS L . 67

followed by multivalent and univalent associa-
tion (Table 3). None of the Pollen mother cells
(PMCs) of C1 tetraploids was found to show dis-
tinct 14 bivalents although exclusive presence of
bivalent association of 28 chromosomes was ex-
hibited in some of the PMCs (5%) of C2 plants
(Fig. 6). The multivalent was exhibited mainly
in the form of trivalent and quadrivalent asso-
ciation. The quadrivalents were ring shaped i.e
symmetrical and the univalent/s was oriented in
close vicinity of other chromosomes (Fig. 7).
Pairing disturbances in the present induced
tetraploids were manifested in different combi-
nation of multivalents, bivalents and univalents
at diakinesis and metaphase-I. In C1 and C2
generations the chromosome paired in major-
ity of cases as bivalents (11II or 12II) in 54%
and 66%, respectively of the PMCs at diakine-
sis and 69.77% and 72.97% PMCs studied at
metaphase-I. The average frequency of bivalent
configuration for one PMC was also estimated
higher than multivalent in tetraploids (Table 3).
Association of one quadrivalent with eleven bi-
valents and two univalents (1IV+11 II+2I) was
predominant (42.00% at diakinesis and 38.40%
at metaphase-I) and was followed by five quad-
rivalents with four bivalents (5IV+4II) and
one quadrivalent with 12 (1IV+12II) bivalents
(12.00% at diakinesis and 9.96% at metaphase-
I) at C1 generation (Figs. 7-11). The number of
cells at metaphase I containing the former two as-
sociations decreased but the latter one increased
(37.00% at diakinesis and 35.63% at metaphase-
I) at C2 generation (Table 3). Occurrence of one
trivalent with twelve bivalents and one univalent
(1III+12II+1I) and clumped bivalents was come
across both in C1 and in C2 generations but tri-
valent configuration decreased marginally in C2
(Figs. 12-14, Table 3).
The present tetraploid plants were also char-
acterized by anomalous nucleolar behaviour at
prophase-I exhibiting nucleolar fragmentation
and budding in varying frequencies while only
one bivalent was found attached with one nu-
cleolus per cell in diploid plants of grass pea
(Figs.4, 15, 16).
Equal separation of 7-7 chromosomes at
anaphase-I was encountered in all the PMCs
of diploid plants (Fig. 17), but various types of
anaphasic disturbances like unequal separation,
laggard, broken bridge and laggard with bridge
were observed in both C1 and C2 tetraploid
plants at anaphase-I and II stages in varying fre-
quencies (Figs. 18, 20-23). Among unequal sepa-
ration 15-13 distributions of chromosomes in
two poles was most common in both generations

TABLE 3 — Chromosome behavior at meiosis in diploid control and induced autotetraploids (C1 and C2 generation)
of grass pea (Lathyrus sativus L).

Meiotic events Tetraploid Diploid

C1 generation C2 generation
Diakinesis – cells studied 100 100 100
7II 00 00 100
12II+1IV 12 37 00
5IV+4II 16 13 00
11II+1IV+2I 42 29 00
Binucleolate 18 11 00
Nucleolar budding 12 10 00
Metaphase-I-cells studied 612 640 500
7II 00 00 500
14II 00 32 00
12II+1IV 61 228 00
5IV+4II 173 133 00
11II+1IV+2I 235 114 00
12II+1III+1I 131 125 00
Clumped bivalents 12 08 00
Average frequency of chromosome configurations per PMC at MI
Univalents 0.98 0.55 00
Bivalents 9.11 10.1 07
Trivalents 0.21 0.19 00
Quadrivalents 1.89 1.57 00
68 TALUKDAR

occurring 48.35% at C1 and 61.62% of PMCs
scored at C2 generations. Equal separation of 28
chromosomes (14-14) has been found increased
to 12.18% of PMCs in C2 progeny (Fig. 19, Ta-
ble 4). Formation of bridge accompanied with
laggard at anaphase-I have been encountered in
18.54% and 16.24% PMCs at C1 and C2 genera-
tions respectively and this frequency decreased at
anaphase-II. Micronuclei formation was also en-
countered in tetraploid, possibly as the fate of the
laggards and/or fragments at telophase II and its
frequency decreased in C2 plants (Figs. 24, 25).
DISCUSSION
The tetraploid plants isolated in the present
investigation exhibited superiority over their
diploid counterparts in terms of increase in col-
our intensity, size and thickness of leaves and
stipules, number of branches, length of tendril,
peduncle and number as well as size of flowers
resulting in luxuriant growth but as compared to
diploids basic arrangement of different vegeta-
tive parts and arrangement, number and colour
of different floral parts remained unmodified.
In tetraploid plants of Lotus sp, a legume from
French Alps, GAUTHIER et al. (1998) explained
that similar nonsize morphological traits such as
flower colour, plant habit etc. between diploid
and tetraploid plants indicated autopolyploid
origin of tetraploidy and the present study fully
agreed with this finding. However, deep green
colour of the larger fleshy leaves in the present
tetraploids might be attributed to the increased
number of large sized chloroplasts with quantita-
tive increase in DNA content in the tetraploids
(BUTTERFASS1983). Stomatal size and frequency
have proved to be reliable indicators of ploidy
level in a number of species and the process is
simple and largely non-destructive (TAN and
DUNN 1973; BECK et al. 2003; GU et al. 2005). In
colchicine-treated Datura autotetraploids, BLAKE-
SLEE and AVERY (1937) opined that guard cells of
the stomata were less influenced by environmen-
tal factors than many other cells of the plant. In
the present tetraploids, enhancement of stomatal
size over diploids was mainly due to increase in
length and width of stomatal guard cell as well
as aperture and this result was in agreement with
other reports on different plants (BECK et al.
2003; OLIVEIRA et al. 2004). Pollen diameter also
increased significantly in tetraploids and BLAKE-
SLEE and AVERY (1937) as well as STEBBINS (1980)
pointed out that the correlation between larger
pollen and higher ploidy number was consistent
only in autotetraploids.
Delayed as well as prolonged duration of flow-
ering in both C1 and C2 generations of the induced
tetraploids was presumably, due to relatively low-
er permeability accompanied with lower rate of
respiration, transpiration and transport of manu-
factured food from the site of production to the
place of utilization (BISWAS and BHATTACHARYA
1976; BISWAS 1998). Increase in the number and
size of flowers had also distinguished the present
tetraploids from diploids and relatively more sec-
ondary branches in tetraploids has been ascribed
for enhanced number of flower. Significant mor-
phological differences between the diploid and
the tetraploid plants due to increased size of cer-
tain vegetative traits and higher number of flower

TABLE 4 — Meiotic chromosome behavior at anaphase and telophase in diploid control and induced autotetraploids
(C1 and C2 generation) of grass pea (Lathyrus sativus L).

Meiotic events Tetraploid Diploid

C1 generation C2 generation
Anaphase-I-cells scored 302 271 300
7-7 separation 00 00 300
14-14 14 33 00
15-13 146 167 00
18-10 35 16 00
Laggard with bridge 56 44 00
Broken bridge 51 11 00
Anaphase-II - total cells studied 100 100 100
Laggard with bridge 10 06 00
Telophase-II -cells scored 55 55 55
Laggard 11 05 00
Micronuclei 21 14 0
CYTOGENETICS OF INDUCED AUTOTETRAPLOIDS IN LATHYRUS SATIVUS L . 69

were also observed in autotetraploid Lotus plants
(GAUTHIER et al. 1998). Number of flowers how-
ever, reportedly reduced in induced autotetra-
ploids of different other legume plants (BISWAS
and BHATTACHARYA 1976).
Considerable pollen sterility contributed re-
duced seed yield in the present tetraploid plants
but the seeds produced by tetraploid plants were
larger than that of diploids. Reduced fertility
and increased cell and organ size (gigas-effects)
were considered as universal effects of artificial
polyploidization (STEBBINS 1980; LEWIS 1980).
Luxuriant vegetative growth, low pollen fertility,
decreased seed setting but increased seed weight
have also been reported in colchicine treated
C1seedlings of Helianthus annuus (SRIVASTAVA
and SRIVASTAVA 2002) and in Vicia faba (JOSHI
and VERMA 2004). High pollen viability but poor
seed setting was observed in pea tetraploids
(KRAVCHENKO 1995) while high seed setting with
38% increase in seed weight and filled pollen
was reported in colchiploids of Plantago media
(DIJK and DELDEN1990).
Patterns of meiosis in polyploids are influ-
enced by their mode of origin (SYBENGA 1996).
Occurrence of multivalents particularly quadri-
valents was conspicuous in varying frequencies
in the present tetraploids, since the latter are
derived from a single genome resulting in four
homologous sets of chromosomes in tetraploids
(STACE 1980). Artificial nature of origin, reduced
fertility and presence of ring shaped quadrivalent
indicated autopolyploid nature of present tetra-
ploid plants. BRUNSBERG (1977) and OLIVEIRA
et al. (2004) suggested autotetraploid nature of
Lathyrus pratensis and Stevia rebaudiana respec-
tively on the basis of quadrivalent configurations
in the meiosis of tetraploids.

Figs. 16-25 — Chromosomal behaviour of diploid and tetraploid plants during anaphase-I, II and telophase-II stages
of meiosis. (16) Nucleolar budding in C1 tetraploid plants. (17) Normal 7-7 separation in diploid. (18) Unequal distri-
bution of 15-13 chromosomes in tetraploids. (19) Equal separation of 14-14 chromosomes. (20) A 18-10 separation.
(21) Bridge accompanied with laggard at anaphase-I. (22) Broken bridge at anaphase-I. (23) Unequal separation, lag-
gard and bridge formation at anaphase-II. (24) Laggard at telophase-II. (25) Micronuclei formation at telophase-II.
Scale bar- 1SD=10 µm for figures 4-25.
70
Chromosome association at the first (I) and
second (II) division of the meiosis underwent
some changes from C1 to C2 generation in the
present tetraploid material of grass pea. Number
of cells containing trivalent association at met-
aphase I and lagging chromosomes and bridges
at anaphase I and II decreased in the C2 genera-
tion. Increased tendency of bivalent association
has been evidenced by predominant occurrence
of chromosome pairing as bivalents (11II or 12
II) in 69.77% at C1 and 72.97% at C2 and also
as 14 II (2n=4x=28) in 5% PMCs at C2 genera-
tion accompanied with decrease in number of
cells containing higher number of quadrivalent
associations (5IV+4II) in latter generations (Ta-
ble 3). In Arabidopsis autotetraplopids, SANTOS
et al. (2003) attributed increased tendency of bi-
valent association to the reduction in the number
of autonomous pairing sites (APSs) and presence
of just a single APS per chromosome resulted in
only bivalents as found in 5% cells in the present
autotetraploids. This bivalent association at met-
aphase-I might be due to chiasma insufficiency
or due to an altered distribution of chiasmata
relative to pairing partner exchanges. In grass
pea aneuploids, BISWAS and BISWAS (2004) and
TALUKDAR and BISWAS (2007) reported reduction
in chiasma formation. No conclusion, however,
can be drawn in the present material although
preliminary investigation indicated that the re-
duction in multivalent frequencies and concomi-
tant increase in bivalent frequencies in C2 gen-
eration presumably resulted from chromosome
size-dependent changes in pairing/synapsis of
homologous chromosomes and in chiasma fre-
quency distribution. Presence of univalents in
the present tetraploids at metaphase I indicated
failure of chiasma formation or due to loss of chi-
asmata with the progress of meiosis, as explained
earlier in grass pea by BISWAS (1998). As shorter
chromosomes are more prone to reduction in
multivalent formation (SANTOS et al. 2003) it
seemed likely that this size dependency increased
in C2 generation in the present autotetraploids
and multivalent has been maintained by involv-
ing relatively longer-sized chromosomes. Efforts
are being made to identify the chromosomes in-
volved in multivalent formation in grass pea au-
totetraploids. Moreover, like lentil and pea, grass
pea genome exhibited chromosome structural
rearrangement (TALUKDAR 2010) and possibil-
ity of its involvement in promotion of bivalent
formation can also not be ruled out. Further in-
vestigation in this regard, however, is absolutely
necessary in the present tetraploids. Studying ex-
TALUKDAR
clusive bivalent association of all chromosomes
in the colchiploid of Nicandra physaloides, GUPTA
and ROY (1986) suggested genetically controlled
pairing of chromosomes irrespective of size in
multivalent formation of polyploids. According
to JACKSON (1982), fewer multivalents and more
bivalents in autopolyploids might be due to pres-
ence of Ph-like (pairing homoeologous) genes
conferring allopolyploid-like behaviour through
preferential chromosome pairing. Among other
leguminous plants, KRAVCHENKO (1995) reported
higher frequency of multivalent formation than
bivalents in Pisum sativum L. while in colchiploid
soybeans, KUMAR and RAI (2007) opined that C2
generation showing increased frequency of biva-
lents was more stable than C1 generation. Higher
frequency of bivalent formation was also report-
ed in colchitetraploids of ornamental plant, Im-
patiens balsamina L (DIKSHIT and KUMAR 2007).
Relationship between multivalents, bivalents and
univalent formation and chromosome differen-
tiation has been critically examined at metaphase
I in colchicine-induced autotetraploid hybrids of
Secale cereale (BENAVENTE and ORELLANA 1991).
Formation of multivalents and univalents at
metaphase-I in different associations brought
about meiotic disturbances leading to high inci-
dence of pollen sterility at C1 generation through
various types of anaphasic irregularities in the
present tetraploid plants. The disjunction of tri-
valents and the behaviour of univalent/s at ana-
phase-I being highly irregular a variable portion
of gametes produced by tetraploids was unbal-
anced and became non-viable resulting in pollen
sterility. However, the degree of pollen sterility
among tetraploid plants was influenced by multi-
valent and bivalent formation during metaphase-
I. Improvement of pollen fertility and seed setting
to some extent at C2 generation of present mate-
rial were, presumably, due to combined action of
higher number of bivalents at metaphase I, some
degree of regularity in quadrivalent disjunction
and chromosome separation during anaphase
I. However, these have been counterbalanced
by the formation of trivalents and univalents at
metaphase I and unequal separation of chromo-
somes in a considerable number of microsporo-
cytes during anaphase I which brought about
meiotic disturbances maintaining pollen sterility
and low seed yield in tetraploid plants in com-
parison to diploid plants. HAZARIKA and REES
(1967) explained that univalents and multiva-
lents due to segregational errors lowered the fer-
tility in autotetraploids. Number of univalents at
metaphase-I was considered as an important fac-
CYTOGENETICS OF INDUCED AUTOTETRAPLOIDS IN LATHYRUS SATIVUS L .
tor in formation of micronuclei at telophase-II in
meiosis and caused genetic and physiological im-
balances leading to pollen sterility and abortion
in tetraploid plants (MENDES-BONATO et al.2002;
CHEN et al. 2007). RAGHUVANSHI and SINGH (1980)
opined that meiotic abnormalities coupled with
genetic factors contributed towards sterility in
autotetraploid Trigonella foenum-graecum
In the first (I) and second (II) division of the
meiosis, bridge formation accompanied with the
fragment/s was exhibited in both C1 and C2 gen-
erations of present autotetraploid plants. Sybenga
(1996) attributed it to the presence of paracentric
inversion. So, after detail cytogenetic analysis
possibility of development of cytological stocks
including both neumerical and structural chro-
mosomal aberration can be explored in grass pea.
Anomalous nucleolar behaviour such as fragmen-
tation, budding and multinucleolate condition in
the present tetraploid might be attributed to the
inherent instability of nucleolar organizing chro-
mosomes and to the activity of more than one
pair of chromosomes, although its exact mecha-
nism is not well understood (KUMAR and TRIPATHI
2007). Clumping of chromosomes at metaphase-I
in tetraploids might have occurred due to longi-
tudinal orientation of spindle particles or due to
alteration in the surface tension around the chro-
mosomes following disappearance of nuclear
membrane (BAJER and MOL-BAJER 1956).
The present set of autotetraploids in grass
pea manifested cytogenetic peculiarities. Us-
ing different cytogenetics data and isozymatic
analysis, GUTIERREZ et al. (1994) differentiated
allopolyploid and autopolyploid origin of dif-
ferent Lathyrus species. Different morphological
and cytological parameters indicated autopoly-
ploid origin of present tetraploid plants which
can be further confirmed by studying tetrasomic
inheritance pattern of different isozyme markers
in the present material as suggested by SOLTIS et
al., (2007). KHAWAJA et al.(1998) used colchicine-
induced autotetraploids as valuable source for
development and maintenance of different aneu-
ploids including trisomics in other Lathyrus spp.
The information gathered from the present study
on induced tetraploids could be useful in future
improvement of grass pea breeding.
Acknowledgments — Author is thankful to Prof.
(retd.) A.K. Biswas, Department of Botany, Univer-
sity of Kalyani, Kalyani, Nadia, India and Dr. S.C.
Biswas, Scientist, Seed Technology Division, BRAC,
Bangladesh for giving valuable suggestions during the
present study.
71
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Received May 28th 2009; accepted March 9th 2010