2. ADVISORY BOARD
• CHAIRMAN :- DR. A. K. ROY
• MEMBER:- DR. D. R. MALAVIYA
• MEMBER:- DR. P. KAUSHAL
• MEMBER:- DR. A. CHANDRA
• MEMBER:- DR. SURESH KUMAR
2
3. Assessment of molecular genetic diversity
among different species of Trifolium and
ecotypes of T. alexandrinum.
Study on interspecific/intervarietal
compatibility and inheritance of traits like
regeneration ability.
3
4. • Genus: Trifolium Tourn (L.).
• Family: Leguminosae.
• Sub-family: Palpilionaceae.
• It comprises of 290 annual and perennial herbs
commonly called clovers including several
cultivated and pasture species of high fodder value.
4
5. The important perennial pasture clover T.
repens, T. hybridum, T. pratense and T.
ambiguum are widely distributed in the
temperate and sub-temperate regions of the
world.
The annual types such as T. resupinatum, T.
subterraneum and T. alexandrinum are
commonly cultivated as winter annuals in
the tropical and subtropical regions.
5
6. The genus name Trifolium refers to the distinctive
leaves usually composed of three leaflets (trifoliolate).
The Mediterranean region is the centre of diversity
of the genus Trifolium (Zohary and Heller, 1984).
Berseem is widely adapted by the farmers of north,
northwest, eastern and central India.
Nearly 2 million hectare arable land is under
cultivation of Berseem with an average national
productivity of 85 t/ha.
6
7. The importance of crop is known for:-
Its high green fodder production (up to 100t/ha).
Its multicut nature (6-7 cuts).
Long duration of green fodder availability
(November to April).
High crude protein content (20-24%).
High digestability.
High palatability .
High nitrogen fixation ability.
7
9. To incorporate desirable traits like early flowering
from Fahli group and regeneration ability from
Mescavi group, inter-varietal crosses were
attempted among three ecotypes of T. alexandrinum
(Berseem) i.e., Mescavi (cv Wardan and BB-2),
Saidi (Ex-8) and Fahli (FAO-5).
F1 hybrids were crossed to male parent and female
parents to obtain BC1 seed.
9
10. All F1s and their parents, were
characterized for 12 qualitative and
quantitative traits.
F2 and BC1 populations were characterized
for 10 different morphological attributes
including metric and non-metric traits.
10
11. Hybrids and their parents were analyzed for
esterase and protein native banding pattern
along with their parents.
F2 progenies and their respective parents
were compared only for esterase isozyme
banding pattern.
11
12. BSA (Michelmore et al, 1991) was used to identify
RAPD markers linked with regeneration and non-
regeneration trait.
BC1 population from contrasting parents Wardan
(regenerating genotype) & FAO-5 (non regenerating
genotype) was developed by backcrossing F1 progenies
with Wardan.
4 bulks representing viz., P1 bulk (Wardan bulk), P1
type bulk (Wardan type plant progenies), P2 bulk (FAO-
5) & P2 type bulk (FAO-5 type plant progenies) were
prepared and screened with 110 RAPD primers for
identification of putative marker linked to regeneration. 12
13. DNA extracted from fresh leaves and PCR
reaction carried out as described by Williams
(1993).
18 decamer RAPD primers were used in analysis
of diversity within and between different ecotypes
of T. alexandrinum. 13
14. A total of 38 SSR primer pairs were used to estimate
polymorphism among 24 species of genus Trifolium.
DNA isolated and PCR reaction was performed in 10µl
volume containing template DNA, Taq polymerase enzyme,
Taq polymerase buffer, dNTPs, forward and reverse
primers.
The thermocycle parameters were used as described by
Barret (2004) with minor modifications.
The PCR products were electrophoresed on 3.3%
agarose gel.
14
15. To identify the stages of pre and post fertilization
barriers where 3 genotypes of T. alexandrinum
(EC511781, Wardan and BB-2) were used as
female parent and crosses were made with
different Trifolium species.
Pollen tube growth was studied at regular
interval of 1hr, 3hrs, 5hrs and 8hrs after
pollination.
The karyotype will be prepared using root –tip
mitotic chromosomes studies based on standard
root –tip squash method after pretreatment,
fixation, hydrolysis and squash method.
15
16. The metric data was analyzed statistically to group
the plants of similar types based on their similarity.
Euclidian cluster analysis for grouping of genotypes
and Mahalanobis Euclidean2 analysis was used.
16
18. Intra and Interspecific variation analyzed in 51
accessions representing 19 species of genus Trifolium.
Wide range of variation observed for most of the
traits like plant height (19-143.89 cm), number of
branches (5.67-55.11), number of node (5.0-16.33) etc.
18
25. Wide variation was recorded for various
qualitative traits among the species studied.
Growth habit:- Most of the Trifolium species were
prostrate in nature while T. alexandrinum showed
erect or semi-erect growth habit.
Flowering period: T. clypeatum and T. dasyrum
flowered in 2nd week of February while T. hybridum
EC425029 followed by T. diffusum EC528538
flowered in 1st week of May. Most of the species
flowered in the month of March. 25
27. Petiole hairiness: varied from hairy to non hairy.
T. spumosum and T. resupinatum had non hairy or glabrous
petiole while dense hairs in few species viz., T. diffusum, T.
hirtum, T. pilulare, T. balansae, T. clypeatum and T.
alexandrinum EC539057.
Leaflet hairiness:- Hairy or dense hairy leaflets were
found in most of the species viz., T. apertum, T.
angustifolium, T. diffusum, T. hirtum, T. balansae and T.
dasyrum but species like T. spumosum, T. repens, T. hybridum
and T. resupinatum had non hairy or glabrous leaflets. 27
28. Leaflet apex shape:- Round leaflet apex was found
to be common however the species such as T.
apertum, T. angustifolium, T. resupinatum, T. pilulare
and T. dasyrum had only tapering leaflet apex.
Leaf shape:-This trait varied from lanceolate,
oval, ovate, elongated, obovate, to rhomboid where
lanceolate leaf shape was seen predominantly.
28
29. Fig: Variation for leaf shape & leaf margin in different species of genus Trifolium.
29
30. Leaflet margin:-Leaflet had either serrate or entire
where entire margin was quite frequent.
Inflorescence apex shape:- Round was observed to
be predominant with some conical or tapering apex.
Flower colour:- A wide range of variation has been
observed for flower colour which varied from white to
dark purple colour, white flower (in 15 accessions) was
most common among the species followed by light
pink flower (in 14 accessions). 30
31. Fig: Variation for flower colour in different species of genus Trifolium.
31
32. T. compestre, had yellow flower and T.
clypeatum, T. hirtum, T. echinatum had
flowers with white + pink petals. Flowers
of T. clypeatum were largest in size as
compared to other species.
32
33. 51 accessions of 19 Trifolium species were grouped
into 7 clusters based on similarities of Mahalanobis’s
D2 values.
Cluster-1 was the largest cluster with 30 accessions
followed by cluster-2 comprising of 8 accessions,
cluster-4 comprising of 6 accessions and cluster-3
comprising of 4 accessions.
Cluster-5 (T. alexandrinum EC418538), 6 (T.
angustifolium EC539064) & 7 (T. angustifolium
EC539058) were monotypic. 33
34. Cluster No. of accessions Species Name
No. per cluster
1 30 T.echinatum EC425075, EC425076, T.lappaceum EC402165, T.echinatum EC425078, T.ligusticum
EC528544, T.lappaceum EC528542, T.resupinatum EC425071, T.echinatum EC425077, T.balansae
EC511775, T.grandiflorum EC528540, T.resupinatum EC425072, T.hybridum EC425029, EC401702,
T.hirtum EC425037, EC425039, T.hybridum EC401701, T.pilulare EC528544, T.diffusum EC528538,
T.subterraneum EC401717, T.spumosum EC528549, EC518781, T.repens EC400984, T. dasyrum
EC511808, T.subterraneum EC401718, T.echinatum EC401714, T.subterraneum EC539068,
T.alexandrinum EC528530, EC539057, EC511781 & EC400773
2 8 T.clypeatum EC511807, EC528536, T.repens EC402155 A, EC400385 B, EC400987 A, EC402155 B,
EC400987 B & EC400385 A
3 4 T.compestre EC425028, EC5425027, EC402155 & T. dasyrum EC528537
4 6 T.resupinatum EC425036, EC425074, EC425073, EC401715, T.apertum EC401712 & T.alexandrinum
EC400976
5 1 T.alexandrinum EC418538
6 1 T.angustifolium EC539064
7 1 T.angustifolium EC539058
Total clusters=7 Total Accession=51 Total species=19 34
37. F1 hybrid: 5 hybrids (named as C-552-1, C-552-2, C-
552-3, 380 and 468) were produced.
Hybrid varied from parents for growth habit,
axillary bud, flowering initiation, leaf wrinkles and
most of the quantitative traits which confirmed the
hybridity.
37
38. The similarity was estimated between 5 hybrids of Wardan
x FAO-5 combinations & their parents using the Jaccard
similarity matrix based on total 20 bands of esterase (10
bands) and native protein (10 bands).
Wardan
71% Cluster-1
468 (79%)
C-552-2
Sub-cluster-2-1
C-552-1 (83%)
78% Sub-cluster-2-2
76% FAO5
C-552-3
Cluster-3 (85%)
380
0.70 0.77 0.85 0.93 1.00 38
41. Only 3 F1 hybrid i.e., C-552-1, C-552-2 and 380
successfully survived and produced 23 F2 individuals
representing 9 F2 individuals of C-552-1, 12 of C-552-2
and 2 of 380 survived.
These 23 F2 individuals were evaluated for
morphological characterization.
All F2 individuals showed segregation of traits. 41
42. In general, these F2 individuals were less vigorous then
F1s.
All F2 individuals showed different vigour and
regeneration.
Out of 23 F2 individuals, 7 were intermediate between the
parents, 7 were P1 type (female type branching) however
remaining 9 individuals were significantly more close to
male parent (P2) regarding flowering and branching.
F2 individuals, showed significant variation for days to
flower initiation ranging from 62 to 100 days as compared to
early flowering (66 to 70 days) in F1 hybrids. 42
43. 9 F2 progenies of this combination including their
respective parents were analyzed for esterase isozyme
system.
Dendrogram generated, based on 11 polymorphic and
1 monomorphic band, divided the progenies in 3 clusters.
Wardan Cluster-1
1 (72%)
2
5
FAO-5 Cluster-2
9
7
(72%)
3
6
Cluster-3
8
4 43
0.60 0.70 0.80 0.90 1.00
44. The genetic similarity was estimated between 11 F2
progenies of hybrid C-552-1 and their parents using the
Jaccard similarity matrix.
Dendrogram revealed presence of 3 main clusters.
Wardan Cluster-1
1
2
(62.1 %) 3
8
10
4 Cluster-2
5 (66.4% to 100%)
6
FAO-5
7
(61%) 11
9 Cluster-3
0.60 0.70 0.80 0.90 1.00
44
45. 33 BC1 plants were survived and evaluated for different
morphological attributes.
For general vigour, BC1 plants were significantly similar to F2
plants.
The days to flowering ranged from 66 to 70 days with an average
of 67.33 days in F1 hybrids while in F2 and BC1 plants, it was 62 to
100 (average 80.78) and 82 to 98 days (average 89.24) respectively.
The traits like early flowering, dense secondary branching and
leaf wrinkles were noticed in F1 hybrids as in Fahli group (male
parent). These traits were more prominent in BC1 plants as
compared to F2 plants. 45
46. Only 7 BC1 plants survived successfully and were evaluated
for various morphological traits.
Vigour of BC1 plants was quite similar to F2 plants.
The broad range for the character days to flowering
initiation was recorded in F2 plants ranging from 62 to 100
days. It was quite contrasting to BC1 plants, where days to
flowering ranged from 97 to 100 days and quite similar to P1
female parent (Wardan) showing a range of 90 to 94 days. In
P2 male parent (FAO-5) days to flowering initiation was
between 62 to 64 days whereas in F1 hybrids the range was 66
to 91 days. 46
47. The regeneration was the characteristic feature of
female parent P1 (Wardan) segregated in F2 plants
(three poor, thirteen medium good and seven good
regeneration) as well as in BC1 plants (two good, two
medium good and two very good regeneration).
F1 hybrids showed dense branching and early
flowering while BC1 plants did not show early
flowering and dense branching, showing their
closeness to P1 parent (Female: Wardan). 47
48. BB-2 x FAO-5 Combination:-
In this combination 4 hybrid plants numbered as C-
579-1, C-579-2, 482 and C-10 were produced.
These hybrids varied significantly for all qualitative
traits except leaflet margin which was entire in all the
hybrids.
However, semi-erect growth habit, hairy leaflet and
dense hairy stipule pubescence were similar in C-579-1,
C-579-2 and 482, while C-10 had erect growth, dense
hairy leaflet and hairy stipule.
Traits like petiole hairiness, leaflet apex shape and
leaflet colour were similar in all the hybrids except C-
579-1.
Variation was observed for quantitative traits among
the hybrids. 48
49. Biochemical characterization of BB-2 x FAO-5
hybrids
4 hybrids of BB-2 x FAO-5 were analyzed for native
protein banding pattern along with parents.
The dendrogram based on 11 bands of native protein
was divided in 3 main clusters.
BB-2
Sub-cluster 1-1
C-579-1
C-579-2 Sub-cluster 1-2
(88%)
C-10
Cluster -2 (100%)
FAO-5
(74%)
482 Cluster -3
0.70 0.77 0.85 0.93 1.00
49
50. 7 F2 individuals representing 5 F2 individuals of
C-10 hybrid and 2 F2 individuals of hybrid 482
survived successfully.
F2 progenies also differed for regeneration (one
very good, four good and two medium).
Flowering initiation was similar in F1 hybrids
and F2 individuals.
50
51. 3 resembled male parent, 1 individual had
male type of high secondary branching and 1
individual was maternal type having less
secondary branches whereas 1 individual was
intermediate among both the parents.
51
52. Similarity among F2 populations of BB-
2 x FAO-5
UPGMA dendrogram was formed on the basis of 15 scorable
bands of esterase isozyme in 7 F2 progenies and their parents .
The population was divided in 2 main clusters; cluster-1 was
comparatively larger where intra-cluster similarity ranged from
66% to 100%.
BB-2
6 Sub-Cluster-1-1
7
(66%-100%)
1
4 Sub-Cluster-1-2
5
(55%)
2
Sub-Cluster-1-3
3
FAO-5 Cluster-2
0.50 0.63 0.75 0.88 1.00
52
53. Ex-8 x FAO-5 combination
3 hybrid were obtained and named as C-560, C-564
and 203 which were similar for qualitative traits like
leaflet margin, leaflet shape, leaflet apex shape, leaflet
colour, stipule pubescence and flower colour.
The hybrid C-560 was more similar to hybrid C-564
than hybrid 203 for qualitative traits.
The hybrid C-564 was more vigorous having higher
values than other hybrids and the parents for most of the
quantitative traits.
On comparing the hybrids with parents, it was
observed that leaflet margin, leaflet shape, axillary bud
and flower colour were similar among the parents and the
hybrids while rest of the qualitative traits varied.
53
54. The dendrogram was based on 24 bands representing
10 bands of esterase and 14 bands of protein native.
Ex-8
Cluster-1-1
FAO-5
88%
79%
203
Cluster-1-2
74% C-560
Cluster-2
C-564
0.70 0.77 0.85 0.93 1.00
54
55. 16 F2 individuals representing 4 F2 individuals from
C-560 and 12 from C-564 were compared with F1
hybrids for some morphological attributes.
Both F1 hybrids showed oval bud apex and dark
green leaf colour whereas in F2 individuals both the
traits varied.
Most of the F2 individuals flowered early like their
respective parents.
55
56. 7 F2 progenies resembled P1 parent, 7
intermediate and 2 were P2 type with respect to
branching behavior.
The range of number of nodes on secondary
branch of 4th node and length of secondary branch
of 4th node was 0 to 8 and 0 to 55cm in F2
individuals respectively.
The average of number of primary branches per
plant was 2.5 in all F2 individuals. 56
57. Dendrogram was based on 5 monomorphic and 6
polymorphic bands and generated by using the
UPGMA algorithm. Ex-8
Cluster-1-1
(94.1%)
2
(85%)
1
3 Cluster-1-2
(77%)
4
FAO-5 Cluster-2
0.75 0.81 0.88 0.94 1.00
57
58. In this combination 2 hybrid viz, 79-1 and 79-2
were produced which were similar for almost all
qualitative traits.
Growth habit, petiole hairiness and axillary bud
in the hybrids were more similar to the female
parent than the male parent.
Hybrids were early flowering, quite similar to
the female parent while male parent flowered late.
58
59. Both the hybrids had oval inflorescence apex and
pentafoliate leaves while in the parents it was
elongated trifoliate respectively.
No. of branches, no. of nodes, plant height, leaflet
length, leaflet width, leaflet no. and stipule length were
higher in the hybrids than the parents while rest of the
traits varied among the parents and the hybrids. 59
60. The genetic similarity was estimated between 4hybrids
(213, 378, 79-1 & 79-2) along with their parents.
Based on 14 bands of protein native, dendrogram was
divided in 2 clusters.
Ex8
Cluster-1-1
83%
213 92%
79-1
79-2 Cluster-1-2
78%
Wardan
378 Cluster-2
0.80 0.85 0.90 0.95 1.00
Coefficient 60
61. 12 F2 individuals (11 from 79-1 and 1 from 79-2 hybrid)
successfully survived. The trait, vigour and regeneration
showed variation among the F2 individuals.
A range of 0 to 6 and 0 to 24cm has been found for the
trait, number of nodes on secondary branch of 4th node and
length of secondary branch of 4th node respectively.
7 were similar to male parent for branching behavior and
1 was more close to female parent while 4 were
intermediate among both the parents.
Flowering initiation was quite late in F2 individuals
(ranging from 51 to 119 days) as compared to F1 hybrids
61
which flowered after 67 days.
62. 4 bulks representing viz., P1 bulk (Wardan
bulk), P1 type bulk (Wardan type plant
progenies), P2 bulk (FAO-5) & P2 type bulk
(FAO-5 type plant progenies) were screened
with 110 RAPD primers.
62
63. Total primers 110
Total amplified primers 92
Total yielded bands 623
Monomorhpic bands 403 (64.68%)
Polymorphic bands 220 (35.32%)
Range of yielded bands
1 band (OPH-1, OPH-8, OPH-15 & OPJ-16) - 14 bands (KITAB-18)
Average of bands/primer 6.77
Not Amplified 18
Monomorphic primers 17
Polymorphic primers 75
Primer generate nonspecific bands 46
Primer generate specific bands 29
63
64. Fig: Screening of four bulks with twenty-nine RAPD primer
generating specific bands:-
64
65. S.No. Primers Specific band no.
1 OPH-2 B5 for regeneration
2 OPH-3 B5 for regeneration and B3 for non-regeneration
3 OPH-6 Three bands, B3, B9 & B10 for non-regeneration
4 OPH-7 B1 for regeneration
5 OPH-19 B8 for non-regneration
6 OPD-2 B8 for regeneration
7 OPD-4 B8 for regeneration
8 OPD-5 B5 for regeneration, B13 for non-regeneration
9 OPD-7 B6 for non-regeneration
10 OPD-8 B6 for regeneration
11 OPD-14 B4 for regeneration
12 OPD-19 B3 for non-regeneration
13 OPD-20 B3 &B6 for regeneration
14 KITAB-4 B2 & B7 for non-regeneration, B8 for regeneration
15 KITAB-5 B9 for regeneration
16 KITAB-7 B10 for regeneration
17 KITAB-9 B3 for regeneration);
18 KITAB-11 B10 for non-regeneration, B7 & B8 for regeneration
19 KITAB-12 B7 for regeneration
20 KITAB-17 B2 for regeneration
21 KITAB-19 B4 for regeneration
22 OPE-15 B1 for regeneration
23 OPJ-9 B3 for regeneration
24 OPJ-7 B2 for non-regeneration, B1 for regeneration
25 OPJ-13 B3 for non-regeneration, B5 for regeneration
26 OPJ-17 B5 for regeneration
27 OPC-4 B7 for regeneration
28 OPC-12 B4 for non-regeneratio 65
29 OPC-20 B4 for regeneration
66. Dendogram based on 623 bands generated by 92
primers was developed.
The dendrogram showed that the genetic similarity
among the bulks was quite high, ranging from 87% to
92%.
P1 bulk
Sub-cluster 1-1
88% P1 type bulk (92%)
87% P2 type bulk Sub-cluster 1-2
P2 bulkbulk
Cluster -2
0.85 0.89 0.93 0.96 1.00 66
72. Genetic variability was calculated among 120 plants
comprising of 24 plants each of 5 genotypes viz., BB-2,
BB-3, Wardan, Ex-8 and FAO-5 by using 13
oligonucleotide primers.
The dendogram showed the presence of 11 main
clusters, which were again divided in total 17 sub-
clusters.
Inter-cluster similarity among 11 clusters was
ranging from 47.0% to 92.0%. 72
74. A total of 38 SSR primer pairs were used to screen for
amplification and polymorphism among 75 accessions
belonging to 24 species of genus Trifolium.
Out of 38 SSR primer pairs, 34 primer pairs amplified
generating 130 polymorphic bands while remaining 4
primer pairs were non-informative.
The number of fragments amplified by individual
primers ranged from 2 to 6. The amplified products size
ranged from 100 bp to 280 bp.
74
75. Fig: SSR profile generated by primer PR-27 (433) in 24 species of
genus Trifolium:-
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 M
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 M 24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 M 1 2 3 4 5 M
75
76. The genetic distance between 75 accessions belonging to
24 species was estimated using the dendogram formed by
SAHN agglomerative clustering.
The grouping based on 130 bands showed the presence of
11 main clusters.
Main clusters were further divided in 25 sub-clusters and
4 sub-sub-clusters.
Inter-cluster similarity ranged from 28% to 96% among
11 clusters.
76
78. Cluster Sub-cluster Total accessions Species
T. alexandrinum (Wardan, BB-3, Giza-6, BB-2, Sakha-4, FAO-5, Serw-1, Helaly, Ex-8,
1-1-1 14
Fahli original, EC400976, EC418536, EC539050 & EC539057)
1-1 1-1-2 2 T. alexandrinum EC539048 and EC539047
1-1-3 3 T. alexandrinum (EC539054 & EC418539) and T. clypeatum EC528536
1
1-1-4 2 T. alexandrinum EC418538 and T. dasyurum EC528537
1-2 1 T. alexandrinum EC528530
1-3 1 T. clypeatum EC511807
1-4 2 T. hirtum EC429039 and EC425037
2-1 2 T. angustifolium EC539063 and EC425062
2
2-2 3 T. angustifolium (EC539058 & EC539064) and T. glomeratum EC401700
3-1 4 T. subterraneum (EC539068, EC401717 & EC539069) and T. pilulare EC528547
3
3-2 1 T. subterraneum EC401718
4 1 T. echinatum EC425075
T. apertum EC401712, T.fragiferum EC528539 and T. vesiculosum (EC401716, EC Ooty &
5-1 5
EC Palampur)
T. echinatum EC425078, T. lappaceum (EC528542 & EC402165), T. grandiflorum EC528540,
5 5-2 6
T. ligusticum EC528544 and T. balanse EC511775
5-3 1 T. diffusum EC528538
5-4 3 T. hybridum EC401702 , EC425029 and EC401701
6-1 1 T. nigrescence
6 6-2 5 T. repens EC400385, EC402155, EC400984, EC400379 and EC401705
6-3 1 T. repens EC401706
7 2 T. pratense EC400980 & EC400979
8-1 3 T. dasyurum EC511808 and T. echinatum (EC425076 & EC425077)
8-2 2 T. spumosum EC511781 and EC528549
8
T. glomeratum EC405033 and T. resupinatum (EC401715, EC425073, EC425074, EC425072
8-3 6
and EC425071)
9 3 T. compestre EC402155, EC425027 & EC425028 78
10 1 T. repens EC400987
79. Compatibility between different species is an indication of
close affinity between two species.
In Trifolium, the incompatibility barriers are very strong.
Evans (1962 b) reported the existence of high degree of
incompatibility between species of Trifolium.
The present study is an attempt to know the genetic
similarity among 10 Trifolium species and their relationship
with T. alexandrinum based on compatibility studies. 79
80. S. No. Female Parent Male Parent Obs-1 Obs-2 Obs-3 Obs-4 P-I P-II P-III P-VI P-V Observations Seed
Setting
Control T.alexandrinum T.alexandrinum 5hrs 10 1.8 1.7 1.9 1.7 2.4 2.4 1.4 Normal seed Normal
cross cv Wardan cv Wardan setting
1 T.alexandrinum T.angustifolium 1hr 10 0.2 0.1 0 0 0 0 0 No withering No
cv Wardan EC539058 5hrs 10 1.8 1.5 0.5 0.3 0.3 0.2 0
8hrs 5 0 0 0 0 0 0 0
2 T.alexandrinum T.clypeatum 1hr 5 0.2 0.2 0 0 0 0 0 No withering No
EC511781 EC528536 3hrs 5 0 0 0 0 0 0 0
5hrs 5 0 0 0 0 0 0 0
8hrs 5 0 0 0 0 0 0 0
3 T.alexandrinum T.grandiflorum 3hrs 5 0.6 1.2 0 1 0.2 0 0 Withering 2 DAP No
cv Wardan ECEC528540 5hrs 5 0.8 0.8 0 0.2 0.4 0.2 0 & shrivel ovaries
8hrs 5 1.2 2.2 1.6 1.2 1.2 1 0.2
4 T.alexandrinum T. compestre 1hr 5 0.6 0.6 0.4 0 0 0 0 No withering up No
cv Wardan EC5425027 3hrs 5 1.2 0.8 0.8 0.2 0 0 0 to 5 days
5hrs 5 0.8 1.4 0.8 0.4 0.2 0 0
5 T.alexandrinum T .repens 1hr 10 0.9 0.6 0.5 0 0 0 0 No withering No
cv Wardan EC400385 5hrs 11 0.82 0.82 0.6 0.18 0 0 0
6 T.alexandrinum T .ligusticum 1hr 10 0.5 0.6 0.5 0.1 0 0 0 Withering 2 DAP No
cv Wardan EC528544 5hrs 10 1.6 0.5 0.2 0.2 0.4 0.1 0
7 T.alexandrinum T. balansae 1hr 10 1.1 0.6 0 0 0 0 0 No withering No
cv Wardan EC511775 5hrs 10 0.5 0.4 0 0 0 0 0
8 T.alexandrinum T. hirtum 1hr 10 0.3 0.2 0 0 0 0 0 No withering No
cv BB-2 EC425039 5hrs 10 0.2 0.4 0.5 0 0 0 0
9 T.alexandrinum T. pilulare 1hr 10 0.2 0.2 0 0 0 0 0 No withering No
cv Wardan EC528547 5hrs 10 0.2 0.3 0.2 0 0 0 0
10 T.alexandrinum T. hybridum 1hr 10 0.2 0.3 0 0 0 0 0 No withering No
cv BB-2 EC41702 5hrs 10 1.3 0.3 0.4 0 0 0 0
Obs-1=Flowers harvested after 5hrs Obs-2=Number of pistils observed.
Obs-3Number of pollen attached (Average). Obs-4=Hairy region of stigma with pollen tubes or pollen germination (Average).
80
81. Fig: Pollen tube growth in different cross combinations
Control cross (A-J)
(Wardan) x T. clypeatum EC528536 (K-L)
(Wardan) x T. hirtum
EC425039 (M)
(Wardan) x T. angustifolium EC539058 (N-X)
81
82. Fig: Pollen tube growth in different cross combinations:-
(Wardan) x T. balansae EC511775 (A-D)
(Wardan) x T. compestre EC5425027 (E-H)
(Wardan) x T. ligusticum EC528544 (I-Q)
(Wardan) x T. repense EC400385 (R-T)
82
83. Good mitotic preparations with well spread
metaphase were studied.
5 cultivars of T. alexandrinum representing 3
ecotypes viz., Mescawi (Wardan, BB-2 & BB-3), Saidi
(Ex-8) and Fahli (FAO-5) were analyzed on the basis
of mitotic preparations.
The number of chromosomes were found to be
2n=16 in Wardan, BB-2, FAO-5 and Ex-8 while in BB-
3, the number of chromosome was 2n=32. 83
84. Mitotic preparations of different ecotypes of berseem (T. alexandrinum)
BB-3 Wardan BB-2 Ex-8 FAO-5
84
