Abelmoschus esculentus (L.) Moench Family : Malvaceae

1. s

2. Doctoral seminar II
ADVANCES IN BREEDING FOR YVMV RESISTANCE
IN OKRA

3. OKRA
Abelmoschus esculentus (L.) Moench
Family : Malvaceae
In India
Area : 0.53 million ha.
Production: 6.36 million tonnes
Productivity : 11.9 tonnes/ha
Okra is constituent for balance food due to its dietary fibers and amino acid. It also contains
iron, calcium, manganese and vitamins A,B,C and K ( USDA National Nutrient Database,
2016)
NHB database 2015

4. Okra yellow vein mosaic virus disease
• 1924 : 1 𝑠𝑡 reported by Kulkarni from India
• 1940 : named as “yellow vein mosaic of okra”
by Uppalet al.
• Epidemic where okra grown
• Feb-March no infection
• Only rainy season crop infected
• Yield loss : 50-94%
• Begomovirus - Geminiviridae - circular
single stranded DNA genome
Diseased plants showed a reduction of 24.9% in Plant ht., 15.5% in root length, 32.1% in
no. fruit/plant and 16.3% in stem girth (sheikh et al. 2013)

5. Electron microscopic view of twinned icosahedral begomovirus particle
Monopertite or bipertite single stranded DNA
Genome organization of Begomoviruses

6. Begomovirus associate with yellow vein mosaic disease of okra

7. SYMPTOMS
Homogenous interwoven network of yellow veins
Plants stunted, fruit become deformed, small and tough

8. YVMV vector
1-2 mm in length
Feed and lay egg on
leaves undersurface
Wide host range

9. White fly (Bemesia tabaci): Life cycle

10. Breeding Approaches
Conventional techniques
1. Evaluation and selection
2. Back crossing
3. Interspecific Hybridization
4. Heterosis breeding
5. Mutation breeding
Advance techniques
1. CRISPR/Cas9 system
2. PTGS system
3. Marker assisted selection
4. Marker assisted backcross breeding
5. Marker assisted gene pyramiding

11. Conventional approaches
Evaluation and selection1

12. Evaluation of okra germplasm for their reaction to whitefly
(Bemisia tabaci) and okra yellow vein mosaic virus (OYVMV)
Grade Reaction PDI (%)
0 Immune 00
1 Highly resistant 1-10
2 Moderately resistant 11-25
3 Tolerant 26-50
4 Moderately susceptible 51-60
5 Susceptible 61-70
6 Highly susceptible 71-100
Scale for scoring the disease reaction
Manjua et al., 2018

13. Reaction of okra germplasm against YVMV in okra under field
conditions during 2015 and 2016
Grade Reaction Genotypes
0 Immune IC344558, PSRJ-12952, RJR-124
(0.00%), (0.00%), (0.00 %)
1 Highly resistant NIC9402, IC433597, IC141020, IC433667 and IC433438
2 Moderately resistant None
3 Tolerant NSJ-401
4 Moderately susceptible IC90402, RJR-45, RJR-110, RJR-670, EC305736,
EC305672, RJR-405, RJR-479 and EC305619
5 Susceptible Parbhani Kranti , RJR-587 (63.00%), RJR 279 (65.49%),
and RJR-265 (67.16%)
6 Highly susceptible PSRJ 13040 (84.16%), RJR-193 (83.33%) and Pusa Sawani
(75.16%)
Manjua et al., 2018

14. Screening of okra germplasm against YVMV during rainy season
2015 at VRC, Pantnagar
Grade Reaction Genotype
0 Highly
resistant (2)
EC 169430 & EC 169435
2 Moderate
Resistant (11)
Arka Anamika (C), VRO-5 (C), VRO-6 (C), EC 169400,
EC 169408, IC 093591, IC 093655, EC 169506, IC
117245, IC 117 351 and IC 117355
4 Susceptible
(5)
IC 117313, IC 117123, IC 117328, Pusa Sawani (C) and
Parbhani Kranti (C)
5 Highly
susceptible
Remaining 167 genotypes showed highly susceptible
Singh, 2018

15. Inheritance study of YVMV resistance in okra
S.
No
.
Resistant
parent (R)
Susceptible
parent (S)
Cross Gene action/ Remarks References
1 A. manihot (L.)
Medik and
A. manihot (L.)
Medik ssp.
manihot
A.
esculentus
cv. Pusa
Sawani
F2, BC, and subsequent
generations
Single dominant gene Jambhale and
Nekar, 1981
2 A. manihot ssp.
manihot
Different generations Two dominant genes Sharma and
Dhillon 1983
3 A. manihot A.
tetraphyllus
Different generations Single dominant gene Dutta 1984
4 A. manihot (L.)
Medikus ssp.
manihot
A.
esculentus
cv. Hisar
Unnat
Different generations Two complimentary
dominant
genes
Sharma and
Sharma 1984;
Dhankhar et
al. 2005
5 BCO-1 and
VNR Green
Pusa
Sawani and
Arka
Anamika
Six generations (P1, P2, F1, F2,
BC1,
BC2) of Tolerant × Tolerant
(T×T),
Tolerant × Susceptible (T×S) and
Susceptible × Susceptible (S×S)
crosses
Two duplicate dominant
genes in
T×T, and 02
complementary
dominant genes in T×S
cross
Seth et al.
2017

16. A. esculentus
(2n=130)
X A. manihot
(2n=66)
F1
F2
F3
X A. manihot
(2n=66)
A. esculentus
(2n=130)
X
BC1 BC2
A. esculentus
(2n=130)
X A. manihot ssp.
manihot (2n=194)
F1
F2
F3
X A. manihot ssp.
manihot
(2n=194)
A. esculentus
(2n=130)
X
BC1
BC2
Jambhale and Nerkar (1981)

17. Generation Resistant Susceptible Total Ratio X2
A. esculentus cv. ‘Pusa
Sawani’
50 50
A. manihot 50 50
F1 50 50
F2 66 16 82 3:1 1.317
BC1 76 68 144 1:1 0.444
BC2 27 16 43 1:1 2.813
Segregation pattern for YVMV resistance in the cross: A. esculentus X A. manihot
Segregation pattern for YVMV resistance in the cross: A. esculentus X A. manihot ssp. manihot
Generation Resistant Susceptible Total Ratio X2
A. esculentus cv. ‘Pusa
Sawani’
50 50
A. manihot 50 50
F1 50 50
F2 180 51 231 3:1 1.052
BC1 41 37 78 1:1 0.205
BC2 31 38 69 1:1 0.710
Jambhale and Nerkar (1981)

18. Backcrossing2

19. 3 Interspecific Hybridization

20. A. ficulneus A. manihot A. moschatus A. crinitus
A. tuberculatusA. esculentus
A. angulosus var.
purpureus
A. angulosus var.
grandiflorus
Wild sources of resistance

21.  The A. esculentus var. MI 7 were crossed with wild species namely A.
angulosus
 The cultivated variety MI 7 was susceptible to YVMV disease, whereas wild
species A. angulosus was completely resistance to this disease.
 MI 7 was selected as female and A. angulosus was selected as male.
Samarajeewa et al., 2007

22. A. esculentus var. MI 7
(♀)
Highly susceptible to
YVMV disease
A. angulosus
(♂)
Highly resistant to YVMV
disease
X
F1XMI 7
B1F1
B1F2
Three back cross plants and their self
progeny were maintained in the PGRC field
The F1 plants were
raised in the pots in
green house and
backcrossed with MI 7
Samarajeewa et al., 2007

23. Screening of parent and progeny lines for virus resistance was done by both graft
inoculations and vector transmission
MI 7 was maintained in the field as virus reservoir
Two to three months old plants were graft inoculated using about 2cm long scions
obtained from YVMV infected plants
Four weeks after grafting plants were observed for disease symptoms and
development of yellowing in veins was recorded
All the three backcross (B1F1) and and 11 plants of (B1F2) showed field resistance
to YVMV disease.
Due to rainy weather condition graft inoculation were not successful hence require
further evaluation.
Samarajeewa et al., 2007

24. Molecular analysis
Preliminary work was carried out
to identify potential RAPD on
parents and their progenies.
Out of the 6 primers tested,
OPCO2, OPA10, OPC10, OPM10
and OPD20 showing unique band.
These unique bands were present
in all segregating individuals
which showed field resistance to
YVMV
Samarajeewa et al., 2007

25. Heterosis breeding4

26. Evidence of Economic Heterosis and Genetic Control of Fruit Yield
and Yellow Vein Mosaic Virus Disease Severity Traits of Okra
Genotypes Species
BCO-1 A. esculentus
VNR Green A. esculentus
VRO-6 A. esculentus
11/RES-6 A. esculentus
10/RES-6 A. esculentus
10/RES-4 A. esculentus
Pusa Sawani A. esculentus
Arka Anamika A. esculentus
IC-140950 A. manihot
IC-433483 A. caillei
Crossing programme: Half-diallele
Eight cultivated spp. and two wild
spp.
45 F1 hybrids
Standard Check: Shakti & Ambatika (hybrid check)
Seth et al., 2016

27. Parents BCO-1 VNR
Green
VRO-
6
11/RES
-6
10/RE
S-6
10/RE
S-4
Pusa
Sawani
Arka
Anami
ka
IC-
140950
IC-
433483
NFPP 25.07 12.17 13.33 16.80 16.40 18.33 5.63 6.47 27.57 7.67
FYPP 177.53 113.88 111.09 170.00 111.80 108.79 48.46 55.18 32.64 95.43
PDI (%) 9.56 17.25 60.23 14.40 45.86 55.40 74.29 64.02 27.72 25.46
Mean (per se) performance of 10 parents of okra
Characters Heterosis% over Shakti Heterosis% over Ambatika GCA effects SCA effects
NFPP BCO-1 ×ArkaAnamika
(48.41%**)
BCO-1 ×ArkaAnamika
(23.03%**)
BCO-1 (2.83** ),
Arka Anamika (-0.64**),
VRO-6 (0.55** ),
11/RES-6 (1.29** )
8.61** (23.40)
VRO-6 × 11/RES-6
(43.55%**)
VRO-6 × 11/RES-6
(25.55%**)
8.21** (22.63)
FYPP BCO-1 ×ArkaAnamika
(4.62%**)
BCO-1 ×ArkaAnamika
(17.59%**)
BCO-1 (34.44**),
Arka Anamika (2.15*),
VRO-6 (14.61**),
11/ RES-6 (21.44**)
108.42**
(257.21)
VRO-6 × 11/RES-6
(2.35%)
VRO-6 × 11/RES-6
(12.00%**)
96.73**
(245.0)
PDI (%) BCO-1 ×ArkaAnamika
(-71.28%**)
BCO-1 ×ArkaAnamika
(-72.28%**)
BCO-1 (-19.20**),
Arka Anamika (6.54**),
VRO-6 (10.82**),
11/ RES-6 (-14.28**)
-22.39**
(7.23)
VRO-6 × 11/RES-6
(-54.24%**)
VRO-6 × 11/RES-6
(-55.82%**)
-27.30**
(11.52)
Selected crosses with high standard heterosis (%), their corresponding gca and sca effects
Seth et al., 2016

28. Seth et al., 2016

29. Correlation comparison matrix between disease causing variables and fruit yield among tolerant
and susceptible crosses of okra.
Parameter Fruit yield per plant
(g)
PDI (%) of YVMV
disease
Average whitefly
population per leaf
Fruit yield per plant
(g)
1.000 -0.976** -0.972**
PDI (%) of YVMV
disease
1.000 0.940**
Average whitefly
population per leaf
1.000
Tolerant/Susce
ptible
Crosses
Number of
plants
inoculated with
whiteflies
Plants infected
at 30
days after
inoculation
Plants infected
at 45
days after
inoculation
Plants infected
at 60
days after
inoculation
Percentage of
plants infected
at 60 days after
inoculation
BCO-1 x Arka
Anamika
20 0 0 1 5.00
VRO-6 x
11/RES-6
20 0 1 3 15.00
BCO-1 × VRO-
6
20 5 11 16 80.00
Per cent infection of YVMV disease in tolerant/susceptible hybrids after cross inoculation.
Seth et al. 2016

30. Mutation breeding
• Not much significant work has been reported in improvement of okra through this
breeding method, due to heavy odds against it.
• Till date, two varieties have been developed through mutation breeding.
MDU1
• Evolved by the TNAU,
Coimbatore in 1978.
• Induced mutant isolated from
Pusa Sawani
• Stem is green with light purple
pigmentation.
• Fruits are light green, about 20
cm long.
• Notified by the Central Seed
Committee in 1985
EMS-8(Punjab 8)
• Developed by PAU, Ludhiana in
1989
• Induced mutant derived from
Pusa Sawani treated with 1%
EMS.
• Plants are tall, with purple
pigmentation on the stem,
petioles, and basal portion of the
lower surface of the leaves.
• Fruits medium long, thin, tender,
green and 5-edged.
• Field resistant to YVMV
5

31. Advance techniques

32. Engineering Plants for virus Resistance with
CRISPR/Cas9 System
• Clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR-associated 9
(Cas9) is a prokaryotic molecular immunity system against invading viruses and has been
harnessed as a powerful tool for targeted genomic editing/Silencing.
• For targeted modification of genomic sites, transgenic production of a single guide RNA
(sgRNA) provides specificity to the Cas9 endonuclease, allowing targeted cleavage of
specific DNA sequences in eukaryotic cells.
• Recent studies demonstrated that the CRISPR/Cas9 system could be harnessed to confer
resistance against viruses in plants by using sgRNAs designed to target viral genomic
DNAs.
Host Source Target virus Reference
Nicotiana benthamiana Bean Bean yellow dwarf virus (BeYDV) Baltes, N. et al. (2015)
N. benthamiana Beet Beet severe curly top virus (BSCTV ) Ji, X. et al. (2015)
N. benthamiana Tomato Tomato yellow leaf curl virus
(TYLCV)
Ali, Z. et al. (2015)
N. benthamiana Merremia Merremia mosaic virus (MeMV) Ali, Z. et al. (2015)
N. benthamiana Beet Beet curly top virus (BCTV) Ali, Z. et al. (2015)
1

33. CRISPR/Cas9 system
• The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Type II system is a
bacterial immune system that has been modified for genome engineering.
• If a viral infection threatens a bacterial cell, the CRISPR immune system can thwart the attack by
destroying the genome of the invading virus.
• CRISPR consists of two components:
1) guide RNA (gRNA)
2) CRISPR-associated endonuclease (Cas9)

34. Engineered CRISPR/cas9 system

36. PTGS as mean to achieve virus resistance
• Post transcriptional gene silencing and RNA interference (PTGS/RNAi) is another strategy to create
viral disease resistance in plants.
• In cross-protection, an initial viral infection generates siRNAs species which provide immunity to
further viral attack.
• Cleavage of (ds RNA) into (si RNAs) of 21-25 nucleotides. It is catalyzed by Dicer, an RNAse III type
enzyme.
• Then these si RNAs guide an RNA induced silencing complex (RISC) to destroy (ssRNA).
• As a result the virus cannot proliferate in the host.
Transgenic Target virus Reference
Tomato Potato spindle viroid Schbind et al. (2008)
Cassava African Cassava mosaic virus (ACMV) Vanderschuren et al. (2009)
Tobacco Tobacco rattle virus (TRV) Xie et al. (2004)
Turnip Turnip crickle virus (TCV) Xie et al. (2004)
Cucumber Cucumber mosaic virus (CMV) Xie et al. (2004)
Cabage Cabage leaf curl virus (CaLCV) Xie et al. (2004)
Turnip Turnip mosaic virus (TMV) Jan et al. (2006)
2

37. 3

38. Markers
Markers can also be used in MAS programs.
RFLP, SSR, RAPD, AFLP, SCAR, and SNP
For efficient MAS:
Small amount of DNA required
Repeatability of results
High rate of polymorphism
Occurrence throughout the genome
Codominance

39. Markers used in okra
Genetic diversity studies in okra using various molecular marker

40. Marker-assisted backcrossing (MAB)
 MAB has several advantages over conventional backcrossing:
Effective selection of target loci
Minimize linkage drag
Accelerated recovery of recurrent parent
4

41. Marker Assisted Gene Pyramiding
Widely used for combining multiple disease resistance genes for
specific races of a pathogen
Pyramiding is extremely difficult to achieve using conventional
methods
Consider: phenotyping a single plant for multiple
forms of seedling resistance – almost impossible
Important to develop ‘durable’ disease resistance against different
races
5

42. Process of combining several genes, usually from 2
different parents, together into a single genotype

43. Achievements
66

44.  Variety identified through XXXII
AICRP (VC) group meeting held
at IGKV, Raipur
 Yield : 150-155 q/ha
 Resistance to YVMV and ELCV
both under field condition
Kashi Vardaan (VRO 25)

45. VRO-115
 Advance breeding line of okra
 Yield : 170-180 q/ha
 Resistance to both YVMV and ELCV.

46. IC 117090 : Nine ridges okra accession
 Screening 1225 okra accession ‘IC 117090’ nine ridges has been identified
 Tolerance to both YVMVand OELCV

47. Pusa A 4
Released in 1995 by
(SVRC, New Delhi)
Resistant to yellow vain
mosaic virus, tolerant to
aphids and jassids; fruits dark
green, 12-15 cm long; first
picking after 45 days.

48. Parbhani Kranti
A. esculentus cv. ‘Pusa Sawani’ X A. manihot
F1XA. esculentus cv. ‘Pusa Sawani’
BC1A. esculentus cv. ‘Pusa Sawani’X
BC2
F8
Evolved by N. D. Jambhale and Y. S. Nerkar in
1985. It was carrying resistance to YVMV.
Now it is susceptible to this disease. It produces
yield 85-90q/ha. in summer and 115q/ha during
rainy season.

49. • A esculentus X A manihot
ssp. teraphyllus
• Resistance to YVMV
• Evolve by O.P. Dutta in 1984
• Yield: 115q/ha
Arka Anamika

50. Released by IIHR, Bangalore to YVMV. It is siter line of Arka Anamika. The plants
resemble Arka Anamika in appearance as well as YVMV resistance.

51. Pusa Sawani
Pusa Makhmali X IC-1542 (field resistance to YVMV)
• Evolved by H. B. Singh in 1957-58
• Yield: 100q/ha

52. Punjab Padmini
A. esculentus
cv. ‘Reshmi’
X A. manihot ssp.
manihot cv. Ghana
F1
A. esculentus cv.
Pusa Sawani
X A. manihot ssp.
manihot cv. Ghana
F1
F2
X
F8

53. Varieties developed from IIVR
Variety Resistant to Breeding method
Shitla Uphar YVMV Heterosis Breeding
Shitla Jyoti YVMV Heterosis Breeding
Kashi Bhairav YVMV Heterosis Breeding
Kashi Mahima YVMV Heterosis Breeding
Kashi Mohini YVMV Selection
Kashi Mangali YVMV Selection
Kashi Vibhuti YVMV Pedigree Selection
Kashi Pragati (NIC 9303 X PK 20) YVMV Pedigree Selection
Kashi Satdhari (PK X IC 111542) X IIVR20 YVMV Pedigree Selection
Kashi lila YVMV Pedigree Selection
Kashi Kranti(VRO-6 X 161012) YVMV Pedigree Selection

54. 67
2

55. Identification of new resistant sources
Pyramiding of genes along with major and minor QTL can
provide a stable resistance.
Use of molecular approaches along with the developmental
technology could help to combat the YVMV in okra.
68

56. 69

57. 69

58. X
IC 117328-X
Pant Okra 1

59. X
Pant Okra 1
IC 117351

60. X
IC 093655
Pant Okra