ADVANCES IN BREEDING FOR YVMV RESISTANCE IN OKRA

Abelmoschus esculentus (L.) Moench
Family : Malvaceae

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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 : reported by Kulkarni from India 1940 : named as “yellow vein mosaic of okra” by Uppal et 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. Conventional approaches Evaluation and selection 1

11. Evaluation of okra germplasm for their reaction to whitefly ( Bemisia tabaci ) and okra yellow vein mosaic virus (OYVMV) Grade Reaction PDI (%) 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

12. Reaction of okra germplasm against YVMV in okra under field conditions during 2015 and 2016 Grade Reaction Genotypes 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

13. Screening of okra germplasm against YVMV during rainy season 2015 at VRC, Pantnagar Grade Reaction Genotype 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

14. 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

15. e sculentus (2n=130) X m anihot (2n=66) F1 F2 F3 X m anihot (2n=66) e sculentus (2n=130) X BC1 BC2 e sculentus (2n=130) X m anihot ssp. manihot (2n=194) F1 F2 F3 X m anihot ssp. manihot (2n=194) e sculentus (2n=130) X BC1 BC2 Jambhale and Nerkar (1981 )

16. 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)

17. Backcrossing 2

18. 3 Interspecific Hybridization

19. A. ficulneus A. manihot A. moschatus A. crinitus A. tuberculatus A. esculentus A. angulosus   var. purpureus A. angulosus   var. grandiflorus Wild sources of resistance

20. 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

21. A. esculentus var. MI 7 ( ♀ ) Highly susceptible to YVMV disease A. angulosus ( ♂ ) Highly resistant to YVMV disease X F 1 X MI 7 B 1 F 1 B 1 F 2 Three back cross plants and their self progeny were maintained in the PGRC field The F 1 plants were raised in the pots in green house and backcrossed with MI 7 Samarajeewa et al., 2007

22. 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 v eins 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

23. 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

24. Heterosis breeding 4

25. 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

26. Parents BCO-1 VNR Green VRO-6 11/RES-6 10/RES-6 10/RES-4 Pusa Sawani Arka Anamika 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

27. Seth et al ., 2016

28. 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/Susceptible 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 1 5.00 VRO-6 x 11/RES-6 20 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

29. 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

30. Advance techniques

31. 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

32. CRISPR/Cas9 system The C lustered R egularly I nterspaced S hort P alindromic R epeats (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)

33. Engineered CRISPR/cas9 system

34. 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 siRNA s 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

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36. 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

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

38. 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

39. 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

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

41. Achievements 66

42. 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)

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

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

45. 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.

46. Parbhani Kranti A. esculentus cv. ‘ Pusa Sawani ’ X A. manihot F1 X A. esculentus cv. ‘ Pusa Sawani ’ BC1 A. 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.

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

48. 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.

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

50. Punjab Padmini A. e sculentus cv. ‘ Reshmi ’ X A. m anihot ssp. m anihot cv. Ghana F1 A. e sculentus cv. Pusa Sawani X A. m anihot ssp. m anihot cv. Ghana F1 F2 X F8

51. 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

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53. 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. Future line of work 68

54. THANK YOU 69

55. THANK YOU 69

56. X IC 117328-X Pant Okra 1

57. X Pant Okra 1 IC 117351

58. X IC 093655 Pant Okra