New Breeding Technologies in fruit crops - seminar-Final.pptx

This ppt contain the information about different new breeding technologies in fruit crops along with some case studies for easy understanding

1. 2 25-06-2024 Dept. of FSC

2. 3 25-06-2024 Dept. of FSC

3. 4 25-06-2024 Dept. of FSC

4. Traits associated with perennial fruit crops 25-06-2024 Dept. of FSC 5

5. 25-06-2024 Dept. of FSC 6 Introduction Selection Hybridization

6. 25-06-2024 Dept. of FSC 7 Mutation

7. 25-06-2024 Dept. of FSC 8 New Breeding Technologies

8. Major Advisor Dr. Venkat Rao Associate Professor Department of Fruit Science COH, Bengaluru Speaker Priyanka Hugar UHS22PGD445, II Ph.D. Department of Fruit Science COH, Bengaluru New breeding techniques for fruit crop improvement UNIVERSITY OF HORTICULTURAL SCIENCES, BAGALKOT COLLEGE OF HORTICULTRE, BENGALURU

9. N ew genetic engineering techniques Genome editing, Cisgenesis , Intragenesis , RNAi technique, Speed breeding and Transgrafting (Anonymous, 2017) Generation of stable and heritable genetic modifications What are new breeding technologies (NBTs) 25-06-2024 Dept. of FSC 10

10. 25-06-2024 Dept. of FSC 11 Advantages (Sattar et al., 2021)

11. 25-06-2024 Dept. of FSC 12 2 4 1 5 3 6 Saves time & energy For parthenocarpy, incompatibility problems Lower regulatory hurdles Avoids the linkage drag and increases the specificity Sustainable fruit production Will not cross the species barrier Advantages

12. 1 Gene silencing, k nockout , a ctivation and overexpression 2 Insertion of desired genomic sequence for desired trait 3 Speed breeding 4 Trans grafting Applications Pest and disease resistance Abiotic stress tolerance Quality and nutrition improvement Creation of variability Early flower induction 25-06-2024 Dept. of FSC 13

13. 25-06-2024 Dept. of FSC 14

14. 25-06-2024 Dept. of FSC 15 1. Cisgenesis and intragenesis Cisgenesis Genes from the recipient species or from a cross-compatible species to create natural variant Intragenesis New gene combination created by in vitro assembly of elements isolated naturally isolated from the sexually compatible Limera et al., 2017

15. 25-06-2024 16 E Jacobson F A.Krens H J.Schouten Dept. of FSC Telam et al. (2013) History 2006 “ Cisgenic ” concept by Schouten, Krens and Jacobson 2010 In apple (Gala) Scab resistance gene HcrVf2

16. 25-06-2024 Dept. of FSC 17

17. Crop Type Promoter Gene Trait Authors Apple Expression Own genes MdMYB10 Induces anthocyanin accumulations Krens et al., 2015 Apple Expression Own genes FB_MR5 Increased Resistance to Fire Blight Kost et al., 2015 Grapevine Expression 35S-CMV/35S-CMV + core promoter VVTL-1 Fungal disease resistance Dhekney et al., 2011, Dalla Costa et al., 2016 Strawberry Over expression Own genes PGIP Grey mould resistance Schaart , 2004 Apple Expression Own genes HcrVf-2 Scab resistance Vanblaere et al., 2011 25-06-2024 18 Dept. of FSC Limera et al., 2017 Table 1: Overview of the c isgenesis study in fruits

18. What is public opinion on cisgenic food ? Holme et al .(2013) 25-06-2024 Dept. of FSC (ISB News Report, 2019) 19

19. 25-06-2024 Dept. of FSC 20 Schlatholter et al., 2022

20. 25-06-2024 Dept. of FSC 21 To evaluate the cisgenic line C44.4.146 for resistance, morphological and biochemical characters Breeding Research, Research Division Plant Breeding, Waedenswil , Switzer land Schlatholter et al., 2023 Field study of the fire-blight-resistant cisgenic apple line C44.4.146 Ina Schlatholter, Giovanni A. L. Broggini, Sebastian Streb, Bruno Studer and Andrea Patocchi Case study: 1

21. Dept. of FSC 22 Gala Galaxy cultivar using the cisgene FB_MR5 from wild apple Malus × robusta 5 (Mr5) with binary vector p9-Dao-FLPi After elimination of the selectable markers through heat-induced recombinase, both PCR and Southern blot analysis did not detect any transgenes. The transformed line C44.4.146 carried just the cisgene FB_MR5 and its native regulatory sequence. Background Kost et al., 2015 25-06-2024

22. Royal Gala (RG) Gala Schniga SchniCo red (GSR) The original Gala (GO), In vitro cultured Gala Galaxy (IVG), Gala Galaxy from a commercial nursery in Switzerland (GG) and Italy (GGB) The cisgenic line C44.4.146 Floral fire blight inoculation and analysis E. amylovora EA222_JKI containing 1x10 7 cfu / ml in phosphate-buffered saline Floral clusters were assessed for fire blight symptoms 21 days post-inoculation (dpi) using the harmonized scale Metabolite extraction of fruit tissue and analysis was done using LC–MS/MS analysis Tree, leaves, flower and fruit related phenotypes were studied according to DUS 01 02 03 Material and methods Planting material on M 9 T337 rootstock Dept. of FSC Schlatholter et al., 2023 25-06-2024 23

23. 25-06-2024 Dept. of FSC 24 Score Severity No infection 1 Floral infection 2 Infection in flowers and peduncle 3 Floral and bourse infection 4 Floral, bourse and bourse shoot infection 5 Infection advanced up to 4 cm along the branch Fig.1: Floral fire blight disease scores of the cisgenic line C44.4.146 (CIS) and Gala Galaxy (GG) Schlatholter et al., 2023 Table 2: Harmonised scale described by Peil et al., 2019 228 flower clusters in C44.4.146 219 flower clusters in Gala Galaxy in two seasons

24. 25-06-2024 Dept. of FSC 25 Schlatholter et al., 2023 Out of 44 characters accessed tree habit , extent of anthocyanin coloration from base of the petiole , position of the stigmas relative to anthers , fruit general shape, flesh color were uniform within and between the genotypes Leaf color , Acidity, Seed germination showed no genotypic in any investigated years Table 3: Summary of the statistical analyses of all field phenotypic traits and number of genotype-specific traits

25. 25-06-2024 Dept. of FSC 26 Schlatholter et al., 2023 Between CIS and IVG 6 out of 21 tree characters and 12 out of 23 fruit characters shown some differences in single year but not significantly differed. CIS was out of the variability in some characters like leaf blade length and width, yellow and red over color , intensity of the color . Table Cont.,

26. 25-06-2024 Dept. of FSC 27 Schlatholter et al., 2023 Fig.2: Representation fruit ground color

27. 25-06-2024 Dept. of FSC 28 Schlatholter et al., 2023 Fig.3 : Representation fruit pattern of over color

28. 25-06-2024 Dept. of FSC 29 Fig. 4: Representative photographs of fruits of the genotypes The cisgenic line C44.4.146, Gala Galaxy from a commercial nursery in Switzerland (GG), Italy (GGB), O riginal Gala (GO), Royal Gala (RG), I n vitro cultured Gala Galaxy (IVG) and Gala Schniga SchniCo red (GSR), Schlatholter et al., 2023

29. 25-06-2024 Dept. of FSC 30 Schlatholter et al., 2023 Fig.5: Principal component analysis (PCA) of the fruit peel metabolomes based on differentially abundant features Table 4: Number of differentially abundant features (DAFs) of the metabolomes of fruit peel extracts among the investigated Gala genotypes 2019 2166 & 1482 1, 2, & 19 were absent in RG, GG 2145 & 1417 metabolomes met filtering criteria used for analysis

30. 335 & 1155 333 & 1146 metabolomes met filtering criteria used for analysis 25-06-2024 Dept. of FSC 31 Schlatholter et al., 2023 Fig.6: Principal component analysis (PCA) of the fruit flesh metabolomes based on differentially abundant features Table 5: Number of differentially abundant features (DAFs) of the metabolomes of fruit flesh extracts among the investigated Gala genotypes 2019 INFERENCE

31. 25-06-2024 Dept. of FSC 32 2. RNA interference

32. 25-06-2024 Dept. of FSC 33 Andrew Fire and Craig C. Mellow Shared the 2006 Nobel Prize in Physiology and Medicine for their work on RNAi published in 1998 RNAi R. Jorgesen PTGS Post translation gene silencing in plants discovered by Jorgesen and Napoli in 1990 History

33. 25-06-2024 Dept. of FSC 34 Components needed OR hpRNA Argonaute proteins

34. Dept. of FSC 35 25-06-2024 Mechanism of RNAi

35. Table 6: Application of RNA interference in fruit crops 36 Fruit crop Target gene Source Target trait Reference P.communis MdTFL1 Apple Early flowering induction Freiman et al . (2012) Malus domestica iaaM and ipt Agrobacterium tumifaciens Crown gall resistance Viss et al . (2003) Citrus paradisi CTV Citrus tristeza virus (CTV) CTV resistance Febres et al . (2008) Malus domestica MdMLO19 Apple Powdery mildew resistance Pessina et al . (2016) Malus domestica Endo-polygalacturonase1 PG1 Apple Improve post-harvest fruit quality Atkinson et al . (2012) Malus domestica MdAG -like genes: MdMADS15 and MdMADS22 Apple The reduction of fertility and the increase of Floral Attractiveness Klocko et al . (2016) Malus domestica MdGA20-ox Apple The obtainment of dwarf varieties Zhao et al . (2016) Fruit crop Target gene Source Target trait Reference Prunus domestica PPV-CP Plum pox virus (PPV) Sharka (PPV resistance) Scorza et al . (2013) Citrus sinensis CPsV -CP Citrus psorosis virus ( CPsV ) Citrus psorosis resistance Reyes et al . (2011) Traditional Golden Delicious apple (left) versus the Arctic variety (right).  25-06-2024 Dept. of FSC Honey Sweet variety of plum resistance against PPV

36. 25-06-2024 Dept. of FSC 37 To produce transgenic PPV resistant European plum Startovaya ( P. domestica  L.) by RNAi technology Russia Research Institute of Agricultural Biotechnology, Russian Academy of Sciences, Russia Sidorova et al., 2019 Agrobacterium -mediated transformation of Russian commercial plum cv. Startovaya ( Prunus domestica  L.) w ith virus-derived hairpin RNA construct confers durable resistance to PPV infection in mature plants Tatiana Sidorova, Roman Mikhailov, Alexander Sergeevich Pushin , Dmitry Miroshnichenko and Sergey Dolgov Case study: 2 NAAS: 11.60

37. 38 1. Plant materials 2. Transformation through agrobacterium  strain AGL0 using  binary vector pCamPPVRNAi   4. Selection of transformants by southern blot hybridization and northern blot hybridization 5. Grafting for virus inoculation and analysis of infected plants 6. RT-PCR detection, DAS-ELISA, western blot analysis and immuno strip test Materials and Methods Startovaya 25-06-2024 Dept. of FSC Sidorova et al., 2019 3. Selection of transformants by reporter genes expression hpt gene under dCaMV 35S promoter uidA under CaMV35S and self complementary fragments of PPV CP gene with enh35S promoter

38. 25-06-2024 Dept. of FSC 39 Sidorova et al., 2019 Experiment No of Plants No of explants produced hygromycin resistant calli No of independent PCR positive lines Transformation efficiency (%) Hyg + GUS+ RNAi+ 1 452 28 5 4 5 1.1 2 119 7 1 1 1 0.8 3 102 3 1 1 1 1.0 Total 673 38 7 6 7 1.0 Table 7:Efficiency of the genetic transformation of European plum “ Startovaya ” by the pCamPPVRNAi vector.

39. 25-06-2024 Dept. of FSC 40 Sidorova et al., 2019 Fig.7: Generation and histochemical GUS analysis of putative transgenic plum plants. A) Wild-type B) Agrobacterium-inoculated leaves on the shoot regeneration medium after 2 months of cultivation on the presence of 5 mg/L hygromycin. C) Differentiation of putative transformants from leaf-derived morphogenic callus on selective medium D) The proliferation of putative transformants on elongation medium supplemented with 5 mg/L hygromycin E) & F) GUS staining of the non-transformed (E) and transformed (F) leaf explants. G) & H) GUS staining of the putatively transformed callus (G) and the putatively transformed shoots (H) in the cluster I) Rooting of putative transgenic plum plants rooting medium supplemented with 3 mg/L hygromycin J, K) The rooted plants were growing in soil L) Expression of uidA gene in leaves of independent transgenic plants

40. 25-06-2024 Dept. of FSC 41 Sidorova et al., 2019 Fig. 8A: PCR analysis of the putative transgenic lines Where, M–DNA marker, P- plasmid pCamPPVRNAi , WT–genomic DNA of wild-type, 1to7–DNA of transgenic lines RNAi1, RNAi2, RNAi3, RNAi4, RNAi5, RNAi6, and RNAi7. Fig. 8: Molecular analysis of putative transgenic plum plants.

41. 25-06-2024 Dept. of FSC 42 Sidorova et al., 2019 Fig. 8B: Southern hybridization analysis of transgenic lines to confirm stable integration of T DNA Where, M–DNA marker, WT–genomic DNA of wild-type, P- plasmid pCamPPVRNAi / Xbal , 1to5–DNA of transgenic lines RNAi1, RNAi2, RNAi3, RNAi4, and RNAi6 Fig. 8: Molecular analysis of putative transgenic plum plants. DNA probe corresponding sequence to the hairpin arm & octopine synthase

42. 25-06-2024 Dept. of FSC 43 Sidorova et al., 2019 Fig. 8C: Northern Blot analysis to transgene derived expression Where, M–DNA marker, WT1 and WT2–RNA of wild-type, 1 to 3–RNA of transgenic lines RNAi2, RNAi3 and RNAi4 Fig. 8: Molecular analysis of putative transgenic plum plants. Alkaline phosphatase labelled DNA probe specific to PPV CP

43. 25-06-2024 Dept. of FSC 44 Sidorova et al., 2019 Fig. 9: Analysis of infected transgenic plum plants in the year 2010. Fig. 9A & B: Leaf symptoms caused by  Plum pox virus   Fig. 9C: RT-PCR assay of transformed plum plants infected by PPV  M – DNA marker, C+ –infected grafting, WT – uninfected wild-type , H 2 O – water; Si1 and Si2 – infected wild-type, 1 to 8 – transgenic individuals from RNAi1-1, RNAi1-2, RNAi2-1, RNAi2-2, RNAi3-1, RNAi3-2, RNAi4, and RNAi6.

44. 25-06-2024 Dept. of FSC 45 Fig. 10A,B, C & D: Visual symptoms Fig. 10: Analysis of infected transgenic plum plants in the year 2014.  RNAi2 RNAi6  Infected wild-type Sidorova et al., 2019

45. 25-06-2024 Dept. of FSC 46 Fig. 10: Analysis of infected transgenic plum plants in the year 2014. Fig. 10E: RT-PCR assay Fig. 10F: Western blot analysis M – marker, H 2 O- Water, C+ – infected wild-type , 1 – uninfected wild-type, 2 – RNAi1, 3– infected graft on RNAi1, 4 – RNAi2 , 5 – infected graft on RNAi2, 6 – RNAi3-1, 7 – RNAi4, 8– RNAi6, 9 – infected graft on RNAi6. Sidorova et al., 2019

46. 25-06-2024 Dept. of FSC 47 Fig. 10: Analysis of infected transgenic plum plants in the year 2014. Fig. 10E: RT-PCR assay Fig. 10F: Western blot analysis M – marker; C+ – infected wild-type; WT – uninfected wild-type; 1 – RNAi1; 2 – infected graft on RNAi1; 3 – RNAi2; 4 – infected graft on RNAi2; 5 – RNAi3-1; 6 – RNAi3-2; 7 – RNAi4; 8 – RNAi6; 9 – infected graft on RNAi6. Sidorova et al., 2019 Rabbit polyclonal antibodies Fig. 10G: DAS-ELISA assay

47. 25-06-2024 Dept. of FSC 48 Fig. 10: Analysis of infected transgenic plum plants in the year 2014. Fig. 10 H : Immuno strip assay Sidorova et al., 2019

48. 25-06-2024 Dept. of FSC 49 Sidorova et al., 2019 Fig. 11: Analysis of infected transgenic plum plants in the year 2018. Fig. 11B: DAS-ELISA assay Fig. 11A: Visual symptoms

49. 25-06-2024 Dept. of FSC 50 Lines/Year of assessment 2010 2014 2018   Visual symptoms RT-PCR Visual Symptoms RT-PCR Western blot/ ELISA/ImmunoStrip Visual symptoms ELISA WT − − − − − − − WT infected + + + + + + + Graft on WT + + + + + n.a. n.a. RNAi1 − − − − − − − Graft on RNAi1 + n.d. + + + n.a. n.a. RNAi2 − − − − − − − Graft on RNAi2 + n.d. + + + + + RNAi3 − − − − − − − Graft on RNAi3 + n.d. n.a. n.a. n.a. n.a. n.a. RNAi4 − − − − − − − Graft on RNAi4 + n.d. n.a. n.a. n.a. n.a. n.a. RNAi6 − − − − − − − Graft on RNAi6 + n.d. + + + n.a. n.a. Table 8: Plum pox virus detection in transgenic plum plants Sidorova et al., 2019 INFERENCE

50. 3. Advanced genome editing tools 25-06-2024 Dept. of FSC 51 Due to the difficulties related to the creation of flexible DNA-binding proteins, new tool CRISR/Cas mostly using

51. 25-06-2024 Dept. of FSC NGG It is the adaptive immune system of bacteria ( Streptococcus pyogenes ) and archaea CRISPR/Cas has sgRNA, trcrRNA in (CRISPR/Cas9) and Cas protein The tracrRNA and crRNA can be replaced by an engineered single guide RNA (sgRNA) containing a designed hairpin. CRISPR/Cas 9 52 CRISPR/Cas9 utilizes a 20 bp guide RNA sequence, which binds to its DNA target through Watson–Crick base pairing.

52. Heat treatment after T-DNA integration Transgene elimination techniques 25-06-2024 Dept. of FSC 53 Flippase (FLP) / Flippase recognition target (FRT) Cre- LoxP chemical (dexamethasone, β - estradiol ) Agrobacterium -mediated transformation Particle bombardment/ Biolistics protoplast transformation viral vectors Indirect Direct

53. Table 9: Overview of published studies on CRISPR/Cas genome editing applied to fruit trees. 25-06-2024 Dept. of FSC 54

54. 25-06-2024 Dept. of FSC 55

55. Plant species Citrus sinensis Citrus paradise C. × sinensis × Poncirus trifoliate Citrus maxima Fortunella hindsii Nuclease Target gene Target trait Cas9 delivery method Reference Cas9 PDS Method optimization Cas9-mediated gene knock-out using two sgRNAs Jia and Wang, 2014 Cas9 WRKY22 Resistance to XCC Cas9-mediated gene knock-out using two sgRNAs Wang et al., 2019 Cas9 LOB1 Resistance to XCC Cas9-mediated gene knock-out by multiplex using a polycystronic gRNA system Huang et al ., 2021 Cas9 ß-LCY2 Lycopene / fruit quality Cas9-mediated gene knock-out using two sgRNAs Salonia et al., 2022 Cas9 ALS resistant to the herbicide Cas9-mediated gene knock-out using two sgRNAs Huang et al., 2022 Cas9 DMR6 Resistance to XCC Cas9-mediated gene knock-out by multiplex using a polycystronic gRNA system Parajuli et al., 2022 Cas12 Cas9 LOB1 FhRWP Resistance to XCC Method optimization Cas12-mediated gene knock-out using a crRNA Cas9-mediated gene knock-out by multiplex using a polycystronic gRNA system Jia et al ., 2022 Song et al ., 2023 25-06-2024 Dept. of FSC 56

56. 25-06-2024 Dept. of FSC 57

57. 25-06-2024 Dept. of FSC 58 To develop the β- carotene-enriched Cavendish banana cultivar (cv.) Grand Naine (AAA genome) using CRISPR/Cas9 National Agri-Food Biotechnology Institute and Punjab Agricultural University, Punjab, India Kaur et al., 2020 CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β- carotene biosynthesis in banana fruit Navneet Kaur, Anshu Aloka , Shivania , Pankaj Kumara, Navjot Kaura, Praveen Awasthia , Siddhant Chaturvedia , Pankaj Pandeya , Ashutosh Pandeya , Ajay K. Pandeya , Siddharth Tiwaria NAAS: 14.40 Case study: 3

58. 59 GGPP- Geranylgeranyl pyrophosphate PSY- Phytoene synthase PDS- Phytoene desaturase ZDS- Zeta-carotene desaturase CRISCO- Carotenoid isomerase LCYε – Lycopene epsilon cyclase LCYβ- Lycopene beta cyclase Fig. 12: Carotenoid biosynthesis pathway Kaur et al., 2020 25-06-2024 Dept. of FSC

59. Isolation and in-silico analysis of LCYε sequence from cv. Grand Naine Selection of target sit and the gene construction was done using vector pRGEB31 Banana transformation, selection, and regeneration Genomic sequencing was done to screen the transformants and mutation study 1 2 3 4 Statistical analysis Quantitative estimation of carotenoids and antioxidant activity Plant population were maintained in containment net-house for further evaluation 9 7 6 Materials and Methods 25-06-2024 Dept. of FSC 60 Kaur et al., 2020 T7 endonuclease I assay was done to know the mutation pattern 5

60. 25-06-2024 Dept. of FSC 61 Kaur et al., 2020 Fig. 13: Structural arrangement of GN- LCYε , design of CRISPR construct and target selection Fig. 13A: Exon and Intron display of the GN- LCYε On chromosome no 1 1596 bp 11 exons 10 introns

61. 25-06-2024 Dept. of FSC 62 Kaur et al., 2020 Fig. 13: Structural arrangement of GN- LCYε , design of CRISPR construct and target selection Fig. 13B: The domain analysis of GN- LCYε Protein sequence of gene LCYε gene has all the domains of carotene-cyc1 superfamily Lycopene_cyc1 and FixC as functional domains.

62. 25-06-2024 Dept. of FSC 63 Kaur et al., 2020 Fig. 13: Structural arrangement of GN- LCYε , design of CRISPR construct and target selection Fig. 13C: Construct CRISPR_ GN-LCY ε

63. 25-06-2024 Dept. of FSC 64 Kaur et al., 2020 Fig. 14: Banana cv. Grand Naine transformation with CRISPR_GN- LCYε construct and different stages of tissue culture Embryogenic callus Embryogenic cell suspension culture Cells after transformation without selection Cells on selection medium (20mg/L hygromycin) Regenerated plants on selection medium (30mg/L hygromycin) Acclimatization of plants in soil pots Plant growth under greenhouse Plant growth in net house

64. 25-06-2024 Dept. of FSC 65 Kaur et al., 2020 Fig. 15: Banana (cv. Grand Naine ) transformants screening and mutation analysis of the GN- LCYε edited lines. GN-1 to GN-12 represent putatively transformed lines. +C represents the positive control). Cas 9 specific primer of 197bp Indels are represented in a black background with red-letters/dashes. Cyan color represents the targeted region Fig. 15A: PCR screening of transformants Fig. 15B Sequence analysis showing indels in the edited lines.

65. 25-06-2024 Dept. of FSC 66 Fig. 15: Banana ( cv. Grand Naine ) transformants screening and mutation analysis of the GN- LCYε edited lines. Kaur et al., 2020 Lines with different types of mutations Fragmentation pattern was observed with or without control Fig. 15C: T7 endonuclease assay

66. 25-06-2024 Dept. of FSC 67 Kaur et al., 2020 Fig. 16: Carotenoid analysis in leaf and fruit pulp of the GN- LCYε mutant Grand Naine lines. Fig. 16A: Carotenoid estimation in leaf tissues of the mutant lines. Fig. 16C: Carotenoid estimation in ripe fruit pulp tissue of the mutant lines. Fig. 16B: Visual observation of the fruit pulp color of the ( i ) unedited control and (ii) edited line GN-9 Fig. 16D: Antioxidants 92.75% to 72.91% 46% to 168% Absence to 69.50% GN-9 – 6 folds higher

67. 25-06-2024 Dept. of FSC 68 Plant Bunch filling time (weeks) Fruit length (mm) Fruit diameter (mm) Number of hands (± 1) Total number of fingers (± 16) Fruit weight (g) Bunch weight (g) Control 24 14.30 ± 2.0 26.50 ± 0.46 10 126 83.00 ± 11.11 13.5 ± 2.0 GN-4 23 12.70 ± 1.30 24.40 ± 0.38 7 106 61.67 ± 8.42 07.50 GN-7 24 15.05 ± 1.15 27.72 ± 0.32 10 142 86.33 ± 9.54 12.80 GN-9 24 15.60 ± 2.20 33.17 ± 0.54 10 135 93.33 ± 8.59 13.20 GN-10 25 15.05 ± 1.25 31.62 ± 0.33 11 138 97.90 ± 10.28 14.00 Kaur et al., 2020 Table 10: Phenotypic and agro -morphological data of edited and unedited (control) lines. INFERENCE

68. 25-06-2024 Dept. of FSC 69 4. Accelerated Breeding

69. 25-06-2024 Dept. of FSC 70

70. 25-06-2024 Dept. of FSC 71 41 days Gubbuk et al., 2004 12 days Furukawa et al., 1 990 16 days Kamiloglu et al., 2011

71. 25-06-2024 Dept. of FSC 72 Hussain et al., 2014

72. 25-06-2024 Dept. of FSC 73 Overexpression of Arabidopsis FT gene in apple leads to perpetual flowering Tanaka et al., 2014

73. 25-06-2024 Dept. of FSC 74 Silencing TFL1 gene Charrier et al., 2019 CENTRORADIALIS4 gene Varkonyi-Gasic et al., 2019

74. 25-06-2024 Dept. of FSC 75 Plant species Gene Homolog to Type of expression Early flowering Reference Pyrus communis La France ACO 1 Antisense Yes Goe et. al., 2007 Spadona Conference MdTFL 1.1 TFL1.1 RNAi Yes - Malus domestica Orin MdTFL1 TFL1 Antisense Yes Kodota et. al., 2003 Holsteiner Cox, Pinova , Galaxy MdTFL1 TFL1 RNAi Yes Szankowski et al., 2009 Orin MdFT FT Overexpr Yes Kodota et. al., 2010 Pinova MdFT FT Overexpr Yes Kodota et. al., 2003 Table 11: Genes responsible for early flowering

75. Song et al ., 2015 76 Trans-grafting Trans-grafting refers to grafting a non GE scion with a GE root stock specifically targeted to nutrient uptake or can involve mobile signals that affect the phenotype of the wild-type tissues Traits like growth, early flowering, disease resistance, cold tolerance etc. The rootstock can alter the phenotype of the scion, for example by reducing its vigor and encouraging more fruit set, but the rootstock and scion retain their genetic integrity, in that the grafted tissues are joined but their genetic materials do not mix. 25-06-2024 Dept. of FSC

76. 25-06-2024 Dept. of FSC 77 Hence the reliable technique in fruit crop improvement Sidorova et al., 2021 Cont., The interspecific rootstock Elita was combined with cv. Startovaya ( Prunus domestica L.) as a scion. The elevated siRNA level enabled the resistance to PPV & blocked the virus movement through the GM tissues into the NT The mobile siRNA signal was not moved from the GM rootstock to the target NT tissue to a level sufficient to trigger silencing

77. Non transgenic scion Transgenic rootstock Trait Movement direction Experimental evidence References Sweet cherry ‘Emperor Francis’ Transgenic cherry rootstocks Gisela 6 and Gisela 7 expressing short hairpin RNAs of genomic RNA3 of Prunus necrotic ringspot virus (PNRSV- hpRNA ) PNRSV resistance in transgenic rootstock as well as non-transgenic scion. Rootstock-to-scion Small RNA sequencing and PNRSV tolerance Song et al., 2013; Zhao and Song, 2014 Sweet orange cv. Tarocco Nucellare Transgenic citrange ‘Troyer’ overexpressing rolABC genes of agrobacterium Transgenic rootstock showed increased rooting ability and reduced plant size NA NA La Malfa et al., 2011 Grape Chardonnay 41B ( Vitis vinifera x V . berlandieri ) expressing the coat protein gene of Grapevine fanleaf virus (GFLV) Non-transgenic scions on three out of 16 independent transgenic rootstock lines showed GFLV resistance Rootstock to scion transfer of the overexpressing coat protein of GFLV NA Vigne et al ., 2004 Walnut ( Juglans regia ) cv. Chandler Juglans hindsii , Juglans regia rootstock expressing rolABC Transgenic rootstock showed phenotype changes but did not affect the phenotype of scion NA NA Vahdati et al ., 2002 Table 12: Overview of the transgrafting studies 78 25-06-2024 Dept. of FSC

78. 25-06-2024 Dept. of FSC 79 To introduce the CTV resistance into citrus germplasm using precocious transgenic trifoliate orange and master assisted selection National Agriculture and Food Research Organization of Institute of Fruit and Tea Tree Science (NIFTS), Shizuoka, Japan Endo et al., 2020 Fast-track breeding system to introduce CTV resistance of trifoliate orange into citrus germplasm, by integrating early flowering transgenic plants with marker assisted selection Tomoko Endo, Hiroshi Fujii , Mitsuo Omura and Takehiko Shimada NAAS: 11.3 Case study: 4

79. 80 Plant materials construction of genetic map using 125-BC 1 G enetic background substitution evaluated in BC 2  and BC 3  progenies by CAPS markers Comparative genome hybridization (CGH) analysis to detect the vector construct sequence in BC progenies Evaluation of CTV resistance of BC 2  null segregants by immunological detection method Materials and Methods Transgenic trifoliate orange harboring  P35S:: CiFT  line No. T 2–11  Hyuganatsu Endo et al., 2020 25-06-2024 Dept. of FSC

80. 25-06-2024 Dept. of FSC 81 Hyuganatsu T –2-11 possessed 35S:: CiFT in the genome 32 seeds 136 Seeds 576 Seeds 146 Seeds 1 st flowering in 3 weeks Endo et al ., 2020 Fig. 17: Schematic diagram of fast-track breeding system to introduce CTV resistance into citrus germplasm

81. Table 6: Segregation of CTV resistance and transgene in BC progenies 82 Endo et al., 2020 25-06-2024 Dept. of FSC 82

82. 25-06-2024 Dept. of FSC 83 Fig. 18: Precocious flowering and fruiting of the BC 2 progenies and precocious flowering of the BC 3 progenies around 3 weeks after seed planting Endo et al., 2020

83. 25-06-2024 Dept. of FSC 84 Fig. 19: Genetic linkage map using the BC 1 population The CTV resistance locus T-DNA integrated locus Trifoliate leaf Long thorns Endo et al., 2020 702.5 cM with 73 loci

84. 85 Fig. 20: Graphical genotyping map for the evaluation of the genetic background substitutions using representative BC 2 and BC 3 progenies. Endo et al., 2020 25-06-2024 Dept. of FSC Haplotype blocks comprising P alleles from trifoliate orange

85. 25-06-2024 Dept. of FSC 86 Endo et al., 2020 BC2 – 5 (null segregant) BC2 – 8 (null segregant) BC2 – 19 (Transgenic line) BC3 – 8 (null segregant) BC3 – 10 (null segregant) Fig. 21: CGH analysis to detect the sequence of the T-DNA insertion regions CNT: Non transgenic trifoliate orange

86. Endo et al., 2020 25-06-2024 Dept. of FSC 87 Fig. 23: Evaluation for CTV resistance of null segregates, using Immuno-strip for citrus tristeza virus (CTV) K- Kiyomi scion T- Trifoliate orange H- Hyuganastu 1- BC 2 -4 scion (CTV S) 2- BC 2 -5 scion (CTV R) 3- BC 2 -8 scion (CTV R) 4- BC 2 -12 scion (CTV R) 5- BC 2 13 scion(CTV S) 6- BC 2 14 scion (CTV S) INFERENCE

87. Possibility of off-target issues Multi functionality of gene leads to so unusual phenotype expression Breakdown of resistance Ethical issues Need of whole genome sequence Transformation and regeneration Disadvantages 25-06-2024 Dept. of FSC 88

88. 25-06-2024 Dept. of FSC 89 Regulatory bodies Monitoring The Recombinant DNA Advisory Committee (RDAC) IBSC- Institutional Biosafety committee RCGM- Review Committee on Genetic Manipulation GEAC- Genetic Engineering Appraisal Committee SBCC- State Biotechnology Coordination Committee DLC- District Level Committee Approval Advisory

89. 25-06-2024 Dept. of FSC 90 Future research scope Need to work on transformation and regeneration protocols 1 2 3 4 5 Need more experimental works to prove the safety of the techniques Need of whole genome sequencing in natural resistant sources Need to work on multiple gene functions and editing Need more definite and standardized marker gene-free approaches for future breeders Need to work on public awareness about the safety of the NBTs 6

90. Extensive time required for the classical approaches is a potential threat to the fruit industry. Future positive research outcomes can boost the efficiency of the NBTs. New breeding technologies can dramatically accelerate fruit tree breeding and allow for the rapid global dissemination of traits. Combination of technologies helps in precise and faster results. For example RNAi and trans-grafting, can be combined to achieve the desired results Conclusion

91. 92 The advance of genetic engineering makes it quite conceivable that will begin to design our own evolutionary progress 25-06-2024 Dept. of FSC

92. 25-06-2024 Dept. of FSC 93 Campa et al., 2024

93. 25-06-2024 Dept. of FSC 94 Honey Sweet 2018 in European countries Aractic Granny 2015 US Aractin Golden 2015 US Aractic Fuji 2016 US Aracic Fuji 2018 in Canada

94. 25-06-2024 Dept. of FSC 95 IBKP – Indian Biosaftey Knowledge Portal IBSC - Institutional Biosafety committee RCGM- Review Committee on Genetic Manipulation