'Embryo rescue technique in fruit crops'

Embryo rescue is used to rescue immature/mature- lethal embryos and hybrid embryos generated from interspecific and intergeneric crosses (Rog et al., 2023)

1. Doctoral Seminar-II Presented to: Dr. Deep Ji Bhat Seminar Incharge Professor (Division of Fruit Science) Presented by: Pradeepti Sharma Reg. No.: J-22-D-05-HF Ph.D Fruit Science Topic : Embryo rescue technique in fruit crops

2. EMBRYO RESCUE TECHNIQUE IN FRUIT CROPS

3. Index ☟ Introduction Importance History Steps in Embryo Rescue Technique Factors Affecting the Success of Embryo Culture Embryo Rescue Applications Case Studies Future Perspective Conclusion

4. Introduction

5. Embryo rescue is used to rescue immature/mature- lethal embryos and hybrid embryos generated from interspecific and intergeneric crosses (Rog et al ., 2023). Rog et al ., 2023 Embryo rescue

6. History of embryo rescue Charles Bonnet (18 th century) first time documented embryo rescue in Phaseolus vulgaris. Later, many scientists started assessing the process with various nutrient media 1890 to 1904. In 1924, Kurt Dieterich rescued embryo from ( solanaceae , linaceae , brassicaceae ), both mature and immature explants and revealed the differences in nutrient requirements. Friedrich Laibach made first embryo rescue interspecific used this Lilium perenne × Lilium austriacum in 1925. Rog et al ., 2023

7. History of embryo rescue in fruit crops The first successful embryo culture of fruit was achieved by Tukey in 1933 who cultured of cherry embryos. Blake was the first to employ embryo culture in a peach breeding program. Emershad and Ramming was first employ embryo culture in grape 1982. Rog et al ., 2023

8. Steps in embryo rescue technique Selection of ex plant Sterilization Excision of embryo Placing embryo on suitable medium Embryo germination Plantlet formation Peeling of the glumes Li et al ., 2020

9. Factors affecting the success of embryo culture The growth conditions of the mother plant are also a consideration in embryo culture. Schaal et al ., 2019

10. Nurse endosperm transplant

11. Developmental stage of embryo Pramanik et al ., 2021

12. Media Hameg et al ., 2020

13. The barriers in embryo rescue techniques 1. Pre-zygotic barriers in hybridization Niedz et al ., 2016

14. Pollen tubes arrest before the stigma half Ferriol et al., 2023 Pollen tubes arrest at the late stigma or the style

15. Pollen tubes arrest at the distal ovary Pollen tubes arrest at the proximal ovary Ferriol et al., 2023

16. 2. Post-zygotic barriers in hybridization Niedz et al ., 2016 Barriers in embryo rescue technique

17. Flow cytometry Principle :-  Scattering of light and emission of fluorescence which occur when a laser beam hits the cells moving in a directed fluid stream. Xiao et al ., 2014

18. Flow cytometry

19. Diploid (peak=50) Triploid (peak=75) Tetraploid (peak=100) Flow cytometer graph

20. Embryo rescue applications Purugganan et al ., 2019

21. Muralikrishna et al., 2017 Case study: 1

22. Germination percentage of embryo in different media

23. Root and shoot length of embryo in different media

24. Root and shoot length of embryo in different media

25. Case study: 2

26. Yannan et al ., 2023 Embryo rescue breeding of new cold-resistant, seedless grapes

27. Cross combinations Number of embryo developed Number of germination Percentage of germination ‘Flame Seedless’ × ‘ Munake ’ 248 44 17.7 ‘ Jingzaojing ’ × ‘ Beichun ’ 941 161 17.1 ‘Ruby Seedless’ × ‘Shine-Muscat’ 65 39 30 ‘Ruby Seedless’ × ‘ Beibinghong ’ 1088 434 39.9 ‘ Wuhe Cuibao ’ × ‘ Xinyu ’ 61 13 21.3 ‘Melissa seedless’ × ‘ Xinyu ’ 140 31 22.1 ‘ Yuehong Wuhe ’ × ‘SP740 ′ 299 45 15.1 Sum 4369 930 19.6 Embryo rescue of hybrid combination

28. Effect of sampling time on embryo rescue Crosses combinations Sampling dates Embryos developed Number Percentage Yuehong Wuhe × SP740 33 30 22.2 35 27 23.9 37 14 27.5 Ruby Seedless × Shine-Muscat 53 7 38.9 55 19 67.9 57 3 34.5

29. Cross combinations Concentration of sucrose (%) Embryos developed Germination Number Percentage Number Percentage Ruby Seedless × Shine-Muscat 1 22 31.4 11 15.7 1.5 13 16.3 6 7.5 2 7 10 2 2.9 3 3 3.8 1 1.3 Yuehong Wuhe × SP740 1 14 87.5 4 25 1.5 12 75 3 18.8 2 9 56.3 3 6 35.3 1 5.9 Effect of different sucrose concentrations on embryo rescue

30. Sahijram et al., 2013 Case study: 3

31. Hybrid embryo rescue in mango cross Sahijram et al., 2013 1. Amrapali X Kerala Dwarf 2. Alphonso X Kerala Dwarf 3.Arka Anmol X Kerala Dwarf 4. Open-pollinated Totapuri (100%) Harvested after 4-6 weeks

32. S.No . Cross combinations Percentage of embryos forming plantlets in vitro culture Percentage of cultures surviving transplant into mini-pot 1. Amrapali × Kerala Dwarf 67.24 56.80 2. Alphonso × Kerala Dwarf 80.73 80.00 3. Arka Anmol × Kerala Dwarf 82.67 79.77 4. Open-pollinated Totapuri 75.00 50.00 Sahijram et al., 2013

33. Case study: 4

34. Sequential embryo culture in grape breeding Sahijram et al., 2013

35. S.No . Pollen-donor parent used Total number of ovules cultured from the cross Number of fully developed hybrid plantlets 1 Vitis tilifolia 165 28 2 Madras Field Court 134 57 3 SV 12309 58 33 4 SV 12364 189 64 5 Vitis assamica 111 26 6 Open-pollinated Thompson Seedless 37 13 Sahijram et al., 2013

36. Hanae et al ., 2022 Case study: 5

37. Optimization of in vitro embryo rescue for mandarins triploids obtained from diploids crosses Hanae et al ., 2022

38. a: Abnormal, b: Normal Hanae et al ., 2022

39. Small seeds Normal seeds Flat seeds Hanae et al ., 2022

40. Mandarins varieties Length means of seeds mm² Germination rate (%) Abnormally developed Normal Flat Small Ortanique 122.15 70.4 23.9 76.66 Murcott honney 116.25 67.1 26.2 83.73 Nadorcott 122.2 55.8 25.2 91.85 Variant of Murcott (M104) 105 61.2 22.15 89.28 Mean (mm²) 116.40 63.63 24.36 83.58 Seed length and percent germination Hanae et al ., 2022

41. Hanae et al ., 2022 Seedlings from immature embryo rescue of three types of seeds a) normal; b) small; c) flat. (A) Nadocott (B) Variant of Murcott M104 (C) Ortanique (D) Murcott Honney

42. Mandarins varieties Percentage of polyploids Seeds shape Diploids Triploids Ortanique Small 40.54 8.11 Flat 45.95 5.41 Variant of Murcott M104 Small 48.65 8.11 Flat 45.95 Nadorcott Small 56.76 2.7 Flat 35.14 2.7 Murcott Honney Small 54.05 Flat 43.24 2.7 Seed ploidy level Hanae et al ., 2022

43. Hanae et al ., 2022

44. Future thrust Embryo rescue coupled with new technologies like genetic engineering the efficiency and productivity of fruits will be increased ( Akkurt et al., 2013; Bergamini et al ., 2013 ). Helps to incorporate many wild genes which are resistant to diseases, pest and tolerant to climate change. The characteristics of newly germinated plants can be characterized at early stage by the use of markers. Embryo rescue can be used in those crop where number of seed is less and fruit drop is more eg. mango

45. Conclusion Embryo culture is a valuable in vitro tool for breeding. It is most often used to rescue embryos from interspecific and intergeneric crosses and from embryos that do not fully develop naturally. It is useful in understanding embryo morphogenesis.

46. Questions: Define embryo rescue? One application of embryo rescue? Who was the first to discovered embryo rescue technique in fruit crops? What are the factors affecting embryo rescue? Name most common media used in embryo rescue ?

47. Reference: Rog U. et al . (2023) Embryo Rescue in Plant Breeding . Plants . 12, 3106. Li, S.; Liu, K.; Yu, S.; Jia, S.; Chen, S.; Fu, Y.; Sun, F.; Luo, Q.; Wang, Y. The process of embryo abortion of stenospermocarpic grape and it develops into plantlet in vitro using embryo rescue. Plant Cell Tissue Organ Cult. 2020, 143, 389–409 Cisneros A. et al. (2010) 174:73–82. Euphytica DOI 10.1007/s10681-010-0135- Schaal, B. Plants and people: Our shared history and future. Plant People Planet 2019, 1, 14–19 Pramanik, K.; Sahoo, J.P.; Mohapatra, P.P.; Acharya, L.K.; Jena, C. Insights into the embryo rescue—A modern in-vitro crop improvement approach in horticulture. Plant Cell Biotechnol . Mol. Biol. 2021, 22, 20–33 Hameg , R.; Arteta , T.A.; Landin, M.; Gallego, P.P.; Barreal , M.E. Modeling and optimizing culture medium mineral composition for in vitro propagation of Actinidia arguta. Front. Plant Sci. 2020, 11, 55490 Moyle L.C and Jewell C.P .2014. Fertile approaches to dissecting mechanisms of premating and postmating prezygotic reproductive isolation. Curr . Opin . Plant Biol. 18, 16–23