PowerPoint Presentation: “ Embryo Rescue Technique: A Tool for Crop Improvement” PowerPoint Presentation: OUTL I NE INTRODUCTION Embryo Embryo culture Embryo rescue History METHOD Embryo culture Ovule culture Ovary culture Utilizaties CASE STUDIES CONCLUSION Introduction: Introduction Embryo culture, sometimes called embryo rescue, is an in vitro technique that has been used to save the hybrid products of fertilization. Breeding – Hybridization and Selection. No offspring does not mean no fertilization took place. Many times formed embryo dies at an early stage due to known or unknown causes. Known as post-fertilization barriers. 1. History: History Plants Observation Scientist Cochleria and raphanus. Viable plants generated from in vitro isolated embryos. Hannig (1904). ==== Skip the stages of normal embryogenesis and without the completion of dormancy period survive plant. Dietrich(1924). Linum perenne and L, austriacum. Crosses without viable seed production, excise their embryo and grew in artificial medium. Laibach (1925, 1929). Datura hybrid Embryos could be grown in culture on media containing coconut milk Van overbeek et al. (1941) 2. PowerPoint Presentation: Important terms Embryo : Bipolar structure, possessing the basic organization of the adult plant. There are two types of embryo culture: 1. Mature embryo culture. 2. Immature embryo culture/embryo rescue. 3. PowerPoint Presentation: The term “embryo rescue” refers to a number of in vitro techniques to promote the development of an inherently weak, immature or hybrid embryos into a viable plant. EMBRYO RESCUE 4. PowerPoint Presentation: The term “embryo rescue” refers to a number of in vitro techniques whose purpose is to promote the development of an inherently weak, immature or hybrid embryos into a viable plant. 5. Cont…. PowerPoint Presentation: Because the endosperm fails to develop properly (Hub and Wang, 1986). In Interspecific crosses, intergeneric crosses, and crosses between diploids and tetraploids, the endosperm often develops poorly or not at all. Need of embryo rescue Embryo abortion occurs due to the endosperm fails to develop properly (Hub and wang, 1986). In interspecific crosses, intergeneric crosses, and crosses between diploids and tetraploids the endosperm often develops poorly or not at all. 6. PowerPoint Presentation: General technique of embryo rescue 7. PowerPoint Presentation: Mostly embryos are located in the sterile environment of the ovule and surface sterilization of embryos is not necessary. Direct disinfection of embryos is needed if seed coats are cracked or if endophytic pathogens exist ex. Corn ( zea mays L.), And dogwood ( cornus spp. L.) Seeds. Small embryos require the use of micro dissecting tools and a dissecting microscope to excise without injury. Embryos are easily damaged when the seed coat is cut; desiccation avoided.( Rang an, 1984). If liquid endosperm surrounds the embryo change in the pressure injure the embryonic tissue. (Hu and wang, 1986). 8. Cont…. PowerPoint Presentation: 9. PowerPoint Presentation: OVULE CULTURE Very young or small-seeded species . 10. Cont…. PowerPoint Presentation: The ovary is surface sterilized and the ovules removed and placed into culture. One technique, ovule perforation , requires making small holes in each ovule just prior to its placement on the culture medium. These perforations, which should be made with care not to damage embryos. Method 11. Cont…. Ovule culture: Ovule culture University of Delhi Papaver rhoeas Maheshwari (1958) Papaver somniferum Mineral salts + Vitamins + Sucrose (5%) Ovules (6DAP) Viable seeds Globular stage Kinetin Maheshwari (1958) and Lal (1961) Ovule culture Cont… 12 PowerPoint Presentation: Ovule support systems 1. The filter paper support system: Culturing ovules on top of filter paper placed over liquid medium . 2. The vermiculite support technique: Placing ovules side down into a sterile vermiculite/liquid media mixture (vermiculite support). 13. Cont…. OVARY CULTURE : OVARY CULTURE Cont…. 14 PowerPoint Presentation: Utilization of embryo rescue Ovary culture 15. Iberis amara mineral salts + sucrose + b- vitamins ovaries (1 dap) fruits Maheshwari and Lal (1961) PowerPoint Presentation: A technique known as ovary-slice culture has been utilized. Ovaries were cut transversely into sections. The basal cut end of the sections placed on the culture medium. Superior of the two techniques because it was less time consuming. Ovary-slice culture 16. PowerPoint Presentation: Factors effecting embryo culture 17. Factors effecting embryo culture: Factors effecting embryo culture Liu 1993 Cont…. 18. Mineral salts : Mineral salts Salts Species K + , CA 2+ (high), NH 4 + (low) Casella monnier medium 1978) NH 4 + Barley, Datura stramonium and D. Tatula NO 3 - Jute Vitamins Not always essential. May even inhibit normal morphogenesis . Cont…. 19. Culture medium: Culture medium Cont…. Media Scientist Murashige and Skoog (MS) media (Murashige and Skoog, 1962) Gamborg’s B-5 media (Gamborg et al ., 1968) White’s media White’s (1934) ph os phate with Glutamine, A lanine, and five other amino acids. (Cameron-Mills and Duffus, 1977) Energy source. Maintaining suitable osmotic potential. Mature embryos 2% to 3% sucrose, immature embryos grow better at 8% to 12%. Sucrose 20. PowerPoint Presentation: Cont…. 21. PowerPoint Presentation: Cont…. Plant Growth Regulators Effect ABA with NH 4 + (ammonical nitrate) Prevents precocious germination of barley immature embryos. Gibberellins Barley immature embryos. Low concentrations of Auxins Somatic embryo induction stimulated. Exogenous Auxins Not effect plant embryo growth in vitro (Norstog, 1979). Cytokinins Not sole hormone butpromote growth with some auxins (Veen, 1963) Medium: Plant Growth Regulators 22. PowerPoint Presentation: Plant sp. Optimal pH Ref. Capsella 5.4 – 7.5 Rijven (1952) Datura tatula (early heart-shaped embryos) 5.0-7.5 Matsubara (1962) Rice (8 day old embryos) 5 and 9 Sapre (1963) Barley (immature embryos) 4.9 Norstog and Smith (1963) PH of Medium Darkness for the first 1 to 2 weeks of culture generally, then transferred to light to allow chlorophyll formation. A high range 25 to 30 0 c is used (Narayanaswamy and Norstog, 1964). Lilium , require a lower temperature, i.e., 17 0 C, and others require a cold treatment of 4 0 C to break dormancy (Pierik, 1987). Temperature and Light 23. PowerPoint Presentation: 24. Cont…. PowerPoint Presentation: Amino Acid Use Scientist Glutamine Mostly used for cultured embryo growth. Monnier, 1978. Asparagine Enhance embryo growth. Hannig, 1904 Casein hydrolysate Widely used to stimulate growth. Burkholder, 1948 Proline, serine, and glutamine Can replace cassin hydrosate. ================ Usually it begins in the late heart-shaped embryo stage (raghavan, 1976). Less (2–3%) sucrose required in media. For interspecific hybrids, it may be useful to develop media that can nurture embryos of one or both parental species. The growth-promoting factor in the coconut milk was referred to as “embryo factor.” B) The Autotrophic Phase 25. Cont…. PowerPoint Presentation: Developmental stage Length of embryo ( m ) Nutritional requirements Early globular 20-60 Unknown for embryos < 40 m Late globular 61-80 Micronutrient salts + vitamins + 2% sucrose + hormones (IAA) Heart-shaped 81-450 Micronutrient salts + vitamins + 2% sucrose Torpedo-shaped 45-700 Macronutrient salts + vitamins + 2% sucrose Walking-stick-shaped and mature embryos 700 & larger Macronutrient salts + 2% sucrose Raghavan (1966) 26. PowerPoint Presentation: Raghavan & Torrey (1963) Sterile capsules in sterile culture medium. A cut in placenta region and halves pulled apart to expose the ovules. Ovule kept in depression of a slide and cut longitudinally. Ovular tissues teased, embryo with suspensor removed. A cut on the side lacking the embryo. Pressure applied with a blunt needle. Technique of embryo excision in Capsella 27. PowerPoint Presentation: Hybrid embryos generally abort at a very early stage. It is difficult to culture very young embryos on an artificial culture medium. Hordeum x Secale Hybrid embryos. Cultured barley endosperm- (an older one is effective) 30-40% survival of hybrid plants vs 1%. Embryo-nurse endosperm transplant technique 28. PowerPoint Presentation: 29. PowerPoint Presentation: Trifolium interspecific hybrids A glass filter is placed in front of the light source. Pods containing hybrid embryos and normal embryos placed in sterile petridishes. One ovule from each pod removed and kept on a moistfilter paper on the sterilized microscope stage. Williams and De Lautour (1980) Modified embryo-nurse endosperm transplant technique 30. PowerPoint Presentation: Williams and De Lautour (1980) 31. Utilization of embryo rescue 1. Obtaining rare hybrids : Utilization of embryo rescue 1. Obtaining rare hybrids Cross between 2 species. Scientist Lilium henryi x L. regale Skirm (1942) L. speciosum-album x L. auratum (embryo-endosperm incompatibility) Emsweller and Uhring (1962) L. esculentum cv. VENT x L. peruvianum cv LA 1283-4 (embryo – callus culture approach) Thomas and Pratt (1981) L. esculentum x L. chilense (embryo – callus culture approach) Scott and Stevens L. esculentum x Solanum lycopersicoides (embryo – callus culture approach) Scott and Stevens Laibach (1929) 32. Table 1. Utilization of embryo rescue Obtaining rare hybrids : Utilization of embryo rescue Obtaining rare hybrids Cross between 2 species. Product Scientist O. sativa x O. australiensis, O. officinalis, O. brachyantha 38-77%, 46-80%, 31-80% Jena and Khush (1984) Tropical japonicas x O. officinalis, O. australiensis, O. grandiglumis, O. punctata Hybrid plants Abdullah and Somantri Cultivated rice x 8 wild rice sp. F 1 s and BC 1 plants Brar, Elloran and Khush V. mungo x V. radiata Hybrid plants Gosal and Bajaj (1983), Verma and Singh (1986) Cont… . 33. Table 2. PowerPoint Presentation: The present results showed that embryo rescue is a potential technique to overcome post fertilization barriers; However, it is no longer effective where abortion of embryo occurs at early stages of development. Therefore, rescue of hybrid embryo should be done . Figure left :7 day old hybrid( O.sativa cv. Kalanamak/O.officinalis ) embryo ready to germinate Figure right: In vitro regenerated hybrid plants, O.sativa cv. Manshara, O.officinalis . Cont… . Interspecific hybrid plants recovered from In vitro embryo rescue in rice . 34. Jena and Khush (1984 ) PowerPoint Presentation: O.sativa Species Seed set Embryos cultured Hybrid plants IR42 O. eichinger 20 18 13 IR64 “ 12 4 1 IR20 O. officinalis 30 6 1 IR70 “ 1 1 1 IR31917-45-3-2 O.rhizomatis 26 19 8 IR20 O. latifolia 64 39 25 IR22 “ 185 44 33 IR24 “ 173 79 36 IR24 “ 40 19 15 IR72 O. ridleyi 3 1 1 IR74 “ 224 40 27 IR31917-45-3-2 “ 128 12 1 IR56 O. granulata 42 2 0 IR31917-45-3-2 “ 45 14 3 IR31917-45-3-2 O.Indandamanica 1 0 0 IR64(4n) “ 26 4 4 Table 1. Interspecific hybrids produced through embryo rescue between cultivated rice ( O. sativa ) and wild species of Oryza. Jena and Khush (1984 ) 35. Table 3. Utilization of embryo rescue : Utilization of embryo rescue Cont… . 37. Utilization of embryo rescue : Cont… . Utilization of embryo rescue 38. PowerPoint Presentation: 39. Cont… . PowerPoint Presentation: Wheat Maize Hybrid zygote Maize Chromosome elimination Merge entire head into 2,4 – D solution. 2,4-D treatment ( 100mM Conc.) Haploid Wheat Haploid embryo Treat with 0.1% colchicine Doubled Haploid Wheat 40. Kaushik et al. PowerPoint Presentation: Utilization of embryo rescue 3).Overcoming seed dormancy 41. Cont… . Utilization of embryo rescue : Seeds of horticultural crops have long Dormancy periods, which may be reduced through embryo culture Iris 2-3 years 1 year Sunflower . 120-150 days 60-75 days. 4).Shortening the breeding cycle Utilization of embryo rescue 42. Cont… . 5. Broadening the genetic base: 5. Broadening the genetic base Chickpea ( Cicer aretinum ) has a narrow genetic base Cicer arietinum x C. bijugum Interspecific hybrids. Embryo rescue Expected to exhibit C. bijugum characters. Utilization of embryo rescue Mallikarjuna (2004) ICRI 43. Cont… . PowerPoint Presentation: 6. Rapid seed viability test Germination of excised embryos of a batch of seeds better than staining methods. Tetrazolium stain. Cont… Utilization of embryo rescue 44. PowerPoint Presentation: 7) . Transfer of disease resistance Utilization of embryo rescue 45. Agrawal (2006) introduced downy mildew resistance in Thompson Seedless and Flame Seedless (grape cv.s ) through embryo rescue technique. PowerPoint Presentation: Arka Manik x Arka Manik (4x) (2x) Micro-cuttings Seedless watermelon (3x) Embryo rescue Micro-cuttings Clonal propagation (10 million plants a year) Utilization of embryo rescue 8). Development of Seedless watermelon 46. PowerPoint Presentation: Sterile seeds not germinate under appropriate conditions e.g. peach, cherry, plum. Sterility may be due to incomplete embryo development, resulting embryo abortion. In some crosses, the transport of water and nutrients to the immature embryo is cut off too soon resulting in abortion, of the embryo. Ex. : Macapuno coconuts. Utilization of embryo rescue 9) Embryo rescue in early ripening stone fruits 47. PowerPoint Presentation: 10.) Clonal Micropropagation Complete plantlets in vitro formed in long-leaved pine ( Pinus palustris ) through embryo culture (Sommer et al. 1975). Followed by plantlet formation and their clonal micropropagation from embryos of P. elliottii, P. radiata, P. regida, P. monticola, P. taeda, P. virginiana and P. sabiniana. 48. PowerPoint Presentation: Case Study 49. PowerPoint Presentation: Nalini and Fred 2005 Cont… 50. PowerPoint Presentation: Chickpea hybridization using in vitro techniques. Cont… Nalini and Fred 2005 51. Transferring genes from tertiary gene pool.: Transferring genes from tertiary gene pool. Cont… Nalini and Fred 2005 52. PowerPoint Presentation: Burun and Esin Oban(2001). Cont…. 54. PowerPoint Presentation: Media Sterilization Method Number of Embryos Cultured Infection of Embryos Contamination (%) Plantlet Development from Embryos (%) RC II 46 2 4.3 95.7 III 46 - 0 78.3 MS II 50 1 2 95.9 III 50 - 0 92.0 Table. Effect of sterilization with Na-hypochlorite supported with antibiotics or sterilant HgCl2 on the plantlet development in RC and MS media. Burun and Esin Oban(2001). Sterilization with sodium hypochlorite followed by treatment of antibiotic (sterilization method II). Sterilization with HgCl2 (sterilization method III). Randolph and cox medium (RC) Murashige and skoog (ms). 55. Table 4. PowerPoint Presentation: Cont…. Romero et al (2006) 56. In vitro rescue of immature avocado (Persea americana Mill.) embryos: In vitro rescue of immature avocado (Persea americana Mill .) embryos Cont…. Romero et al (2006) 57. Effect of embryo developmental stage on germination on solid M1 medium was not significantly affected by embryo developmental stage.: Effect of embryo developmental stage on germination on solid M1 medium was not significantly affected by embryo developmental stage. Embryo size (mm ) Germination (%) 4.0 5.48 10.5 10.17 16.5 11.76 20.5 5.26 25.5 0 30.5 0 40.0 0 4.0 5.48 10.5 10.17 Romero et al (2006) Germinated embryos of smaller sizes (4–10.5 mm) developed only shoots while complete germination (development of shoot and root) was only observed in 20.5 mm long embryos. 58. Cont…. Table 5. PowerPoint Presentation: Cont…. Niroula et al 2005 59. Interspecific hybrid plants recovered from In vitro embryo rescue in rice.: Interspecific hybrid plants recovered from In vitro embryo rescue in rice. Cont…. Niroula et al 2005 60. PowerPoint Presentation: Parental combination Seed set Seeds without embryo No. of embryos cultured Germination % Died after germination No. of seedlings grown Hardening No. of F1 plants obtained Crossability % IR 72 / O . officinalis 36 21 15 20.00 3 - - - 0 IR 64 / O . officinalis 11 8 3 66.67 2 - - - 0 Manshara / O . officinalis 44 24 20 55.00 6 5 5 4 1.97 Pokhreli / O . officinalis 26 13 13 61.54 4 4 4 3 1.72 Jethobudo / O . officinalis 10 2 8 37.50 3 - - - 0 Jethobudo / O . granulata 75 60 15 53.33 7 1 1 - 0 Kalanamak / O . officinalis 60 42 18 33.33 2 4 4 4 1.99 Table : Results of embryo rescue and crossability among O. sativa, O. officinalis, and O. granulata Niroula et al 2005 61. PowerPoint Presentation: 62 Conclusion: Embryo rescue procedures have been successfully used for producing interspecific and intergeneric hybrids and progeny of other incompatible crosses. It leads to successful application of this highly valuable in vitro procedure to additional crop species. The method can be useful to rescue seedless triploid embryos, produce haploids, overcome seed dormancy, determining seed viability, understanding embryo morphogenesis and precocious germination. Success application of the technique depends on the procedure we follow, therefore continued efforts are required for crop improvement by using wild relatives.