EMBRYO CULTURE.pptx

Embryo culture and it's significance, introduction about embryo culture, types of embryo culture, mature embryo culture, immature embryo culture, procedure of embryo culture, technique of embryo culture, significance of embryo culture, application for embryo culture.

1. EMBRYO CULTURE AND ITS SIGNIFICANCE

2. INDEX INTRODUCTION EMBRYO CULTURE MATURE EMBRYO CULTURE IMMATURE EMBRYO CULTURE SIGNIFICANCE CONCLUSION

3. INTRODUCTION Embryo culture is a technique used for more than half a century to save hybrid fertilization products when they could otherwise degenerate. Success was first achieved in 1904 by Hanning who obtained viable plants from mature embryos of two cruciferous plants which were aseptically isolated and grown on a mineral salt medium supplemented with sugar ( Norstog , 1979). Dietrich cultivated mature and immature embryos of various plant species to determine if they could still germinate without ending the dormant period in 1924. He reported that the mature embryos grew immediately, by passing dormancy. Immature embryos germinated early without further embryonic development. Since the early 1940s, embryo culture has been used increasingly to understand the physical and nutritional requirements for embryonic development, bypass seed dormancy, shorten the breeding cycle, test seed viability, provide material for micro propagation, and rescue immature hybrid embryos from incompatible crosses ( Hu and Wang, 1986). In this study learn about the type, requirements for success, media, technique and significance of embryo culture.

4. EMBRYO CULTURE Embryo culture involves isolating and growing an immature or mature zygotic embryo under sterile conditions on an aseptic nutrient medium with the goal of obtaining a viable plant. The basic principle of this technique is that the integrity of the hybrid genome is preserved in an embryo that is stunted or aborted and that its potential for resumption of normal growth can be realized if it is fed with the appropriate growth substances. It is based on isolating the embryo without injury, formulating an appropriate nutrient medium and inducing embryogenic growth and continuous seedling formation. Embryo culture is of two types.

5. Mature embryos culture The culture of mature embryos from ripened seeds is used to eliminate seed germination inhibitors or to shorten the breeding cycle if, for example, dormancy is a problem. This culture is easy and only requires a simple nutrient medium with agar, sugar, and minerals. Immature embryo culture The culture of immature embryos is used to rescue embryos that would normally abort or that would not undergo the progressive sequence of ontogeny. This process is difficult due to the tedious dissection necessary and the complex nutrient medium requirements. Success with this type of culture depends strongly on the

6. developmental stage of the embryo when it is isolated ( Monnier , 1976; Raghavan , 1980). Culture technique for Embryo Rescue: the aseptically isolated of embryos can be grown in a suitable medium under optimal conditions. In general, a complex nutrient medium is required for culture methods involving embryo rescue. For adequate nutritional support of immature embryos, embryo-endosperm transplant it used in the figure below. Figure1 Embryo-endosperm transplant technique used in embryo rescue (or immature embryo culture)

7. Figure1 Embryo-endosperm transplant technique used in embryo rescue (or immature embryo culture)

8. Requirements for success The successful development of an embryo depends on many factors. The plant genotype greatly influences success. Embryos of some species are easier to grow in culture than are others, and differences sometimes occur between closely related cultivars (Collins and Grosser, 1984; Rangan , 1984). Light and temperature are two environmental factors that are of major concern in embryo culture. Embryos sometimes grow best when maintained in darkness for the first 1 to 2 weeks of culture and then transferred to light to allow chlorophyll formation. Isolated embryos frequently germinate in a wider temperature range than the intact seeds. The optimum temperature depantds on plant species,but normaly a high range of 25 to 30 C is used ( Narayanaswamy and Norstog , 1964). Some embryos, from species such as Lilium , require a lower temperature, i.e.,17 C,and others require a cold tretment of 4 C to break dormancy(Pierik,1987).

9. Media In 1924, Dieterich showed that mature embryos could grow normally on a semisolid medium containing only Knop’s minerl salts and 2.5% to 5% sucrose. However, many scientists believe that the most important aspects of embryo culture is medium selection. Several formulations of mineral salts have been used for embryo culture without much critical evaluation of the role of individual elements ( Bhojwani and Razdan , 1983). Murashige and Skoog (1962) and Gamborg’s B5 medium, with certain degrees of modification,are the most widely used basal media in embryo culture. The exact nutritional requirements depend on the stage of the embryo development. Raghavan identified two phases of the embryonic development in 1966. In the heterotrophic phase, the young embryo depends on the endosperm and surrounding maternal tissue, and requires a more complex environment and higher osmotic pressure than older embryos. The continuous development of young embryos requires complex environments supplemented by combinations of vitamins, amino acids, growth hormones such as auxine (which is encourages plant growth by increasing the elongation of cells and proliferation). The transport of auxins synthesized in meristematic tissues, such as leaves, upper buds and flowers, down. Indole-3-acetic acid (IAA) is the only hormone naturally synthesized in plants; however, many synthetic materials were shown to have similar effects to IAA (Williams, 2011). And cytokine (which is unlike other hormones produced in plant tissues especially during cell division, they are organic substances in quinine structure found both in plants and animals .

10. Cytokinins are divided into two main groups: ( i ) synthetic phenyl urea derivatives, thidiazuron (TDZ), urea and (ii) naturally occurring adenine derivatives, kinetin (KN) and 6- benzyl adenine (BA) ( Sezgin and Kahya 2018) (Figure 2). Figure 2 Responses of Plant Tissue culture to Cytokinin and Auxin

11. Technique Embryos are located in a sterile environment of the ovum and surface sterilization of the embryos is not necessary. Instead, whole eggs or ovaries are sterilized on the surface, and then the embryos are aseptically removed from surrounding tissue. Dissecting embryos can cause problems. Large embryos are not difficult to excise. However, small embryos require the use of micro dissection tools and a dissecting microscope to excise without injury. Embryos are easily damaged when the seed coat is cut; it is also important that the excised embryo does not become desiccated during culture ( Rangan , 1984).

12. SIGNIFICANCE Applications of embryo culture: This way of culturing is needed for: 1. Prevention of embryo abortion Incompatibility barriers in interspecific and inter-generic hybridization programs leading to embryo abortion can be successfully overcome by embryo rescue. In fact, many distant hybrids have been obtained through embryo rescue techniques. Some distant plant species crossed and the resistance traits developed by employing embryo rescue. 2. Overcoming seed dormancy Seed dormancy is caused by several factors—endogenous inhibitors, embryo immaturity, specific light and temperature requirements, dry storage requirements etc. Further, in some plants the natural period of seed dormancy itself is too long. Embryo culture is successfully applied to overcome seed dormancy, and to produce viable seedlings in these plant species.

13. 3. Shortening of breeding cycle Some of the plants in their natural state have long breeding cycles. This is mostly due to seed dormancy attributed to seed coat and/or endosperm. The embryos can be excised and cultured in vitro to develop into plants within a short period. For instance, Hollies, a Christmas decoration plant can be grown in 2-3 weeks through embryo cultures in contrast to 3 years period required through seed germination. 4. Overcoming seed sterility Certain plant species produce sterile seeds that do not germinate e.g. early ripening varieties of cherry, apricot, and plum. Seed sterility is mostly associated with incomplete embryo development which leads to the death of the germinating embryo. Using embryo cultures, it is possible to raise seedlings from sterile seeds of early ripening fruits e.g. apricot, plum. 5. Clonal Propagation Clonal propagation refers to the process of asexual reproduction by multiplication of genetically identical copies of individual plants. The term clone is used to represent a plant population derived from a single individual by asexual reproduction. Embryos are ideally suited for in vitro clonal propagation. This is due to the fact that embryos are juvenile in nature with high regenerative potential.

14. Applications:- Embryo culture can shorten the breeding cycle by overcoming dormancy in seeds. Dormancy may be caused by endogenous inhibitors, light requirements, low temperatures, dry storage requirements, and embryo immaturity ( Yeung et al., 1981). Seed dormancy factors may be localized in the seed coat, the endosperm, or both. By removing the embryos from the influences of these factors, the embryos germinate and grow quickly and the breeding cycle is shortened. Isolated embryos can also be vernalized and may, in some instances, reduce the generation time by 40 days (Sharma and Gill, 1983). In addition to the applied uses of embryo culture, the procedure is useful in basic studies. Growing embryos outside the ovule (ex ovulo ) is an excellent way to study the nutrition and metabolism of the embryos at various stages of development. The technique can also be used to examine the growth requirements of embryos, the effects of phytohormones and environmental conditions on zygotic embryogenesis, and the regeneration potentials of whole embryos and their segments ( Yeung et al., 1981). Embryo culture can be used to localize sites of germination promoters and inhibitors, for studies of

15. embryogenesis, and for cryopreservation (Grout, 1986). Embryo culture can be used to produce haploids through eliminating chromosomes following distant hybridization. This can occur by rescuing haploid maternal embryos in which the paternal chromosomes have been eliminated. In these situations, fertilization occurs, but the pollen parent chromosomes are subsequently eliminated by the seed parent. The viability of the haploid embryo can only be achieved through embryo culture. Chromosome doubling of the rescued embryo produces a homozygote monoploid .

16. Conclusion Embryo culture is a useful technique to study the effects of nutrients, plant growth regulators and other chemical and physical factors on embryonal growth and differentiation. In addition, hybrids between species and genera can be obtained by developing plants from immature embryos of forest species, garden plants, and forest species, as agronomically . Thus, the application area of biotechnological methods in the improvement of products is significantly expanded. Overcoming seed dormancy in some species is also accomplished with this technique.

17. REFERENCE Biological and Chemical Sciences ( EurasianBioChem 2020) March 19-20,2020\ Ankara,Turkey . Pp: 634-637. https://www.google.com/search?q=STEPS+IN+embryo+culture&sxsrf=AOaemvJPa9XQojzWszXIv7N8d- https://scholar.cu.edu.eg/?q=drrehabhafez/files/theoretical_course_plant_biotechnology_-_p4.pdf

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