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 300C is used (Narayanaswamy and Norstog, 1964).
• Some embryos, from species such as Lilium, require a lower temperature,
i.e.,170C,and others require a cold tretment of 40C 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

12. 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).

13. 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.

14. 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.

15. 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

16. 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.

17. 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.

18. 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

19. THANK YOU