Embryo Culture - Aspects, Types & Application

The term embryo culture means excision of embryos regardless of age, size & developmental stage from their natural environment and growing them under artificial environmental conditions.

1. EMBRYO CULTURE Anugya Jaiswal BSc . (Hons.) Biotechnology

2. INTRODUCTION Embryo culture is the sterile isolation and growth of an immature or mature embryo in vitro, with the goal of obtaining a viable plant. In 1904, Hannig first attempt to grow the embryos of angiosperms to obtained viable plants. In 1924, Dietrich grew embryos of different plant species and established that mature embryos grew normally In 1925, Laibach demonstrated the practical application of technique by isolating and growing the embryos of interspecific cross that aborted in vivo.

3. ASPECTS OF EMBRYO CULTURE Excision of Embryo The mature embryos can be isolated by splitting open the seed with hard seed coat are dissected after soaking them in water. To excise immature embryos from single ovule, the ovule is split longitudinally to isolate the half containing the embryo. Cultural requirements The most important aspects of embryo culture is the selection of a suitable medium that would support progressive and orderly development of embryos excised at different stages of development. Pro embryo Globular stage Hearted shaped stage Torpedo stage cotyledonary stage

4. TYPES OF EMBRYO CULTURE Mature Embryo culture It is culture of mature embryo derived from ripe seeds .it requires simple medium. This is done when embryos 1. Do not survive in vitro 2. Become dormant for longer periods of time 3. To eliminate the inhibition of seed germination 4 . For converting sterile seeds to viable seedlings Embryos excised from the developing seed at or near the mature stage are autotrophic and grow on a simple inorganic medium with a supplemental energy source.

5. Immature Embryo Culture/ Embryo Rescue Culture of immature embryos to rescue the embryo of wild crosses is used to avoid embryo abortion and produce viable plants . It requires complex media which includes special amino acids, hormones, endosperm extract like coconut milk etc. Embryo abortion can be avoided by isolating and culturing the hybrid embryos prior to abortion. Application of embryo rescue is the production of interspecific and inter-generic hybrids from wild plant species. The endosperm transplant technique used for culturing immature embryos.

6. ENDOSPERM TRANSPLANT TECHNIQUE The hybrid embryo from the ovule in which endosperm development has failed is taken out by excision. Another normally developed ovule with endosperm enclosing an embryo is chosen. This ovule is dissected and the normal embryo is pressed out. This leaves a normal endosperm with an exit hole . Now, the hybrid embryo can be inserted into the normal endosperm through exit hole. This results in embryo-endosperm transplant which can be cultured in a suitable medium. By using embryo-endosperm transplant, many interspecific and inter-generic plants have been raised e.g., hybrid plants of legumes.

7. BARRIERS TO DEVELOP HYBRIDS Pre fertilization barriers – Differences in flowering time of two parents Lack of stigma receptivity and pollen viability Inability of pollen to germinate Failure of pollen tube to reach the ovule due to slow growth Cross incompatibility due to any other reason. Post – fertilization barriers – Degeneration of endosperm leading to starvation and abortion of hybrid embryo. Embryo, endosperm incompatibility ,where the endosperm produces toxic substances that kill Embryo. Malfunctioning of endosperm due to abnormal behavior of antipodal cells, nucleus ,integuments around the Embryos.

8. NUTRITION REQUIREMENTS Carbohydrates Amino acid and vitamins Mineral salts Natural plant products Growth regulators p H of the medium Culture Environment

9. MEDIUM COMPOSITION Inorganic constituents of MS, B5 or White’s media are adequate. Sucrose is most commonly used energy source. Ammonium nitrate is the preferred source of nitrogen. Casein hydrolysate, rich in various amino acids is frequently used. Certain natural plant extracts with embryo factor promote embryo cultures e.g. liquid endosperm of coconut milk. The embryo factor is believed to supply certain amino acids, sugars, growth regulators etc. In general, growth regulators are not required, as they induce callus formation. Embryos grow well in the pH range of 5-7.5. An incubation temperature of 24-26°C is ideal. Better growth of embryos is observed in darkness which are then transferred to light for germination. During the culture conditions, the embryos are grown into plantlets, and then transferred to sterile soil for full-pledged growth to maturity.

10. APPLICATIONS Production of rare hybrids from inter-generic and interspecific crosses. Development of disease resistant plants Production of Haploids To overcome seed dormancy I n vitro clonal propagation Prevention of embryo abortion Rapid seed viability testing Shortening of breeding cycle Germination of seeds of obligatory parasites

11. DISADVANTAGES The number of hybrid seedlings rescued in many instances is extremely low due to the difficulty in growing very young embryos Viability decreases with age of embryo in most of the incompatible crosses. Efforts are needed to identify requirements for embryos of progressively younger stages inn major crop species

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