This document discusses embryo culture techniques. It begins by defining embryo culture as the sterile isolation and growth of immature or mature zygotic embryos in vitro to obtain viable plants. Embryo culture is commonly used for embryo rescue in intergeneric plant crosses. The document then discusses types of embryo culture including mature and immature embryo culture. It provides details on excising embryos and the cultural requirements for embryo growth, such as suitable media, carbohydrates, amino acids, and growth regulators. Finally, the document outlines several applications of embryo culture including producing rare hybrids, developing disease resistance, propagation of rare plants, and shortening breeding cycles.

1. SUBMITTED BY- ANAND KUMAR
ROLL NO- 8/VKSCOA/2021-22
SEMESTER- 5th
COURSE CODE- AHT 311
COURSE TITLE- MICROPROPAGATION TECHNOLOGIES
SUBMITTED TO- Dr NANDITA MAM
INTRODUCTION
▪ The term embryo culture means excision of embryos regardless of age, size and developmental stage from their
natural environment and growing them under artificial environmental conditions.
▪ Embryo culture is the sterile isolation and growth of an immature or mature zygotic embryo in vitro with the
goal of obtaining a viable plant.
▪ Most commonly used technique is that of embryo rescue which produces an adult plant following an
intergeneric crosses with plants (Wheat with Barley, Pearl millet with elephant grass, oats with wild oats,
peanuts with wild Arachnis and Soyabean with wild glycine.
▪ Embryo culture is a relatively simple in that the embryo is a minature complete plant and therefore no de novo
differentiation of shoots or roots is needed.
▪ In 1904, Hannig first attempt to grow the embryos of angiosperms to obtained viable plants.
▪ In 1924, Dietrich grew embryos of different plant species & established that mature embryo grew normally.
▪ Laibach in 1925 isolated inter specific hybrid embryos of Linum perenne x Linum austriacum, Seeds are greatly
shriveled, very light and incapable of germination when planted in soil.
▪ By excising embryos from such seed and growing them in moist filter paper or cotton soaked in 15% glucose
solution, he was able to rise the hybrid plant.
▪ Aseptic method of clonal propagation is called as Micro-propagation & it offers the advantage of large number
of true-to-type plantlets can be produced with relatively short time and space from a single individual.
▪ It is the fact that micro-propagation is the only commercially viable method of clonal propagation of most of the
horticultural crops. E.g. Orchids
▪ The first commercial use of plant propagation on artificial media was in the growth of orchid plants in 1920.

2. Types of Embryo culture
▪ 1) Mature Embryo culture
▪ 2) Immature embryo culture
1. Mature embryo culture
▪ It is culture of mature embryo derived from ripe seeds. It requires simple medium.
▪ This is done when embryos
▪ i) Do not survive in vitro
▪ ii) Become dormant for longer periods of time
▪ iii) To eliminate the inhibition of seed germination
▪ Some species produce sterile seeds which may be due to incomplete embryo development. Such embryos can
be cultured and viable seedlings can be produced.
▪ Eg: Iris, orchids
Reasons for carrying Mature Embryo culture:
▪ When the embryos remain dormant for long periods.
▪ Low survival of embryos in vivo.
▪ To avoid inhibition in the seed for germination.
▪ For converting sterile seeds to viable seedlings.
1. Immature embryo culture (or) embryo rescue technique:-
Culture of immature embryos to rescue the embryos of wild crosses is used to embryo abortion and produce viable
plants.
It requires complex media which includes special amino acids, hormones, endosperm extract like coconut milk etc.
It is the culture on immature embryos to rescue the embryos of wide crosses.
In less than optimum medium, the immature embryos may fail to survive, turn into undifferentiated callus, or
germinate prematurely (precocious germination).

3. Culturing of embryos:-
The most important aspects of embryo culture are
1) Excision of embryo
2) Cultural requirements
Excision of embryo:-
▪ a) Material:- Artificial pollination of freshly opened flower is necessary when embryos are to be cultured at the
specific stage of development.
▪ The mature embryos can be isolated by splitting open the seed. Seeds 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.
▪ For excising older embryo a small incision is made in the ovule on the side lacking the embryo and than a slight
pressure is applied with a blunt needle to release the intact 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.
▪ The zygotic embryos develop through the following stages:-
PRO EMBRYO → GLOBULAR → HEART SHAPED STAGE → TORPEDO STAGE → COTYLEDONARY STAGE
▪ Embryo culture:- A fully developed embryo undergoes a period of maturation during which the embryo
becomes hardy, Up to a certain stage Ex:- up to globular stage in Capsella the embryo is heterotrophic as it
derives some part of its nutrition from endosperm.
▪ Beyond this stage the embryo becomes autotrophic and is able to synthesis its biochemical needsc from simple
nutrients like salt and sugar.

4. ▪ In general the older as embryo the simpler its nutritional needs. M.S medium supports maximum growth of
embryos but survival frequency is low. Monnier’s medium favors both good growth and survival.
NUTRITION REQUIREMENTS
▪ Carbohydrates: Sucrose is the best carbon source, produces energy and also maintains suitable osmolarity.
▪ Mature embryos: grow well with 2% sucrose.
▪ younger embryos: high level of sucrose conc. (8-12%).
▪ for heart shaped embryos: Mannitol at 120g/lit is suitable.
▪ Amino acids and vitamins: Reduced Nitrogen in form of glutamine 400mg/lit. (or) Aspargine is beneficial
particularly for younger embryos.
▪ Mineral salts: Monnier (1978) modified the M.S. medium and it favors higher survival rate of cultured immature
embryos.
▪ Natural plant products: Vanover beak et al 1941 reported the importance of coconut milk (liquid endosperm of
coconut) in promoting the growth of certain immature embryos.
▪ Growth regulators: Auxins and cytokinins are not generally used in embryo cultures since embryos are usually
autotrophic. In this respect ABA checks precocious germination and promoted embryo development and
maturation.
▪ PH
of the Medium: It is critical for Organogenic differentiation. The range of PH for growth of excised embryos is
5 to7.5
▪ Culture Environment: Embryo cultures are kept at temperature ranging from 25°-30°C. Embryos developed into
plantlets in 14-30 days, if there is appropriate medium.
Applications of embryo culture:
1. Production of rare hybrids from Intergeneric and interspecific crosses.
▪ The hybrid embryo failed to develop due to poor (or) abnormal development of endosperm.
▪ Hordeum vulgare x secale cereale
▪ Triticum durrum x secale cereale
▪ Triticum aestivum x Agiliops speltoides
▪ Triticum aestivum x Agropyron repens
▪ Hordeum vulgare x Triticum species

5. ▪ Legumes:-
▪ Arachis hypogaea x Arachis monticola
▪ Phaseolus vulgaris x Phaseolus. Vitensis
▪ Development of disease resistant plants
2. Embryo culture has been useful in evolving disease resistant plants.
▪ Eg: Tomato resistance to virus, fungi and nematode.
▪ Lycopersican esculentum x Lycopersican peruvianum
3. Production of haploids:
▪ Kasha and Kao (1970) reported a novel technique for the production of haploids in barley. The method involves
crossing of Hordeum vulgare (2n = 14) with H. bulbosum (2n = 14).
4.Overcoming seed dormancy: Iris seeds may take two to several years to germinate. This dormancy can be eliminated
by embryo culture technique.
5.Shortening of Breeding cycle: Rose plants take normally one year to come to flowering but embryo cultured plants
come to flowering in 6 months.
6. Propagation of rare plants: Ex: banana, colacasia.
7. Propagation of orchids: Eg: Orchids
8. Prevention of embryo abortion: Eg: Early ripening varieties, of peach, cherry, apple, plum etc.
9. Clonal Micro propagation
10. Rapid seed viability testing
11. For development of callus culture.
12. Acceleration of hybridization programme in crops having heavy fruit drop problem.
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 in major crop species.

6. DIAGRAMATTIC REPRESENTATION OF EMBRYO CULTURE:-
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