Ppt on embryo culture

1. Embryo culture

2. Theory:
▪ 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.
▪ 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.
▪ Types of Embryo culture
▪ 1) Mature Embryo culture
▪ 2) Immature embryo culture

3. ▪ 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

4. ▪ Immature embryo culture (or) embryo rescue technique:-
▪ Culture of immature embryos to rescue the embryos of wild crosses
is use d to avoid embryo abortion and produce viable plants.
▪ It requires complex media which includes special amino acids,
hormones, endosperm extract like coconut milk etc.
▪ Culturing of embryos:
▪ The most important aspects of embryo culture are
▪ 1) Excision of embryo
▪ 2) Cultural requirements

5. ▪ 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.

8. ▪ 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
stage
Hearted
shaped stage
Torpedo
stage
Coteledonary
stage

10. ▪ 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 needs from simple nutrients like salt and
sugar.
▪ 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.

11. 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.

12. ▪ 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.

13. ▪ Applications of embryo culture:
▪ Production of rare hybrids from Intergeneric and interspecific crosses.
The hybrid embryo failed to develop due to poor (or) abnormal development
of endosperm.
▪ Cereals:-
▪ 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
▪ Legumes:-
▪ Arachis hypogaea x Arachis monticola
▪ Phaseolus vulgaris x Phaseolus. Vitensis

14. ▪ Development of disease resistant plants
▪ Embryo culture has been useful in evolving disease resistant plants.
▪ Eg: Tomato resistance to virus, fungi and nematode.
▪ Lycopersican esculentum x L peruvianum
▪ Potato - resistance to potato leaf roll virus
▪ Solanum tuberosum x S. etuberosum

15. ▪ 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).
Hordeum vulgare x H. Bulbosum
2n=14 2n-14
(VV) (BB)
Zygote
2n=14 (VB)
Chromosome elimination of H. bulbosum
Young embryo (n = 7-V)
(Embryo culture + Chromosome Doubling)
Haploid plants of H. vulgare (VV=14)

16. ▪ Overcoming seed dormancy: Iris seeds may take two to several years to
germinate. This dormancy can be eliminated by embryo culture technique.
▪ Shortening of Breeding cycle: Rose plants take normally one year to come
to flowering but embryo cultured plants come to flowering in 6 months.
▪ Propagation of rare plants: Ex: banana, colacasia.
▪ Propagation of orchids: Eg: Orchids
▪ Prevention of embryo abortion: Eg: Early ripening varieties, of peach,
cherry, apple, plum etc.
▪ Clonal Micro propagation
▪ Rapid seed viability testing

17. ▪ 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.