1. Embryo Culture

2. Embryo Culture
Isolation & growth of an immature or mature embryo in vitro, with the goal of obtaining a viable plant.
The first attempt to grow the embryos of angiosperms was made by Hannig (1904) from two Crucifers Cochleria & Raphanus.

3. Types of Embryo culture
Mature Embryo Culture
Immature Embryo Culture/ Embryo rescue

4. Mature Embryo Culture
It is the culture of mature embryos derived from ripe seeds.
This type of culture is done when embryos do not survive invivo or become dormant for long periods of time.
This culture is done to eliminate the inhibition of seed germination.

5. Immature Embryo Culture
It is the culture of immature embryos to rescue the embryos of wide crosses.
This type of culture is mainly used to avoid embryo abortion with the purpose of producing a viable plant.

6. Media Requirement For Mature embryos-
Basal salt medium with a carbon energy source.
For Immature embryos-
Different vitamins, amino acids & growth regulators are required.

7. Culture-medium factors
mineral salts – K, Ca, N most important
carbohydrate and osmotic pressure
- 2% sucrose works well for mature embryos
- 8-12% for immature embryos
- transfer to progressively lower levels as
embryo grows

8. Culture-medium factors
Amino acids
- reduced N is often helpful
- up to 10 amino acids can be added to
replace N salts, incl. glutamine, alanine,
arginine, aspartic acid, etc.
- requires filter-sterilizing a portion of the
medium

9. Culture-medium factors
natural plant extracts
- Coconut milk (liquid endosperm of
coconut)
- enhanced growth attributed to undefined
hormonal factors and/or organic
compounds
- others – extracts of dates, bananas, milk,
tomato juice

10. Culture-medium factors
Plant Growth Regulators
- globular embryos – require low conc. of
auxin and cytokinin
- heart-stage and later – moderate level of
auxin & low level of cytokinin
- GA and ABA regulate "precocious
germination"

11. Embryo Rescue
In immature embryo culture, wide hybrids often suffer from early spontaneous abortion due to several barriers which operate at pre- & post- fertilization levels.
Pre- fertilization barriers include all factors that hinder effective fertilization .
(due to inhibition of pollen tube growth)

12. Embryo Rescue
Post fertilization barriers retard the development of the zygote after fertilization and normal development of the seed.
(due to failure of endosperm)
This is known as embryo-endosperm incompatibility where the endosperm produces toxins that kills the embryo.

13. EMBRYO CULTURE OF
MAIZE

14. SCIENTIFIC CLASSIFICATION OF MAIZE
Kingdom - Plantae
Order Poales
Family Poaceae
Genus Zea
Species mays

15. The Kernels of Maize has a pericarp of fruit fused with the seed coat and the entire Kernel is reffered as seed
The Cob is close to a multiple fruit in structure, except that the individual fruits (Kernels) never fused to a single mass

16. Procedure Immature cob is taken from the healthy field ↓
Its Immature seeds (Kernels) were extracted out
Surface sterilize the immature seeds by treating them with
0.1% mercuric chloride solution for 7 minutes
Give three washings with autoclaved distilled water
Dissect out the embryo from the seed under totally aseptic
conditions
Culture the embryos in the jars containing MS media
Incubate the jars at temperature 28 ̊-30 ̊C

17. EMBRYO CULTURE OF MAIZE

18. Applications of Embryo Culture
Production of haploids
Prevention of embryo abortion in wide crosses
Overcoming Seed dormancy
Shortening of breeding cycle
Prevention of embryo abortion with early ripening stone fruits

19. Production of haploids
Production of monoploids
- useful for obtaining "haploids" of barley,
wheat, other cereals.
- the barley system uses Hordeum bulbosum
as a pollen parent.

20. Production of haploids
Hordeum vulgare is the seed parent.
zygote develops into an embryo with elimination of HB chromosomes.
eventually, only HV chromosomes are left
embryo is "rescued" by culturing 10 PP to avoid abortion.

21. Prevention of embryo abortion
Embryo rescue from a cross between an early Japanese cv.- Sunago wase & a Chinese cv.- Yuhualu obtained “Zaoxialu” an extra early maturing peach cultivar.

22. Wide crosses in embryo culture
Resistance traits
Potato leaf roll virus
Triazine resistance
Bacterial blight
Crossing species
1. Solanum tuberosum x S. etuberosum
2. Brassica oleracea x B. napus
3.Oryza sativa x O. minuta

23. Overcoming Seed dormancy
Embryo rescue technique is applied to break seed dormancy.
Production of seedlings from seed of naturally vegetatively propagated plants such as Banana & Colocasia, whose seeds do not germinate in nature.

24. Shortening of breeding cycle
Under proper germination conditions, 3 years are required for seeds from mature berries to complete their embryonic development.
The excised embryo goes through all the developmental sequences in vitro & plants can be obtained in 2-3 weeks time.

25. Prevention of embryo abortion in stone fruits
Some species produce sterile seeds that will not germinate under appropriate conditions e.g.- Peach, Nectarine, Plum.
Embryo culture has been practised as a general method in horticultured crops including Peach, Nectarine & Plum.