Endosperm and embryo culture

history
Lampe & Mills (1933) were the first to report the proliferation of immature endosperm tissue of Maize, grown on medium containing extract of potato.
La Rue (1947) observed that in nature, in maize , the pericarp ruptured & the endosperm exhibited a white tissue mass.

1. Endosperm Culture

2. History Lampe & Mills (1933) were the first to report the proliferation of immature endosperm tissue of Maize, grown on medium containing extract of potato. La Rue (1947) observed that in nature, in maize , the pericarp ruptured & the endosperm exhibited a white tissue mass.

3. Introduction In flowering plants, nutrition of the embryo is an important and vital aspect of the life cycle. In angiosperms, it is accomplished by the formation of a new structure, the endosperm, produced by a unique event of double fertilization.

4. Double Fertilization Double fertilization occurs: One sperm nucleus (1n) fertilizes the egg, producing a zygote (2n)  which becomes the plant embryo inside the seed. Another sperm nucleus fuses with the polar nuclei, resulting in a triploid endosperm (3n). Endosperm is a source of food for the young embryo.

5. Endosperm

6. Endosperm Culture Sehgal (1974) cultured the immature endosperm of Hordeum & Triticum . In organogenesis was successfully induced in culture of immature endosperm of Oryza sativa by Nakano et al. (1975).

7. Endosperm Culture The age of endosperm at culture is critical for its growth in vitro. In Maize, Wheat & Barley, endosperm tissue younger than 8 or older than 12 days after pollination did not grow in cultures. The endosperm of Cucumis can be grown only when excised 4-7 days after pollination.

8. Procedure of endosperm culture Explant source. Inoculation of explant. Maintenance of culture. Shoot but differentiation or embryogenesis. Plantlet development.

9. Media Requirement The mature endosperm of parasitic taxa shows optimal growth on a medium containing either a cytokinin or a cytokinin & an auxin . In autotrophic members, casein hydrolysate or yeast extract is also essential.

10. Endosperms of Santalum album , Croton bonplandianum & Ricinus communis could be grown on White’s medium containing 2,4-D, kinetin & yeast extract. Organ formation has been demonstrated mainly in parasitic species ( Exocarpus , Taxillus ) and in some autotrophic species ( Croton, Santalum , Citrus )

11. Differentiation In all parasitic members - Differentiation of shoot buds or roots without callusing. In autotrophic members - Callus mass followed by differentiation of shoot buds or roots.

12. Organogenesis in Culture Organogenesis observed only in few species such as Rice, Apple, Citrus, Santalum , Croton , etc. In Ricinus communis , Zea mays & Cucumis mature endosperm proliferation resulted in a callus tissue of unlimited growth but organogenesis did not occur.

13. Factors affecting endosperm culture Explant storage. Nutrient medium. Physical factors. Embryo factors.

14. Applications Triploid plants can be raised through endosperm culture. Wang & Chang (1978) produced triploid plantlets from Citrus. Laxmi sita et al. (1980) developed triploid plants of Santalum album . Presently there are number of crop species ( banana, apple, beet, tea, mulberry ) in which triploids are in commercial use. Endosperm can be used as a nurse tissue for raising hybrid embryos.

15. Embryo Culture

16. 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.

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

18. 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.

19. 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.

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

21. 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

22. 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

23. 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"

24. 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)

25. 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.

26. 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

27. EMBRYO CULTURE OF MAIZE

28. 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

29. 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.

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

31. 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.

32. 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 invitro & plants can be obtained in 2-3 weeks time.

33. 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.

34. Plum Peach Nectarine

35. Thank you