Embryo Culture

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.

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

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.

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.

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

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

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

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

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"

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)

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.

EMBRYO CULTURE OF MAIZE

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

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

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

EMBRYO CULTURE OF MAIZE

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

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.

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.

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.

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

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.

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.

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.