Organ culture in plant biotechnology

Organ Culture

Importance

Production of virus free germplasm

EMBRYO CULTUREEmbryo culture is the culturing of embryos excised from the ovaries at earlier stages of their development. This technique helps to overcome problems associated with embryo development. Embryos are prevented from development by different factors like incompatibility with the female tissue, absence of endosperm etc. Hybrids produced by wide crosses usually fail to develop inside the ovaries of the mother plants. In such cases, the embryos can be rescued (the technique is called embryo rescue) and grown in culture media so as to produce viable progeny.

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

• Culture of surgically dissected embryo

• Immature Embryo Culture/ Embryo rescue

• Culture of intact seed containing undifferentiated embryo

• Culture of adventive embryos from proembryonic seeds

• Culture of inviable or abortive embryo

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.

• Mature embryo culture: to analyse the various parameters of embryonic growth and the metabolic and biochemical aspects of dormancy and germination

• Culture of surgically disected embryos:

- the mature seed embryo can be dissected surgically into a number of segments

- Such embryo segments are cultured to analyse the relationship of different parts of the embryo to its final form in culture

Immature Embryo Culture

• It is the culture of immature embryos or proembryos to rescue the embryos of wide crosses. Globular and heart shaped- proembryo stage

• This type of culture is mainly used to avoid embryo abortion with the purpose of producing a viable plant.

• To understand the control of differentiation and the nutritional requirements

• Culture of intact seeds containg undifferentiated embryo:

• Spherical mass of tissue lacking both radicle and plumule

• No seed coat, seed coat is reduced to a membraneous structure

• Culture of adventive embryos from polyembryonic seeds

• Besides the zygotic embryo produced from egg cell some embryos are produced from nucellar tissue

• Such additional abortive embryos can be exploited in culture for clonal propogation

• Culture of inviable or abortive embryos:

Inviable or abortive embryos may develop due to unsuccessful crosses

Possible to raise hybrid plant by culturing the inviable embryos in vitro

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

Procedure

Immature cob is taken from the healthy field

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Its Immature seeds (Kernels) were extracted out

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Surface sterilize the immature seeds by treating them with

0.1% mercuric chloride solution for 7 minutes

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Give three washings with autoclaved distilled water

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Dissect out the embryo from the seed under totally aseptic

conditions

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Culture the embryos in the jars containing MS media

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Applications of Embryo Culture

• Prevention of embryo abortion in wide crosses

• Overcoming Seed dormancy

• Shortening of breeding cycle

• Prevention of embryo abortion with early ripening stone fruits

• Helps determining the factors that regulate the growth of primordial organs of the seedling plant

• Study the metabolic and biochemical aspects of dormancy and germination

• Analysis of various parameters of embryonic growth

• To study the nutritional requirements

• Changes in the nutritional requirement of embryo at different stages

• Effect of aminoacids, vitamins and plant hormones can be observed on embryo culture

Applied applications

• To raise a healthy plant from abortive or non-viable embryos

• To overcome seed dormancy• Shortening the breeding cycle• To overcome self-sterility of seeds• Seed testing

Other applications

• To study evolutionary relationship• To study the host parasite interaction• To study mutagenic effect