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  Section: General Biotechnology / Plant Biotechnology
 
 
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In Vitro Culture Techniques : The Biotechnological Principles

 
     
 
Embryo Culture
In addition to root, shoot, and pollen culture, embryo culture has also been done for the production of haploid plants. Embryo culture is used for the recovery of plants from distinct crosses. Embryo culture is useful where embryo fails to develop due to degeneration of embryonic tissues. It is being used extensively in the extraction of haploid barley (Hordeum vulgare) from the crosses H. vulgare x H. bulbosum. Embryo culture is also a routine technique employed in orchid propagation and in breeding of those species that show dormancy. Das and Barman (1992) developed the method of regeneration of tea shoots from embryo callus. The embryo callus produced somatic embryoids within 8 weeks of culture in the second medium which differentiated into buds after 2 weeks. Several shoots with 4-6 leaves developed after 16 weeks of culture. 

Culturing method

The general method of embryo culture follows the following steps.

(i)

Pluck healthy and mature fruits from the field and wash thoroughly in running water for about an hour.

(ii)

Surface sterilize with 0.01% Tween-20 for 15 min, rinse seeds several times with distilled water and finally treat with 0.01% HgCl2 solution for 10-15 min. Finally rinse it for six times with sterile distilled water.

(iii)

Break seeds aseptically and isolate the embryo.

(iv)

Culture embryo on callus proliferation medium. Supplement the basal medium of Murashige and Skoog (1962) with different combinations and concentrations of sugar, vitamins, hormones and other growth adjuvants for callus proliferation and shoot regeneration.

(v)

Incubate the cultures at 22-25°C under a 16 h photoperiod of 2000 lux luminous intensity.

(vi)

After two weeks of inoculation the embryo begins to swell on callus proliferation medium. Distinct callus growth is observed after 4 weeks.

(vii)

After 8 weeks of inoculation transfer the callus on shoot regeneration medium. Within 4 weeks of transfer into second medium the callus turns green and produces soft spongy tissue. Some of these tissues are differentiated into embryoids.

(viii)

The embryoids produce cluster of budlets when subcultured onto shoot regeneration medium. The budlets grow into shoots and produce 2-3 leaf appendages within 12 weeks. Thereafter, they are separated into individual shoots and then subcultured into a fresh medium of the same composition until shoots develop.
 

Content

Totipotency

Historical background

Requirements for cell and Tissue Cultures

 

A tissues culture laboratory

 

Nutrient media

 

 

Inorganic chemicals

 

 

Growth hormones

 

 

Organic constitutents

 

 

Vitamins

 

 

Amino acids

Culture of plant materials

 

Explant culture

 

Callus formation and its culture

 

Organogenesis

 

Root culture

 

Shoot culture and micropropagation

 

Cell culture

 

 

Benefits from cell culture

 

Somatic embryogenesis

 

Somaclonal variation

 

Protoplast culture

 

 

Isolation

 

 

Regeneration

 

Protoplast fusion and somatic hybridization

 

 

Fusion products

 

 

Method of somatic hybridization

 

Anther and pollen Culture

 

 

Culturing techniques

 

In vitro androgenesis (direct and indirect androgenesis)

 

Mentor pollen technology

 

Embryo culture

 

Embryo rescue

 

Protoplast fusion in fungi

 
     
 
 
     



     
 
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