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In Vitro Culture Techniques : The Biotechnological Principles

 
     
 
Anther and Pollen Culture (Production of Haploid Plants)
Anther, a male reproductive organ, is diploid in chromosome numbers. As a result of microsporogenesis, tetrads of microspores are formed from a single spore mother cell. They are known as pollen grains after release from tetrads (Bhojwani and Bhatnagar, 1974). The aim of anther and pollen culture is to get haploid plants by induction of embryogenesis. Haploid plants have single complete set of chromosomes that in turn may be useful for the improvement of many crop plants (Sunderland, 1979). Moreover, chromosome set of these haploids can be doubled by mutagenic chemicals (e.g. colchicine) or regeneration technique to obtain fertile homozygous diploi4Su(Vasil and Nitsch, 1975).
 

Tulecke (1951) cultured pollen grains of Ginkgo biloba (gymnosperm) and succeeded to induce the development of haploid callus. Guha and Maheswari (1964) made a remarkable discovery by culturing pollen grains of an angiospermic plant, Datura innoxia on the nutrient agar medium and also developed torpedo- shaped embryoids that metamorphosed into plantlets through the process. 

Sunderland (1979) has described that the anthers to be cultured should be one of the three categories i.e. premitotic, mitotic and postmitotic. In premitotic anthers, where the microspores have completed meiosis but not started first pollen division, the best response is achieved e.g. Hordeum vulgare. In mitotic anthers where first pollen division has started the optimum responses are achieved e.g. N. tabacum and D. innoxia. In post mitotic anthers, the early bicellular stage of pollen development is the best time to culture e.g. Atropa belladona.
 

Haploid plants are very useful in (i) direct screening of recessive mutation because in diploid or polyploid screening of recessive mutation is not possible, and (ii) development of homozygous diploid plants following chromosome doubling of haploid plant cells. 

In China, the most widely grown wheat is a doubled haploid produced through homozygous diploid lines. Anther culture of rice is also successfully grown. Haploid plants have been produced in tobacco, wheat and rice through pollen culture. These are used for the development of disease resistant and superior diploid lines. 

At present, more than 247 plant species and hybrids belonging to 38 genera and 34 families of dicots and monocots have been regenerated using anther culture technique. They include economically important crops and trees such as rice, wheat maize, coconut, rubber trees, etc, (Maheswari et al.1983).
 

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


Culturing techniques
Methods of anther culture are shown in Fig. 8.9. Anthers are superficially sterilized and washed with double distilled sterile water. They are excised from the flower buds and their proper developmental stages are determined under microscope. On confirmation of a stage, (a) the anthers are directly transferred on nutrient agar or liquid medium where induction of embryogenesis occurs, or (b) the pollen grains are aseptically removed from the anthers and cultured on liquid medium (Reinert and Bajaj, 1977).
  Methods of anther culture, and production of haploid and diploid plants in vitro.
 

Fig. 8.9. Methods of anther culture, and production of haploid and diploid plants in vitro.

 
     
 
 
     



     
 
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