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


Historical Background
During 19th century the idea of development of callus (a disorganized proliferated mass of actively dividing cells) from isolated stem fragments and root apices came into existence. Callus could also be developed from buds, and root and shoot fragments of about 1.5 mm in size without using nutrient medium. For the first time in Berlin, Hamberlandt (1902) originated the concept of cell culture. He attempted to cultivate the isolated plant cells in vitro on an artificial medium (Knop's solution, peptone, asparagin and sucrose).

The term 'Tissue Culture' can be applied to any multicellular culture growing on a solid medium (or attached to substratum and nurtured with a liquid medium) that consists of many cells in protoplasmic continuity. But in organ culture (e.g. excised roots) the cultured plant material maintains its morphological identity, more or less, with the same anatomy and physiology as in vitro of the parent plants (Doods and Roberts, 1985).

Until the early 1930s, R.P. White (USA), Gautheret (France) and Nobercourt (France) independently cultured tissues excised from several plants on the defined nutrient media for a long period. Gautheret (1939) cultured cambium tissue of carrot on Knop's solution supplemented with other chemicals in trace amount. White (1939) cultured tobacco tumor tissue from the hybrid Nicotiana glduca, and N. Langsdorjfii.

In the 1950s several important achievements were made in the field of plant physiology. The understanding of plant growth hormone in rapid multiplication of totipotent cell was developed. At the university of Wisconsin, Skoog and co-workers found out the role of pytokinins in tissue culture. Consequently, several chemicals were tested which stimulated callus. Adenine in the presence of auxin was found to induce callus growth and bud formation in tobacco cultures (Skoog and Tsui, 1948). Eventually a potent cell division factor from degraded DNA preparations was isolated, identified and named as kinetin (Miller et al., 1955). The term cytokinin was given to this group after substituting the aminopurine compounds that stimulate cell division in cultured plant tissue and behave physiologically similar to kinetin. Later on other cytokinins (the naturally occurring plant hormones) such as zeatin, and isopentyl adenin were discovered. Skoog and Miller (1957) advanced the hypothesis of organogenesis in cultured callus by varying the ratio of auxin and cytokinin in the growth medium. The shoot was formed with keeping the ratio of kinetin higher and root developed when ratio was lower.




Historical background

Requirements for cell and Tissue Cultures


A tissues culture laboratory


Nutrient media



Inorganic chemicals



Growth hormones



Organic constitutents






Amino acids

Culture of plant materials


Explant culture


Callus formation and its culture




Root culture


Shoot culture and micropropagation


Cell culture



Benefits from cell culture


Somatic embryogenesis


Somaclonal variation


Protoplast culture








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

When fragments of callus are transferred into a liquid medium and aerated on a shaker, it gives a suspension of single cell and aggregate of cells. A cell can be propagated by subculturing. Muir (1953) developed a successful technique for the culture of single isolated cells which is commonly known as paper-raft nurse technique (placing a single cell on filter paper kept on an actively growing nurse tissue). Later on, attempts were also made for single cell culture by hanging drop and agar plate method. During this period phenomenon of totipotency was fully developed by demonstrating that a single isolated cell can divide and regenerate a whole plant (Vasil and Hilderbrantt, 1965).

Cell wall creates a barrier in plant protoplast culture. In 1960s, the role of enzymes e.g. cellulase and pectinase in dissolution of cell wall in buffer solution at suitable pH, and isolation and culture of protoplast was developed (Cocking, 1960). During this period extensive study of plant tissue culture was done by using the explants (excised plant parts ) from different parts of gymnosperms and angiosperms. Ajuha and Maheshwari (1966) developed techniques for the production of vast numbers of embryos from cultures of pollens and sporogenous tissues of anther. Nitsch (1974) gave methods to double the chromosome number in microspores of Nicotiana and Datura, and collected seeds from the homozygous diploid plants within 5 months. However, the benefits from protoplast culture currently availed are :


Intraspecific, interspecific and intergeneric protoplast fusion and some times between plant and animal,


Transfer of mitochondria and plastids into protoplast,


Uptake of certain beneficial genes of blue-green algae, bacteria and viruses by protoplasts, and


Transfer of genetic informations into isolated protoplasts.

Vasil (1982) has emphasized to develop the following techniques of cell culture and somatic genetics of mainly grasses and cereals:

(i) Rapid clonal propagation, (ii) Regeneration from single cell and protoplast (iii) Androgenetic haploids, (iv) Mutant/variant cell lines and plant regeneration, (v) Somatic hybridization, (vi) Transformation, and molecular biology.


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