Tissue culture is a technique whereby small pieces of viable tissue (called 'explants') are isolated from parent plants and grown in a defined nutritional and controlled environment for prolonged period under aseptic conditions. For successful plant tissue culture it is best to proceed with an explants rich in undermined cells, e.g. those from the cortex or meristem, because such cells are capable of rapid proliferation.
The explants to proceed with tissue culture technique in plants are usually taken from leaves, buds, root apex, shoot-spex, nodal segments or germinating seeds. These explants are transferred onto the suitable culture media under specific conditions where they grow into an undifferentiated mass of cells called 'callus'.
To develop a callus from the explants it is essential that the nutrient medium used contains phytohormones such as auxins (e.g. IAA), cytokinins. These hormones vary for different tissue explants from different parts of the same plant and for the same explants from different genera of plants. Thus there is no 'ideal' medium, but, however, the typical medium used in plant tissue culture technology is Murashige-Scoog (MS) medium.
There are four main stages involved in plant tissue culture technology. These are:
(a) Initiation of Culture:
The most important factor in tissue culture technology is the maintenance of aseptic conditions. To meet this condition, the plant material which usually contains bacterial or fungal spores has first to be surface sterilized by the use of chlorine water or mercuric chloride or some other surface-sterilizing agents to eliminate microorganisms.
This is necessary because the culture medium on which these tissue pieces are inoculated contains mineral salts, vitamins, sugars, phytohormones and other growth regulators on which microbial contaminants would grow at a faster rate than plant tissue. After inoculation, the cultures are incubated under controlled conditions at 25°C in light.
(b) Multiplication or Sub-Culture Stage:
After 2-3 weeks, the explants show visible growth by forming either callus or differentiated organs like shoots, roots, or complete plant depending upon the composition of the medium.
If one desires to obtain large number of plantlets from the callus, he subcultures the callus. What is done is that the callus produced from the explants is taken out, cut into small pieces, and each piece is transferred to fresh medium in a separate tube where the callus pieces grow into big mass of callus. This is called sub-culture stage. Sub-culturing may be repeated if required.
(c) Development and Differentiation of Subculture:
The sub-cultured callus is now induced to proceed for further development and, finally, differentiation. If the concentration of phytohormones in the medium are altered then the callus can be induced to differentiate.
A very high auxin to cytokinin ratio induces root formation and a high cytokinin-to-auxin ration induces shoot formation. This is referred to as 'organogenesis'. Alternatively, a structure called embryoid may develop if media concentration is so altered, this process is called 'somatic embryo gene sis'. If the hormonal conditions are correctly balanced then an entire plantlet can be induced to grow on culture medium, this process is called 'regeneration'.
(d) From Test-Tubes to Field:
In the last stage, which is followed only when full-grown plants are to be obtained, the test-tube rooted plantlets are first subjected to "aclamatization" so that they can adjust later in the field conditions, and then transferred to the field. The plantlet is takes out from the rooting medium, is washed slowly with running water for an hour or more to ensure that no piece of agar is left on the surface of the plantlet.
If agar remains there would invite microbes to grow and destroy the plantlet. Now the plantlet is put to low minimal salt medium (LMSM) for 24-48 hours and then transferred to a pot that contains autoclave-sterilized mixture of clay- soil, core sand leaf moults in 1 : 1 : 1 proportion.
The plantlet-containing pot is covered generally with transparent polythene (beakers may also be used) to maintain the humidity. This condition is maintained for about 15-30 days.
At this stage the plantlet is fully acclimatized and is able to withstand open environment conditions. The plantlet then is transferred to soil and the techniques employed are similar to normal agricultural practices.
(a) Improvement of crops and ornamentals:
Plant tissue culture technology bears tremendous potential for the improvement of crops and ornamentals. Some major areas of this application are :
(i) Large Scale Plant Regeneration:
Clonal Propagation (Micropropagation) : One of the most important aspects of plant tissue culture technology is its use in clonal propagation of selected species of crops and ornamentals. The clonal propagation via tissue culture technique (i.e. in vitro clonal propagation) is popularly called "micropropagation ".
In most cases, in vitro clonal propagation has been achieved using shoot apex tissue or axillary bud tissues. The apical shoot tip or axillary bud is surface sterilized and placed onto a culture medium that promotes formation of additional shoot (the medium is called shooting medium).
Subsequently, multiple shoots are separated and transferred onto such a medium that induces root formation (the medium is called rooting medium). Plantlets are then aclamatized and transferred to the green-house or field.
Interest in vitro clonal propagation of plants originated from the success of G. Morel (1960) with orchids. At present, however, in vitro clonal propagation is the only commercially viable method of propagation of orchids and almost all economically important orchids are clonable in vitro. Certain species of ornamentals or parental breeding lines are often difficult or time consuming to propagate using conventional sexual methods. In either case, mass clonal propagation using tissue culture technique could be commercially useful.
Shoot apex clonal propagation has already been used to commercially propagate a large number of marketable ornamentals. For instance, ornamental plants such as Gerbera, Cordyline, Dracaena, Hemerocallis are now commercially propagated via cloning.
[Clonal propagation, which is popularly called 'micropropagation' represents the technique in which vegetative tissue are used to produce plants that are genetically identical to their parents. A clone is however, the genetically alike progeny of plant derived from its vegetative tissue.]
Clonal propagation is, however, being used commercially for large-scale plant propagation in certain crop species (e.g. potato) by the production of minitubers. When the axillary shoot cultures of potato are cultivated in the presence of appropriate levels of phytohormones such as cytokinin and gibberellin, they result in large number of small tubers called 'minitubers'. The later are transferred directly to the field and generate normal plants. One can see from the following table that the method of minituber- oriented plant propagation is advantageous.
The significant advantages offered by in vitro clonal propagation over the conventional methods of clonal propgation (methods of vegetative propagation) in vivo are:
(1) Over a million plants can be regenerated from a small even microscopic, piece of plant tissue within a year. Contrary to it, none of the in vivo methods of clonal propagation can result in such a prolific rate of multiplication.
(2) In vitro clonal propagation can continue throughout the year irrespective of the season.
(3) An enhanced rate of in vitro clonal propagation reduces considerably the period between the selection and the release of a new culture.
(4) In vitro clonal propagation provides reliable and economic methods of maintaining pathogen free plants in a state that can achieve rapid multiplication when needed, irrespective of the time of the year.
(ii) Non-Clonal Production:
Contrary to the shoot apex propagation which faithfully produces clones, there are sizable number of evidences which suggest that the regeneration of plants from callus, leaf explants, protoplasts does not result in production of clones, the regenerated plants bear genetic variability.
Regeneration of plants from long-term callus culture is associated with in vitro chromosome instability and recovery of aneuploid plants. Regeneration of plants directly from leaf explants has resulted in morphological variation. It has been reported that the regenerates of potato and sugarcane varied in response to diseases. Plants regenerated from callus of sugarcane were examined in detail and have been found resistant to eye-spot diseases.
Fiji disease and downy mildew for which the parent was susceptible. These regenerate-lines are being incorporated into a conventional breeding programme. In this way the large scale plant regeneration using callus, leaf explants etc. may lead to the production of genetically variable crop species which may prove novel agriculturally. However, no new crop varieties have yet been produced using the culture induced genetic variability although this method will probably produced new varieties shortly.
(b) Production of Virus Free Plants:
Virus infection has been a major problem of crop and ornamental plants. Such infections greatly reduce the yield, vigour and quality of plants and their products. For instance, virus infection of ornamental plants reduces the size and number of blooms produced and infection of fruit crops reduce yield by upto 90%.
The traditional method of virus elimination is by heat-treatment, but, only a few viruses can be eliminated by this method. It has been observed in a large number of species, however, that the concentration of infective virus is low in the apical meristems of a plant.
It is so because the lack of vacular differentiation in the meristem prevents intercellular movement of viruses and the active metabolism of mitotic cells precludes viral infection.
It is considered, therefore, that if a small enough piece of apical meristem is exised and cultured, it is often possible to obtain virus-free plant regenerates. Genetic stability as measured by phenotypic and chromosomal stability is usually preserved in plant regenerates recovered from apical meristem cultures.
G. Morel and C. Martin (1952) succeeded for the first time in obtaining virus-free Dahlia plants by culturing apical meristem from infected plants. Since then virus have been eliminated using shoot-apex culture from a number of economical species. These species include the elimination of mottle virus from strawberry, potato Virus-X from potato, mosaic virus from cassava, and cauliflower mosaic virus from cauliflower.
Virus-free plants regenerated from shoot- apex cultures have resulted in significant increase in yield when compared to virus infected plants. Result have been particularly favourable in potato where yield of virus-free potatoes is 60% higher than that of infected plants.
Besides apical meristems, callus from tissue explants is also used to regenerate virus free plants, callus is considered similar to apical meristems as mitosis occurs rapidly and the vascular differentiation is incomplete. In callus, viral infection may be completely eliminated via repeated subculture.
Virus free plants have been regenerated from callus in tobacco, germanium, gladiolus, potatoes, Although plants regenerated from callus are virus- free, it has been found that the chromosomal abnormalities occurs frequently among callus derived plant regenerates. Simultaneously, it has also been demonstrated that the populations of callus- derived regenerates are not as uniform as apical meristem-derived regenerates.
Plants regenerated from viral infection-free leaf tissue explants have also been found virus-free. Murakishi and Carlson (1976) regenerated tobacco plants from dark green uninfected explants of a TMV- infected leaf. They observed that approximately half of the regenerated plants were virus-free. Similarly, Shepard (1977) reported that 7.5% of plants regenerated from potato virus-X infected tobacco leaves were free of viruses.
One of the most important aspects of tissue culture technology is rapid regeneration of forest plants. These have been successfully demonstrated in case of orchids, herbaceous plants, and some fruit tree species. However, realization of the importance of tissue culture in accelerating improvement programmes for forest tree spp. is a recent origin.
A number of reports on methods of producing plants by tissue culture of a whole range of forest tree belonging to angiosperms such as teak, eucalyptus, sandalwood, populus, birch, tamarind, rubber, oil palm, and gymnosperms such as Picea abies, Pinus radiata, Sequoia sempervirens, Thuja plicata have recently been published.
In India, the scientists of National Chemical Laboratory, Pune have developed tissue culture methods by which hundreds of plants of teak (Tectona grandis) and Eucalyptus citriodora could be obtained from a single bud in a year.
Over 600 plants of teak produced by this method have been supplied to Forest Development Corporation, Maharashtra for field trials. National Botanical Research Institute (NBRI), Lucknow has perfected a method for the mass propagation of Dioscorea floribunda by tissue culture technique.
(d) Production of Secondary Metabolities:
Plant tissues cultured in vitro produce some or all of the primary and secondary metabolites produced by mature plants. The primary metabolities consists of proteins, carbohydrates, fats, vitamins etc. which are essential for growth, while secondary metabolities like alkaloids, steroids, phenolics, flavonoids may be produced as defence mechanisms by the plant. Secondary metabolities have a wide range of applications in the pharmaceuticals, chemical and food industries.
Commercial production of these high cost secondary metabolities by plant tissue cultures is attracting greater attention now than before. This is evident from the large number of commercial companies taking keen interest to exploit the potential. Till recnetly, only a few groups in W. Germany, Japan and Canada were actively involved in the production of metabolities from tissue culture, but now, many companies have actively entered this area of biotechnology which offers great promise for the days to come.