Steps Involved in Variant-Selection through Single Cell Cultures

(a) Crop Improvement:

It has been found that when plants are regenerated by growing somatic explants (other than those of apical meristems and auxiliary bud tissue) in vitro, they usually show genetic variations (called ‘somaclonal variations’). The high degree of their variations is being exploited in mutant (variant) selection in relation to crop improvement.

Two approaches have been followed to recover such variant plants: first, plants are regenerated from somatic explants cultured for different periods and screened for the desired traits, second for a number of somalclonal variations, the screening is required to be done at single cell level. The merit of screening of plant variants at single cell level is that it permits screening millions of cells with a comparatively small input of effort and resources.

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Isolation of real variants through single cell cultures requires a series of step-wise investigations (Umiel, 1978). These are as follows:

(i) Characterization of isolated variant cell lives.

(ii) Phenotypic expression of the trait in tissue culture.

(iii) Production of plants from the variant cells.

(iv) Test in tissue culture for the transmission of the variation through plant regeneration.

(v) Production of seeds and progenies.

(vi) Tests in tissue cultures for the transmission of the variation through seeds,

(vii) Tests for the expression of the variation at the plant level in plants derived from seeds.

(viii) Genetic studies and the establishment of seed stock for the variant lines.

(ix) Studies on the nature of the variations.

(b) Resistance to Diseases:

Sections of cell lines resistant to diseases may be of direct practical values. Resistance to most plant diseases is not mediated by single gene and hence is not detectable in culture.

However, sometimes the toxic effect of a phytotoxin on tissue, cell or protoplast cultures is equivalent to its effect on the whole plant. Furthermore, if the phytotoxin is the only reason for pathogenecity direct selection for disease resistance in vitro using the phytotoxin may come to reality.

It was Carlson (1973) who extended experimental support to this hypothesis he reported that plants regenerated from tobacco protoplasts selected for resistance to methionine sulfoximine (MSO) showed enhanced resistance to Pseudomonas tabaci. Though methionine sulfoximine has not been analogous to the phytotoxin of P. tabaci, this observation opened the door of selecting phytotoxin-resistant cell lines.

(c) Resistance to Herbicides:

Similar to the disease resistance, selection of cell lines resistance to herbicides may also be of direct practical value. In the recent past fertile plants resistant to herbicides such as amitrol, 2, 4-D, sodium chlorate, picloram, chlorsulfuron and sulfometuron methyl have been selected from cultivated cells of Nicotiana.

However, further research on the metabolism of herbicide action on cells and plants is required if the potential for isolating herbicide-resistant phenotypes using tissue/single cell techniques is to be exploited fully.

(d) Resistance to Amino Acids and their Analogues:

Selection of cell lines resistant to amino acids or their analogoues has been stimulated by the prospect of improving nutritional quality of plants. A potato cell line resistant to 5-methyl-tryptophan (5MT) has been used to demonstrate the presence of two possible isoenzymes of anthranilate enthuse the first regulatory enzyme unique to tryptophan biosynthesis. Further 5 MT resistant cell lines were isolated by Jacobsen et al (1985). The cells accumulated free tryptophan and phenylalanine and tyrosine demonstrating the possiblity of manupulating the free amino acid pools of potato cells.

(e) Tolerance to Environmental Stress:

High salt levels in the soil and the low temperatures are the main environmental stress on the distribution of sites where a particular crop can be grown. Scientists are busy in isolating stress-tolerant cells-lines and demonstrating this characteristics in plants regenerated from such cell-lines.

For instance, salt tolerant cell lines of Nicotiana tabacum and N. sylvestric have been isolated. Salt tolerance in N. tabacum is transmitted sexually although at low frequency, but no plants were regenerated from salt tolerant N. sylvestris cells lines. Similarly, low temeprature (cold) tolerant cell lines of N. Sylvestris have been isolated but the characteristic is yet not transmitted sexually.

(f) Secondary Metabolite Production:

A few but well established examples of secondary metabolites production in high amounts by selected cell lines are known. Following table indicates the increased yields so far achieved with products in selected cell-line suspension cultures.

Anthraquinone is commercially obtained from the cortical cells and bark cells of the roots of Morinda citrifolia. This alkaloid is now being obtained from cell line suspension cultures. If calculated, in percentage dry weight, the cell line cultures result in about eight times higher alkaloid content than the whole plant yield.

High amount synthesis of serpentine and ajmalicine by selected cell lines of Catharanthus roseus has been reported. The total content of these two alkaloids in the roots of C. roseus is generally less than 0.5% of their dry weight. The selected cell lines of some strains of this species, however, show an alkaloid content as high as 2%.

Suspension cultures of Coleus blumei have been reported to accumulate rosmarine acid upto 15% of dry weight of the cells which stands five times higher than the alkaloid content in the intact plant.

The suspension culture of Glycoyrrhiza glabra can produce glycoyrrhizin in a fair amount of at least 3-4% of dry weight.

(g) Biotransformation:

Biotransformation of particular substrates to more useful compounds by plant cell is now considered to be one of the most promising area in the biotechnological application of plant cell culture. The projected potentials of the biotransformation lie in the conversion of less expensive and bulk product to value added high cost and low volume products.

It is expected that specific modifications of chemical structures of certain compounds may be performed more easily by cultured plant cells than by microorganisms or by chemical synthesis. The biotransformation of ‘digitoxin’ or ‘(3- methyldigitoxini’ to ‘digoxin’or ‘B-methyldigoxin’ by specific hydroxylation of suspension cultures of ‘Digitalis lantana can carry out biotransformation at 15% conversion in 24 hours and 70% in 7 days. Digoxin is used in cardiac treatment.

Cultured cells of Datura posses a remarkably high capability for glycosylation of hydroquinone to form ‘arbutin’ which finds its use as a diuretic and urinary antiseptic. The diterpenoid stevioside is widely used as natural sweetener (it is 100 times sweeter than cane sugar) but not its aglycone steviol. The cell cultures of Stevia rebaudiana and Digitalis purpurea can biotransform the Steviol to steviolbiocide and stevioside.

(h) Plant Cell Culture and Cotton:

Cotton plants are no longer the only source of cotton. Biotechnologists at Texas University Lubbock (USA) have successfully explored a technique to convert isolated cotton plant cells of any type into fibre cells and to grow cotton fibres in solution.

The resulting cotton is a microorganism free fibre of controllable length, thickness and quality for specific uses. Individual cotton plant cells have been teased apart from plant parts in water solutions.

These isolated cells have been cultured in nutrient solutions containing the hormones auxins, cytokinins and gibberelliris. There has been found a balance of these hormones that caused cells to lose differentiation. Further, changes in the hormonal balance caused undifferentiated cells to become fibre cells. Thousands of such cells on cotton seed surfaces normally grow into individual cotton fibres.