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In Vitro Culture Techniques : The Biotechnological Principles

 
     
 
Somaclonal Variation
The genetic heterogeneity of cells in a population represents continuity of genotypes, whereas phenotypically the population is represented as a discrete sum of subclones (Carlson, 1983). After cloning a single cell, from the population of a strain of Dioscorea deltoidea, there developed a sub populations that differed in their growth rate and sapogenin (diosgenin and yamogenin) content. The traits varied independently. In one clone of 12 studies a high growth rate was combined with a high diosgenin content (Butenko, 1985).

The genetic nature of variability in cultured cells explains the appearance of a large number of somaclonal variants with heterosis among the regenerated plants (Table 8.3). In 1981, P.J.Larkin and W.R.Scowcroft at the Division of Plant Industry, C.S.I.R.O., Australia gave the name somaclonal variation to genetic variability generated during tissue culture. They explained that it may be due to (i) reflection of heterogeneity between cells and explant tissue, (ii) a simple representation of spontaneous mutation rate, or (iii) activation by culture environment of transposition of genetic materials. The mass occurrence of somaclonal variants, increase in the resistance, productivity and vital force of the plant (the heterotic effects) have been explained by Carlson (1983). This would be due to dominance of nonlethal mutations that lead to heterozygosis with a wild type allelle which is thus phenotypically expressed as hybrid heterosis.

One of the most complex and difficult problems involving the population of cultured cells is the problem of retention and expression of the trait of totipotency i.e. the ability to realize the programme of development from cell to plant. The genetic variability of cultured cells is the basis for obtaining somaclonal variants of the plants that have valuable traits. But this variability makes it difficult to realize the results of cell and genetic engineering as the trait may be lost in the row of cell generations leading to the regeneration of the plant. Stability is an essential requirement for clonal micropropagation and stabilizing selection in plant breeding (Butenko, 1985). 

Most significant achievement of somaclonal variation was made by Shepard and co-workers in old variety of potatoes. Shepard et al. (1980) screened about 100 somaclones produced from leaf protoplasts of Russet Burbank and found a singnificant and stable variation in compactness of growth habit, maturity, date, tuber uniformity, tuber skin color and photoperiodic requirements. The characters of greater, tuber uniformity and early onset of tuberization were agronomic improvements over the parent variety. Moreover, somaclonal variation is applicable for seed propagated plants only e.g. rice, wheat, maize, tobacco, etc. not for vegetatively propagated species. 

Thus, somaclonal variation has proved an alternative tool to plant breeding for generating new varieties that can exhibit disease resistance and improvement in quality and yield in plants such as cereals, legumes, oil seeds, tuber crops, fruit crops, etc. (Bhaskaran, 1987).
 
 

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

In 1984, P.J.Larkin and coworkers have performed a detailed genetic analysis of. wheat somaclonal variation, coupled with qualitative and quantitative assessment for yield and other attributes of commercial importance. The main findings are as below : (i) heritable variation is encountered for characters e.g. height, awns, tiller number, grain color, gliadin protein and amylase regulation, (ii) besides morphological variation, numerical and structural aberration in chromosome occur, (iii) morphological variations may not be correlated with chromosomal variation, (iv) the occurrence of translocations has a major scope for exploitation by introgression of genes from wild relatives by culture of immature embryos of wide crosses, and (v) the somaclonal variation holds great scope for creating utilizable germplasm for crop improvement.

Table 8.3. Somaclonal variation in some plants.

Species

Explants

Variant traits

Brassica spp.

(Brassicaceae)

Anther, embryo, meristem

Flowering time, growth habit, wax

Nicotiana tabacum

(Solanaceae)

Anther, protoplast, leaf callus

Plant height, leaf size, alkaloids

Oryza sativa

(Poaceae)            

Embryo

Plant height, tillering number, panicle size, seed fertility, flowering date

Saccharum officinarum

(Poaceae)            

Various

Auricle length, sugar yield, pathogenic disease

Solarium tuberosum

(Solanaceae)

Leaf callus, protoplast

Plant habit, disease resistance and shape, yield and maturity date of tubers

Triticum aestivum

(Poaceae)

Immature embryo

Plant height, spike shape, maturity tillering, cc-amylase, leaf wax

Zea mays

(Poaceae)

Immature embryo

Endosperm and seedling mutant, mtDNA sequence rerrangement

Source : Scowcroft (1984).

One of the most complex and difficult problems involving the population of cultured cells is the problem of retention and expression of the trait of totipotency i.e. the ability to realize the programme of development from cell to plant. The genetic variability of cultured cells is the basis for obtaining somaclonal variants of the plants that have valuable traits. But this variability makes it difficult to realize the results of cell and genetic engineering as the trait may be lost in the row of cell generations leading to the regeneration of the plant. Stability is an essential requirement for clonal micropropagation and stabilizing selection in plant breeding (Butenko, 1985).

Most significant achievement of somaclonal variation was made by Shepard and co-workers in old variety of potatoes. Shepard et al. (1980) screened about 100 somaclones produced from leaf protoplasts of Russet Burbank and found a singnificant and stable variation in compactness of growth habit, maturity, date, tuber uniformity, tuber skin color and photoperiodic requirements. The characters of greater, tuber uniformity and early onset of tuberization were agronomic improvements over the parent variety. Moreover, somaclonal variation is applicable for seed propagated plants only e.g. rice, wheat, maize, tobacco, etc. not for vegetatively propagated species.

Thus, somaclonal variation has proved an alternative tool to plant breeding for generating new varieties that can exhibit disease resistance and improvement in quality and yield in plants such as cereals, legumes, oil seeds, tuber crops, fruit crops, etc. (Bhaskaran, 1987).

In 1984, P.J.Larkin and coworkers have performed a detailed genetic analysis of. wheat somaclonal variation, coupled with qualitative and quantitative assessment for yield and other attributes of commercial importance. The main findings are as below : (i) heritable variation is encountered for characters e.g. height, awns, tiller number, grain color, gliadin protein and amylase regulation, (ii) besides morphological variation, numerical and structural aberration in chromosome occur, (iii) morphological variations may not be correlated with chromosomal variation, (iv) the occurrence of translocations has a major scope for exploitation by introgression of genes from wild relatives by culture of immature embryos of wide crosses, and (v) the somaclonal variation holds great scope for creating utilizable germplasm for crop improvement.

In Hawaii, tissue culture is integrated in the breeding programme and somaclonal variation has been exploited for the isolation of clones resistant to certain diseases, for example viruses, downy mildew and eye spot disease. Some of the resistant clones have advantages in having higher sugar content and yield than parental ones.

In India, Sugarcane Breeding Institute, Coimbatore has released varieties produced through the process of somaclonal variation.

Bio-13. In India, a somaclonal variant of Citronella Java, a medicinal plant has been released as 'Bio-13' for commercial cultivation by Central Institute for Medicinal and Aromatic Plants (CIMAP), Lucknow. Bio-13 yields 37% more oil and 39% more citronellon than the control variant.

Supertomatoes. Heinz Co. and DNA Plant Technology Laboratories (USA) developed Supertomatoes with high solid component by screening somaclones which reduced shipping and processing costs. According to an estimate made in 1986, in the USA alone, for every percent increase in tomato solid processors would save about 100 million dollars per annum.

Oxalate removal. Through selection of somaclonal variants, nutritional stress factors have been estimated in some vegetables. First application of this technique was shown in Amaranthus gangeticus, a leafy vegetable.

 
     
 
 
     



     
 
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