SOMACLONAL VARIATION

Somaclonal variation is defines as the genetic variability present among cultured somatic cells.
Plants derived from such cells or progeny of such plants are called somalcones.
The term somaclonal variation was first used by Larkin and Scowcroft in 1981.
Somaclonal variations can be selected for disease resistance, improvement of nutritional quality, adaptation of plants to stress conditions, resistance to herbicides etc.
Somaclonal variation has been observed in plants such as apple, sugarcane, potato, tomato etc.

PROCEDURE FOR ISOLATION OF SOMACLONAL VARIANTS


Isolation of somaclonal variants can be grouped into two broad categories. They are:
1. Screening
2. Cell selection Screening

This involves the observation of large number of cells/plants from tissue culture and detection of variants.
In general R1 progeny (progeny of regenerated R0 plants) are used for the identification of variant plants.
R2 progeny (progeny of R1 plants) are used for confirmation. This has been employed for a number of plants.
Computer based automated cell sorting devices have been used to screen as many as 1000-2000 cells/second from which variant cells can be automatically separated. These variant cells are further regenerated to produce complete plantlets.
This approach is widely used for the isolation of variants which produce high yield and desirable traits.
Also used to obtain cell clones that produce higher quantities of certain biochemical agents.

Cell Selection
In this method an appropriate selection pressure is applied which permits the survival/growth of vibrant cells only during culture.
When the selection pressure allows only the variant (mutagenic) cells to survive, it is called positive selection.
In negative selection, the selection pressure allows only the wild type cells to survive. These wild type cells are later killed by counter selection pressure. The variant cells are rescued by the removal of counter selection agents.
Positive selection approach maybe further sub-divided into 4 categories,

a) Direct selection
b) Rescue selection
c) Step wise selection
d) Double selection

Direct selection
In this method the selection agents kills the wild type cells. The mutant (variant) cells remain unaffected. The mutant cells continue growing and in dividing the medium. This is the most common method employed to obtain variants that are resistant to toxins, herbicides, antibiotics, high salt concentration etc.
Rescue selection
In this method the selection agent kills the wild type cells. The mutant (variant) cells remain alive but do not divide due to unfavourable environment created by the selection agent. The selection agent is then removed to recover the variants. This method is used to obtain variants that are resistant to aluminium, cold temperature etc.

Stepwise selection

In this method the concentration of selection agent is increased in a stepwise manner. Eg, to obtain variants those are resistant to high salt concentration. At first low concentration of salt is added to the medium. Those cells which survive are then subjected to higher salt concentration, those cells which are survive at this concentration are further subjected to higher salt concentration and so on.

Double selection

In this method variants with two traits are selected simultaneously with the same selection agent. E.g.; Selection of a variant, which shows antibiotic resistance (streptomycin resistance) and development of chlorophyll. Here streptomycin resistance is the first trait and development of chlorophyll in the cells is the second trait.

ADVANTAGES OF SOMACLONAL VARIATIONS

1. Somaclonal variations are stable and occur at high frequencies.
2. Somaclonal variations may show novel mutations.
3. Can be performed in all types of cells, ie; vegetatively or sexually or asexually propagated plants.
4. Somaclonal variations may reduce two years the time required for the release of new variety compared to mutation breeding.
5. Only approach for the isolation of biochemical mutatants.
6. It is an effective method.

DISADVANTAGES OF SOMACLONAL VARIATIONS

1. Somaclonal variation is applicable to only those species which can regenerate complete plants
2. Many Somaclonal variants show undesirable features such as reduced fertility, growth rate etc.
3. The variation is not always heritable
4. The variation is generally cultivar dependent
5. Selected clones show unpredictable and uncontrollable variants.

BASIS OF SOMACLONAL VARIATION

A number of factors are responsible for somaclonal variation.
Gene mutations such as translocations, deletions, inversions
Pre-existing chromosomal ploidy in the explants
Number fragmentation at callus induction stages.
Changes in gene expression and gene amplification.

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MICROPROPAGATION

Generally plants propagate by sexual and asexual methods.
Sexual method: here fertilization of ovary taken lace pollen grains and the resulting plants show high degree of heterogenecity.
Asexual Methods: here the cells undergo mitosis and the resulting plants will be genetically identical to parents.
Multiplication of genetically identical copies of plants is known as clonal propagation.
Clonal propagation of plants through tissue culture is known as Micropropagation.
Explants (starting material used to initiate tissue culture
The explants widely used for tissue culture include
a) Meristem
b) Shoot-tip
c) Auxiliary buds
a) Meristem
This is the terminal portion of shoot tip containing a group of actively dividing cells.
b) Shoot-tip
Shoot-tip/ shoot apex also contains a group of actively dividing cells with one-three leaf premordia.
c) Auxiliary buds
These are actively dividing cells present in the axile portion of the node.
Murashige in 1978 recognized four stages (stage I, II, III, IV) for Micropropagation. Stage I, II, III is performed in-vitro. Later in 1981, Debergh and Maene introduced stage 0 for Micropropagation.
The stages include:
a) Stage 0:- selection of stock plant
b) Stage I: - Establishment of aseptic culture.
c) Stage II: - multiplication of explant on defined medium.
d) Stage III:- Rooting
e) Stage IV: - hardening
Stage 0
Stock plants having derived charades are selected.
Maintained in controlled environment conditions for 3 months
They are grown in low humidity, irrigation and without systematic microbial infection.
Stage I
The selected explants (derived from stock plants) are prepared for inoculation.
The explants are surface sterilized by using chemicals such as 0.1% Hgcl2 or 5% sodium hypochlorite or 70% alcohol or a combination of all these chemicals.
These surface sterilized explants are then inoculated onto MS medium supplemented with vitamins, sucrose and growth regulators.
These cultures are incubated at 3000-10000Lux light intensity with 16hrs photoperiod.
Note: Auxin stimulates callus formation.
Cytokinin (1 – 3 mg/l BAP) is good for Micropropagation.
Stage II
This is the longest period.
Single shoots develop from apical shoots.
These are excised into nodal explants.
The nodal explants are further inoculated on cytokinin medium to proliferate multiple shoots.
Note: Multiple shoots can also be obtained directly from explants by organogenesis or somatic embryogenesis)
In most plants, explants are known to produce 1-3 shoots in 4-5 weeks. This would give upto 510-612 plants in one year if all plants survive.
Stage III
Individually produced shoots in stage II are inoculated in fresh medium (with auxins) for rooting.
Or in some cases rooting is induced directly in the soil in high moisture condition.
In case of somatic embryos, they are allowed to germinate in the medium and then transferred to soil.
The plantlets obtained are slowly transferred to soil for hardening.
Stage IV
The plantlets are first prepared for soil conditions by keeping them in medium containing peat /vermiculite/fearlite which holds more moisture.
This makes plants to become resistant to moisture, stress and disease making plants completely autotrophic from their heterotrophic.
The plantlets are protected from direct sunlight.
Humidity is gradually decreased.
During this period, plants will form well developed roots and the aerial tissues will for cuticular wax.
Thus, the plants acclimatize themselves and become suitable for transfer into the field.
Note:
Some species grow in vitro (in tab) from brittle, glassy and water soaked shoots and this is known as vitrification.
Vitrification is due to poorly developed vascular bundles, abnormal functioning of stomata etc.
This can be overcome by addition of high concentration of agar (1%), bottom cooling of culture tubes etc.
Applications of Micropropagation
1. Alternative method of vegetative propagation.
2. A small amount of plant tissue is sufficient to produce millions of clones in a year
3. Requires less space for large number of plants.
4. Plants with high yield and vigour can be obtained.
5. Disease free plants are produced from this method.
6. Helps in germplasm storage and saving of endangered species.
7. Provides speedy international exchange of plant material.

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Basic Structure of an Amino Acid

Basic Structure

All amino acids found in proteins have this basic structure, differing only in the structure of the R-group or the side chain.

The simplest, and smallest, amino acid found in proteins is glycine for which the R-group is hydrogen (H).

L-isomer
In proteins, only the L-isomer is found normally.

As you travel onward (from the carbonyl carbon to the amino group), the R group of L-amino acids will be on the left as shown in the molecular graphic on the right

Essential amino acids

Humans can produce 10 of the 20 amino acids. The others must be supplied in the food. Failure to obtain enough of even 1 of the 10 essential amino acids, those that we cannot make, results in degradation of the body's proteins—muscle and so forth—to obtain the one amino acid that is needed. Unlike fat and starch, the human body does not store excess amino acids for later use—the amino acids must be in the food every day.

The 10 amino acids that we can produce are alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine. Tyrosine is produced from phenylalanine, so if the diet is deficient in phenylalanine, tyrosine will be required as well. The essential amino acids are argentine (required for the young, but not for adults), histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. These amino acids are required in the diet. Plants, of course, must be able to make all the amino acids. Humans, on the other hand, do not have all the enzymes required for the biosynthesis of all of the amino acids.

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