SUSPENSION CULTURE

Individual cells or cell aggregates dispersed and growing in moving liquid media is known as suspension culture.

Explants used in suspension culture
In regular practice suspension culture is initiated by transferring piece of undifferentiated and friable cells to a liquid medium which is continuously agitated by a rotator shaker.
Suspension cultures have also been started from sterile seedlings or imbibed embryos or leaves. Leaves and other tissues can be gently grinded using homogenizer. This homogenate containing living cells, dead cells and cell debris is cleared by filtration and centrifugation and then transferred to a liquid medium. This is the mechanical method
In enzymatic method, pectinases (enzymes which digest pectin cell wall) are used for isolation of single cells.
Method
At first stage culture are initiated by placing freshly cut sections of plant organs (root, stem, leaves) on a solidified nutrient medium
In this condition explants is transferred a liquid medium to obtain callus.
The callus is transferred to a liquid medium and agitated to obtain a fine suspension of cells.

Medium for suspension culture
A wide variety of media compositions have been used for suspension culture. These include MS, B5, LS (linsmaier and skoog) and Blaydes medium. For these media vitamins, inositol, glucose and growth regulators are incorporated.
Orbital shakers
In suspension cultures, orbital shakers are widely employed for initiation and serial propagation of plant cells. They should have a variable speed control (30-150rpm). They serve three main purposes.
They exert a mild pressure breaking the cell aggregates into single cells.
Agitation maintains uniform distribution of cells in the medium.
Movement of the medium provides good gaseous exchange.
Culture vessels
The Erlenmeyer flasks are commonly used as culture vessels. The volume of the culture medium should be appropriate to the size of the culture vessel:
Eg; 20ml/100ml flask
70ml/250ml flask
The flasks are normally sealed with aluminium foil.
Types of suspension culture:
1. Batch culture
2. Continuous culture
1. Batch culture:
The cell suspension culture grown in a fixed volume of nutrient medium is taken as batch culture. This is also known as closed system because the cells are incubated within a single batch of medium. The cells exhibit 5 phases of growth cycle. They are:
a. Lag phase
b. Log phase
c. Linear phase
d. Deceleration phase
e. Stationary phase

Lag phase:
Here the cells prepare to divide. It is the initial period pf the batch culture, where no cell division occurs, but the cells are metabolically active and the cell size increases, due to the synthesis of various components.
Log phase:
Here the rate of cell division is highest; here the cell division is exponential as the result there is an increase in cell number.
Linear phase
Here the cell division slows, but the rate of cell expansion (cell elongation) increase. After 3-4 generations, the cell growth declines.
Deceleration phase:
Here the rate of cell division and cell elongation decreases.
Stationary phase:
Here the number and size of cells remains constant. The doubling time in suspension culture varies for 24-48hrs. Cells should be sub cultured at weekly intervals.

2.Continuous culture:
Is one in which in flow of fresh medium is balanced by outflow of culture. In continuous culture the growth rate of cells and cell density are held constant by a fixed rate of addition of growth limiting nutrients and removal of cells and spent medium is; as there is increase in the cells, there is depletion of nutrients, therefore fresh medium is added and simultaneously equal volume of culture is removed. Hence this is also called as open system.



Continuous culture systems are of 2 types
1. Chemostat 2. Turbidostat

1. Chemostat
In this system growth rate and cell density are held constant by a fixed rate of a growth stimulating nutrient (nitrogen, phosphorous, glucose). In such a medium all constituents other than growth limiting nutrients other than growth limiting nutrient are present at a higher concentration than that is required.

2. Turbidostat
When there is an increase in turbidity of the culture, fresh medium is added and an equal volume of culture is removed.

Applications:
1. Production of secondary metabolites
2. Major source for obtaining individual cells which can be used for protoplast fusion and hybrid production.
3. Suspension cultures are used for induction of mutation and genetic manipulation of plant cells.
4. They can be preserved easily.

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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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