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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Posted in: Biotechnology

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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Posted in: Biotechnology

Chemical composition of eukaryotic chromosome

The eukayotic chromosome is composed of DNA, basic proteins called histones, non-histone proteins(involved in transcription, replication, repair and recombination of DNA) and RNA . The histone proteins are basic proteins rich in arginine and lysine. They are of 5 types-H1, H2A, H2B, H3 and H4.
Eight molecules two each of H2A, H2B, H3 and H4. From an ellipsoid core (11nm long and 6.5-7nm in diameter) around which about 147-166bp of DNA coils in 13/4 turns. This DNA histone complex is called nucleosome, the building block of chromosome, the building block of chromosome found as repeating units. The nucleosomes appear as a string of beads in the chromatin. About 14-100bp of DNA between these beads forms the LINKER region.
The hsitone H1 associates with the linker to aid folding of DNA into a more complex chromatin (in the form of 10nm zigzag or 30nm solenoild fibire). DNA enters and exits the nucleosome at sites close to each other and two turns of turns of DNA are stabilized and “sealed off” by H1, during cell division there is maximum folding of the chromatin and hence visible as chromosomes.

Polythene chromosomes:

In certain tissues of insects belonging to the order dipteral (flies, mosquitoes). The cell nuclei have reached a high degree of enlargement accompanied by many extra replications of each new chromosome within a single nucleus (endopolyploidy), however instead of each new chromosome separating as an individual unit all replicates of the same chromosome are lined up together in parallel fashion. This parallel duplication or polyteny, results in very think chromosomes that magnify any differences in density along their length (eg chromosomes). The numbers of bands varies between different species but are constant for the member of any particular sp.
In polyteny the two homologous chromosomes of each diploid pair are also often lined up side by side (somatic pairing) so that if the total diploid number of chromosomes id eight (4 pairs). Only four very thick and long chromosomes appear. The same chromomere in many paired chromatids may expand to form “puffs”. Polytene chromosomes were alilized in genetic research first by painter in 1933.

Lampbrush Chromosomes

The oocytes of some vertebrates with large yolky eggs expand greatly during their growth period, forming correspondingly large nuclei at these stages. In some aphibia the meiotic prophase chromosomes of such nuclei can reach about 1000 μm in length with long lateral loops giving a hairy “lampbrush”. Each pair of loops arise from single chromosomes located at short intervals along the very thin and double stranded chromosome. Towards the end of meiotic prophase the loops begin to disappear and the chromosomes contract, so that the metaphase bivalents are of the usual small size.

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Posted in: Microbiology

Chromosome of Cyanobacteria

The DNA in cyanobacteria is organized into a complex helical and folded structure and is distributed uniformly throughout the cytoplasm. It is likely that DNA is associated with histone like proteins and RNA. The size of the genome varies widely in cyanobacteria with molecular weight ranging between 1.6x109 and 8x109 daltons. Most of the unicellular cyanobacteria possess genomes of about 1.6x109 – 2.7x109 daltons. Filamentous cyano bacteria how ever have larger genomes.
Viral Genome
The core of the virion is made up of nucleic acid, either DNA or RNA never both. Four types of nucleic acid are formed in viruses with reference to number of strands. They formed in viruses with reference to number of strands.
They are:
1- Single stranded DNA ( ssDNA) Eg.Ø X 174
2- Double stranded DNA (dcDNA) Eg. Herpes virus
3- Single Stranded RNA (ssRNA) eg. TMV
4- Double stranded RNA (dsRNA) eg. Reovirus
The ssDNA may be linear (Parvovirus) or circular (Ø X 174). The ssDNA becomes double-stranded during replication when it is called replicative form (RF).
Double stranded DNA (dsDNA) is found in many animal viruses and bacteriophages. It maybe linear (in bacteriophages), cross-linked (Vaccinia virus) or closed circular duplex (Papova virus)
Single- stranded RNA (ssRNA) found in a variety of animal viruses and plant viruses maybe plus (infectious) RNA as in RNA bacteriophage, togaviruses etc or minus (non- infectious) as in rhabdoviruses and paramyxoviruses. Plus ssRNA directly acts as mRNA and is translated to proteins on host ribosomes where as minus ssRNA first transcribe in mRNA through an RNA/DNA intermediate and then gets translated to proteins.
Double- stranded RNA is found in animal viruses like reovirus.
In general, plant viruses have RNA (ss/ds) as genetic material except for canlimovirus and geminivirus which contain DNA. Animal viruses have DNA (ds) and RNA (ss/ds). Bacteriophages have DNA (ss/ds) or RNA (ss/ds). Most phages are DNA viruses.

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Posted in: Microbiology

CHROMOSOME

The term chromosome was introduced by waldeyer in 1888. The identification of chromosomes as vehicles of hereditary was put forth by Sutton, boven and others in 1903 - chromosome theory of hereditary. The chromosomes are seen as microscopic thread-like structures in the nucleus of eukaryotes and in the nucleotide region of prokaryotes.

Prokaryotic chromosome

Prokaryotic cell contains neither a distinct membrane around nucleus nor a mitotic apparatus. The Prokaryotic chromosome, almost always a single circle of double stranded DNA is located in an irregularly shaped region called the nucleoid (also called the nuclear body, chromatin body, nuclear region). The nucleoid can be observed under the electron microscope and also under the light microscope after staining with the Feulgen stain, which specifically reacts with DNA. A cell can have more than one nucleoid when cell division occurs after DNA replication. Some bacteria like Agrobacterium tumefaciens are found to have more than 4 chromosomes.

Sinorhizobium meliloti is found to have 3 chromosomes. But usually, most prokaryotes are haploid with only a single chromosome.

In actively growing bacteria, the nucleoid has projections that extend into the cytoplasmic matrix. These may contain DNA that is being actively transcribed to produce mRNA.

Electron microscopic studies have shown the nucleoid in contact with either the mesosome or the plasma membrane which maybe involved in separation of DNA into daughter cells during division. Chemical analysis of nucleoids reveals that they are composed of about 60% DNA, some RNA and a small amount of protein.

In E.coli, a rod shaped bacterium of about 2-6 m length and 0.5- 1.5 m diameter, the closed DNA circle measures approximately 1400 m representing 4x10⁶ bp. Therefore it needs to be efficiently packaged to fit within the nucleoid. The DNA is looped and coiled extensively (about 45 supercoiled loops) probably with the aid of nucleoid proteins which differ from the histone like protein (Hu) have been found to be associated with isolated DNA. Perhaps these proteins act as repressors and prevent some sections of the chromosome from being transcribed.

Recently electron microscope observations have shown the presence of nucleosome-like structure in the E.coli chromosome. DNA is found to constitute 80% by weight of the chromosome and in addition proteins (10% largely RNA polymerase enzyme) and RNA (30% newly transcribed mRNA, tRNA, rRNA) core is present which acts as a scaffold and holds the loops and determines their position. The chromosome appears to be a highly regular. Then negative charge of the DNA neutralized by polyamines such as spermine and spermidine and by Mg²⁺ as well as basic proteins. A dense region containing membranous material is seen in the central part and is likely to represent fragments of plasma membrane,(mesosomes), to which the chromosomes is attached in the intact cell. The DNA has in addition to the four usual bases small amounts of methylated bases such as 6-methylaminopurine and 5-methylcytosine.

In archaeobecteria like Thermoplasma acidophilus, DNA is found condensed by wrapping around a proteinaceous core, quite unusual for a prokaryote. It contains a single, small, basic histone-like protein (termed HTa) that forms nucleosome like structure containing a core of four molecules of HTa around which a 40 base pair length DNA is wrapped.

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Posted in: Microbiology