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