Mycorrhizas as biofertilizers

Mycorrhiza (fungus roots) is a distinct morphological structure which develops as a result of mutualistic symbiosis between some specific root – inhabitating fungai and plant roots. Plants which suffer from nutrient scarcity, especially P and N, develop mycorrhiza i.e. the plants belong to all groups e.g. herbs, shrubs, trees, aquatic, xerophytes, epiphytes, hydrophytes or terrestrial ones. In most of the cases plant seedling fails to grow if the soil does not contain inoculum of mycorrhizal fungi.

In recent years, use of artificially produced inoculum of mycorrhizal fungi has increased its significance due to its multifarious role in plant growth and yield, and resistance against climatic and edaphic stresses, pathogens and pests.

Mechanism of symbiosis:
The mechanism of symbiosis is not fully understood. Bjorkman (1949) postulated the carbohydrate theory and explained the development of mycorrhizas in soils deficient in available P and N, and high light intensity. Slankis (1961) found that at high light intensity, surplus carbohydrates are formed which are exuded from roots. This in turn induces the mycorrhizal fungi of soil to infect the roots. At low light intensity, carbohydrates are not produced in surplus, therefore, plant roots fail to develop mycorrhizas.

Types of Mycorrhizas:

By earlier mycologists the mycorrhizas were divided into the following three groups:
i) Ectomycorrhiza: It is found among the gymnosperms and angiosperms. In short roots of higher plants generally root hairs are absent. Therefore, the roots are infected by mycorrhizal fungi which, in turn, replace the root hairs (if present) and form a mantle. The hyphae grow intercellularly and develop Hartig net in cortex. Thus, a bridge is established between the soil and root through the mycelia.
ii) Endomycorrhiza: The morphology if endomycorrhizal roots, after infection and establishment, remain unchanged. Root hairs develop in a normal way. The fungi are present on root surface individually. They also penetrate the cortical cells and get established intracellularly by secreting extracellular enzymes. Endomycorrhizas are found in all groups of plant kingdom.

iii) Ectendomycorrhiza: In the roots of some of the gymnosperms and angiosperms, ectotropic fungal infection occurs. Hyphae are established intracellularly in cortical cells. Thus, symbiotic relation develops similar to the ecto- and endo-mycorrhizas.

Marks (1991) classified the mycorrhizas into seven types on the basis of types of relationships with the host

(i) Vesicular-arbuscular (VA) mycorrhizas (coiled, intracellular hyphae, vesicle and arbuscules present),

(ii) Ectomycorrhizas (sheath and inter-cellular hyphae present),

(iii) Ectendomycorrhizas (sheath optional, inter and intra-cellular hyphae present).

(iv) Arbutoid mycorrhizas (sheath, inter- and intra-cellular hyphae present).

(v) Ericoid mycorrhizas (only coiled intracellular hyphae, long coiled hyphae present)

(vi) Monotropid mycorrhizas (sheath, inter-and intra-cellular hyphae and peg like haustoria present) and

(vii) Orchidaceous mycorrhizas (only coiled intracellular hyphae present).

Type (i) is present in all groups of plant kingdom; Types (ii) and (iii) are found in gymnosperms and angiosperms. Types (iv), (v) and (vi) are restricted to Ericales, Monotropaceae and Ericales respectively. Types (vii) is restricted to Orchidaceous only. Types (iv) and (v) were previously grouped under ericoid mycorrhizas.

Application of PCR technology

The PCR technology is extensively applied in the following areas of molecular biology, medicines, and biotechnology.

• Amplification of DNA and RNA

• Diagnosis of diseases and casual microorganisms. For example, PCR-based diagnostic tests for AIDS, chlamydia, tuberculosis, hepatitis, human papilloma virus, and other infectious agents and diseases are being developed. The tests are rapid, sensitive and specific.

• Determination of orientation and location of restriction fragments relative to one another.

• The PCR is important in detection of genetic diseases such as sickle cell anemia, phenylketonuria and muscular dystrophy.

• It is most applicable in forensic science where it is being used in search of criminals through DNA fingerprinting technology. However, the feasibility of fingerprinting is now being challenged in court of law. In these cases only small samples of biological materials are required.

• It is also applied in diagnosis of plant diseases. A large number of plant pathogens in various hosts or environmental samples are detected by using PCR, for example, viroids (associated with hops, apple, pear, grape, citrus, etc), viruses (such as TMV, cauliflower mosaic virus, bean yellow mosaic-virus, plum pox virus, potyviruses), mycoplasmas, bacteria (Agrobacterium tumifaciens, pseudomonas solanacearum, Rhizobium leguminosarum, Xanthomonas compestris, etc), fungi (e.g. Colletotrichum gloeosporioides, Glomus spp; Laccaria spp., Phytophthora spp, Verticillium spp), and nematodes (e.g Meloidogyne incoginta, M. javanica, etc) (Henson and French, 1993; Chawla, 1998)

PCR is finding considerable and unique use in archaeology; it is doubtful whether scientists will be able to resurrect woolly mammoth and dinosaurs from the remains of ancient animals as epitomised in Michael Crighton's Jurassic Park.

Antenatal diagnosis of haemoglobinopathies

There are as many as 2,500 genetic defects known to occur in human beings. Some of them (e.g. phenylketonuria and haemophilia) have been cured but most of them are not understood. If a pregnant woman bears a child with a genetic defect, often she is advised to abort it but not to give birth to genetically defected child. Thus, the technique of diagnosis and thereby suggestion for abortion of a genetically defected child is known as antenatal diagnosis. The genetically determined defects of function or synthesis of human haemoglobin is known as haemoglobinopathies.

The inherited haemoglobinopathies are:

1. The structural variants where till now 300 amino acid variants are identified, and
2. The thalassaemias, a condition characterized by reduced or absent synthesis of α and β-globin chain, α and β-thalassaemia respectively.

Methods of antenatal diagnosis:

Antenatal diagnosis is done by taking a few milliliters of amniotic fluid from the foetus of about 16-18 weeks pregnant woman. Foetal blood samples are analyzed by testing for the ability to synthesize globin chains (alpha and beta). Difficulties with the antenatal diagnosis are that the foetus of less than 3 months of age synthesize very little α or β- globin chains. At this stage the foetal blood contains normal level of α and low level of β-chain. Synthesis of β-chain takes place in low amount in foetus of 8 weeks age, and therefore, it is possible to analyze the globin in a foetus but difficult to analyse β-chains (Little, 1981).

The foetus fluid also contains foetally derived fibroblasts which serve, with or without additional culture, as a source of tissue for preparation of foetal DNA. Structural analysis of DNA is done by (a) isolation of all the globin genes as recombinant DNA molecules, and cloning in bacteria, or (b) without cloning the recombinant DNA in bacteria by “Southern Blotting Techniques” (Southern, 1975), and (c) linked polymorphism. A detailed account of DNA analysis of structural genes is given by Little (1981). There are about 35 diseases which have been identified by antenatal diagnosis. This method helps in suggesting the abortion of defected baby, and if possible, to cure. It can be recommended for gene therapy as well. Thus, the antenatal diagnosis is applied for both genetic counseling and gene therapy.

Genetic counseling:

Genetic counseling is a technique which carried out in detail based on antenatal diagnosis and suggested for the possibilities of future progeny. A genetic disease is first identified by a genetic counselor who understands the family history of genetic disease, and then suggests for the possibilities of giving birth to the child or aborting the child. The child may be normal, genetically defected or carrier of genetic disease. Take the case of thalassaemia. Alpha thalassaemia is caused by the presence of two chromosomes each of them carries one dysfunctional globin gene or one normal chromosome and other dysfunctional genes. Undoubtedly, a person carrying two chromosomes each with single functional α-genes donates one normal genes to the progenies. It is, therefore, impossible to suffer their progenies from ‘hydrops fetalis’ as it occurs due to complete dysfunctioning of α-genes. DNA analysis of thalassaemia has revealed that non-function of both a genes on α single chromosome is only due to deletion of both genes from the chromosome.

Recent techniques have made it possible to detect α-thalassaemic couple for the presence of no gene chromosome. However, there is risk for giving birth to hydropic fetuses if both of the couple have α-thalassaemia due to no gene chromosome (Little, 1981). If there lies any possibility for gene therapy the patients are also suggested for the same. It has also been suggested that one should go to genetic counselor before marriage for safe and normal progeny.

Electrophoresis - Seperation and purification of DNA fragments

Electrophoresis refers to carrying something by applying electricity. It is an analytical device commonly used for separation and purification of DNA fragments. A gel is used in electrophoresis which is either polyacrylamide or agarose. The former is preferred for smaller DNA fragments and the latter for larger ones. Agarose is a purified powder isolated from agar, a gelatinous material of sea weeds. Agarose powder when dissolved in water and boiled results into gel form. The gel prepared in a mixture of salt and water becomes a good conductor of electricity. The gel forms small pores the size of which varies depending on its amount in a given water. These pores act as molecular sieve. These allow the larger molecules to move slowly than the smaller molecules.

The electrophoresis box consists of a positive and a negative electrode, a shelf designed to held the gel, a comb used to form the wells within the gel, and a power supply. The DNA to be electrophoresed is digested with restriction enzymes which yields DNA fragments of unequal length. The fragments are mixed with sucrose and a dye (ethidium bromide or methylene blue) which altogether is known as loading dye. Sucrose increases the density of DNA preparation and dye increases the visibility of the preparation.

The preparation is loaded into wells at one end of the gel. At least one well is filled with reference DNA (i.e. DNA fragments of known length) for comparison with those of unknown length. Electric current is applied at opposite ends of electrophoresis chamber. A current is generated between a negative electrode at the top of loading end of the gel and a positive electrode at the bottom of the end of gel resulting in movement of fragments through pores of the gel. DNA molecules have a negative electric charges due to PO4(4-) which alternate with sugar molecules. Opposite electric charges tend to attract one another. The small DNA molecules move at faster speed as compared to larger ones. All DNA molecules of a given length migrate nearly the same distance into the gel and form bands. Each band represents many copies of DNA fragments having about the same length. After completion of electrophoresis gel is removed from the chamber and stained to make bands easily seen either with ethidium bromide (EB) or methylene blue. When gel is illuminated with UV light, fluorescent orange bands appear due to EB; methylene blue results in blue bands under normal room temperature.