Plant Biotechnology & Nitrogen Cycle Processes
Plant Tissue Culture: Techniques and Applications
Plant tissue culture is the technique of growing plant cells, tissues, and organs in an artificial medium under aseptic conditions.
Applications of Plant Tissue Culture
(a) Clonal Multiplication (Micropropagation)
Tissue culture is a rapid and cost-effective method of multiplication. Many agricultural and horticultural plants are multiplied by this method, also known as micropropagation. Raising plants by this method offers the following advantages:
- Disease-free stocks can be prepared.
- It is a rapid and economical method for preparing clones.
- Hybrids can be raised by micropropagation without the risk of segregation.
- In cases where seeds are not formed, it is the only and ideal method of multiplication.
(b) Elimination of Pathogens
Molecular probes and monoclonal antibodies are used for the detection of pathogens. ELISA (Enzyme-Linked Immunosorbent Assay) is a technique for virus detection applicable in both plants and animals. ELISA tests are economical, rapid, and readily adaptable for qualitative measurements. Virus-free plants have been produced by this method in more than 80 species.
(c) Isolation of Desirable Mutants
Successful and effective isolation of mutants has been achieved in many cases. Disease resistance was the first trait of agronomic importance to be achieved using in vitro culture. Herbicide resistance is another significant area in this field. Resistant cell lines have been developed for phenoxy herbicides. Resistant cell lines and plants are also used for cloning a resistance gene and inserting it into a crop species. Cell lines with increased cold resistance have been obtained in Nicotiana and Daucus roots.
(d) Somaclonal and Gametoclonal Variations
The variations observed in cultured somatic cells or tissues are termed somaclonal variations. Variations observed in gametes, such as microspores, are called gametoclonal variations. Somaclonal variations in sugarcane have been obtained for cultivars resistant to diseases such as eye spot disease (Helminthosporium oryzae) and downy mildew (Sclerospora sacchari). Variations have been successfully obtained in Geranium, leading to the release of a new cultivar, ‘Velvet Rose’. In gametoclonal variations, recessive mutations are immediately expressed. However, in gametoclonal cultures, the chromosome number is typically required to be doubled.
Nitrogen Fixation in Plants
Members of the family Leguminosae, such as beans, gram, groundnut, and soybean, bear small nodule-like swellings on their secondary, tertiary, and sometimes primary roots. The size of these nodules depends upon cell division and cell enlargement. The nodules have a limited lifespan. Rhizobium penetrates into the cortex of the root through an infection thread. Simultaneously, cortical cells of the root are stimulated to divide more vigorously to form nodules on the root. Neither the bacterium nor the plant alone can fix nitrogen in such cases; nitrogen fixation is actually the outcome of a symbiotic relationship between the two.
The pigment leghaemoglobin imparts a pinkish color to nodules. This pigment is closely related to hemoglobin and is helpful in creating an optimal condition for nitrogen fixation. Like hemoglobin, leghaemoglobin is an oxygen scavenger. Fixation of nitrogen is achieved with the help of nitrogen-fixing genes (nif genes) that produce a special enzyme, nitrogenase. This enzyme functions under anaerobic conditions. Leghaemoglobin combines with oxygen and protects nitrogenase.
Enzyme Nitrogenase
Nitrogenase is a special enzyme produced by nif genes in plants. It functions under anaerobic conditions. The anaerobic environment in the nodule is created by leghaemoglobin, which acts as an oxygen scavenger.
Nitrogenase is a complex of two enzymes: a molybdenum-iron protein and an iron protein. The iron protein part gets damaged in the presence of oxygen. In this process, the dinitrogen molecule is progressively reduced by the addition of pairs of hydrogen atoms. Depending upon the type of nitrogen fixer, respiration or photosynthesis may be the source of ATP and reducing agents like ferredoxin. Ammonia thus formed is used for the synthesis of amino acids.
Nitrification Process
Plants derive nitrogen from the soil in inorganic forms such as nitrates (NO3–) or ammonium (NH4+). The soil typically contains about 0.05% nitrogen. Once ammonia has been produced, it is converted into nitrates by nitrifying bacteria, a process called nitrification.
Soil bacteria such as Nitrosomonas and Nitrococcus convert ammonia into nitrites:
2NH3 + 3O2 → 2NO2– + 2H+ + 2H2O (catalyzed by Nitrosomonas)
Nitrites are then oxidized into nitrates by Nitrobacter:
2NO2– + O2 → 2NO3– (catalyzed by Nitrobacter)
These nitrifying bacteria are actually chemoautotrophs. They utilize the energy released during oxidation for their various metabolic activities.