STRESS TOLERANT PLANTS
This project is the result of the result of many people's dedication. I Vishal Sehgal of B.Tech Biotechnology would like to appreciate the valuable advice, suggestion and cooperation received from Mr. Harsh Kumar (lecturer), as I am able to complete this project under her guidance. Finally, I will like to thank all of those who helped me and stood by me in completion of this project.
Agriculture in INDIA has to maximize its efficiency because INDIA has to support 16% of word food needs in the available less than 2% land of the country. This can only be done by understanding and engineering the plants to make them survive in adverse conditions.soil has been called the "placenta of life" , because it supplies essential nutrients to all land plants and plants in turn serves the entire terrestrial ecosystem.
Plants have evolved genetically based physiological strategies to react to the changing environmental conditions, be they biotic or abiotic. Most plants complete their life cycle in a single location and are therefore plagued by challenges such as nutrient acquisition, pathogen attack, and environmental stresses.
Environmental factors which limit plant exploitation of the environment include Light, Oxidative Stress, Cold, Heat, Nutrition, Water, Salinity, Toxic concentrations of Metals and Pathogens.
Agricultural plant science has two main goals:
1. To increase the yield and,
2. To improve the protection of crops from diseases or limitations caused by pathogens , insects, nutrients and stress.
One way of doing this is to develop crops that are more tolerant to such stresses as drought , flooding, heat, radiation, salinity, chilling, and freezing, so that new land can be brought under cultivation
- With the use of stress tolerant plants, there will be the possibility of using a lower quality of irrigation water with a higher salt content.
- It may be possible to use less water over the course of the season which would be an important factor as farmers face increasing competition for water resources from municipal users. There are plants such as cacti and mosses that are naturally capable of surviving in hot, dry regions.
Most crops are salt sensitive or hypersensitive (glycophytes) in contrast to halophytes, which are native flora pf saline environment, halophytes have the capacity to accommodate extreme salinity, because of various special anatomical and morphological and physiological adaptations or avoidance mechanisms.
Plants which grow and complete their life cycle in a habit with a high salt concentration ae known as halophytes and are commonly found near sea shore where the concentration of salts are relatively high.
Halophytes are physiologically dry but physically wet habitants because they grow in areas well saturated with water but water absorption is a difficult process. Due to this reason they have undergone morphological and physiological changes.
1. Root :
In addition to normal roots , many stilt or prop roots develops from the aerial branches of the stem. For example Rizophora mucronata.
Sometimes a large number of roots develop from the basal part of the tree trunks. For example Dischidia numularia.
In order to compensate lack of soil aeration they develop special type of negatively geotropic roots , called pneumatophores , this peg like structures causes numerous lenticels inner surface.
2. Stem :
- Stems of several halophytes are succulent.
- They are either hard or tough or swollen or fleshy and are usually covered with hairs.
3. Leaves :
- The leaves of most halophytes are thick , succulent , genrally small sized and glassy in appearance
- Leaves of aerohalopytes are densely covered with trichomes on their surface
- Leaves of submerged marine halophytes are thin , thorny with thick cutinized cuticle.
1. Epidermis :
- highly cutinized
- covered with epidermal outgrowths like hairs which prevents transpiration and salt spray into the plant body
2. Cortex :
- Shows Mucilage cavities, , spicule , lacuna , schlerides
- salt glands which are very important characteristic modification of the cortical regions in such plants which are adapted in this saline environment.
3. Vascular bundles :
- very poorly developed
- they are conjoint collateral with exarch xylum strands.
4. Stele :
it is lignified
5. Cell walls :
6. Mesophyll cells:
differtiated into palisade and spongy parenchyma.
7. Chlorophyll :
Very low within the cells among these halophytes.
1. Salinity : reduces the rate of cell division which promotes the rate of cell elongation
2. The cells of free ions: which improves its turgidity and increases its adaptability from salinity
3. The plants show high rates of transpiration which is helpful to tolerate saline condition and to maintain normal rate of metabolism.
4. They show exudation of sap that contains dissolved salts.
5. Some have salt secreting glands and water storage tissues.
DEVELOPMENT OF STRESS TOLEANT PLANTS:
CLASSICAL DEVELOPMENT TECHNIQUES:
Classical methods were based on crosses and selection schemes but the problem with the traditional plant breeding is that :
1. It is time consuming.
3. It is difficult to modify single traits
4. It relies on existing genetic variability.
THE USE OF GENOMICS:
Genomics makes use of a variety of technologies that allow organisms to be dscribed at DNA , mRNA, protein, and metabolite levels and allow relationships within and among these levels to be deciphered.
Isolation of the single genes responsible for stress tolerance and testing them in a new genetic context can be obtained by molecular genetics.
Different strategies to reveal the basic parameters of stress tolerance:
1. One strategy is to take a stress tolerant plant identify which molecules are responsible for the basis of tolerance. Using this method, an array of stress-induced genes have been isolated.
2. Second strategy is to take non-tolerant plants and transform them with given genes and assess the effect of these genes on stress tolerance.
A crucial point in this procedure is the selection of the genes used for transformation, and this is where the two strategies overlap. The genes can also come from several soucres such as animals, bacteria or yeast.
Two main types of method for plant transformation are:
1. The use of Agrobacterium as a biological vector for foreign gene transfer,
- The most widely used method for the introduction of new genes into plants is based on the natural DNA transfer capacity of Agrobacterium tumefaciens.
- Agrobacterium tumefaciens is a soil-borne bacterium.
- Like Rhizobium, the nitrogen-fixing bacterial genus, Agrobacterium has developed a way of living and deriving nourishment from plant tissues. However, unlike Rhizobium, Agrobacterium is a parasite and provides no benefit to the plant that it colonises. Instead, it causes Crown Gall disease .
- A. tumefaciens has been used extensively for genetic engineering of plants. This is achieved by engineering selected genes into the T-DNA of the bacterial plasmid so that they become integrated into the plant chromosomes when the T-DNA is transferred.
2. Direct gene transfer techniques, in which DNA is introduced into cells by the use of physical, electrical or chemical means.
Direct transformation implies that the cells take up the foreign gene of interest without the help of any vector.
These methods are species and genotype independent in terms of DNA transfer.
Their efficiency is influenced by the type of target cell , and their utility for the production of transgenic plants in most cases depends on the ease of regeneration from the targeted cells.
DIRECT GENE TRANSFER TECHNIQUES:
1. Direct gene uptake by protoplasts:
- Protoplasts are cells without rigid cellulose walls.
- Plant protoplasts treated with polyethylene glycol, commonly used to induce protoplast fusion, will take up DNA from their surrounding medium
- It can be stably integrated into the plant chromosomal DNA.
A delivery system that involves the direct injection of foreign DNA into plant cells using minute needles.
This is a technique using electrical fields to make protoplasts temporarily permeable to DNA, and offers an effective alternative to vectors.
In electroporation plant cell protoplasts are suspended in a small chamber with electrodes at opposite ends. The suspension medium contains the DNA or other material to be inserted into the cells. Pulses of high voltages are applied to the electrodes. The high voltage produces pores in the plasma membranes, allowing the DNA (or other material) to diffuse into the cells. The membranes reseal after a short period. If properly treated, the cells can then regenerate cell walls, divide to form callus, and finally regenerate new plants.
4. Liposome mediated DNA delivery:
- Liposomes are small artificial lipid vesicles prepared from phosphatidyl choline and stearylamine by a process known as reverse-phase evaporation
- Nucleic acid entrapped in such liposomes renders them highly tolerant to attack by nucleases.
5. Microprojectile gun method:
To overcome the limitations of protoplast regeneration, high velocity microprojectiles are being used to deliver nucleic acids directly into intact plant cells or tissues.
In this method DNA is coated on the surface of tungsten particles which are projected by means of a particle gun into intact cells or tissues. The particles can penetrate through several layers of cells and can transform cells within explants. For example; soybean, tobacco.
APPLICATIONS OF THE TECHNOLOGY:
1. DIFFERENT APPROACHES TO IMPROVING STRESS TOLERANCE
a. Production of osmoprotective compounds:
- The accumulation of osmoprotectants and osmolytes such as quarternary amines, amino acids, is an important strategy that plants use to overcome the environmental stress.
- Some species are able to accumulate such compounds more than others.
- For example: rice potato and tobacco accumulate limited amounts of glycine betain which is an osmoprotective compound. This makes them excellent targets for introducing osmoprotectant or osmolyte producing enzyme systems.
- Two mechanisms are involved in the activity of these substances:
1. The ability to raise the osmotic potential of the cell, thus balancing the osmotic potential of an externally increased osmotic pressure.
2. the ability to stabalise the membranes and macromolecular structures.
b. Improved membrane flexibility :
The presence of centrally positioned cis- double bonds in the membrane lipid lowers the phase transition temperature to approximately 0 degrees.
c. Stress induced proteins:
The transcription of genes encoding the late-embryogenesis-abundant(LEA) proteins is activated under abiotic stress. This protein has a protective effect and it was tested by introducing the HVA1 gene, encoding a group 3 LEA protein from barley, into rice.
2. IMPROVED PLANTS:
- To withstand osmotic stress, which is a common feature of drought, salinity, low and high temperatures, certain plants have evolved high capacity to synthesize and accumulate osmoprotectants or compatible solutes.
- Being non-toxic, the osmoprotectants are accumulated to osmotically significant levels without disrupting plant metabolism.
- Their function range from acting as an energy source to help the cells overcome oxidative stress to acting as an osmoprotectant by stabalising both the quaternary structure of proteins and the highly ordered structure of membranes against the adverse effects of high salinity and extreme temperatures.
a. Contamination tolerant plants that can grow in the presence of high levels of toxic compounds and accumulate them in their tissues:
- The removal from the environment of many potentially toxic compounds is complicated by the numerous classes and types of these chemicals.
- For example: many soils are contaminated with one or more metals , radioactive or inorganic compounds.
- The metals may include lead, zinc, cadmium, etc. the radioactive compounds may be uranium , cesium, and other inorganic compounds such as sodium, nitrate, ammonium, etc.
- Phytoremediation is a relatively new approach to removing contaminants from the environment. It is defined s the use of plants to remove, destroy the hazardous substances from the environment.
- Unfortunately, even plants that are relatively tolerant of various environmental contaminants often remain small in the presence of the contaminant.
- Some plants have been especially bio-engineered to enable them remove toxic waste from the environment.
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