plant_biotechnology_تنافسي_2024_2025_د_اسماء_كاطع_تم_التحويل.pdf

Definition Biotechnology- Bio means life and technology means the application of knowledge for practical use ie., the use of living organisms to make or improve a product. Other definitions for the term Biotechnology The use of living organisms to solve problems or make useful products. The u...

Definition Biotechnology- Bio means life and technology means the application of knowledge for practical use ie., the use of living organisms to make or improve a product. Other definitions for the term Biotechnology The use of living organisms to solve problems or make useful products. The use of cells and biological molecules to solve problems or make useful products. Biological molecules include DNA, RNA and proteins. The commercial application of living organisms or their products, which involves the deliberate manipulation of their DNA molecules. Make a living cell to perform a specific task in a predictable and controllable way..‫اصنع خلية حية ألداء مهمة محددة بطريقة يمكن التنبؤ بها والتحكم فيها‬ (https://pravara.in/wp- Plant Biotechnology Plant biotechnology is a set of techniques used to adapt plants for specific needs or opportunities. Situations that combine multiple needs and opportunities are common. For example, a single crop may be required to provide sustainable food and healthful nutrition, protection of the environment, and opportunities for jobs and income. Finding or developing suitable plants is typically a highly complex challenge. Plant biotechnology /Agricultural biotechnology A process to produce a genetically modified plant by removing genetic information from an organism, manipulating it in the laboratory and then transferring it into a plant to change certain of its characteristics. In Nutshell it’s the manipulation of plants for the benefit of mankind Plants are mainly manipulated for two major objectives A. Crop improvement Herbicide tolerance (in use) Pest resistance (in use) Drought tolerance Nitrogen fixing ability 5 Acidity and Salinity tolerance B. Nutritional value of crops Improving food quality and safety Healthier cooking oils by decreasing the conc. of saturated fatty acids in vegetable oils 3 Functional foods: foods containing significant levels of biologically active components that impart health benefits Genetic engineering Manipulation of genes is called genetic engineering or recombinant DNA technology. It removes gene(s) from one organism and either Transfers them to another Puts them back in the original with a different combination Various gene transfer techniques used in genetic engineering includes In direct gene transfer (Agrobacterium mediated gene transfer): Desired trait is isolated from DNA of original organism, inserted into Agrobacterium, target plant is infected. Cells that accept the DNA are grown into plants with the new trait. Direct gene transfer ex; Gene gun that the DNA that codes for the desired trait is coated onto tiny particles of tungsten and fired into a group of plant cells. Cells that accept the DNA are grown into plants with the desired trait.  Current interest in genetic engineering centres on its various applications, such as: Isolation of a particular gene, part of a gene, or region of a genome Production of particular RNA and protein molecules in quantities formerly thought to be unobtainable Improvement in the production of biochemicals (such as enzymes and drugs) and commercially important organic chemicals ً ‫املهمة تجاريا‬ Production of varieties of plants having particular desirable characteristics (for example, requiring less fertilizer or being resistant to disease) Correction of genetic defects in higher organisms 6 Creation of organisms with economically important features (for example, plants capable of maturing faster or having greater yield). The basic requirements for successful genetic engineering are Restriction enzymes Cloning vehicles (vectors) to carry the genes of interest Detection and selection of cloned genes. A model genetic engineering of a plant comprises the following general steps: Selection of a plant gene whose introduction in other plants would be of positive agricultural value; Identification and isolation of such genes; Transference of isolated genes to the plant cell; Regeneration of complete plants from transferred cells or tissues.  Some of the goals of plant genetic engineers include production of plants that are Resistant to herbicide, insect, fungal and viral pathogens Improved protein quality and amino acid composition Improved photosynthetic efficiency, ٤ Improved post harvest handling. This technology could provide an additional tool for the plant breeder who is trying to improve crops by traditional methods. In addition, plants can be viewed as a genetic resource, genes being cloned into, and expressed in bacteria. These bacteria may then be used to produce desirable plant products on an industrial scale using fermenter. The first transgenic plants expressing engineered foreign genes were recovered in 1984. Dramatic progress has been made in the last few years in the development of a gene transfer system for higher plants. About 20 crops can be genetically engineered at present. Rapid progress is being made in the genetic manipulation of many species and almost every month another successful plant transformation is reported. Methods for the Development of Genetically engineered plants for Production of Natural Products Indirect Methods ✓ Agrobacterium - mediated genetic transformation Structure of Ti Plasmid Use of Ti plasmid in genetic transformation Steps involved in Agrobacterium- mediated genetic transformation of plants by ‘Wounded explant’ method Direct Method Microprojectile/particle Bombardment Gen gun (biolistics) Electroporation Microinjection Chemical mediated gene transfer Liposome mediated gene transfer Silicon carbide method ✓ Selection of transformants Selectable marker Screening marker Direct Methods Direct methods are those methods which do not use bacteria as mediators for integration of DNA into host genome. These methods include microprojectile bombardment, electroporation and microinjection ect….. Microprojectile/particle Bombardment (biolistics): Is a method where cells are physically impregnated with nucleic acids or other biological molecules. Abiolistic particle delivery system is a device for plant transformation where cells are bombarded with heavy metal particles coated with DNA/RNA. This technique was invented by John Stanford in 1984 for introduction of DNA into cells by physical means. Agrobacterium-mediated genetic transformation system works well for dicotyledonous plants but has low efficiency for monocots. Biolistic particle delivery system provides an effective and versatile way to transform almost all type of cells. It has been proven to be a successful alternative for creating transgenic organisms in prokaryotes, mammalian and plant species. In this process, construct having gene of interest is coated on the surface of tiny particles of gold or tungsten (0.6 – 1 mm in size). Prior to coating, DNA is precipitated with calcium chloride, spermidine and polyethylene glycol. These coated microparticles are loaded on to the macro- carrier and accelerated to high speed by using pressurized helium gas. Plant cell suspensions, callus cultures, or tissues could be used as the target of these microprojectiles. As the microprojectiles penetrate the plant cell walls and membranes to enter the cells, coated DNA is released from its surface and incorporated into the plant’s genome. In biolistics, use of binary vectors with T-DNA border sequences is not required. This method is especially important for monocots, for which efficiency of other transformation methods is not satisfactory. A wide range of tissues such as apical and floral meristems, embryos, seedlings, leaves, cultured cells and floral tissues could be used as target in this method. Figure: Particle bombardment method of Plant transformation (1) Isolation of protoplasts. (2)Injection of DNA-coated particles using particle gun. (3) Regeneration of transformed protoplasts into plantlets. (4) Acclimatization of regenerated plantlets in a greenhouse. A number of parameters should be carefully considered before using particle bombardment. These can be classified under three categories: Physical parameters Nature, chemical and physical properties of the metal particles utilized to carry the foreign DNA. The nature and preparation of DNA, binding of DNA on the particles and target tissues.  Environmental parameters Variables such as temperature, photoperiod and humidity of donor plants, explants, and bombarded tissues affect physiology of tissues and influence receptiveness of the target tissue. Biological parameters Choice and nature of explants, pre- and post bombardment culture conditions, osmotic pre- and post-treatment of explants Electroporation Electroporation is a method of transformation via direct gene transfer. In this technique mixture containing cells and DNA is exposed to very high voltage electrical pulses (4000 – 8000 V/cm) for very brief time periods (few milliseconds). It results in formation of transient pores in the plasma membrane, thorough which DNA seems to enter inside the cell and then nucleus. Figure: Electroporation (A) Diagram showing formation of transient pores in cell membrane on applying electrical pulse, entry of DNA inside the cell and sealing of pores afterwards. A suspension of cells with DNA is taken in an electroporation cuvette placed between electrodes and electrical pulses are applied. Temporary micropores are formed in cell membranes which allow cells to take up plasmid DNA leading to stable or transient DNA expression. Figure:(A) Main components of an electroporator. (B) Cuvettes used for electroporation. These are plastic cuvettes with lid and aluminium electrodes, having a maximum capacity of 400µl. Electoporation as a transformation method is fast, convenient, simple, and inexpensive and has low cell toxicity. The disadvantage associated with this technique is difficulty in regenerating plants from protoplasts, if protoplast is used for electroporation. Microinjection The process of using a fine glass micropipette to manually inject transgene at microscopic or borderline macroscopic level is known as microinjection. Figure: Illustration of micrinjection method Microinjection involves direct mechanical introduction of DNA into the nucleus or cytoplasm using a glass microcapillary injection pipette. The protoplasts are immobilized in low melting agar, while working under a microscope, using a holding pipette and suction force. DNA is then directly injected into the cytoplasm or the nucleus. The injected cells are then cultured in vitro and regenerated into plants. Successful examples of this process has been shown in rapeseed, tobacco and various other plants. There are two types of microinjection systems; constant flow system and pulsed flow system. In the constant flow system the amount of sample injected is determined by the duration for which needle remains in the cell. The constant flow system is relatively simple and inexpensive but outdated. The pulsed flow system has greater control over the volume of substance delivered, needle placement and movement and has better precision. This technique results in less damage to the receiving cell, however, the components of this system are quite expensive. Chemical mediated gene transfer Cells or protoplasts can be stimulated to take up foreign DNA using some chemicals. Polyethylene glycol (PEG) is the most commonly used chemical for this purpose. It helps in precipitation of DNA, which can then be taken up by the cells through the process of endocytosis. Liposome mediated gene transfer Plasmid containing foreign desired gene can be enclosed in small lipid bags called liposomes, which can then be fused with protoplasts using chemicals like PEG. Silicon carbide method In this method, fibres of organic material like silicon carbide are used for gene transfer. These fibres, when mixed with plasmid DNA and plant tissue or cells, help in penetration of the foreign DNA into the plant tissue. Selectable and screenable markers are important tools in genetic engineering. Selection of transformants In a genetic transformation experiment, only one in a several million to billion cells may take up the transgene depending upon the efficiency of transformation. Rather than checking every single cell/organism, a selective agent that kills or gives a different phenotype to all the cells not carrying foreign DNA can be employed. These selective genes are called as marker genes. These genes also help in assessing the success rate of a genetic transformation study. Marker gene is a gene introduced into cell along with the transgene. It is used to determine if the transgene has been successfully inserted into host organism's genome as marker gene’s presence can be seen or detected. There are two types of marker genes: Selectable markers are the genes present in the cloning vectors. It Selectable marker Screening marker Selectable marker A selectable marker is a gene that confers a trait suitable for artificial selection as it protects the organism from a selective agent that would normally kill it or prevent its growth. In most of the genetic transformation experiments only one in a several million or billion cells will take up the transgene. In order to find out transformed cell/organism a selective agent is utilized which kills all the cells without transgene, leaving only the transformed ones. Antibiotics or herbicides are the most common selective agents. When grown on medium containing antibiotic or herbicide the non- recombinants die due to lack of resistance. Screening marker A marker for screening is one which will make cells containing the gene look different and allows the researcher to distinguish between wanted and unwanted cells/organisms is known as screening marker. These markers do not provide a cell with a selective advantage, but are used to identify transgenic events by manually separating transgenic and non-transformed material. Reporter systems have been used to determine the intracellular localization of a gene product, efficiency of gene delivery systems, detection of protein-protein or protein-DNA interactions and activity of promoter What is the difference between selectable marker and screenable marker? Unlike a selectable marker, a screenable marker is a non-antibiotic marker. A screenable marker is used to con rm transformation and to establish its ef ciency. fi fi