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METHODS OF GENE TRANSFER IN PLANTS
Dr. Thirunahari Ugandhar Assistant Professor in Botany Govt Degree College Mahabubabad
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Sufficient, Nutritionally Adequate & Culturally Acceptable Food for an Active, Healthy Life.
10/11/2020
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Introduction The “sharing” of DNA among living forms is well documented as a natural phenomenon. For thousands of years, genes have moved from one organism to another. For example, Agrobacterium tumefaciens, a soil bacterium known as 'nature's own genetic engineer', has the natural ability to genetically engineer plants.
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Manipulation of Plants
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Conventional breeding Plants Tissue culture Genetic engineering
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Why Gene transfer? What is Gene transfer
Insertion of copies of a gene into living cells in order to induce synthes is of the gene's product. Why Gene transfer? To add a desired trait to a crop, a foreign gene (transgene) encoding the trait must be inserted into plant cells, along with a “cassette” of additional genetic material. The cassette includes a DNA sequence called a “promoter,” which determines where and when the foreign. Gene is expressed in the host, and a “marker gene” that allows breeders to determine which plants contain the inserted gene by screening or selection.
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Why genetically Engineer Plants?
To improve the agricultural, horticultural or ornamental value of a crop plant. To serve as a living bioreactor for the production of economically important proteins or metabolites To provide a renewable source of energy To provide a powerful means for studying the action of genes (and gene products) during development and other biological processes
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Desired Traits Pest resistance Herbicide tolerance Viral resistance
Drought resistance Increased nutritional value Improved Fruit Altered ripening Economical benefits: Money is saved on pesticides. Huge money upfront, but with a shorter amount of time to produce desired product, preciseness, and no unwanted genes. Herbicide-resistance: GM crops can be produced to be herbicide resistant . This means farmers could spray these crops with herbicide and kill the weeds, without affecting the crop. IN effect, the amount of herbicide used in one season would be reduced, with a subsequent reduction in costs. In some cases pesticide use is not needed, which saves money and protects the environment. (experimenting to make drought or salt-tolerant or less reliance on fertilizer) Better quality foods-Animals can be genetically modified to be leaner, grow faster, and need less food.
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Genetic Engineering Genetic engineering, also called genetic modification, is the direct manipulation of an organism's Genome using Biotechnology New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using Molecular Cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism.
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Engineer An Engineer is a Professional Practitioner of Engineering, concerned with applying Scientific Knowledge and ingenuity to develop solutions for technical problems. Engineers design materials, structures, and systems while considering the limitations imposed by practicality, regulation, safety, and cost. The word Engineer is derived from the Latin roots ingeniare ("to contrive, devise") and ingenium ("cleverness")
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TRANSGENIC PLANTS The process of introducing new genes into plants to produce new traits is called Plant Genetic Engineering. The product of this process is called Transgenic Plants. Transgenic plants contain a gene or genes which have been artificially inserted. The inserted genes are known as transgenic. 10/11/2020
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TRANSGENIC PLANTS – AN INTRODUCTION
Transformation – The process of obtaining transgenic plants. Transgenic plant – a plant with a foreign gene (or genes) from another plant/animal that is incorporated into its chromosome Marc Van Montagu and Jeff Schell, discovered the gene transfer mechanism between Agrobacterium and plants, which resulted in the development of methods to alter the bacterium into an efficient delivery system for genetic engineering in plants. Most common genes (and traits) in transgenic or biotech crops herbicide resistance Insecticide resistance, Bt genes in field corn (maize) virus-resistance (coat-protein) genes
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WHY HAVE GM CROPS? Growing Human Population Loss of Farmable Land
Remediation of Soil Enrich Nutrient Content Transgenic Wild-type
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Plant Transformation Methods
Physical Chemical Biological In planta Microinjection Pressure Biolistics - gene gun/ particle bombardment Electroporation Silica/carbon fibers Lazer mediated SAT PEG DEAE-dextran Calcium phosphate Artificial lipids Proteins Dendrimers A. Tumefaciens Rhizogenes Virus-mediated 10/11/2020
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Genetic Transformation
PLANTS - PHYSICAL METHODS Microinjection Electroporation Biolistics - gene gun Silica/carbon fibers Lazer mediated SAT 10/11/2020
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1. MICROINJECTION 10/11/2020
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2. Electroporation This electroporator is for low-current applications such as those using small electrodes 10/11/2020
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3. Biolistic / Gene Gun 10/11/2020
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3. Biolistic / Gene Gun
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4. SILICA/CARBON FIBERS
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Streptothricin Acetyl Transferase (SAT)
TRANSFORMATION 10/11/2020
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Chemical Gene Transfer
PEG DEAE-dextran Calcium phosphate Artificial lipids Proteins Dendrimers
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1. PEG Gene Transfer
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2. Dead Dextran Mediated Gene Transfer
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3. Calcium phosphate Transfer
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Artificial lipoplex Mediated Gene Transfer
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Biological Transformation
Agrobacterium tumefaciens & Agrobacterium rhizogenes 10/11/2020
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BIOLOGICAL TRANSFORMATION
Possible plant compounds, that initiate Agrobacterium to infect plant cells. Acetosyringone, ferulic acid, gallic acid, Hydroxybenzoic acid, pyrogallic acid, vanillin etc. In monocot – not efficient Transformation frequencey – very less 10/11/2020
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Agrobacterium Agrobacterium (disease symptomology and host range)
radiobacter - “avirulent” species A. tumefaciens - crown gall disease rhizogenes - hairy root disease rubi cane gall disease vitis galls on grape and a few other plant species 10/11/2020 Otten et al., 1984
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Cellular process of Agrobacterium–host interaction
Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3), 10/11/2020
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Plant Transformation Methods
Virus-mediated gene transfer (Plant viruses as vectors) Caulimo viruses – ds DNA – CaMV Gemini viruses - 2ss DNA – maize streak virus RNA plant viruses - TMV 10/11/2020
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POLYMERASE CHAIN REACTION
PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. Kary Banks Mullis (Born December 28, 1944) is a Nobel Prize -winning American Biochemist, author, and lecturer. In recognition of his improvement of the Polymerase Chain Reaction (PCR) technique
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PCR Animation Denaturation: DNA melts Annealing: Primers bind
This is an animation of one step in the PCR process. Take a few minutes and let the animation run through a number of times. It will recycle on its own. This step will show the denaturation (converting the DNA from single- to double-stranded state). The second step is annealing (the binding of the primer to the single-stranded DNA). The final step is extension (the duplication of a strand from the end of the primer). Denaturation: DNA melts Annealing: Primers bind Extension: DNA is replicated
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Recombinant DNA & Plasmids Combining genes from different sources and/or species
Circular DNA from bacteria = plasmids Target DNA recombined in plasmid Bacteria rapidly reproduces many clones
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Gene transfer techniques…
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Production of transgenic plants
Isolate and clone gene of interest Add DNA segments to initiate or enhance gene expression Add selectable markers Introduce gene construct into plant cells (transformation) Select transformed cells or tissues Regenerate whole plants 10/11/2020
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PLANT TRANSFORMATION Plants are the easiest of higher organisms to transform Both physical and biological methods exist for transformation Until recently, only transgenic organisms in wide public release were plants 10/11/2020
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Which foods contain GM Product?
India Approval for GM food crops Corn Soy Papaya Canola Potato Chicory Rice Squash Sugarbeet Tomatoes Approval does not necessarily mean these crops are distributed Database of GM crops: Bolded crops are widely grown & sold in US.
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Genetically Engineered Foods
India approval for GM foods: Corn Soy Papaya Canola Potato Rice Tomatoes India approval does not mean that these crops are distributed. 20% of India grocery food contains GM ingredients Some current trials: apples that resist insect attack, bananas free of viruses and worm parasites, coffee with a lower caffeine content, cabbage that resists caterpillar attacks, melons that have a longer shelf life, sunflowers that produce oil with lower saturated fat.
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Molecular agriculture makes new gene combinations possible
Peas (on the left) that make a genetically engineered bean protein are insect-resistant and do not need to be sprayed with pesticides.
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Molecular agriculture makes new gene combinations possible
Peas (on the left) that make a genetically engineered bean protein are insect-resistant and do not need to be sprayed with pesticides.
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Some GM crops will improve the nutritional quality of foods
Some GM crops will improve the nutritional quality of foods. Such foods are now in India.
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The approach is to down regulate the expression of the gene encoding the major allergenic protein (antisense)
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Truthful labels can be misleading or meaningless
There are no GM apples anywhere! Is food labeled this way nutritious?
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How to Label? GMO Conventionally grown
Conventional grown GMO Conventionally grown Pesticides, twice a week Pesticide free
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Genetic change resulting from crop domestication took 10,000 years.
Teosinte (top) and corn or maize (bottom)
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Genetically modified crops in future
Cauliflower 10/11/2020
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Purple tomatoes high in anthocyanins
High anthocyanin purple tomato and red wild-type tomato 10/11/2020
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Conclusion Consumer fears are all potential risks, some more than others, and use of GM crops should only be continued with extreme care and intense long term research on the topic should be continued. With most of the cases the use of GM crops can only be justified when the conventional methods are worse and pose even higher risks to the environment. Also, the labeling that is enforced in much of the world should also be mandatory here in the United States.
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Transgenic and Nontransgenic Crops
Global Area (mil ha) Transgenic (mil ha) % Transgenic Soybean 91 58.6 64 Cotton 35 13.4 38 Canola 27 4.8 18 Maize 148 25.2 17 Total 301 102 34 James, 2006
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Prof. N. Rama swamy My grateful acknowledgement is due to Prof. N. Rama swamy (Teacher of my supervisor) Head Department of Bio-technology, University PG College Kakatiya University Warangal for their kind encouragement and blessing. 10/11/2020
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tugandharbiotech@gmail.comin
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