GENETICALLY MODIFIED PLANTS AND ANIMALS

BY: RAHUL ANDHARIA (MSIWM001)

GM Plants: To prepare a GM plant, new DNA is transferred into plant cells. Genetic engineering techniques are used to modify DNA. Genetic composition of the plant is altered by adding specific useful genes. Once the new DNA is inserted, than the cells are taken and grown in tissue culture using appropriate medium where they develop into new plants and will inherit the new DNA which was inserted.

History:

Tobacco plant, first genetically modified plant was produced in the year 1982. These plants were modified for antibiotic resistance. The first country to commercialise use of transgenic plants was China. The tobacco plants were made antibiotic resistant by creating a chimeric gene which joined the antibiotic resistant gene present on the T1 plasmid of Agrobacterium.  Bt cotton was the first commercialised genetically modified crop used in India which was made by Maharashtra hybrid seeds company along with Monsanto company from USA.

Process of developing GM Plants/Crops:(6 steps)

  • Isolating Gene of interest- Gene of interest is isolated from other plants or organism. Information like structure, function and location of chromosomes is useful in identifying gene of interest in an organism. Information about gene function and it’s regulation in donor organism(organism from which gene is taken) and in recipient organism(organism in which the gene is inserted) must be known fully before starting the experiment in order to minimize the adverse effects.

Gene insertion by using Transfer vector and plant transformation-  Plasmid from Agrobacterium Tumifaecins, are used as common transfer tools. Using rDNA technology, the gene of interest is inserted into the plasmid.  Plant cells or explants are mixed with Agrobacterium cells containing the plasmid with new genes. T-DNA, piece of plasmid is taken up by the cells. Desired genes are inserted into one of the plant chromosome by Tumifaecins, and now the plant is called Genetically modified. Other method used for transfer is Gene gun method or particle bombardment method. In this method, the relevant metal surfaces are coated with small DNA fragments and these particles are bombarded into plant cells. Mostly Tungsten or gold particles are used for coating DNA (called microprojectiles). This method is a bit costly but can be effective.

  • Selection and Regeneration of modified plant cells to form a whole plant:  Generally, a small fraction of plant cells take up the gene of interest after transformation. Hence, selectable marker genes, that favours antibiotic and herbicide resistance are used to favour growth of transformed cells. After the process, the transformed cells are regenerated into a whole plant using plant tissue culture method.
  • Plant transformation verification: The gene inserted has to inherit normally, so this needs to be verified. For this purposes, tests are performed to determine number of copies inserted, intactness of the copies inserted and effects of inserted gene with the other genes. In this, gene expression (mRNA-proteins) is also checked to confirm whether gene is functional or not.
  • Testing of plant performance: checking plant performance is vital. After transformation, only fraction of plant cells have the copies of inserted gene. So selective markers are used to favour growth of transformed cells. The resistance genes along with genes for desired traits are transferred using a suitable vector. So when cells are exposed to antibiotics or herbicides, only transformed cells with this selective markers will grow. This way performance is checked and only transformed cells are taken to regenerate and create a whole plant by using tissue culture.
  • Safety assessments: safety assessments are necessary in terms of food and environment. There are different tests to determine whether the released modified plant is safe for consumption or for  cultivation to produce  higher yields without  damaging the environment.

GM Crops in India:

  1. Bt cotton:
    1. Bt cotton was developed to tackle the boll worm infection in cotton plants. The Bt cotton variety was developed by Maharashtra hybrid seeds company along with Monsanto, USA.
    1. GEAC(genetic engineering approval Committee), in 2002 approved Bt cotton making it the first genetically modified plant in India to receive the approval.
    1.  Bt cotton is an  insect resistant genetically modified crop.(made resistant to cotton boll worms, that destroys cotton plants).
    1. Bt is a protein from bacillus thureingenesis, bacteria which has 200 different types of Bt toxins. Each toxin affects and works on different types of insects.

Cry group of endotoxins in Bt cotton are modified by inserting a gene with toxin crystals, when organisms(insects) ingests this genes with toxins, toxins dissolves the gut lining of the insects leading to its death. Thus, the crop plant is protected from boll worms.

  • Bt Brinjal:
    • It was developed to give resistannce against lepidopteran insects, specifically to leucinodes orbonalis, which is a fruit and shoot borer in Brinjal plants.
    •  Bt Brinjal was developed by Maharashtra hybrid seeds company in collaboration with Tamilnadu agriculture University and Dharwad institute of agricultural sciences.
    •  GEAC approved commercialisation of Bt Brinjal in the year 2007, however , due to lack of proper safety and efficacy and lack of scientific consensus, it was banned in the year 2010.
  • HT Mustard:
    •  DMH(Dhara Mustard) was created to reduce the demands of edible oil imports of India.
    • It was created by Delhi University professor, Deepak Pental.
    •  DHM-11 was created using transgenic technologies in particularly involving Barstar/Barnase gene technologies.
    •   Male fertility is conferred by Barnase gene, while Barstar, restores fertile seeds producing abilities of DHM-11.
    • GEAC approved it in the year 2017.

  Advantages of GM crops:

  • Crop Protection: Resistance to diseases, pests, insects and herbicides. Resistance is achieved by using Genetic engineering methods and by using toxins in case of Bt cotton.
  • Economic Benefits: GM plants increases the yield double times compared to the normal plants.
  • With increasing demands of quality food, GM crops can be beneficiary in providing and supplying food at much faster rates.

Concerns with GM crops:

  • Health concerns: Transfer of antibiotic resistance markers or allergens are some of the potential risks. Example- In HT Soya areas, in Argentina, birth defects and childhood Cancers were increased by threefold. This was a report based study.
  • Environmental concerns: can reduce diversity of species. For example, if insects that are not be killed gets killed by developing modified crops can reduce species diversity. Super weeds(transfer of genes from one crop to other creates super weeds) which are resistant to most of common control methods.
  • Economic concerns: Launching of GM crop to market is costly and time consuming process. Also violation of ethical issues have been raised as a concern, for example- organisms intrinsic natural values have been violated by mixing it with other species.

GEAC(Genetic engineering approval Committee)– It was approved under ministry of environment, forest and climate change for manufacturing, use, import, export of GMOs (genetically modified organisms). This committee is also responsible for giving technical approvals for proposed GMO products including field trials.

Safety of GM crops and it’s related products is monitored by Institutional Biosafety Committee (IBSCs).

GM Animals:

GM animals can be created by inserting a foreign gene of interest into their genomes. rDNA technology is used for construction of foreign gene. Along with gene, DNA is also modified and contain different sequences in order to incorporate and express into the host cells.

History:

Mice embryos in-vitro manipulation was first reported in the year 1940 using a culture system. In Angora Rabbits, first successful transfer of embryos was achieved in the year 1891 by Walter Heape. Modern genetic modifications began in the year 1973, when Herbert Boyer and Stanley Cohen first discovered and demonstrated that gene from one organism can be cut, and pasted to other organism. Mouse is the first genetically modified animal which was developed by Rudolph Jaenisch, in the year 1974.

Examples of GM Animals:

  1. Mice: GM mouse models are used extensively as models for studying and understanding  different diseases. Two methods are well known for developing GM mice, Embryonic stem cell method and Pronucleus method.

Embryonic stem cell Method: (ES)(method 1)

  • Mouse blastocyst has inner cell mass from which ES are harvested.
  • rDNA technology is used to make the DNA containing the desired gene of interest, vector and promoter and enhancer sequences.
  • ES cells are transferred in culture. When ES cells are exposed to DNA, it gets incorporated into it.Transformed cells are selected and injected into inner cell mass of mouse blastocyst.
  • Transfer of embryo. Pseudo pregnant mouse is created and the embryo is transferred into its uterus.
  • Offspring produced by it is tested. For testing, remove small piece of tissue from tail and examine it’s DNA. It should be present in 10-20% and it should be heterozygous for the gene.
  • Transgenic strain is established by mating two heterozygous mice and screening their offspring’s.

Pronucleus method:(method 2)

  • rDNA technology is used for preparing DNA with desired gene, vector and promoter and enhancer sequences.
  • Freshly fertilized eggs are harvested before sperm head becoming Pronucleus.
  • The male Pronucleus is injected with DNA that is prepared.
  • Zygote formed by pronuclei fusion is allowed to divide by mitosis to form 2 cell embryo.
  • The embryo is than implanted into pseudo pregnant foster mother.
  • Than the rest of the steps are common with respect to ES method. Offspring test is performed followed by establishing Transgenic strain.
  • GM chicken:
  • Embryos are infected with viral vectors carrying human gene with  a therapeutic protein and promoter sequences.
  • Human gene is transformed with the rooster sperm or appropriate promoter.
  • Check for transgenic offspring’s..
  • The method is cost effective.

C.   GM Sheep’s:

  • Connective tissue cells of sheep are treatedwith a vector, which has 2 homologous regions to that of COL1a1 gene of sheep, alpha 1 Anti-trypsin coded by human gene, antibiotic neomycin resistant gene, beta lacto globulin gene promoter site and ribosome binding sites for beta lacto globulin to be translated.
  • Transform the cells and fuse with enucleated(without nucleus) sheep cells.
  • Next step is implantation into uterus of female sheep(called ewe).
  • Lambs produces large amounts of milk when treated with hormones.

However, this method implemented by one of the company in 2000, abandoned it in 2003 because for purification of protein from sheep’s milk, the cost of expenses were almost doubled.

Applications of GM Animals:

  • GM animals are used as models to understand the disease process and it’s progression.
  • They can be used as models to test new therapeutics that are being developed for treatment of diseases.
  • GM animal models are also used to study gene function. For example- animals with certain genes being turned off or non functional genes can be studied to understand how turning off of this genes can lead to diseases and the mechanisms behind it.
  • Can be used in agriculture to confer resistance to diseases against pathogens.
  •  Knockout mice- used extensively in research to understand genes for which mutant strains are not available.
  • Knock-in mice: It removes certain DNA sequences that otherwise blocks transcription. So the target gene can be turned on as per wish. Also new gene can be introduced by replacing one of the mouse gene.

SCOPE

Scope and appliance of microbiology

About

  • Microbiology is a discipline of biology which deals with the study of microscopic organism, their interaction with other organisms and with environment.
  • It includes microscopic level organisms like bacteria, algae, fungi, protozoa and the infectious agent viruses too.
  • Microorganisms present all over the globe form high altitude to the deep seas include hot springs, extremely cold climates, and high pressure even in the salty lakes.
  • Microorganisms are both beneficial and harmful to human. I.e. required in the industrial production of food stuff (bread, yogurt, beer, wine, etc), antibiotics(penicillin, chloromycetin, streptomycin) vaccine, enzymes, vitamins and many more products along with it is harmful in the way by causing fatal disease like small pox, plague, malaria, cholera, HIV, influenza and more.
  • They plays important role in maintaining the stability of ecosystem by recycling the organic and inorganic substance in carbon, nitrogen, sulphur and phosphorus cycle.
  • There were many events in history that tells us how infectious microbes put the human population in danger. Like black death(1346),  yellow fever(1793), Spanish flu(1918), SARS(2002), H1N1 flu(2009), MERS(2014), ebola(2014), etc.
  • In addition to the disease outbreaks microorganism plays major role in food spoilage, detoriation on materials like paper, wood, metal and plastics.
  • In agriculture nowadays genetically improved crops are used to get more yields and disease resistant crop which can be obtained by the involvement of microorganism.
  • As microbes are present everywhere it enhances the scope and contribute in many fields like pharma, agriculture, dairy, food industries, research, nanotechnology, water industry, chemical industry.
  • Microbiologists are the person who studies these microorganisms and their morphology, behaviour, metabolic activity, habitat, reproduction, nutritional requirement, their application and pathogenicity, improvemnet and modifications which leads to the high demand of microbiologist globally.

Fields:

Dairy and food industry:

  • Deals with the microbial production for the food stuff, prevention of spoilage of food and transmission of food borne disease.  

Agricultural microbiology:

  • It include the study of microbial strains which are used to obtain genetically modified crops which are resistant to many diseases and higher yields. Production of bio-fertilizers and maintenance of the rhizo-flora.

Medical microbiology:

  • Study of disease their causative agents, prevention, diagnosis and treatment. In addition in includes various clinical appliance of microbes oh human health.  

Environmental microbiology:

  • The study of microbes and their interaction with environment, role in geochemical cycles, microbial diversity, bio-remediation

Genetic engineering:

  • It deals with the study of modifying microorganism at gene level and engineered microbes are used to produce hormones, enzymes, vaccines, vitamins, antibiotic and other products.

Microbial physiology:

  • Includes the study of microbial morphological structure, metabolism and growth.

Industrial microbiology:

  • Deals with the production of antibiotic, fermented food, aminoacids, vitamins, steroids, enzymes, alcohol. In addition with the strain improvement and process of enhancing product quantity.

Soil microbiology:

  • The study of soil flora and role of microorganism in soil fertility

Water microbiology:

  • Major part of this field is the waste water management as is it a challenging condition for the world with industrial waste discharged in the water bodies and leading to the pollution causing threat for aquatic life.

Applications:

The diversity of microbes on the globe makes microbiology the most complex and largest discipline.

Food:

  • There are a majority of microbes used in the food and dairy industries for the production of food from wine, beer through the cheese, yogurt to manufacturing of bread.
  • Include Process of fermentation, pasteurization, industrial production, processing of food its packaging, food preservation and storage.
  • Microbial spoilage of food production and their prevention.

Environmental microbiology:

  • working of biogeocycle(carbon, nitrogen, sulphur and phosphorus) done by microorganism
  • microorganism are present in free living state and in association with plants in symbiotic relationship.
  • Maintaining the soil fertility without exhausting soil nutrients.
  • Responsible for cleaning toxic substance from the environment. 
  • Some are pathogenic to the plant but there are few strain which act as biological control agents and protect plant against this diseases.

Medical microbiology:

  • Disease causing microbes i.e. bacteria, algae, fungi, protozoa and virus responsible for causing numerous types of diseases ranging from  acute to severe life threatening.
  • Examples are cholera, influenza, malaria, HIV, tuberculosis, plague, etc
  • Their diagnosis, transmission, prevention and cure are the major part of the medical microbiology
  • In contrast to the pathogenicity there are some strains inhibit the growth of other diseases causing microbes by producing antibiotic, hence, used for the production of antibiotics.

Biotechnology:

  • Genetically engineered strain used for the production of therapeutic substance like human growth hormone, insulin, etc
  • Also contribute in the commercial production of  acetone, alcohol, drugs etc

Research:

  • Diversity and unicellular structure of microbes make them easy to study and research over multicellular structure.
  • In addition they can produce millions of copies from a single cell rapidly with very low cost which is good for experiments performed.
  • Short generation time leads to quick result analysis.

Future aspects of microbiology:

  • Due to population explosion in the world there could be scarcity of food in near future in that condition single cell protein can be an alternative.
  • Newly and highly resistant species of diseases causing microbes is a challenge for the present and in the future so r DNA technology is useful to overcome this problem.
  • Treatment of cancer and HIV like diseases
  • Food preservation methods for highly perishable food items