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.
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:
- Bt cotton:
- 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.
- GEAC(genetic engineering approval Committee), in 2002 approved Bt cotton making it the first genetically modified plant in India to receive the approval.
- Bt cotton is an insect resistant genetically modified crop.(made resistant to cotton boll worms, that destroys cotton plants).
- 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 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.
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:
- 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.