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Gene transfer and biosafety issues
- 1. Different methods ofgene transfer and biosafety issues related to GM crops Pl.Path 604 Molecular basis of host pathogen interaction Kiran Mohan (2019-27-003)
- 2. Content • Introduction • Methodsof gene transfer • Biosafety issues related to gene transfer • Conclusion
- 3. • Gene –Segment of nucleic acid that encodes a functional protein or RNA and is the unit of inheritance. • Genetic engineering - Considerable efficiency and skill in gene transfer into organisms and desirable expression have been achieved. • Transgenic animals and plants can be obtained by introduction of exogenous DNA into targeted animals and plants accompanied by the stable expression.
- 4. Methods of genetransfer • There are mainly two methods; 1. Vector-Mediated Gene Transfer 2. Direct or Vector-less DNA Transfer
- 5. Direct / Vectorless gene transfer 1. Natural methods 1. Conjugation 2. Bacterial transformation 3. Transposition 4. Phage transduction 5. Retroviral transduction 2. Artificial methods Physical methods • Macroinjection • Microinjection • Protoplast fusion • Biolistic transformation / Microprojectile bombardment • Pollen tube pathway Chemical methods • DNA transfer by calcium phosphate method • Polyethylene glycol mediated • Polybrene/DMSO assisted • DEAE-Dextran mediated • Liposome mediated • Silicon carbide mediated transformation Electrical methods • Electroporation Horizontal gene transfer / Lateral gene transfer
- 6. Conjugation • Conjugation isa natural microbial recombination process. • During conjugation, two live bacteria (a donor and a recipient) come together, join by cytoplasmic bridges and transfer single-stranded DNA (from donor to recipient). • Conjugation can occur among the cells from different genera of bacteria. Bacterial transformation / Bactofection • It is a method of direct gene transfer using bacteria into the target cell, tissue, organ or organism. • The genes located on the plasmids of the transformed bacterial strains are delivered and expressed into the cells. • Strains that are invasive and having better cell to cell spread are more efficient.
- 7. Transduction • Transduction isthe process by which DNA is transferred from one bacterium to another by a virus. • Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA. • Movement of DNA from one location to another. • Segments of DNA with this ability to move are called transposable elements or transposon. • Transposition can be either "replicative" or "conservative" in nature. • Method of gene silencing. Transposition
- 8. Macroinjection • Used inrye (Secale cereale) plants. • Needles used for injecting DNA are with the diameter greater than cell diameter. • DNA injected with conventional syringe into region of plant which will develop into floral tillers. • Timing of injection is important and should be fourteen days before meiosis. • Dela Pena and coworkers in 1987. • Used to study the cellular functions of plant cells and plastid physiology, e.g. in tobacco, alfa alfa and Vicia faba • Very fine needles or glass micropipettes having the diameter of 0.5 to 10 μm are used. • Computerized control of holding pipette, needle, microscope stage and video technology has improved the efficiency. • Crossway and Reich in l986. Microinjection
- 9. Macroinjection Advantages • This techniquedoes not require protoplast. • Instrument will be simple and cheap. • Methods may prove useful for gene transfer into cereals which do not regenerate from cultured cell easily. • Technically simple. Disadvantages • Less specific. • Less efficient. • Frequency of transformation is very low. • Only proved on rye. Microinjection Advantages • Frequency of stable integration of DNA is far better as compare to other methods. • Method is effective in transforming primary cells as well as cells in established cultures. • The DNA injected in this process is subjected to less extensive modifications. • Mere precise integration of recombinant gene. • Disadvantages • Costly & Skilled person required. • Limited use in plants due to characteristic of plant cell wall.
- 10. Protoplast fusion • Protoplastsare the cells of which cell walls are removed and cytoplasmic membrane is the outermost layer. • Protoplast fusion is a physical phenomenon, reported successful b/w Brassica napus and Diplotaxis harra. • Normally isolated protoplast do not fuse with each other because the surface of isolated protoplast carries negative charges (-10mV to - 30mV) • Some agents required to reduce the electronegativity of the isolated protoplast and allow them to fuse with each others. Mechanical fusion • Under the microscope and using micromanipulator or perfusion micropipette, protoplasts brought into intimate contact Chemical fusion • Sodium nitrate, polyethylene glycol, Ca2+ commonly used. • Non specific, in expensive and cause massive fusion product having less frequency Electofusion • Electric field of low strength 10 Kvm-1 is used. • Easy to control with a fusion frequency 100 % • Sophisticated and expensive procedure.
- 12. • Developed bySanford & co- workers in 1987. • The use of DNA-coated microprojectiles (tungsten or gold) for the delivery of genes into intact cells and tissues. • Successfully used for transformation in soybean, tobacco, maize, rice, wheat, oats and tall fescue. • These microprojectiles, normally 1-3pm in diameter and the acceleration is achieved either by an explosive charge (cordite explosion) or by using shock waves initiated by a high voltage electric discharge. Advantage • Used to transform all plant species. • No binary vector is required. • Transformation protocol is relatively simple. Disadvantage • Difficulty in obtaining single copy transgenic events. • High cost of the equipment and microcarriers. • Intracellular target is random. • Transfer DNA is not protected. Biolistic transformation / Microprojectile bombardment
- 14. • It willbe ideal to introduce the transgene into the microspore when it is uninucleate, so that the transgene is passed on to both the generative cells. • Touraev and co-workers pioneered this method in tobacco during 1997. • Although scientists were pondering over pollen transformation for a long time without success, it can possibly become a successful technique in future if right efforts are made Advantage • Fast and regeneration independent method • Avoids somaclonal variation • Genotype independent • Cheap method • No need for skilled workers • No need of in vitro conditions • Can be routinely employed • Availability of large number of pollen grains Disadvantage • Not demonstrated in many species • Genes have to be introduced into pollen at correct stage • Frequency of transformation is low Pollen tube Transformation
- 16. • DNA isallowed to mix with calcium chloride solution and isotonic phosphate buffer to form DNA calcium phosphate precipitate. • When the actively dividing cells in culture are exposed to this precipitate for several hours, the cells get transformed. • The success of this method dependent on the high concentration of DNA and the protection of the complex precipitate. • Addition of dimethyl sulfoxide (DMSO) increase the efficency of transformation. Limitations • Frequency is very low • Integrated gene undergo substantial modification • Many cells do not like having the solid precipitate adhering to them and the surface of their culture vessel • Integration with host cell chromosome is random Calcium phosphate mediated transformation
- 18. Polybrene/DMSO assisted transformation •Simple and versatile method • Capable of producing high number of stable transfectants with low quantities of exogenous DNA. • Two step procedure 1. Adsorption 2. internalization • Adsorption is mediated by a polycation polymer called polybrene • Unifrom coating of target cells with polybrene-DNA complexes • Internalization of DNA complexes is by the exposure to dimethyl sulfoxide (DMSO) . • During 25-30% DMSO treatment, cell membrane permeability increases which enhance the uptake. Advantage • Less toxic than other polycations. • High transformation efficency requires very small quantities of plasmid DNA to be used.
- 19. • This methodis only for protoplast • Protoplast kept in a solution containing PEG and plasmid DNA. • Calcium chloride is added and sucrose and glucose acts as osmotic buffering agent. • PEG (15-25%) in the presence of Ca2+ , destabilize plasma membrane of the protoplast renders it permeable to naked DNA • Which stimulates uptake of DNA by endocytosis without any gross damage to protoplasts. • After transfer of DNA to protoplast, PEG is allowed to get removed Advantage • A large number of protoplast can be simultaneously transformed • Can successfully use for a wide range of plant species Limitations • DNA is susceptible for degradation and rearrangements. • Random integration of foreign DNA into genome may result undesirable traits. • Regeneration of plants from transformed protoplasts is a difficult task. Polyethylene glycol mediated transformation
- 20. • DNA complexedto the high molecular weight polymer Di Ethyl Amino Ethyl Dextran (DEAE) is used as a method of gene transfer. • If DEAE-Dextran treatment is coupled with Dimethyl Sulphoxide (DMSO) shock, then upto 80% transformed cell can express the transformed gene. • Treatment with chloroquinine increase transient expression of DNA Advantage • It is cheap and simple • Can be used for transient cells which cannot survive even short exposure of calcium phosphate Disadvantage • Stable expression is very difficult to obtain • After transformation, further improvement of plant tissue culture is not good • Truncation, recombination, rearrangement and silencing may occur. DEAE – Dextran mediated transformation
- 21. • Liposomes arespheres of lipids which can be used to transport molecules into the cells called lipofection. • Cationic liposomes fuse with cell surface resulting in the delivery of the DNA directly across the plasma membrane. • These are commercially available lipids that are sold as an in vitro-transfecting agent. • Cationic liposomes can be produced from a number of cationic lipids like DOTAP and DOTMA Advantages • Protection of DNA/RNA from nuclease digestion. • Low cell toxicity. • Stability and storage of nucleic acids due to encapsulation in liposomes. • High degree of reproducibility. • Applicability to a wide range of cell types. • Simplicity. Liposome mediated transformation
- 23. • Plant materialslike cell suspension culture, embryos etc introduced into a buffer containing DNA and the silicon fibres which is then vortexed. • The fibres (0.3 – 0.6 micro meter in dia. and 10-100 micro meter long) penetrate the cell wall and plasma membrane, allowing the DNA to gain access to the inside of the cells • Cellular penetration is likely enhanced by frequent and forceful intercellular collision encountered in the vortexed millue. Advantage • Improvement in yield • Herbicide resistance • Improvement in quality • Insect resistance • Resistance to abiotic stress • Rapid and accurate technique Disadvantage • Inherent dangers of fibres, require careful handling • Many cereals produce embryonic callus which is hard and compact. Silicon carbide mediated transformation
- 24. • This methodwas introduced by Fromm and his coworkers in 1986. • High field strength electrical impulses used to reversibly permeabilize the cell membranes for the uptake of DNA. • DNA then migrates through high- voltage-induced pores in the plasma membrane called electrospores. • Low voltage long pulses (300-400 V cm-1 for 10-50 ms) or High voltage short pulses (1000-1500 V cm-1 for 10 µs) used. • Reported in Tobacco, petunia, maize, rice, wheat and sorghum etc. Advantage • Both intact cell and tissue can be transformed. • Less costly. • Applied for a number of cell types. • Simultaneously a large number of cell can be treated. • High percentage of stable transformants can be produced Disadvantage • About 50% of the transformed cells can survive. • No proof was available for transfer of gene into walled cells through this method. Electroporation
- 26. Vector-mediated gene transfer •Agrobacterium mediated transformation • Virus mediated transformation
- 27. Agrobacterium mediated transformation •It is rod shaped and motile, and belongs to the bacterial family of Rhizobiaceae • It is known as natural genetic engineer A. tumefaciens that induces crown gall disease. A. rhizogenes that induces hairy root disease.
- 28. Organization of Tiplasmid
- 29. T-DNA transfer andintegration 1. Signal induction to Agrobacterium 2. Attachment of Agrobacterium to plant cells 3. Production of virulence proteins 4. Transfer of T-DNA out of Agrobacterium 5. Transfer of T-DNA into plant cells and integration
- 31. Ti plasmid derivedvector system • Ti plasmid of Agrobacterium has the ability to genetically transform plants. • Insert a desired DNA sequence (gene) into the T-DNA region (of Ti plasmid). • Use A. tumefaciens to deliver this gene(s) into the genome of plant cell. • There are some limitations to use Ti plasmid directly as vector. • Ti plasmid- based vectors with suitable modifications are used for transgenesis. • Two types of Ti plasmid-derived vectors are used for genetic transformation of plants— co- integrate vectors and binary vectors Co-integrate vector • T-DNA genes for hormone biosynthesis are removed. • In place of the deleted DNA, a bacterial plasmid (pBR322) DNA sequence is incorporated - intermediated cloning vector. Binary vector • Binary system is a pair of plasmids consisting of a binary plasmid and a helper plasmid. • Binary plasmid: shuttle vector • Helper plasmid: Entire T-DNA region including borders deleted while vir gene is retained.
- 32. Steps for Agrobacteiummediated transformation
- 33. Advantages • This isa natural method of gene transfer. • Agrobacterium can conveniently infect any explant (cells/tissues/organs). • Even large fragments of DNA can be efficiently transferred. • Stability of transferred DNA is reasonably good. • Transformed plants can be regenerated effectively. Limitations • There is a limitation of host plants for Agrobacterium, since many crop plants (monocotyledons e.g. cereals) are not infected by it. • The cells that regenerate more efficiently are often difficult to transform.
- 34. Virus mediated transformation •Natural vectors for genetic engineering. • They can infect the intact plants and amplify the transferred genes through viral genome replication. • They can introduce the desirable gene(s) into almost all the plant cells since the viral infections are mostly systemic. • Plant viruses are non-integrative vectors: They spread systemically within the host plant where the new genetic material is expressed. • The three groups of plant viruses used – Caulimo viruses, Gemini viruses, RNA viruses
- 35. Caulimo viruses • Thecaulimo viruses contain circular double- stranded DNA, and are spherical in shape. • Widely distributed and Cauliflower mosaic virus (CaMV) is the most important for gene transfer. • Limited capacity for insertion of foreign genes. Gemini viruses • Twin and paired capsid structures with one or two single-stranded circular DNAs. • They can infect a wide range of crop plants. • But it is very difficult to introduce purified viral DNA into the plants. RNA plant viruses • Two type of single stranded RNA viruses. 1. Mono-partite viruses - Tobacco mosaic virus 2. Multipartite viruses - Brome mosaic virus – good efficiency to infect cells – and spread to different tissues. – high level of gene expression - difficulty of joining RNA molecules in vitro.
- 36. Biosafety issues relatedto GM crops
- 38. Bt cotton • Btis a naturally-occuring soil borne bacterium found worldwide. • Shigetane Ishiwattari in 1901 first isolated. • Gram positive, facultative aerobic, rod-like, motile and sporulating bacterium. • Produces crystals of endotoxin (cry protein or delta toxin)- toxic to insect mainly in the larval stage . • These crystal protein (cry protein) are insect stomach poisons. • Thus they act as insecticide. • Insect stop feeding within two hours of bite,if enough toxin is eaten, die with in two or three days. Mechanism 1. Ingestion. 2. Solublization & proteolytic activation. 3. Binding to target site. 4. Formation of toxic lesions.
- 40. Biosafety issues relatedto GM crops 1. Bio-safety of human and animal health 2. Labelling of GM and non GM food 3. Ecological concerns 4. Public attitude 5. Socio economic and ethical consideration
- 41. 1.Bio-safety of humanand animal health a. Safety of source organism and genes b. Effectiveness of antibiotics c. Potential toxicity and allergenicity d. Changes in nutritional level 2. Labelling of GM and non GM food a. Keeping GM and non GM crops seperately. 3. Ecological concerns a. Creation of weediness b. Erosion of biodiversity • Pollution of gene pools 4. Public attitude • Consumer response depends on perceptions about risks and benefits of GM crops • Communicate to the customers and stake holders • Media, individuals, scientists , administrators, politicians and NGO have the responsibility. 5. Socio economic issues • Due to increasing seed market, developing countries may get dependent on few suppliers • Countries with effective and efficent technology will obtain and sustain advantage of international markets
- 42. Direct effect onnon- target organism In may 1999, reported that pollen from Bt resistant corn had a negative impact on Monarch butterfly larvae This report raised concern about Monarchs and other non target organisms. Issue of accidental cross breeding • A recent study of Jean- Francois Arnaud published by Royal society reported the cross breeding of GM crops. • He studied three separate crops of commercial sugar beet, wild sea beet and weed beet . • From the DNA matching he found that , weed beet act as a bridge between the wild and commercial crops via accidental seed flow.
- 43. Impact of BtBrinjal in Bengladesh • Brinjal (Solanum melongena L.), also known as eggplant, is a popular multiuse vegetable cultivated in Asian countries • The eggplant fruit and shoot borer (FSB) is responsible for the chronic and widespread infestation • In severe cases, the infestation levels may exceed 90% and causing yield loss of up to 86% in Bangladesh. • On 30 October 2013, Bangladesh approved the official release of four genetically modified, varieties of insect-resistant Bt- brinjal for seed production and initial commercialization. Impact of the Bt brinjal • This survey found that 89% of the farmers believe the cultivation of Bt-brinjal improved quality of brinjal • 59% of farmers opined that price was reduced due to Bt- brinjal cultivation because of higher production • The farmers (97%) informed that Bt-brinjal cultivation reduced pesticide use. • 96% of farmers consider Bt- brinjal is safe for human health
- 44. Possibility of pestshift • Northern China reported long term ecological effect of Bacillus thuringiensis on non target pest. • Over a period of 10 years, Bt cotton cultivation become increased and insecticide use in this crop dropped considerably. • Mirid bugs (Heteroptera: Miridae) have progressively increased the population size and acquired pest status.
- 45. Regulatory mechanism inIndia • Biosafety regulations cover assessment of risks and the policies and procedures adopted to ensure environmentally safe applications of biotechnology. Rules and policies 1. Rules, 1989 under Environment Protection Act (1986). 2. Seed Policy, 2002. Guidelines 1. Recombinant DNA guidelines, 1990 2. Guidelines for research in transgenic crops, 1998 Agency for implementation • Ministry of Environment, Forests and Climate Change. • Department of Biotechnology, Government of India. Competent authorities 1. Recombinant DNA Advisory Committee (RDAC) 2. Review Committee on Genetic Manipulation (RCGM) 3. Genetic Engineering Approval Committee (GEAC) 4. Institutional Biosafety Committees (IBSC) 5. State Biosafety Coordination Committees (SBCC) 6. District Level Committees (DLC).
- 46. Conclusion • Different typesof methods are available for gene transfer. • The choice of methods of DNA transfer depends upon the target cells and objectives of gene manipulation. • Agrobacterium mediated transfer is reported to be widely adopted one. • Biosafety issues should be addressed before releasing GM crops.
- 47. Reference • https://bio.libretexts.org/@go/page/9297 • http://www.ejbiotechnology.info/content/vol1/issue3/full/1/figur e1.html •https://bio.libretexts.org/@go/page/9298 • https://vikaspedia.in/agriculture/crop-production/advanced- technologies/transgenic-crops-biosafety-concerns-and- regulations-in-india • Khan, K.H. 2010. Gene transfer technologies and their applications: roles in human disease. Asian J. Biol. Sci. 1: 208-218. • Khan, K.H.2009. Gene transfer technologies in plants: roles in improving crops. Recent Res. Sci. Technol. 1(3): 116-123. • Shelton A. M., Sarwer, S. H., Hossain, Md. J., Brookes, G and Paranjape, V. 2020. Impact of Bt Brinjalcultivation in the market value chain in five districts of Bangladesh. Front. Bioeng. Biotechnol. https://doi.org/10.3389/fbioe.2020.00498
Editor's Notes
- #6 Horizontal gene transfer also known as lateral gene transfer is the process by which an organism incorporates genetic material from another organism without mating.
- #8 Transduction is especially important because it explains one mechanism by which antibiotic drugs become ineffective due to the transfer of antibiotic-resistance genes between bacteria. Commonly used virus are bacteriophage viruses, retroviruses are also used. In replicative transposition, the transposable element is copied and and the copy is relocated, leaving behind the original. This results in duplicate copies of the TE existing within the genome. In conservative transposition, the transposable element is excised from its original location then reinserted at a new location. Because transposons can damage genomes by interfering with essential gene expression.
- #9 Its importance for plant transformation is rather limited due to the characteristics of plant cell walls, which contain a thick layer of lignins and cellulose. The plant cell wall is a barrier for glass micro tools.
- #10 The presence of lignin and cellulose in the plant cell wall reduce the efficiency and success rate.
- #11 Protoplast can be obtained by specific lytic enzymes to remove cell wall - Cellulase, pectinase or macerozyme for breaking plant cell wall. During fusion two or more protoplasts come in contact and adhere with one another either spontaneously or in presence of fusion inducing agents. Brassica napus – Rapeseed, Diplotaxis harra – leaf vegetable Negative charge around the outside of the plasma membrane. And thus their is a strong tendency in the protoplast to repel each other due to their same charges .So this type of fusion needs a fusion inducing chemicals which actually reduce the electronegativity of the isolated protoplast and allow them to fuse with each others.
- #14 A handy Helios Gene gun used for gene delivery into target host plants, tissues in the gene house or under standard lab condition. This gun uses biolistic particle bombardment where DNA coated gold particles are located into the gun. A low pressure helium pulse delivers the coated gold particles into virtually any target cell or tissue.
- #15 Pollen transformation is conducted on the basis of the assumption that DNA uptake by pollen will lead to integration of transgene into the germline with subsequent transmission to the progeny. Two decades back, transformation of ‘cereals’ was considered very difficult (Potrykus 1990b), but today it is a routine technique. Mature pollen grain contains a vegetative cell and two sperm cells. Since only one of the sperm cells fuse with the egg cell giving rise to the zygote and the second sperm cell fuses with the central cell to produce the endosperm (Berger et al. 2008), the introduction of DNA into the sperm cell, destined to fuse with the egg cell is important in determining the development of transgenic seed, while DNA transferred to vegetative cell and to the second sperm cell involved in triple fusion to produce endosperm will not produce transgenic plants, with subsequent transmission of the trait to the progeny. Hence, it will be ideal to introduce the transgene into the microspore
- #22 These are artificial vesicles can act as exogenous materials including transgene Cationic liposomes are those having positive charge, There are also liposomes with uncharged.
- #24 Vortexing is an eppendorf tube containing a mixture of plasmid DNA encoding a selectable or screen able marker gene, si fibre and the explants tissue. Vortexing can occur when liquids move in a circular motion forming a cavity, it's most prevalent in situations where a suction force is applied. eppendorf tube - microcentrifuge tubes
- #25 Low voltage long pulses for produce high rate of transient formation. High voltage short pulses for produce high rates of stable transformation.
- #28 In 1907, Smith and Townsend postulated that a bacterium was the causative agent of crown gall tumors. A. tumefaciens infects wounded or damaged plant tissues and induces the formation of a plant tumor called crown gall. The entry of the bacterium into the plant tissues is facilitated by the release of certain phenolic compounds (acetosyringone, hydroxyacetosyringone) by the wounded sites. Bacterium releases its T-DNA into the plant cell cytoplasm. T-DNA, gets integrated into the plant cell chromosome (i.e. host genome). Thus, crown gall disease is a naturally evolved genetic engineering process.
- #29 Ti plasmids (~200 kb ) exist as independent replicating circular DNA molecules within the Agrobacterium cells. The T-DNA (transferred DNA) is variable in length in the range of 12 to 24 kb, which depends on the bacterial strain from which Ti plasmids come. Nopaline strains of Ti plasmid have one T-DNA with length of 20 kb Octopine strains have two T-DNA regions referred to as TLand TR that are respectively 14 kb and 7 kb in length. This region has the genes for the biosynthesis of auxin (aux), cytokinin (cyt) and opine (ocs), and is flanked by left and right borders. It is now clearly established that the right border is more critical for T-DNA transfer and tumorigenesis. Responsible for the transfer of T-DNA into the host plant Located outside T-DNA Referred as vir or virulence region. Codes for proteins involved in T-DNA transfer. At least nine vir-gene operons have been identified- vir A, vir G, vir B1, vir C1, vir D1, D2 and D4, and vir E1, and E2. Opine catabolism region:This region codes for proteins involved in the uptake and metabolisms of opines. Ori region: responsible for the origin of DNA replication which permits the Ti plasmid to be stably maintained in A. tumefaciens
- #30 1. The wounded plant cells release certain chemicals- phenolic compounds and sugars which are recognized as signals by Agrobacterium. The signals induced result in a sequence of biochemical events in Agrobacterium that ultimately helps in the transfer of T-DNA of T-plasmid. 2. Attaches through polysaccharides, particularly cellulose fibres produced by the bacterium. Several chromosomal virulence (chv) genes responsible for the attachment of bacterial cells to plant cells. 3. Signal induction by phenolics, stimulates vir A which in turn activates (by phosphorylation) vir C. This induces expression of virulence genes of Ti plasmid to produce the corresponding virulence proteins (D1, D2, E2, B etc.). Certain sugars (e.g. glucose, galactose, xylose) also induce virulence genes. 4. The right and left borders of T-DNA are recognized by vir D1/vir D2 proteins. These proteins are involved in the production single-stranded T-DNA (ss DNA), its protection and export to plant cells. 5. The ss T-DNA — vir D2 complex in association with vir G is exported from the bacterial cell. 6. T-DNA-vir D2 complex crosses the plant plasma membrane. In the plant cells, T-DNA gets covered with vir E2 to protect T-DNA from degradation by nucleases vir D2 and vir E2 interact with a variety of plant proteins which influences T-DNA transport and integration.The T-DNA-vir D2-vir E2 — plant protein complex enters the nucleus through nuclear pore complex. Gets integrated into the plant chromosome through illegitimate recombination.
- #32 The desired foreign gene (target-gene) is first cloned in the multiple cloning site of the intermediate vector. The cloning process is carried out in E. coli, the bacterium The intermediate vector is mated with Agrobacterium so that the foreign gene is mobilised into the latter. Binary plasmid: shuttle vector (E. coli and A. tumefaciens). T-DNA: left and right border sequences, transgene, plant selectable marker (PSM) Outside T-DNA: bacterial selectable marker (BSM) and an origin of replication (ori) for bacteria Helper plasmid: Entire T-DNA region including borders deleted while vir gene is retained It may be noted that both of them are (or integrated). not physically linked. The target (foreign) gene of interest is inserted into the multiple cloning site of the binary vector. Between the right and left border repeats and cloned in E. coli. By a mating process, the binary vector is mobilised from E. coli to Agrobacterium. Now, the virulence gene proteins of helper plasmid facilitate the transfer of T-DNA of the vector into plant cells.
- #35 i. Must be capable of spreading from cell to cell through plasmodesmata ii. Viral genome should be able to replicate in the absence of viral coat protein and spread from cell to cell (insertion of foreign DNA will make the viral genome too big to be packed) iii. Should have a broad host range & must elicit little or no disease symptoms in the infected plants. v. Virus with DNA genome is preferred since the genetic manipulations involve plant DNA.
- #36 Gemini virus can infect a wide range of crop plants (monocotyledons and dicotyledons). Mono-partite viruses: Large viruses and contain undivided genomes for all the genetic information e.g. Tobacco mosaic virus (TMV). 2. Multipartite viruses: Genome is divided into small RNAs which may be in the same particle or different particles e.g. Brome mosaic virus (BMV). With virus vectors, this is not possible unless the virus is seed-transmitted However, in case of vegetatively propagated plants, transmission of desired traits can be done e.g. potatoes. Even in these plants, there is always a risk for the transferred gene to be lost anytime.
- #37 Organisms produced by various gene transfer techniques. They have some improved characters like, Drought resistance, yield improved, herbicide tolerant etc.
- #42 a. Danger from eating foreign DNA in GM crops b. Antibiotic resistance marker reduce effectiveness of antibiotic to fight disease when these crops are taken with the meal. 2. For illiterate people color codes should be used.
- #46 IBSC - set-up at each institution for monitoring institute level research in genetically modified organisms. RCGM - set-up at DBT to monitor ongoing research activities in GMOs and small scale field trials. GEAC- set-up in the Ministry of Environment, Forests and Climate Change to authorize large-scale trials and environmental release of genetically modified organisms. The Recombinant DNA Advisory Committee (RDAC) constituted by DBT takes note of developments in biotechnology at national and international level and prepares suitable recommendations. The State Biotechnology Coordination Committees (SBCCs) set up in each state where research and application of GMOs are contemplated, coordinate the activities related to GMOs in the state with the central ministry. SBCCs have monitoring functions and therefore have got powers to inspect, investigate and to take punitive action in case of violations. Similarly, District Level Committees (DLCs) are constituted at district level to monitor the safety regulations in installations engaged in the use of GMOs in research and application.