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pBR322 is one of the most used cloning vectors in molecular Biology. Cloning vectors, best-known as plasmids, are autonomously replicating DNA units into which DNA fragments can be inserted for gene cloning. Genes taken up by these plasmids are multiplied (or cloned) as the vector replicates, to yields numbers suitable for molecular analysis. The most versatile and well-known plasmid is certainly pBR322 (in fact was one of the first ever used in gene cloning techniques) and has genetically tailored cutting sites into which DNA can be inserted without affecting plasmid self-replication. pBR322 general characteristics are: a) Size: 4.3 kb; b) Replicon: ColE1, relaxed; c) Selective markers (resistance): Amp and Tet; d) Single sites (enzymatic restriction single sites): Ava I, Pst I, BamHI, PvuII, ClaI, SalI, EcoRI, and HindIII.
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What is the cloning capacity of pBR322 vector?
The cloning capacity of pBR322 vector is 1-5kb.
Why pBR322 plasmid have high copy number?
I think pBR322 has a replication module from E coli plasmid colE1 ,which permits plasmid replication even when chromosome replication and cell division are inhibited by amino acid starvation and chloramphenicol, as a result, under such condition each cell accumulates several thousands copies of the plasmids up to 3000, so that one litre of bacterial culture easily yields a milligram of plasmid DNA.
Explain the functioning of pBR322 vector with diagram?
FUNCTIONG OF pBR322
What is the cloning capacity of pBR322 vector?
The cloning capacity of pBR322 vector is 1-5kb.
How many bands were produced after pBR322 was digested with EcoR1?
pBR322 has one EcroR1 site so 1 band however if it was not fully digested you will find 2 or 3 (Linear- [cut], Supercoiled-, Round-Plasmid).
What is the advantages and disadvantages of pBR322 as a cloning vector?
pBR322 advantages is it widely used for the analysis of prokaryotic transcription and translation as well as topological changes in DNA conformation. then the disadvantage is it has only few cloning sites and the selection procedure is therefore time consuming.
Why pBR322 plasmid have high copy number?
I think pBR322 has a replication module from E coli plasmid colE1 ,which permits plasmid replication even when chromosome replication and cell division are inhibited by amino acid starvation and chloramphenicol, as a result, under such condition each cell accumulates several thousands copies of the plasmids up to 3000, so that one litre of bacterial culture easily yields a milligram of plasmid DNA.
What is universal primer?
Universal primers are really not 'universal' in the sense that they will bind to anything. Universal is kind of a misnomer. Really, universal primers are PCR/sequencing primers that bind to a sequence found in many plasmid cloning vectors, most of which are derived from pUC vectors (which in turn come from pBR322). These sequences were defined as good PCR and sequencing sites as they flank the multiple cloning site where an inserted DNA sequence would be put. You can now buy these universal primers from various companies. You can see that these primers are called universal because they can be used to amplify or sequence any insert that is put in the multiple cloning site.
How do restriction enzymes create recombinant DNA?
There are many methods, though one of the most common is the use of restriction endonucleases. These enzymes can be used to cut DNA fragments at specific locations. Cut DNA fragments will recombine into new orders, which are sealed using DNA ligase. A selection process must be used to locate the desired recombinant DNA, since it will be in a mixture of various undesired recombinations.
Can antibiotics cause resistance in bacteria?
A simple way to explain it is:When you have an infection and take antibiotics for it, the weaker bacteria are killed first, with the stronger ones surviving, or taking longer to destroy. So when you don't take the full round of antibiotics, the stronger bacteria are the ones left in your body, and they are the ones that will continue to multiply, resulting in a bacteria resistant to the antibiotic. This is why you should always take the full prescribed course of antibiotics.Also, when antibiotics are prescribed, the body's natural defense system (the good bacteria) are destroyed along with the bad bacteria. This is why you should always eat yogurt with active cultures or drink buttermilk while on antibiotics - the active cultures put the "good" bacteria back in the body.
What is the role of a plasmid in genetic engineering?
A plasmid vector available today is made with a specific host in mind. For example, if you decide to express a gene in a bacteria, there will be plasmids available with features that suit the particular organism that you wish to transform and they will be different from plasmids used to transfect for example, yeast. However, generally, a plasmid will have at the very least an origin of replication recognizable by the desired organism, a promoter upstream of the multiple cloning site that is recognizable by the organisms, and a selection marker such as an antibiotic resistance gene.The process of expressing a gene from one organism in another host via plasmid vectors begin with the isolation of the gene from the original organism. For the sake of this example, suppose the insulin gene in humans is the gene of interest. First, beta cells from the Islets of Langerhans will have to be lysed and total RNA will be isolated from the cell. Because DNA is filled with many introns that are hard to get rid of, gene isolation from higher eukaryotes almost always start from the mRNA stage because the introns were already sliced out in mRNA processing. The RNA will be then subjected to reverse-transcriptase polymerase chain reaction with primers specific for the insulin gene. The insulin gene will subsequently be selectively amplified and the reaction mixture can then be purified to contain only cDNA of the insulin gene.With the purified cDNA, a process called molecular cloning is used to get the gene into the plasmid. The plasmid and the gene are both cut with compatible restriction enzymes. The cuts on the plasmid has to be in the multiple cloning site the the cuts on the gene has to be outside of the open reading frame for the cloning to produce an effective vector. (Review molecular biology for the necessity of promoters and an intact open reading frame) The cut plasmid and gene fragments are then placed together and ligated. The ligated product should theoretically now contain the gene inside the multiple cloning site directly following the promoter. The promoter may express the gene constitutively or it may be inducible, requiring certain conditions to be met before it is turned on.The plasmid with the cloned insulin gene can now be transformed into competent bacteria hosts (or yeast if desired, however it will not be as efficient). Competence describes the ability of bacteria to take up DNA from its surroundings. The most commonly used host, E. coli, are artificially made to be competent by treatment with a high concentration calcium solution in a cold environment, while others, such as B. subtilis, are naturally competent. All bacteria can be made competent with electroporation but E. Coli is most often used because of its easily satisfied nutrient requirements and very short generation time. The plasmid and competent E. coli is placed together in a cold environment to initiate the uptake of the plasmid into E. coli cells. The mixture is then heat shocked and bacterial growth medium with the necessary selection agent is added to start the incubation process. If the selection marker on the plasmid is an antibiotic resistance gene, for example ampicillin resistance, a medium with ampicillin will be used to incubate the bacterial culture because only the cells that contain the plasmid will be resistant to the antibiotic while cells that have failed to take up the plasmid will die. The cells can then be incubated for as long as needed and split into different cultures if needed because they now contain the plasmid and will express the gene carried on the plasmid.A plasmid can be considered as a suitable vehicle for cloning, because 1. It can be isolated from the cells2. It possesses a single restriction site for one or more restriction enzymes.3.Insertion of a linear molecule at one of these sites does not alter its replication properties.4.Reinsertion of these vectors to the host cell can identified and selectable.5.They do not occur free in nature and found in bacterial cells.Ex: for plasmid cloning vectors are pBr322,pACYC18,pUC,pUN121.
Another science word that starts with a P?
P element P1 P1-derived artificial chromosome P1-derived artificial chromosome (PAC) P53 PAC pachynema Paleontology palindrome Palindromic sequence pAMP Pan balance Panel testing panmictic papilla papillate paracentric inversion paralogous genes paramecin parameters parapatric speciation Parasegment border parasexual cycle parasite Parasitism Parasitology parasympathetic nervous system paratope parent generation parental ditype (PD) parenteral parietal lobes parthenogenesis partial digest particle particulate inheritance parts per billion (ppb) Parts per million (ppm) Pascal's triangle passive diffusion passive transport paternally path diagram pathogen pathogenesis pathogenic pathogenicity pathology pathovar patient patroclinous inheritance pattern formation pBR 322 pBR322 PCR PCR amplicons pedigree pelagic pellet Pelvic inflammatory disease (PID) penetrance pent- peptide peptide bond peptidyl site peptidyl transferase per- percent coefficient of variation percent concentration percent error percent yield peri- peri-natal pericentric inversion pericentromere perinatal period periodic law peripheral membrane protein peripheral nervous system (PNS) peripheral neurons peripheral neuropathy periphyton permissive condition permissive temperature peroxidase Persistence Pest sequence Pesticide petite petite mutation petrifaction pH pH scale phagocytes phagocytosis pharmacotyping pharyngeal arches pharynx phasmid Phencyclidine hydrochloride (PCP) phenocopy phenotype phenotypic sex determination phenotypic variance Phenylalanine phenylketonuria (pku) pheromone Philadelphia chromosome phloem Phosphatase Phosphate group phosphodiester bond phosphodiesterase Phospholipase A2 (PLA2) phospholipids Phosphorus (P) phosphorylate Phosphorylation photic zone photoautotroph photoheterotroph photon photoreactivation Photorespiration phragmospore phyletic gradualism phylloplane phylogenetic tree physical change physical chemistry Physical map phytoplankton phytoplasma phytotoxic phytotoxin piebald pilus (plural pili) Pipettes Pituitary pK pKa placebo placenta planet plankton plant plant breeding plaque plasma Plasma membrane plasmalemma plasmid plasmid suicide vector plasmogamy plasmolysis plastid plate platelet platelet-activating factor (PAF) Pleiotrophy pleiotropic mutation pleiotropy pleo- plerome plesionecrosis plexus ploidy Pluripotency PMA poikilothermal point mutation Poisson distribution poky mutation polar polar body polar covalent bond polar effect polar gene conversion polar granules polar molecule polar mutation polarity polarity gene Pole cells Pollen grain pollinator Poly(A) polymerase poly- poly-A tail poly-dA/poly-dT technique polyacrylamide Polyacrylamide gel electrophoresis polyadenosine tail polyatomic polycistronic polycistronic mRNA Polyclonal antibodies polydactyly polyethylene polygene polygenic Polygenic disorder polygenic inheritance polyinvagination islands Polylinker Polymer polymerase (DNA or RNA) polymerase chain reaction polymerase slippage polymerase slippage model polymerize polymodal polymorphism polynucleate Polynucleotide polynucleotide phosphorylase polynucleotide polymerase polyolefin polypeptide polyphenism polyphyletic polyploid polysaccharide polysome polyspermy polytene chromosome Polyvalent vaccine pons population population density Population genetics position effect position-effect variegation Positional cloning Positional information positive assortative mating positive control positive interference post- post-transcriptional modification posterior neuropore postmortem postreplicative repair Postsynaptic Membrane potential energy potentiometric titration pre- pre-mRNA pre-symptomatic pre-synaptic terminal precipitate precision precocious predation predator preemptor stem preformationism Premarket Approval prey Pribnow box Primary cell primary consumer primary oocyte primary spermatocyte primary structure primary transcript primase primer primitive folds primitive streak primosome prion prion rods pro-inflammatory cytokines probability probability theory probe Probe Amplification processivity producer product product of meiosis product rule proflavin progeny testing prokaryote prokaryotic cell prolepsis proliferate Proline promoter Pronucleus proofread prophage prophase proplastid propositus prosencephalon prostaglandins prostate gland protamine protease proteasome protein protein aggregate protein synthesis Proteolytic Proteome Proteomics proto-oncogene protocorm proton proton acceptor proton donor proton gradient protoplast protostomes prototroph provirus prox- proximal PrP pseudo- pseudoallele pseudoautosomal gene pseudodominance pseudogene pull down assays pull-down assays pulse-chase experiment pulsed-field gel electrophoresis punctuated equilibrium Punnett square pure pure-breeding line or strain purines Purkinje cells putamen pycnosis pygmism pyknosis pyramidal nerve cells pyriform pyrimidine pyruvate pyruvic acid
What is transformational mediation?
INTRODUCTIONInduction of crown gall and hairy root diseases in several dicot plants by the common soil borne Gram-negative bacteria Agrobacterium tumefaciens and A.rhizogenes, respectively , are example of natural transformation of plants wherein the bacterial genes are stably introduced into the genome of higher plants. This system represents the only known natural case in which a prokaryotic organism transfers genetic information to a eukaryotic host. This capability underlies the biotechnological uses of Agrobacterium , mostly employed for the genetic transformation of numerous plants species. Recent discoveries have expanded the potential biotechnological uses of Agrobacterium; indeed , under laboratory condition , Agrobacterium is able to transfer DNA and proteins to numerous non.plant species , including several species of yeast and other fungi as well as sea urchin embryos , human cells in cultureAgrobacterium-mediated transformation is the easiest and most simple plant transformation. Plant tissue are cut into small pieces , eg. 10×10 mm, and soaked for 10 min. in a fluid containing suspended Agrobacterium . Some cells along the cut will be transformed by the bacterium that inserts its DNA into the cell . Placed on selectable rooting and shooting media , the plants will regrow .Some plants species can be transformed just by dipping the suspension of Agrobacterium and then planting the seeds in a selective medium. Unfortunately , many plants are not transformable bt this method .1. DETAIL DESCRIPTIONRecombinant DNA technology is based on the insertion of a DNA fragment ( Gene) of interest into a suitable cloning vector and then its introduction into a suitable host to propagate the recombinant DNA.Fig: generalized method of gene transfer in cells1.1. GENE CARRIER VEHICLE -1. If a gene is to be introduced into a host cell, a carrier moleculethat can transport the gene into the host cell is required Sucha molecule is called a cloning vehicle , carrier molecule or avector.1.2. FOLLOWING ARE A FEW GENE CARRIER VEHICLES1.Plasmids2. Bacteriophages3. Viruses2.1.AGROBACTERIUM TUMEFACIENS1. Agrobacterium tumecaiens is a soil borne gram negativebacterium.2 .It invades many dicot plants when they are injured at the soillevel and causes grown gall disease3.The bacterium enters the plant through a fresh wound andattaches itself to the wall of the intact cell.4.This cell is genetically transformed by bacterium .5. This transformation result in a tumour which synthesizesOPINES :A. The Tumors develops only at site of the wound.B. Such tumours can be removed from the plant and culteredin-vitro where they continue to grow indefinitely.C. Continued presence of agrobacterium is not required for tumorProfileration.D. Agrobacterium induced tumours synthesize a variety of unusalCompound called opinesE. Opines are of 3 types -a). Octapineb). Nopalinec). AgropineThese opines are catabolised by Agrobacterium to obtain energy,AT genectically engineers the plant cell for its won purpose.3.1. TUMOUR INDUCING PRINCIPLE1. The tumour inducing principle of AT is a plasmid calles tumourinducing plasmid or Ti Plasmid .a). 200 kb long.b). Has two essential regions : T-DNA and vir region.c). These two regions are essential for the transformation process.3.2. TRANSFER OF TUMOUR INDUCING PRINCIPLE1.T-DNA(transferred DNA ) is excised from the Ti-Plasmid andtransferred to the nucleus of the plant cell.2. Here the T-DNA gets integrated into the DNA which is stable.3. The T-DNA can be passed on to daughter cells as an integralPart of the plant chromosome.Figure. Induction of crown gall on a dicot plant by agrobacteriumtumefaciens.4.1 Ti PLASMIDS1. An extra chromosomal double stranded circular DNA molecule.2. Tumour inducing .3. 200 kb in size and conjugative type.4. Encodes enzymes responsible for the synthesis and catabolism of certain opnies.5. One of the opines is nopaline.6. pTiC58 is present in Agrobacterium strain C58. it is 192 kb long7. Only a small segment of the Ti Plasmid is transferred to the host plant cell and gets integrated with the grnome . This is the T-DNA.Figure. Ti plasmid pTic 58 having 192 kb5.1. T-DNA1.Only a small segmented of the Ti Plasmid is transferred to thehost plant cell and gets integrated with the genome.2.This is the T-DNA3.It contains gene for tumour formation (Tum) and nopalinebiosynthesis (Nos).4.The genes encodes enzymes that catalyse the synthesis ofphtohormones like the IAA and the cytokinin , isopentenyladenosine that cuse tumerous growth of cells in crown galls.5.The T-DNA is bordered by 25 bp repeats, required for theexcision and transfer of T-DNA.6.1. NOPALINE Ti PLASMID pTiC581. The Vir region of the Ti-plasmid contains the genes required forthe T-DNA transferprocess . the genes in this region encode theDNA processing enzymes required for excision , transefer andintegration of the T- DNA segmented.7.1. TUMOUR INDUCTION BY AGROBACTERIUM1. Recognition of susceptible wounded plant cell:a). Plant exudates: act as signal by inducing genes in the Vir Genesof the Ti Plasmid.b). Acetosyringone (as) , alfa- hydroxy acetosyringone (OH-HS)2. Binding to wound cells : controlled by two chromosomal genes ofagrobacterium : chv-A and chv-B.3. Excision , transfer and integration :a). The border repeats of T- DNA play an important role .b). Any DNA sequence located between the border repeats istransferred to the host plant.c). The T-DNA region is excised from the plasmid by the enzymesencoded by the vir- region.4.These enzymes specifically recognize the T-DNA borders.5.The T-DNA enters the plant cell and integrates into the hostgenome , mediated by host enzymes.8.1. Ti-PLASMID AS A VECTOR1. The Ti- plasmid has an innate ability to transmit bacterial DNAinto plant cells .2. This potential is explited by the genetic engineers to use as avector.3.The gene of a donor organism can be introduces into the Ti-plasmid at the T-DNA region4. This plasmid now becomes a recombinant plasmid.5. By agrobacterium infection , the donor genes can be transferredfrom the recombinant Ti-Plasmid and integrated into the genotypeof the host plant.6. This results in the production of transgenic plant.Pic. Ti-Plasmid mediated transfer of gene into a plant9.1. DISARMED Ti PLASMID1.Disarmed Ti-plasmida).Deletion of T-DNA regionb). PGV3850 is constructed from pTiC58.2. It has pBR 322 with AmpR3.It border repeats and NOS genes4. Agrobacterium having this PGV3850 can transfer the modifiedT-DNA into plant cells.5. But the recipient cell will not produce tumour , but could producenopaline.6. This can be used as a efficient vector for introducing foreign geneinto plants.10.1. CONTRUCTION OF A COINTEGRATE1. A foreign gene cloned into an appropirate plasmid (pBR322) canbe integrated with the disarmed Ti-Plamid by a homologousrecombination2. A compound plasmid called a cointegrate is formed.Figure. Cointegrate Plasmid .11.1. TRANSFORMATION OF TISSUE EXPLANTS BYCO-CULTIVATION WITH AGROBACTERIUM1. A co-integrate plasmid derived by recombination of pGV3850 and pBR322 loaded with foreign gene is now used to transfer the foreign gene into many crop plants.2. Small disc (a few mm diameter ) are punched from leaves of petunia, tobacco, tomato or other dicot plants.3. These disc are incubated in a medium containing Agrobacterium carrying the recombinant disarmed T-DNA as a co-ingerate .The cointegrate plasmid has the foreign gene and also the gene for resistance to kanamycin (KmR).4. The disc are cultered for two days . the agrobacterium infects the cut edges of the disc.5. The disc are then transferred to a shoot inducing solid medium (high cyokinin ) containing kanamycin to select the transferred kanamycin to select the transferred kanamycin gene. Corbenicillin in the medium kills agrobacterium.6. After 2-4 weeks the shoot develops.7. The callus having the shoot is transferred to root inducing solid medium (high in auxin content).8. After 4-7 weeks roots appear.9. The rooted plantlets are transferred to soil.Pic. Transformation of leaf disc explants by co-cultivation with agrobacteriumhaving the cointegrate Ti plasmidREVIEW OF LITERATUREAgrobacterium mediated transformation has been a method of choice in dicotyledonous plant species where plant regeneration system are well established (Van Wordragen and Dons , 1992 ; dale et al. 1993 ). The host range of this pathogen includes about 60% of gymnosperms and dicotyledonous angiosperms. Besides , transformation success has also been achieved in some monocots like Asparagus officinalis , Chlorophytum , Narcissus (hernalstees et al. 1984, hookyaasvan slogteren et al. 1984 ) .It was believed that monocots lack wound response i.e. factors that are required to initiate of `vir` genes. (Schafer et al. 1987) achieved success in transforming another monocot ,yam (Diosscorea bulbifera) from potato tubers .likewise , the use of acetosyringone (synthetic phenolic compound ) either during bacterial growth or during co-cultivation has been found to be beneficial in the transformation of other monocots. There are several reports on successful transformation of rice using Agrobacterium (Hiei et al. 1994 ; Li et al. 1994 ; vijaychandra et al. 1995 ). This method has also been extended to barley, wheat, maize and sugarcane.APPLICATIONGenetically modified plants have been developed commercially to improve shelf life, disease resistance, herbicide resistance and pest resistance. Plants engineered to tolerate non-biological stresses like drought, frost and nitrogen starvation or with increased nutritional value (e.g. Golden rice) were in development in 2011. Future generations of GM plants are intended to be suitable for harsh environments, produce increased amounts of nutrients or even pharmaceutical agents, or are improved for the production of bioenergy and biofuels. Due to high regulatory and research costs, the majority of genetically modified crops in agriculture consist of commodity crops, such as soybean, maize, cotton and rapeseed. However, commercial growing was reported in 2009, of smaller amounts of genetically modified sugar beet, papayas, squash sweet pepper, tomatoes, petunias, carnations, roses and poplars. Recently, some research and development has been targeted to enhancement of crops that are locally important in developing countries, such as insect-resistant cowpea for Africa and insect-resistant brinjal (eggplant) for India. In research tobacco and Arabidopsis thaliana are the most genetically modified plants, due to well developed transformation methods, easy propagation and well studied genomes.They serve as model organisms for other plant species. Genetically modified plants have also been used for bioremediation of contaminated soils. Mercury, selenium and organic pollutants such as polychlorinated biphenyls (PCBs) have been removed from soils by transgenic plants containing genes for bacterial enzymes.PRESENT STATUSToday , agrobacterium mediated gene transfer method used in variousfield like1. Agriculture: Crops having larger yields, disease- and drought- resistancy; bacterial sprays to prevent crop damage from freezing.2. Temperatures; and livestock improvement through changes in animal traits.3. Industry: Use of bacteria to convert old newspaper and wood chips into sugar; oil- and toxin-absorbing bacteria for oil spill or toxic waste clean-ups; and yeasts to accelerate wine fermentation.4. Medicine: Alteration of human genes to eliminate disease (experimental stage); faster and more economical production of vital human substances to alleviate deficiency and disease symptoms (but not to cure them) such as insulin, interferon (cancer therapy), vitamins, human growth hormone ADA, antibodies, vaccines, and antibiotics.5. Research: Modification of gene structure in medical research, especially cancer research. Food processing: Rennin (enzyme) in cheese aging.FUTURE PROSPECTUSThe field of Agrobacterium research is increasing our understanding of the bacterium itself; in particular, the mechanism by which the T-DNA is translocated into the plant cell and exactly which bacterial virulence proteins accompany it. This understanding is not only vital for the biotechnological application of Agrobacterium, the plant factors used by Agrobacterium to ensure transport across the plant wall, membrane, and cytoplasm, nuclear import, and finally integration of the T-DNA. The application of this knowledge to improve transformation rates will bring gene technology to species that, at present, are recalcitrant to Agrobacterium and not transformation competent using a biolistic approach. The improvement of transformation protocols is a practical advance, but the really exciting advances will be in the types of modification and the application therein. Current research foci include biodegradable plastics in plants (Mittendorf et al., 1998), vaccination against common human diseases administered by eating the plant (Staub et al., 2000), and plants as indicators of environmental toxins (Kovalchuk et al., 2003). With so much promise, Agrobacterium could be the key to future agricultural progress. It can only be hoped that regular, constructive debate can lead to legislative solutions for the ethical, health, and political issues that are likely to play such an influential role in the development of our society.CONCLUSIONTransformation is an important topic in plant biology and transgenic plants have become a major focus in plant research and breeding programs. Agrobacterium-mediated transformation as a practical and common method for introducing specific DNA fragments into plant genomes is well established and the number of transgenic plants produced using this method is increasing. Despite the popularity of the method, low efficiency of transformation is a major challenge for scientists. Modification of different genetic and environmental aspects of transformation method may lead to better understanding of the system and result in high efficiency transformation. In this review, we deal with recent genetic findings as well as different environmental factors which potentially influence Agrobacterium-mediated transformation.
What is agrobacterium mediated transformation?
INTRODUCTIONInduction of crown gall and hairy root diseases in several dicot plants by the common soil borne Gram-negative bacteria Agrobacterium tumefaciens and A.rhizogenes, respectively , are example of natural transformation of plants wherein the bacterial genes are stably introduced into the genome of higher plants. This system represents the only known natural case in which a prokaryotic organism transfers genetic information to a eukaryotic host. This capability underlies the biotechnological uses of Agrobacterium , mostly employed for the genetic transformation of numerous plants species. Recent discoveries have expanded the potential biotechnological uses of Agrobacterium; indeed , under laboratory condition , Agrobacterium is able to transfer DNA and proteins to numerous non.plant species , including several species of yeast and other fungi as well as sea urchin embryos , human cells in cultureAgrobacterium-mediated transformation is the easiest and most simple plant transformation. Plant tissue are cut into small pieces , eg. 10×10 mm, and soaked for 10 min. in a fluid containing suspended Agrobacterium . Some cells along the cut will be transformed by the bacterium that inserts its DNA into the cell . Placed on selectable rooting and shooting media , the plants will regrow .Some plants species can be transformed just by dipping the suspension of Agrobacterium and then planting the seeds in a selective medium. Unfortunately , many plants are not transformable bt this method .1. DETAIL DESCRIPTIONRecombinant DNA technology is based on the insertion of a DNA fragment ( Gene) of interest into a suitable cloning vector and then its introduction into a suitable host to propagate the recombinant DNA.Fig: generalized method of gene transfer in cells1.1. GENE CARRIER VEHICLE -1. If a gene is to be introduced into a host cell, a carrier moleculethat can transport the gene into the host cell is required Sucha molecule is called a cloning vehicle , carrier molecule or avector.1.2. FOLLOWING ARE A FEW GENE CARRIER VEHICLES1.Plasmids2. Bacteriophages3. Viruses2.1.AGROBACTERIUM TUMEFACIENS1. Agrobacterium tumecaiens is a soil borne gram negativebacterium.2 .It invades many dicot plants when they are injured at the soillevel and causes grown gall disease3.The bacterium enters the plant through a fresh wound andattaches itself to the wall of the intact cell.4.This cell is genetically transformed by bacterium .5. This transformation result in a tumour which synthesizesOPINES :A. The Tumors develops only at site of the wound.B. Such tumours can be removed from the plant and culteredin-vitro where they continue to grow indefinitely.C. Continued presence of agrobacterium is not required for tumorProfileration.D. Agrobacterium induced tumours synthesize a variety of unusalCompound called opinesE. Opines are of 3 types -a). Octapineb). Nopalinec). AgropineThese opines are catabolised by Agrobacterium to obtain energy,AT genectically engineers the plant cell for its won purpose.3.1. TUMOUR INDUCING PRINCIPLE1. The tumour inducing principle of AT is a plasmid calles tumourinducing plasmid or Ti Plasmid .a). 200 kb long.b). Has two essential regions : T-DNA and vir region.c). These two regions are essential for the transformation process.3.2. TRANSFER OF TUMOUR INDUCING PRINCIPLE1.T-DNA(transferred DNA ) is excised from the Ti-Plasmid andtransferred to the nucleus of the plant cell.2. Here the T-DNA gets integrated into the DNA which is stable.3. The T-DNA can be passed on to daughter cells as an integralPart of the plant chromosome.Figure. Induction of crown gall on a dicot plant by agrobacteriumtumefaciens.4.1 Ti PLASMIDS1. An extra chromosomal double stranded circular DNA molecule.2. Tumour inducing .3. 200 kb in size and conjugative type.4. Encodes enzymes responsible for the synthesis and catabolism of certain opnies.5. One of the opines is nopaline.6. pTiC58 is present in Agrobacterium strain C58. it is 192 kb long7. Only a small segment of the Ti Plasmid is transferred to the host plant cell and gets integrated with the grnome . This is the T-DNA.Figure. Ti plasmid pTic 58 having 192 kb5.1. T-DNA1.Only a small segmented of the Ti Plasmid is transferred to thehost plant cell and gets integrated with the genome.2.This is the T-DNA3.It contains gene for tumour formation (Tum) and nopalinebiosynthesis (Nos).4.The genes encodes enzymes that catalyse the synthesis ofphtohormones like the IAA and the cytokinin , isopentenyladenosine that cuse tumerous growth of cells in crown galls.5.The T-DNA is bordered by 25 bp repeats, required for theexcision and transfer of T-DNA.6.1. NOPALINE Ti PLASMID pTiC581. The Vir region of the Ti-plasmid contains the genes required forthe T-DNA transferprocess . the genes in this region encode theDNA processing enzymes required for excision , transefer andintegration of the T- DNA segmented.7.1. TUMOUR INDUCTION BY AGROBACTERIUM1. Recognition of susceptible wounded plant cell:a). Plant exudates: act as signal by inducing genes in the Vir Genesof the Ti Plasmid.b). Acetosyringone (as) , alfa- hydroxy acetosyringone (OH-HS)2. Binding to wound cells : controlled by two chromosomal genes ofagrobacterium : chv-A and chv-B.3. Excision , transfer and integration :a). The border repeats of T- DNA play an important role .b). Any DNA sequence located between the border repeats istransferred to the host plant.c). The T-DNA region is excised from the plasmid by the enzymesencoded by the vir- region.4.These enzymes specifically recognize the T-DNA borders.5.The T-DNA enters the plant cell and integrates into the hostgenome , mediated by host enzymes.8.1. Ti-PLASMID AS A VECTOR1. The Ti- plasmid has an innate ability to transmit bacterial DNAinto plant cells .2. This potential is explited by the genetic engineers to use as avector.3.The gene of a donor organism can be introduces into the Ti-plasmid at the T-DNA region4. This plasmid now becomes a recombinant plasmid.5. By agrobacterium infection , the donor genes can be transferredfrom the recombinant Ti-Plasmid and integrated into the genotypeof the host plant.6. This results in the production of transgenic plant.Pic. Ti-Plasmid mediated transfer of gene into a plant9.1. DISARMED Ti PLASMID1.Disarmed Ti-plasmida).Deletion of T-DNA regionb). PGV3850 is constructed from pTiC58.2. It has pBR 322 with AmpR3.It border repeats and NOS genes4. Agrobacterium having this PGV3850 can transfer the modifiedT-DNA into plant cells.5. But the recipient cell will not produce tumour , but could producenopaline.6. This can be used as a efficient vector for introducing foreign geneinto plants.10.1. CONTRUCTION OF A COINTEGRATE1. A foreign gene cloned into an appropirate plasmid (pBR322) canbe integrated with the disarmed Ti-Plamid by a homologousrecombination2. A compound plasmid called a cointegrate is formed.Figure. Cointegrate Plasmid .11.1. TRANSFORMATION OF TISSUE EXPLANTS BYCO-CULTIVATION WITH AGROBACTERIUM1. A co-integrate plasmid derived by recombination of pGV3850 and pBR322 loaded with foreign gene is now used to transfer the foreign gene into many crop plants.2. Small disc (a few mm diameter ) are punched from leaves of petunia, tobacco, tomato or other dicot plants.3. These disc are incubated in a medium containing Agrobacterium carrying the recombinant disarmed T-DNA as a co-ingerate .The cointegrate plasmid has the foreign gene and also the gene for resistance to kanamycin (KmR).4. The disc are cultered for two days . the agrobacterium infects the cut edges of the disc.5. The disc are then transferred to a shoot inducing solid medium (high cyokinin ) containing kanamycin to select the transferred kanamycin to select the transferred kanamycin gene. Corbenicillin in the medium kills agrobacterium.6. After 2-4 weeks the shoot develops.7. The callus having the shoot is transferred to root inducing solid medium (high in auxin content).8. After 4-7 weeks roots appear.9. The rooted plantlets are transferred to soil.Pic. Transformation of leaf disc explants by co-cultivation with agrobacteriumhaving the cointegrate Ti plasmidREVIEW OF LITERATUREAgrobacterium mediated transformation has been a method of choice in dicotyledonous plant species where plant regeneration system are well established (Van Wordragen and Dons , 1992 ; dale et al. 1993 ). The host range of this pathogen includes about 60% of gymnosperms and dicotyledonous angiosperms. Besides , transformation success has also been achieved in some monocots like Asparagus officinalis , Chlorophytum , Narcissus (hernalstees et al. 1984, hookyaasvan slogteren et al. 1984 ) .It was believed that monocots lack wound response i.e. factors that are required to initiate of `vir` genes. (Schafer et al. 1987) achieved success in transforming another monocot ,yam (Diosscorea bulbifera) from potato tubers .likewise , the use of acetosyringone (synthetic phenolic compound ) either during bacterial growth or during co-cultivation has been found to be beneficial in the transformation of other monocots. There are several reports on successful transformation of rice using Agrobacterium (Hiei et al. 1994 ; Li et al. 1994 ; vijaychandra et al. 1995 ). This method has also been extended to barley, wheat, maize and sugarcane.APPLICATIONGenetically modified plants have been developed commercially to improve shelf life, disease resistance, herbicide resistance and pest resistance. Plants engineered to tolerate non-biological stresses like drought, frost and nitrogen starvation or with increased nutritional value (e.g. Golden rice) were in development in 2011. Future generations of GM plants are intended to be suitable for harsh environments, produce increased amounts of nutrients or even pharmaceutical agents, or are improved for the production of bioenergy and biofuels. Due to high regulatory and research costs, the majority of genetically modified crops in agriculture consist of commodity crops, such as soybean, maize, cotton and rapeseed. However, commercial growing was reported in 2009, of smaller amounts of genetically modified sugar beet, papayas, squash sweet pepper, tomatoes, petunias, carnations, roses and poplars. Recently, some research and development has been targeted to enhancement of crops that are locally important in developing countries, such as insect-resistant cowpea for Africa and insect-resistant brinjal (eggplant) for India. In research tobacco and Arabidopsis thaliana are the most genetically modified plants, due to well developed transformation methods, easy propagation and well studied genomes.They serve as model organisms for other plant species. Genetically modified plants have also been used for bioremediation of contaminated soils. Mercury, selenium and organic pollutants such as polychlorinated biphenyls (PCBs) have been removed from soils by transgenic plants containing genes for bacterial enzymes.PRESENT STATUSToday , agrobacterium mediated gene transfer method used in variousfield like1. Agriculture: Crops having larger yields, disease- and drought- resistancy; bacterial sprays to prevent crop damage from freezing.2. Temperatures; and livestock improvement through changes in animal traits.3. Industry: Use of bacteria to convert old newspaper and wood chips into sugar; oil- and toxin-absorbing bacteria for oil spill or toxic waste clean-ups; and yeasts to accelerate wine fermentation.4. Medicine: Alteration of human genes to eliminate disease (experimental stage); faster and more economical production of vital human substances to alleviate deficiency and disease symptoms (but not to cure them) such as insulin, interferon (cancer therapy), vitamins, human growth hormone ADA, antibodies, vaccines, and antibiotics.5. Research: Modification of gene structure in medical research, especially cancer research. Food processing: Rennin (enzyme) in cheese aging.FUTURE PROSPECTUSThe field of Agrobacterium research is increasing our understanding of the bacterium itself; in particular, the mechanism by which the T-DNA is translocated into the plant cell and exactly which bacterial virulence proteins accompany it. This understanding is not only vital for the biotechnological application of Agrobacterium, the plant factors used by Agrobacterium to ensure transport across the plant wall, membrane, and cytoplasm, nuclear import, and finally integration of the T-DNA. The application of this knowledge to improve transformation rates will bring gene technology to species that, at present, are recalcitrant to Agrobacterium and not transformation competent using a biolistic approach. The improvement of transformation protocols is a practical advance, but the really exciting advances will be in the types of modification and the application therein. Current research foci include biodegradable plastics in plants (Mittendorf et al., 1998), vaccination against common human diseases administered by eating the plant (Staub et al., 2000), and plants as indicators of environmental toxins (Kovalchuk et al., 2003). With so much promise, Agrobacterium could be the key to future agricultural progress. It can only be hoped that regular, constructive debate can lead to legislative solutions for the ethical, health, and political issues that are likely to play such an influential role in the development of our society.CONCLUSIONTransformation is an important topic in plant biology and transgenic plants have become a major focus in plant research and breeding programs. Agrobacterium-mediated transformation as a practical and common method for introducing specific DNA fragments into plant genomes is well established and the number of transgenic plants produced using this method is increasing. Despite the popularity of the method, low efficiency of transformation is a major challenge for scientists. Modification of different genetic and environmental aspects of transformation method may lead to better understanding of the system and result in high efficiency transformation. In this review, we deal with recent genetic findings as well as different environmental factors which potentially influence Agrobacterium-mediated transformation.
