Agrobacterium mediated transformation, its mode of action and applications in crop improvement
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The document discusses Agrobacterium-mediated transformation, a key method in genetic engineering for plant improvement, allowing for the introduction of beneficial genes from diverse sources. It outlines the mechanisms of gene transfer, types of vectors, and steps in the transformation process, emphasizing the advantages of genetic engineering over traditional breeding methods. Applications include enhancing crop traits such as yield, disease resistance, and nutritional quality through the development of transgenic crops.
Agrobacterium mediated transformation, its mode of action and applications in crop improvement
- 1. Agrobacterium mediated transformation, its mode of action and applications in crop improvement 1 Surbhaiyya Shobha Devidas Ph.D Scholar (Agricultural Biotechnology) Biotechnology centre, DR. P.D.K.V, AKOLA
- 2. Outlines Introduction Strategies to improve crop production Steps in Genetic engineering Basics of Genetic Engineering Co-integrative and binary vectors Genetic modification of plants using genetic engineering General transformation steps Agrobacterium as a tool for plant genetic engineering ◦ Agrobacterium species ◦ Gene Transfer using Agrobacterium ◦ Characteristics of Agrobacterium tumefaciens ◦ Ti-plasmid features ◦ T-DNA ◦ Forms of T-DNA that are found in Agrobacterium Model of Agrobacterium-mediated genetic transformation Overview of the Infection Process: Generation of the T-strand, T-DNA transfer and integration Factors affecting transformation frequency Gene construction and visible markers Selectable marker and promoter General transformation protocol Applications References
- 3. Traditionally, a plant breeder tries to exchange genes between two plants to produce offspring that have desired traits. In cross breeding, however, is limited to exchanges between the same or very closely related species. It can also take a long time to achieve desired results and frequently, characteristics of interest do not exist in any related species. Genetic engineering enables plant breeders to bring together in one plant useful genes from a wide range of living sources, not just from within the crop species or from closely related plants. Marc Van Montagu and Jeff Schell, discovered the gene transfer mechanism between Agrobacterium and plants, which resulted in the development of methods to alter the bacterium into an efficient delivery system for genetic engineering in plants. Introduction
- 4. Plants Conventional breeding Tissue culture Genetic engineering Strategies to improve crop production
- 5. Genetic engineering, also called genetic modification, is the direct manipulation of an organism's genome using biotechnology. DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism. Crops developed through genetic engineering are commonly known as transgenic crops or genetically modified (GM) crops. The gene can be introduced into the cells of the plant being modified through a process called transformation. The most common methods used to introduce the gene package into plant cells include biolistic transformation (using a gene gun) or Agrobacterium-mediated transformation. Agrobacterium tumefaciens mediated transformation is the most commonly used method for obtaining transgenic plants. Genetic engineering process and uses:
- 7. •R.E: Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonucleases. •Vectors: A vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. •Types of Vectors: •Plasmids (5-10 kb) •Bacteriophage (10-15 kb) •Cosmids (50 kb) •BACs & YACs (300 kb, up to 1,000 kb) •Plasmid as a good vector: •A plasmid is a small, circular, double-stranded DNA molecule that is distinct from a cell's chromosomal DNA. Plasmids naturally exist in bacterial cells, and they also occur in some eukaryotes. •Binary vectors: •A T-DNA binary system is a pair of plasmids consisting of a binary plasmid and a helper plasmid. Basics of Genetic Engineering
- 8. Binary vector t-DNA VIR genes Plasmid DNA Bacterial Chromosome Bacterial ORI Ampicillin resistance LB RB Co-integrative Co-integrative and binary vectors
- 9. 1. DNA is cut with a restriction enzyme 2. Fragments are mixed with vector molecules cut by the same enzyme DNA ligase joins recombinant DNA molecules 3. Plasmid vectors with inserted DNA fragments are transferred into bacterial cells 4. Colonies carrying cloned recombinant DNA molecules are identified, collected, and grown Steps in genetic engineering
- 10. Transformation : The genetic alteration of a cell resulting from the introduction, uptake and expression of foreign genetic material (DNA) in molecular biology. All stable transformation methods consist of three steps: 1. Delivery of DNA into a single plant cell. 2. Integration of the DNA into the plant cell genome. 3. Regeneration of the transformed cell into a whole plant. Genetic modification of plants using genetic engineering
- 11. Physical Chemical Biological •Macroinjection •Biolistics - gene gun/ particle bombardment •Electroporation •Microinjection •Silica/carbon fibers •Lazer mediated •PEG •DEAE-dextran •Calcium phosphate •Artificial lipids •Proteins •Dendrimers A.tumefaciens A.rhizogenes Virus-mediated In planta Plant Transformation Methods
- 12. Agrobacterium (disease symptomology and host range) A. radiobacter - “avirulent” species A. tumefaciens - crown gall disease A. rhizogenes - hairy root disease A. rubi - cane gall disease A. vitis - galls on grape and a few other plant species Agrobacterium species
- 13. Agropine-type (strain EHA105::pEHA105): Carry genes for agropine synthesis and catabolism. Tumors do not differentiate and die out. Octopine-type (strain LBA4404::pAL4404): Carry genes(3 required) to synthesize octopine in the plant and catabolism in the bacteria. Tumors do not differentiate, but remain as callus tissue. Nopaline-type (strain GV3101::pMP90 (pTiC58)): Carry gene for synthesizing nopaline in the plant and for utilization (catabolism) in the bacteria. Tumors can differentiate into shooty masses (teratomas). Agro types of Agrobacterium
- 14. – Plant parasite that causes Crown gall disease – Encodes a large (~250kbp) plasmid called Tumor-inducing (Ti) plasmid – Portion of the Ti plasmid is transferred between bacterial cells and plant cells T-DNA (Tumor DNA) – T-DNA integrates stably into plant genome – T-DNA ss DNA fragment is converted to dsDNA fragment by plant cell – Then integrated into plant genome Characteristics of Agrobacterium tumefaciens
- 15. Size is about 200 kb Has a central role in Crown-gall formation Contains T-DNA region that is integrated into the genome of host plants Contain a vir region ~ 40 kb at least 8~11 vir genes Has origin of replication Has genes for the catabolism of opines Ti-plasmid features
- 16. Size~ 12 to 24 kb Left and right border sequence(24-bp) which will be transferred into genome of host plant Oncogenes e.g Auxin, cytokinin, opines tm1 gene for determining the tumour size crown gall tumorigenesis is due to the "activation" of unregulated phytohormone synthesis in the transformed cells T-DNA
- 17. ds circles - found only in induced bacteria, not (apparently) in plant cells. ds linear T-DNA - found only in induced bacteria, not (apparently) in plant cells. ss linear T-DNA - found in bacteria and plant cells. Forms of T-DNA that are found in Agrobacterium
- 18. Chromosomal and Vir Genes Chromosomal and vir genes of bacterial cells are both involved in T-DNA transfer Virulence genes vir A Chemoreceptor, activator of vir G vir B Transmembrane complex vir C Host-range specificity vir D Site-specific endonuclease vir E T-DNA processing and protection vir F Host range specificity vir G Positive regulator of vir B, C, D, E, F Mode of Action of Agrobacterium tumefaciens
- 19. Overview of the Infection Process
- 20. 1. Signal recognition by Agrobacterium: -Agrobacterium perceive signals such as sugar and phenolic compounds which are released from plants 2. Attachment to plants cells: Two step processes: i) initial attachment via polysaccharide ii) mesh of cellulose fiber is produced by bacteria. Virulence genes (chv genes) are involved in the attachment of bacterial cells to the plants cells. 3. Vir gene induction: VirA senses phenolics ans subsequently phosphorylating and thereby activating VirG. VirG then induces expression of all the vir genes. 4. T-strand production:VirD1/virD2 complex recognises the LB and RB. virD2 produces single-stranded nicks in DNA. Then virD2 attached to ssDNA. virC may assist this process. Process of T-DNA transfer and integration
- 21. 5. Transfer of T-DNA out of bacterial cells: T-DNA/VirD2 is exported from the bacterial cell by “T-pilus” composed of proteins encoded by virB operon and VirD2. VirE2 and VirF are also exported from bacterial cells. 6. Transfer of the T-DNA and Vir proteins into the plant nuclear localization: T-DNA/VirD2 complex and other Vir proteins cross the plasma membrane through channels formed from VirE2. VirE2 protect T-DNA from nucleases, facilitate nuclear localization and confer the correct conformation to the T-DNA/virD2 complex for passage through the nuclear pore complex (NPC). The T-DNA/VirD2/VirF2 /plant protein complex the nucleus through nuclear pore complex. And integrated into host chromosome. Conti……..
- 22. Ti plasmid type and binary vector Selectable marker Bacterial strains: A. tumefaciens (LBA4404 and EHA101, EHA105 from EHA101, AGL0 and AGL1 from LBA4404). and A. rhizogenes (LBA9402 and Ar2626) Inoculation and co-cultivation medium, culture conditions prior to during inoculation . Activation of T-DNA transfer process by exogenously added acetosyringone. Genotype and explant are considered to be the major limiting factors. Factors for transformation frequency
- 23. Plant specific promoter Gene of interest Antibiotic resistance Signal peptides PolyA-tail Gene construction
- 24. B-glucuronidase (GUS) The UidA gene encoding activity is commonly used. Gives a blue colour from a colourless substrate (X-glu) for a qualitative assay. Green Fluorescent Protein (GFP) Fluoresces green under UV illumination Non-destructive Problems with a cryptic intron now resolved. Has been used for selection on its own. Visible markers
- 25. Marker genes enable the transformed cells to survive on medium containing the selective agent, while non-transformed cells and tissues die. Usual to use a positive selective agent like antibiotic resistance. The NptII gene encoding Neomycin phospho-transferase II phosphorylates kanamycin group antibiotics and is commonly used. Selectable markers
- 26. Selectable marker genes commonly used in cereal transformation
- 27. A promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located near the genes they transcribe, on the same strand and upstream on the DNA. Promoters can be about 100–1000 base pairs long. CaMV35S, rice ACT1 and maize UBI1 gene promoters have been used extensively to drive high and constitutive expression of transgene in rice. Inducible or tissue or stages specific promoter Promoters of rice POXA and POXN were found to be root-specific and their expression was also enhanced in the leaves on UV and wounding treatment. Endosperm-specific, root/shoot-specific, seed-specific, anther-specific, and early development stage specific promoters. Seed storage glutelin promoters, GluA,B,C, containing endosperm specificity-determining motifs(GCN4,AACA,and P-box). Promoters
- 28. O/N culture Sterile explants with dividing cells Inoculate (mins-hrs) (bacterial attachment) Co-cultivate (days) Transfer of t-DNA Transfer to medium with bactericidal antibiotics (days) Kill off Agrobacterium Transfer to medium with bactericidal antibiotics plus selective antibiotics Kill off Agrobacterium and select transgenic cells Transfer to regeneration medium plus selective antibiotics Regeneration of transgenic plants Transformation Recovery of transgenic plants Wash General transformation protocol
- 29. Insect resistance: proteinase inhibitor, lectin, Insecticidal protein( cry). Disease resistance: Xa1 to Xa29, OSWRKY71,resistance (R) gene, Pathogenesis-related (PR) genes, CP or replicase-mediated resistance to virus infection. Abiotic stresses: various compatible solutes (glycine betaine, trehalose, proline, and polyamines), Late embryogenesis proteins, membrane transporters, regulators of signal transduction or transcription. Herbicide Tolerance: bar; P450 monooxygenase and glutathione S- transferase. Enhancement of stress tolerances
- 30. Nutritional Enhancement: Milled rice contains no β-carotene (provitamin A) daffodil phytoene synthase gene. Golden rice, phytoene synthase (psy) and lycopene β-cyclase (lcy) genes from daffodil and carotene desaturase (crt1) from bacterium, Erwinia uredovora Alteration of Starch Content: WX genes. Improvement of grain quality
- 31. ADP-glucose pyrophosphorylase in transgenic rice had been shown to increase seed weight per plant. Transferring of C4 plant genes in C3 plants Arabidopsis phytochrome A gene could increase the seed yield by 6–21% in transgenic rice . A link between phytohormones (GA, cytokinin, brassinosteroids) metabolism and grain yield has been established in rice . Yield improvement
- 32. Flower development: OSMADS Overexpression of floral control gene LEAFY in Arabidopsis is sufficient for the transformation of lateral shoots into flowers and causes early flowering Plant architecture:OSTB1, teosinte branched 1 homologue in rice, has been found to be a negative regulator for lateral branching in rice MONOCULM 1 Histone acetylase Expansins Control of Plant Development
- 33. Tissue browning and necrosis after Agrobacterium infection are still major obstacles in the genetic transformation of cereals. On pathogen infection, one of the earliest defence mechanisms activated is the production of reactive oxygen species, referred to as an oxidative burst, which activates programmed cell death (HR). A correlation between the reduction in cell death and the improved transformation frequency has been demonstrated. Necrosis inhibiting agents, such as silver nitrate, increased efficiency of transformation. Suppression of the host defence response is a prerequisite to successful plant transformation. Existing problems and future prospects
- 34. Glufosinater herbicide Sethoxydimr herbicide Bromoxynilr herbicide Glyphosater herbicide Sulfonylurear herbicide Male-sterility Modified fatty acid Flower colour Flower life Delayed fruit ripening Virus resistance Bt insect resistance Approved Traits
- 35. Tzfira and Citovsky, (2006)Agrobacterium-mediated genetic transformation. Current Opinion in Biotechnology, 17:147–154. Gelvin SB,(1998)The introduction and expression of transgenes in plants. Curr Opin Biotechnol, 9:227-232. Roa-Rodriguez C, Nottenburg C, (2003) Agrobacterium-mediated transformation of plants.; URL: CAMBIA technology landscape paper http://www.bios.net/Agrobacterium. H.S Chawala Text Book Of Plant Biotechnology,Second edition. References
- 36. 36 Thank You
Editor's Notes
- #9: Co-integrative vectors require the genes that are transferred from bacteria to go into the plasmid DNA by homologous recombination. For binary vectors, the plasmid containing the t-DNA is able to replicate in E.coli and can be mobilised into Agrobacterium by a triparental mating with a helper strain of E.coli. This greatly simplifies the process of plasmid construction.