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Plant transformation methods

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the presentation is provided with information about how the foreign genes are transferred to plant genome.

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Plant transformation methods

  1. 1. 1BY-MD SAMIYUDDINJr. M.Sc. (CIB)
  2. 2. INTRODUCTION Plant genetic engineering has become one of the mostimportant molecular tools in the modern molecularbreeding of crops. Over the last decade, significant progress has been made inthe development of new and efficient transformationmethods in plants. Despite a variety of available DNA delivery methods,Agrobacterium- and biolistic-mediated transformationremain the two predominantly employed approaches.
  3. 3.  In particular, progress in Agrobacterium-mediatedtransformation of cereals and other recalcitrant dicotspecies has been quite remarkable. In the meantime, other transgenic-enabling technologieshave emerged, including generation of marker-freetransgenics, gene targeting, and chromosomal engineering.
  4. 4.  Although transformation of some plant species or elitegermplasm remains a challenge, further advancement intransformation technology is expected because themechanisms of governing the regeneration andtransformation processes are now better understood andare being creatively applied to designing improvedtransformation methods or to developing new enablingtechnologies.
  5. 5.  The genetic constitution of plants can be altered in thelaboratory by a process called transformation, whereby asegment of DNA (deoxyribonucleic acid) is introducedthat becomes inserted in one of the plant chromosomes. Several methods to accomplish plant transformation havebeen devised. In all these methods, single cells aretransformed and thereafter regenerated into complete,fertile plants by tissue culture procedures. Some of the transformation methods can only be appliedto protoplasts.
  6. 6.  Nowadays, particle bombardment and the natural vectorAgrobacterium tumefaciens are preferred because they canalso cope with whole plant tissues such as roots and leaves,which are easier to handle, more stable and require less ofthe lengthy steps that are required for plant regeneration. Transgenes are integrated at random positions in the plantgenome, but procedures for targeted integration becomemore and more efficient.
  7. 7.  Production of transgenic plantsIsolate and clone gene of interestAdd DNA segments to initiate orenhance gene expressionAdd selectable markersIntroduce gene construct into plantcells (transformation)Select transformed cells or tissuesRegenerate whole plants
  8. 8. Plant Transformation Methods(DIRECT)Physical Chemical(INDIRECT)BiologicalIn Planta•Microinjection•Pressure•Biolistics – genegun/particlebombardment•Electroporation•Microinjection•Silica/carbon fibers•Laser mediated•SAT•PEG•DEAE- dextran•Calcium phosphate•Artificial lipids•Proteins•DendrimersA.TumefaciensA. Rhizogenes• Virus-mediated•Meristemtransformation•Floral dipmethod•Pollentransformation
  9. 9. Techniques for plant genetictransformation Indirect method- Agrobacterium mediated genetransfer Direct methods- Particle bombardment (biolistics) Microprojectile gun method Electroporation Silicon carbide fibres Polyethylene glycol (PEG)/protoplast fusion Liposome mediated gene transfer
  10. 10. Properties of good host1. Easy to transform2. Supports the replications of recombinant DNA3. Is free from elements that interfere with replication ofrecombinant DNA4. Lacks active restriction enzymes ex: E.coli k12 substrainHB 1015. Does not have methylases6. Is deficient in normal recombination function : so thatit is not altered by recombination events.
  11. 11. Transformation vector requirements Origin of replication Bacterial selectable marker Gene constructs of interest T-DNA borders and other Agrobacterium genes if usingAgrobacterium Compatible with helper plasmid if using Agrobacterium
  12. 12. Agrobacterium mediated gene transferAgrobacterium- Soil borne, gram negative, rod shaped, motile found inrhizosphere Causative agents of “Crown gall” disease of dicoltyledones Have ability transfer bacterial genes to plant genome Attracted to wound site via chemotaxis in response to chemicals(sugar and Phenolic molecules: acetosyringone) released fromdamaged plant cells Contains Ti plasmid which can transfer its T-DNA region intogenome of host plants
  13. 13. Ti-plasmid features Two strains of Ti-plasmid:-Octopine strains- contains two T-DNA region: TL (14kb) and TR ( 7 kb)-Nopaline strains- contain one T-DNA region(20 kb) Size is about 200 kb Has a central role in Crown-gall formation Contains one or more T-DNA region that is integratedinto the genome of host plants Contain a vir region ~ 40 kb at least 8~11 vir genes Has origin of replication Contains a region enabling conjugative transfer Has genes for the catabolism of opines
  14. 14. Nopaline OctopineAcetosyringone
  15. 15. Forms of T-DNA that are found in Agrobacterium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 plantcells.what is not found - Ti plasmids with evidence thatT-DNA has been precisely deleted.
  16. 16. Process of T-DNA transfer and integration1. Signal recognition by Agrobacterium:-Agrobacterium perceive signals such as sugar and phenolic compoundswhich are released from plants.2. Attachment to plants cells:Two step processes: i) initial attachment via polysaccharide ii) mesh ofcellulose fiber is produced by bacteria.Virulence genes (chv genes) are involved in the attachment of bacterialcells to the plants cells.3. Vir gene induction:VirA senses phenolics ans subsequently phosphorylating and therebyactivating VirG. VirG then induces expression of all the vir genes.4. T-strand production:VirD1/virD2 complex recognises the LB andRB. virD2 produces single-stranded nicks in DNA. Then virD2 attachedto ssDNA. virC may assist this process.
  17. 17. Process of T-DNA transfer and integration (continu)5. Transfer of T-DNA out of bacterial cells: T-DNA/VirD2 isexported from the bacterial cell by “T-pilus” composed ofproteins encoded by virB operon and VirD2. VirE2 and VirF arealso exported from bacterial cells.6. Transfer of the T-DNA and Vir proteins into the plantnuclear localization: T-DNA/VirD2 complex and other Virproteins cross the plasma membrane through channels formedfrom VirE2. VirE2 protect T-DNA from nucleases, facilitatenuclear localization and confer the correct conformation to theT-DNA/virD2 complex for passage through the nuclear porecomplex (NPC). The T-DNA/VirD2/VirF2 /plant proteincomplex the nucleus through nuclear pore complex. Andintegrated into host chromosome.
  18. 18. Agrobacterium tumefaciens mediatedtransforamtion in plantsExplant{ decapitated seedlings, cells, protoplast, leaf discs}Addition of woundingAcetosyringone Agrobacteriaco cultivation to allow infectionantibiotics to kill bacteriatransformed and non transformed tissueselective media to kill the non transformed tissuesi.e, addition of kanamycin and hygromycin etctransformed tissue / callustransformed shootsrooted shootsadult plantsSEEDS
  19. 19. Practical application of Agrobacterium-mediatedplant transformation 1. Agrobacterium mediated transformationmethods are thought to induce lessrearrangement of the transgene. Lower transgene copy number that direct DNAdelivery methods. Successful production of transgenic plantsdepends on the suitable transformationprotocols.
  20. 20. The basic protocol used for any Agrobacteruimmediated transformation experiments1. Identify a suitable explants: Suitable plant tissue isremoved and sterilized. Leaf is used for Tobacco.2. Co-cultivate with the Agrobacterium: Leaf tissue is cutinto small pieces and placed into a culture of Agrobacteriumfor about 30 mins. The explants are subsequently removedfrom the bacterial culture and placed on to the MS mediumthat contain no selective agent. The incubation of explantswith Agrobacterium is allowed to continue for 2 days to allowtransfer of the T-DNA transfer to the plant cells.3. Kill the Agrobacterium with a suitable antibiotic: Theexplants are removed from the medium and washed inantibiotic (cefotaxime) solution that kill Agrobacterium cells.
  21. 21. The basic protocol used for any Agrobacteruimmediated transformation experiments (Conti-)4. Select for transformed plant cells: The explantare transferred to fresh solid medium supplementedwith a selective agent (kanamycin). It also containscefotaxime. Auxin, Cytokinin are used to encourage theregeneration of by organogenesis. High cytokinin toauxin ratio promotes shoot formation from theexplants.5. Regeneration of whole plant :The shoot can berooted by placing them on solid medium containing ahigh auxin to cytokinin ratio.
  22. 22. Advantages of A.bacterium mediated transformation It is a natural mean of transfer and is perceived as more acceptabletechnique to those who feel natural is best. It is capable of infecting intact plant cells, tissues and organs. As resultof tissue culture limitations are much less of a problem. Transformedplants can be regenerated more rapidly. It is having a capable of transferring large fragments of DNA veryefficiently without substantial rearrangements. Integration of T-DNA is relatively precise process, it serves as an idealinsertional mutagenesis vehicle as it introduces one to several copiesof the transferred DNA into the intact genome at one or a few loci. The stability of gene transferred is excellent. Integrated T- DNA givesconsistent maps and appropriate segregation ratios. Introduces traitshave been found to be stable over many generations. This at ability is critical to commercialization of transgenic plants.
  23. 23. Disadvantages It has limitation of host range, Some of the important food crops can not be infected bythis although, lately much progress has been achieved toover come this limitation by the development of highlyvirulent strains of Agrobacterium.Sometimes, the cells in a tissue that are able to regenarate,are difficult to transform. it might be that these are inlayers too deep to be reached by Agrobacterium, orsimply are not targets for T-DNA transfer.
  24. 24. Virus mediated Vectors based on viruses are desirable because of high efficiencyof gene transfer that can be obtained by infection and because ofthe amplification of transferred gene that occurs via viral genomereplication. Vectors for transferring gene into plants are based on DNA orRNA molecules, that naturally express their genetic informationin the plant cells. The replicating genomes of plant viruses are non-integrativevectors, as compared to vectors based on the T-DNA of A.Tumefaciens which are integrative gene vectors.
  25. 25.  The plant virus vectors do not integrate into theplant cells or host genome , rather they spreadsystematically within a plant and accumulate tohigh copy number in their respective target cells. In most of the cases , the viral genome has modifiedto accommodate the insertion of foreign sequences,which are transferred, multiplied and expressed inplant as part of recombinant virus genome.
  26. 26.  Plant viruses as vectors Caulimoviruses – ds DNA – CaMV Geminiviruses - 2ss DNA – maize streak virus RNA plant viruses - TMV
  27. 27. Vectorless or Direct DNA transfer The trem “Direct gene transfer” is used to discriminatebetween the methods of plant transformation that rely onAgrobacterium (indirect method) and those that do not (directmethods). Direct gene transfer methods all rely on the deliveryof large amount of naked DNA whilst plant is transientlypermeabilised.Direct methods-Particle bombardment (biolistics)Microprojectile gun methodElectroporationSilicon carbide fibresPolyethylene glycol (PEG)/protoplastfusionLiposome mediated gene transfer
  28. 28. Physical methods DMGT: DNA Mediated Gene TransferA general scheme for production of transgenic plants usingvarious direct DNA delivery methods have beencommercialized. Electroporation Particle bombardment / Micro projectile / Biolistics Electro injection Micro injection macro injection Liposome mediated Silicon carbide fibre mediated ultra sound mediated DNA transfer via pollen
  29. 29. Electroporation technique. Is the process where by electrical impulses of highfield strength are used to reversibly permeabalize cellmembrane to facilitates uptake of large molecules,including DNA. It has been used for long time for transient andintegrative transformation of protoplasts. 1 to 1.5 k V. so uses low capacitance hence shortdecay time .
  30. 30. Electroporation It can be used to deliver DNA into plant cells and protoplasts. The genes of interest require plant regulatory sequence. Plant materials is incubated in a buffer solution containingDNA and subjected to high-voltage electric pulse. The DNA then migrates through high-voltage-induced pores inthe plasma membrane and integrates into the genome. It can be used to transform all the major cereals particularlyrice, wheat, maize. Advantages and disadvantages: Both intact cells and tissue can be transformed. The efficiency of transformation depends upon the plantmaterials, electroporation and tissue treatment conditions usedfor transformation. ~40 to 50% incubated cells receive DNA ~50% of the transformed cells can survive
  31. 31. DuracellDNA containingthe gene of interestPlant cellProtoplastElectroporation TechniquePower supplyDNA inside theplant cellThe plant cell withthe new gene
  32. 32. Biolistic/Particle bombardment High velocity micro projectile were utilize todeliver nucleic acids into loving cells. Advantages :Transformation of organized tissueUniversal delivery systemTransformation of recalcitrant sppStudy of basic plant development processes.
  33. 33. Particle bombardment Why Biolistics or Biolistic bombardment? Is the most powerful method for introducing nucleic acidsinto plants, because the helium pressure can drivemicrocarriers through cell walls Is much easier and less time consuming than microinjectingnucleic acids into plant cells or embryos Allows transformation of animal cells that have uniquegrowth requirements and that are not amenable to genetransfer using any other method Requires less DNA and fewer cells than other methods, andcan be used for either transient or stable transformation
  34. 34. Principle The gold or tungsten particles are coated with the DNA thatis used to be transform the plant tissue. The particles are propelled at high speed into the targetplant material where the DNA is released within then celland can integrate into the genome. Two types of plant tissues are used for particlebombardment: a) Primary explants that are bombarded and then inducedto become embryogenic b) Proliferating embryonic cultures that are bombardedand then allowed to proliferate further and subsequentlyregenerate.
  35. 35. PDS-1000/He bombardment SystemFig: Schematic representation of the PDS-1000/Hesystem upon activation. The arrows indicate thedirection of helium flowFig: The PDS-1000/He system, shown herewith magnified view of the Hepta adaptor.
  36. 36. How the PDS-1000/He System Works The sample to be transformed is placed in the bombardment chamber,which is evacuated to subatmospheric pressure The instrument is fired; helium flows into the gas acceleration tube and isheld until the specific pressure of the rupture disk is reached The disk bursts, and the ensuing helium shock wave drives themacrocarrier disk (which carries the coated microparticles) a shortdistance toward the stopping screen The stopping screen retains the macrocarrier, while the microparticlespass through the screen into the bombardment chamber and penetratethe target cells The launch velocity of microcarriers depends on a number of adjustableparameters: the helium pressure (rupture disk selection, 450–2,200 psi),the amount of vacuum, the distance from the rupture disk to themacrocarrier, the distance from the microcarrier launch assembly to thestopping screen, and the distance between the stopping screen and targetcells. Adjusting these parameters allows you to produce a range ofvelocities to optimally transform many different cell types.
  37. 37. The Helium Gas Gun – Circa 2000Particle Gun
  38. 38. Microinjection Under a microscope, a cell is manipulated to a bluntcapillary. Gentle suction holds the cell in place. With a micromanipulator, a very fine tipped pipet isinserted into the cytoplasm or nucleus. DNA or RNA is injected directly into the nucleus orcytoplasm. Microinjection has been successfully used with large frogeggs, cultured mammalian cells, mammalian embryos,and plant protoplasts and tissues
  39. 39. MicroinjectionMicroinjection techniques for plant protoplasts utilize a holdingpipette for immobilizing the protoplast while an injection pipette isutilized to inject the macromolecule. In order to manipulate the protoplasts without damage, theprotoplasts are cultured for from about 1 to 5 days before theinjection is performed to allow for partial regeneration of the cellwall.It was found that injection through the partially regenerated cellwall could still be accomplished and particular compartments of thecell could be targeted. The methods are particularly useful for transformation of plantprotoplasts with exogenous genes.
  40. 40. Microinjection of GOI
  41. 41. Silicon carbide fibres-Whiskers Plant materials (Cells in suspension culture, embryos andembryo-derived callus) is introduced into a buffer containingDNA and the silicon fibers which is then vortexed. The fibers (0.3-0.6 μm in diameter and 10-100μm long)penetrate the cell wall and plasma membrane, allowing theDNA to gain access to the inside of the cells. Disadvantages and advantages The drawbacks of this technique relate to the availability ofsuitable plant material and the inherent dangers of the fibers,which require careful handing. Many cereals, produce embryonic callus that is hard andcompact and not easily transformed with this technique. Despite the some disadvantages, this method is recently usedfor successful transformation of wheat, baerly, and maizewithout the need to cell suspension.
  42. 42. Silicon carbide fibre mediated 0.6 micro m dia, 10 – 80 micro m lth. vortexing in an eppendorf tube a mixture of plasmid DNA encodinga selectable or screen able marker gene, si fibre and the explantstissue. DNA delivery in his system is presumably due to cell wall penetrationby DNA coated si C F. cellular penetration is likely enhanced by frequent and forcefulintercellular collisions encountered in the votrexed millue. DNA bound to the fibre surface is carried into the penetrated cell andbecomes integrated into the nuclear genome and stably transformplant cells with plant regeneration capacity will unable to theapplication of Si c f mediated.
  43. 43. Silicon carbide fiber
  44. 44. Ultrasound mediated Immersion of explant in sonication buffer containing plasmidDNA and is then sonicated with an ultra sonic pulseregenerater at 0.5 w/cm2 aucostic intensity for 30 mins.Then the samples are rinsed in buffer soln, and then culturedfor Gth and deferentiation. Advantage: Being simple method Inexpensive and multi functional equipment. There is no need of tissue culturists.
  45. 45. DNA transfer via pollen Y pollen as v? Fertilization of zygotic embryogenesis, and ovules aredifficult to isolate and injection of DNA into embryo sac ininsitu seemed to be too tedious and unpredictable.. Ex: DNA treated pollen as vector for pollinating fertileplants of Maize by ohta.
  46. 46.  Gene transfer through pollens
  47. 47. Chemical gene transfer method PEG mediated Calcium phosphate Co-precipitation The polycation DMSO technique DEAE dextran procedure
  48. 48. Calcium phosphate Co-precipitation DNA is mixed with CaCl2 soln & isotonic buffer to formDNA CaPO4 ppt. this ppt is allowed to react with activelydividing cells for several hours, washed and then incubatedin fresh culture medium. The efficiency of transformation though low can be increasedby giving them a physiological shock with DMSO. relative success depends upon high DNA concentration andits apparent protection in the ppt.
  49. 49. The polycation DMSO technique Involves polycation, polybrene to increase theadsorption of DNA to the surface followed by abrief treatment with 25-30% of DMSO to increasethe membrane permeability and enhance uptake. Advantage : Less toxic than other polycations, High transformation efficiency and allows verysmall quantities of plasmid DNA to be used.
  50. 50. DEAE dextran procedure Transformation of cells with DNA complexed to the high molecularweight polymer Di Ethyl Amino Ethyl dextran is used obtainefficient transient expression. The efficiency increases to 80% whenDMSO shock id given, but it wont produce stable transformants. Advantage: Direct gene transfer method Ex: cereals Disadvantage After transformation the further improvement of plant tissueculture is not good so.. It show unpredictable pattern of foreign DNA integration During their passage into the nucleus , the DNA are subjected tonucleolytic cleavage resulting in truncation, recombination,rearrangement or silencing of DNA occurs.
  51. 51. Tomatoes comes in manyvarieties, colors and shapesTransgenic tomatoes -expressing different malarialantigens
  52. 52. Normal and mutant tomato fruithigh-pigment 1 (hp1/hp1), high-pigment2 (hp2/hp2), Never-ripe (Nr/Nr),Green-ripe (Gr/Gr), Colorless non-ripening (Cnr/Cnr) &ripening-inhibitor (rin/rin) mutations
  53. 53. Delivery of a corn-based ediblevaccineTransgenic corn kernels (a)Corn snack (b) orEmbryo or germ cells (c)
  54. 54. Dr EadyCrop & Food Research in New Zealand andhis collaborators in JapanTearless Onion
  55. 55. http://www.dailymail.co.uk/news/article-514799/The-orange-purple-green-cauliflowers-scientists-claim-healthier-you.html
  56. 56. High anthocyanin purple tomato and redwild-type tomatoPurple tomatoes high in anthocyaninhttp://news.bbc.co.uk/2/hi/health/7688310.stm
  57. 57. http://www.seedquest.com/News/releases/2008/october/24091.htmAnthocyanins offerprotection against certaincancers, cardiovascular diseaseand age-related degenerativediseases. Anthocyanins alsohave anti-inflammatory activity,promote visual acuity and hinderobesity and diabetes.
  58. 58. Worlds First Blue Roses On Display In JapanThe Blue Rose was developed by SuntoryFlowersOctober 31, 2008,
  59. 59. Thank you„Urs Sami

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