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Chlorop l ast T rans f ormat i on and its Applications to Plant Improvement

Explore the applications and advantages of chloroplast transformation in plant improvement, including high protein levels and the ability to express multiple proteins. Learn about the unique features of chloroplasts, their organization, and the method used in chloroplast genetic engineering.

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Chlorop l ast T rans f ormat i on and its Applications to Plant Improvement

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  1. ChloroplastTransformationandits Applications to PlantImprovement

  2. What is Chloroplast? • Chloroplast is a plastid containing chlorophyll and other pigments occurring in plants and eukaryotic algae and which possess their own genome or plastome, besides nuclear genome that carry out photosynthesis. Fig- Diagrammatic Presentation ofChloroplast

  3. What is Chloroplast? • Genetic materials in plants is distributed into nucleus,plastids • and mitochondria. • There are up to 300 plastids in one plantcell. • In most angiosperm plant species (80%) plastids are strictly maternallyinherited.

  4. Organization of Chloroplast Genome • - Size ~120–150 (30-201) kb that encode ~120 genes, responsiblefor • gene expression, photosynthesis andmetabolism • Example- 107 kb in Cathaya argyrophylla , 218 kb inPelargonium • 1,000-10,000 copies of Genomes inasingle cell • In case of Arabidopsis thaliana nucleus encode about 2100 chloroplast proteins and the whole chloroplast genome encodes for 117proteins.

  5. Organization of Chloroplast Genome • Circular double-stranded DNA through construction of- • complete genomemaps, • a large single copy (LSC), • a small single copy (SSC),and • duplication of a large (~25 kb) Inverted region(IRs) • The number of copies of plastomes per leafcell- • -1000 to 1700 in Arabidopsis thalianaand • - up to 50,000 in Triticumsp.

  6. Why Chloroplastis a Unique Transformation tool? • Sources of EnormousAdvantages • -sequestration of carbon, production of starch, and evolution of oxygen, synthesis of amino acids, fatty acids, and pigments, and key aspects of sulfur and nitrogenmetabolism • Have diversefunctions • Chromoplasts– for pigment synthesis andstorage • Gerontoplasts – control dismantling of photosynthetic apparatus duringsenescence • Leucoplasts –– monoterpenesynthesis • An attractive alternative to nuclear genetransformation • -High protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs, and gene containment through the lack of pollentransmission.

  7. Why Chloroplastis a Unique Transformation tools? • Precursor for Photosynthesis • Getaway to high-throughput genomesequencing • - more than 230 photosynthetic organisms including 130 higherplants. • An excellent tool for phylogenetic and evolutionarystudies • -Endosymbiosis -cyanobacterial cell - engulfed by heterotrophiceukaryote • -Chloroplast organelle -evolved from photosyntheticbacteria • Storage Compartment for Biosynthetic Pathways.

  8. Why Chloroplastis a Unique Transformation tools? • Protein accumulator - soluble proteins and intrinsic membraneproteins. • Cellular location for valuable recombinantproducts • Own genetic systems and genomes, high transcription translationmachinery. copy number, • Plastid possesprokaryotic gene expressionmachinery.

  9. Why Plastid transformation isPreferred? • High level of transgene expression and proteinaccumulation • The possibility of co-expressing several transgenes inoperons • The precise transgene integration by homologous recombination. • The feasibilityofexpressingmultipleproteinsfrompolycistronic mRNAs • Regeneration ofcropplantswithhigherresistancetobioticand abiotic stresses and molecular pharming.

  10. Why Plastid transformation isPreferred? • Absence of epigeneticeffects • Uni-parental inheritance is commerciallyfavored • Easy transgene stacking inoperons • Foreign protein accumulation of up to > 40% of TSP, 70% inTobacco • Absenceofpositioneffectsduetolackofacompact chromatinstructure • Efficient transgene integration byhomologous

  11. Plastid Transformation-Superior than NuclearTransformation

  12. Comparison of the nuclear and plastidgenome

  13. Milestones of Chloroplastengineering

  14. Chloroplast transformationrequires A chloroplast specific expressionvector A method for DNAdelivery An efficient selection for thetransplastome

  15. key conditions to achieve plastidtransformation Generally, three key conditions have to be full-filled to achieve plastid transformation: A robust method of DNA delivery into the chloroplast The presence of active homologous recombination machinery in the plastid,and The availability of highly efficient selection and regeneration protocols for transplastomic cells

  16. Steps in chloroplast geneticengineering • Aseptic growthofplant on MSmedium • Biolistic particle treated with vector and otherchemicals • Injection of recombinant DNA Plasmid into chloroplast using Gene gun or othermethods. • After 2 days, leaves cut into section and transferred to medium containing an antibiotic ,for recombinantselection.

  17. Steps in chloroplast geneticengineering • Greencalliformedonthebleachedleafaresub-culturedonthe • same medium Calli formedshoots • These shoots were rooted on MS medium to obtain plants, express the desiredprotein.

  18. METHODS USED IN GENE DELIVERY INTOPLASTIDS • Presently, both Biolistic and PEG (polyethylene Glycol) treatment of protoplasts have been used to DNAdelivery • The first one consists in bombarding of tissue or cells with DNA coated particles. • The second method treats isolated protoplasts with PEG. • Micro injection is also used (femtosyringe)

  19. METHODS USED IN GENE DELIVERY INTOPLASTIDS Table 1- Chloroplast transformation methods and selection conditions for different plantspecies

  20. Fig. Steps in volves in Plant plastid engineering by Genegun

  21. BIOLISTIC METHODSOF GENEDELIVERY • Advantages • Simple operation and high efficiency makes it afavorable • No need to obtain protoplast as the intact cell wall can be • penetrated. • This device offers to place DNA or RNA exactly where it is needed into anyorganism. • Disadvantages • The transformation efficiency may be lower than Agrobacterium- mediatedtransformation. • Associated cell damage canoccur. • The target tissue should have regenerationcapacity.

  22. PEGMETHODSOF GENEDELIVERY • PEG-mediated transformation of plastids requires enzymatically removing the cell wall to obtain protoplasts, then exposing the protoplasts to purified DNA in the presence ofPEG. • The protoplasts first shrink in the presence of PEG, then lyse due to disintegration of the cell membrane. Removing PEG before the membrane is irreversibly damaged reverses theprocess. • Treatment of freshly isolated protoplasts with PEG allows permeabilization of the plasma membrane and facilitates uptake of DNA.

  23. PEGMETHODSOF GENEDELIVERY • Plasmid DNA passes the plastid membranes and reaches the stroma where it integrates into the plastome as during biolistic transformation. • A relatively small number of species have been transformed using this approach, mainly because it requires efficient isolation, culture and regeneration of protoplasts, a tedious and technically demanding in vitrotechnology.

  24. PEGMETHODSOF GENEDELIVERY • Advantages • A large number of protoplasts can be simultaneouslytransformed. • Thiscanbesuccessfullyusedforawiderangeofplantspecies • with adequatemodifications. • Disadvantages • The DNA is susceptible for degradation and rearrangement. • Random integration of foreign DNA into genome may result in undesirabletraits. • Regeneration of plants from transformed protoplasts is adifficult • task.

  25. A List of Some Transplastomic Plants that has Engineered for Various AgronomicTraits Plantspecies Geneintroduced rrn16, nptII, uidA, hST, cry, cry9Aa2, Bar & aadA, rbcL, DXR,gfp, Cor 15a-FAD7, Delta(9) desaturase , AsA2, PhaG &PhaC Nicotianatabacum aadA &gfp Solanumtuberosum aadA &gfp Oryzasativa aadA,Lyc Solanumlycopersicon Brassicanapus aadA & cry1Aa10,aadA Daucuscarota dehydrogenase(badh) Gossypiumhirsutum aphA-6 Glycinemax aadA Lactucasativa gfp Brassicaoleracea gus &aadA Lettuce gfp Brassicaoleracea aadA &uidA Betavulgaris aadA &uidA Solanummelongena aadA Arabidopsisthaliana pre-Tic40-His Zeamays ManA

  26. Vector Design for ChloroplastTransformation Fig. Basic design of a typical vector for transforming the plastidgenome P-Promoteranddirectionoftranscription,T-Terminators,Whitecircles-UTRs,The thin dotted lines with arrows indicate homologousrecombination.

  27. Vector Design for ChloroplastTransformation • Selectable Markergenes- • Spectinomycin resistance- The most efficient and routinelyused • 16S rRNA (rrn16) gene- Initially used and selected by spectinomycin resistance with lowefficiency. • aadA (aminoglycoside 3′ adenylyltransferase) gene- • marker gene that confers resistance to streptomycin and spectinomycin by Dominant • inactivation ofantibiotics. • Plastid expressed GFP (green fluorescent protein)- a visualmarker • for identification of plastid transformants at the early stage of selection and shoot regeneration. • The npt II- Transformation efficiency was low, i.e. about one transplastomic line per 25 bombardedsamples

  28. Vector Design for ChloroplastTransformation • Selectable Markergenes- • neo gene-yielded 34 kanamycin resistant clones out of Bombardment of 25 leaves • The bacterial bar gene, encoding phosphinothricin acetyltransferase (PAT)- it was not goodenough • Betaine aldehyde dehydrogenase(BADH) gene- efficiency was 25- • fold higher with betaine aldehyde (BA) selection than with spectinomycin in tobacco

  29. Vector Design for ChloroplastTransformation • Insertionsites- • Insertion of foreign DNA in intergenic regions of the plastid genomehadbeenaccomplishedat16sites,mostcommonlyused • insertion sites are - trnV-3'rps12 ,trnI-trnA andtrnfM-trnG • The trnV-3'rps12 and trnI-trnA sites- located in the 25 kb inverted repeat (IR) region of ptDNA and a gene inserted into these sites would be rapidly copied into two copies in the IRregion.

  30. Vector Design for ChloroplastTransformation • Insertionsites- • The trnfM-trnG site- Located in the large single copy region of the ptDNA, and the gene inserted between trnfM and trnG should have only one copy perptDNA. • The pPRV series vectors- Targeting insertions at thetrnV-3'rps12 • intergenic region, the most commonly used vectors in tobacco and yield high levels of proteinexpression • The trnI and trnA genes-These two tRNAs are located between the • small(rrn16)andlarge(rrn23)rRNAsubunitgenesandtheoperonis transcribed from promoters upstream ofrrn16.

  31. Vector Design for ChloroplastTransformation • Regulatorysequences • The level of gene expression in plastids is predominately • determined by regulatory sequences such as promoter as well as 5′ • UTR elements. • Strong promoter is required to ensure high mRNA level for high- level of protein accumulation e.g. rRNA operon (rrn) promoter(Prrn). • Most commonly used cauliflower mosaic virus expression indicots. promoter is which drives CaMV high 35S level promoter of of transgene

  32. Vector Design for ChloroplastTransformation • Reporter genes used inplastids • GUS (β-glucuronidase), chloramphenicol acetyltransferase (CAT), • and GFP(Green FluorescentProtien) • The enzymatic activity of GUS can be visualized by histochemical staining • GFP is a visual marker that allows direct imaging of the fluorescent gene product in livingcells. • GFP has been used to detect transient geneexpression. • GFP has also been fused with AadA and used as a bi-functional visual and selectable marker

  33. How Chloroplasts are Transformed? Fig- Sorting ptDNA at the organelle andcellular

  34. How Chloroplasts are Transformed?

  35. Selection ofTransplastomic • Common selection marker used for plastid transformation is the bacterial spectinomycin resistancegeneaadA (3´aminoglycoside- • adenyltransferase). • Transplastomic clones are identified as green shoots on spectinomycinmedium. • Spectinomycin inhibits greening and shoots regeneration of wild type. • After integration, Homoplastomic cells obtained by several • rounds of cell division and organelle segregation.

  36. Confirmation of transgene integration into chloroplastgenome • Integration of transgenes into the chloroplast genome can be confirmed by PCR using internal primers, first primer anneals to the flanking sequence and second primer anneals to the transgeneregion. • An expected size of PCR product was amplified and this confirmed integration of the transgenes in different cell cultures of plant • Integrationofthetransgenesintoplastidgenomecanbe • investigated by Southern blotanalysis.

  37. Confirmation of transgene integration into chloroplastgenome • Genomic DNA from transformed and untransformed cultures Can be digested with appropriate restriction enzymes, transferred to nitrocellulose membrane and probed with P32- radio-label . • Transformed chloroplast genomic DNA digested with restriction enzymes yielded an expected 3.3 kb size hybridizing fragment.

  38. Applications of chloroplast Transformation to Plant Improvement • The expression of foreign genes in chloroplasts offers several advantages over their expression in thenucleus: • Improvement of Agronomictraits • Biotic stresses or Insect and Diseasesresistance • Abiotic stresses or Drought and Salinitytolerance • Production of biopharmaceuticals and vaccines inplants • Metabolic pathwayengineering • Research on RNAediting • Phytoremediation • Production of Industrial enzymes andBiofuels

  39. Agronomic trait development through ChloroplastTransformation

  40. Production of biopharmaceuticals and vaccines inplants • Protein drugs made by plant chloroplasts overcome most of these challenges like expensive fermentation systems, prohibitively expensive purification from host proteins, the need for refrigerated storage andtransport. • E7 HPV type 16 protein is an attractive candidate for anticancervaccine • development inTobacco. • Plastid transformation systems became successful in the oral deliveryof • vaccine antigens against cholera, tetanus, anthrax, parvovirus. • Above 7.6% Protein accumulation . Example-OspA plague, and canine

  41. Phytoremadiation • Phytoremediationisasafeandcost-effectivesystemforcleaningup • contaminated environments usingplants. • Two bacterial genes encoding two enzymes, mercuric ion reductase (merA) and organomercurial lyase (merB), were expressed as an operon in transgenic tobaccochloroplasts. • Phytoremediation of toxic mercury was achieved by engineering of tobacco chloroplast with metallothioneinenzyme.

  42. Production of industrial enzymes andbiomaterials • To produced the highest level of the poly (p-hydroxybenzoic acid (pHBA) polymer (25% dry weight) in normal healthy plants poly hydroxy butyrate (PHB) was designed using an operon extension strategy • To date, the highest levels of PHB have been achieved in plastids due to the high flux of the PHB pathway substrate acetyl-CoA through this organelle during fatty acidbiosynthesis

  43. Metabolic PathwayEngineering • Plastid- ‘biosynthetic centre of the plantcell’ • The most complex metabolic pathway- synthesis of the bioplastic polyhydroxybutyrate (PHB) , cause male sterility (b-ketothiolase expression ) and severe growthretardation • 1st in Tobacco, Recent- in tomato to alter carotenoid biosynthesis • towards producing fruits with elevated contents ofβ-carotene. • Successfulexampleofengineeringanutritionallyimportantbiochemical • pathway in non-green plastids by transforming the chloroplastgenome.

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