Unit 3: Transfer Techniques

Unit-3 gene Transfer techniques:- The uptake of transgene or foreign DNA into the Wells is called gene tranfer. plant veelts ② There are genes can be these techniques introduced by uchich trans- introduced into plant. ant cells. techniques These ter are grouped into a types 14. Agrobacterium mediated gene transfer facthit By Direct gene transfer cxcctodeans) Cveck bansformation nay. stable hans in case Paltegrative or integrative. be non-integrative Dan non-integrative asan the tra entra chromosonial transfexmation Ento the the Lant type is maintain DNAN but in integrah transgend is integrated genome The integrative table bre transformation is. meritchle achile non-integrative type of transformetiors i non - heritable - Autgro vector mediated gens transfer. nifer are also by viruses & Transponible elements- O Agrobacterium gene hamfer Hesa -ve gram bastexla, rod shaped. in the soil. bacterium focung? Ost has 3 species • A. tumofaciens shieogenes induces urocon gall decass - induces hairy root disease. 1. Radiobacter - dviveulent strain (non- pathogenic type). Agrobacterium infect the crouens, Rool stenu of several dicat 4 gymnosperms. @at induces tumerous growth infections at the site The tunerous growth in the crocen plants is called urocongall kalled erown gall Whease. gall and ts Agrobacterium never infeels monocot plants. Zainen etal 1974 first noted that virulense is due to its plasmid. agrobactriu and agrobacterium There are large plasmid sonich are plasmid) 4 walked Tiplamids Ri (tumour inducry the tumour formation is due to the and feinctional entegration of Ti platning calleg transfer of a small party + DNA inds the Igenome (Chilton-etal), 1977. plant nuclier Organisation of Ti plasmid :- of Ther are Large etrculas as DNA molecules 200kb in length and 120-160mg in molecules @ecight The toplasmid have many regions like virulense Megion, anigin of replication, conjugation, encogenesity ang There is catabolism es a segmont q T transfers okly achich is transfers only last well. D prasmeig healeeq DNA prom plasmid into the Based on are the type of opine synthesis the Ti | 2 typer plasm By octopline. Nopalaine all this Tr-plasmid ercept are identical En struckers in their sequences for and opine catapolisms. opine synthesis structure of actopine - Nopaline Tiplasmids The actopine Tiplarning encode the pyntheids of ectoping wchich is antisual da synthesized from arginine an example Teplasmid of PIA PTEAcht aan ectopine Toplasmid. of actopine Border requsnes TDNA PTIC 68 (210000 hp) Jane Tom region Kodes for the auxin & bytokinins" me for the 8(4) - Gess reap af Nopoline pesin CATI 152) synthesis of auxi helps in tilmour formation region helps in the transfer & one bacterin A TRA Maginn ferjugates transfer of The TRA conjugation D The occ region is of actopine. catabolism agrow on indoparation. @ The ne locus es forced incompatibility amory plasmid it a bacteric. oni d Ies formed for replication of plasmid. The vir Vresión es por transfer integration. T-DNA Vento tul chrokiosonmal of plant wells. ber NOC sformed for Kataboliem I ropaline. Structure of 7-DNA- The Portion of tegrated into the Ti plasmid ive transfer f into the chromosomal DNA plant cell is called IDNA. the on either side of T.. DNA Here is short sequenzeige walled border sequence are essential are present colich for tumorxu induction. -The T-DNA has FEIM sequence aurin 4 cytokinin which syntherine It has also Noc/oes sequences. nopaline of octopine for synthesise of TUM Region border surin cytokinin , Nopaling/ocloping Syntheich region Right border T-DNA (RIKDP) Mechaniam EDNA transferase. @ The T-DNA hamper begin with the the introduction of bacteria ined a plant won wound. @wounged plant well syntherise acetosyringone ang B-hydroxyacetosyringone. @This phytochemical induce the expression of the ti-plasmid of agrobacterium. The vir genes epresented by 8 operons He walled VirA-Mir-H vir aperons are not transcribed during rmal vegetative groveth. They ang products proteins achvate them. wehen ne are activated phytochemical The proteins og v vir exegion provide endemisione that initiates the mans fore process T-DNA atspecific site. By nicking @ Nicking takes place beth sind 14th base of bottoms. strand, of The nick at right border provides a priming eng ang synthesize a nevo DNA thin nick from 53' direction. strang pem new from strand displaces strang the old T-DNA single. proteini produced by vir-Boperon forma Lonjugallipore re or or pilus pilus in the membrane through which cell. T-DNA is qelivesof ento the plane 12 The T-DNA closely samd baute solowed to bacteri the event resembles in bacterial confugation. The t-DNA single strand forme a coneplex with Jets transported to plant Vir e protein and gets nucleus @Agrobacterium mediated gent manipulation in higher plant et not so simple because ti plasmid the ti is too large ge and difficult to fing out unique reestriction site to insert DNA. foreign yat involve 3 phages - ef construction 1 of agrobacterium strain that has recombinant ti plasmid. 12) co-cultivation og agrobacterium of plant tissues that has to be pansformed sety ferexeration at plantelets pom "infected pealt inues. the agrobacterium Direct gens transfor method: The The process Introduction of genes into plant wells without the involvencent of agents leading to stable as direct ological bio transformation gene transfer neetholy. welled 2 Direct ect gene transfer was mage! Uplasmid Integrated. bacterial plasmid The mechanism of integration not known in detail. in 1984 when ento plant genome. and transfer coas The spontanences uptake og gene into plant cellk es very methods Loco. Hence are plant adaptad physical and chemical to gene into bansfer gene ④ the various methods for direct gene transfer aht as followsー 8) chemical methods 4 physical methods bike- By electroporation. Evy Microprojectile bombard- microinfectiow meant Lipofection etc. chemical method:- certain chemicals like REG (polyethylene • (polyethylene glycol), polyvinyl alcohol, calcium phosphates enhance the uptake protoplast. of foreign gene fredres into play plant protoplast are suspended in a trans- formation medium 47 wing Mg" containing Ma cons, followusing PEG addition with pff's van penes Ento protoplast. transfer gener Housever chemical method yield only transformant wells. 1.528%, transfor that to in certain plant species. physical method physical method - gene transfer includes transfer Electroporation Microingbehan Microprojectile bombardment Lépofection:- af Electroporation method:- Dentroduction of genes (R-DNA) into wells electric treatment called electroporation by @There are 2 system of electroporation. of Low voltage lang pulse method by High け shoot method 2 for Low voldage Long pulse method 300-400 vem -peed for 10-15 msee es giver for high nottage short pulse method 1000-1500 Vem for tomsee is applied for electroporation Ploch montaining prasmid genes. suspended Recombinant this electroporation mirture is exposed to desired voltage ang pulses for required no. of cycles The protoplast ars then cultured to obtain well kolonies ang plants The optimal voltage and timme depend on plants species, houres of protoplast and reexistantance of medium. This methods gives high rate of This method She applied stable transformation (in plant transformathensp. elke tobacco, petunia, maize, rice, ueheat etc. In tobacco the transformation frequency En presence 7% PEG. Limitation & electroportation:- 2-87. This method is adapted cannot not adapted to Introduce R-DNA into intact plant cells. duinial cells are sensitive to electric treatment. - af electric strength is strong then protoplast Loose thetr viability. of Micro injection method The infection of branefer of wells whing cappisary miles DNA directly into micropippettes with the help of mulromanipulator is called micro- enfection method. Cappilas The micropippette used here is a small expirat tube weith open ends. Its one end is very sharp 40.3mm? in diameter. @Before microinjection. is plasmolyzed the cell is gently by keeping it in hypertonic som, then it is Hansfered on to a microscopic slide unger the microscope. M es belof the sell is a in a paritiers meinga pippette one end of cucking peppette is postin and a gentel gentle on the surface of the kell and a of ruccion pressure is applied. Q The R-gene DNA/gene is on its other end. R-gene DNA/gene is sucked in micropippelter ang the sharp and of pippette is gently inserted in the well ④ the gure is delivered into me well throwpe.. gene the sherp eng ang through ette drawn back the micropippette cerefully. The Wells arl then transfer the fresh mediun to estabilish well culture. Successful transformation by microinjection. has been done in tobacco, alpha-alphя, brassica sp. asith transformation frequency of 14-66% Limitation:- -8-DNAS feus Genes van cells at a time. miers injected only into a - Microinjection needs technical skill of experienced wworkeru microprojectile bombardment @shooting He plant (or) animal cell by DNA-Cool. tangstein particles for es caused micropre or by DNAS into the welle bombarqueent cor) qun gene biostatistic particle gun gene transfer method. This method is suitable for plant wells. animal wells + cell organells and mitochondria such as fungal well chloroplay The T-DNA 1qere is mixed weith in or of 1-4rum fit is treated usith calz gold particle firing pin → Blank charse → Nylon microprojectile Microprojectile Air vends projectile shopping chamber plate with plans wells. 2 Cacly precipitate the R-DNA finto the metal particles. @re DNA woated the to be microprojectiles. particles are said The microprojecthier are animel vells with the fered Into the plant or speed of 800-dooms uning an instrument satused called micro- projectile gun cor) particle grun Gun powder electric power Is used to provide propelling force to the giun to dry the micron projectile Iinto the cells Othe genes integrated & transform @ta ureful method wells transfer noble genes cribryo, embryo callus, epidermal wells of man monocot /dicat plant. & many DH is also an effechve affective method to transfer of to transfer gene A organs enk mammalian celle 4 fig- A microprojectile guun (simplified) alipofection Introduction gene into selle by liposonces in of gene called lipofection - A liposome a small up of phospholipids ical vesicle nage spherica lipofection method is suitable for delivery of genes ints animal well culture invitro. It delivery futo antially wieg for gene delivery plant pratoplast. The transformation frequency. equency in to be yox10 this method Section of transgenic: In presence be Manegere in the target cells cen detected rapidly by by per method. - the transgene integration is usually confirmed by southan hybridi sahen. transgene is the targetvell & detected by per but it doesnot endicate, PER gere entegration, into the plant genome Southern hybridization shown single of multile, - cus insection of genes luto the host genomme - In this method selectable marker gene 4 reporter genes confirme the Integration of gene Insection. genic antibione to select the recombinant transgenic reesistant gene tumerogente geres & reporter Olke Luciferase ves fo GEP etc are qus re wed. a geres transgene by selectable marker. • Selection & trave -The Ti stred factor plasare desired resistant gene 4 kanamyckne contains auphicilin resitant gene - If is a foreign gene is inserted into vector the recombinant aprobacterium can't growin the presence of these antibiotice: Not all plant cells / in a culhere are enfected by esconebinant aprobacterium. Some in the sulbore. vell emath winpected wchen planta are -This deprecated in this culture some transgenic 4 orkes seen to be non-transgenic. mansgeneic plant grow faster then the non- tranegenic plants. -The fast growing cells of tissue are choosen as reconibinant 1 transgenic. Reporter Gene: Did gene which codes for an be enzyme that can n enzyme. eally assayed and recoursed the presence of cloned gene in a wecombinant is called exporter gene Reporter genes genes lies ufor. and nodes detected. He along was th the desered gene enxyme that can easily positive result of enzyme assay reports the presence of gene chiexed in a reconibinant. The gene for p-D-glucoranidase (Gus), vodevy for uciferdse nopaline synthare firefly etc are Luciferase, used as to select the recombinant plant celu. porter. ened ①p-D glucoranidae (Gus): - there is no detectable endogenase Gus activity higher plantene in most gene is isolated from E.coliand Gus gene inserted Jat just upstream of desired gene quchile constructing K-BNA. @Theo-DNA is Entroduced into protoplast con calls. The protoplast multire is treated with brand-4-chloro-3-indole B-D gliceronic acid. the recombinant produce proglicronidase enxyme that breaks BCIGA into a bleue colourag Deampound ang appear beve They be selected unger a microscope. by riensing the protoplast нехе, the GUS achwity Report the presence recombinants. 24ecombinants have no GDS activi ② desired gene in thts The non- @Luciferave Thuxgene 19- Certain bacters, possess the enxynce luciferent substrate such as 14 fengi and firefly! Qluesferase acts on ang Luntniscence visible light. Luciferase is encodeq LUX gene by a jene called 2 ritgher plants wells have no leux gere. Hence, this gene is used as select recombinants. Reporter gene to Bacterial Lux gene lias in 2 sequences, this 2 sequences are isolated forn together and insextelof just docon stream of desired gene. R-DNA & constructed in vitro and introduced prekplast or callus. the protoplast sulture is then treated with decanol and FMN H₂ The bacterial luciferase in the recombinant on decand plant wells act on by whilining the reducing power & FMN H₂. Hence the reconibinant cell luniiniscence recombinant eight. @they can be selected to regenerate transgende plants. green florescent protein & IGFP] one of the that emit uchen wit absorb visible light spectra. @this proteins are composed of 238 an with molecular weight 9 26 kαίν 26 kdavitible to UV Spectra. an well the and molecular biology GERL frequently used as This protein. neporter expression. wan be used in modified form in biosenser & meny animals have belt jcreated that express GFP. Gppicen introduced in animal through hansgenée technique