ecambinont ene os sirwct_ DNA Technoloqy - L) Conctrnuct s an arti id aly- gene ort DnlA goeoSeament o DNa botne ona veAa Hhout can be mol piieo aA DNA ncospor0ote genekc materrias înto Jartoet 4ive t (ell- se construdt Conhens Vîa a ered transfrsmation T sertd, caes Dnln a a Aransgem whicn aalous Ahe n Serk vetox do be rrepicatd ancos oxprojed n he arge cel S gene con be 4 Cloned thecauy cosn structed TJ c the veekus usec aA odgin SSelertable o foom a onA iepicodn, naturauy OtU a a RnDNA COnGArwoSs moiaiphe ctonin hec, & a Congtrwet Omanee% DNA Conswcts a be as Smou as a few thous and base Con DNP ok (areying Sinq e qene, Usi velkos Svcdh o Pla mid or bactemo phaqes ort as large/| hund neds oKb Fort lárae -Scaue qenomiC uolies u s i an a t t s i a l chromesome PPicodion- Ranion kumart CakeoT chann el" ONIH Consttwchs Canbe use prodwce prodoins,înduolira/ bolh notturouly turinoq proteins and Can be These Proteins a. PoroeOn8 make kKerareuhe Poduchs, Svch Bngineemed mutont aanH hbadieg. PharmaceU{call and ONA neAr e 2h Cong truds Con aso Change, the expresion level ob Ghe enes b exnesingneakury Seqpenees Sudh ag PRomolera and D I A COnstrwcts Can be used Po researnh Such ag crueatna aenomi C brarues Sequenurq clamec DNA Conc tudyinay RnIA and PoAeho ex Presirn Consrwctios ok Ghenomicibrar d DAI A aenomie library is a Colleuiom o the to Heu enomh rom Sinafe ggoniSTn 0 OR Agenomc library iS a coltethon ok ovenlapinsamendso gene o ONA 7hot CLOnedinto a b&K bone veekor, (unow as bnlA hsert oo gene o intene) SAraisticauly inc tudes a regions O the Jenom e OTt uhic, Gan ganisn Aaenomic ubraruy Con be dekne ag he colleuhion d the ctone bearury the to-la enome ONA ok Ovaani COnstrwcAion n order to Cmstrwot a aenomBe Jhe oraons m's DN ibrasuy xoeut don Ces and the oqegted wi-th a ArcHon to Co4 DNA into Proagment S oa Spesic Sixe. vetto USínay DNA 1iqa se inMo e The frcegmeaS arrehen nsertt o q a n i s - Comnanly NRxt, h e vettos DnI Can be taken up by a host Es chei Chia (colí on Yeout u h eath en containiny porulcUon ok One vectso molewie n a and meie va ok Spettre clones, Cuhen host ce) to army h e vety allou fo eoy amp1iecation Fom Hhe libray fr analygis. d i r e t Foom he aenormie 4he c tones ane deru The c lone Se} o CaJri by veatns hey ane Calee gnomie Con es The Sum total tibnay. genamic aj Frouments is caud i knouun collettoro idauy emsents the en Hne in 4his DNA Inserds aenome 6 the Concertned doganin, Rks Yr channe melhed 3 V Oheromic Library othe ovqani Sm Ocu Shotqun exreimen Mo Psaqments 9nthis method he enhne qendme esonape sixe b restition (nsentedin e D N endo Unoleases. These ONA Fraqmenls a r t e hen OSu Hble loning vettos t Poldce a poPuladion ak 7econbinat DNA. This Consti-ute hshotaun arproauh to ene Clonirg. Such Tecombirant vecstoss ane hen insertt into hosl colt &ie e.co. Nod eauh (ey Con taininey e Cabinant DNA S coltused Sets Separsadeuy to obtain cloned These Ocioned Ces Posm enomiC ibray Sn i s method lambda Phage. vetdoy 0 coSmidS arne mest Commonl e y d i zalian Method chen genes ane Pnesent in moltple copies, his Y mehad USed fos theirr iso laBira ,uakch Comruse d foltouwíng Steps I s e l a t i m s h e entine ONA foom the deyirned doenm iy akalne cigez-n Or hea ONA (ds DN)S rteament,the0 doutle Sranded Converted t o Sirgee Srand DNA (S DNNn). (iö DNA RNA hylmd S Co Produred m SDND & mRNA (Hnans cribd oy the gene) T h e s DNA sArend ukich Cmtents- conHans nucleohde Seguente fov ttrans Crs pHons 3eponcted Ond isolate Fsom DNA XNA nybid ) The SS DNA is onvex Ad fo dsDN A by the enyme brN Pely- merase-T. Leapenue agene o0 Aethniqee Pertiwlar DalASeapuenue is isolatedson he cDNNA or qenomi ibnaa3 knoun a by a Coloay hyrndi katin hpicat1on L. AHerr a libraruy i Cnealed Ahe Jenome an Osganigm can e Sespenre to elucidae how 3eney akect an daanim at tocom pane Similart ortqantm athe genomí Level aheromic lxarty Construchon & he Cirst Sep in any-bND Seuenirg Paojeds nendmic 1ibanie hers in identi ffcadkm od the noveA phaRma/euticoalhy irnprdank Jene, 4 hes in e us in ondenslanding 4he opexidy 5Servirg ag Sounre o 3enme genomic seuvente FogenetaBim o rang en ic animals thrzough ngineeny 2eneA1 CDNR Library DIn ruchon tibrardsacoltethon NA the gene3 Clone d Hhod arne expro)ed Partculoro peniod ype at a N A tepresents CCmple meake CDNNA & ComLainino afven cen wi4hin Colletimn DAIA) epreents tibrad es tepreents Seuenes Aisve a dk Sire Pe o cell mRNAComplemets e ONA reprents pesd tron Sttptes ater Ony anscro)phon modificotios (i-e Spifedng nkpn) dvanleqe o cOB ibga istha Com Beuns o O iegion oka genme. heCodirg NR s prroducad om Funy monsvibed mRNe adi Found in ucles andthere fore conteins ony eoer ed geneg oan Onaan tm. eeNA ibro Constrwction3e Gbray A The bauic bnarhe Constrwltm Tnuvde toom LSo latiom ok mRNA eukary o4t fnAion mRN Snthess e Ce DNA mRAA sleps o DNa doom dhis InSvi-labie e t u r 1íkR Clnin o Synihesixed eDNIA R k YT Cam ne Plosmi os Phog 3. Svidabie ho ike bottemm, o nDducfm Scveenir&mainttnante sTudio ve DAÐ s oSet o Clme thoe Pooeedue RND mature Creahede vfoom e u e toans Captase a Condain Pooma cbNA. eukanyit Cey Use o oa lomgevenue o neukary o4es , a poly A) Haá\ (ConsisHr om tRNA &RNA distinouigties mRN adenine hucled tides ) site fos everse fes eused a aprumex anc Con theirr gieth the 4ruansiption. pNA furele Fo r Piratty, he mRNA ob]ained ore patin Severta methodlo froom-the Tet o the RNAs Colum) SUch og RNA r zol exlracHfon onc\ PuriticoHon ucDNA CongtTucion Once mRNA s p ied,otigo-dT(a shat Seuente ok deoxy-hymidine hodeotides ) i agg ag a Complemen tary Povidna a Frrae Pruimers which bin tho-Ahe poto Poly- A jase 2'6H end Ahad con be eplendeA by rteverse tons t(rneate Compementa DNA Snanc Noo the m RNA Sirale SArand a Thi S emove i r on KNAASe enAyme, leqvin CDNA (es cDNA ssc DNA converted no coubte-Stsence DNA with he hetpo DNA polymesose Aawever , for DNA sAtand , A the Ce 3oy end s needod Thu is Pobvidod SeDNA i sel by coing poly megale to Synthe Sixes a Comptemenfa On i4 by generading a Sel sel lcop Chdupi by at he 370ond 1aler me loopat soH end se eslends he 3o4 end, ane og S nudease is penabthe Scidosing aon a n e {hon Ue Kestruction endon uctease s ard Drd A ltaase The polymereg do Clone h e Seqpenes into batkriae Plagmids. baetemas a r e t h e Selettea,Commomtyrouh the se o The clom i anti biotie selertion c e SetetedeStocks ok brary eane Crneatto cONAA uhtoh con ladtes be grooun kseqven vato Cmile n e ibram ss o ONA Library o non Codiny torog2 o teduceamount o preyjed in pookanypfc poibms ex dinety boe Con Hrarie) cDNA heLe the aoddi timou CDNA libnaries Qune usekui în n e v e r u e genetig informahan due to the Usee gonomic ínformakion is dk 1es CONA + li branie is oge fon f r MRNA nemoua Codes Fos PexHCula aene that iLolatng OSefui tuol in the anea CDNA library is a pood Pouesfuj ond Rks VT Channet" bioteth nolo 631 iS helpo0 FuI a ex predin eukaryoticgenes in D kanyo t,which PsdCO og Piukamyotes. helps in 4he toons Cipti 4 a u o 0selo1 Fo4he Speisic DNA é DNA o Prutems mo noc lonal Can anti boclies scneerning 3enomic iraalg to isolade acilitate to genesale anh bodies is ínposten4 arpicatian aCDN isary to Stud-y rexdn o mRNG mRNA LBis k cols and Pu ticakoy ek The mos ane 4be . AAARA 3 mRNA Poly At ybroidize cwth PoLy T) Poimes Reverse AsansCap}e 85 Pouy T (Poimer) AAAAn mRNA Alkalune hydotysis tremove mpNÐ TTTITS CDNA ExBende hobk S(Oa ss c DN) hairupin oop ONA DulplEx with hodk NoCLeae AtemenA Aooemove hook 2 DitIINIIIIuL RegosenBafino Synthesis o To veo ON Dupex 3 (for Clonirg&Fosm cDMe {ibran) Vsing xexese Revere Hse cDNA fom mRrna ScrLeenirg DNA1brerie p oblonene o inieres etlc rteny creeniro s Ahe pocena of identHfleaten o the clones cay"g the gene ok nterat est. ba sed7 Det btanios Can be dme hy Fouewiry appoathes edirq a porhaulos DNA Seuente an hened 2X presion Omethools Fos Screeniray boyeol n delehiryDn Sevene cneenirgyhyicaton i s a Common matha Used in DNA Ii bsamy emeenmnay to detet DNB Seauente s in cA-frsbes . 9 OSe eileg on the nat sonFepte-e 4hod siroe a usinq radio cuh we Gnanded DNA moleto l e to 14s Compenen lay aPoobe hytoidi xe euen ce ano lden1y the Spe ui sE9pente. this method is quick , can handte a veruy larae numbert ob Cloneg Ond hen vsed in the idenh hcoutton) o 0 oN clones uhicn OFLe no foll- 1eno h Commoo1 Utecl melhods o yhndation as con colon Zation 3 (olony hyondi zakun is he troenir oaorary witha tebae PsD be Cradto cutie, ec). Ho identt aSpetic sequenre DrNA, RAA,ejme, (n ten o anhibod The plaqueR arte stneen bg tech bay-e om he yrihzoton o t fg Nudesti de mo be do Sn his Case A tngct DNA DNA Ssansferr foom 4he Pdlsh to #he itey uhch iy hen 1ato incubate wih lebeles y oadioqrop n by auto RkO Vt chonne eyPRPaiymernve charn reachm Pr) 'Sas 3oc aghyk ibrranio) Pre pobes ons theo a a y dieHy dizadio RhniaeF Sureemr DNA bU ocleuae înfmmautim must be Avaitalh1e porme poimey for his melhod. The colonia are mcún ÀeÁnad wel a s Ccnoen.eo muldiwau,Plae, paihn y p c R and he PoSie ueng arre ideni fre ScreonigMelhodb FneYPenia (a)Iromuno togiCas Scrooenîr3 Can be us f u y the dletdtim mmuno logi cou techaique oa pu&4em os a poy Pepetide Sgndhosizoo bja 9ene Sn this melhod Jhe ceu amee obun as (olsnîe dn q hes ma Sde piaseg Whin are troas Aerst fo a o i d madiy ((e nitgo-enlk The onle ame n Sobjete to lsis Ondhe 4he neete nelese Proteins bovne t0-Ihe maiy Thce PrDent ane oJreoked wi hkn speufically a pmimomy andtbedy binds to he 'poD 4eins ncooo b lhe tarqet bNA ayalny, a Second an4i body is un bovne anibody Ater hemovingythe, which speihcauly nds h the int anti body added áne nemo ved by waainqs Aqfn the nbound anti bodîes The secmd rreoldih anti bodeg Carrie an enXyme lebetlebe C e ho rg e Pesoxidase d alkouine Phos phaetase) bovnel to it bstoate The detctim Pocexis So deviseo -ha og a Coja ley Pais ac ted up b hi's enxyme a Coloused Poodut s foome The Colonies uhich ive PositHe p rteult ( i e co(seo p o ) are S0 Hontiied oSpeati colany The ce Pla Sconin Synthei King a is capaole o that i ten auivi ty not en enXume ) A Cu lkrd be sob drrom the meger e h exproim9he targe tanqst orp ot the gene ti brany Ahe Can Can SpecstC Sobstrsche fo uSe avticotany Pobduceod by he hot cel nasc maly be ro ten oroeni Ond its utii2atim bya Cojony ok5 is Useel cen ndcakg qhe reente ot an emyme that ads on he ubitate. e x a m p e the gene codira Fer enxyme d-amylase s P-ucosidase Can e dentif e iqyfuntiosneCope o n c i m a Compemenlatn byhttechniqe mendatioa Rk yT ChannL he process ot Compen sat nq a mitsi ng funtan ina mutant ce the wi teston e b Peruicdas ONA Sebne ford pwenotyre mudent Cens ore non-viable, he cey c caruin 6the heClone 0 Tnteres t Con be2 posi hveu SaeuCd and isolateo St is a voru, Powey ful hethod aepresion eloni ne and aso K uselui setu FFos dentifi coutian hat o having Same rol e detective The U Sorenon end ident ication On ethers the2 UnGian f phenoyre 9n PlemenBation StTTOUns of an osqani S gene in argan ism Gogani Sm. anolhedK fositive clone i viSibie bted Chbnge in two with dißterent homo Zugous recenive muadhon thet genettg,complemenlatin Prroducothe Same moBanB Oceury cuhen phenotjre have osrra na hed prres he wild e e henope uhe maed or CYoseo Acowplemenlaian 4est Con b too Strtains a e n Deiaaian COmp lem endation ed do Hy uhehenhe mutAions in terent ones os om Ahe. Same gene Telers to a rmlatonRhip betueen tuo dileren stn o oo ovgani sm tuhich boh hawe homoyaus e e s necesive mulahHo Aha Proapce Same, pheanotyre, bul ihith olonoA rte side on 4he Sane gne. U o n i n , Cmplemen.hhon is a method P6oqmen and Fov ideni-fi caton a DN Setetion o he odelective cel e upon whih it Confers nesstante do a Seletil ve pneyre de cel h a ane hyrerc ensitHve to modi afion. -Colon hybiclizahon is Ahe blot analyis jni i dization teehntae aherte the bocema Cels are a n s fe redArrom Sotid nuten medlu t h e absoy bent mamat. Foy isolaim dhe spei hic be delined as the the Can DNA Sen\nenue meMo O gene foomthe bateria cel| Conasn ina hybi DNA USîng a nio Cenul ose Memboane f1te. The A 14e Parer) 1s Hhen trans ferirgmedivm ( nttro cenv lose Subjethe to J Seweros Chemicou & rhystcaL treaBments oD Cewloseler raper- st is the ans ferrinay medium the CoVony hybiei zahon hak tosms the mast 0 Plate's nepicos.0 The nitmelwlose acds as a memfane, (daingi the NiBoo act eeuuoseCopies ok the gene o dhod slk Mak Pa i Dpes t ilHespes ies 1. S1is 100. PUe o1o 44irg Pa niooceltulo oovides high 1pasity troansfer O.4s p Poresixe, fati jae elhitien+ runs torsim a . Collony hybndizoathon Op acks as 4he SchenaiUs+s method Frut hrunsten Or Hognesin the Yean 1 5 îs deveapea15 he Colo ne hybidizoton volveg neplico Pepasahon on thne nit Vcelw ose membane 14ex papes in Volve3, the in SiHw trrans ey o bactes Celis' frsom he ma Ber pla ke Dthe nitsocetu ]ose membrane The nlhn Cetulase r e Paethough Lome uwahinay mehod, Uv ey posure and the desi ned ene's hyriclilzaion with SretiHe Po be oThe Dala hyondxaton OCUrS by siro rtadioach ve RNA Pollowea y outp o The yhyi dixed DNA neevente ithen pidck ed f r +he nefevene 66 masld Plae Po furthe Stuc Rka yt Chaone BRks t cdhoann e hybidizaH Colony re hepmoces a Colony hybn'7ochior) nvo lve the doiuowing SHeps e a n a i a n o Mettd biate - Sn Hhese Slep the batterc Ceu suspen Cio are inocolate at oo 4he Soid aga PrLepa h e mastey plae . Colnie wil| e hatema mastr On the U5 t mediim to 1ndtulation', 4he nombes ok d e e o p uith direrenk Pumids rLeerence plate. 2. Froma f on ok Rerilcas overoDiDCaolase h1te Then, e Plcte the bauteriaa Ces tothe arnefzans fer ed from membsane Ur hl Rs Usi a mate maytey n i t c o n ) oSe Filten parex pre e FMtos. then itroot otlulose Overs Ahe Suridar e t the malen Plafe Thf Compne Nim o the FRIle membene 4oil o nepi Cag o do the malèy attunaL cet) meste plote Coptosels twe Treal mept olte pes meoliuno oith Spg 3-ALeytha, niBroocelluese Pil4er popers tead uith 4he sDS (Solim dodecy Sulfhate de tengen to lse We balena cells. h e Filen a mend e e p medium wih allkaui- Tne tp parvale Hhe DNA into LOTth Ihe alkau?( Rive Sodium hydwyide) 6e Sgte SAawnds. DNA Onto the. Flehelum T o fx the DHo Cetlujose FH} e r paper elher bak the Orr e poseîre, Hb 4he Uv ttght Xyhe he onto DN Pilte papes a 8oe vAdditiom o radioacve Prove- bru diae the nitroLelld 1ose FAten Ppox ConHoiainay mpriots o 4he Plagmi DNA Suy addirey the tadioatke RNA Pyave. po be. Ths radiocdie RNA Poue win ode me desined gene ok Seqyenue from he bateia celIs, vin A d Temovee Ao nadioraph_9nAkT Slep Un band the Ho paper i coaseked to he probe pocide. AHe 4hat Fi Aes pmpers s Expose to the x ray Fi|m by he method calle toacl109rsapkh a e wnoun a hecolonie that appear alt aubroo'o rPhy1 auto racis qnem Vy Tdenti-tiEdion ohe deined Then 4he deveuopo auto radiogam deto idenity he Colon ie Contairtiry SCompame_awih mastei pate eúninayJAhe desined gone, ui l| aeneontere)t.The cen arto n the tigia mediom and the Can be futex Procbmed foy he i s o lahm o recombinant Plajmic DNG. cen Concwde thad the Coluny CGNCWSloN Therte ore, we hbndi Xaion methsd is the Scneeiay echniqwe thod vseg the toalioactue Pmbe. The tadioattive 1ebeued pors Pm be Sneens o6 isolates the perticu gena foom he numhe ot baulona Colonie Probes Prabe ane Smaul(l5-20 to deted the rregente sk Samptes bSes long) nucteo4ide Seqence csed Comple menBaky Sequenue in nucelcaid RKg 0 chenne fs athieved by penmititng Pavbes lo ase pai WHh Ahe Sample Oudeic ad deni fying he Samples that suds en 0 baje pasru ney wHh he o b i e hymdi zohi om. Habri dizotha Con octu uken he babe Seqpente ola poo be is pRejent A i n 4he aene or ONA Segmen uKich t aimel to deett Poth DNA and RNA orue osed o nobe. This The The RNA ro0 bes may bebrroacluy Pobe (Ribopesobe). The DNA(Gs) Pabe ds b) cDNA pnuCiesdide probes DAA O RANA O Awo Aypeg * (0) DNn DNA Probe Povbe. may be Po be ond ) Aen dmic These One hocB Stneches oSingte Stoendeol to deje he preente s Complementany nucteic auid Seyente (fdrgcd seapence) hybridizoti oligono Ceo Hde prrobez ane osuaily o rtadio isoAores'epi topes, bioin han dAe hectj o te labeted Fos exemple width fuuDrhohes D enabie ologous probes Heterro loqgoi prst loe is a Povbe thaTS bunot exauy Complementay to the nudeicae i SRmilar to Fo h{Uh we arte Sereh Searschiro Hetead loqous loqo vs Pro be s a lb-eleol nucteic attd hawi r O gneten togous 0o non- identicay Senjente csedo rto moletule Tleni y ever Lelateomoletules b homolo loqous Pooe the homologous ay dentkcoR 4 ybidi ?xatin ooobing Probe thoat Comtain htemest bot PrEsbes icaions ok Priobes - Fom DNA How molecue (labeileol) hag Seqpene s- bNIA whtch en codes Porhe ene ano there Spei os Coulee Pn be hae a Jar to to 4he employe atPo bes arue ag Follo&os ProDbes heteo o prica Since 4hey Permit a highy riel table kVariedy eydremey kia entdeteti detettion ok nucleic áoiSesvened complemen r Sohe c arpi cottn Sdentificatiom ol he necmbl nanef (lneg carîrhe desino DNA insertt 7 ConFirmatim inteaaim a Dln insen4 into he host aenom e, and iAs ex prex iom in rans Fosmeo cels Developmen t ok RFLP (rgBaichan Proament leng poty imovphim maps DNA Plant Fo he fingorPntir vafieties , 'criminas, Uneapi voca identi Aca Hon ok Partentou uelalimhiPa et. S9 SiH hybidhkation Foi dekermiira the ocaAion o Specikic Sevence in Speei fie chono&orme. Pueparatin genome map okeukanyoteg,înctwclirq mon thuna Mlhod Y aene Tnans fere battorium meoliateo Aaao- (W Genome Pje4) 1 RK Y Channe ene rans fex Dîtetd Gene tronS ter Agro- Batlenm mecliahe Gene Transfer TntTRODuhon Agrto boltemum is a toomd PlontS though opPlar Plant a vertu Phytorouthog-entha infecks disease. s4 is sites causirg Cmown l Aransf matidm pol tos se Fox enskor gene o in krret sAne soil Hhogen hadss oHuze ens Agno baterion me fati Tv secne fion em s h e fans er o bacheria tyre 11 hostreil s 4s T DNA into the Cies 0 Aln Agto bautoaum Consists og diBherenSpe AheRr Aiseae Sympt mojo 2 ho a n g depend ioon Iucle, A 4umerfacionce Some Speties oagobatemom inc nc(od nhixogens e A.ub1, Aadio batho, Avit The 9nus The hest rangt okhe batemae r delerminea dbreren bachevia ag well ag piant Padrss. accemau Patos Incde viw lene vin) oene Cnd T-DAIA Oncogones, here a3 the plant faui inctude genes teapíne fox Hroms fos matios) and tumas Foimahm The natval diversity o he bad terda is defermine adeo the Prmegence ok the Pruimony Pathogenie detoruminant'he TilR Plomid The eente o dilherent pasmidg and he abiity h e ooanism do ran Ros a DNA Seqmen 4he ethe hos4 Celî Aarobatenum in pant usith u mes indotig pla mi Prvmay fattus ans fosmafionm behind hto h e genome he se o hao battenUm is a oram - negafive rod shaped bacenum naang in sixe b 1-5 3m in tenqth and 0 o.6 to l-6 Am in width .The baterium iS mofle with oned - chan T h e kasis o Flagollo Agnobattonm-medialed gene tansfox ts the abilito he o s n i ceil úentiy. O me mechanism Into the plan The tans for ils T-D int hre hos baue on the rars fer oa piez e ok flaJmid kyihe. oacheria nFUtin cel dun paimíd 4hen PrteqO Trate foto the nudear jenome inoroler fo ex pres 4s own genég ord afeut he hosmal balanie in he hot4 CeLl esi des, the battono ouo poodure a umbey o enxymeg thad ane nvolv in Ahe &yn-hesis o opine hat is hen sed by bncfeia a nut-oend he exentias CogmponenBAs olythe bettena 90me învotuedn infecton ane -DNA PTLe3ent on 4he wth oth e Plajmid calle Ti plamid al funcHonds Componenls tike Virtu len(e (vin) (onjvgalimCon) & Oriin o renilaim i ) . Rke yT Chamet ne r e i tooducea He PRnt sin Agobatonom3- The Ihe abituty d Hgrroba deu ver DNA 1nto Plam cells apen e abuJ nt baCHeium engineemy,culowing Neuo doGs the Pmcluctin o geneticaly mocified Joats Pents (rans qende flanBs). ans Pme involved in (rons mediakro tinayhevansdey oT-DNh wh HiTg e Coqni ye fho boide seaperhe Hhe T-DND negin herte fore i s Poysibie Foy SCiontHfs Ao Ure TDNG pondler to seguene Fosk or dlagined Se9 venve o gene o interest Con hen &usUcha Product Ube incertdinto a pogmid Ond intToelwre into Hgrobaemum ceul ent Thene, the bordes sepente will be rtecoqni xed b the oan fery aparaatus okA. tumefaciene ondte detiverted in a andarse momey inpthe lanqe Plant cei) s honeoversby leavirg behind cnly 4he b0bder Seuente of he T- DN A the rreu| rreu| titg tirq prod&ct wiy edi the Plan gnome iahouA Couasírg an Uness nPlan ts. /n?smemod ha been used tb modiftedy esevat Crnop Planas ntto din e, baa le and uheak PrOce duse r ptocol The Poutocol or Poceduse Por t n e Ybauewm-mediedhed aene ronyFen migh A eme 4 dces. The Followi Por hve 4hetre o xplank seteta trons e x aene the rTatoco AgnoboAGin meoticHod Fos n Case o on embryo h e n e dvons Fex in et o Cose an emby Stend ixotiamand gpramminadn o Seed Steruiized uHh Cz F nuate fur 2hoirs 2 a T h e feeol a r e houss ond the Cee are nen Soake RoOm Hem ina Petm di%. Ihe eed Coats r carue nenov twih FoTceps fvorn he Seed and aLe fotAhers with 51. aidonol Thejeteo then Steitizo TnSee wth 20ml o 2t Cadi Um hypo chLomte he steiie Zed &ge ane plaled On Peti Plateg con-onning e 3eri natiro medium and ae incu boHed o r ° c Fos 2doays. nOculum merabafion 3- Aml o Le mid.m Comaun apicin 4 kanamy ci n is inoculated h a Sirgte ayo bcen The cooR at 28e she incubate in 4h8holkery in(dbat h e h e agrebatteium Coltured Lolomin. Preporred U Centnfugo at q009 O The Supeynant isrelmaned are.cleane with a rereraon olthe Img Ms (iuid nenoved,and h e pellek Mediunn go Pan he sce s ane hertaren out oHhe gerumineim tulbe omedim CePat e n emply SBeile PeBoiPlat wi4th a AaIk o fiHe Pafer ThWen ttadide is nemavad ane Soeo Cut ema Ve lh o Coy ledon. By RKs Yi channes. The delauhad Cotyledom arre Colletted into ms liqvid melkm-Then his medium CarLa Poured into S liqpid meim Conini d aas ba cAeium) and Ahen Shaken it e Pven) The glax beakei is coveme to4ha en Cootasney and Seale ith im, and Put into the desiccatrs atatheo to 9enH'y the vactum PUmP n e x p lonts ane e ht on he medium fos S min. af te aruu inFiItodion. Shoile P1Hedparapedp A tos ses io paked on he Co- Culivation mediom a n d h e infetted x planas arne Ploeo On the RHe papes with fosceps. The odaja Lusdace,oh he =he Pe Cokyledon is t be Kert upuads. he pelru ptclo arne {hen Sele and hw baled Fos ada at 28°. îh he da nk &nooinhahom -The e plant S ane hen tron fesrd Sveofirg medioo Con}ainiaKanamytin and Canbeoicitio n arde to inhibid 4he gDuth o Ag baderim Ando)hey Cnnansfer to -Ande 1ndue v oLos makim. roorymeoim D aro ncubotnler Then he pe eveny plaje arne iaht ntubeA Fos 2-3 doys a aDu ok_Plant- AFeler AeaimatHzatim aeetm gnadualy Plan tHes a n e ConcltHa Once &hGat l/thesen envinom menlau nitHatfon ok e au în imoukize rood hadbi tveled in h e enu r m merntal Conoi n aken b e d Condl ithom Panis e e aRe hplants a n e Hnalty.the Plant that becau hey Caruuy Plant Poodute is knoun uoans qenic qene o inere)* vans Feeo to the plonis an rnans gene on 4his wo he gore ok in teres Using Aro battwm) r? Oired Gene Transrer CYm - The terun odiret 4rans Per o ene ir i tiretty ntoboluce ndo h e Plant aenomé Y DNA Is uhen he Rnforeign vovement og a biologtognt 1 e AgTobadenim o without Leod to Stabie fsans FUmathon).0 Direet DNA tronc Pen method tey on-the Delivern ob hake DNA in tu Hhe ptand ceus The spomtaneoos uptae ok OnNg by Plant ebce ig te 1ou. Thene fore , dilferent chermied cal & Physau fneaments ame employe *o faciitate Ahe entry ok DNIA e nto Plant cells. dinet gene teas fes can be bricaduy divided into fuwo qmops 4phutiecaut te qene tsonsPen bombondment Chemica Aene trons Fes ) maiateo, eledo povaion, micro injetim , Petue metho - Poly- ethytene giyeo1 (PE6)- DEAE-Cdiehyl amins etny dexrran - medieted a1tm Phosphaté Precdpia tion Physical leu ene trons frsa Method Rke yT ehannes poratim_- Th method invo in a Soitabie jnic Sotuti Concuniray Plasmic DNA e This mîxture Long po1 Se or high vol4age O " uspensian oh plant prona lenearu Xed tuecombinant is Ahen expojed to low- Voltage ehost pu \fey Fos4he desire numbes yes The e e i cal ane though do ihdwe Azansien + porel pulse Plasmoa lemna throuh uhich he DAWA mole cules are entere oo Pot and fncspovaAted înte nuceef au o te în 4he Hhe Proho plos The the Arreated Pro to plaJAg ae hen Cultreo Ho obtein Cen Coloniog and PloniS This Medhod e catte eleutoo ponoation ob DNA nt PLant teil 'by max eîng. minute parte inMe IntouduHton Plan0+ ce membrane Jelettos+ciny heneroai lo vol tage on puse Hran PumoNon, uhite high Vottege - Produre high Tato transiend Sho} p\se giws high oees S4able sans fos main, Eretko poadim a been ed to Pdue stubi 4rons Fumed cen tineg l e Plant in & Seveaa Plan Spetie oba(Yo, PoHunia, 'maize, Tice ,ulead elc. eMiCxo-neution methoe Tnto a Porn thgioL DNA isa d r e toesirabe involviry methaaicos înSention ols the Hangel 0 cell. The anqe eu may be one idenHjieo meris Hem e* ntau ces, mioioinjedion C s the o mico-în Jetton embryes, PTZOHo plag-A9, aulu5, Foy t h e trang fen ok Cellulas roanel mani Pulchon ok hroumo Sorme holqe of mioooînjechn învo veg 4he ran Pey okn D N g sotHn +hrm ugh a miupipetde OS Lo-0 Pm tothe Cyto plasm/ nucleus o a Pland Ce o pooto playt wih in A hoip ot mloro manipálatus ok a micbo injedto asemblf e t h e gene' rreuns Pex is Kept n done, the recipien } Ceu 9ane immobi xedin agasS e embeddlhoy and hedl loy SuCtion holdrga Pipee. t h e Poocen Ua midoD înjettiy campete, the rvenifarme Ces olturo and groun to cdeOAOP Thdo a Aoansgoni Plonhr Sueh Soceem Pol tran Posmatihon by miGovi njeti Prroto plasts hag beea ahievedin Brragsica Spf. e The ma imitehon o o cen to bacco micrav inettion ane tha i+ l s o pensi e , time Consumina ( maximum ok A-50 o oplas Con be mico»înjettd n one hour N ha to e be Perfromed buy rahed and &xill Pensbnb. Micooroehie Pombarndment pahhe Oombandment aun methed dr Biotís tis Ranankimaraho tdanneo partice bUmbzardmony is the mesB elfeutiue methed o fd gene drraasfe anc Creadiom o °sonsqenic vensaite olue the P(anisThis method is fat hed it can be SoC eXFuily OSeo Fos the DNA oosS Fers mamnalian CesMiso-onenism The miso projectiie bomandlment l meBhoe wog indially amec a3 biotistia u its inven to fasd (tui). Bio tis tics is a Combinion obioloi caSenfrSan an anisrC hTs method, 1-2 Am qold os tungslen partide Coohed wth DN a r e shoo n Hhe flana (-e) b USi a heuium PTLe UIe paruiUe qun device . iotistic dechniaueha been wed to povduce slale Jene chransfes coton , tnoúze TiOR , Soaghun, Soubean , sugrrcane Po plas ett , Papaya, halco uhead This eneoansfer mettod quite at¥saulie because DNA Canbe detived ntp cels o ghoo mrskms og Shoo 41sOmys uKch mak gene tsans fes icependen TeeneraHon ab?uty o he Speires 91s Chtek timialm sthe need ot Pos a panticte m By Ranon kumun Sahos Y Cos{ly device ehanne e)ChenicakeneJaPe methoe e n gohoPE )meoliated ranE Fer DNIA moteCule con be forced to enteo înto he host genome mbj m hose cels which do not n dhis methoo 4he nav poge Celual Plant Poppiasls are mixed eih mole cule DNA Containin the freign gene. The hoo Plamid mixedna rans fo mation meel{Oum ihin ma2t ong in place olk Ca21 ons Poltowiry whih ao Polyethy tene ayot (Peo) Solutio tnearizel arne i adde Ate theU Aoeat ment teduteand ca we PEG Cmt emsatian Con centoation is enhanced 94 Promoteg he re onety atoangformadhon. The chemica melhed lo volviny roan& foomatin n presente opo lyetny) Ene ycol is Convenient aneld Simpe but therte arte, .hoùeveY, SomeAIsacluantoges Meny cells ate So snti tive that 4he chemicau method eom can (4 be apr0tieahoreas Some Cew die duig e e a t mdnt. This method S no Pen fect beause many fneoBed ceu donof Concin any droansfers DNA Some Ai meg he foseign DNA i degsaded n h e yto pasm bekove teadhirA Ahe nuLeus. Some ahe Chemicoos Rike. Cacim phoshode., Poly Viny](oho| efe areen hanue he utave a DN Utee 1 9ene t zun S fer in Plan ecin "Rhe yr Channe ansgenics i s J r a s gene naturall o bpant Poaplayt Ondare AAranigene isa gene -had ho been trran ferred by any o a number o Cme cngaism to ono tho n ondON to enei Chamge nin eerirg techniqed Fono h e phenotyfe st oraanifm ar Pde Povterg in organlbm. The pmcen o mtoduction oa panigene 1s Known a3 ron{ge n ens This hon naive Seqmen t o Drp (cs tans gene) ne }ein t h abilittD poodlure RNA ds foojein înthe ansqonic Osgoniy or al Hex to runal, Trransgenic FunÄin Os o e sonsgenic, crgonibro doansaonic Goonism tefeng hb COndean enetic rademca into uhih ym an Onneladed orqanikm heg been ait gene Ht Code. canis muhfun Dn Ten gene) simp1-the oo nin uhich Canynonsaehe Ransqenic, OrqorKism o toansgenic 1s catlesa Seedion anaoni & Tronsaenics con be seleted Aoom non troanigeni mauy be ) SeLecAabie maxkes gene manker qenes cdhich 2 epesteUs or sase gene Seleehrg Ou roansqonic ces is dlone h Co- sanj Fermir he ceK wi ans10ene hus Chn odoltHooau2ene CottemMADkeg 0gene