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Current Trends in Biotechnology and PharmacyVol. 5 (2) 1149-1156 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)may over come two limitations of currenttransgenic technology i.e., unpredictabletransgene expression and effect of the mutationinduced by transgene integration. Selection ofmultiple copy gene as targeted site for HR basedintegration may result in multicopy transgenics.However, this is a rare possibility, when chancesof getting one HR event is in an order of 10 -4chances of getting two integration events in asingle cell will be in an order of 10 -8 .Identification of gene targeted eventsLow frequency of homologousrecombination in comparison to random T-DNAintegration is a major limitation to find a rare genetargetedtransgenic event. Earlier Terada et al.,has used a positive-negative selection foridentification of six gene targeting events in rice(30). However, use of negative selection such asdiphtheria toxin (DT) has a disadvantage becausethe presence of even single toxin moleculeproduced by transient expression is sufficient tokill a cell. Losing any cell having gene integrationby HR, because of transiently expressed DT willbe counterproductive when GT frequency is inan order of 10 -4 . To overcome this problem anegative screening strategy in combination with1153a positive selection marker nptII was employedin the present study. The T-DNA carries GUSgene expression cassette, which is placed out ofF3F gene sequence used for homologousrecombination (Fig. 1). Thus the randomintegration transgenic events will have a functionalGUS expression cassette while transgenicdeveloped by homologous recombination will bedevoid of GUS gene (Fig. 1, 3). Randomintegration events were screened out by GUSassay at an early stage of plant regeneration.However, with this approach non-transgenic andpartial T-DNA integration (without GUS) eventsalso show GUS-negative results. Hence, putativetransgenic plants need further molecular analysis.This limitation was overcome by employing a PCRscreening method earlier developed in ourlaboratory (29) that overcomes the limitation offalse positive amplification arising fromcontaminating Agrobacterium. Combination ofnegative screening with modified PCR protocolgreatly simplified the identification of rare HRevents. Use of negative screening methodologymay prove useful in plants that have long callusphase during regeneration. Random integrationevents can be screened out during early callusphase.Fig. 3 GUS assay of leaf from putative transgenic brinjal plants. A. Transgenic plants developed throughrandom T-DNA integration (GUS positive), B. Transgenic plants developed through targeted gene integration(GUS negative).Bt gene targeting in Brinjal
Current Trends in Biotechnology and PharmacyVol. 5 (2) 1149-1156 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1154Fig. 4: PCR amplification of cry1F, nptII and GUS gene for confirmation of putative transgenic brinjal forhomologous recombination mediated gene integration. M. Marker 1 kb DNA ladder, Lane 1, 3, 4, 8 and 10putative transgenic brinjal obtained through random integration, lane 6 and 9 putative GT events and lane 2, 5and 7 escapes.At the first step of screening, out of 956kanamycin resistant shoots obtained throughAgrobacterium-mediated genetic transformation,189 were found GUS negative. The GUS positiveshoots were eliminated from the study and GUSnegative shoots were further analyzed for HRbased gene targeting events. At the second stepof screening, integration of nptII and cry1Fwithout GUS gene was confirmed by PCR (Fig.3). Out of the 189 shoots screened by PCR, 75shoots showed integration of all the three genes(possibly low expressing lines) while 112 shootsshowed integration of none or only nptII (possibleescapes or truncated T-DNA integration). Twotransgenic lines were found positive for nptII andcry1F integration but without GUS gene (putativeGT events) (Fig. 4).HR based gene targeting in plants has beenused only to demonstrate either induction ofmutation or replacement of endogenous DNAfragment (23). Development of GT rice cell linestolerant to the acetolactate synthase (ALS)inhibiting herbicide bipyribac (BS) have beenreported (31). Terada et al. (2007) Improved GTprocedure to obtain nine independent transformedcalli in rice having Alcohol dehydrogenase2 (Adh2)gene modified with the frequency of 2% persurviving callus. Shukla et al. (2009) used ZFNsto modify endogenous loci in maize. Insertionaldisruption of one target locus, IPK1, resulted inboth herbicide tolerance and the expectedalteration of the inositol phosphate profile indeveloping seeds. Herbicide-resistance mutationswere introduced into SuR loci by ZFN-mediatedgene targeting at frequencies exceeding 2% oftransformed cells for mutations as far as1.3 kilobases from the ZFN cleavage site. Morethan 40% of recombinant plants had modificationsin multiple SuR alleles (20, 21)In this report, an important vegetable crophas been transformed by targeted gene integrationand the heterologous gene introduced (syntheticcry1F) to provides protection against its majorinsect pest BFSB (L. orbonalis). The presentstudy provides initial leads in the developmentDipty Shrivastava et al
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(December 7-9, 2011)Venue: Karunya
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