full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (2) 1149-1156 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1154Fig. 4: PCR amplification <strong>of</strong> cry1F, nptII <strong>and</strong> GUS gene for confirmation <strong>of</strong> putative transgenic brinjal forhomologous recombination mediated gene integration. M. Marker 1 kb DNA ladder, Lane 1, 3, 4, 8 <strong>and</strong> 10putative transgenic brinjal obtained through r<strong>and</strong>om integration, lane 6 <strong>and</strong> 9 putative GT events <strong>and</strong> lane 2, 5<strong>and</strong> 7 escapes.At the first step <strong>of</strong> screening, out <strong>of</strong> 956kanamycin resistant shoots obtained throughAgrobacterium-mediated genetic transformation,189 were found GUS negative. The GUS positiveshoots were eliminated from the study <strong>and</strong> GUSnegative shoots were further analyzed for HRbased gene targeting events. At the second step<strong>of</strong> screening, integration <strong>of</strong> nptII <strong>and</strong> cry1Fwithout GUS gene was confirmed by PCR (Fig.3). Out <strong>of</strong> the 189 shoots screened by PCR, 75shoots showed integration <strong>of</strong> all the three genes(possibly low expressing lines) while 112 shootsshowed integration <strong>of</strong> none or only nptII (possibleescapes or truncated T-DNA integration). Twotransgenic lines were found positive for nptII <strong>and</strong>cry1F integration but without GUS gene (putativeGT events) (Fig. 4).HR based gene targeting in plants has beenused only to demonstrate either induction <strong>of</strong>mutation or replacement <strong>of</strong> endogenous DNAfragment (23). Development <strong>of</strong> 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 <strong>of</strong> 2% persurviving callus. Shukla et al. (2009) used ZFNsto modify endogenous loci in maize. Insertionaldisruption <strong>of</strong> one target locus, IPK1, resulted inboth herbicide tolerance <strong>and</strong> the expectedalteration <strong>of</strong> the inositol phosphate pr<strong>of</strong>ile indeveloping seeds. Herbicide-resistance mutationswere introduced into SuR loci by ZFN-mediatedgene targeting at frequencies exceeding 2% <strong>of</strong>transformed cells for mutations as far as1.3 kilobases from the ZFN cleavage site. Morethan 40% <strong>of</strong> recombinant plants had modificationsin multiple SuR alleles (20, 21)In this report, an important vegetable crophas been transformed by targeted gene integration<strong>and</strong> 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