Target-specific gene delivery in plant systems and their expression: Insights into recent developments

ABSTRACT

High-yielding Indian cotton varieties are not amenable for regeneration and transformation because they arerecalcitrant in nature. In this work, we have developed Narasimha (NA1325) cotton variety by introducing threeCrygenes driven by three different promoters conferring insect resistance. The meristematic region of embryo axisexplants were infected and co-cultivated with Agrobacterium tumefacience (LBA4404) harbouring pMDC100vector with three Cry gene cassettes (a-globulin Cry2Ab, DECaMV35s Cry1F and nodulin Cry1Ac) with Npt IIas a selectable marker gene. Out of 1010 embryo axes explants infected, 121 (T0) regenerated under two rounds ofkanamycin selection medium. About 2551 T1 seeds were collected from 111 T0 plants and these seeds screened againwith kanamycin at seedling stage. The transgenic plants were characterized by PCR, real time quantitative PCR,lateral flow strip protein assay and insect bioassay. Out of 145 kanamycin resistant plants (T1), twelve showedamplification of all four transgenes Npt II, Cry2Ab, Cry1F and Cry1Ac through PCR with expected amplicons as395, 870, 840 and 618 bp, respectively. Further, lateral flow strip test revealed Cry1F and Cry1Ac proteinsaccumulated in 12 plants, whereas Cry2Ab protein was detected in eight only. The transcripts of all three Cry geneswere accumulated significantly higher in transgenic plants at T2 generation. The transgenic lines showed effectiveresistance against Helicoverpa armigera and Spodoptera litura larvae. The T2 line L-3 exhibited highest percentageof insect mortality, in which transcripts of all cry genes were accumulated higher than other plants. The transgeniccotton plants carrying triple Cry genes could be an excellent germplasm resource for the breeders for introgressions.