Characterisation of synthetically developed cry1Ab gene in transgenic tobacco chloroplasts

Abstract

Synthetic cry1Ab gene was developed with optimised codons and expressed in the tobacco plastome. Characterisation of the synthetic cry1Ab gene was carried out on homoplasmic transgenic shoots. To purify the homoplasmic shoots, the proliferating heteroplasmic shoots were subjected to various rounds of selection and regeneration on spectinomycin-containing regeneration medium of plant medium. The PCR-screened homoplasmic shoots were placed on MS medium for root induction and proliferation. The functionally expressed CRY protein was detected using ImmunoStrips, and the amount of toxin protein in transplastomic tobacco plants was measured by DAS-ELISA, specific for cry1Ab gene product. The accumulation of Bacillus thuringiensis (Bt) toxin protein in transgenic chloroplasts confirmed that the synthetic gene could be expressed in other crop plants using constitutive and tissue specific regulatory sequences.