Indian Society of Genetics & Plant Breeding

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Reconstruction and validation of three different binary vectors suitable for generation of genetically engineered Helicoverpa protected crops

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Availability of a suitable plant transformation binary vector
is necessary for the generation of transgenic crops with an
adequate expression of transgenic proteins. Therefore,
three binary vectors were constructed viz., pBK204, pBK205,
and pBK206 harboring either a truncated or a full-length
version of a Cry1Ac gene for the generation of Helicoverpa
protected crops. Two different promoters viz., Arabidopsis
Rubisco small subunit (AtSSU) gene promoter or CaMV35S
promoters were used to regulate the various versions of
Cry1Ac gene. The binary vectors were reconstructed either
by the Gibson assembly method and others by ligating the
restriction enzyme digested fragments. The reconstructed
binary vectors were mobilized into Agrobacterium strain
AGL1 and validated by Agrobacterium infiltration assays
of Nicotiana benthamiana. The amount of Cry1Ac protein
accumulated in the Agroinfiltrated tobacco leaves was
quantified using the quantitative ELISA assay. The
expression of the Cry1Ac protein in the tobacco leaves
ranged from 0.25 to 0.26 µg /g fresh weight (FW) when
transformed with these three constructs. Thus, the vectors
constructed in this study appeared to be suitable for
generation of Helicoverpa resistant transgenic crops by
Agrobacterium-mediated genetic transformation method.
 

Keywords: Binary vector, Cry1Ac, agroinfiltration, transformation

Info

Year: 2019
Volume: 79
Issue: 1
Article DOI: https://doi.org/10.31742/IJGPB.79.1.15
Print ISSN: 0019-5200
Online ISSN: 0975-6906

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