Molecular Characterization of Transgenic Tomato (Lycopersicon esculentum Mill.) for fruit borer [Helicoverpa armigera Hübner] resistance
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Date
2015
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
CCSHAU
Abstract
Tomato (Lycopersicon esculentum M.) is the most imperative vegetable crop cultivated across
the globe. In India, the tomato productivity is stumpy as one of the major yield constraint is the tomato
fruit borer (Helicoverpa armigera Hübner). At present there is no source for genetic resistance in
tomato germplasm against this pest. The conventional methods for management of this particular pest
are often futile. Therefore, Bt transgenic technology provides a secure and consistent means for
management of this pest. Bt transgenic tomato cv. Hisar Arun carrying cry1Ac gene was developed
through Agrobacterium- mediated transformation for providing resistance against fruit borer in our
laboratory. In the present study, the developed transgenics were evaluated in three generations for
transgene integration, expression and bioefficacy against Helicoverpa armigera. Transgenic plants
carrying cry1Ac gene were analyzed by PCR for both the cry1Ac and nptII gene (selectable marker).
Out of 96 T1 plants, 68 plants were found positive for cry1Ac and nptII gene confirming the presence of
transgene in transgenic tomato plants. Similarly, in T2
and T
3
generations, screening of transgenic
plants showed amplification of nptII and cry1Ac gene with amplification product of 1050 bp and 533
bp, respectively. Semi-quantitative RT-PCR analysis also revealed transcripts of 533 bp for cry1Ac and
1050 bp for nptII verifying the expression of respective genes in the transgenic plants in T
1
and T
2
generations. The study proved the stability of introduced gene in all three generations. Further,
quantitative assessment of Cry protein using enzyme-linked immunosorbent assay (ELISA) proved the
expression of Cry protein with the average value 357.5 ng/g, 231.5 ng/g and 216.9 ng/g FW in leaves
of 60 days old transgenic plants in T
1
, T2
and T
3
generations respectively. The efficacy of transgenic
tomato plants expressing cry1Ac gene against the target pest was required to judge the performance of
these plants. Therefore, insect bioassay was performed with in vitro reared freshly hatched second
instar larvae of Helicoverpa armigera. Transgenic plants showed resistance to Helicoverpa armigera
showing significant mortality of H. armigera after 72 hours in all the three generations. Few promising
tomato plants were selected on the basis of expression and bioefficacy analysis, which could be further
used for developing Helicoverpa armigera resistant tomato lines/cultivars.
Description
Keywords
Tomato, Helicoverpa armigera, Agrobacterium, ELISA.