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Molecular Plant Breeding 2013, Vol.5, No.5, 24
-
28
http://mpb.biopublisher.ca
24
Research Report Open Access
Development of Insect Resistant Transgenic Lines Mediated by
Agrobacterium
in
Cotton (
Gossypium hirsutum
L.)
N.Kumari Vinodhana , N.Meenakshi Ganesan , S.Rajeswari
Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India
Corresponding authors email: soundhini@yahoo.co.in
Authors
Molecular Plant Breeding, 2014, Vol.5, No.5 doi: 10.5376/mpb.2014.05.0005
Received: 21 Apr., 2014
Accepted: 10 May, 2014
Published: 28 Mar., 2014
Copyright
©2014 Vinodhana et al. This is an open access article published under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Vinodhana et al., Development of Insect Resistant Transgenic Lines Mediated by
Agrobacterium
in Cotton (
Gossypium hirsutum
L.), Molecular Plant Breeding,
2014, Vol.5, No. 5 24
-
28 (doi: 10.5376/mpb.2014.05.0005)
Abstract
In order to produce transgenic cotton resistant to insects, five months old embryogenic calli of Coker 310 were
transformed with
Agrobacterium tumefaciens
strain LBA4404 harbouring the plasmid pBI 121 containing cry2Ab gene under the control of
CaMv 35S promoter.
Neomycin phosphotransferase
(nptII) gene was used as a selectable marker. Infected embryogenic calli were
incubated in dark at 24°C in co-cultivation medium containing 100
M acetosyringone for three days. Transformed calli were
selected on selection medium containing 25 mg/L kanamycin and 500 mg/L cefotaxime. After four rounds of selection (each round
with 15 days duration) the induction of kanamycin resistant embryogenic calli was at an average frequency of 37.0% and further two
subcultures (each with one month duration) of the kanamycin resistant embryogenic calli in the same media resulted in the formation
of somatic embryos at an average frequency of 23.7%. The matured cotyledonary stage embryos were germinated on the plantlet
development medium without kanamycin selection. Out of 500 explants co-cultivated, 5 plants (1.0% transformation efficiency)
showed the presence of genes nptII and cry2Ab through PCR analysis. Insect bioassay against
Helicoverpa armigera
performed with
the five T
0
plants showed 33.0% to 67.0% larval mortality.
Keywords
Embryogenic calli; Transformation; Co-cultivation;
Agrobacterium tumefaciens
; Insect bioassay
Background
Cotton has attracted much interest in the field of
genetic engineering and gene transfer for the development
of transgenic plants with economically important new
traits. The main goal of gene transfer in cotton is the
development of bollworm resistant varieties especially
with
Bt
crystal toxin producing (cry) genes.
Considerable progress so far made in developing
transgenic cotton for bollworm resistance utilized the
Bt
genes cry1Ab and cry1Ac (Perlak et al., 1990),
cry1Ac (Benedict et al., 1996), cry1Ac and cry2Ab
(Stewart and Knighten, 2000), cry1Ac (Kategari et al.,
2007) and cry1Ec (Kumar et al., 2009). The end goal
of
Bt
technology is to obtain a durable protection,
which requires in particular the stability of gene
expression during the course of selfing or back crosses
and also requires reducing the probability of
appearance of resistant insects. However, the potential
of insects to evolve resistance against
Bt
toxins is a
serious threat to this technology. To combat from this
problem, cry2A genes are considered as promising
candidates for management of insects in crop plants
owing to their difference in structure and insecticidal
mechanism (Jain et al., 2006).
Of the gene delivery systems,
Agrobacterium
mediated transformation via somatic embryogenesis
remains the method of choice for the development of
transgenic cotton because of single cell origin of the
somatic embryos thus reducing the chimeric
transformation events (Khan et al., 2010). However,
the recalcitrance of elite cotton genotypes to
regeneration through somatic embryogenesis hinders
the use of gene transfer technology to improve this
crop. As only Coker varieties were found to respond
better for gene transfer, most of the desirable genes
are introduced initially into Coker and backcrossed
into other varieties later. The objective of the study
was to transform the
Gossypium hirsutum
cultivar
Coker 310 with cry2Ab gene using the
Agrobacterium
system for enhancing resistance to Lepidopteran insects.
1 Results and Discussion
Agrobacterium
-mediated transformation was carried
out using
cry2Ab gene
in
five months old
embryogenic calli, since embryogenic calli provide
a large population of embryogenic competent cells