Development of Insect Resistant Transgenic Lines Mediated by Agrobacterium in Cotton (Gossypium hirsutum L.)
N. Kumari Vinodhana
N. Meenakshi Ganesan
Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India
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 May, 2014
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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)
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 100mM 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 T0 plants showed 33.0% to 67.0% larval mortality.
Embryogenic calli; Transformation; Co-cultivation; Agrobacterium tumefaciens; Insect bioassay