Agrobacterium -mediated transformation of cotton (Gossypium hirsutum) using a heterologous bean chitinase gene

Abstract

Cotton (Gossypium hirsutum L., var. Coker 312) hypocotyl explants were transformed with three strains of Agrobacterium tumefaciens, LBA4404, EHA101 and C58, each harboring the recombinant binary vector pBI121 containing the chi gene insert and neomycin phosphotransferase (nptII) gene, as selectable marker. Inoculated tissue sections were placed onto cotton co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg l )1 kanamycin and 200 mg l )1 cepotaxime. Putative calli were subsequently regenerated into cotton plantlets expressing both the kanamycin resistance gene and b-glucuronidase (gus) as a reporter gene. Polymerase chain reaction was used to confirm the integration of chi and nptII transgenes in the T 1 plants genome. Integration of chi gene into the genome of putative transgenic was further confirmed by Southern blot analysis. 'Western' immunoblot analysis of leaves isolated from T 0 transformants and progeny plants (T 1 ) revealed the presence of an immunoreactive band with MW of approximately 31 kDa in transgenic cotton lines using anti-chitinase-I polyclonal anti-serum. Untransformed control and one transgenic line did not show such an immunoreactive band. Chitinase specific activity in leaf tissues of transgenic lines was several folds greater than that of untransformed cotton. Crude leaf extracts from transgenic lines showed in vitro inhibitory activity against Verticillium dahliae. Transgenic plants currently growing in a greenhouse and will be bioassayed for improved resistance against V. dahlia the causal against of verticilliosis in cotton.

Transgenic Indian Cotton (Gossypium hirsutum) Harboring Rice Chitinase Gene (Chi II) Confers Resistance to Two Fungal Pathogens

“…These results represent a convincing confirmation of Agrobacterium-mediated transformation of cotton and show the potential of this technique for genetic engineering of Indian cotton. The frequencies of transformation obtained in this study seem to be equal to other systems of transformation (protoplast and biolistic) used to obtain transgenic cotton, although genotypic differences must be considered [9] . Selection markers mediated inheritance studies on T 1 progeny showed that the chitinase gene is transmitted to the T 1 progeny, demonstrating stable incorporation of T-DNA into the cotton nuclear DNA and the 3:1 segregation ratio suggested that the chitinase gene was integrated at a single locus.…”

“…Previously, three reports were published on transgenic cotton with chitinase gene. Those studies, they used tobacco chitinase [36] : Fungal chitinase of Trichoderma virens [37] and bean chitinase [9] to control the fungal pathogens Verticillium dahliae, Rhizoctonia solani, Alternaria macrospora and A. alternate respectively [38] . These reports are relevant to American and Turkey cotton varieties due to genotype dependent responses.…”

“…Various protocols have been explored for the transformation of cotton such as meristem transformation [6] , particle bombardment [7] and Agrobacterium-mediated transformation [8] . Of these methods, only the Agrobacterium-mediated and microprojectile bombardment methods are routinely used in cotton transformation studies [9] . Agrobacteriummediated transformation via somatic embryogenesis has been the most common method for transgenic cotton development.…”

Factors influencing high callusing proliferation in cotton (Gossypium hirsutum L.)

“…Callus maturation was achieved on MS basal medium in duration of 2 months. Most of the published works had also reported MS based medium containing 2,4-D and kinetin as the best for callus induction Goodin, 1987 and1988a, b;Kumria et al, 2003;Choudhary et al, 2003;Haq and Zafar, 2004;Tohidfar et al, 2005;Zhao et al, 2006).…”

Clonal variation in Scots pine (Pinus sylvestris) and in transgenic silver birch (Betula pendula)

“…Transferred chitinase genes originate either from plants (Lin et al 1995, Kishimoto et al 2004, Chye et al 2005, Takashaki et al 2005, Tohidfar et al 2005, Vellicce et al 2006, Xiao et al 2007, He et al 2008, fungi (Terakawa et al 1997, Mora and Earle 2001, Kumar et al 2009, Kern et al 2010, Prasad et al 2012 or viruses (Corrado et al 2008). …”