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

Abstract

Problem statement: The present investigation described a simple and reproducible protocol for transgenic cotton regeneration and characterization of chitinase (Chi II) gene expression against two different fungal pathogens in cotton. Approach: Transgenic cotton (Gossypium hirsutum cv. SVPR2) plants were produced by pCambia-bar-Chi II (13.8 kb) under the control of the CaMV 35S promoter, harbored in the strain LBA 4404 Agrobacterium tumefaciens by using shoot tip explants. Results: Finally, from the 10 experiments, 21.8% of transformation frequency was recorded. Segregation ratio of 3:1 was recorded in the T 0 plant seeds. Polymerase chain reaction and southern blotting analysis were used to confirm the integration of Chi II transgene in the T 0 plants genome of putative transgenics. Quantitiave and qualitative (SDS-PAGE) analyses were also carried out to confirm the expression of chitinase enzyme in T 0 plants. Further, randomly selected transgenic plants (T 0 ) were analyzed for disease tolerance by evaluating them with spores of Fusarium oxysporum and Alternaria macrospora. All the selected PCR positive plants showed enhanced disease resistance against Fusarium wilt. The plants selected randomly showed an enhanced survival rate compared with the control when they were grown in earthen pots inoculated with 1×10 5 spores 100 −1 g of soil mixture.Another four randomly selected plantlets were sprayed with spores of Alternaria macrospora in order to test their tolerance to Alternaria leaf spot disease. After 20 days of culture, the number of lesions per leaf and the lesion length per leaf spot in non-transferred leaves increased. In the case of transgenic plantlets, lesion formation was completely absent. Conclusion: The disease resistance against Fusarium wilt and Alternaria leaf spot in cotton strains would serve as good breeding materials for producing fungal disease resistant cotton varieties.

IN VITRO REGENERATION AND TRANSFORMATION OF APPLE (Malus domestica Borkh.) ROOTSTOCK MALLING 7 USING RICE CHITINASE GENE

“…Among these, it is one of the diagnostic tool for transformed cells detection. Likewise, in the present investigations the presence of rice chitinase gene (chiII) in transformed plants was confirmed by amplifying 584 bp and 237 bp size fragments through PCR as previously reported by Ganeshan et al, 2009;Sharma et al, 2012 who also used the same construct in case of cotton and apple rootstock 'MM106' respectively.…”

Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to Fusarium oxysporum f. sp. lycopersici

“…In a very first demonstration, the expression of bean chitinase in transgenic tobacco confers protection against Rhizoctonia solani (Brogue et al 1991). Furthermore, in a recent study, Shin et al (2008) has demonstrated resistance to Fusarium graminearum in transgenic wheat that expresses barley chitinase II, while the protection against two fungal pathogens, Fusarium oxysporum and Acarosporina microspora, was observed in transgenic Indian cotton that expresses the rice chitinases ChiII gene (Ganesan et al 2009). However, there are only a few reports on the successful engineering of tomato resistance against Fusarium wilt using transgenic methods.…”

Molecular cloning and expression of drought-induced protein 3 (DIP3) encoding a class III chitinase in upland rice

“…Different structure and roles of plant chitinases had been found during plant evolution. Till now, several genes encoding chitinases have been cloned from many plants (Fortunato et al, 2007;Baranski et al, 2008;Ganesan et al, 2009;Liu et al, 2010;Taira et al, 2011). Several plant chitinases are constitutive, developmentally regulated and tissue-or organ-specific.…”