Muhammad TARIQ, Tayyab HUSNAIN, Idrees Ahmad NASIR, Bushra TABASSUM, Nida TOUFIQ, Muhammad Umar BHATTI, Saman RIAZ, Anwar KHAN

Antifungal activity of chitinase II against Colletotrichum falcatum Went. causing red rot disease in transgenic sugarcane

We evaluated transgenic lines of sugarcane modified with the barley chitinase class-II gene to create resistance against the redrot causative agent Colletotrichum falcatum Went. Local sugarcane cultivar SP93 was transformed with a 690-bp coding sequence of thechitinase-II gene under the influence of a polyubiquitin promoter. Transgenic sugarcane lines ($T_0$) overexpressing the chitinase genewere obtained through a particle bombardment method with 13.3% transformation efficiency. Four transgenic sugarcane lines, SCT–03,SCT–05, SCT–15, and SCT–20, were tested for resistance against red rot by in vitro antifungal assays. Crude protein extracts fromtransgenic sugarcane plants SCT–03, SCT–05, SCT–15, and SCT–20 inhibited the mycelial growth of C. falcatum by 49%, 40%, 56%,and 52%, respectively, in a quantitative in vitro assay. Our findings revealed that two transgenic lines, SCT–15 and SCT–20, exhibitedthe highest endochitinase activity of 0.72 and 0.58 U/mL, respectively. Furthermore, transgenic lines SCT–15 and SCT–20 exhibitedstrong resistance against inoculated C. falcatum in an in vitro bioassay, as they remained healthy and green in comparison with thecontrol sugarcane plants, which turned yellow and eventually died 3 weeks after infection. The mRNA expression of the transgene inthe C. falcatum-inoculated transgenic sugarcane lines increased gradually compared to the control plant. The mRNA expression was thehighest at 72 h in both transgenic lines and remained almost stable in the subsequent hours.

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