Genetic transformation of cry1EC gene into cotton (Gossypium hirsutum L.) for resistance against Spodoptera litura

Abstract

Cotton is the chief fibre crop of global importance. It plays a significant role in the national economy. Cotton crop is vulnerable to a number of insect species, especially to the larvae of lepidopteron pests. 60% insecticides sprayed on cotton are meant to control the damage caused by bollworm complex. Transgenic technology has become a popular option for the development of bollworm resistant cotton. Spodoptera litura is one of the notorious emerging pests in cotton. Introduction of cry1EC gene into commercial variety have very significant importance. This research work was carried out to transform chimeric δ-endotoxin Cry1EC into cotton. The tobacco cutworm (Spodoptera litura) is a polyphagous foliage insect which is susceptible to the chimeric δ-endotoxin Cry1EC. Six month-old highly friable embryogenic calli derived from cotyledonary explants of Coker 310 were used for transformation using Agrobacterium tumefaciens strain LBA4404 harboring plasmid pBI101.1 carrying the marker gene neomycin phosphotransferase II (npt II) and a synthetic cry 1EC gene under a constitutive 35 S promoter. Agrobacterium treated calli were selected on MS medium containing, 50 mg/L kanamycin, 500 mg/L cefotaxime, 30 g/L maltose and 0.4% phytagel. Embryos developed on kanamycin resistant calli were maintained on the same medium till somatic embryos matured. The cotyledonary stage embryos (3 to 5 mm size) were germinated on MS basal slat with 0.1 mg/L GA3 + 1.0 mg/L IAA, 30 g/L sucrose and solidified with 0.4% phytagel. The regenerated putative transgenic plants were hardened and transferred to the transgenic green house. Transgenic plants were confirmed by polymerase chain reaction (PCR) amplification of 800 bp npt II fragment, and 578 bp amplification of cry1Ec gene. Transgenic plant with single copy insertion of cry1EC was selected in T₀ by southern blot hybridization. Insect bioassay using Spodoptera litura larvae of first instar stages on T₀ plants showed 70% mortality. Not much data has been published on the toxicity of the endotoxins to S. litura, which is a common pest in warm and humid climates. Efforts are aimed at recovering more efficient transgenic plants through efficient transformation system and developing high resistant transgenic cotton against S. litura and will paved a way for promising future in cotton production.

Keywords: Agrobacterium transformation, cry 1EC, molecular analysis, insect bioassay

African Journal of Biotechnology Vol. 12(15), pp. 1820-1827