Production of transgenic indica rice resistant to yellow stem borer and sap-sucking insects, using super-binary vectors of Agrobacterium tumefaciens

Abstract

Indica rice ( Oryza sativa L) varieties are known to be recalcitrant to Agrobacterium-mediated genetic transformation. We have used the pSB111 super-binary vectors containing Bacillus thuringiensis δ-endotoxin synthetic cry1Ab and cry1Ac genes driven by maize ubiquitin promoter, and snowdrop lectin gene gna driven by rice sucrose synthase promoter along with the herbicide resistance gene bar driven by cauliflower mosaic virus 35S promoter, to transform various indica rice lines susceptible to major insect pests, viz. yellow stem borer and three sap-sucking insects. The present transformation protocol has been optimized to enhance the frequency of T-DNA transfer into the rice callus cells during co-cultivation with Agrobacterium. Transformation studies with the gusA containing pTOK233 vector revealed substantial increase (61.54–133.33%) in the number of calli showing transient GUS expression when the calli were treated with 100 mM acetosyringone before co-cultivation. After co-cultivation with Agrobacterium, embryogenic calli were selected on the medium containing phosphinothricin. Southern blot analyses of primary transformants revealed the stable integration of bar, gna and cry coding sequences into rice genome with a predominant single copy integration and without any rearrangement of T-DNA. Northern blot analyses revealed the expression of cry and gna genes in different transformants. Molecular analyses of T 1 transgenics showed a monogenic (3:1) pattern of transgene segregation. Furthermore, co-segregation of bar– cry and bar– gna in T 1 lines confirms that bar– cry and bar– gna are integrated at single sites in the rice genome. Transgenic lines expressing cry and gna exhibited substantial resistance against yellow stem borer as well as three major sap-sucking insects of rice. This is the first report dealing with the successful introduction of three exotic resistant genes into diverse indica rice lines using pSB111 super-binary vectors of Agrobacterium tumefaciens.