Abstract
BackgroundRice (Oryza sativa) productivity is adversely impacted by numerous biotic and abiotic factors. An approximate 52% of the global production of rice is lost annually owing to the damage caused by biotic factors, of which ~21% is attributed to the attack of insect pests. In this paper we report the isolation, cloning and characterization of Allium sativum leaf agglutinin (asal) gene, and its expression in elite indica rice cultivars using Agrobacterium-mediated genetic transformation method. The stable transgenic lines, expressing ASAL, showed explicit resistance against major sap-sucking pests.ResultsAllium sativum leaf lectin gene (asal), coding for mannose binding homodimeric protein (ASAL) from garlic plants, has been isolated and introduced into elite indica rice cultivars susceptible to sap-sucking insects, viz., brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper (WBPH). Embryogenic calli of rice were co-cultivated with Agrobacterium harbouring pSB111 super-binary vector comprising garlic lectin gene asal along with the herbicide resistance gene bar, both under the control of CaMV35S promoter. PCR and Southern blot analyses confirmed stable integration of transgenes into the genomes of rice plants. Northern and western blot analyses revealed expression of ASAL in different transgenic rice lines. In primary transformants, the level of ASAL protein, as estimated by enzyme-linked immunosorbent assay, varied between 0.74% and 1.45% of the total soluble proteins. In planta insect bioassays on transgenic rice lines revealed potent entomotoxic effects of ASAL on BPH, GLH and WBPH insects, as evidenced by significant decreases in the survival, development and fecundity of the insects.ConclusionIn planta insect bioassays were carried out on asal transgenic rice lines employing standard screening techniques followed in conventional breeding for selection of insect resistant plants. The ASAL expressing rice plants, bestowed with high entomotoxic effects, imparted appreciable resistance against three major sap-sucking insects. Our results amply demonstrate that transgenic indica rice harbouring asal exhibit surpassing resistance against BPH, GLH and WBPH insects. The prototypic asal transgenic rice lines appear promising for direct commercial cultivation besides serving as a potential genetic resource in recombination breeding.
Highlights
Rice (Oryza sativa) productivity is adversely impacted by numerous biotic and abiotic factors
Isolation of A. sativum agglutinin gene and construction of pSB111-bar-asal plant expression vector Coding sequence of the asal was isolated from garlic plants through the synthesis of cDNA followed by PCR using gene specific primers
The recombinant clone was introduced into Agrobacterium strain LBA4404 by triparental mating and the resultant super-binary vector was designated as pSB111-bar-asal (Fig. 1)
Summary
Rice (Oryza sativa) productivity is adversely impacted by numerous biotic and abiotic factors. An approximate 52% of the global production of rice is lost annually owing to the damage caused by biotic factors, of which ~21% is attributed to the attack of insect pests [3]. Insects belonging to Delphacidae and Cicadellidae contain a large group of sap-sucking planthoppers and leafhoppers, respectively. As such, they are difficult to control and manage, resulting in huge yield losses occurring in most of the rice growing areas. Three major sap-sucking pests of rice, viz., brown planthopper (Nilaparvata lugens, BPH), green leafhopper (Nephotettix virescens, GLH) and whitebacked planthopper (Sogatella furcifera, WBPH) are known to cause severe damage to the rice plant besides acting as vectors for major viral diseases
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