Bean [alpha]-Amylase Inhibitor Confers Resistance to the Pea Weevil (Bruchus pisorum) in Transgenic Peas (Pisum sativum L.)

Abstract

Bruchid larvae cause major losses of grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil and the azuki bean weevil, are pests that damage stored seeds. Others, such as the pea weevil (Bruchus pisorum), attack the crop growing i n the field. We transferred the cDNA encoding the a-amylase inhibitor (a-AI) found in the seeds of the common bean (Phaseolus vulgaris) into pea (Pisum sativum) using Agrobacferium-mediated transformation. Expression was driven by the promoter of phytohemagglutinin, another bean seed protein. The a-amylase inhibitor gene was stably expressed in the transgenic pea seeds at least to the T, seed generation, and a-AI accumulated i n the seeds up to 3% of soluble protein. This level is somewhat higher than that normally found in beans, which contain 1 to 2 % a-AI. In the 1 , seed generation the development of pea weevil larvae was blocked at an early stage. Seed damage was minimal and seed yield was not significantly reduced in the transgenic plants. These results confirm the feasibility of protecting other grain legumes such as lentils, mungbean, groundnuts, and chickpeas against a variety of bruchids using the same approach. Although a-AI also inhibits human a-amylase, cooked peas should not have a negative impact on human energy metabolism.The common bean (Phaseolus vulgaris L.) contains a family of structurally related seed proteins: PHA-E and -L, arcelin, and a-AI. PHA-E and PHA-L are strong agglutinins, i.e. classical lectins that bind carbohydrate, and arcelin, which is found only in certain wild accessions of the common bean, may be a weak agglutinin (Hartweck et al., 1991). The bean a-AI has 65 to 70% amino acid sequence identity with the other three but lacks at least one of the conserved residues needed for lectin activity. Its biochemical mode of action is to form a one-to-one complex with certain amylases (for reviews, see Chrispeels and Raikhel, 1991;Rouge et al., 1993).

Molecular and functional characterization ofcry1Actransgenic pea lines

“…sponsor) lines Negawo 2012 were used for molecular and functional characterizations. The transgenic lines were developed using the routine pea transformation protocol using Agrobacterium tumefaciens strain EHA105 harboring a pSoup pGII35SCry1Ac vector Schroeder et al, 1993 with modification as described in Richter et al Richter et al, 2006 The vector contains bar gene as selectable marker …”

“…Sharma et al, 2010 In pea, 10 to 70 % yield losses can be incurred depending on the insect pests. Schroeder et al, 1995;Clement, Hardie, Elberson 2002;Legowski, Gould 1960;Williams, Schotzko, Okeeffe 1995;Biddle, Cattlin 2001;Korth 2008 When a desired trait is not available in a given gene pool, one way to complement conventional breeding is to apply biotechnological techniques to access and transfer genes for novel traits from other sources. This approach has been used to develop not only varieties resistant to production constraints (such as insect and diseases) but also to improve the nutritional value of different crops.…”

“…Korth, Stewart 2008 Pea is one of the economically important legume crops that have been extensively studied under in vitro conditions during the last few decades. Today, against the odds of many grain legumes which are recalcitrant to in vitro conditions, there is a well established and routinely used regeneration and transformation protocol Schroeder et al, 1993 for pea. Transgenic pea lines against different production constraints such as insect pests and diseases have been developed.…”

“…Transgenic pea lines against different production constraints such as insect pests and diseases have been developed. Schroeder et al, 1995;Shade et al, 1994;Hassan, Meens, Jacobsen, Kiesecker et al, 2009;Richter 2006 Despite the successful application of transgenic approaches in pea, little attention has been given to insect resistance development using cry genes from Bacillus thuringiensis (Bt). The Cry proteins are targeted to the larval stage of the insect, they are activated in the midgut of larvae and bind to the receptors on the epithelial cell membrane leading to the formation of membrane pores.…”

Expression analysis of recombinant Vigna radiata plant defensin 1 protein in transgenic tobacco plants

“…However, content of VrPDF1 in mungbean seeds is very low, so increasing the content of VrPDF1 in mungbean seeds to enhance alpha-amylase inhibition in larvae and bruchids is necessary for the study of improving bruchid resistance of mung beans and plants in general. Transformation has been a successfully applied technique to enhance recombinant protein expression in seeds [16], [17]. Swathi et al (2008) analyzed expression of defensin gene in tobacco and peanut to enhance antifungal in transgenic plants [18].…”