Engineering sugarcane cultivars with bovine pancreatic trypsin inhibitor (aprotinin) gene for protection against top borer (Scirpophaga excerptalis Walker)

Abstract

The inhibitory activity of bovine pancreatic trypsin inhibitor (aprotinin), a natural polypeptide and a proteinase inhibitor, was demonstrated on gut proteinases of three lepidopteran borers of sugarcane using commercially available aprotinin. A synthetic gene coding for aprotinin, designed and codon optimized for better expression in plant system (Shantaram 1999), was transferred to two sugarcane cultivars namely CoC 92061 and Co 86032 through particle bombardment. Aprotinin gene expression was driven by maize ubiquitin promoter and the plant selection marker used was hygromycin resistance. The integration, expression and functionality of the transgene was confirmed by Southern, Western and insect bioassay, respectively. Southern analysis showed two to four integration sites of the transgene in the transformed plants. Independent transgenic events showed varied levels of transgene expression resulting in different levels (0.16-0.50%) of aprotinin. In in vivo bioassay studies, larvae of top borer Scirpophaga excerptalis Walker (Lepidoptera: Pyralidae) fed on transgenics showed significant reduction in larval weight which indicated impairment of their development. Results of this study show the possibility of deploying aprotinin gene for the development of transgenic sugarcane cultivars resistant to top borer.

Genetic Engineering of Saccharum

“…Insect tolerant transgenic sugarcane engineered with the Bacillus thuringiensis ( Bt ) toxin genes cry1A(b) and cry1A(c) (Arencibia et al 1997(Arencibia et al , 1999Arvinth et al 2010 ;Braga et al 2003 ;Weng et al 2006 ) , the carbohydrate binding lectin gene gna and the proteinase inhibitor genes pinII , SKTI , SBBI , and aprotinin (Christy et al 2009 ;Legaspi and Mirkov 2000 ;Nutt et al 1999 ;Sétamou et al 2002 ) were also generated (Table 11.1 ), and fi eld or greenhouse trials have demonstrated encouraging levels of insect tolerance.…”

Transgenic sugarcane plants expressing high levels of modified cry1Ac provide effective control against stem borers in field trials

“…Since 1990s, several methods for generation of transgenic sugarcane plant have been established, such as microprojectile bombardment and electroporation of embryogenic cells (Arencibia et al 1995;Bower and Birch 1992). The progress in transformation methodology has facilitated incorporation of several useful genes into sugarcane genomes, including a bacterial toxin degradation gene (Zhang et al 1999), three virus resistance genes (Gilbert et al 2009;Ingelbrecht et al 1999;McQualter et al 2004), a bovine pancreatic trypsin inhibitor gene (Christy et al 2009), and several insect resistance genes (Arencibia et al 1997;Falco and Silva-Filho 2003;Weng et al 2006). However, a common problem associated with these transgenic sugarcane plants is the low expression levels of transgenes (Zhang et al 2007), which might account for the less than satisfactory performance of elite transgenic sugarcane lines in field trials (Arencibia et al 1999).…”

“…These sugarcane insects are often hard to control using chemical insecticides because of their ability to tunnel into and feed inside the sugarcane stems. Moreover, the insect-resistant germplasm is not available in sugarcane varieties (Christy et al 2009). The annual loss caused by insects has been estimated to be more than 10% of the sugarcane yield losses worldwide (Ricaud et al 1989).…”

Advances in Transgenic Research for Insect Resistance in Sugarcane

“…They then studied the expression of Cry content and resistance to sugarcane stem borer P. venosatus, a major sugarcane borer pest in China. The expression level of this engineered cry1Ac Christy et al (2009) ranged between 1 and 10 ng mg −1 total soluble proteins in the leaf and 0.2-6.0 ng mg −1 in the stem, which is sevenfold higher than that of cry1A(b) reported by Arencibia et al (1997). In both in vitro and in vivo insect bioassays, transgenic lines having higher Cry1Ac content showed greater resistance to stem borer.…”

“…In a study with PI of animal origin, Christy et al (2009) demonstrated through gut assays that aprotinin more effectively inhibited the gut proteinases of S. excerptalis than those of C. infuscatellus and C. sacchariphagus indicus. In subsequent studies, the gene coding for aprotinin driven by the maize ubi-1 promoter was transferred to sugarcane lines which showed expression levels of 0.16-0.50% of the total soluble leaf proteins.…”