Genetic transformation and pyramiding of aprotinin-expressing sugarcane with cry1Ab for shoot borer (Chilo infuscatellus) resistance

Abstract

We evaluated the insecticidal toxicity of Cry1Aa, Cry1Ab and Cry1Ac toxins against neonate larvae of sugarcane shoot borer Chilo infuscatellus Snellen (Lepidoptera: Crambidae) in vitro on diet surface. With the lowest LC(50) value, Cry1Ab emerged as the most effective among the three toxins. Sugarcane cultivars Co 86032 and CoJ 64 were transformed with cry1Ab gene driven by maize ubiquitin promoter through particle bombardment and Agrobacterium-mediated transformation systems. Gene pyramiding was also attempted by retransforming sugarcane plants carrying bovine pancreatic trypsin inhibitor (aprotinin) gene, with cry1Ab. Southern analysis confirmed multiple integration of the transgene in case of particle bombardment and single site integration in Agrobacterium-mediated transformants. The expression of cry1Ab was demonstrated through Western analysis and the toxin was quantified using ELISA. The amount of Cry1Ab protein in different events varied from 0.007 to 1.73% of the total soluble leaf protein; the events transformed by Agrobacterium method showed significantly higher values. In in vivo bioassay with neonate larvae of shoot borer, transgenics produced considerably lower percentage of deadhearts despite suffering feeding damage by the borer compared with the untransformed control plants. Expressed Cry1Ab content was negatively related to deadheart damage. Aprotinin-expressing sugarcane pyramided with cry1Ab also showed reduction in damage. The potential of producing sugarcane transgenics with cry1Ab and aprotinin genes resistant to early shoot borer was discussed in the light of the results obtained.

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

“…The amounts of Cry1Ab protein in various transgenic lines varied from 0.007 to 1.73% of the total soluble leaf protein. The expressed Cry1Ab content was found to be negatively related to deadheart damage in in vivo bioassay with neonate larvae of shoot borers (Arvinth et al 2010).…”

Genetic Engineering of Saccharum

“…The ef fi ciency of recovery of transgenic plants using Agrobacterium -mediated system in sugarcane has been reported to be relatively low, in the range of 1-5 % (Arencibia and Carmona 2006 ;Arvinth et al 2010 ;Wang et al 2005b ) . Approaches that can signi fi cantly improve transformation ef fi ciencies are still under investigation.…”

“…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.…”

Truncated Ubiquitin 5′ Regulatory Region from Erianthus arundinaceus Drives Enhanced Transgene Expression in Heterologous Systems

“…Initially, Agrobacterium-mediated transformation was performed in rice [26], tobacco [27], and sugarcane [28], with pEriubi 3.2-GUS. Subsequently, to evaluate the functional role of different domains, rice and tobacco were transformed with twelve different deletion constructs of Eriubi 3.2.…”