No 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.

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.

The first phase of transgenic research in sugarcane concentrated on the development and evaluation of transgenic lines transformed for resistance to biotic stresses, particularly diseases and insect pests. Sugarcane is attacked by a range of insects including tissue borers, sucking pests and canegrubs. Losses due to these pests are estimated to be around 10%. Although chemical control and integrated pest management are regularly practiced for the control of insect pests, success is often limited due to practical difficulties. The genetic complexity of sugarcane coupled with the nonavailability of resistance genes in the germplasm has made conventional breeding for insect resistance difficult. In this context, transgenic technology has become a handy tool for imparting insect resistance to an elite variety which is otherwise superior for most other agronomic traits. A number of transgenic sugarcane lines have been developed with genes expressing Cry proteins, proteinase inhibitors or lectins resistant to borers, sucking insects or grubs. While commercializing transgenic lines, issues such as higher and stable transgene expression, preparedness for resistance management and non-target effects need to be addressed. To manage the constant threat of resistance development in target insects, it is imperative to deploy field-level strategies taking clues from other crops coupled with the search for new potent replacement molecules for transformation.

SUMMARYAccessions of four Saccharum spp. from the world collection of sugarcane germplasm maintained at the ICAR-Sugarcane Breeding Institute Research Center, Kannur, Kerala State, India, were screened against Chilo sacchariphagus indicus (Kapur) (Lepidoptera: Crambidae), commonly known as internode borer. Observations on the progression of borer attack in the most susceptible Saccharum officinarum indicated that the incidence began in the first fortnight of July, increased in the next 2 months and reached its peak in September. Thereafter, the incidence decreased in the next 2 months but reached its overall peak in December. Simple correlation coefficients between mean monthly weather parameters and borer incidence were not significant. Percent of canes attacked was significantly lower in 2011 than in 2012 for S. officinarum, Saccharum barberi and Saccharum sinense but did not differ between the years for Saccharum robustum. Amongst the four Saccharum spp., S. robustum showed the lowest borer incidence whereas S. officinarum recorded the lowest attack intensity. Whilst infestation index showed the same trend as percent canes attacked, percent deadhearts did not differ amongst the four Saccharum spp. Considering the 171 accessions evaluated, 29 (16.9%) accessions were resistant, 39 (22.8%) moderately resistant and 103 (60.2%) susceptible to internode borer. Out of the 39 accessions of S. officinarum, none occupied the resistant category whereas 17.9% were in the moderately resistant category. In S. robustum, whilst nearly half (44.5%) the accessions emerged as resistant, a considerable number occupied the moderately resistant category. In S. barberi, no accession was resistant to the borer. In S. sinense, only one accession each represented resistant and moderately resistant categories. Plant morphological characters, yield and quality parameters did not show clear-cut relationship with the three infestation parameters.