Abstract
Genetically modified crops which had been commercial applied extensively majorly are the insect resistance and herbicide tolerance events. In this study, the Bt insecticidal gene Cry1Ab, the glyphosate-tolerant gene EPSPS, and the selection marker gene PMI were combined into a single transferred DNA fragment and introduced into sugarcane by Agrobacterium-mediated transformation. Thirty-three resistant plantlets were obtained after selection using a PMI/mannose selection system. Thirty of these resistant plantlets were PCR positive for the three target genes. Southern blot assay revealed that the copy number of the integrated fragment in the transformed plantlets varied from 1 to 7. ELISA analysis showed that 23 of the 33 resistant plantlets expressed Cry1Ab and EPSPS protein. Five single-copy and ELISA-positive transgenic lines were tested under laboratory and field conditions to determine their resistance to insects and herbicides, and also evaluated their agronomic characteristics and industrial traits. Results showed that larvae fed with fodder mixture containing stem tissues from single-copy transgenic lines were weak and small, moreover, pupation and eclosion were delayed significantly during voluntary feeding bioassays. None of transgenic sugarcane was destroyed by cane borer while more than 30% of wild type sugarcane was destroyed by cane borer. For herbicide resistance, the transgenic plantlets grew healthy even when treated with up to 0.5% roundup while wild type plantlets would die off when treated with 0.1% roundup. Thus demonstrate that these transgenic lines showed strong insect resistance and glyphosate tolerance under both laboratory and field conditions. But in the field most of the transgenic plants were shorter and more slender than non-transformed control plants. So they presented poor agronomic characteristics and industrial traits than non-transformed control plants. Thus, a considerable number of embryogenic calli should be infected to obtain transgenic lines with potential for commercial use.
Highlights
Herbicide tolerance and insect resistance are important traits considered in genetic improvement of various crops (James, 2013)
ROC22 represents the largest acreage of sugarcane cultivars in China; Scholars have focused on improving the resistance of this cultivar to herbicides and insect borers
The two target genes were placed under the control of the constitutively expressed promoter Ubi1 and the selectable marker gene PMI was placed under the control of the promoter Act1
Summary
Herbicide tolerance and insect resistance are important traits considered in genetic improvement of various crops (James, 2013). Transgenic plants stacked with multiple genes are mostly obtained by cross-hybridization among different transgenic plants (Cao et al, 2002; Datta et al, 2002; Zhao et al, 2003) or re-transformation with different genes (Jobling et al, 2002; Rosati et al, 2003; Singla-Pareek et al, 2003; Qi et al, 2004) These methods for combining transgenes present several significant limitations. Cross-hybridization is time consuming and labor intensive, transgenic lines segregate again in subsequent generations (Halpin et al, 2001; Halpin, 2005) In this regard, scholars have developed a fast and cost effective method of introducing one single transferred DNA (T-DNA) containing multiple genes, with each gene possessing its own promoter and terminator (Slater et al, 1999; Bohmert et al, 2000, 2002). This method must be further improved (Poirier et al, 2000; Ye et al, 2000; Goderis et al, 2002; Thomson et al, 2002)
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