Abstract
Sugarcane borer is the most common and harmful pest in Chinese sugarcane fields, and can cause damage to the whole plant during the entire growing season. To improve borer resistance in sugarcane, we constructed a plant expression vector pGcry2A0229 with the bar gene as the marker and the cry2A gene as the target, and introduced it into embryogenic calli of most widely cultivated sugarcane cultivar ROC22 by particle bombardment. After screening with phosphinothricin in vitro and Basta spray, 21 resistance-regenerated plants were obtained, and 10 positive transgenic lines harboring the cry2A gene were further confirmed by conventional PCR detection. Real-time quantitative PCR (RT-qPCR) analysis showed that the copy number of the cry2A gene varied among different transgenic lines but did not exceed four copies. Quantitative ELISA analysis showed that there was no linear relationship with copy number but negatively correlated with the percentage of borer-infested plants. The analysis of industrial and agronomic traits showed that the theoretical sugar yields of transgenic lines TR-4 and TR-10 were slightly lower than that of the control in both plant cane and ratoon cane; nevertheless, TR-4 and TR-10 lines exhibited markedly lower in frequency of borer-infested plants in plant cane and in the ratoon cane compared to the control. Our results indicate that the introduction of the cry2A gene via bombardment produces transgenic lines with obviously increased stem borer resistance and comparable sugar yield, providing a practical value in direct commercial cultivation and crossbreeding for ROC22 has been used as the most popular elite genitor in various breeding programs in China.
Highlights
Sugarcane is the most important sugar crop, with sucrose accounting for 80% of the total sugar production in the world and accounting for more than 92% of total sugar production in China
Sugarcane is a perennial crop and, to save costs, ratooning is usually conducted for over three years or even up to five years or more, during which sucrose content reduction and wind broken stalk increases can be caused by stem borer [4,5]
The GNA gene was integrated into sugarcane genome via Agrobacterium-mediated transformation by Zhangsun et al (2007), and the results showed that transgenic sugarcane plants had a significant resistance to the woolly aphid [25]
Summary
Sugarcane is the most important sugar crop, with sucrose accounting for 80% of the total sugar production in the world and accounting for more than 92% of total sugar production in China. The bar gene has been widely used as a screening marker gene [40,41], and its application to sugarcane has been described in several reports [22,29,42,43]. To obtain cry2A transgenic sugarcane, a plant expression vector pGcry2A0229 with the bar gene as a screening marker and cry2A as a target gene was constructed and genetically transformed into sugarcane by particle bombardment. SSiinnggllee--eennzzyymmee ddiiggeessttiioonn ooff ppGGccrryy22AA00222299 uussiinngg rreessttrriiccttiioonn eennddoonnuucclleeaassee HHiinndd IIIIII ggeenneerraatteedd tthhee eexxppeecctteedd ssiinnggllee bbaanndd wwiitthh aa ssiizzee ooff 88221133 bbpp. EElleeccttrroopphhoorreessiiss aannaallyyssiiss ooff ddoouubbllee eennzzyymmee ddiiggeessttiioonn wwiitthh HHiinndd IIIIII aanndd EEccooRR II sshhoowweedd tthhee eexxppeecctteedd ttwwoo bbaannddss wwiitthh ssiizzeess ooff aabboouutt 44443366 aanndd 33777777 bbpp,, rreessppeeccttiivveellyy ((FFiigguurree 22)).
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