Development of an intragenic gene transfer and selection protocol for sugarcane resulting in resistance to acetolactate synthase-inhibiting herbicide

Abstract

Sugarcane is a prime crop for commercial production of bioethanol and table sugar. Genetic engineering is complementing traditional breeding in sugarcane improvement. Costs and delays associated with regulatory approval for a transgenic event are substantial impediments to the commercialization of transgenic crops. Generation of intragenic sugarcane events carrying additional sequences from sexually compatible sorghum resembles traditional plant breeding and may facilitate regulatory approval. Acetolactate synthase (ALS), is a key enzyme in the biosynthetic pathway of branched-chain amino acids, and also a target for ALS-inhibiting herbicides including sulfonylureas and imidazolinones. In this study, we developed an intragenic minimal expression cassette encoding a mutant sorghum acetolactate synthase gene (mALS) under transcriptional control of the sorghum ubiquitin promoter and sorghum HSP 3′UTR. mALS was delivered into tissue cultures of sugarcane cultivar CP 88-1762 by biolistic gene transfer. A suitable selection protocol for recovery of intragenic events was developed for the herbicide bispyribac sodium. Intragenic sugarcane events were identified by PCR, and qRT-PCR analysis revealed events with different levels of mALS expression. Expression of the mALS gene in sugarcane resulted in chlorsulfuron resistance, as demonstrated by lower injury level and increased plant height following application of chlorsulfuron compared to the control cultivar. The results demonstrated that the mALS gene from sorghum supports the production of intragenic, herbicide resistant sugarcane. This is the first report of an intragenic selectable marker for sugarcane.