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The successful development of genetically engineered monocots using Agrobacterium-mediated transformation has created an increasing demand for compatible vectors. We have developed a new expression vector, pGVG, for efficient transformation and expression of different constructs for gene overexpression and silencing in sugarcane. The pCAMBIA2300 binary vector was modified by adding Gateway recombination sites for fast gene transfer between vectors and the maize polyubiquitin promoter Ubi-1 (ZmUbi1), which is known to drive high gene expression levels in monocots. Transformation efficiency using the pGVG vector reached up to 14 transgenic events per gram of transformed callus. Transgenic plants expressing the β-glucuronidase (GUS) reporter gene from pGVG showed high levels of GUS activity. qRT-PCR evaluations demonstrated success for both overexpression and hairpin-based silencing cassettes. Therefore, pGVG is suitable for plant transformation and subsequent applications for high-throughput production of stable transgenic sugarcane. The use of an expression cassette based on the ZmUbi1 promoter opens the possibility of using pGVG in other monocot species.

Chemically inducible gene switches can provide precise control over gene expression, enabling more specific analyses of gene function and expanding the plant biotechnology toolkit beyond traditional constitutive expression systems. The alc gene expression system is one of the most promising chemically inducible gene switches in plants because of its potential in both fundamental research and commercial biotechnology applications. However, there are no published reports demonstrating that this versatile gene switch is functional in transgenic monocotyledonous plants, which include some of the most important agricultural crops. We found that the original alc gene switch was ineffective in the monocotyledonous plant sugar cane, and describe a modified alc system that is functional in this globally significant crop. A promoter consisting of tandem copies of the ethanol receptor inverted repeat binding site, in combination with a minimal promoter sequence, was sufficient to give enhanced sensitivity and significantly higher levels of ethanol inducible gene expression. A longer CaMV 35S minimal promoter than was used in the original alc gene switch also substantially improved ethanol inducibility. Treating the roots with ethanol effectively induced the modified alc system in sugar cane leaves and stem, while an aerial spray was relatively ineffective. The extension of this chemically inducible gene expression system to sugar cane opens the door to new opportunities for basic research and crop biotechnology.

Orientador: Adilson Leite Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Doutorado