OPTIMIZATION OF GENETIC TRANSFORMATION IN SUGARCANE AND DEVELOPMENT OF GENOTYPES TOLERANT TO HERBICIDES AT ESTACIÓN EXPERIMENTAL OBISPO COLOMBRES (TUCUMÁN, ARGENTINA)

NOGUERA, A. S.; RACEDO, J.; PERERA, M. F.; FILIPPONE, M. P.; CASTAGNARO, A. P.

MACEIO

Workshop; 10th GERMPLASM AND BREEDING - 7th MOLECULARBIOLOGY WORKSHOPS; 2011

ISSCT - International Society of Sugar Cane Technologists

Conventional breeding has always involved genetic manipulation of crops through crossing and selection cycles. However, several interesting traits are not found in the cultivated germplasm or the one directly related to it, which thus limits their transfer through sexual transmission. Recent development of genetic transformation methods, which allow transferring one or a few traits to improved cultivars, overcomes this limitation. In sugarcane breeding, transgenesis is a valuable tool, especially when considering its high ploidy level and vegetative propagation, which enables the transfer and stable propagation of transgenic material. There are different methods to transform plants and the choice depends on the species, the plant material, its regeneration capacity and transformation efficiency. The biobalistic technique has been successfully used with several plant species, including sugarcane. It involves a process in which DNA-coated microparticles are accelerated by compressed gas and introduced into plant cells. Since 2006, EEAOC Biotechnology Department has been working on sugarcane breeding so as to introduce traits of agronomic importance into local commercial varieties RA87-3 and TUCCP77-42. To evaluate the efficiency of the biobalistic technique, expression assays of a reporter gene (uidA) were carried out to visually detect the introduction of the gene into the plant genome. This allowed adjusting involved parameters, not only optimizing the transformation process of calli (undifferentiated tissue) with the gene of interest, but also the regeneration of plants from these calli. To obtain embryogenic calli, discs of immature sugarcane leaves were cultured in vitro. Calli were bombarded with tungsten particles, in which a lineal segment of plasmid DNA containing the EPSPS and NPTII genes was precipitated. The former codes for an enzyme that confers tolerance to the herbicide glyphosate, and the latter confers resistance to geneticin, which enables in vitro selection of transformed cells. Transgene presence was evaluated by PCR using specific primers. Transgenic plants were micropropagated and later planted and kept in the greenhouse, in accordance with the legislation passed by Comisión Nacional Asesora de Biotecnología Agropecuaria in Argentina (CONABIA; Exp Nº S01-0231880/2007). To determine levels of tolerance to glyphosate, several doses of herbicide were tested and the different events were classified according to herbicide tolerance and multiplied for field evaluation tests.