INVESTIGADORES
CASTAGNARO Atilio Pedro
congresos y reuniones científicas
Título:
Optimization of genetic transformation in sugarcane and development of genotypes tolerant to herbicides at the Estación Experimental Obispo Colombres (Tucumán, Argentina).
Autor/es:
NOGUERA, A.S.; RACEDO, J.; PERERA, M.F.; FILIPPONE M.P. Y CASTAGNARO A.P. 2011.
Reunión:
Workshop; 10th Germplasm and Breeding and 7th Molecular Biology Workshop. ISSCT; 2011
Resumen:
The conventional breeding have involved the genetic manipulation of crops through crossing and selection cycles. However, several interesting traits are not found in the cultivated germplasm or directly related, which avoid its transfer through sexual transmission. The recent development of genetic transformation methods, wich allows the transfer of one or a few traits to cultivars improved, overcomes this limitation. In sugarcane breeding, the transgenesis is a valuable tool especially taking into account its high ploidy level and its vegetative propagation wich makes possible the transfer and stable propagation of the transgenic material. There are different methods to transform plants; the choice depends on the species, the plant material, the regeneration capacity and the transformation efficiency. Biobalistic technique has been successfully employed in several plant species, including sugarcane. It involves a process in which DNA-coated microparticles are accelerated by a compressed gas and introduced into plant cells. The Biotechnology Area of the EEAOC has been working since 2006 in sugarcane transformation in order to introduce traits of agronomic importance in the local commercial varieties RA 87-3 y TUCCP 77-42. 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 in the plant genome. This allowed to adjust the involved parameters and not only to optimize the transformation process of calli (undifferentiated tissue) with the interesting gene but also the regeneration of plants from these calli. To obtain embryogenic calli, used as explants, disc of immature leaves of sugarcane were in vitro cultured. Then, the calli were bombarded with tungsten particles in which a lineal segment of plasmid DNA that carries the EPSPS and NPTII genes, was precipitated. The first gene codes for an enzyme that confers tolerance to the herbicide glyphosate and the later confers resistance to geneticin, which allows the in vitro selection of the transformed cells. The transgene presence was evaluated through the PCR technique by using specific primers. Transgenic plants were micropropagated to transplant then to soil and keep in the greenhouse according to the legislation of Comisión Nacional Asesora de Biotecnología Agropecuaria in Argentina (CONABIA; Exp Nº S01-0231880/2007). To determine the different levels of tolerance to glyphosate, several doses of herbicide were checked and the different events were classified according to their herbicide tolerance and multiply to carry out the field evaluation tests.