The purpose of the present research was to establish a routine procedure for Agrobacterium tumefaciens-mediated transformation of important Italian rice cultivars (Oryza sativa L. ssp. japonica). A detailed investigation on in vitro culture, plant regeneration and susceptibility to infection from various strains of Agrobacterium tumefaciens was performed. Twenty-six genetically differentiated rice cultivars, chosen for outstanding grain quality and agronomical characters, were established as tissue cultures from somatic and gametic cells. Regenerative callus cultures were obtained from both mature and immature embryos, and from microspores through anther culture. We identified highly responsive varieties to tissue culture and plant regeneration; several genotypes showed high embryogenic callus induction frequency which was genetically determined. The interaction between the callus systems and different Agrobacterium tumefaciens strains characterized by different chromosomal background and Ti-plasmid content was subsequently investigated. Five Agrobacterium tumefaciens strains, all carrying the marker cassette -INTGUS- for early detection of transformation, were co-cultivated with rice tissues in the presence of acetosyringone according to standard procedures. The tissues were histochemically stained for GUS activity and evaluated using a visual scoring scale. In all cases we identified a good combination Agrobacterium X rice genotype, and the interaction was genotype-dependent. In some cases the combination explored gave a very high rating of transient expression, thus confirming the reliability of the system for transformation of the genotypes considered. Good combination Agrobacterium X rice genotypes were found for both somatic and gametic systems and, in several cases, the combination efficiency for an Agrobacterium strain was different depending on the cell system. In most cases, the infection process did not hamper the subsequent capability of the rice cultures to promote plant regeneration. These indications are of major interest for the identification of key parameters in the establishment of efficient routine procedures for Agrobacterium tumefaciens-mediated genetic transformation of Italian rice cultivars.
Agrobacterium tumefaciens-mediated trasformation of rice (Oryza sativa L. ssp. japonica) Italian cultivars. 1. Interaction among Agrobacterium strains and rice genotypes in embryogenic callus of somatic and gametic origin
PERUGINI, Iolanda;REALI, Alessia;
1998-01-01
Abstract
The purpose of the present research was to establish a routine procedure for Agrobacterium tumefaciens-mediated transformation of important Italian rice cultivars (Oryza sativa L. ssp. japonica). A detailed investigation on in vitro culture, plant regeneration and susceptibility to infection from various strains of Agrobacterium tumefaciens was performed. Twenty-six genetically differentiated rice cultivars, chosen for outstanding grain quality and agronomical characters, were established as tissue cultures from somatic and gametic cells. Regenerative callus cultures were obtained from both mature and immature embryos, and from microspores through anther culture. We identified highly responsive varieties to tissue culture and plant regeneration; several genotypes showed high embryogenic callus induction frequency which was genetically determined. The interaction between the callus systems and different Agrobacterium tumefaciens strains characterized by different chromosomal background and Ti-plasmid content was subsequently investigated. Five Agrobacterium tumefaciens strains, all carrying the marker cassette -INTGUS- for early detection of transformation, were co-cultivated with rice tissues in the presence of acetosyringone according to standard procedures. The tissues were histochemically stained for GUS activity and evaluated using a visual scoring scale. In all cases we identified a good combination Agrobacterium X rice genotype, and the interaction was genotype-dependent. In some cases the combination explored gave a very high rating of transient expression, thus confirming the reliability of the system for transformation of the genotypes considered. Good combination Agrobacterium X rice genotypes were found for both somatic and gametic systems and, in several cases, the combination efficiency for an Agrobacterium strain was different depending on the cell system. In most cases, the infection process did not hamper the subsequent capability of the rice cultures to promote plant regeneration. These indications are of major interest for the identification of key parameters in the establishment of efficient routine procedures for Agrobacterium tumefaciens-mediated genetic transformation of Italian rice cultivars.
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