CONICET | Buscador de Institutos y Recursos Humanos

The aim of this work was to improve on existing transformation protocols and to transform specific genotypes of P. notatum for functional analyses of reproductive candidate genes. Three different explants were assayed for in vitro plant regeneration: mature seeds, mature embryos and shoot meristems. Regeneration percentages ranging from 9.0 to 78.0 were obtained from all explant types, but mature seeds produced the optimal (78.0%) value and were easiest to manipulate. A method based on serial re-induction of calli from meristems of the regenerated lines was also developed. It could be useful in plant breeding strategies pursuing somaclonal variation. Transient transformation experiments were performed on calli obtained from mature seeds using a compressed helium gene gun. Transient transformation constructs included anthocyanin-synthesis genes cloned under the CAMV 35S promoter and an enhanced green fluorescent protein gene (egfp) driven by the rice actin1 (act1) promoter. Selection curves for ammonium glufosinate were developed in order to determine the better selective pressure for stable transformation (1.0 mg/L). Stable co-transformation experiments were carried out with two different constructs containing: 1) the reporter egfp gene cloned under the rice act1 promoter and 2) the selector bar gene driven by the ubi promoter. A total of 27 (64.3%) transgenic plants out of 42 resistant plants analysed were obtained. The presence of the transgenes in regenerated plants was confirmed by PCR and DNA gel blot analysis. Gene expression was demonstrated by eGFP fluorescence detection and in vivo assays for ammonium glufosinate tolerance. This platform is being used to generate transgenic plants of P. notatum to analyze the function of apomixis-associated candidate genes.

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