Development of a transformation platform for the study of apomixis in Bahiagrass (Paspalum notatum).

WOITOVICH N; MANCINI M; PERMINGEAT H; ORTIZ JPA; QUARIN C; PESSINO SC; FELITTI S

Buenos Aires

Simposio; The 6th International Symposium of the Molecular Breeding of Forage and Turf; 2010

Molecular Breeding of Forage and Turf international organizing committee

Apomixis is an asexual mode of reproduction through seeds that occurs naturally in some angiosperms. Several genes possibly involved in the apomictic development have been identified in different species. Particularly, a unigene set of 65 candidates differentially expressed in inflorescences of sexual and apomictic P. notatum was identified. The objective of this work was to develop a platform of in vitro tissue culture and transformation of P. notatum that allowed for the functional characterization of candidate genes. Apomictic and sexual polyploid genotypes were used. Seeds and embryos were cultured on IM medium with different hormone concentrations depending on the explant. Embryogenic calli were sub-cultured 4 to 6 weeks later on the same medium. Calli were transferred to induction medium (IM + BAP, GA3 and CuSO4) 2 to 4 weeks later for regeneration of tillers. In order to promote root development, calli with tillers longer than 5 mm were transferred to a medium containing SH + NAA and B5 vitamin. Embryogenic calli were transformed using biolistics with pressures of 800-1100 psi. Plasmid pDP687 (containing 2 genes for anthocyanin synthesis) was used in transient transformation experiments. Bombarded tissues were examined 48 hs after the experiment in order to detect colored cells indicating the success of transformation. Also, selection curves were obtained using glufosinate and kanamycin. Stable transformation was carried out using plasmid pAHC25 (containing the cassettes Ubi-gus-nos and Ubi-bar-nos). This platform together with a set of Gateway technology-based vectors, will allow for the functional characterization of candidate genes using anti-sense and RNAi-mediated gene silencing.