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

WOITOVICH, N.; MANCINI, M.; PERMINGEAT, H.; ORTIZ, J.P.A.; QUARIN, C.; PESSINO, S.C.; FELITTI, S.A.

Buenos Aires

Simposio; The 6th International Symposium on the Molecular Breeding of Forage and Turf - MBFT 2010.; 2010

<!-- /* Font Definitions */ @font-face {font-family:"MS Mincho"; panose-1:2 2 6 9 4 2 5 8 3 4; mso-font-alt:"ＭＳ 明朝"; mso-font-charset:128; mso-generic-font-family:roman; mso-font-format:other; mso-font-pitch:fixed; mso-font-signature:1 134676480 16 0 131072 0;} @font-face {font-family:"@MS Mincho"; panose-1:0 0 0 0 0 0 0 0 0 0; mso-font-charset:128; mso-generic-font-family:roman; mso-font-format:other; mso-font-pitch:fixed; mso-font-signature:1 134676480 16 0 131072 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-fareast-language:ES;} @page Section1 {size:595.3pt 841.9pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:35.4pt; mso-footer-margin:35.4pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Development of a transformation platform for the study of apomixis in Bahiagrass (Paspalum notatum) Apomixis is an asexual mode of reproduction through seeds that occurs naturally in some angiosperms. Several genes possibly involved in 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 genetic transformation of P. notatum to start functional characterization of candidate genes in the species. Apomictic and sexual tetraploid genotypes were used in tissue culture experiments. Mature seeds and isolated embryos were cultured 4-6 weeks on IM medium with different hormone concentrations, depending on the explant. Embryogenic calli were sub-cultured every 2 weeks on the same medium. Sections of calli of 5 mm in diameter were transferred to shoot induction medium (IM + BAP, GA3 and CuSO4) for 3-6 weeks for plant regeneration. In order to promote root development, calli with shoots longer than 5 mm were transferred to a medium containing SH salts + NAA and B5 vitamin. On average the best media combination used produced a 47% of calli (apomictic genotype) that responded to plant regeneration. Embryogenic calli were transformed using biolistics with pressures between 800-1100 psi and a 5 cm distance. Plasmid pDP687 (containing 2 genes for anthocyanin synthesis) was used for transient transformation experiments. Bombarded tissues were examined 72 hs after transformation in order to detect colored cells. The best transformation condition consisted of 1100 psi and generated 145 transformed cells per plate. Moreover, selection strategies were obtained using glufosinate or kanamycin as selective agents. Stable transformation will be optimized using plasmid pAHC25 (containing the cassettes Ubi-gus-nos and Ubi-bar-nos). This platform together with a set of Gateway technology-based vectors containing candidate genes, will allow for the functional characterization of apomixis-related genes using anti-sense and RNAi-mediated gene silencing.