Development of a modified transformation platform for apomixis candidate genes research in Paspalum notatum (bahiagrass)

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

IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY. PLANT

SPRINGER

Lugar: Berlin; Año: 2014 vol. 50 p. 412 - 424

1054-5476

The aim of this work was to improve existingtransformation protocols and to transform specific genotypesof Paspalum notatum (bahiagrass) for functional analyses ofcandidate genes involved in reproduction. Three differentexplants were assayed for in vitro plant regeneration: matureseeds, mature embryos, and shoot meristems. Plant regenerationwas achieved with all explant types, but mature seedsproduced the optimal rate (78.0%) and were easiest to manipulate.A method based on serial re-induction of calli frommeristems of the regenerated lines was also developed, whichcould be useful in plant breeding strategies pursuingsomaclonal variation. Transient transformation experimentswere performed on calli obtained from mature seeds using acompressed helium gene gun. Transient transformation constructsincluded anthocyanin-synthesis genes cloned under theCAMV 35S promoter and an enhanced green fluorescentprotein gene (egfp) driven by the rice actin1 (act1) promoter.Selection curves for ammonium glufosinate were developedin order to determine the optimal selective pressure for stabletransformation (1.0 mg/L). Stable co-transformation experimentswere carried out with two different constructs containing:(1) the reporter egfp gene cloned under the rice act1promoter and (2) the selector bar gene driven by the ubiquitinpromoter. A total of 27 (64.2%) transgenic plants out of 42resistant plants analyzed were obtained. The presence of thetransgenes in regenerated plants was confirmed bypolymerase chain reaction and DNA gel blot analysis. Geneexpression was demonstrated by eGFP fluorescence detectionand in vivo assays for ammonium glufosinate tolerance. Thisplatform is being used to generate transgenic plants ofP. notatum to analyze the function of apomixis-associatedcandidate genes.