RESUMO
Plant defense responses can lead to altered metabolism and even cell death at the sites of Agrobacterium infection, and thus lower transformation frequencies. In this report, we demonstrate that the utilization of culture conditions associated with an attenuation of defense responses in monocot plant cells led to highly improved Agrobacterium-mediated transformation efficiencies in perennial ryegrass (Lolium perenne L.). The removal of myo-inositol from the callus culture media in combination with a cold shock pretreatment and the addition of L-Gln prior to and during Agrobacterium-infection resulted in about 84 % of the treated calluses being stably transformed. The omission of myo-inositol from the callus culture media was associated with the failure of certain pathogenesis related genes to be induced after Agrobacterium infection. The addition of a cold shock and supplemental Gln appeared to have synergistic effects on infection and transformation efficiencies. Nearly 60 % of the stably transformed calluses regenerated into green plantlets. Calluses cultured on media lacking myo-inositol also displayed profound physiological and biochemical changes compared to ones cultured on standard growth media, such as reduced lignin within the cell walls, increased starch and inositol hexaphosphate accumulation, enhanced Agrobacterium binding to the cell surface, and less H(2)O(2) production after Agrobacterium infection. Furthermore, the cold treatment greatly reduced callus browning after infection. The simple modifications described in this report may have broad application for improving genetic transformation of recalcitrant monocot species.
Assuntos
Agrobacterium tumefaciens/genética , Glutamina/farmacologia , Inositol/farmacologia , Lolium/genética , Oryza/genética , Transformação Genética , Agrobacterium tumefaciens/fisiologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/fisiologia , Parede Celular/metabolismo , Temperatura Baixa , Meios de Cultura , Técnicas de Transferência de Genes , Vetores Genéticos , Proteínas de Fluorescência Verde , Peróxido de Hidrogênio/metabolismo , Lignina/metabolismo , Lolium/efeitos dos fármacos , Lolium/imunologia , Lolium/fisiologia , Oryza/efeitos dos fármacos , Oryza/imunologia , Oryza/fisiologia , Ácido Fítico/metabolismo , Imunidade Vegetal , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/imunologia , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas , Regeneração , Sementes/efeitos dos fármacos , Sementes/genética , Sementes/imunologia , Sementes/fisiologia , Amido/metabolismo , Técnicas de Cultura de TecidosRESUMO
The ion chromatographic method for the quantification of phytate (InsP(6)) in foods was adapted for the analysis of roots and tubers. To maximize sensitivity, ultraviolet (UV) detection following postcolumn derivatization was compared with evaporative light-scattering detection (ELSD). Detection limits for phytate were 0.5 and 1 microg for UV and ELSD, respectively. Unidentified peaks eluting close to and after InsP(6) were removed by solid-phase extraction. Phytate was detected in 11 of 15 roots or tubers. The highest phytate levels were 0.169 and 0.133% of the fresh weight of taro (Colocasia esculenta) and yuca (Manihot esculenta), respectively. Potatoes (Solanum tuberosum) contained 0.035-0.073% phytate, whereas no phytate at a detection limit of 0.003% of fresh weight was observed in sweet potatoes (Ipomoea batatas).