Articulo
Development of grapevine somatic embryogenesis using an air-lift bioreactor as an efficient tool in the generation of transgenic plants
JOURNAL OF BIOTECHNOLOGY;
J. Biotechnol.
Registro en:
0
D01I1064
D01I1064
WOS:000262795600015
0168-1656
Autor
Prieto-Encalada, Humberto
Castro-Oyarzún, Álvaro
Acevedo-Bonzi, Fernando
López-Planes, Reinaldo
Montes, Christian
Sequeida, Álvaro
Tapia, Eduardo
Zamora, Pablo
Institución
Resumen
The grapevine genetic transformation programs have relayed on the use of solid media-based somatic embryogenesis. To reach a high throughput of candidate gene evaluation in 'Thompson Seedless', a semi-automatic system allowing Viable transformation of explants was designed. An intermediate procedure using liquid media and agitated flasks was first characterized, leading to reduction in the biomass duplication time of pro-embryogenic (PE) cells from 30 d in dishes to 14 d. The oxygen transfer coefficient value in this system was 213 h(-1) at 120 rpm and 25 degrees C with a 16/8-h (light/darkness) photoperiod. The scaling-up to the air-lift bioreactor decreased the biomass duplication time of PE cells up to 5.3 d post-inoculation (pi) and an average volumetric productivity of 1.6 g/(d x L). Although slight browning was seen in the explants during the phase of 8-14 d pi. no losses in their viability and regenerative capability were observed. Cultured cells showed normal elongation in the transition from heart- to the torpedo-shape and finally to advanced developmental stages, with radicle emergence and whole plant generation. Agrobacterium-mediated transformation of cells was efficiently incorporated after this Multiplication process by use of conventional procedures in dishes, allowing the generation of transgenic plantlets confirmed by PCR. (c) 2008 Elsevier B.V. All rights reserved. To Catalina Alvarez, Blanca Olmedo and Marisol Mufioz for the plant tissue culture work and Ljubomir Papic for his valuable help and discussion. This work was funded by grants Innova-Chile-FDI-05CR11PAT-19:, FONDEF-Chile D99I1001; and FONDEF-Chile D01I1064. This work was also funded by Brown Group and Biotecnologia Fruticola S.A. (PBCT-Chile). 5 FONDEF hprieto@inia.cl Innova-Chile [FDI-05CR11PAT-19]; FONDEF-Chile [D99I1001, D01I1064]; Brown Group and Biotecnologia Fruticola S.A. (PBCT-Chile) 1 FONDEF 139