Development of grapevine somatic embryogenesis using an air-lift bioreactor as an efficient tool in the generation of transgenic plants.
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 213h(-1) at 120rpm 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 1.6g/(dxL). 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.
Tapia
Eduardo
E
Biotechnological Engineering Department, Universidad Tecnológica de Chile INACAP, Santiago de Chile, Chile.
Sequeida
Alvaro
A
Castro
Alvaro
A
Montes
Christian
C
Zamora
Pablo
P
López
Reinaldo
R
Acevedo
Fernando
F
Prieto
Humberto
H
D000704:Analysis of Variance; D018533:Biomass; D019149:Bioreactors; D002478:Cells, Cultured; D004867:Equipment Design; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D010100:Oxygen / Q000378:metabolism; D030821:Plants, Genetically Modified; D013395:Sucrose / Q000378:metabolism; D046509:Tissue Culture Techniques; D014170:Transformation, Genetic; D027843:Vitis / Q000166:cytology / Q000196:embryology* / Q000235:genetics*
D013395:Sucrose; D010100:Oxygen