Abstract:
: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.
journal_name
J Biotechnoljournal_title
Journal of biotechnologyauthors
Tapia E,Sequeida A,Castro A,Montes C,Zamora P,López R,Acevedo F,Prieto Hdoi
10.1016/j.jbiotec.2008.09.009subject
Has Abstractpub_date
2009-01-01 00:00:00pages
95-101issue
1eissn
0168-1656issn
1873-4863pii
S0168-1656(08)02404-8journal_volume
139pub_type
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