Analysis of conditions forAgrobacterium-mediated transformation of tobacco cells in suspension

Abstract

We have developed an Agrobacteriurn-mediated transformation system, using tobacco cell suspensions, that permits evaluation of factors affecting transformation within seven days of co-cultivation. Tobacco cell transformation was determined by monitoring fl-glucuronidase (GUS) activity detected in plant cell extracts. The use of a chimeric gene construct, 35S-GUS/INT, containing a portable intron in the uidA reading frame, assured only plant-specific GUS expression. During the co-cultivation period, induction of the bacterial vir-reNon was monitored using a heterologous gene construct composed of a virB promoter fragment from pTiC58 fused to the chloramphenicol acetyltransferase (CAT) gene of Tn9. Tobacco cell transformants were confirmed by antibiotic selection of transformed plant cells and by X-Gluc staining. Maximum transformation was obtained when plant suspension cultures were growing rapidly which also was coincidental with elevated levels of bacterial vir-region expression. One week after co-cultivation, the transformed cultures exhibited a stable pattern of GUS activity which remained constant without antibiotic selection. The system was used to compare the virulence of a number of Agrobacterium strains. GUS activity of plant cells co-cultivated with a strain containing a cointegrate plasmid was 3-fold higher than that of one with a binary configuration of the T-DNA. When the co-cultivating Agrobacterium strain also carried the plasmid used to monitor vir induction, the frequency of transformation was reduced by as much as 97%.

Stable expression of human β1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns

“…Tobacco BY2 suspension-cultured cells (Nicotiana tabacum L. cv. Bright Yellow 2) were transformed as described (21). Transformants were selected and maintained on modified Linsmaier and Skoog medium (14,22) supplemented with 150 g͞ml of kanamycin and 250 g͞ml of carbenicillin.…”

A Bright Future for the Bright Yellow-2 Cell Culture

“…BY-2 cells are readily transformed after protoplastation (Mathur and Koncz, 1998) or directly via particle bombardment or cocultivation with Agrobacterium tumefaciens (An, 1985;Klein et al, 1988;Rempel and Nelson, 1995). Although A. tumefaciens-mediated BY-2 transformation is performed routinely in many laboratories, we found that the efficiency in obtaining transgenic calli varies between experiments and mainly depends on the quality of the BY-2 cell culture.…”

Genetic transformation of gentian Gentiana tibetica (King) leaf explants with Agrobacterium tumefaciens strain C58C1

“…It should be emphasized that cell suspension, regardless of its type, requires a considerably longer time of the bacteria incubation, for example: Arabidopsis thaliana 48 h (Gallego et al 1999) and Nicotiana tabacum even 72 h (Rempel and Nelson 1995). Most probably, it is the reason that cell wall of external cells of aggregate is thicker than the internal walls of any explant and what is more important is not damaged, so bacteria virulantial action comes cross with obstacles, which takes longer.…”