Biological Activity of thetzsGene of NopalineAgrobacterium tumefaciensGV3101 in Plant Regeneration and Genetic Transformation

Abstract

Agrobacterium tumefaciens has been widely used in plant genetic transformation. Hormone-encoding genes residing in the T-DNA region have been removed, resulting in disarmed Agrobacterium strains that are used in various transformation experiments. Nopaline Agrobacterium strains, however, carry another hormone gene, trans-zeatin synthesizing (tzs), that codes for trans-zeatin in the virulence region of the tumor-inducing plasmids. We investigated the activity and function of the tzs gene of a nopaline Agrobacterium sp. strain GV3101 in plant in vitro regeneration. Leaf explants of tobacco and Nicotiana benthamiana co-cultured with strain GV3101 exhibited active shoot regeneration in media without added plant growth regulators. On medium without plant growth regulators, transgenic shoots were also induced from explants co-cultured with GV3101 containing a binary vector. Enzyme-linked immunosorbent assay showed that cell-free extracts of Agrobacterium sp. strain GV3101 culture contained the trans-zeatin at 860 ng/liter. Polymerase chain reaction using tzs-specific primers showed that the tzs gene was present in strain GV3101 but not in other Agrobacterium strains. The study showed that the tzs gene in GV3101 was actively expressed, and that trans-zeatin produced in the Agrobacterium strain can promote plant shoot regeneration.

Agrobacterium-derived cytokinin influences plastid morphology and starch accumulation in Nicotiana benthamiana during transient assays

“…This is in line with previously published data suggesting that octopine strains lack the tzs gene on their Ti-plasmids, and are thus incapable of secreting high levels of trans -zeatin in culture [30,32]. …”

“…Cytokinin accumulation in culture was found to be the direct result of the activation of the tzs ( trans-zeatin synthase ) gene [30], an isopentenyl transferase (IPT) gene situated outside of the wild type T-DNA boundaries, in very close proximity to the vir region of the Ti plasmid [30,31]. The tzs gene was not removed during the creation of the disarmed Ti-plasmid pMP90 [17] harbored by GV [32]. The ability of GV cells to stay viable in leaf tissue for more than two weeks after infiltration [9], coupled with our observation that infiltrated tissue developed strong physiological symptoms characteristic of high cytokinin levels (Figure 2H and 2K), suggests that GV is also a constant and significant source of cytokinin in plant tissue in the days following infiltration.…”

Establishment and optimization of a hemp (Cannabis sativa L.) agroinfiltration system for gene expression and silencing studies

“…The genetic background of the Agrobacterium can influence the T-DNA transfer into plant cellular nuclei, and some Agrobacterium strains are more virulent than others based on the target species 29,30 . In this study, different transformation rates were observed for three avirulent strains.…”

An orphan protein of Fusarium graminearum modulates host immunity by mediating proteasomal degradation of TaSnRK1α

“…P CaMV 35S -Osp24 ΔSP was cloned into pGR106 and transformed into A. tumefaciens strain GV3101 90 expressing the BAX and INF1 constructs 91 . For infiltration assays with N. benthamiana leaves, A. tumefaciens cells were resuspended to OD 600 of 0.8 in infiltration solution (10 mM MES, 10 mM MgCl 2 , and 150 μM acetosyringone) 90 .…”