T-DNA Transfer to Maize Cells: Histochemical Investigation of beta- Glucuronidase Activity in Maize Tissues (original) (raw)
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T-DNA transfer to maize plants
Molecular Biotechnology, 1999
Agrobacterium-mediated transformation is the method of choice to engineer desirable genes into plants. Here we describe a protocol for demonstrating T-DNA transfer from Agrobacterium into the economically important graminaceous plant maize. Expression of the T-DNA-located GUS gene was observed with high efficiency on shoots of young maize seedlings after cocultivation with Agrobacterium.
Mechanisms of crown gall formation: T-DNA transfer fromAgrobacterium tumefaciens to plant cells
The Botanical Magazine Tokyo, 1989
Agrobacterium tumefaciens harbouring the Ti plasmid incites crown gall tumor on dicotyledonous species. Upon infection of these plants, T-DNA in the Ti plasmid is transferred by unknown mechanisms to plant cells to be integrated into nuclear DNA. When Agrobacterium is incubated with protoplasts or seedlings of dicotyledonous plants, circularization of T-DNA and expression of vir (virulence) genes on the Ti plasmid are induced. The circularization event is efficiently induced by mesophyll protoplasts of tobacco which are highly competent for transformation by the T-DNA, and is also induced by diffusible phenolic compounds excreted from the protoplasts. The circularization and formation of crown gall both require the expression of the v/rD locus, one of the inducible vir genes. These results suggest that the circularization of T-DNA reflects one of steps of the T-DNA transfer during formation of crown gall. In contrast to dicotyledonous plants, monocotyledonous plants are thought to be unresponsive to infection by Agrobacterium. We showed that monocotyledonous plants do not excrete diffusible inducers for the expression of v/r genes, while the); contain a novel type of a signal substance(s). This inducer is not detected in the exudates of seedlings of monocotyledonous plants, but is found in the extracts from the seedlings, and also those from the seeds, bran and germ of wheat and oats. This finding suggests that T-DNA processing, and possibly its transfer, should take place when Agrobacterium invades seedlings and seeds of monocotyledonous plants.
Delivery of T-DNA from the Agrobacterium tumefaciens chromosome into plant cells
The EMBO journal, 1984
The intact T-region of the B6Ti plasmid of Agrobacterium tumefaciens was stepwise cloned into a site in transposon Tn3. In this way a suitable vehicle (Tn1882) was obtained for translocating the T-region to different replicons, i.e., to other plasmids or the chromosome. The IncP plasmid R772::Tn1882 conferred tumorigenicity on Agrobacterium if the virulence genes were provided in trans in the same cell. This result showed that the T-region present on Tn1882 was transferred efficiently to plant cells. Normal tumor development also occurred if the T-region was placed in the chromosome of A. tumefaciens and an R' plasmid was present carrying virA-E or virA-F. We conclude that the plasmid location of the T-region is not a prerequisite for transfer to the plant cell. The apparently normal delivery of the T-DNA from a bacterial chromosomal location supports a model involving a processing step within Agrobacterium effecting transfer of the T-region as a separate entity.
Association of single-stranded transferred DNA from Agrobacterium tumefaciens with tobacco cells
Proceedings of the National Academy of Sciences, 1994
During the inception of crown gall tumorigenesis, the traferred DNA (T-DNA) is processed from the Ti (tumor Indc n)pl d of Agrobacterium twfaciens and is transferred to plant cells. T-DNA processing and tr er require the induction of vir (virulence) genes by phenolic compounds se by wounded plant cells. After vir gene induction, both single-stranded (T-strands) and doublestranded forms of proessd T-DNA ac Late in the bacteria. Although current models favor the trnser of T-strands to plants, there has yet been no experinental evidence to show this. In this paper, we show that T-strands disappear from acetosyringone-induced A. twmefaciens within 30 min of bacterial cocultivation with tobacco protoplasts. PCR analysis of T-DNA associated with protoplasts indicates that singlestranded, but not double-stranded, T-DNA can be detected in the plant cells within 30 min of bacterial cocultivation. Control
Journal of Plant Physiology, 2000
Reproducible, efficient Agrobacterium-mediated transformation has been established for cultivars of Indica, Japonica and Javanica rice. Embryogenic calli derived from mature seed scutella were cocultivated with A. tumefaciens LBA4404 carrying (1) the binary vector pVOH65 [T-ONA encoding ~-glucuronidase (gus-intron) and neomycin phosphotransferase genes; strain 0065], (2) pVOH65 and the supervirulent pTOK47 (strain 1065), or (3) the super-virulent binary vector pTOK233 [T-ONA encoding the neomycin phosphotransferase, ~-glucuronidase (gus-intron) and hygromycin phosphotransferase genes]. GUS activity was observed in callus following co-cultivation with strains LBA4404(pTOK233) and 1065, but not with strain 0065. Regeneration of phenotypically normal transgenic plants occurred from 12-21 %, 16-31 %, and 10-19 % of transformed tissues of the cultivars Pusa Basmati 1 (Indica rice), Taipei 309 (Japonica rice), and Tinawen (Javanica rice) respectively, following co-cultivation with LBA4404 (pTOK233) and selection on hygromycin-containing medium. Single T-ONA inserts were rare in hygromycin-resistant transformants. However, T-ONA inserts were stably inherited and expressed in T1 seed generation plants of Taipei 309, with transgenes being expressed in a 3: 1 ratio in T1 progeny, indicating the presence of active T-ONA at a single locus. Comparison of T2 generation hemizygotes and homozygotes revealed a positive correlation between transgene dosage and GUS activity.
Transfer and Integration of T-DNA without Cell Injury in the Host Plant
Plant Cell, 1997
Agrobacterium colonizes plant cells via a gene transfer mechanism that results in plant tumorigenesis. Virulence (vi4 genes are transcriptionally activated in the bacteria by plant metabolites released from the wound site. Hence, it is believed that agrobacteria use injuries to facilitate their entrance into the host plant and that the wounded state is required for plant cell competence for Agrobacterium-mediated gene delivery. However, our experiments using vir gene-activated bacteria sprayed onto tobacco plantlets demonstrated that cells in unwounded plants could also be efficiently transformed. The condition of the plant cells was monitored using P-glucuronidase under the control of a wound-inducible promoter. lnfection of leaf tissue is light dependent, and it is drastically reduced when abscisic acid is exogenously applied to the plant. Under these experimental conditions, stomatal opening seems to be used by Agrobacterium to circumvent the physical barrier of the cuticle. These results thus show that the proposed cellular responses evoked by wounding in higher plants are not essential for Agrobacterium-mediated transformation.
Deviating T-DNA transfer fromAgrobacterium tumefaciens to plants
Plant Molecular Biology, 1996
We analyzed 29 T-DNA inserts in transgenicArabidopsis thaliana plants for the junction of the right border sequences and the flanking plant DNA. DNA sequencing showed that in most lines the right border sequences transferred had been preserved during integration, corroborating literature data. Surprisingly, in four independent transgenic lines a complete right border repeat was present followed by binary vector sequences. Cloning of two of these T-DNA inserts by plasmid rescue showed that in these lines the transferred DNA consisted of the complete binary vector sequences in addition to the T-region. On the basis of the structure of the transferred DNA we propose that in these lines T-DNA transfer started at the left-border repeat, continued through the vector part, passed the right border repeat, and ended only after reaching again this left-border repeat.