Agrobacterium -mediated transient expression of vacuolar GFPs in Petunia leaves and petals (original) (raw)
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Agrobacterium-mediated transient expression system in banana immature fruits
2010
Transient expression assays are useful for saving time in studies of gene function, particularly ones expressed in fruit. Transient expression system of a reporter gene in immature fruit of banana was developed. Agrobacterium tumefaciens, whose plasmid contains an intron-containing β-glucuronidase (gusA) gene under regulatory control of the CaMV 35S promoter, was vacuum-infiltrated into sliced fruits and co-cultured. GUS histochemical assay was performed three days after co-cultivation, and a high level of GUS expression was observed. This transient expression system is useful for routine transient assays to validate genes expressed in banana fruit.
Agroinjection of Tomato Fruits. A Tool for Rapid Functional Analysis of Transgenes Directly in Fruit
PLANT PHYSIOLOGY, 2005
Transient expression of foreign genes in plant tissues is a valuable tool for plant biotechnology. To shorten the time for gene functional analysis in fruits, we developed a transient methodology that could be applied to tomato (Solanum lycopersicum cv Micro Tom) fruits. It was found that injection of Agrobacterium cultures through the fruit stylar apex resulted in complete fruit infiltration. This infiltration method, named fruit agroinjection, rendered high levels of 35S Cauliflower mosaic virus-driven b-glucuronidase and yellow fluorescence protein transient expression in the fruit, with higher expression levels around the placenta and moderate levels in the pericarp. Usefulness of fruit agroinjection was assayed in three case studies: (1) the heat shock regulation of an Arabidopsis (Arabidopsis thaliana) promoter, (2) the production of recombinant IgA antibodies as an example of molecular farming, and (3) the virus-induced gene silencing of the carotene biosynthesis pathway. In all three instances, this technology was shown to be efficient as a tool for fast transgene expression in fruits.
Biotechnology Progress, 2008
The reporter gene -glucuronidase was transiently expressed in a 51-L bioreactor-grown plant cell suspension culture of Nicotiana glutinosa at a yield of approximately 1.1 mg through coculture with an auxotrophic strain of Agrobacterium tumefaciens. The three order of magnitude scale-up involved the investigation of factors contributing to transient expression including the timing of Agrobacterium inoculation relative to the plant cell growth phase, plant tissue culture hormonal triggers and plant cell cycle synchronization. The co-culture process was simplified to facilitate implementation in a pilot-scale bioreactor. At the shake flask scale it was determined that elevated concentrations of oxygen in the headspace were detrimental to transient expression levels and the addition of acetosyringone to the co-culture had a negligible effect. The bacterial preparation process was also streamlined, permitting the direct transfer of the Agrobacterium culture from a bench-scale fermentor to the pilot-scale plant cell culture bioreactor. Increasing expression levels and overcoming batch-to-batch variability despite extensive procedure systemization remain the major technical hurdles.
An Agrobacterium-mediated transient gene expression system for intact leaves
Plant Science, 1997
,411 efficient and reproducible AgrohLlc~teriut,l-mediated transient gene expression system for intact leaf tissue was developed. A high level of transient expression was observed when bacteria. which were pretreated in C?Y gene-inducing conditions. were infiltrated into complete leaf tissue. Histochemical /I-glucuronidase assays showed large transgene-expressing sectors comprising of up to 90% of the leaf area. As a consequence of infiltration, the induced bacteria entered into the intercellular spaces, thus enabling T-DNA transfer in all cell layers of the leaf. The protocol was optimized for Phasrolus wlgaris leaves. but similar results were obtained with other plant species. such as P/mroh~.s uc,utifdim. poplar, and tobacco. A /I-glucuronidase chimeric gene interrupted by an intron was used as a marker for histological detection of the sectors. Copyright :CJ 1997 Elsevier Science Ireland Ltd.
Biotechnology Progress, 2008
A viral vector based on the bean yellow dwarf virus was investigated for its potential to increase transient gene expression. An intron-containing GUS reporter gene and the cis-acting viral regulatory elements were incorporated in the viral vector and could be complemented by the viral replication-associated proteins provided on a secondary vector. All vectors were delivered to Nicotiana glutinosa plant cell suspension or hairy root cultures via auxotrophic Agrobacterium tumefaciens. Cell culture generated greater yield of reporter gene expression than did root culture, as a result of the limitation imposed on roots to express the protein only in surface tissue containing actively dividing cells. Reporter gene expression increased for cell culture when the reporter gene construct was co-delivered with the construct supplying both viral replication associated proteins (REP and REPA); gene expression decreased when the construct supplying only the viral REP protein was co-delivered. Reporter protein expression increased from 0.091% for the reporter construct alone to 0.22% total soluble protein (% TSP) when the viral Repsupplying vector was co-delivered with the reporter gene construct. Reporter protein was generated 3 days after the initiation of bacterial co-culture, providing for rapid generation of heterologous protein in cell culture.
Objective. Assess transient gene expression of GUS in cassava (Manihot esculenta Crantz) leaves using Agrobacterium tumefaciens infiltration. Materials and methods. A. tumefaciens strains GV3101 and AGL1 containing pCAMBIA1305.2 were used to evaluate transient gene expression of β-glucuronidase (GUS). A. tumefaciens infiltration (agroinfiltration) was made using both leaves from in vitro and 1 month old greenhouse plants. Leaves were incubated in X-GLUC buffer, stained and photographed to detect GUS activity. Results. Agroinfiltration assays showed GUS transient expression in leaves of cassava varieties widely cultivated in the north coast and eastern savannah, MCOL2215 (Venezuelan) and CM6438-14 (Vergara), respectively. A. tumefaciens agressive strain AGL1 showed high efficiency inducing GUS expression in cassava leaves. Conclusions. We recommend using A. tumefaciens agressive strain AGL1 for agroinfiltration to assess transient expression in cassava leaves.
Plant Cell, Tissue and Organ Culture, 2006
To determine the optimum conditions for Agrobacterium-mediated gene transfer, peach explants including cotyledons, embryonic axes and hypocotyl slices from non-germinated seeds and epicotyl internode slices from germinating seeds were exposed to Agrobacterium-mediated transformation treatments. The GUS (uidA) marker gene was tested using two different A. tumefaciens strains, three plasmids and four promoters [CaMV35s, (Aocs)3AmasPmas (''super-promoter''), mas-CaMV35s, and CAB]. GFP was tested with six A. tumefaciens strains, one plasmid (pLC101) and the doubleCaMV35s (dCaMV35s) promoter. The CaMV35s promoter produced more GUS expression than the CAB promoter. A. tumefaciens strains EHA105 and LBA4404 harboring the same plasmid (pBIN19) differed in their effects on GUS expression suggesting an interaction between A. tumefaciens strain and plasmid. A combination of A. tumefaciens EHA105, plasmid pBIN19 and the CaMV35s promoter produced the highest rates of transformation in peach epicotyl internodes (56.8%), cotyledons (52.7%), leaves (20%), and embryonic axes (46.7%) as evaluated by the percentage of explants expressing GUS 14 days after co-cultivation. GFP expression under the control of the dCaMV35s promoter was highest for internode explants but only reached levels of 18-19%. When GFP-containing plasmid pCL101 was combined with each of five A. tumefaciens strains the highest levels of transformation were 20-21% (internode and cotyledons, respectively). When nine peach genotypes were co-cultivated with A. tumefaciens strain EHA105 and GFP-containing plasmid pCL101 the highest levels of transformation were 26-28% (cotyledons and internodes, respectively). While GFP represents a potentially useful transformation marker that allows the non-destructive evaluation of transformation, rates of GFP transformation under the conditions of this study were low. It will be necessary to optimize expression of this marker gene in peach.
A rapid Agrobacterium -mediated Arabidopsis thaliana transient assay system
Plant Molecular Biology Reporter, 2004
Stable transformation of Arabidopsis thaliana is a lengthy process that involves up to 3 mo of plant growth and seed selection. We have developed a rapid, 3-wk transient assay system to test the functionality of cis-regulatory regions controlling expression of a reporter gene in plants before undertaking stable transformation. Two-week-old Arabidopsis seedlings were vacuum-infiltrated with Agrobacterium tumefaciens cultures carrying various upstream regulatory regions controlling uidA (~-glucuronidase [GUS]) expression. Seedlings were fixed and stained for GUS activity 3-5 d following infiltration. Regulatory regions tested in this system include the cauliflower mosaic virus (CaMV) 35S promoter, the upstream regulatory region of ribosomal protein gene L23A-1, and a temperature-inducible regulatory region (HSPIO1B) also from Arabidopsis. The percentage of seedlings positive for GUS activity varied depending on the construct used, with the CaMV 35S promoter producing the highest number of GUS-positive seedlings. Temperature induction treatments elicited increased GUS expression in seedlings transformed with the HSPIO1B regulatory region. Regardless of construct, GUS expression levels were higher in seedlings collected 5 d following Agrobacterium infiltration than those collected 3-4 d postinfiltration.