A Novel Non-wounding Transient Expression Assay for Cereals Mediated by Agrobacterium tumefaciens

Abstract

A novel Agrobacterium tumefaciens-mediated transient expression assay (AmTEA) was developed for young plants of different cereal species and the model dicot Arabidopsis thaliana. AmTEA was evaluated using five promoters (six constructs) and two reporter genes, gus and egfp. The constitutive 35S promoter and the promoter of the rice glutaredoxin gene showed gus and egfp expression in the cereals analyzed in the present study. A promoter for the DEAD-box RNA helicase family protein gene from Arabidopsis showed similar expression patterns of reporter genes in stable transgenic lines as well as in transient expression lines of Arabidopsis. Agrobacterium tumefaciens co-cultivation and plant incubation times were optimized using 35S and the rice expressed protein gene promoter (R2-273). The possibility of non-specific expression of the reporter genes was ruled out by using the antibiotic carbenicillin and the comparison of expression of the reporter genes driven by full-length and truncated R2-273 promoters. AmTEA considerably reduced time, space, labor, and cost requirements. Ease of use with stress treatments is another major advantage of this method. AmTEA can be automated and used for large-scale studies to decipher promoter and gene functions with the ultimate goal to enhance the performance of cereal crops against biotic and abiotic stresses.

Molecular characterization of the promoter of the stress-inducible ZmMYB30 gene in maize

“…The plates were then placed vertically in a greenhouse (22 °C, 16-h photoperiod, 70 % humidity, 120 μmol m −2 s −1 irradiance) for 2 weeks. The 2-week Arabidopsis seedlings were used in an Agrobacterium tumefaciens-mediated transient expression assay (AmTEA) (Dhadi et al 2012). After co-cultivation with Agrobacterium GV3101 for 20 h, the seedlings were exposed to dehydration and salinity stresses for 4, 8, and 12 h by substituting the MS medium with 20 % (m/v) polyethylene glycol 6000 (PEG6000) and 150 mM NaCl, respectively.…”

“…The recombinant constructs were confirmed by sequencing and then transformed into the A. tumefaciens GV3101 strain using the freeze-thaw method. The transient transformation experiments were carried out using the AmTEA protocol (Dhadi et al 2012). For co-cultivation procedures, 10 cm 3 of the overnight grown in Lurie and Bertani (LB) broth Agrobacterium GV3101 cultures harbouring different constructs of interest (absorbance approximately 1) were diluted with 30 cm 3 of LB broth.…”

Identification and validation of organ-preferential genes and analysis of corresponding upstream tissue-specific promoters in wheat

“…All organs or tissues were immersed in GUS staining solution, which contained 50 mM sodium phosphate buffer (pH 7.0), 0.5 mM each of potassium ferrocyanide and potassium ferricyanide, 0.5 mg cm -3 5-bromo-4-chloro-3-indolylβ-D-glucuronide (X-Gluc), 0.1 % (v/v) Triton X-100 and 20 % (v/v) methanol. Carbenicillin (500 mg dm -3 ) was added to the staining solution to avoid non-specific GUS expression from residual Agrobacterium and endophytes in wheat tissues (Dhadi et al 2012). After incubation in the dark at 37 °C overnight, the chlorophyll was cleared off from the samples by gradient treatment with 70 -90 % (v/v) ethanol.…”

Recent Advancements in Gene Expression and Enabling Technologies in Crop Plants

“…Although GUS expression could be detected in the switchgrass leaf cells after 3 days incubation, leaf decay was observed by 6 days of incubation (VanderGheynst et al 2008). In a more recent report, a method was described for Agrobacterium-mediated transient expression in several cereals (namely rice, barley, maize, oats, rye, sorghum and wheat), which allows functional analyses of genes involved in various abiotic stress responses and which eliminates the need for wounding of plants (Dhadi et al 2012). In this procedure, young 12-15-day--old plantlets were uprooted, cleaned, co-cultivated with Agrobacterium harbouring the expression construct in half strength Murashige and Skoog (0.5 MS) basal salt medium supplemented with acetosyringone, a natural phenolic wound response product, at 28 °C for 15 h on a rotating platform.…”