Cold acclimation is the major process that prepares plants for freezing tolerance. In addition to extensive transcription regulation by coldinducible master transcription factors, oxidative stress signaling has been postulated to play a role in freezing tolerance. Activation of oxidative signaling through the expression of an active mitogenactivated protein kinase kinase kinase provided benefits in transgenic tobacco at freezing temperature bypassing cold acclimation. Because involvement of the mitogen-activated protein kinase cascade in oxidative stress signaling is evolutionarily conserved in eukaryotes from yeast to mammals, we tested the effect of expressing a heterologous tobacco mitogen-activated protein kinase kinase kinase (Nicotiana PK1), which can mimic H2O2 signaling, in a major cereal crop. We demonstrate that low-level but constitutive expression of the Nicotiana PK1 gene enhances freezing tolerance in transgenic maize plants that are normally frost sensitive. Our results suggest that a new molecular approach can be designed to genetically enhance freezing tolerance in important crops.

Conditions affecting Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merr.], including seed vigor of explant source, selection system, and cocultivation conditions, were investigated. A negative correlation between seed sterilization duration and seed vigor, and a positive correlation between seed vigor and regenerability of explants were observed in the study, suggesting that use of high vigor seed and minimum seed sterilization duration can further improve transformation efficiency. Selection schemes using glufosinate or bialaphos as selective agents in vitro were assessed. Glufosinate selection enhanced soybean transformation as compared to bialaphos. The use of 6mgL−1 glufosinate during shoot induction and shoot elongation stages yielded higher final transformation efficiency ranging from 2.0% to 6.3% while bialaphos at 4 to 8mgL−1 gave 0% to 2.1% efficiency. Including cysteine and DTT during cocultivation increased the transformation efficiency from 0.2-0.9% to 0.6-2.9%. This treatment also improved T-DNA transfer as indicated by enhanced transient GUS expression. Shoot regeneration and Agrobacterium infection were attained in twelve soybean cultivars belonging to maturity groups I-VI. These cultivars may be amenable to genetic transformation and may provide a valuable tool in soybean improvement programs.

Particle bombardment and Agrobacterium-mediated transformation are two popular methods currently used for producing transgenic maize. Agrobacterium-mediated transformation is expected to produce transformants carrying fewer copies of the transgene and a more predictable pattern of integration. These putative advantages, however, tradeoff with transformation efficiency in maize when a standard binary vector transformation system is used. Using Southern, northern, real-time PCR, and real-time RT-PCR techniques, we compared transgene copy numbers and RNA expression levels in R1 and R2 generations of transgenic maize events generated using the above two gene delivery methods. Our results demonstrated that the Agrobacterium-derived maize transformants have lower transgene copies, and higher and more stable gene expression than their bombardment-derived counterparts. In addition, we showed that more than 70% of transgenic events produced from Agrobacteriummediated transformation contained various lengths of the bacterial plasmid backbone DNA sequence, indicating that the Agrobacterium-mediated transformation was not as precise as previously perceived, using the current binary vector system.

Drought is one of the most important abiotic stresses affecting the productivity of maize. Previous studies have shown that expression of a mitogen-activated protein kinase kinase kinase (MAPKKK) gene activated an oxidative signal cascade and led to the tolerance of freezing, heat, and salinity stress in transgenic tobacco. To analyse the role of activation of oxidative stress signalling in improving drought tolerance in major crops, a tobacco MAPKKK (NPK1) was expressed constitutively in maize. Results show that NPK1 expression enhanced drought tolerance in transgenic maize. Under drought conditions, transgenic maize plants maintained signi

50011 We have achieved routine transformation of maize (Zea mays) using an Agrobacterium tumefaciens standard binary (non-super binary) vector system. Immature zygotic embryos of the hybrid line Hi II were infected with A. tumefaciens strain EHA101 harboring a standard binary vector and cocultivated in the presence of 400 mg L-1 l-cysteine. Inclusion of l-cysteine in cocultivation medium lead to an improvement in transient β-glucuronidase expression observed in targeted cells and a significant increase in stable transformation efficiency, but was associated with a decrease in embryo response after cocultivation. The average stable transformation efficiency (no. of bialaphos-resistant events recovered per 100 embryos infected) of the present protocol was 5.5%. Southern-blot and progeny analyses confirmed the integration, expression, and inheritance of the bar and gus transgenes in R0, R1, and R2 generations of transgenic events. To our knowledge, this represents the first report in which fertile, stable transgenic maize has been routinely produced using an A. tumefaciens standard binary vector system.

Soybean pollen-tube pathway transformation Abstract. The interest in developing tissue culture-independent geneStihcoturaentsfaolr. mation methods for plants has been growing. The pollen-tube pathway transformation technique is one method; however, this method is controversial because it is difficult to duplicate and produces insufficient molecular evidence to confirm transformation. Our objective was to evaluate the robustness of the soybean pollen-tube pathway technique (Glycine max L. Merr.). Solutions of purified DNA constructs carrying a bar marker gene and a gus reporter gene or a gene of interest (npk1) were applied to severed styles of flowers 6-8h after self-pollination. The experiment was repeated 3 summers in the field, in which 4 DNA constructs and 7 soybean genotypes were tested. A total of 4793 progeny seeds were harvested from 5590 individually treated soybean flowers. All seeds were germinated and screened for transformants with herbicide spray, histochemical GUS assay, and Southern blot analysis. Although 2% of progenies showed partial resistance to the herbicide, no positive plants were identified from GUS assay and Southern analysis. Our results indicate that soybean pollen-tube pathway transformation is not reproducible.