Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells

Early detection of plant transformation events is necessary for the rapid establishment and optimization of plant transformation protocols. We have assessed modified versions of the green fluorescent protein (GFP) from Aequorea victoria as early reporters of plant transformation using a dissecting fluorescence microscope with appropriate filters. Gfp-expressing cells from four different plant species (sugarcane, maize, lettuce, and tobacco) were readily distinguished, following either Agrobacterium-mediated or particle bombardment-mediated transformation. The identification of gfp-expressing sugarcane cells allowed for the elimination of a high proportion of non-expressing explants and also enabled visual selection of dividing transgenic cells, an early step in the generation of transgenic organisms. The recovery of transgenic cell clusters was streamlined by the ability to visualize gfp-expressing tissues in vitro

PEA-CLARITY: 3D molecular imaging of whole plant organs

Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration. Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in Tobacco and Arabidopsis leaves. Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material, thus facilitating protein localisation of intact tissue in 3D whilst retaining cellular structure

Growth and metabolism in sugarcane are altered by the creation of a new hexose-phosphate sink

An efficient in planta sugarcane-based production system may be realized by coupling the synthesis of alternative products to the metabolic intermediates of sucrose metabolism, thus taking advantage of the sucrose-producing capability of the plant. This was evaluated by synthesizing sorbitol in sugarcane (Saccharum hybrids) using the Malus domestica sorbitol-6-phosphate dehydrogenase gene (mds6pdh). Mature transgenic sugarcane plants were compared with untransformed sugarcane variety Q117 by evaluation of the growth, metabolite levels and extractable activity of relevant enzymes. The average amounts of sorbitol detected in the most productive line were 120 mg/g dry weight (equivalent to 61% of the soluble sugars) in the leaf lamina and 10 mg/g dry weight in the stalk pith. The levels of enzymes involved in sucrose synthesis and cleavage were elevated in the leaves of plants accumulating sorbitol, but this did not affect sucrose accumulation in the culm. The activity of oxidative reactions in the pentose phosphate pathway and the non-reversible glyceraldehyde-3-phosphate dehydrogenase reaction were elevated to replenish the reducing power consumed by sorbitol synthesis. Sorbitol-producing sugarcane generated 30%−40% less aerial biomass and was 10%−30% shorter than control lines. Leaves developed necrosis in a pattern characteristic of early senescence, and the severity was related to the relative quantity of sorbitol accumulated. When the Zymomonas mobilis glucokinase (zmglk) gene was co-expressed with mds6pdh to increase the production of glucose-6-phosphate, the plants were again smaller, indicating that glucose-6-phosphate deficiency was not responsible for the reduced growth. In summary, sorbitol hyperaccumulation affected sugarcane growth and metabolism, but the outcome was not lethal for the plant. This work also demonstrated that impressive yields of alternative products can be generated from the intermediates of sucrose metabolism in Saccharum spp