An improved and efficient method of Agrobacterium syringe infiltration for transient transformation and its application in the elucidation of gene function in poplar

Background Forest trees have important economic and ecological value. As a model tree, poplar has played a significant role in elucidating the molecular mechanisms underlying tree biology. However, a lack of mutant libraries and time-consuming stable genetic transformation processes severely limit progress into the functional characterization of poplar genes. A convenient and fast transient transformation method is therefore needed to enhance progress on functional genomics in poplar. Methods A total of 11 poplar clones were screened for amenability to syringe infiltration. Syringe infiltration was performed on the lower side of the leaves of young soil-grown plants. Transient expression was evaluated by visualizing the reporters β-glucuronidase (GUS) and green fluorescent protein (GFP). The experimental parameters of the syringe agroinfiltration were optimized based on the expression levels of the reporter luciferase (LUC). Stably transformed plants were regenerated from transiently transformed leaf explants through callus-induced organogenesis. The functions of Populus genes in secondary cell wall-thickening were characterized by visualizing lignin deposition therein after staining with basic fuchsin. Results We greatly improved the transient transformation efficiency of syringe Agrobacterium infiltration in poplar through screening for a suitable poplar clone from a variety of clones and optimizing the syringe infiltration procedure. The selected poplar clone, Populus davidiana × P. bolleana, is amenable to Agrobacterium syringe infiltration, as indicated by the easy diffusion of the bacterial suspension inside the leaf tissues. Using this technique, we localized a variety of poplar proteins in specific intracellular organelles and illustrated the protein–protein and protein–DNA interactions. The transiently transformed leaves could be used to generate stably transformed plants with high efficiency through callus induction and differentiation processes. Furthermore, transdifferentiation of the protoxylem-like vessel element and ectopic secondary wall thickening were induced in the agroinfiltrated leaves via the transient overexpression of genes associated with secondary wall formation. Conclusions The application of P. davidiana × P. bolleana in Agrobacterium syringe infiltration provides a foundation for the rapid and high-throughput functional characterization of Populus genes in intact poplar plants, including those involved in wood formation, and provides an effective alternative to Populus stable genetic transformation.

Characterization of the belowground microbial community in a poplar-assisted bioremediation strategy for restoring a multi-contaminated soil

Background: Due to their widespread use in industrial applications in recent decades, Polychlorobiphenyls (PCBs) and heavy metals (HMs) are the most common soil contaminants worldwide, posing a risk for both ecosystems and human health.Results: a poplar-assisted bioremediation strategy has been applied for more than four years to a historically contaminated area (PCBs and HMs) in Southern Italy using the Monviso poplar clone. This clone was effective in promoting a decrease in all contaminants and an increase in soil quality in terms of organic carbon and microbial abundance. Moreover, a significant shift in the structure and functioning of the belowground microbial community was also observed when analysing both DNA and cDNA sequencing. In fact, an increase in bacterial genera belonging to Proteobacteria and able to degrade PCBs and resist HMs was observed. Moreover, the functional profiling of the microbial community predicted by PICRUSt2 made it possible to identify several genes associated with PCB transformation (e.g. bphAa, bphAb, bphB, bphC), response to HM oxidative stress (e.g. catalase, superoxide reductase, peroxidase) and HM uptake and expulsion (e.g. ABC transporters).Conclusions: This work demonstrated the effectiveness of the poplar clone used in stimulating the natural belowground microbial community to remove contaminants (phyto-assisted bioremediation) and improve the overall soil quality. It is a practical example of a nature based solution involving synergic interactions between plants and the belowground microbial community.

Comparative analysis of the assortment structure of poplar clones I-214 and Pannonia

Poplars occupy about 31.4 million ha in the world, while in Serbia poplars spread over the area of 48.000 ha. The subject of this research are artificially raised poplar plantations, consisting of poplar clone I-214 (Populus?euramericana (Dode) Guinier cl. I-214) and poplar clone Pannonia (Populus?euramericana (Dode) Guinier cl. Pannonia). Field activities of collecting data required for this research were conducted in two phases. The first phase of data collection included measurement of tree diameter. Also, the numbering, marking and recording of poplar rows, as well as each poplar in the row, was conducted. The second phase of data collection was conducted after the felling of trees that were selected for detailed measurement of the elements required for theoretical cross cutting. In accordance with the general principles of cross cutting, as well as the principles of maximum financial effect, the qualitative partition of trunks into several variants was performed. The classification of wood assortments was performed on the basis of SRPS wood standards. The share of technical wood for veneer (F and L class) in the analyzed poplar trees clone I-214 is 47.54% of the total volume of wood assortments. When it comes to the clone Pannonia, logs for cutting (quality class II), have the greatest share in total volume of wood assortments with 44.08. There is no statistically significant difference between the total volume and the value of the assortments of the two analyzed poplar clones, except when it comes to assortments for chemical exploitation where statistical differences exist.

Microcosm Experiment to Assess the Capacity of a Poplar Clone to Grow in a PCB-Contaminated Soil

Polychlorinated byphenyls (PCBs) are a class of Persistent Organic Pollutants extremely hard to remove from soil. The use of plants to promote the degradation of PCBs, thanks to synergic interactions between roots and the natural soil microorganisms in the rhizosphere, has been proved to constitute an effective and environmentally friendly remediation technique. Preliminary microcosm experiments were conducted in a greenhouse for 12 months to evaluate the capacity of the Monviso hybrid poplar clone, a model plant for phytoremediation, to grow in a low quality and PCB-contaminated soil in order to assess if this clone could be subsequently used in a field experiment. For this purpose, three different soil conditions (Microbiologically Active, Pre-sterilized and Hypoxic soils) were set up in order to assess the capacity of this clone to grow in the polluted soil in these different conditions and support the soil microbial community activity. The growth and physiology (chlorophyll content, chlorophyll fluorescence, ascorbate, phenolic compounds and flavonoid contents) of the poplar were determined. Moreover, chemical analyses were performed to assess the concentrations of PCB indicators in soil and plant roots. Finally, the microbial community was evaluated in terms of total abundance and activity under the different experimental conditions. Results showed that the poplar clone was able to grow efficiently in the contaminated soil and to promote microbial transformations of PCBs. Plants grown in the hypoxic condition promoted the formation of a higher number of higher-chlorinated PCBs and accumulated lower PCBs in their roots. However, plants in this condition showed a higher stress level than the other microcosms, producing higher amounts of phenolic, flavonoid and ascorbate contents, as a defence mechanism.