Liriodendron is a genus of the magnolia family comprised of two flowering tree species that produce hardwoods of great ecological and economic value. However, only a limited amount of genetic research has been performed on the Liriodendron genus partly because transient or stable transgenic trees have been difficult to produce. In general, transient expression systems are indispensable for rapid, high-throughput screening and systematic characterization of gene functions at a low cost; therefore, development of such a system for Liriodendron would provide a necessary step forward for research on Magnoliaceae and other woody trees. Herein, we describe an efficient and rapid protocol for preparing protoplasts from the leaf mesophyll tissue of a Liriodendron hybrid and an optimized system for polyethylene glycol–mediated transient transfection of the protoplasts. Because the leaves of the Liriodendron hybrid are waxy, we formulated an enzyme mix containing 1.5% (w/v) Cellulase R-10, 0.5% (w/v) Macerozyme R-10, and 0.1% (w/v) Pectolyase Y-23 to efficiently isolate protoplasts from the Liriodendron hybrid leaf mesophyll tissue in 3 h. We optimized Liriodendron protoplast transfection efficiency by including 20 μg plasmid DNA per 104 protoplasts, a transformation time of 20 min, and inclusion of 20% (w/v) polyethylene glycol 4000. After integrating the Liriodendron WOX1 gene into pJIT166-GFP to produce a WOX1-GFP fusion product and transfecting it into isolated protoplasts, LhWOX1-GFP was found to localize to the nucleus according to its green fluorescence.
Intensive irrigation coupled with excessive nitrogen (N) fertilizer input has resulted in high soil greenhouse gas (GHG) emissions in vegetable cropping systems. Biochar as a soil amendment has been advocated as a desirable option to reduce GHG emissions in agricultural systems, but its interactive effects with soil N availability in vegetable systems have yet to be clarified. We performed a field study to examine how biochar interacts with N fertilizer in driving annual methane (CH4) and nitrous oxide (N2O) emissions from an intensively-irrigated greenhouse vegetable cropping system acting as both sources of atmospheric CH4 and N2O in subtropical China. Biochar amendment significantly increased soil CH4 emissions by 33% and 85%, while it decreased soil N2O emissions by 22% and 12% with and without N fertilizer input, respectively. Fertilizer N combination weakened the positive response of CH4 to biochar while it enhanced the mitigation potential of biochar for N2O. Annual direct emission factors of fertilizer N for N2O were estimated to be 1.35% and 1.94% for the fields with and without biochar amendment, respectively. Annual flux-sustained global warming potential (SGWP) and greenhouse gas intensity (GHGI) were significantly decreased by biochar amendment, and this mitigation effect was enhanced with fertilizer N combination. Altogether, we highlight that biochar can reconcile higher yield and lower climatic impact in intensive vegetable cropping systems in subtropical China, particularly in vegetable soils with high N availability.
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