Design principles for synthetic control systems to engineer plants.

KEY MESSAGE: Synthetic control systems have led to significant advancement in the study and engineering of unicellular organisms, but it has been challenging to apply these tools to multicellular organisms like plants. The ability to predictably engineer plants will enable the development of novel traits capable of alleviating global problems, such as climate change and food insecurity. Engineering predictable multicellular phenotypes will require the development of synthetic control systems that can precisely regulate how the information encoded in genomes is translated into phenotypes. Many efficient control systems have been developed for unicellular organisms. However, it remains challenging to use such tools to study or engineer multicellular organisms. Plants are a good chassis within which to develop strategies to overcome these challenges, thanks to their capacity to withstand large-scale reprogramming without lethality. Additionally, engineered plants have great potential for solving major societal problems. Here we briefly review the progress of control system development in unicellular organisms, and how that information can be leveraged to characterize control systems in plants. Further, we discuss strategies for developing control systems designed to regulate the expression of transgenes or endogenous loci and generate dosage-dependent or discrete traits. Finally, we discuss the utility that mathematical models of biological processes have for control system deployment.

GRF-GIF chimeric proteins enhance in vitro regeneration and Agrobacterium-mediated transformation efficiencies of lettuce (Lactuca spp.).

KEY MESSAGE: GRF-GIF chimeric proteins from multiple source species enhance in vitro regeneration in both wild and cultivated lettuce. In addition, they enhance regeneration in multiple types of lettuce including butterheads, romaines, and crispheads. The ability of plants to regenerate in vitro has been exploited for use in tissue culture systems for plant propagation, plant transformation, and genome editing. The success of in vitro regeneration is often genotype dependent and continues to be a bottleneck for Agrobacterium-mediated transformation and its deployment for improvement of some crop species. Manipulation of transcription factors that play key roles in plant development such as BABY BOOM, WUSCHEL, and GROWTH-REGULATING FACTORs (GRFs) has improved regeneration and transformation efficiencies in several plant species. Here, we compare the efficacy of GRF-GIF gene fusions from multiple species to boost regeneration efficiency and shooting frequency in four genotypes of wild and cultivated lettuce (Lactuca spp. L.). In addition, we show that GRF-GIFs with mutated miRNA 396 binding sites increase regeneration efficiency and shooting frequency when compared to controls. We also present a co-transformation strategy for increased transformation efficiency and recovery of transgenic plants harboring a gene of interest. This strategy will enhance the recovery of transgenic plants of other lettuce genotypes and likely other crops in the Compositae family.

Molecular Determinants of in vitro Plant Regeneration: Prospects for Enhanced Manipulation of Lettuce (Lactuca sativa L.).

In vitro plant regeneration involves dedifferentiation and molecular reprogramming of cells in order to regenerate whole organs. Plant regeneration can occur via two pathways, de novo organogenesis and somatic embryogenesis. Both pathways involve intricate molecular mechanisms and crosstalk between auxin and cytokinin signaling. Molecular determinants of both pathways have been studied in detail in model species, but little is known about the molecular mechanisms controlling de novo shoot organogenesis in lettuce. This review provides a synopsis of our current knowledge on molecular determinants of de novo organogenesis and somatic embryogenesis with an emphasis on the former as well as provides insights into applying this information for enhanced in vitro regeneration in non-model species such as lettuce (Lactuca sativa L.).

Fast Fluorescence Titration Quantification of Plasmid DNA with DNA Attractive Magnetic Nanoparticles.

Fluorescence titration using magnetic nanoparticles (FTMN) was performed as a rapid, inexpensive, and simple method for quantifying the amount of fluorophore-intercalated plasmid DNA on these DNA attractive nanoparticles. Binding of the propidium iodide (PI)-intercalated DNA (PI/DNA) to polyethylenimine (PEI)-coated monodisperse iron oxide magnetic nanoparticles (PEI-MNs) was confirmed with transmission electron microscopy after the two species were mixed in water for less than a minute. The amount of DNA on PEI-MNs in aqueous solution, however, could not be easily determined using direct fluorescence measurements due to strong scattering by aggregated MNs, especially at high nanoparticle concentrations. Instead, fluorescence measurements were taken immediately after the solution of PI/DNA and PEI-MN mixtures was treated with a magnet to pull the PEI-MNs out of the solution. The detected fluorescence signal of the remaining free PI/DNA in the solution decreased as the concentration of PEI-MNs in the pre-treated solutions increased, resulting in a titration curve, which was used to determine the amount of DNA on MNs, the dissociation constant, and binding energy after the concentration of PEI-MNs was calibrated with microwave-plasma atomic emission spectroscopy. Quantitative polymerase chain reaction was used to understand the binding of DNA to MNs and to measure the amount of free PI/DNA in solution, and the results were similar to those obtained with the FTMN method.