Comparative Review of Plant Transformation Techniques

Plant transformation is now an important biotechnological tool in plant biology and a practical tool for transgenic plant development. There are many verified methods for stable introduction of novel genes into the nuclear genomes of diverse plant species. As a result, gene transfer and regeneration of transgenic plants are no longer the factors limiting the development and application of practical transformation systems for many plant species. However, the desire for higher transformation efficiency has stimulated work on not only improving various existing methods but also in inventing novel methods. Different methods of transferring the gene into plant cells have been developed and continuous efforts have been made to increase its efficiency. Both direct and indirect methods of gene transfer have their own merits and demerits. Efforts have been made continuously to eliminate drawbacks and to develop an easy and eco-friendly method to transfer foreign genes. Many methods of genetic transformation have been proposed and tried in the laboratories, but most of them result to transient expressions. However, transformation work based on particle bombardment with DNA coated micro projectiles and Agrobacterium mediated transformation have proved to be promising in producing stable transgenic plants from a range of plant species.

Infinite-state graph transformation systems under adverse conditions

We present an approach for modeling adverse conditions by graph transformation systems. To this end, we introduce joint graph transformation systems which involve a system, an interfering environment, and an automaton modeling their interaction. For joint graph transformation systems, we present notions of correctness under adverse conditions. Some instances of correctness are expressible in LTL (linear temporal logic), or in CTL (computation tree logic), respectively. In these cases, verification of joint graph transformation systems is reduced to temporal model checking. To handle infinite state spaces, we incorporate the concept of well-structuredness. We discuss ideas for the verification of joint graph transformation systems using results based on well-structuredness.

Natural transformation in Gram-negative bacteria thriving in extreme environments: from genes and genomes to proteins, structures and regulation

Extremophilic prokaryotes live under harsh environmental conditions which require far-reaching cellular adaptations. The acquisition of novel genetic information via natural transformation plays an important role in bacterial adaptation. This mode of DNA transfer permits the transfer of genetic information between microorganisms of distant evolutionary lineages and even between members of different domains. This phenomenon, known as horizontal gene transfer (HGT), significantly contributes to genome plasticity over evolutionary history and is a driving force for the spread of fitness-enhancing functions including virulence genes and antibiotic resistances. In particular, HGT has played an important role for adaptation of bacteria to extreme environments. Here, we present a survey of the natural transformation systems in bacteria that live under extreme conditions: the thermophile Thermus thermophilus and two desiccation-resistant members of the genus Acinetobacter such as Acinetobacter baylyi and Acinetobacter baumannii. The latter is an opportunistic pathogen and has become a world-wide threat in health-care institutions. We highlight conserved and unique features of the DNA transporter in Thermus and Acinetobacter and present tentative models of both systems. The structure and function of both DNA transporter are described and the mechanism of DNA uptake is discussed.

Frontiers in the Standardization of the Plant Platform for High Scale Production of Vaccines

The recent COVID-19 pandemic has highlighted the value of technologies that allow a fast setup and production of biopharmaceuticals in emergency situations. The plant factory system can provide a fast response to epidemics/pandemics. Thanks to their scalability and genome plasticity, plants represent advantageous platforms to produce vaccines. Plant systems imply less complicated production processes and quality controls with respect to mammalian and bacterial cells. The expression of vaccines in plants is based on transient or stable transformation systems and the recent progresses in genome editing techniques, based on the CRISPR/Cas method, allow the manipulation of DNA in an efficient, fast, and easy way by introducing specific modifications in specific sites of a genome. Nonetheless, CRISPR/Cas is far away from being fully exploited for vaccine expression in plants. In this review, an overview of the potential conjugation of the renewed vaccine technologies (i.e., virus-like particles - VLPs, and industrialization of the production process) with genome editing to produce vaccines in plants is reported, illustrating the potential advantages in the standardization of the plant platforms, with the overtaking of constancy of large-scale production challenges, facilitating regulatory requirements and expediting the release and commercialization of the vaccine products of genome edited plants.

Formal testing of timed graph transformation systems using metric temporal graph logic

Embedded real-time systems generate state sequences where time elapses between state changes. Ensuring that such systems adhere to a provided specification of admissible or desired behavior is essential. Formal model-based testing is often a suitable cost-effective approach. We introduce an extended version of the formalism of symbolic graphs, which encompasses types as well as attributes, for representing states of dynamic systems. Relying on this extension of symbolic graphs, we present a novel formalism of timed graph transformation systems (TGTSs) that supports the model-based development of dynamic real-time systems at an abstract level where possible state changes and delays are specified by graph transformation rules. We then introduce an extended form of the metric temporal graph logic (MTGL) with increased expressiveness to improve the applicability of MTGL for the specification of timed graph sequences generated by a TGTS. Based on the metric temporal operators of MTGL and its built-in graph binding mechanics, we express properties on the structure and attributes of graphs as well as on the occurrence of graphs over time that are related by their inner structure. We provide formal support for checking whether a single generated timed graph sequence adheres to a provided MTGL specification. Relying on this logical foundation, we develop a testing framework for TGTSs that are specified using MTGL. Lastly, we apply this testing framework to a running example by using our prototypical implementation in the tool AutoGraph.

An Improved Transformation System for Phytophthora cinnamomi Using Green Fluorescent Protein

Phytophthora cinnamomi is a destructive pathogen causing root rot and dieback diseases on hundreds of economically and ecologically important plant species. Effective transformation systems enable modifications of candidate genes to understand the pathogenesis of P. cinnamomi. A previous study reported a polyethylene glycol and calcium dichloride (PEG/CaCl2)-mediated protoplast transformation method of P. cinnamomi. However, the virulence of the transformants was compromised. In this study, we selected ATCC 15400 as a suitable wild-type isolate for PEG/CaCl2 transformation using the green fluorescent protein after screening 11 P. cinnamomi isolates. Three transformants, namely, PcGFP-1, PcGFP-3, and PcGFP-5, consistently displayed a green fluorescence in their hyphae, chlamydospores, and sporangia. The randomly selected transformant PcGFP-1 was as virulent as the wild-type isolate in causing hypocotyl lesions on lupines. Fluorescent hyphae and haustoria were observed intracellularly and intercellularly in lupine tissues inoculated with PcGFP-1 zoospores. The potential application of this improved transformation system for functional genomics studies of P. cinnamomi is discussed.