Plant transformation: Problems and strategies for practical application

Plant transformation is now a core research tool in plant biology and a practical tool for cultivar improvement. There are verified methods for stable introduction of novel genes into the nuclear genomes of over 120 diverse plant species. This review examines the criteria to verify plant transformation; the biological and practical requirements for transformation systems; the integration of tissue culture, gene transfer, selection, and transgene expression strategies to achieve transformation in recalcitrant species; and other constraints to plant transformation including regulatory environment, public perceptions, intellectual property, and economics. Because the costs of screening populations showing diverse genetic changes can far exceed the costs of transformation, it is important to distinguish absolute and useful transformation efficiencies. The major technical challenge facing plant transformation biology is the development of methods and constructs to produce a high proportion of plants showing predictable transgene expression without collateral genetic damage. This will require answers to a series of biological and technical questions, some of which are defined.

Review: intellectual property aspects of plant transformation

One of the recurring themes of the debates concerning the application of genetic transformation technology has been the role of Intellectual Property Rights (IPR). This term covers both the content of patents and the confidential expertise usually related to methodology and referred to as 'Trade Secrets'. This review explains the concepts behind patent protection, and discusses the wide-ranging scope of existing patents that cover all aspects of transgenic technology, from selectable markers and novel promoters to methods of gene introduction. Although few of the patents in this area have any real commercial value, there are a small number of key patents that restrict the 'freedom to operate' of new companies seeking to exploit the methods. Over the last 20 years, these restrictions have forced extensive cross-licensing between ag-biotech companies and have been one of the driving forces behind the consolidation of these companies. Although such issues are often considered of little interest to the academic scientist working in the public sector, they are of great importance in any discussion of the role of 'public-good breeding' and of the relationship between the public and private sectors.

“Agrolistic” transformation of plant cells: Integration of T-strands generated in planta

We describe a novel plant transformation technique, termed “agrolistic,” that combines the advantages of the Agrobacterium transformation system with the high efficiency of biolistic DNA delivery. Agrolistic transformation allows integration of the gene of interest without undesired vector sequence. The virulence genes virD1 and virD2 from Agrobacterium tumefaciens that are required in bacteria for excision of T-strands from the tumor-inducing plasmid were placed under the control of the CaMV35S promoter and codelivered with a target plasmid containing border sequences flanking the gene of interest. Transient expression assays in tobacco and in maize cells indicated that vir gene products caused strand-specific nicking in planta at the right border sequence, similar to VirD1/VirD2-catalyzed T-strand excision observed in Agrobacterium. Agrolistically transformed tobacco calli were obtained after codelivery of virD1 and virD2 genes together with a selectable marker flanked by border sequences. Some inserts exhibited right junctions with plant DNA that corresponded precisely to the sequence expected for T-DNA (portion of the tumor-inducing plasmid that is transferred to plant cells) insertion events. We designate these as “agrolistic” inserts, as distinguished from “biolistic” inserts. Both types of inserts were found in some transformed lines. The frequency of agrolistic inserts was 20% that of biolistic inserts.

Optimization of particle bombardment parameters for the genetic transformation of Brazilian maize inbred lines

The objective of this work was to develop a genetic transformation system for tropical maize genotypes via particle bombardment of immature zygotic embryos. Particle bombardment was carried out using a genetic construct with bar and uidA genes under control of CaMV35S promoter. The best conditions to transform maize tropical inbred lines L3 and L1345 were obtained when immature embryos were cultivated, prior to the bombardment, in higher osmolarity during 4 hours and bombarded at an acceleration helium gas pressure of 1,100 psi, two shots per plate, and a microcarrier flying distance of 6.6 cm. Transformation frequencies obtained using these conditions ranged from 0.9 to 2.31%. Integration of foreign genes into the genome of maize plants was confirmed by Southern blot analysis as well as bar and uidA gene expressions. The maize genetic transformation protocol developed in this work will possibly improve the efficiency to produce new transgenic tropical maize lines expressing desirable agronomic characteristics.

Derivatives of the Antimicrobial Peptide BP100 for Expression in Plant Systems

Production of antimicrobial peptides in plants constitutes an approach for obtaining them in high amounts. However, their heterologous expression in a practical and efficient manner demands some structural requirements such as a minimum size, the incorporation of retention signals to assure their accumulation in specific tissues, and the presence of protease cleavage amino acids and of target sequences to facilitate peptide detection. Since any sequence modification may influence the biological activity, peptides that will be obtained from the expression must be screened prior to the synthesis of the genes for plant transformation. We report herein a strategy for the modification of the antimicrobial undecapeptide BP100 that allowed the identification of analogues that can be expressed in plants and exhibit optimum biological properties. We prepared 40 analogues obtained by incorporating repeated units of the antimicrobial undecapeptide, fragments of natural peptides, one or two AGPA hinges, a Gly or Ser residue at the N-terminus, and a KDEL fragment and/or the epitope tag54 at the C-terminus. Their antimicrobial, hemolytic and phytotoxic activities, and protease susceptibility were evaluated. Best sequences contained a magainin fragment linked to the antimicrobial undecapeptide through an AGPA hinge. Moreover, since the presence of a KDEL unit or of tag54 did not influence significantly the biological activity, these moieties can be introduced when designing compounds to be retained in the endoplasmic reticulum and detected using a complementary epitope. These findings may contribute to the design of peptides to be expressed in plants

Regeneration and Transformation of Millets

Millets are versatile in tolerating to diverse climatic and soil conditions such as poor soil fertility and moisture deficit. Establishing optimum regeneration method for each millet type and ecotype is a pre-requisite prior to embarking on plant transformation as successes in plant transformation is largely dependent on the efficiency of regeneration. Various studies made to identify optimum regeneration and transformation methods as well as prospects of applying advanced techniques to these vital but under-studied crops of developing world are discussed.

Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium

Import of DNA into mammalian nuclei is generally inefficient. Therefore, one of the current challenges in human gene therapy is the development of efficient DNA delivery systems. Here we tested whether bacterial proteins could be used to target DNA to mammalian cells. Agrobacterium tumefaciens, a plant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells. Agrobacterium-mediated T-DNA transfer to plant cells is the only known example for interkingdom DNA transfer and is widely used for plant transformation. Agrobacterium virulence proteins VirD2 and VirE2 perform important functions in this process. We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA) in vitro. These complexes were tested for import into HeLa cell nuclei. Import of ssDNA required both VirD2 and VirE2 proteins. A VirD2 mutant lacking its C-terminal nuclear localization signal was deficient in import of the ssDNA–protein complexes into nuclei. Import of VirD2–ssDNA–VirE2 complexes was fast and efficient, and was shown to depended on importin α, Ran, and an energy source. We report here that the bacterium-derived and plant-adapted protein–DNA complex, made in vitro, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway. This demonstrates the potential of our approach to enhance gene transfer to animal cells.

Stable transfer of intact high molecular weight DNA into plant chromosomes.

In conjunction with an enhanced system for Agrobacterium-mediated plant transformation, a new binary bacterial artificial chromosome (BIBAC) vector has been developed that is capable of transferring at least 150 kb of foreign DNA into a plant nuclear genome. The transferred DNA appears to be intact in the majority of transformed tobacco plants analyzed and is faithfully inherited in the progeny. The ability to introduce high molecular weight DNA into plant chromosomes should accelerate gene identification and genetic engineering of plants and may lead to new approaches in studies of genome organization.

A bacterial artificial chromosome library of Lotus japonicus constructed in an Agrobacterium tumefaciens-transformable vector

We constructed a BAC library of the model legume Lotus japonicus with a 6-to 7-fold genome coverage. We used vector PCLD04541, which allows direct plant transformation by BACs. The average insert size is 94 kb. Clones were stable in Escherichia coli and Agrobacterium tumefaciens.

Towards modification of Medicago truncatula epigenome: genome editing with engineered nucleases

Periodic drought is the primary limitation of plant growth and crop yield. The rise of water demand caused by the increase in world population and climate change, leads to one of the biggest challenges of modern agriculture: to increase food and feed production. De novo DNA methylation is a process regulated by small interfering RNA (siRNAs), which play a role in plant response and adaptation to abiotic stress. In the particular case of water deficit, growing evidences suggest a link between the siRNA pathways and drought response in the model legume Medicago truncatula. As a first step to understand the role of DNA methylation under water stress, we have set up several bioinformatics and molecular methodologies allowing the design of Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems and the assembly of TALENs (transcription activator-like effector nucleases), to target both dicer-like 3 (MtDCL3) and RNA-Dependent RNA polymerase (MtRDR2), enzymes of the RNA-directed DNA methylation pathway. TALENs efficiency was evaluated prior to plant transformation by a yeast-based assay using two different strategies to test TALENs activity: Polyacrylamide gel electrophoresis (PAGE) and Single strand conformation polymorphisms (SSCP). In this assay, yeast cells triple transformation emerged as good and rapid alternative to laborious yeast mating strategies. PAGE analysis might be a valuable tool to test TALENs efficacy in vivo if we could increase TALENs activity. SSCP-based approach proved to be ineffective due to the generation of several false positives. TALENs and CRISPR/Cas9 system constructed and designed in this work will in the future certainly enable the successful disruption of DCL3 and RDR2 genes and shed the light on the relationship between plant stress resistance and epigenetic regulation mediated by siRNAs in M.truncatula.

A simple and reliable method for the screening of transgenic tobacco plants

Even though much improvement has been made in plant transformation methods, the screening of transgenic plants is often a laborious work. Most approaches for detecting the transgene in transformed plants are still timeconsuming, and can be quite expensive. The objective of this study was to search for a simpler method to screen for transgenic plants. The infiltration of kanamycin (100 mg/mL) into tobacco leaves resulted in conspicuous chlorotic spots on the non-transgenic plant leaves, while no spots were seen on the leaves of transformed plants. This reaction occurred regardless of age of the tested plants, and the method has proven to be simple, fast, non-destructive, relatively cheap, and reliable. These results were comparable to those obtained by the polymerase chain reaction (PCR) amplification of the transgene using specific primers.

GUS gene expression driven by a citrus promoter in transgenic tobacco and 'Valencia' sweet orange

The objective of this work was the transformation of tobacco and 'Valencia' sweet orange with the GUS gene driven by the citrus phenylalanine ammonia-lyase (PAL) gene promoter (CsPP). Transformation was accomplished by co-cultivation of tobacco and 'Valência' sweet orange explants with Agrobacterium tumefaciens containing the binary vector CsPP-GUS/2201. After plant transformation and regeneration, histochemical analyses using GUS staining revealed that CsPP promoter preferentially, but not exclusively, conferred gene expression in xylem tissues of tobacco. Weaker GUS staining was also detected throughout the petiole region in tobacco and citrus CsPP transgenic plants.

Traditional and transgenic strategies for controlling tomato-infecting begomoviruses

Viruses of to the family Geminiviridae are considered some of the most important pathogens in tropical and subtropical regions of the world. Members of one Geminiviridae genus, Begomovirus, have been causing severe losses, particularly in tomato (Lycopersicon esculentum) production in the Americas and the Caribbean. Several new begomoviruses have been reported in the region and, at least one, Tomato yellow leaf curl virus (TYLCV), has been brought in from the Old World via infected transplants. In addition, the recombination events that are playing an important role in Begomovirus diversity have increased the complexity of their control. This scenario has led to the search for control measures that go beyond traditional host genetic resistance, chemical controls and cultural practices. In this review, besides the recommended classical control measures, transgenic approaches will be discussed, as well as the mechanisms involved in their successful control of viruses.

A heavy metal P-type ATPase OsHMA4 prevents copper accumulation in rice grain

Acknowledgements We thank B. Lahner, E. Yakubova and S. Rikiishi for ICP-MS analysis, N. Komiyama, Iowa State University Plant Transformation Facility and Prashant Hosmani for generation of transgenic rice, K. Wang for providing pTF101.1 vector and N. Verbruggen for providing pYES2 and pYEC2/CT-GFP vectors. We also thank Rice T-DNA Insertion Sequence Database center for providing the T-DNA insertion line and X. Wang, T. Zheng and Z. Li for accessing 3 K rice genome sequence, and Graeme Paton for helpful discussions on Cu bioavailability in water-logged soils. This research was supported by a Grant-in-Aid for Specially promoted Research (JSPS KAKENHI Grant Number 16H06296 to J.F.M), and the US National Science Foundation, Plant Genome Research Program (Grant #IOS 0701119 to D.E.S., M.L.G. and S.R.M.P.).