RESUMEN

This study provides a comprehensive investigation into the structure-dependent uptake, distribution, biotransformation, and potential toxicity effects of alkyl organophosphate esters (OPEs) in hydroponic lettuce (Lactuca sativa L.). Trimethyl, triethyl, and tripropyl phosphates were readily absorbed and acropetally translocated, while tributyl, tripentyl, and trihexyl phosphates accumulated mainly in lateral roots. The acropetal translocation potential was negatively associated with log Kow values. Trimethyl and triethyl phosphates are less prone to biotransformation, while a total of 14 novel hydrolysis, hydroxylated, and conjugated metabolites were identified for other OPEs using nontarget analysis. The extent of hydroxylation decreases from tripropyl phosphate to trihexyl phosphate, but multiple hydroxylations occurred more frequently on longer chain OPEs. Further comparative toxicity test revealed that hydrolyzed and hydroxylated metabolites have stronger toxic effects on Ca2+-dependent protein kinases (CDPK) than their parent OPEs. Dibutyl 3-hydroxybutyl phosphate particularly induces upregulation of CDPK in lateral roots of lettuce, probably associated with adenine reduction that may play an important role in the self-defense and detoxification processes. This study contributes to understanding the uptake and transformation behaviors of alkyl OPEs as well as their associations with a toxic effect on lettuce. This emphasizes the necessary evaluation of the environmental risk of the use of OPEs, particularly focusing on their hydroxylated metabolites.

RESUMEN

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas 9 (CRISPR-associated protein 9) is a robust DNA-encoded, RNA-mediated sequence-specific nuclease system widely used for genome editing of various plants. Although there are many reports on the assembly of gRNAs and plant transformation, there is no single resource for the complete gene editing methodology in tomato. This chapter provides a comprehensive protocol for designing gRNAs, their assembly into the vector, plant transformation, and final mutant analysis in tomato.

Asunto(s)

RESUMEN

The rapid advancement of genetic technologies has made it possible to modify various plants through both genetic transformation and gene editing techniques. Poplar, with its rapid in vitro growth and regeneration enabling high rates of micropropagation, has emerged as a model system for the genetic transformation of woody plants. In this study, Populus × berolinensis K. Koch. (Berlin poplar) was chosen as the model organism due to its narrow leaves and spindle-shaped crown, which make it highly suitable for in vitro manipulations. Various protocols for the Agrobacterium-mediated transformation of poplar species have been developed to date. However, the genetic transformation procedures are often constrained by the complexity of the nutrient media used for plant regeneration and growth, which could potentially be simplified. Our study presents a cheaper, simplified, and relatively fast protocol for the Agrobacterium-mediated transformation of Berlin poplar. The protocol involved using internode sections without axillary buds as explants, which were co-cultivated in 10 µL droplets of bacterial suspension directly on the surface of a solid agar-based medium without rinsing and sterile paper drying after inoculation. We used only one regeneration Murashige and Skoogbased medium supplemented with BA (0.2 mg·L-1), TDZ (0.02 mg·L-1), and NAA (0.01 mg·L-1). Acetosyringone was not used as an induction agent for vir genes during the genetic transformation. Applying our protocol and using the binary plasmid pBI121 carrying the nptII selective and uidA reporter genes, we obtained the six transgenic lines of poplar. Transgenesis was confirmed through a PCR-based screening of kanamycin-selected regenerants for the presence of both mentioned genes, Sanger sequencing, and tests for detecting the maintained activity of both genes. The transformation efficiency, considering the 100 explants taken originally, was 6%.

RESUMEN

With the decline of cultivated land and increase of the population in recent years, an agricultural revolution is urgently needed to produce more food to improve the living standards of humans. As one of the foundations of synthetic biology, artificial chromosomes hold great potential for advancing crop improvement. They offer opportunities to increase crop yield and quality, while enhancing crop resistance to disease. The progress made in plant artificial chromosome technology enables selective modification of existing chromosomes or the synthesis of new ones to improve crops and study gene function. However, current artificial chromosome technologies still face limitations, particularly in the synthesis of repeat sequences and the transformation of large DNA fragments. In this review, we will introduce the structure of plant centromeres, the construction of plant artificial chromosomes, and possible methods for transforming large fragments into plant cells.

Asunto(s)

RESUMEN

Attenuated strains of the naturally occurring plant pathogen Agrobacterium tumefaciens can transfer virtually any DNA sequence of interest to model plants and crops. This has made Agrobacterium-mediated transformation (AMT) one of the most commonly used tools in agricultural biotechnology. Understanding AMT, and its functional consequences, is of fundamental importance given that it sits at the intersection of many fundamental fields of study, including plant-microbe interactions, DNA repair/genome stability, and epigenetic regulation of gene expression. Despite extensive research and use of AMT over the last 40 years, the extent of genomic disruption associated with integrating exogenous DNA into plant genomes using this method remains underappreciated. However, new technologies like long-read sequencing make this disruption more apparent, complementing previous findings from multiple research groups that have tackled this question in the past. In this review, we cover progress on the molecular mechanisms involved in Agrobacterium-mediated DNA integration into plant genomes. We also discuss localized mutations at the site of insertion and describe the structure of these DNA insertions, which can range from single copy insertions to large concatemers, consisting of complex DNA originating from different sources. Finally, we discuss the prevalence of large-scale genomic rearrangements associated with the integration of DNA during AMT with examples. Understanding the intended and unintended effects of AMT on genome stability is critical to all plant researchers who use this methodology to generate new genetic variants.

Asunto(s)

RESUMEN

Meristems are stem cell-containing structures that produce all plant organs and are therefore important targets for crop improvement. Developmental regulators control the balance and rate of cell divisions within the meristem. Altering these regulators impacts meristem architecture and, as a consequence, plant form. In this review, we discuss genes involved in regulating the shoot apical meristem, inflorescence meristem, axillary meristem, root apical meristem, and vascular cambium in plants. We highlight several examples showing how crop breeders have manipulated developmental regulators to modify meristem growth and alter crop traits such as inflorescence size and branching patterns. Plant transformation techniques are another innovation related to plant meristem research because they make crop genome engineering possible. We discuss recent advances on plant transformation made possible by studying genes controlling meristem development. Finally, we conclude with discussions about how meristem research can contribute to crop improvement in the coming decades.

Asunto(s)

RESUMEN

Objective: Understanding the regulation and function of plant genes is essential for addressing the challenges faced by modern agriculture. Plant transformation, in conjunction with fluorescence microscopy, offers a powerful approach to investigate the dynamic behavior of plant genes and the proteins they encode. We previously developed a set of Gateway-compatible tissue-specific plant transformation vectors. In this paper we aim to expand the toolkit of vectors available for Agrobacterium-mediated plant transformation and protoplast transfection. Results: Here, we introduce new Agrobacterium-mediated plant transformation vectors by introducing additional fluorophores to create the pJRA vector series. Additionally, we introduce the pLCS series of vectors, a new set of modular Gateway- and Gibson assembly-compatible vectors designed for protoplast transfection. All described vectors are available from Addgene to serve as a resource for the plant research community.

RESUMEN

The ever-increasing food requirement with globally growing population demands advanced agricultural practices to improve grain yield, to gain crop resilience under unpredictable extreme weather, and to reduce production loss caused by insects and pathogens. To fulfill such requests, genome engineering technology has been applied to various plant species. To date, several generations of genome engineering methods have been developed. Among these methods, the new mainstream technology is clustered regularly interspaced short palindromic repeats (CRISPR) with nucleases. One of the most important processes in genome engineering is to deliver gene cassettes into plant cells. Conventionally used systems have several shortcomings, such as being labor- and time-consuming procedures, potential tissue damage, and low transformation efficiency. Taking advantage of nanotechnology, the nanoparticle-mediated gene delivery method presents technical superiority over conventional approaches due to its high efficiency and adaptability in different plant species. In this review, we summarize the evolution of plant biomolecular delivery methods and discussed their characteristics as well as limitations. We focused on the cutting-edge nanotechnology-based delivery system, and reviewed different types of nanoparticles, preparation of nanomaterials, mechanism of nanoparticle transport, and advanced application in plant genome engineering. On the basis of established methods, we concluded that the combination of genome editing, nanoparticle-mediated gene transformation and de novo regeneration technologies can accelerate crop improvement efficiently in the future.

Asunto(s)

RESUMEN

The uptake, translocation, and transformation of 2,2',4,4'-tetra brominated diphenyl ether (BDE-47) in wheat (Triticum aestivum L.) were comprehensively investigated by hydroponic experiments using compound-specific stable isotope analysis (CSIA) and transcriptome analysis. The results indicated that BDE-47 was quickly adsorbed on epidermis of wheat roots and then absorbed in roots via water and anion channels as well as an active process dependent on energy. A small fraction of BDE-47 in roots was subjected to translocation acropetally, and an increase of δ13C values in shoots than roots implied that BDE-47 in roots had to cross at least one lipid bilayer to enter the vascular bundle via transporters. In addition, accompanied by the decreasing concentrations, δ13C values of BDE-47 showed the increasing trend with time in shoots, indicating occurrence of BDE-47 transformation. OH-PBDEs were detected as transformation products, and the hydroxyl group preferentially substituted at the ortho-positions of BDE-47. Based on transcriptome analysis, genes encoding polybrominated diphenyl ether (PBDE)-metabolizing enzymes, including cytochrome P450 enzymes, nitrate reductases, and glutathione S-transferases, were significantly upregulated after exposure to BDE-47 in shoots, further evidencing BDE-47 transformation. This study first reported the stable carbon isotope fractionation of PBDEs during translocation and transformation in plants, and application of CSIA and transcriptome analysis allowed systematically characterize the environmental behaviors of pollutants in plants.

Asunto(s)

RESUMEN

Agrobacterium T-DNA integration into the plant genome is essential for the process of transgenesis and is widely used for genome engineering. The importance of the non-homologous end-joining (NHEJ) protein DNA polymerase Θ, encoded by the PolQ gene, for T-DNA integration is controversial, with some groups claiming it is essential whereas others claim T-DNA integration in Arabidopsis and rice polQ mutant plant tissue. Because of pleiotropic effects of PolQ loss on plant development, scientists have previously had difficulty regenerating transgenic polQ mutant plants. We describe a protocol for regenerating transgenic polQ mutant rice plants using a sequential transformation method. This protocol may be applicable to other plant species.

RESUMEN

Agrobacterium tumefaciens-mediated plant transformation is the most dominant technique for the transformation of plants. It is used to transform monocotyledonous and dicotyledonous plants. A. tumefaciens apply for stable and transient transformation, random and targeted integration of foreign genes, as well as genome editing of plants. The Advantages of this method include cheapness, uncomplicated operation, high reproducibility, a low copy number of integrated transgenes, and the possibility of transferring larger DNA fragments. Engineered endonucleases such as CRISPR/Cas9 systems, TALENs, and ZFNs can be delivered with this method. Nowadays, Agrobacterium-mediated transformation is used for the Knock in, Knock down, and Knock out of genes. The transformation effectiveness of this method is not always desirable. Researchers applied various strategies to improve the effectiveness of this method. Here, a general overview of the characteristics and mechanism of gene transfer with Agrobacterium is presented. Advantages, updated data on the factors involved in optimizing this method, and other useful materials that lead to maximum exploitation as well as overcoming obstacles of this method are discussed. Moreover, the application of this method in the generation of genetically edited plants is stated. This review can help researchers to establish a rapid and highly effective Agrobacterium-mediated transformation protocol for any plant species.

RESUMEN

Viroids are small, non-coding, pathogenic RNAs with the ability to disturb plant developmental processes. This dysregulation redirects the morphogenesis of plant organs, significantly impairing their functionality. Citrus bark cracking viroid (CBCVd) causes detrimental developmental distortions in infected hops (Humulus lupulus) and causes significant economic losses. CBCVd can infect cells and tissues of the model plant tobacco (Nicotiana tabacum), provided it is delivered via transgenesis. The levels of CBCVd in tobacco were enhanced in plant hybrids expressing CBCVd cDNAs and either the tobacco or hop variant of TFIIIA-7ZF, a viroid-mediated splicing derivative of transcription factor IIIA, which is important for viroid replication by DNA-dependent RNA polymerase II. The TFIIIA-7ZF variants can change the tobacco morphogenesis if expressed in leaves and shoots. In addition to the splitting of shoots, the "pathomorphogenic" network in hybrid plants expressing CBCVd and HlTFIIIA-7ZF induced leaf fusions and malformations. Moreover, CBCVd can dramatically change another morphogenesis into teratomic and petal-like tissues if propagated above some limit in young transgenic tobacco microspores and anthers. By comparative RNA profiling of transgenic tobacco shoots bearing TFIIIA-7ZFs and CBCVd-transformed/infected anthers, we found a differential expression of many genes at p < 0.05. As the main common factor showing the differential up-regulation in shoot and anther tissues, a LITTLE ZIPPER 2-like transcription factor was found. We propose that this factor, which can interact as a competitive inhibitor of the also dysregulated homeobox-leucin zipper family protein (HD-ZIPIII) in apical meristem, is essential for a network responsible for some morphological changes and modifications of plant degradome within shoot meristem regulation and secondary xylem differentiation.

Asunto(s)

RESUMEN

Plants provide not only food and feed, but also herbal medicines and various raw materials for industry. Moreover, plants can be green factories producing high value bioproducts such as biopharmaceuticals and vaccines. Advantages of plant-based production platforms include easy scale-up, cost effectiveness, and high safety as plants are not hosts for human and animal pathogens. Plant cells perform many post-translational modifications that are present in humans and animals and can be essential for biological activity of produced recombinant proteins. Stimulated by progress in plant transformation technologies, substantial efforts have been made in both the public and the private sectors to develop plant-based vaccine production platforms. Recent promising examples include plant-made vaccines against COVID-19 and Ebola. The COVIFENZ® COVID-19 vaccine produced in Nicotiana benthamiana has been approved in Canada, and several plant-made influenza vaccines have undergone clinical trials. In this review, we discuss the status of vaccine production in plants and the state of the art in downstream processing according to good manufacturing practice (GMP). We discuss different production approaches, including stable transgenic plants and transient expression technologies, and review selected applications in the area of human and veterinary vaccines. We also highlight specific challenges associated with viral vaccine production for different target organisms, including lower vertebrates (e.g., farmed fish), and discuss future perspectives for the field.

RESUMEN

Fluorescent protein reporters have been widely used for monitoring the expression of target genes in various engineered organisms. Although a wide range of analytical approaches (e.g., genotyping PCR, digital PCR, DNA sequencing) have been utilized to detect and identify genome editing reagents and transgene expression in genetically modified plants, these methods are usually limited to use in the late stages of plant transformation and can only be used invasively. Here we describe GFP- and eYGFPuv-based strategies and methods for assessing and detecting genome editing reagents and transgene expression in plants, including protoplast transformation, leaf infiltration, and stable transformation. These methods and strategies enable easy, noninvasive screening of genome editing and transgenic events in plants.

Asunto(s)

RESUMEN

Agrobacterium tumefaciens-mediated plant transformation has become routine work across the world to study gene function and the production of genetically modified plants. However, several issues hamper the transformation process in a profound way, both directly and indirectly. One of the major concerns is the overgrowth of Agrobacterium, which occasionally appears after the co-cultivation phase of the explant. This phenomenon is reported in several species and seems to spoil the whole transformation process. There are multiple approaches being employed to counter this unwanted growth of bacteria in a few plant species. In reality, once the overgrowth appears, it becomes nearly impossible to cure it. Hence, for the prevention of this phenomenon, numerous factors are regulated. These factors are: explant nature, A. tumefaciens strain, T-DNA vector, co-cultivation (time and condition), acetosyringone, washing medium, antibiotics (type, concentration, combination, incubation period), etc. In this article, we discuss these factors based on available reports. It can be of immense help in formulating viable strategies to control A. tumefaciens overgrowth.

Asunto(s)

RESUMEN

KEY MESSAGE: Efficient selectable marker gene autoexcision in transgenic plants of soybean, cotton, canola, and maize is achieved by effective Cre recombinase expression. Selectable marker genes are often required for efficient generation of transgenic plants in plant transformation but are not desired once the transgenic events are obtained. We have developed Cre/loxP autoexcision systems to remove selectable marker genes in soybean, cotton, canola and maize. We tested a set of vectors with diverse promoters and identified promising promoters to drive cre expression for each of the four crops. We evaluated both the efficiency of generating primary transgenic events with low transgene copy numbers, and the frequency of marker-free progeny in the next generation. The best performing vectors gave no obvious decrease in the transformation frequency in each crop and generated homozygous marker-free progeny in the next generation. We found that effective expression of Cre recombinase for marker gene autoexcision can be species dependent. Among the vectors tested, the best autoexcision frequency (41%) in soybean transformation came from using the soybean RSP1 promoter for cre expression. The cre gene expressed by soybean RSP1 promoter with an Arabidopsis AtpE intron delivered the best autoexcision frequency (69%) in cotton transformation. The cre gene expressed by the embryo-specific eUSP88 promoter from Vicia faba conferred the best marker excision frequency (32%) in canola transformation. Finally, the cre gene expressed by the rice CDC45-1 promoter resulted in 44% autoexcision in maize transformation. The Cre/loxP recombinase system enables the generation of selectable marker-free transgenic plants for commercial product development in four agriculturally important crops and provides further improvement opportunities for more specific and better marker excision efficiency.

Asunto(s)

RESUMEN

Fiber growing inside the cotton bolls is a highly demandable product and its quality is key to the success of the textile industry. Despite the various efforts to improve cotton fiber staple length Pakistan has to import millions of bales to sustain its industrial needs. To improve cotton fiber quality Bacterial cellulose synthase (Bcs) genes (acsA, acsB) were expressed in a local cotton variety CEMB-00. In silico studies revealed a number of conserved domains both in the cotton-derived and bacterial cellulose synthases which are essential for the cellulose synthesis. Transformation efficiency of 1.27% was achieved by using Agrobacterium shoot apex cut method of transformation. The quantitative mRNA expression analysis of the Bcs genes in transgenic cotton fiber was found to be many folds higher during secondary cell wall synthesis stage (35 DPA) than the expression during elongation phase (10 DPA). Average fiber length of the transgenic cotton plant lines S-00-07, S-00-11, S-00-16 and S-00-23 was calculated to be 13.02% higher than that of the non-transgenic control plants. Likewise, the average fiber strength was found to be 20.92% higher with an enhanced cellulose content of 22.45%. The mutated indigenous cellulose synthase genes of cotton generated through application of CRISPR/Cas9 resulted in 6.03% and 12.10% decrease in fiber length and strength respectively. Furthermore, mature cotton fibers of transgenic cotton plants were found to have increased number of twists with smooth surface as compared to non-transgenic control when analyzed under scanning electron microscope. XRD analysis of cotton fibers revealed less cellulose crystallinity index in transgenic cotton fibers as compared to control fibers due to deposition of more amorphous cellulose in transgenic fibers as a result of Bcs gene expression. This study paved the way towards unraveling the fact that Bcs genes influence cellulose synthase activity and this enzyme helps in determining the fate of cotton fiber length and strength.

Asunto(s)

RESUMEN

Agrobacterium tumefaciens microbe-associated molecular pattern elongation factor Tu (EF-Tu) is perceived by orthologs of the Arabidopsis immune receptor EFR activating pattern-triggered immunity (PTI) that causes reduced T-DNA-mediated transient expression. We altered EF-Tu in A. tumefaciens to reduce PTI and improved transformation efficiency. A robust computational pipeline was established to detect EF-Tu protein variation in a large set of plant bacterial species and identified EF-Tu variants from bacterial pathogen Pseudomonas syringae pv. tomato DC3000 that allow the pathogen to escape EFR perception. Agrobacterium tumefaciens strains were engineered to substitute EF-Tu with DC3000 variants and examined their transformation efficiency in plants. Elongation factor Tu variants with rarely occurred amino acid residues were identified within DC3000 EF-Tu that mitigates recognition by EFR. Agrobacterium tumefaciens strains were engineered by expressing DC3000 EF-Tu instead of native agrobacterial EF-Tu and resulted in decreased plant immunity detection. These engineered A. tumefaciens strains displayed an increased efficiency in transient expression in both Arabidopsis thaliana and Camelina sativa. The results support the potential application of these strains as improved vehicles to introduce transgenic alleles into members of the Brassicaceae family.

Asunto(s)

RESUMEN

The high economic value of wood requires intensive breeding towards multipurpose biomass. However, long breeding cycles hamper the fast development of novel tree varieties that have improved biomass properties, are tolerant to biotic and abiotic stresses, and resilient to climate change. To speed up domestication, the integration of conventional breeding and new breeding techniques is needed. In this review, we discuss recent advances in genome editing and Cas-DNA-free genome engineering of forest trees, and briefly discuss how multiplex editing combined with multi-omics approaches can accelerate the genetic improvement of forest trees, with a focus on wood.

Asunto(s)

RESUMEN

Transient transformation in plants is a useful process for evaluating gene function. However, there is a scarcity of minimally perturbing methods for gene delivery that can be used on multiple organs, plant species, and non-excised tissues. We pioneered and demonstrated the use of vertically aligned carbon nanofiber (VACNF) arrays to efficiently perform transient transformation of different tissues with DNA constructs in multiple plant species. The VACNFs permeabilize plant tissue transiently to allow molecules into cells without causing a detectable stress response. We successfully delivered DNA into leaves, roots and fruit of five plant species (Arabidopsis, poplar, lettuce, Nicotiana benthamiana, and tomato) and confirmed accumulation of the encoded fluorescent proteins by confocal microscopy. Using this system, it is possible to transiently transform plant cells with both small and large plasmids. The method is successful for species recalcitrant to Agrobacterium-mediated transformation. VACNFs provide simple, reliable means of DNA delivery into a variety of plant organs and species.