Search

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. [ABSTRACT FROM AUTHOR]

Genetic engineering of plants can boost disease resistance, enhance crop traits, and ultimately improve agricultural productivity. Several approaches to plant bioengineering have been successful in recent decades. Nanomaterials (NMs) can be customized and fabricated with targeting capabilities, making them well-suited for bioengineering applications. These NMs include organic, inorganic, and composite materials with many different structures, including nanofibers, nanoparticles (NPs), and nanomembranes. Protoplasts are often used as target cells because they lack a cell wall and are more likely to endocytose NM. In this review, the efficacy of NMs in delivering genetic material to protoplasts is examined. The challenges associated with protoplast generation and optimization of protocols for transformation are explored and the possible advantages of NMs in this process are identified. The chemical properties of these NMs in relation to their potency is briefly discussed. Ultimately, this technology is evolving and our understanding of NMs and the requirement for migration through the cellular membrane is still missing several key pieces of information. The next decades will likely produce important new insights that will have important impacts in this field.

Millets, the sixth most-grown group of crops in the drylands, support the livelihood of many small-holder farmers in the region. Being one of the most nutritious groups of crops, their production has been increasing since the last decade to meet the demands of the world's ever-increasing population. Since its discovery, CRISPR/Cas-mediated gene editing technology has revolutionized trait improvement in numerous crops by enabling targeted insertions and deletions at specific gene sequences. With advancements like base editing and prime editing, which offer precise modifications at the nucleotide level, this technology holds great promise for enhancing millets by targeting genes responsible for key traits. The updated sequence information in the public domain makes it possible to modify certain genic regions using the CRISPR/Cas-mediated gene editing technology to develop millet crops with improved agronomical properties. The review explores each component of the editing toolbox in millets, including the gRNA designing tools, types of Cas nucleases, and promoters to be considered for enhanced and efficient gene editing in millets. We have discussed fundamental information available to successfully employ CRISPR/Cas-mediated gene editing in millets, such as the availability of genomic information and plant transformation methods. Finally, we have highlighted the limitations of employing this novel technology in millet crops by providing future directions and immediate candidate genes that could be targeted to improve various traits in millet crops. [ABSTRACT FROM AUTHOR]

The economic important medicinal plant, Panax ginseng, encompasses a diverse array of pharmacologically beneficial ginsenosides governed by complex metabolic pathways. The cultivation of P. ginseng over an extended period of time poses many challenges to fulfill market requirements. The utilization of in vitro tissue culture presents a viable alternative approach for the generation of P. ginseng root biomass and metabolites. Harnessing the root inducing plasmid (Ri plasmid) of Agrobacterium rhizogenes in the transformation process to create hairy roots in P. ginseng could be a useful metabolic engineering technique. In this investigation, the transformation efficiency and biomass production of five distinct ginseng genotypes were evaluated. Of them, the ‘Yunpoong’ cultivar, and a local landrace ‘Ganghwa’ had the highest transformation efficiencies of 66.11% and 65.00%, respectively. The biomass production of transgenic hairy roots was 1.5–2.1 times faster than that of non-transgenic adventitious roots without hormone supplementation. Various ginsenosides such as Rg1, Rf, Rh1, Rb1, Rb2, Rd, F2, and Rg3, were found to be similar or greater in the hairy roots when compared to the concentrations observed in adventitious roots. Furthermore, the ginsenoside contents of cultivated roots are similar to those cultured in a bioreactor. The findings provide fundamental insights into the metabolic engineering of ginseng, facilitating the in vitro production of ginsenosides.Key message: In vitro root culture is an ideal method for the large-scale production of biomass for the slowly growing ginseng plant. We report the improved transgenic hairy root culture system for production of biomass and ginsenosides via Ri plasmid transformation using five different ginseng genotypes. This transformation system provides a basic tool for the future metabolic engineering of ginseng. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: Agrobacterium‐mediated transformation (AMT) of plants is used extensively as a tool to create new genetic variants for research purposes or agricultural needs. In this work, we summarize the molecular mechanisms involved in AMT and the effects on plant genomes resulting from the integration of exogenous DNA through this method. [ABSTRACT FROM AUTHOR]

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. This review discusses developmental regulators that affect meristem organization and plant form. The authors provide several examples showing how these regulators have been manipulated by crop breeders to enhance agronomic traits and highlight the significance of plant transformation techniques for crop genome engineering. Precisely fine-tuning meristem regulators will expand our ability to improve crop performance under various environmental conditions in the future. [ABSTRACT FROM AUTHOR]

In the majority of plant biotechnology laboratories throughout the world, plant transformation is a common practice to improve several traits of plants, particularly grain yield. During the experiments, only a small percentage of cells transform in the targeted population. For selection of transformed cells, it is necessary to use the selectable markers such as Neomycin phosphotransferase (nptII), Chloramphenicolacetyle transferase (Cat), Hygromycin phosphotransferase (hph), Streptomycin phosphotransferase (spt), Phosphinothrycin acetyletransferase (pat) and Dihydrofolate reductase (dhf). The majority of these and other markers detoxify different antibiotics including paromomycin, kanamycin, hygromycin, neomycin, and streptomycin etc. But as these antibiotics become resistant, most of the markers raise serious safety concerns to human health. Additionally, in case of multiple genes transfer, it also limits the availability of these selectable markers. Keeping in view the limitations of selectable markers, production of marker-free transgenic plants is becoming the global trend. Current review explored the probabilities and prospects to cope with these issues. This review describes in detail the various plant transformation systems for the development of selectable marker gene (SMG) free transgenic plants. Replacement of selectable marker with screenable marker and some worldwide examples of SMG free crop plants produced by these strategies are also discussed. [ABSTRACT FROM AUTHOR]

Die Entwicklung nachhaltiger Anbaumethoden für Medizinalpflanzen zur Deckung des steigenden Bedarfs an qualitativ hochwertiger Rohware ist für die pharmazeutische Industrie von großer Bedeutung. Gleichzeitig gilt es Umweltauswirkungen zu minimieren und die Biodiversität zu schützen. Im Rahmen der Initiative BioökonomieREVIER zur Bewältigung des Strukturwandels aufgrund des Braunkohleausstiegs soll das Rheinische Revier zu einer Modellregion für nachhaltige Bioökonomie entwickelt werden. Diesbezüglich werden Projekte gefördert, in denen durch eine enge Zusammenarbeit zwischen Wissenschaft und Wirtschaft innovative Lösungen in Form von Innovationslaboren etabliert und umgesetzt werden. Ein Beispiel ist das Projekt „Circular PhytoREVIER“, das die nachhaltige Wertschöpfung mit Medizinalpflanzen im Rheinischen Revier fokussiert. Dafür haben die drei beteiligten Wissenschaftspartner Fraunhofer IME, Fraunhofer UMSICHT und Forschungszentrum Jülich, IBG-2, ihr jeweiliges Fachwissen, ihre Ressourcen, Infrastrukturen sowie Vorarbeiten gezielt zusammengeführt, um im Rahmen des Projekts ertrags- und standortoptimierte Medizinalpflanzen zu etablieren und diese sowohl im Freiland als auch in geschlossenen Indoor-Systemen anzubauen. Darüberhinaus werden innovative Verfahren zur gezielten Steuerung und Erhöhung des Wirkstoffgehalts durch biologische, chemische und physikalische Stressgabe sowie zur nicht-invasiven Erfassung von Wachstums- und Ernteparametern entwickelt. Die Arbeiten des Fraunhofer IME umfassen dabei die Entwicklung molekularer Marker zur Züchtungsunterstützung und die Generierung von Zell- und Gewebekulturen ausgewählter Medizinalpflanzen. Eine unserer Modellpflanzen ist Arnica montana, die unter anderem Sesquiterpenlactone (SL) als wertgebende Inhaltsstoffe produziert. Unsere Arbeiten konzentrierten sich zunächst auf die Identifizierung der bisher unbekannten SL-Biosynthesegene und zukünftig auf deren Charakterisierung. Dafür fokussieren wir uns zurzeit auf die Etablierung der in vitro-Vermehrung von Arnika-Pflanzen sowie die pflanzliche Regeneration aus Einzelzellen und schließlich auf die genetische Modifikation von Arnica montana, um zukünftig Biosynthesegene des SL-Stoffwechsels charakterisieren und den SL-Gehalt in der Pflanze oder auch Zellkultur optimieren zu können.

Plants are adversely affected by abiotic stress conditions such as heat, drought, cold, and salinity. The physiological determinants of cotton stress tolerance and its processes were investigated to mitigate the effect of abiotic stress on cotton growth and development. The DEHYDRATION-RESPONSIVE ELEMENT BINDING protein (DREB) transcription factors are stress-responsive and regulate the expression of downstream stress-inducible genes, leading to subsequent resistance to several abiotic stressors. Advanced omics technologies have recently improved our understanding of the complex molecular events of cotton that precede abiotic stress responses. We highlight the central abiotic stress resistance mechanisms in cotton, elaborating on the identified and unidentified regulatory variables. The evolutionary relationship between DREB transcription factors (TFs), divided into six subtypes in Gossypium hirsutum, Gossypium raimondii, and Arabidopsis thaliana, was reviewed. Moreover, the evolutionary antecedents, conserved motifs, and gene structure of the cotton DREB gene family have been discussed. This review offers an in-depth perspective on the contribution of DREB TFs to cotton tolerance to abiotic stress conditions, such as cold, drought, salt, heat, and heavy metals. Overall, this review identified essential genes in Gossypium spp and other species that could hasten applied studies toward their engineering into plants to mitigate abiotic stress.

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. [ABSTRACT FROM AUTHOR]

Commonly used reporters rely on a single property, such as the fluorescence of GFP and visible color of anthocyanins, therefore these reporters hardly handle the complicated condition in practice. Betaxanthins are a group of plant natural products derived from the amino acid tyrosine. Its visible yellow-orange color and green fluorescence under blue light make it a promising new reporter. Only two enzymatic reactions are required to convert tyrosine into betaxanthins. Here, we synthesized an open reading frame named Bx that contained all the betaxanthins biosynthetic genes and demonstrated its use as a powerful and efficient reporter in tobacco, carrot, and tomato.

Copyright of Revista Colombiana de Ciencias Hortícolas is the property of Revista Colombiana de Ciencias Horticolas and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Biolistic delivery of biomolecular cargoes to plants with micron-scale projectiles is a well-established technique in plant biotechnology. However, the relatively large micron-scale biolistic projectiles can result in tissue damage, low regeneration efficiency, and create difficulties for the biolistic transformation of isomorphic small cells or subcellular target organelles (i.e., mitochondria and plastids). As an alternative to micron-sized carriers, nanomaterials provide a promising approach for biomolecule delivery to plants. While most studies exploring nanoscale biolistic carriers have been carried out in animal cells and tissues, which lack a cell wall, we can nonetheless extrapolate their utility for nanobiolistic delivery of biomolecules in plant targets. Specifically, nanobiolistics has shown promising results for use in animal systems, in which nanoscale projectiles yield lower levels of cell and tissue damage while maintaining similar transformation efficiencies as their micron-scale counterparts. In this chapter, we specifically discuss biolistic delivery of nanoparticles for plant genetic transformation purposes and identify the figures of merit requiring optimization for broad-scale implementation of nanobiolistics in plant genetic transformations.

Copyright of Horticulture / Kertgazdaság is the property of Herman Otto Intezet Nonprofit Kft. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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. [ABSTRACT FROM AUTHOR]

Serendipita indica is a root endophytic fungus and can symbiose with many plant species. Effector proteins secreted by fungi show many functions during symbiosis or infection. To investigate the role of the protein PIIN 05330 secreted by S. indica, the gene PIIN_05330 was cloned and its transgenic Arabidopsis lines were constructed. The transgenic lines were evaluated for their drought tolerance. The results showed that these transgenic Arabidopsis lines were sensitive to drought stress during the germination stage. Compared with Arabidopsis wild type (WT) and the empty vector (VT) lines seedlings, the transgenic lines had higher total root surface area, root diameters, total root volumes, relative root elongation rates, total root length, and root tip numbers. In addition, the transgenic lines showed higher aboveground biomass and chlorophyll levels. Compared with WT and VT lines, activities of SOD, POD and CAT increased in the transgenic Arabidopsis lines under drought condition, especially their CAT activities. Under drought stress, levels of AsA and GSH in the transgenic lines were higher than those in control WT and VT, and concentrations of H2O2 and MDA were significantly lower than those in control WT and VT. Under drought stress, proline concentrations increased, and total concentrations of soluble sugars and proteins significantly increased in transgenic lines, compared with control WT and VT, indicating that the transgenic lines had a higher ability to reduce plant damage and protect plant cell membrane system. All these results suggest that the effector protein PIIN_05330 improved drought tolerance of the transgenic Arabidopsis lines. [ABSTRACT FROM AUTHOR]

Agrobacteriumtumefaciens-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Key message: An optimal RNAi configuration that could restrict gene expression most efficiently was determined. This approach was also used to target PTGS and yielded higher rates of gene-editing events. Although it was characterized long ago, transgene silencing still strongly impairs transgene overexpression, and thus is a major barrier to plant crop gene-editing. The development of strategies that could prevent transgene silencing is therefore essential to the success of gene editing assays. Transgene silencing occurs via the RNA silencing process, which regulates the expression of essential genes and protects the plant from viral infections. The RNA silencing machinery thereby controls central biological processes such as growth, development, genome integrity, and stress resistance. RNA silencing is typically induced by aberrant RNA, that may lack 5′ or 3′ processing, or may consist in double-stranded or hairpin RNA, and involves DICER and ARGONAUTE family proteins. In this study, RNAi inducing constructs were designed in eleven different configurations and were evaluated for their capacity to induce silencing in Nicotiana spp. using transient and stable transformation assays. Using reporter genes, it was found that the overexpression of a hairpin consisting of a forward tandem inverted repeat that started with an ATG and that was not followed downstream by a transcription terminator, could downregulate gene expression most potently. Furthermore, using this method, the downregulation of the NtSGS3 gene caused a significant increase in transgene expression both in transient and stable transformation assays. This SGS3 silencing approach was also employed in gene-editing assays and caused higher rates of gene-editing events. Taken together, these findings suggested the optimal genetic configuration to cause RNA silencing and showed that this strategy may be used to restrict PTGS during gene-editing experiments. [ABSTRACT FROM AUTHOR]

The major challenges that agriculture is facing in the twenty-first century are increasing droughts, water scarcity, flooding, poorer soils, and extreme temperatures due to climate change. However, most crops are not tolerant to extreme climatic environments. The aim in the near future, in a world with hunger and an increasing population, is to breed and/or engineer crops to tolerate abiotic stress with a higher yield. Some crop varieties display a certain degree of tolerance, which has been exploited by plant breeders to develop varieties that thrive under stress conditions. Moreover, a long list of genes involved in abiotic stress tolerance have been identified and characterized by molecular techniques and overexpressed individually in plant transformation experiments. Nevertheless, stress tolerance phenotypes are polygenetic traits, which current genomic tools are dissecting to exploit their use by accelerating genetic introgression using molecular markers or site-directed mutagenesis such as CRISPR-Cas9. In this review, we describe plant mechanisms to sense and tolerate adverse climate conditions and examine and discuss classic and new molecular tools to select and improve abiotic stress tolerance in major crops. [ABSTRACT FROM AUTHOR]

Uptake, translocation, and transformation mechanisms of tris(2-chloroethyl) phosphate (TCEP) in hydroponic wheat ( Triticum aestivum L.) were systematically investigated using compound-specific stable isotope and multiomics analyses in this study. Results showed that TCEP was quickly adsorbed on root epidermis and then absorbed in roots via water and anion channels as well as an active process dependent on energy. Active process and anion channel preferentially translocated TCEP-containing light carbon isotopes and dominated the transmembrane transport of TCEP to enter vascular bundle. Transcriptomic and metabolomic analyses indicated gene-encoding ATP-binding cassette (ABC) transporters and purple acid phosphatases (PAPs) and glutathione S-transferases (GSTs) involved in TCEP transport and transformation, respectively. Molecular docking simulations showed that TCEP bound to the hydrophilic cavity of ABC transporter/PAP and hydrophobic cavity of GST, and hydrogen bonding was the important driving force. The results of this study offered insights for future effective mitigation of TCEP risk in edible plants.

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 K ow 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 Ca 2+ -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.

The global market of a total 42.4 billion US dollars of ornamental plants owes its worth to the color and fragrance associated with these plants. A lot of work has been done to understand the physiological, biochemical, genetic, and environmental factors responsible for the development of color and fragrance in flowers. The aim of these studies about the floral biology was to achieve the desired characters by manipulating all these factors to fulfill the changing demand of the global market. While, modifying flower color and fragrance, scientists moved from the traditional breeding methods to the comparatively modern genetic engineering techniques, yet limitations in results always remained a big issue for researchers. However, genome editing tools look promising to achieve the desired level of efficiency to modify the color and fragrance in the flowers. This review gives insight into the significance of floral characters, molecular factors responsible for these characters and applications of modern genome editing technologies for desirable modification.

The avocado, Persea americana Mill., is one of the most traded tropical fruits in the international market, furthermore, demand has significantly grown over the last decade, increasing its prices and expanding its production. In the current scenario of increasing concern about environmental impact due to the expansion of avocado orchards, climate change and epidemics of invasive pathogens, there is a need to accelerate avocado breeding programmes. Developing new avocado cultivars with novel traits is an important goal for breeders, all in a time- and cost-efficient manner. Desirable traits include the development of dwarf rootstocks, increased postharvest shelf life, salinity, and drought stress tolerance as well as resistance to major pathogens such as Phytophthora cinnamomi, Rosellinia necatrix, and Raffaelea lauricola. At the same time, genetic improvements done in avocado by the agency of traditional breeding have been moderately successful, which is why even today main cultivars still come from seedlings from open pollination. However, advancements on molecular biology and genetic engineering techniques have expanded knowledge on breeding different plants, including some perennial species, in a more efficient, precise, and targeted manner. This review summarises the available literature on avocado genetic transformation works, focusing on the methodologies, highlighting achievements and challenges. Key message: This review summarises the results of recent studies on avocado genetic transformation, highlighting milestones and challenges. It also discusses some of the new genetic engineering techniques with potential to boost functional gene characterisation and breeding programmes in avocado. [ABSTRACT FROM AUTHOR]

Abstract Background Protocols for Agrobacterium rhizogenes-mediated hairy root transformation of the model legume Lotus japonicus have been established previously. However, little efforts were made in the past to quantify and improve the transformation efficiency. Here, we asked whether effectors (nodulation outer proteins) of the nodule bacterium Sinorhizobium sp. NGR234 can promote hairy root transformation of L. japonicus. The co-expressed red fluorescent protein DsRed1 was used for visualization of transformed roots and for estimation of the transformation efficiency. Results Strong induction of hairy root formation was observed when A. rhizogenes strain LBA9402 was used for L. japonicus transformation. Expression of the effector gene nopP in L. japonicus roots resulted in a significantly increased transformation efficiency while nopL, nopM, and nopT did not show such an effect. In nopP expressing plants, more than 65% of the formed hairy roots were transgenic as analyzed by red fluorescence emitted by co-transformed DsRed1. A nodulation experiment indicated that nopP expression did not obviously affect the symbiosis between L. japonicus and Mesorhizobium loti. Conclusion We have established a novel protocol for hairy root transformation of L. japonicus. The use of A. rhizogenes LBA9402 carrying a binary vector containing DsRed1 and nopP allowed efficient formation and identification of transgenic roots.

The remarkable ability of Agrobacterium tumefaciens to transfer DNA to plant cells has allowed the generation of important transgenic crops. One challenge of A. tumefaciens-mediated transformation is eliminating the bacteria after plant transformation to prevent detrimental effects to plants and the release of engineered bacteria to the environment. Here, we use a reverse-genetics approach to identify genes involved in ampicillin resistance, with the goal of utilizing these antibiotic-sensitive strains for plant transformations. We show that treating A. tumefaciens C58 with ampicillin led to increased β-lactamase production, a response dependent on the broad-spectrum β-lactamase AmpC and its transcription factor, AmpR. Loss of the putative ampD orthologue atu2113 led to constitutive production of AmpC-dependent b-lactamase activity and ampicillin resistance. Finally, one cell wall remodeling enzyme, MltB3, was necessary for the AmpCdependent β-lactamase activity, and its loss elicited ampicillin and carbenicillin sensitivity in the A. tumefaciens C58 and GV3101 strains. Furthermore, GV3101 ΔmltB3 transforms plants with efficiency comparable to that of the wild type but can be cleared with sublethal concentrations of ampicillin. The functional characterization of the genes involved in the inducible ampicillin resistance pathway of A. tumefaciens constitutes a major step forward in efforts to reduce the intrinsic antibiotic resistance of this bacterium. [ABSTRACT FROM AUTHOR]

The standard stalwart Agrobacterium-mediated transformation protocols for sorghum use differentiating embryogenic callus induced from immature embryos in the genotype BTx430. While reliable, the standard protocols are lengthy, and both time and resource expensive. We have investigated the use of altruistic morphogenic regulator mediated transformation (MRMT) to improve sorghum transformation for use in genome editing. Here, we show a method for rapid transformation and plant regeneration of sorghum (Sorghum bicolor L. cultivar BTx430) mediated by altruistic Baby boom and Wuschel2. Confirming and extending previous recent work using altruistic MRMT, we show that the altruistic MRMT rapid protocol results in a higher transformation frequency expressed as a function of the number of independent events per immature embryos infected and requires less time in a transformation pipeline which translates to an increased number of constructs one full-time equivalent (FTE) can manage per yr. This approach is compatible with genome editing requirements and should help address the transformation bottleneck for functional genomics and genome editing studies in sorghum. [ABSTRACT FROM AUTHOR]

Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a Cas12a nuclease and four gRNAs was used for Agrobacterium-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors. [ABSTRACT FROM AUTHOR]

Chalcone isomerase (CHI), catalyzing isomerization of chalcones, is a crucial enzyme in flavonoid biosynthesis. Three CHI genes were isolated from Clivia miniata and designated as CmCHI1, CmCHI2 and CmCHI3, respectively. Multiple sequence alignments and phylogenetic analysis showed that CmCHI1 and CmCHI2 were members of type I CHI proteins, whereas CmCHI3 belonged to type IV CHI proteins. Subcellular localization analysis found that all three CmCHIs had diffused distribution in the cytoplasm similar to green fluorescent protein (GFP). Anthocyanin biosynthesis and gene expression analysis demonstrated that CmCHIs were highly expressed in anthocyanin accumulated tissues. To further functionally characterize the role of CmCHIs, an in vitro enzymatic activity assay was carried out using the purified recombinant proteins. Results showed that CmCHI1 and CmCHI2 could completely convert the substrate naringenin chalcone (NC) into the product naringenin (NA), whereas CmCHI3 seemed nonfunctional as no increment of NA was detected. Further genetic transformation of Arabidopsis tt5-1 mutant validated that CmCHI1 and CmCHI2 rather than CmCHI3 could complement the chi deficient phenotypes. In summary, CmCHI1 and CmCHI2 are the real active CHI genes in Clivia miniata. The results not only broaden our knowledge on flavonoid biosynthesis in C. miniata but also lay a new foundation for further flavonoid modification in C. miniata.

Plants are a rich source of bioactive compounds, many of which have been exploited for cosmetic, nutritional, and medicinal purposes. Through the characterization of metabolic pathways, as well as the mechanisms responsible for the accumulation of secondary metabolites, researchers have been able to increase the production of bioactive compounds in different plant species for research and commercial applications. The intent of the current review is to describe the metabolic engineering methods that have been used to transform in vitro or field-grown medicinal plants over the last decade and to identify the most effective approaches to increase the production of alkaloids. The articles summarized were categorized into six groups: endogenous enzyme overexpression, foreign enzyme overexpression, transcription factor overexpression, gene silencing, genome editing, and co-overexpression. We conclude that, because of the complex and multi-step nature of biosynthetic pathways, the approach that has been most commonly used to increase the biosynthesis of alkaloids, and the most effective in terms of fold increase, is the co-overexpression of two or more rate-limiting enzymes followed by the manipulation of regulatory genes.

Selectable markers help the transformed cell/tissue to survive in an otherwise lethal exposure to an antibiotic or herbicide. Unfortunately, almost all the traditional selectable markers are antibiotic and herbicide resistance genes, which are controversial on human health concerns and environmental impact. Novel plant-derived, non-antibiotic, and non-herbicide selectable markers are urgently needed in plant transformation. Our previous work showed that the seedlings of overexpression Arabidopsis lines of AtGASA6 survived on medium with a high concentration of sugar, which leads to the hypothesis that AtGASA6 could be a selectable marker on media with high or low sugar content. In this study, leaf explants of AtGASA6 overexpression tobacco lines regenerated shoots on sugar-free shooting medium while those of wild type could not. Moreover, the seeds of AtGASA6 overexpression tobacco lines germinated and grew into normal seedlings on sugar-free MS medium while those of WT could not. Attractively, no developmental defects were observed in AtGASA6 transgenic progenies. Using AtGASA6 as a selectable marker, overexpression tobacco lines of GAI, which restrains plant size, were created on sugar-free media. The GAI overexpression lines had a smaller plant size than that of control. Considering its plant-derived and non-antibiotic nature, GASA6 is promising to be used as a selectable marker in plant transformation. Key message: This research identifies a novel non-antibiotic selectable marker GASA6 using sugar-free selection medium, which could address ecological concerns and food safety issues. [ABSTRACT FROM AUTHOR]

Millets are small seeded cereal crops predominantly cultivated and consumed by resource-poor farmers in the semi-arid tropics of Asia and Africa. Millets possess rich nutrients and a climate resilience property when compared to the other cereals such as rice and wheat. Millet improvement using modern genetic and genomic tools is falling behind other cereal crops due to their cultivation being restricted to less developed countries. Genome editing tools have been successfully applied to major cereal crops and, as a result, many key traits have been introduced into rice, wheat and maize. However, genome editing tools have not yet been used for most millets although they possess rich nutrients. The foxtail millet is the only millet utilised up to now for genome editing works. Limited genomic resources and lack of efficient transformation systems may slow down genome editing in millets. As millets possess many important traits of agricultural importance, high resolution studies with genome editing tools will help to understand the specific mechanism and transfer such traits to major cereals in the future. This review covers the current status of genome editing studies in millets and discusses the future prospects of genome editing in millets to understand key traits of nutrient fortification and develop climate resilient crops in the future. [ABSTRACT FROM AUTHOR]

Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important ornamental ground cover plant. In this study, seeds of D. spiculifolius were used as explants for callus induction, adventitious bud differentiation, and rooting by adding different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyl aminopurine (6-BA), and naphthaleneacetic acid (NAA) to Murashige and Skoog medium. The calli generated were co-cultured with Agrobacterium tumefaciens EHA105 containing pBI121-GUS or pBI121-GFP plasmids for 30 min, and transgenic regenerated plants were obtained by kanamycin (30 mg · L−1) screening. RT-PCR confirmed the stable expression of the exogenous GUS and GFP genes in the D. spiculifolius. The β-glucuronidase (GUS) histochemical staining confirmed GUS gene expression in transgenic calli, adventitious buds, and regenerated plants of D. spiculifolius. The green fluorescent protein (GFP) visual analysis showed GFP gene expression in transgenic calli. Furthermore, subcellular localization analysis showed that the three organelle marker proteins were not only successfully expressed but also accurately localized to their corresponding organelles in D. spiculifolius callus cells. These results indicated a successful establishment of a reliable and efficient A. tumefaciens-mediated genetic transformation system, which will contribute to functional gene research and genetic improvement of D. spiculifolius.

Abstract Background Drought stress is one of the major factors limiting wheat production globally. Improving drought tolerance is important for agriculture sustainability. Although various morphological, physiological and biochemical responses associated with drought tolerance have been documented, the molecular mechanisms and regulatory genes that are needed to improve drought tolerance in crops require further investigation. We have used a novel 4-component version (for overexpression) and a 3-component version (for underexpression) of a barley stripe mosaic virus-based (BSMV) system for functional characterization of the C2H2-type zinc finger protein TaZFP1B in wheat. These expression systems avoid the need to produce transgenic plant lines and greatly speed up functional gene characterization. Results We show that overexpression of TaZFP1B stimulates plant growth and up-regulates different oxidative stress-responsive genes under well-watered conditions. Plants that overexpress TaZFP1B are more drought tolerant at critical periods of the plant’s life cycle. Furthermore, RNA-Seq analysis revealed that plants overexpressing TaZFP1B reprogram their transcriptome, resulting in physiological and physical modifications that help wheat to grow and survive under drought stress. In contrast, plants transformed to underexpress TaZFP1B are significantly less tolerant to drought and growth is negatively affected. Conclusions This study clearly shows that the two versions of the BSMV system can be used for fast and efficient functional characterization of genes in crops. The extent of transcriptome reprogramming in plants that overexpress TaZFP1B indicates that the encoded transcription factor is a key regulator of drought tolerance in wheat.

Abstract The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing technology has led to great progress in monocot research; however, several factors need to be considered for the efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes, many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA ribonucleoproteins (RNPs) is one strategy that can be used to prevent transgene issues with the expression of sgRNA and Cas proteins. RNPs can be used to efficiently generate transgene-free genome-edited crops that can reduce transgene issues related to the generation of genetically modified organisms. In this review, we introduce new techniques for genome editing and identifying marker-free genome-edited mutants in monocot crops. Four topics are covered: the design and construction of plasmids for genome editing in monocots; alternatives to SpCas9; protoplasts and CRISPR; and screening for marker-free CRISPR/Cas9-induced mutants. We have aimed to encompass a full spectrum of information for genome editing in monocot crops.

Perilla (Perilla frutescens), a traditional medicinal and oilseed crop in Asia, contains extremely high levels of polyunsaturated α-linolenic acid (ALA) (up to 60.9%) in its seeds. ALA biosynthesis is a multistep process catalyzed by fatty acid desaturases (FADs), but the FAD gene family in perilla has not been systematically characterized. Here, we identified 42 PfFADs in the perilla genome and classified them into five subfamilies. Subfamily members of PfFADs had similar exon/intron structures, conserved domain sequences, subcellular localizations, and cis-regulatory elements in their promoter regions. PfFAD s also possessed various expression patterns. PfFAD3.1 was highly expressed in the middle stage of seed development, whereas PfFAD7/8.3 and PfFAD7/8.5 were highly expressed in leaf and later stages of seed development, respectively. Phylogenetic analysis revealed that the evolutionary features coincided with the functionalization of different subfamilies of PUFA desaturase. Heterologous overexpression of PfFAD3.1 in Arabidopsis thaliana seeds increased ALA content by 17.68%–37.03%. These findings provided insights into the characteristics and functions of PfFAD genes in perilla. [ABSTRACT FROM AUTHOR]

Methylglyoxal (MG) is ubiquitously produced in all living organisms as a byproduct of glycolysis, higher levels of which are cytotoxic, leading to oxidative stress and apoptosis in the living systems. Though its generation is spontaneous but its detoxification involves glyoxalase pathway genes. Based on this understanding, the present study describes the possible role of MG as a novel non-antibiotic-based selection agent in rice. Further, by metabolizing MG, the glyoxalase pathway genes viz. glyoxalase I (GLYI) and glyoxalase II (GLYII), may serve as selection markers. Therefore, herein, transgenic rice harboring GLYI-GLYII genes (as selection markers) were developed and the effect of MG as a selection agent was assessed. The 3 mM MG concentration was observed as optimum for the selection of transformed calli, allowing efficient callus induction and proliferation along with high regeneration frequency (55 ± 2%) of the transgenic calli. Since the transformed calli exhibited constitutively higher activity of GLYI and GLYII enzymes compared to the wild type calli, the rise in MG levels was restricted even upon exogenous addition of MG during the selection process, resulting in efficient selection of the transformed calli. Therefore, MG-based selection method is a useful and efficient system for selection of transformed plants without significantly compromising the transformation efficiency. Further, this MG-based selection system is bio-safe and can pave way towards better public acceptance of transgenic plants. [ABSTRACT FROM AUTHOR]

Glyphosate-resistant crops developed by the CP4-EPSPS gene from Agrobacterium have been planted on a massive scale globally, which benefits from the high efficiency and broad spectrum of glyphosate in weed control. Some glyphosate-resistant (GR) genes from microbes have been reported, which might raise biosafety concerns. Most of them were obtained through a hygromycin- HPT transformation system. Here we reported the plant source with 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from goosegrass endowed rice with high resistance to glyphosate. The integrations and inheritability of the transgenes in the rice genome were investigated within two generations. The EiEPSPS transgenic plants displayed similar growth and development to wild type under no glyphosate selection pressure but better reproductive performance under lower glyphosate selection pressure. Furthermore, we reconstructed a binary vector pCEiEPSPS and established the whole stage glyphosate selection using the vector. The Glyphosate-pCEiEPSPS selection system showed a significantly higher transformation efficiency compared with the hygromycin- HPT transformation system. Our results provided a promising alternative gene resource to the development of GR plants and also extended the plant transformation toolbox. [ABSTRACT FROM AUTHOR]