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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]

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.

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.

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.

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., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)