Genomic consequences associated with Agrobacterium-mediated transformation of plants.

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.

Structure-Dependent Distribution, Metabolism, and Toxicity Effects of Alkyl Organophosphate Esters in Lettuce (Lactuca sativa L.).

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.

Efficient CRISPR-mediated base editing in Agrobacterium spp.

Agrobacterium spp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. Although Agrobacterium spp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis of Agrobacterium has been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of both Agrobacterium tumefaciens and Agrobacterium rhizogenes As an example, we generated EHA105 strains with loss-of-function mutations in recA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development by A. rhizogenes K599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of "engineering the engineer," leading to improved Agrobacterium strains for more efficient plant transformation and gene editing.

Engineered Agrobacterium improves transformation by mitigating plant immunity detection.

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.

Reasons and riddance of Agrobacterium tumefaciens overgrowth in plant transformation.

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.

Novel Insights into Uptake, Translocation, and Transformation Mechanisms of 2,2',4,4'-Tetra Brominated Diphenyl Ether (BDE-47) in Wheat (Triticum aestivum L.): Implication by Compound-Specific Stable Isotope and Transcriptome Analysis.

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.

Cre-mediated autoexcision of selectable marker genes in soybean, cotton, canola and maize transgenic plants.

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.

Application of Nanotechnology in Plant Genetic Engineering.

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.

"Pathomorphogenic" Changes Caused by Citrus Bark Cracking Viroid and Transcription Factor TFIIIA-7ZF Variants Support Viroid Propagation in Tobacco.

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.

Induction of AmpC-Mediated ß-Lactam Resistance Requires a Single Lytic Transglycosylase in Agrobacterium tumefaciens.

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 ß-lactamase production, a response dependent on the broad-spectrum ß-lactamase AmpC and its transcription factor, AmpR. Loss of the putative ampD orthologue atu2113 led to constitutive production of AmpC-dependent ß-lactamase activity and ampicillin resistance. Finally, one cell wall remodeling enzyme, MltB3, was necessary for the AmpC-dependent ß-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. IMPORTANCE Agrobacterium tumefaciens, a significant biotechnological tool for production of transgenic plant lines, is highly resistant to a wide variety of antibiotics, posing challenges for various applications. One challenge is the efficient elimination of A. tumefaciens from transformed plant tissue without using levels of antibiotics that are toxic to the plants. Here, we present the functional characterization of genes involved in ß-lactam resistance in A. tumefaciens. Knowledge about proteins that promote or inhibit ß-lactam resistance will enable the development of strains to improve the efficiency of Agrobacterium-mediated plant genetic transformations. Effective removal of Agrobacterium from transformed plant tissue has the potential to maximize crop yield and food production, improving the outlook for global food security.

Identification of Novel Organophosphate Esters in Hydroponic Lettuces (Lactuca sativa L.): Biotransformation and Acropetal Translocation.

The absorption, translocation, and biotransformation behaviors of organophosphate esters (OPEs) and diesters (OPdEs) in a hydroponic system were investigated. The lateral root was found as the main accumulation and biotransformation place of OPEs and OPdEs in lettuce. The nontarget analysis using high-resolution mass spectrometry revealed five hydroxylated metabolites and five conjugating metabolites in the OPE exposure group, among which methylation, acetylation, and palmitoyl conjugating OPEs were reported as metabolites for the first time. Particularly, methylation on phosphate can be a significant process for plant metabolism, and methyl diphenyl phosphate (MDPP) accounted for the majority of metabolites. The translocation factor values of most identified OPE metabolites are negatively associated with their predicted logarithmic octanol-water partitioning coefficient (log Kow) values (0.75-2.45), indicating that hydrophilicity is a dominant factor in the translocation of OPE metabolites in lettuce. In contrast, palmitoyl conjugation may lead to an enhanced acropetal translocation and those with log Kow values < 0 may have limited translocation potential. Additionally, OPE diesters produced from the biotransformation of OPEs in lettuce showed a higher acropetal translocation potential than those exposed directly. These results further emphasize the necessity to consider biotransformation as an utmost important factor in the accumulation and acropetal translocation potential of OPEs in plants.

Genome-editing in millets: current knowledge and future perspectives.

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.

Increased rates of gene-editing events using a simplified RNAi configuration designed to reduce gene silencing.

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.

An Efficient Agrobacterium-Mediated Transformation Method for Hybrid Poplar 84K (Populus alba × P. glandulosa) Using Calli as Explants.

A highly efficient Agrobacterium-mediated transformation method is needed for the molecular study of model tree species such as hybrid poplar 84K (Populus alba × P. glandulosa cv. '84K'). In this study, we report a callus-based transformation method that exhibits high efficiency and reproducibility. The optimized callus induction medium (CIM1) induced the development of calli from leaves with high efficiency, and multiple shoots were induced from calli growing on the optimized shoot induction medium (SIM1). Factors affecting the transformation frequency of calli were optimized as follows: Agrobacterium concentration sets at an OD600 of 0.6, Agrobacterium infective suspension with an acetosyringone (AS) concentration of 100 µM, infection time of 15 min, cocultivation duration of 2 days and precultivation duration of 6 days. Using this method, transgenic plants are obtained within approximately 2 months with a transformation frequency greater than 50%. Polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR) and ß-galactosidase (GUS) histochemical staining analyses confirmed the successful generation of stable transformants. Additionally, the calli from leaves were subcultured and used to obtain new explants; the high transformation efficiency was still maintained in subcultured calli after 6 cycles. This method provides a reference for developing effective transformation protocols for other poplar species.

Having a Same Type IIS Enzyme's Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis.

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.

Biotechnological Advances to Improve Abiotic Stress Tolerance in Crops.

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.

Crocus transcription factors CstMYB1 and CstMYB1R2 modulate apocarotenoid metabolism by regulating carotenogenic genes.

KEY MESSAGE: Two MYB genes have been identified which regulate apocarotenoid metabolism in Crocus sativus. Apocarotenoids like crocin, picrocrocin and safranal are restricted to genus Crocus and are synthesized by oxidative cleavage of zeaxanthin followed by glycosylation reactions. In Crocus sativus, these apocarotenoids are synthesized in stigma part of the flower in developmentally regulated manner. Most of the genes of apocarotenoid pathway are known, however, the mechanism that regulates its tissue and stage specific biosynthesis remains elusive. MYB family was identified as the largest transcription factor family from Crocus transciptome which indicated its possible role in apocarotenoid regulation besides regulating other metabolic pathways. Towards this, we started with identification of 150 MYB genes from Crocus transcriptome databases. The phylogenetic analysis of Crocus MYB genes divided them into 27 clusters. Domain analysis resulted in identification of four groups of MYBs depending upon the number of R repeats present. Expression profiling indicated that 12 MYBs are upregulated in stigma out of which expression of four genes CstMYB1, CstMYB14, CstMYB16 and CstMYB1R2 correlated with crocin accumulation. Transient overexpression of two nuclear localized MYB genes (CstMYB1 and CstMYB1R2) in Crocus confirmed their role in regulating carotenoid metabolism. Yeast-one-hybrid confirmed that CstMYB1 binds to carotenoid cleavage dioxygenase 2 (CCD2) promoter while CstMYB1R2 binds to phytoene synthase (PSY) and CCD2 promoters. Overall, our study established that CstMYB1 and CstMYB1R2 regulate apocarotenoid biosynthesis by directly binding to promoters of pathway genes.

Perspectives on new opportunities for nano-enabled strategies for gene delivery to plants using nanoporous materials.

MAIN CONCLUSION: Engineered nanocarriers have great potential to deliver different genetic cargos to plant cells and increase the efficiency of plant genetic engineering. Genetic engineering has improved the quality and quantity of crops by introducing desired DNA sequences into the plant genome. Traditional transformation strategies face constraints such as low transformation efficiency, damage to plant tissues, and genotype dependency. Smart nanovehicle-based delivery is a newly emerged method for direct DNA delivery to plant genomes. The basis of this new approach of plant genetic transformation, nanomaterial-mediated gene delivery, is the appropriate protection of transferred DNA from the nucleases present in the cell cytoplasm through the nanocarriers. The conjugation of desired nucleic acids with engineered nanocarriers can solve the problem of genetic manipulation in some valuable recalcitrant plant genotypes. Combining nano-enabled genetic transformation with the new and powerful technique of targeted genome editing, CRISPR (clustered regularly interspaced short palindromic repeats), can create new protocols for efficient improvement of desired plants. Silica-based nanoporous materials, especially mesoporous silica nanoparticles (MSNs), are currently regarded as exciting nanoscale platforms for genetic engineering as they possess several useful properties including ordered and porous structure, biocompatibility, biodegradability, and surface chemistry. These specific features have made MSNs promising candidates for the design of smart, controlled, and targeted delivery systems in agricultural sciences. In the present review, we discuss the usability, challenges, and opportunities for possible application of nano-enabled biomolecule transformation as part of innovative approaches for target delivery of genes of interest into plants.

Expression of lavender AGAMOUS-like and SEPALLATA3-like genes promote early flowering and alter leaf morphology in Arabidopsis thaliana.

MAIN CONCLUSION: The expression of full-length cDNAs encoding lavender AGAMOUS-like (LaAG-like) and SEPALLATA3-like (LaSEP3-like) transcription factors induces early flowering and impacts the leaf morphology at a strong expression level in Arabidopsis. Lavandula angustifolia is widely cultivated as an ornamental plant due to its attractive flower structure, and as a source of valuable essential oils for use in cosmetics, alternative medicines, and culinary products. We recently employed RNA-Seq and transcript profiling to describe a number of transcription factors (TFs) that potentially control flower development in this plant. In this study, we investigated the roles of two TFs, LaAGAMOUS-like (LaAG-like) and LaSEPALLATA3-like (LaSEP3-like), that exhibited substantial homology to Arabidopsis thaliana floral development genes, AGAMOUS and SEPALLATA3, respectively, in flowering initiation in Arabidopsis. We stably and constitutively expressed LaAG-like and LaSEP3-like cDNAs in separate Arabidopsis plants. All transgenic plants flowered earlier than the wild-type controls. However, plants that modestly overexpressed the gene were phenotypically normal, while those that strongly expressed the transgene developed curly leaves. We also assessed the expression of five endogenous flowering time regulating genes, from which high expression of Flowering Locus T (AtFT) mRNA in both LaAG-like (type-I and -II) and LaSEP3-like (type-I), and Leafy (AtLFY) mRNAs in LaSEP3-like (type-I) transgenic plants were detected, compared to wild-type controls. Our results suggest that with controlled expression, lavender AG-like and SEP3-like genes are potentially useful for the regulation of flowering time in commercial lavender species, and could be used for plant improvement studies through molecular genetics and targeted breeding programs.

Agrobacterium T-DNA integration in somatic cells does not require the activity of DNA polymerase θ.

Integration of Agrobacterium tumefaciens transferred DNA (T-DNA) into the plant genome is the last step required for stable plant genetic transformation. The mechanism of T-DNA integration remains controversial, although scientists have proposed the participation of various nonhomologous end-joining (NHEJ) pathways. Recent evidence suggests that in Arabidopsis, DNA polymerase θ (PolQ) may be a crucial enzyme involved in T-DNA integration. We conducted quantitative transformation assays of wild-type and polQ mutant Arabidopsis and rice, analyzed T-DNA/plant DNA junction sequences, and (for Arabidopsis) measured the amount of integrated T-DNA in mutant and wild-type tissue. Unexpectedly, we were able to generate stable transformants of all tested lines, although the transformation frequency of polQ mutants was c. 20% that of wild-type plants. T-DNA/plant DNA junctions from these transformed rice and Arabidopsis polQ mutants closely resembled those from wild-type plants, indicating that loss of PolQ activity does not alter the characteristics of T-DNA integration events. polQ mutant plants show growth and developmental defects, perhaps explaining previous unsuccessful attempts at their stable transformation. We suggest that either multiple redundant pathways function in T-DNA integration, and/or that integration requires some yet unknown pathway.