Agrobacterium-mediated Transformation Method Research Articles

Transcription Factor McHB7 Improves Ice Plant Drought Tolerance through ABA Signaling Pathway.

As global climate change continues, drought episodes have become increasingly frequent. Studying plant stress tolerance is urgently needed to ensure food security. The common ice plant is one of the model halophyte plants for plant stress biology research. This study aimed to investigate the functions of a newly discovered transcription factor, Homeobox 7 (HB7), from the ice plant in response to drought stress. An efficient Agrobacterium-mediated transformation method was established in the ice plant, where ectopic McHB7 expression may be sustained for four weeks. The McHB7 overexpression (OE) plants displayed drought tolerance, and the activities of redox enzymes and chlorophyll content in the OE plants were higher than the wild type. Quantitative proteomics revealed 1910 and 495 proteins significantly changed in the OE leaves compared to the wild type under the control and drought conditions, respectively. Most increased proteins were involved in the tricarboxylic acid cycle, photosynthesis, glycolysis, pyruvate metabolism, and oxidative phosphorylation pathways. Some were found to participate in abscisic acid signaling or response. Furthermore, the abscisic acid levels increased in the OE compared with the wild type. McHB7 was revealed to bind to the promoter motifs of Early Responsive to Dehydration genes and abscisic acid-responsive genes, and protein-protein interaction analysis revealed candidate proteins responsive to stresses and hormones (e.g., abscisic acid). To conclude, McHB7 may contribute to enhance plant drought tolerance through abscisic acid signaling.

In-silico analysis and transformation of OsMYB48 transcription factor driven by CaMV35S promoter in model plant – Nicotiana tabacum L. conferring abiotic stress tolerance

ABSTRACT Global crop yield has been affected by a number of abiotic stresses. Heat, salinity, and drought stress are at the top of the list as serious environmental growth-limiting factors. To enhance crop productivity, molecular approaches have been used to determine the key regulators affecting stress-related phenomena. MYB transcription factors (TF) have been reported as one of the promising defensive proteins against the unfavorable conditions that plants must face. Different roles of MYB TFs have been suggested such as regulation of cellular growth and differentiation, hormonal signaling, mediating abiotic stress responses, etc. To gain significant insights, a comprehensive in-silico analysis of OsMYB TF was carried out in comparison with 21 dicot MYB TFs and 10 monocot MYB TFs. Their chromosomal location, gene structure, protein domain, and motifs were analyzed. The phylogenetic relationship was also studied, which resulted in the classification of proteins into four basic groups: groups A, B, C, and D. The protein motif analysis identified several conserved sequences responsible for cellular activities. The gene structure analysis suggested that proteins that were present in the same class, showed similar intron-exon structures. Promoter analysis revealed major cis-acting elements that were found to be responsible for hormonal signaling and initiating a response to abiotic stress and light-induced mechanisms. The transformation of OsMYB TF into tobacco was carried out using the Agrobacterium-mediated transformation method, to further analyze the expression level of a gene in different plant parts, under stress conditions. To summarize, the current studies shed light on the evolution and role of OsMYB TF in plants. Future investigations should focus on elucidating the functional roles of MYB transcription factors in abiotic stress tolerance through targeted genetic modification and CRISPR/Cas9-mediated genome editing. The application of omics approaches and systems biology will be indispensable in delineating the regulatory networks orchestrated by MYB TFs, facilitating the development of crop genotypes with enhanced resilience to environmental stressors. Rigorous field validation of these genetically engineered or edited crops is imperative to ascertain their utility in promoting sustainable agricultural practices.

Control of Plant Height and Lateral Root Development via Stu-miR156 Regulation of SPL9 Transcription Factor in Potato.

MicroRNAs (miRNAs) are a class of endogenous, non-coding small-molecule RNAs that usually regulate the expression of target genes at the post-transcriptional level. miR156 is one of a class of evolutionarily highly conserved miRNA families. SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor is one of the target genes that is regulated by miR156. SPL transcription factors are involved in regulating plant growth and development, hormone response, stress response, and photosynthesis. In the present study, transgenic potato plants with overexpressed miR156 were obtained via the Agrobacterium-mediated transformation method. The results showed that the expression levels of the target gene, StSPL9, were all downregulated in the transgenic plants with overexpressed Stu-miR156. Compared with those of the control plants, the plant height and root length of the transgenic plants were significantly decreased, while the number of lateral roots was significantly increased. These results revealed that the miR156/SPLs module was involved in regulating potato plant height and root growth.

Disruption of UDP-galactopyranose mutase expression: A novel strategy for regulation of galactomannan biosynthesis and monascus pigments secretion in Monascus purpureus M9

The impact of the cell wall structure of Monascus purpureus M9 on the secretion of extracellular monascus pigments (exMPs) was investigated. To modify the cell wall structure, UDP-galactopyranose mutase (GlfA) was knocked out using Agrobacterium-mediated transformation method, leading to a significant reduction in the Galf-based polysaccharide within the cell wall. Changes in mycelium morphology, sporogenesis, and the expression of relevant genes in M9 were also observed following the mutation. Regarding MPs secretion, a notable increase was observed in six types of exMPs (R1, R2, Y1, Y2, O1 and O2). Specifically, these exMPs exhibited enhancement of 1.33, 1.59, 0.8, 2.45, 2.89 and 4.03 times, respectively, compared to the wild-type strain. These findings suggest that the alteration of the cell wall structure could selectively influence the secretion of MPs in M9. The underlying mechanisms were also discussed. This research contributes new insights into the regulation of the synthesis and secretion of MPs in Monascus spp..

Production of functional recombinant roseltide rT1 antimicrobial peptide in tobacco plants

Plant-derived peptides represent a promising group of natural compounds with broad industrial and pharmaceutical applications. Low-efficiency production level is the major obstacle to the commercial production of such bioactive peptides. Today, recombinant techniques have been developed for fast and cost-effective production of high-quality peptides for various applications in the chemical and food industries. The roseltide rT1 is a plant peptide with different antimicrobial properties and therapeutic applications in the prevention and treatment of inflammatory lung diseases by inhibiting human neutrophil elastases. Here, we report the expression of functional recombinant roseltide rT1 peptide in tobacco plants. Transgenic plants were generated by the Agrobacterium-mediated transformation method followed by molecular analysis of transgenic plants to demonstrate successful integration and expression of recombinant rT1 peptide. Protein extracts of transgenic plants expressing a single-copy rT1 gene showed efficient antimicrobial properties as verified by growth inhibition of different bacterial strains. Our results illustrate that plant-derived recombinant rT1 peptide is a promising alternative for rapid and cost-effective production of this important antimicrobial peptide for application in therapeutic and food industries.

Development of an efficient Agrobacterium-mediated transformation method and its application in tryptophan pathway modification in Catharanthus roseus.

The biosynthetic pathway of Catharanthus roseus vinca alkaloids has a long research history, including not only identification of metabolic intermediates but also the mechanisms of inter-cellular transport and accumulation of biosynthesized components. Vinca alkaloids pathway begins with strictosidine, which is biosynthesized by condensing tryptamine from the tryptophan pathway and secologanin from the isoprenoid pathway. Therefore, increasing the supply of precursor tryptophan may enhance vinca alkaloid content or their metabolic intermediates. Many reports on the genetic modification of C. roseus use cultured cells or hairy roots, but few reports cover the production of transgenic plants. In this study, we first investigated a method for stably producing transgenic plants of C. roseus, then, using this technique, we modified the tryptophan metabolism system to produce transgenic plants with increased tryptophan content. Transformed plants were obtained by infecting cotyledons two weeks after sowing with Agrobacterium strain A13 containing a plant expression vector, then selecting with 1/2 B5 medium supplemented with 50 mg l-1 kanamycin and 20 mg l-1 meropenem. Sixty-eight regenerated plants were obtained from 4,200 cotyledons infected with Agrobacterium, after which genomic PCR analysis using NPTII-specific primers confirmed gene presence in 24 plants with a transformation rate of 0.6%. Furthermore, we performed transformation into C. roseus using an expression vector to join trpE8 and aroG4 genes, which are feedback-resistant mutant genes derived from Escherichia coli. The resulting transformed plants showed exactly the same morphology as the wild-type, albeit with a marked increase in tryptophan and alkaloids content, especially catharanthine in leaves.

DcGA20ox2 and DcGA2ox1 alter endogenous gibberellin contents and adjust lignin accumulation in carrot

Gibberellins (GAs) are a class of plant hormones that can affect plant growth and development. GA-oxidases are rate-limiting enzymes, which play a direct role in GA accumulation in plants. However, the roles of GA-oxidase on carrot (Daucus carota L.) taproot development are still unclear. In this study, two GA-oxidase genes, DcGA20ox2 and DcGA2ox1, were identified in carrot. Transgenic carrot plants were obtained by using Agrobacterium-mediated genetic transformation method. The results showed that overexpression of DcGA20ox2 significantly promoted the accumulation of active GAs in carrot, increased plant height, generated more branches, and enhanced xylem development. Overexpression of DcGA2ox1 significantly reduced the total contents of active GAs compared with the control group, resulting in a dwarf phenotype and markedly increased lignin content of the transgenic carrot. The expression profiling showed that the genes of GA metabolic pathway responded to the negative feedback regulation mechanism. At the same time, the expression of most genes in lignin biosynthesis and polymerization process was up-regulated, corresponding to the massive accumulation of lignin. These findings indicated that DcGA20ox2 and DcGA2ox1 affected carrot growth and development by regulating the levels of endogenous GAs. The results from current work might shed light on further studies aimed to regulate lignification in carrot and other crops.

Obtaining genetically modified winter wheat plants with partial suppression of the prolin dehydrogenase gene

Aim. Production of genetically modified plants of new promising winter wheat genotypes with partial suppression of the proline dehydrogenase gene in in vitro culture and determination of proline content in transgenic and control plants. Methods. Agrobacterium-mediated transformation in culture in vitro, molecular genetic analysis; biochemical determination of proline content; of mathematical statistics. Results. Using the method of Agrobacterium-mediated transformation of callus cultures of new promising genotypes of winter wheat, transgenic plants carrying a double-stranded RNA suppressor of the proline dehydrogenase gene were obtained. The frequency of transformation with the use of the LBA 4404 strain in the studied genotypes was 1.7-2.0 %, and with the use of the AGL0 strain, it was 2.0-2.3 %. It was established that plants with reduced activity of proline dehydrogenase are characterized by a significantly higher content of free L-proline compared to the control. Conclusions. The relatively greater efficiency of using the AGL0 strain for obtaining transgenic plants of winter wheat genotypes with partial suppression of the proline dehydrogenase gene in in vitro culture is shown. The presence of a double-stranded RNA suppressor of the ProDH gene in transgenic plants leads to an increase in the level of free L-proline accumulation.

Enhancing Maize Transformation and Targeted Mutagenesis through the Assistance of Non-Integrating Wus2 Vector.

Efficient genetic transformation is a prerequisite for rapid gene functional analyses and crop trait improvements. We recently demonstrated that new T-DNA binary vectors with NptII/G418 selection and a compatible helper plasmid can efficiently transform maize inbred B104 using our rapid Agrobacterium-mediated transformation method. In this work, we implemented the non-integrating Wuschel2 (Wus2) T-DNA vector method for Agrobacterium-mediated B104 transformation and tested its potential for recalcitrant inbred B73 transformation and gene editing. The non-integrating Wus2 (NIW) T-DNA vector-assisted transformation method uses two Agrobacterium strains: one carrying a gene-of-interest (GOI) construct and the other providing an NIW construct. To monitor Wus2 co-integration into the maize genome, we combined the maize Wus2 expression cassette driven by a strong constitutive promoter with a new visible marker RUBY, which produces the purple pigment betalain. As a GOI construct, we used a previously tested CRISPR-Cas9 construct pKL2359 for Glossy2 gene mutagenesis. When both GOI and NIW constructs were delivered by LBA4404Thy- strain, B104 transformation frequency was significantly enhanced by about two-fold (10% vs. 18.8%). Importantly, we were able to transform a recalcitrant inbred B73 using the NIW-assisted transformation method and obtained three transgene-free edited plants by omitting the selection agent G418. These results suggest that NIW-assisted transformation can improve maize B104 transformation frequency and provide a novel option for CRISPR technology for transgene-free genome editing.

Construction of versatile yeast plasmid vectors transferable by Agrobacterium-mediated transformation and their application to bread-making yeast strains

Agrobacterium-mediated transformation (AMT) potentially has great advantages over other DNA introduction methods: e.g., long DNA and numerous recipient strains can be dealt with at a time merely by co-cultivation with donor Agrobacterium cells. However, AMT was applied only to several laboratory yeast strains, and has never been considered as a standard gene-introduction method for yeast species. To disseminate the AMT method in yeast species, it is necessary to develop versatile AMT plasmid vectors including shuttle type ones, which have been unavailable yet for yeasts. In this study, we constructed a series of AMT plasmid vectors that consist of replicative (shuttle)- and integrative-types and harbor a gene conferring resistance to either G418 or aureobasidin A for application to prototrophic yeast strains. The vectors were successfully applied to five industrial yeast strains belonging to Saccharomyces cerevisiae after a modification of a previous AMT protocol, i.e., simply inputting a smaller number of yeast cells to the co-cultivation than that in the previous protocol. The revised protocol enabled all five yeast strains to generate recombinant colonies not only at high efficiency using replicative-type vectors, but also readily at an efficiency around 10-5 using integrative one. Further modification of the protocol demonstrated AMT for multiple yeast strains at a time with less labor. Therefore, AMT would facilitate molecular genetic approaches to many yeast strains in basic and applied sciences.

An efficient and reproducible Agrobacterium-mediated genetic transformation method for the ornamental monocotyledonous plant Ornithogalum dubium Houtt

Ornithogalum is a genus from the Hyacinthaceae (Asparagaceae) family that comprises about 200 species with remarkable white, yellow, or orange flowers that display exceptional vase life. These properties have made it a popular cut flower and pot plant. Forward genetics approaches may be advantageous to generate novel phenotypes, but the Agrobacterium-mediated transformation of plants from this genus remains challenging. Here, a stable and efficient Agrobacterium-mediated transformation system was established for O. dubium. We found that the timing of transformation with respect to light exposure of the tissue affected transformation rates more than other tested parameters. In the transgenic plants obtained, T-DNA integrations were confirmed by polymerase chain reactions and positive plants were established in the greenhouse and displayed weak transgene expression. This study exposed an efficient platform for gene function research and germplasm improvement in O. dubium plants. The present protocol is now available for the development of novel improved O. dubium varieties.

A modified Agrobacterium-mediated transformation for two oomycete pathogens.

Oomycetes are a group of filamentous microorganisms that include some of the biggest threats to food security and natural ecosystems. However, much of the molecular basis of the pathogenesis and the development in these organisms remains to be learned, largely due to shortage of efficient genetic manipulation methods. In this study, we developed modified transformation methods for two important oomycete species, Phytophthora infestans and Plasmopara viticola, that bring destructive damage in agricultural production. As part of the study, we established an improved Agrobacterium-mediated transformation (AMT) method by prokaryotic expression in Agrobacterium tumefaciens of AtVIP1 (VirE2-interacting protein 1), an Arabidopsis bZIP gene required for AMT but absent in oomycetes genomes. Using the new method, we achieved an increment in transformation efficiency in two P. infestans strains. We further obtained a positive GFP transformant of P. viticola using the modified AMT method. By combining this method with the CRISPR/Cas12a genome editing system, we successfully performed targeted mutagenesis and generated loss-of-function mutations in two P. infestans genes. We edited a MADS-box transcription factor-encoding gene and found that a homozygous mutation in MADS-box results in poor sporulation and significantly reduced virulence. Meanwhile, a single-copy avirulence effector-encoding gene Avr8 in P. infestans was targeted and the edited transformants were virulent on potato carrying the cognate resistance gene R8, suggesting that loss of Avr8 led to successful evasion of the host immune response by the pathogen. In summary, this study reports on a modified genetic transformation and genome editing system, providing a potential tool for accelerating molecular genetic studies not only in oomycetes, but also other microorganisms.

Establishment of an Efficient Agrobacterium-Mediated Genetic Transformation System to Enhance the Tolerance of the Paraquat Stress in Engineering Goosegrass (Eleusine Indica L.)

Eleusine indica (goosegrass) is a problematic weed worldwide known for its multi-herbicide tolerance/resistance biotype. However, a genetic transformation method in goosegrass has not been successfully established, making a bottleneck for functional genomics studies in this species. Here, we report a successful Agrobacterium-mediated transformation method for goosegrass. Firstly, we optimized conditions for breaking seed dormancy and increasing seed germination rate. A higher callus induction rate from germinated seeds was obtained in N6 than in MS or B5 medium. Then the optimal transformation efficiency of the gus reporter gene was obtained by infection with Agrobacterium tumefaciens culture of OD600 = 0.5 for 30 min, followed by 3 days of co-cultivation with 300 μmol/L acetosyringone. Concentrations of 20 mg L-1 kanamycin and 100 mg L-1 timentin were used to select the transformed calli. The optimal rate of regeneration of the calli was generated by using 0.50 mg L-1 6-BA and 0.50 mg L-1 KT in the culture medium. Then, using this transformation method, we overexpressed the paraquat-resistant EiKCS gene into a paraquat-susceptible goosegrass biotype MZ04 and confirmed the stable inheritance of paraquat-resistance in the transgenic goosegrass lines. This approach may provide a potential mechanism for the evolution of paraquat-resistant goosegrass and a promising gene for the manipulation of paraquat-resistance plants. This study is novel and valuable in future research using similar methods for herbicide resistance.

Transformation and expressional studies of GaZnF gene to improve drought tolerance in Gossypium hirsutum

Drought stress is the major limiting factor in plant growth and production. Cotton is a significant crop as textile fiber and oilseed, but its production is generally affected by drought stress, mainly in dry regions. This study aimed to investigate the expression of Zinc finger transcription factor’s gene (GaZnF) to enhance the drought tolerance in Gossypium hirsutum. Sequence features of the GaZnF protein were recognized through different bioinformatics tools like multiple sequence alignment analysis, phylogenetic tree for evolutionary relationships, Protein motifs, a transmembrane domain, secondary structure and physio-chemical properties indicating that GaZnF is a stable protein. CIM-482, a local Gossypium hirsutum variety was transformed with GaZnF through Agrobacterium-mediated transformation method with 2.57% transformation efficiency. The integration of GaZnF was confirmed through Southern blot showing 531 bp, and Western blot indicated a 95 kDa transgene-GUS fusion band in transgenic plants. The normalized real-time expression analysis revealed the highest relative fold spatial expression of cDNA of GaZnF within leaf tissues at vegetative and flowering stages under drought stress. Morphological, physiological and biochemical parameters of transgenic cotton plants at 05- and 10-day drought stress was higher than those of non-transgenic control plants. The values of fresh and dry biomass, chlorophyll content, photosynthesis, transpiration rate, and stomatal conductance reduced in GaZnF transgenic cotton plants at 05- and 10-day drought stress, but their values were less low in transgenic plants than those of non-transgenic control plants. These findings showed that GaZnF gene expression in transgenic plants could be a valuable source for the development of drought-tolerant homozygous lines through breeding.

Engineering of cry genes “Cry11 and Cry1h” in cotton (Gossypium hirsutum L.) for protection against insect pest attack

Cotton is white gold and contributes significantly to the economy of the countries. The process of evolution of resistance in various cotton insects directly affects the life of people. Among the cotton insect pests, pink bollworm (Pectinophora gossypiella) and army bollworm (Spodoptera litura) have turned out to be destructive. The development of seed using gene stacking approach has tremendously reduced the applications of agrochemicals making the technology greener for society. A gene cassette containing Cry11 and Cry1H genes was constructed. The cassette was cloned into the pCAMBIA2300 plant expression vector using the CAMV3-35S promoter. The construct was transformed into cotton line FBS-222 using the shoot apex-cut Agrobacterium-mediated transformation method. The expression of the Cry genes was 3-fold higher in transgenic than the non-transgenic cotton plants using qRT-PCR. Finally, the insect bioassay with transgenic cotton showed 90% mortality against pink bollworms and 80% against cotton army bollworms. The results demonstrate the development of seeds using the gene pyramiding approach is an effective strategy to control insect pest attacks.

Subchronic Toxicological Evaluation of Xiushui 134Bt Transgenic Insect-Resistant Rice in Rats

In this study, Xiushui 134Bt, a highly insect-resistant rice transgenic material with stable expression of CryIAc1 gene in rice plants and no significant changes in main agronomic traits, was obtained by means of Agrobacterium-mediated transformation method using japonica rice Xiushui 134 as receptor material. Biosecurity of the transgenic rice Xiushui 134Bt was assessed by rat feeding experiments. Wistar rats were fed a nutritionally balanced diet supplemented with 35% Genetically Modified (LGM) or 70% Genetically Modified (HGM) Xiushui 134Bt, respectively, for 90 days. Compared with the wild-type group (Xiushui 134), there were no significant differences in body weight, total food intake, food conversion rate, relative organ weight, blood routine, blood physiological or biochemical parameters, or histopathological examination between LGM and HGM Bt groups (p > 0.05). These results indicate that the transgenic rice Xiushui 134Bt is non-toxic in laboratory animals and provide guidance for the future commercial release of the transgenic rice Xiushui 134Bt in China.

Efficient Regeneration and Agrobacterium-Mediated Transformation Method For Cultivated and Wild Tomato

Efficient Regeneration and Agrobacterium-Mediated Transformation Method For Cultivated and Wild Tomato

Genetic engineering of complex feed enzymes into barley seed for direct utilization in animal feedstuff.

Currently, feed enzymes are primarily obtained through fermentation of fungi, bacteria, and other microorganisms. Although the manufacturing technology for feed enzymes has evolved rapidly, the activities of these enzymes decline during the granulating process and the cost of application has increased over time. An alternative approach is the use of genetically modified plants containing complex feed enzymes for direct utilization in animal feedstuff. We co-expressed three commonly used feed enzymes (phytase, β-glucanase, and xylanase) in barley seeds using the Agrobacterium-mediated transformation method and generated a new barley germplasm. The results showed that these enzymes were stable and had no effect on the development of the seeds. Supplementation of the basal diet of laying hens with only 8% of enzyme-containing seeds decreased the quantities of indigestible carbohydrates, improved the availability of phosphorus, and reduced the impact of animal production on the environment to an extent similar to directly adding exogenous enzymes to the feed. Feeding enzyme-containing seeds to layers significantly increased the strength of the eggshell and the weight of the eggs by 10.0%-11.3% and 5.6%-7.7% respectively. The intestinal microbiota obtained from layers fed with enzyme-containing seeds was altered compared to controls and was dominated by Alispes and Rikenella. Therefore, the transgenic barley seeds produced in this study can be used as an ideal feedstuff for use in animal feed.

Multiplex CRISPR/Cas9 gene-editing platform in oil palm targeting mutations in EgFAD2 and EgPAT genes

BackgroundCRISPR/Cas9 is the most powerful and versatile genome-editing tool that permits multiplexed-targeted gene modifications for the genetic enhancement of oil palm. Multiplex genome-editing has recently been developed for modifying multiple loci in a gene or multiple genes in a genome with high precision. This study focuses on the development of high-oleic oil palm, the primary target trait for healthy low-saturated oil. To achieve this, the fatty acid desaturase 2 (FAD2) and palmitoyl-acyl carrier protein thioesterase (PAT) genes, both of which are associated with fatty acid metabolism biosynthesis pathways in oil palm, need to be knocked out. The knockout of FAD2 and PAT leads to an accumulation of oleic acid content in oil palms. ResultsA total of four single-guide RNAs (sgRNAs) were designed in silico based on the genomic sequences of EgFAD2 and EgPAT. Using robust plant CRISPR/Cas9 vector technology, multiple sgRNA expression cassettes were efficiently constructed into a single-binary CRISPR/Cas9 vector to edit the EgFAD2 and EgPAT genes. Each of the constructed transformation vectors was then delivered into oil palm embryogenic calli using the biolistic, Agrobacterium-mediated, and PEG-mediated protoplast transformation methods. Sequence analysis of PCR products from 15 samples confirmed that mutations were introduced at four target sites of the oil palm EgFAD2 and EgPAT genes. Single- and double-knockout mutants of both genes were generated, with large and small deletions within the targeted regions. Mutations found at EgFAD2 and EgPAT target sites indicate that the Cas9/sgRNA genome-editing system effectively knocked out both genes in oil palm. ConclusionThis technology is the first in oil palm to use CRISPR/Cas9 genome-editing to target high-oleic-associated genes. These findings showed that multiplex genome-editing in oil palm could be achieved using multiple sgRNAs. Targeted mutations detected establish that the CRISPR/Cas9 technology offers a great potential for oil palm.

Base Editing in Poplar Through an Agrobacterium-Mediated Transformation Method.

CRISPR-Cas9 systems have revolutionized genome editing in plants and facilitated gene knockout and functional genomic studies in woody plants, like poplar. However, in tree species, previous studies have only focused on targeting indel mutations through CRISPR-based nonhomologous end joining (NHEJ) pathway. Cytosine base editors (CBEs) and adenine base editors (ABEs) enableC-to-T and A-to-G base changes, respectively. These base editors can introduce premature stop codons and amino acid changes, alter RNA splicing sites, and edit cis-regulatory elements of promoters. Base editing systems have only been recently established in trees. In this chapter, we describe a detailed, robust, and thoroughly tested protocol for preparing T-DNA vectors with two highly efficient CBEs, PmCDA1-BE3 and A3A/Y130F-BE3, and the highly efficient ABE8e as well as delivering the T-DNA through an improved protocol for Agrobacterium-mediated transformation in poplar. This chapter will provide promising application potential for precise base editing in poplar and other trees.